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The present study aimed to assess the association between the cribriform pattern (CP)/intraductal carcinoma (IDC) and the adverse pathological and clinical outcomes in the radical prostatectomy (RP) cohort. A systematic search was performed according to the Preferred Reporting Items for Systematic Review and Meta-Analysis statement (PRISMA). The protocol from this review was registered on the PROSPERO platform. We searched PubMed(r), the Cochrane Library and EM-BASE(r) up to the 30th of April 2022. The outcomes of interest were the extraprostatic extension (EPE), seminal vesicle invasion (SVI), lymph node metastasis (LNS met), risk of biochemical recurrence (BCR), distant metastasis (MET) and disease-specific death (DSD). As a result, we identified 16 studies with 164 296 patients. A total of 13 studies containing 3254 RP patients were eligible for the meta-analysis. The CP/IDC was associated with adverse outcomes, including EPE (pooled OR = 2.55, 95%CI 1.23-5.26), SVI (pooled OR = 4.27, 95%CI 1.90-9.64), LNs met (pooled OR = 6.47, 95%CI 3.76-11.14), BCR (pooled OR = 5.09, 95%CI 2.23-11.62) and MET/DSD (pooled OR = 9.84, 95%CI 2.75-35.20, p < 0.001). In conclusion, the CP/IDC belong to highly malignant prostate cancer patterns which have a negative impact on both the pathological and clinical outcomes. The presence of the CP/IDC should be included in the surgical planning and postoperative treatment guidance. prostate cancer cribriform pattern intraductal carcinoma meta-analysis radical prostatectomy This research received no external funding. pmc1. Introduction Prostate cancer (PCa) is the second most common male malignancy worldwide . The introduction of the prostate-specific antigen (PSA) at the end of the 1980s has revolutionized both the diagnosis and the management of the disease. The subsequent years brought a constant decline in prostate cancer-specific mortality in the US . At the same time, numbers of indolent PCa soared and resembled overdiagnosis. It was followed by overtreatment. The mainstay therapy for localized PCa remains either a radical prostatectomy (RP) or radiotherapy. Unfortunately, the two modalities are widely known to be associated with significant morbidity. Therefore, the protocols of active surveillance (AS) aiming at postponing the treatment were eagerly implemented across the world . AS oncological safety has been shown in many studies, and it has become the pivotal therapy of low-risk disease. The success has encouraged a number of investigators to explore the role of AS in intermediate-risk PCa and expand the indications above the lowest risk category. To avoid harm, potential predictive markers of the AS outcome in this specific group of PCa patients are currently under scrutiny. The major advantage of RP is the postoperative histopathological assessment that provides the highest possible accuracy of the staging and grading of PCa. A detailed report embraces valuable information regarding a patient's prognosis which aids in postoperative clinical decision making. The pathological features in the RP specimen that have a confirmed impact on the treatment outcome include the cancer grade and stage, positive surgical margins (PSM), and lymph node metastasis (LNs met) . In addition to the prognosticators, there are other emerging pathological features that appear to compromise the oncological outcomes. The 2014 International Society of Urological Pathology Consensus Conference on the Gleason Grading of Prostatic Carcinoma stated that the cribriform pattern (CP) should be considered part of the spectrum of the Gleason Grade 4 pattern along with glomeruloid, fused and poorly formed glands . Previous studies indicate its association with the extraprostatic extension (EPE), PSM, biochemical recurrence (BCR), distant metastasis (MET) and disease-specific death (DSD). Another pathological PCa entity that may have a dismal prognosis is intraductal carcinoma (IDC). In 2006, Guo and Epstein published the most commonly used morphological description of IDC . The WHO Classification of Prostatic Tumors in 2016, for the first time, recognized IDC as a new entity and provided a detailed histopathological description of it. . The incidence of IDC in an RP specimen ranges from 20-40%, depending on the tumor grade and stage . The studies conducted so far associate the presence of IDC with the Gleason pattern (GP) 4 and 5, a more advanced tumor stage, an increased risk of BCR and shorter MET-free survival . From a pathological point of view, IDC and the CP show architectural similarities and, as a result, can sometimes be misinterpreted. It has been confirmed that they might coexist in the same tumor . Therefore, in equivocal cases, additional immunohistochemical staining for basal cells is recommended. Despite that, sometimes it is impossible to distinguish between the two patterns. In the present study, we aimed to investigate the correlation of both the CP and IDC with pathological as well as clinical outcomes by performing a systematic review and meta-analysis of the published data. 2. Materials and Methods 2.1. Pathological Definition In the present study, only articles with pathological assessment based on the 2005 or 2014 ISUP guidelines were analyzed. The 2005 ISUP guidelines recommended CP to be graded as GP4 but allowed some cribriform glands to be included as GP3 . Among various changes that the 2014 ISUP conference brought to the pathological assessment of the RP specimen, one of the most important was the recommendation to include all cribriform glands as one of the GP4 spectrum morphologies . However, until 2021, there was no uniform definition of CP. Nonetheless, a study by Kweldam et al. proved that CP had good interobserver reproducibility among pathologists, unlike other Gleason 4 patterns, such as ill-formed or fused glands. To further improve the pathological assessment of CP, in 2021, ISUP provided consensus definition of CP. Currently, CP is defined as a confluent sheet of contiguous malignant epithelial cells with multiple glandular lumina that are easily visible at low power. There should be no intervening stroma or mucin separating individual or fused glandular structures . In 2016, WHO recognized IDC as a new entity, not included in the Gleason classification, and defined it as an intra-acinar and/or intraductal neoplastic epithelial proliferation that has some features of high-grade prostatic intraepithelial neoplasia (HGPIN), exhibiting much greater architectural and/or cytological atypia, typically associated with high-grade and/or high-stage PCa . The 2022 EAU Prostate Cancer Guidelines require reporting of both patterns in prostate biopsy and RP specimens. These guidelines, however, do not specify whether immunohistochemical staining is demanded to distinguish those entities . Even the absence of basal cells cannot be considered pathognomonic for invasive CP as they may not be visible on a particular slide . CP and IDC can morphologically mimic each other and even belong to a pathological and biological continuum . Kryvenko et al. found that the frequency of IDC was related to the proportion of CP. Kweldam et al. reported that CP and IDC tend to coexist in both biopsy and RP specimens. Therefore, we decided to assess both entities CP and IDC together. 2.2. Evidence Acquisition This systematic review and meta-analysis were conducted in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-analyses (PRISMA) guidelines . The protocol was registered in PROSPERO (CRD42020183346). The research question for this systematic review was established in the PICO (population, intervention, comparison and outcomes) framework, which was as follows: What is the prognostic value of PCa patterns: CP/IDC for clinical and pathological outcomes in men who underwent radical prostatectomy? 2.3. Literature Search A thorough literature search was conducted, PubMed(r), the Cochrane Library and EMBASE(r) databases, with the following keywords: ("prostatic neoplasms" OR "prostate cancer" OR "pca" OR "prostate" OR "prostatic") AND ("intraductal" OR "intraductal cancer" OR "intraductal carcinoma" OR "IDC" OR "cribriform" OR "cribriforming"). The search was restricted to English language publications and included every article fulfilling the inclusion criteria from the inception of the databases until 30 April 2022. The Mendeley Desktop version 1.19.4 citation manager was used to store records and remove duplicates. For completeness, references to incorporated articles and review articles were also extensively searched. Two investigators (R.O. and M.K.) independently screened and assessed article eligibility based on their titles and abstracts, adopting the PICO approach. Any disagreements were resolved by discussion with a senior author (J.D.). Eligible were original studies with a cohort of patients with PCa, whose initial treatment modality was RP (surgical approach was not considered). Additional inclusion criteria were a comparison of adverse pathological/clinical outcomes between PCa with and without CP/IDC. Both investigators extracted data from full-text publications and cross-checked values. In the case of multiple reports of the same cohort, the most complete data were selected. We excluded articles not in English, reviews, editorials, case reports and animal or in vitro studies. Additionally, references where CP/IDC was evaluated in a prostate biopsy, or where data were not stratified, or the authors were not able to extract data by the presence of CP/IDC, or the record did not provide data on prognosis/adverse outcome were also excluded. The selection process is shown in Figure 1. 2.4. Data Extraction Data extracted from eligible studies and study characteristics are presented in Supplementary Table S1. Despite direct e-mail contact with authors, it was not possible to obtain more precise data from 3 studies . The adverse outcomes reported in the analyzed studies included EPE, SVI, LNs met, BCR, local recurrence (LR), clinical recurrence (CR), MET, DSD, cancer-specific survival (CSS), overall survival (OS). The PSM as an outcome measure was excluded from this systematic review and meta-analysis because it does not necessarily confer non-organ-confined disease and may result from surgical flaws, postsurgical tissue handling or misinterpretation. Quantitative analysis was performed wherever data for calculations were available. 2.5. Assessment of Study Quality The assessment of the risk of bias in the studies included in the meta-analysis was conducted with the Newcastle-Ottawa Quality Assessment Scale (NOS), which was developed to assess the risk of bias in observational studies . We decided to dismiss the response rate in the exposure domain in case-control studies as this subdomain was not applicable to this article. Each study could receive a maximum of 8 stars for case-control studies and 9 stars for cohort studies. Two investigators (R.O. and M.K.) independently evaluated each article, any disagreement was discussed with and resolved by the third senior investigator (J.D.). Observational comparative studies with scores of <4, 4-6 and >6 were considered, having a high, intermediate and low risk of bias, respectively. According to the NOS, 11 studies were considered high quality and 2 were evaluated as intermediate quality (Supplementary Tables S2 and S3). 2.6. Statistical Analysis Statistical analyses were performed with the use of Review Manager software (RevMan version 5.4 Cochrane, UK) and StatsDirect 3 link software (version 3.3.5; StatsDirect Ltd. Wirral, UK). The strength of associations between CP/IDC and pathological as well as clinical outcomes were analyzed by a calculation of the pooled odds ratios (OR) with 95% confidence interval (CI). The degree of heterogeneity between included studies was estimated with the I2 test which describes the proportion of variance (from 0% to 100%). The random effects method (DerSimonian-Laird; REM) was used to calculate the pooled OR with the 95% CI. The obtained results were summarized in tables as well as illustrated using forest plots. Potential publication bias was analyzed with Egger's regression and Begg's rank correlation tests for all comparisons. In addition, sensitivity analyses were made by sequential exclusion of each study to evaluate the stability and in turn reliability of the results. 3. Evidence Synthesis Characteristics of Included Studies The article selection process was conducted according to the PRISMA statement, and a total of 1798 references were identified. Of 16 studies included in the systematic review, 13 articles provided sufficient data for a quantitative evaluation. The studies included in this systematic review were published between 2011 and 2020. The systematic review encompassed 164,296 patients. The sample sizes varied significantly among the studies, ranging from 28 to 159,777 participants. It is noteworthy that cohorts from two studies partially overlapped, but the endpoint measures were different; therefore, both studies were included and assessed separately . The statistical analysis was based on 3254 patients; of those, 1405 had the CP/IDC morphology (43%). All the included studies were retrospective (16/16). Most of them were case-control studies (14/16), two of which were designed as paired and nested paired case-control studies . Additionally, there was one population-based study and one cohort study . Eight studies focused on the CP , whereas five evaluated IDC . Three studies allowed for a combined assessment of both patterns . The pathological evaluation of resected specimens was conducted according to the ISUP 2005 criteria in six studies . The ISUP 2014 Consensus Conference Working Group criteria were adopted by the authors of six studies . In the study by Kweldam et al. , the adopted histopathological criteria differed depending on the time of the RP. Two articles did not include detailed information regarding a pathological assessment . In the study by Dinerman et al. , the data on the histopathological evaluation were not provided. Two studies investigated the large (LC)/expansile CP (EC) . According to the 2021 WHO recommendation, the CP should be reported without subtypes. Therefore, we considered the LC as the CP. Luo et al. focused on invasive cribriform lesions (ICLs), which we also included as the CP. In the studies which focused on IDC, this pattern was defined using the Guo and Epstein criteria in four studies . Kato et al. adopted the McNeal and Yemoto definition of IDC. Miyai et al. defined IDC as one or both of the following patterns: (1) solid or dense cribriform (less than 50% lumen in a duct) intraductal lesions or (2) loose cribriform (50% and more than 50% lumen in a duct) or micropapillary intraductal lesions with prominent nuclear pleomorphism (nuclear size greater than 6x normal) and/or non-focal comedonecrosis. In the study by Hollemans et al. , IDC was identified if the cribriform structures were clearly continuous with the pre-existing glands lined by normal basal epithelium or containing corpora amylacea. The presence or absence of the CP/IDC in the evaluated specimen was reassessed by at least two specialized genitourinary pathologists in 62.5% of the studies (10/16). Luo et al. stated only that the slides were reviewed for the purpose of the study. In two other studies, the diagnosis of the CP/IDC was extracted from pathology reports . In the remaining articles, this information was either not disclosed or there was a single reviewer . Dinerman et al. extracted data on the presence of IDC from the SEER database. In equivocal cases, the authors of nine papers used immunohistochemical staining (IHC) to distinguish IDC from the CP or HGPIN , whereas the authors of three papers relied solely on the cancer cell morphology . In four articles, the information regarding the use of immunohistochemical staining was not disclosed . The median follow-up period in the studies that assessed the BCR varied significantly from 360.5 days to 130.6 months. For the MET or DSD, the median follow-up duration also differed across the studies and ranged from 20 to 120 months. The BCR definitions varied among the studies with a serum PSA level >= 0.2 ng/mL being the most commonly used threshold (6/9) . In two studies, the BCR was defined as a PSA >= 0.1 ng/mL (2/9) , and in one case, the definition differed depending on the period of the sample and data collection . Although Kweldam et al. in their study investigated the BCR, the presented data were insufficient for a quantitative analysis. The LR was investigated by two authors . Both authors assessed either the CP or IDC in RP specimens. Trinh et al. defined the LR as a peri-prostatic involvement after surgery as confirmed by imaging, biopsy and/or suspect results from a digital rectal examination (DRE), followed by exclusive radiotherapy to the prostatic bed leading to a serum PSA-level reduction without concurrent hormone therapy. Luo et al. did not elaborate on the LR definition. MET was also diagnosed differently. In the studies by Kweldam et al. and Trinh et al., the diagnosis of MET required either radiological or pathological confirmation . Dong et al. relied only on imaging and Hollemans et al. either on a biopsy or multidisciplinary consensus . In one study, the diagnostic criteria for MET were not defined . None of the authors included information regarding the imaging modality used for the confirmation of metastatic spread. Two studies provided additional value for this systematic review. Luo et al. conducted a systematic review and meta-analysis on the impact of the CP in prostate biopsy/RP specimens on adverse outcomes. The studies included in the analysis by Luo and the current systematic review had some overlap, but the addition of articles evaluating IDC in the RP cohort extended the scope of the current study (see Discussion). Greenland et al. assessed the DECIPHER score in case it was either positive for the CP or the glomerulation pattern. The DECIPHER assay was developed to predict the risk of metastases within 5 years after a prostatectomy and measures the expression levels of the RNA of 22 genes in the radical prostatectomy specimen. A DECIPHER score < 0.45 was interpreted as low risk, 0.45-0.6 corresponded to average risk and > 0.6 was assessed as high risk. The authors of that study emphasize that the assay was performed at the discretion of the treating physician, and patients with negative pathological risk factors were more likely to undergo genetic testing. Nonetheless, CP-positive patients were characterized by higher mean DECIPHER scores than patients with glomerulation patterns (0.61 vs. 0.47; p = 0.02; SD = 0.18, 0.17). Studies by Dinerman et al., Kato et al. and Trudel et al. were qualitatively assessed in the systematic review, but because of either different study concepts or a lack of detailed statistical data, they were disqualified from the meta-analysis (see Discussion) . 4. Results 4.1. Correlation between EPE and CP/IDC The correlation between the EPE and CP/IDC was analyzed in eight studies, with a total number of 903 patients with CP/IDC and 1285 without CP/IDC. Significant heterogeneity was observed between the studies (I2 89%). The random effect models analysis revealed that the EPE was significantly more common in patients with than without CP/IDC (41.3% vs. 17.7%, respectively: pooled OR = 2.55 95%CI 1.23-5.26, p = 0.01) . 4.2. Correlation between SVI and CP/IDC The authors of seven studies presented information regarding the relationship between SVI and the CP/IDC. In this meta-analysis, there were 788 patients with CP/IDC and 1215 without CP/IDC, respectively. SVI was significantly more common in patients with CP/IDC than without (19.9% vs. 4.9%), respectively, which resulted in a pooled OR = 4.27 (95%CI 1.90-9.64, p < 0.001) . 4.3. Correlation between LNs met and CP/IDC Seven studies provided data for the analysis of the LNs met in correlation with the CP/ICD, with a total of 639 patients with CP/IDC and 1217 patients without CP/IDC. The LNs met were significantly more prevalent in patients positive for CP/IDC (21.6% vs. 5.8%), with a pooled OR = 6.47 (95%CI 3.76-11.14, p < 0.001) . 4.4. Correlation between BCR and CP/IDC Nine studies analyzed the association between the BCR and CP/IDC. The total number of patients with CP/IDC was 1023 and there were 1498 cases without CP/IDC. The random effect models analysis showed that the BCR was significantly more frequent in individuals positive for CP/IDC (32.2% vs. 8.0%), with a pooled OR = 5.09 (95%CI 2.23-11.62, p < 0.001) . 4.5. Correlation between MET/DSD and CP/IDC MET/DSD as an adverse outcome was analyzed in five studies, with a total number of 581 positive for CP/IDC and 411 without CP/IDC. The random effect models analysis revealed that MET/DSD was also significantly more common in patients with CP/IDC (23.4% vs. 2.9%), which resulted in a pooled OR = 9.84 (95%CI 2.75-35.20, p < 0.001) . In the sensitivity analysis, no changes in the OR value were demonstrated in all the performed comparisons after excluding the subsequent studies. Therefore, the above-described analyses were considered stable and reliable. 4.6. Publication Bias The publication bias tests indicated that there is no publication bias in the studies on the relationship between the EPE, SVI, LNs met, BCR, MET/DSD and CP/IDC. The exact results of Egger's regression and Begg's rank correlation tests, i.e., the intercept with a 95%CI and Kendall's tau are shown in Supplementary Table S4. In addition, the publication bias was evaluated visually with the inspection of funnel plots, which were eventually found to be symmetric . 5. Discussion We performed this systematic review and meta-analysis to investigate the impact of the CP/IDC on the adverse pathological and clinical outcomes after an RP. To the best of our knowledge, this is the first study to systematically and quantitively assess the prognostic factors of CP/IDC in the RP cohort. The results of this study indicate a relationship between the CP/IDC and unfavorable prognostic factors, such as the advanced stage of the disease, BCR, MET and DSD. The aim of a radical prostatectomy is to remove the whole prostatic gland, seminal vesicles and distal part of the vas deferens with the adjacent periprostatic tissue in select cases to ensure the best oncological outcome. Proper surgical planning includes neurovascular bundle (NVB)-sparing techniques in order to restore continence and erectile function after surgery . To date, no recommendations have been released regarding the extent of the surgery in cases with CP/IDC morphology. The results of the meta-analysis clearly indicate a close relationship between the EPE/SVI and CP/IDC (OR = 2.55 and OR = 4.27, respectively). Although we decided to exclude the PSM from this systematic review and meta-analysis, as explained earlier, the PSM has an added value with regard to an oncological prognosis. The articles included in the systematic review reported inconclusive results on the association between the CP/IDC and PSM. According to Sarbay et al. , GG1 cases with a CP have been found to have a much higher incidence of PSM (23.1% vs. 5.1%, p < 0.011), but no such correlation was observed in patients with GG2 and 3 PCa (p < 0.331). Dong et al. and Kweldam et al. also did not report an association between the CP and PSM. However, in two other studies, PSMs were more commonly found after an RP in the presence of the CP . PSMs in the presence of IDC were also investigated. In a retrospective population-based analysis, the PSM was found to be more commonly encountered in the presence of IDC (25.6% vs. 19.5%, p = 0.02) . According to Trinh et al. , there was not any significant correlation between IDC and the PSM. Contrary to our results, Flammia et al. found no correlation between the CP and the local disease stage, the SM in GG 2-5 RP patients. The EAU recommends offering NVB-sparing surgery to patients with a low risk of EPE, which is, among other factors, based on the GG . GP 4 itself does not exclude the NVB-sparing approach, but those guidelines do not take into consideration the GP4 submorphologies or IDC. The results of our meta-analysis indicate a higher prevalence of both the EPE and SVI in CP/IDC cases. Therefore, in the presence of the CP/IDC, intrafascial NVB preservation may pose a risk of a PSM. We also identified the CP/IDC in the radical RP as being associated with LNs met. These results are consistent with our previous study, which investigated the clinical importance of the CP in a prostate biopsy . Nodal metastases, especially in greater numbers, are a known negative prognostic factor associated with a worse BCR-free survival, MET-free and OS . Unfortunately, the included studies did not provide detailed data on the extent of the LN dissection (LND) and the number of dissected LNs, which made it impossible to draw meaningful conclusions regarding the true significance of the LNs met in the presence of the CP/IDC. At the same time, especially extending the LND during an RP provides more tissue for a histopathological analysis. Detailed information on the disease stage may help guide the adjuvant treatment. The extent of the LND and the role of adjuvant therapy in the presence of the CP/IDC and other postprostatectomy negative prognostic factors have yet not been fully understood. The results of our meta-analysis clearly indicate the negative impact of the CP/IDC on the long-term oncological outcomes such as the BCR, MET and DSD and are consistent with the results presented in the literature . Moreover, a meta-analysis by Luo et al. also revealed the negative impact of the CP on the BCR, MET and DSD, which is understandable as the articles included by Luo et al. partially overlapped with those evaluated in our study. However, a study by Flammia et al. showed that no association between the CP and BCR was found in the GG 2-5 subjected to an RP with a median follow-up of 22 months. A comprehensive systematic review and meta-analysis conducted by Miura et al. on the clinical importance of IDC in localized and advanced PCa showed that in the case of localized PCa, the presence of IDC in either a biopsy or radical prostatectomy specimen was associated with worse BCR-free survival (pooled HR 2.09) and CSS ((pooled HR = 2.93). Additionally, there was a correlation between IDC and shorter OS in advanced PCa (pooled HR = 2.93). Dinerman et al. found a 3-fold higher risk for DSD in the presence of IDC but reported no association between IDC and OS. BCR-free survival in more than 1000 RP patients was significantly worse in any ISUP with IDC than without in a study by Kato et al. Moreover, GG2 men with IDC had a prognosis similar to GG4 or GG5 patients without this morphology . Trudel et al. , who investigated the BCR-free rate, concluded that in the presence of LC/IDC, the BCR occurred more commonly, and again, any GG without LC/IDC had a better prognosis. In a subgroup analysis, Kaplan-Mayer curves for the BCR-free survival were similar in men with LC and in men with LC/IDC. IDC as an isolated morphology was, on the other hand, associated with worse outcomes. Nonetheless, the authors highlighted that isolated IDC was present in only 10 men and this cohort was too small to draw meaningful conclusions . Trinh et al. investigated the effect of adjuvant radiotherapy (ART) on the BCR in patients with IDC and found that the worst BCR-free survival was in men positive for IDC, who did not undergo ART (log-rank p = 0.023). AS oncological safety has been shown in many studies. As a result, it has become the pivotal therapy of low-risk disease, acknowledged by many international and national urological associations . The success has encouraged a number of investigators to explore the role of AS in intermediate-risk PCa and expand the indications above the lowest risk category. Decisions regarding AS are made based on the results of a prostate biopsy. This systematic review and meta-analysis refers to the RP histopathological assessment which is more detailed and provides the most accurate evaluation of the disease stage and grade. In our study, CP/IDC emerge as negative prognosticators in terms of both pathological and clinical outcomes after an RP. Considering our results, the negative impact of the CP/IDC should be discussed with every patient qualified for AS. This statement is supported by international urological associations. The current EAU guidelines allow AS in highly selected GG2 cases (<10% GP4, PSA< 10 ng/mL, cT2a); the identification of CP/IDC in a biopsy sample is considered as an absolute contraindication for this type of deferred treatment . Similarly, the PCa guidelines provided by the America Urological Association (AUA) recognize CP/IDC in intermediate-risk patients as unfavorable characteristics and therefore do not recommend AS in those cases Several factors could not be included in the meta-analysis due to insufficient data. In the systematic review by Luo et al. , we found that the risk of LR is more than twice as high in patients with the CP (OR 2.32). Trinh et al. showed that IDC was linked to distant metastasis (mainly bone metastases) as the initial site of the CR on both univariate and multivariate analyses (OR = 6.27), but surprisingly, the time to the CR and CSS was not significantly different between men with and without IDC. A subgroup analysis revealed that radiotherapy (adjuvant or salvage) was superior to pre-RP ADT in respect to the CSS in men with IDC (170 months, 95%CI (124-215) vs. 159 months, 95%CI (122-196)). That was one of the first studies to highlight the detailed evaluation of a pathological specimen and IDC detection in terms of post-RP management. A single study included in this meta-analysis evaluating the impact of the CP on the OS found the OS in GS7 (includes both GG 2 and 3) men with CP to be shorter (log-rank p = 0.001) . The malignant potential and basis for the joint assessment of CP/IDC are reflected in the genomic and epigenetic alternation observed in these histopathological patterns. Elevated SChLAP1 expression and well as genomic instability were found in PCa with CP/IDC . Mehra et al. indicated that increased SChLAP1 expression was associated with a higher risk of BCR, DSD and MET. Based on the whole slide images of the TGGA database and dataset from CPCGN, Bottcher et al. found that IDC and the CP were associated with an increased genomic instability that affected specific regions: the chromosomal deletions of 3p13, 6q15, 8p21-23, 10q23, 13q14 16q21-24 and 18q21-23, and the amplification of chromosome 8q24. Those regions are known to be involved in aggressive PCa, for example, the BRCA 2 gene is located on the long arm of chromosome 13. In CP/IDC-positive cases, genomic alternations also affect the epigenetic profile. Olkhow-Mitsel et al. reported that CP/IDC cases were characterized by DNA hypermethylation in the APC, RASSF1 and TBX15 genes. Hypermethylation in those genes has already been linked to more aggressive PCa . The authors also suggested the utilization of methylation profiles in those genes as a marker of CP/IDC . In a study by Taylor et al. , who investigated the correlation between the CP and IDC with the DECIPHER genomic classifier, found that the predominance of CP in RP specimens significantly increased the DECIPHER score. IDC was non-significantly associated with an increased score (OR 1.92 p = 0.24). On the other hand, the presence of CP/IDC did not reach statistical significance in terms of high-risk categorization (OR = 4.53, p = 0.08), which could be due to the small sample size (11 men) . In a study by Risbridger et al. , which investigated patients-derived xenografts, BRCA 2 germline mutations were more common in IDC-positive men with localized PCa. In contrast to those results, Lozano et al. found no association between the germline BRCA 2 mutations and CP/IDC morphology. In the same study, however, the bi-allelic BRCA2 loss in primary PCa was significantly correlated with the CP and IDC. The authors, therefore, advised against the routine germline BRCA2 testing in the presence of CP/IDC but felt that further research was needed to investigate the relevance of CP/IDC as a marker of the bi-allelic BRCA2 loss in primary PCa. The present study has several limitations. The key one is the different diagnostic criteria applied by authors for the identification of CP/IDC. Various definitions of IDC used by authors and high interobserver variation in the identification of IDC might have contributed to its overdiagnosis in the selected articles. Additionally, the histopathological evaluation of the RP specimens was conducted according to either the ISUP 2005 or 2014 diagnostic criteria. Therefore, the true prevalence of CP/IDC might differ from what was reported. Moreover, the definitions of BCR and MET varied across the studies, which in turn might have had an influence on the presented results. Our conclusion to exclude patients with CP/IDC from the AS is based on studies in which the RP specimen was assessed and not the prostate biopsy. Nonetheless, this attitude is supported by international urological associations. Although a statistical analysis revealed that CP/IDC compromise the long-term oncological outcome in the RP cohort, an analysis of the impact of radiotherapy on patients with CP/IDC might contribute meaningful insight into the prognosis in the setting of different radical treatment modalities. 6. Conclusions The CP and IDC should be considered as highly aggressive histopathological morphologies of the prostatic adenocarcinoma, which have a negative impact on both the pathological and oncological outcomes. The presence of CP/IDC should be taken into account in the planning of surgery and proper postoperative counselling. In intermediate-risk patients with either of the two entities, a detailed explanation of their higher malignant potential is required and AS should be discouraged, in line with international urological guidelines. Supplementary Materials The following supporting information can be downloaded at: www.mdpi.com/article/10.3390/cancers15051372/s1, Table S1: Characteristics of included studies; Table S2: Risk of bias studies; Table S3: Risk of bias cohort study; Table S4: The results of Egger's and Begg's tests. Click here for additional data file. Author Contributions Conceptualization, R.O. and M.K.; methodology, R.O. and M.K.; statistical analysis and manuscript preparation, B.S.-H.; software, B.S.-H.; writing--original draft preparation, R.O.; writing--review and editing, M.K. and M.P.; supervision, J.D. All authors have read and agreed to the published version of the manuscript. Conflicts of Interest The authors declare no conflict of interest. Figure 1 Literature search, screening and selection for inclusion in systematic review.; CP = cribriform pattern; IDC = intraductal carcinoma. Figure 2 Forest plot of odds ratios (ORs) in the studies analyzing the relationship between EPE and CP/IDC . M.-H. = Mantel-Haenszel; CI = confidence interval; I2 = heterogeneity; df = degrees of freedom; EPE = extraprostatic extension; CP = cribriform pattern; IDC = intraductal carcinoma. Each blue square in Figure 2 represents an effect size of a study and the area of the square represents the magnitude of a related study in the effect size. The lines on either side of the squares indicate the lower and upper limits in a 95%CI of the calculated effect sizes. The black rhombus at the bottom of the plot shows the calculated overall effect size. Figure 3 Forest plot of odds ratios (ORs) in the studies analyzing the relation between SVI and CP/IDC . M.-H. = Mantel-Haenszel; CI = confidence interval; I2 = heterogeneity; df = degrees of freedom; SVI = seminal vesicle invasion; CP = cribriform pattern; IDC = intraductal carcinoma. Each blue square in Figure 3 represents an effect size of a study and the area of the square represents the magnitude of a related study in the effect size. The lines on either side of the squares indicate the lower and upper limits in a 95%CI of the calculated effect sizes. The black rhombus at the bottom of the plot shows the calculated overall effect size. Figure 4 Forest plot of odds ratios (ORs) in the studies analyzing the relation between LNs met and CP/ICD . M.-H. = Mantel-Haenszel; CI = confidence interval; I2 = heterogeneity; df = degrees of freedom; LNs met = lymph node metastasis; CP = cribriform pattern; IDC = intraductal carcinoma. Each blue square in Figure 4 represents an effect size of a study and the area of the square represents the magnitude of a related study in the effect size. The lines on either side of the squares indicate the lower and upper limits in a 95%CI of the calculated effect sizes. The black rhombus at the bottom of the plot shows the calculated overall effect size. Figure 5 Forest plot of odds ratios (ORs) in the studies analyzing the relation between BCR and CP/ICD . M.-H. = Mantel-Haenszel; CI = confidence interval; I2 = heterogeneity; df = degrees of freedom; BCR = biochemical recurrence; CP = cribriform pattern; IDC = intraductal carcinoma. Each blue square in Figure 5 represents an effect size of a study and the area of the square represents the magnitude of a related study in the effect size. The lines on either side of the squares indicate the lower and upper limits in a 95%CI of the calculated effect sizes. The black rhombus at the bottom of the plot shows the calculated overall effect size. Figure 6 Forest plot of odds ratios (ORs) in the studies analyzing the relation between MET/DSD and CP/IDC . M.-H. = Mantel-Haenszel; CI = confidence interval; I2 = heterogeneity; df = degrees of freedom; MET = distant metastasis; DSD = disease-specific death; CP = cribriform pattern; IDC = intraductal carcinoma. Each blue square in Figure 6 represents an effect size of a study and the area of the square represents the magnitude of a related study in the effect size. The lines on either side of the squares indicate the lower and upper limits in a 95%CI of the calculated effect sizes. The black rhombus at the bottom of the plot shows the calculated overall effect size. Figure 7 Funnel plots of the correlation between extraprostatic extension (A); seminal vesicle invasion (B); lymph node metastasis (C); biochemical recurrence (D); distant metastasis/disease-specific death (E) and cribriform pattern/intraductal carcinoma in the studies included. Each circle represents a study included in the analysis. Disclaimer/Publisher's Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

PMC10000113

Early weaning is an effective strategy to improve cow feed utilization and shorten postpartum intervals in cows; however, this may lead to poor performance of the weaned calves. This study was conducted to test the effects of supplementing milk replacer with Bacillus licheniformis and a complex of probiotics and enzyme preparations on body weight (BW), size, and serum biochemical parameters and hormones in early-weaned grazing yak calves. Thirty two-month-old male grazing yaks (38.89 +- 1.45 kg body weight) were fed milk replacer at 3% of their BW and were randomly assigned to three treatments (n = 10, each): T1 (supplementation with 0.15 g/kg Bacillus licheniformis), T2 (supplementation with a 2.4 g/kg combination of probiotics and enzymes), and a control (without supplementation). Compared to the controls, the average daily gain (ADG) from 0 to 60 d was significantly higher in calves administered the T1 and T2 treatments, and that from 30 to 60 d was significantly higher in calves administered the T2 treatment. The ADG from 0 to 60 d was significantly higher in the in the T1-treated yaks. The concentration of serum growth hormone, insulin growth factor-1, and epidermal growth factor was significantly higher in the T2-treated calves than in the controls. The concentration of serum cortisol was significantly lower in the T1 treatment than in the controls. We concluded that supplementation with probiotics alone or a combination of probiotics and enzymes can improve the ADG of early-weaned grazing yak calves. Supplementation with the combination of probiotics and enzymes had a stronger positive effect on growth and serum hormone levels, compared to the single-probiotic treatment with Bacillus licheniformis, providing a basis for the application of a combination of probiotics and enzymes. early weaning probiotics enzyme preparations yak calves growth performance The National Key Research Program2021YFD1600200 The Lhasa Comprehensive Experimental Station of National Cattle and Yak Industry Technology SystemCARS-37 This study was funded by the National Key Research Program (2021YFD1600200) and the Lhasa Comprehensive Experimental Station of National Cattle and Yak Industry Technology System (CARS-37). pmc1. Introduction Yaks (Bos grunniens) occur on the Qinghai-Tibet Plateau at high altitudes and with long cold seasons and limited pasture resources. This species is a unique product of long-term natural selection, providing local herders with the most basic living materials and livelihood resources, such as meat, milk, shelter (hides and furs), and fuel (dung), and is an indispensable part of the ecology and economy of the Qinghai-Tibetan Plateau . However, the low reproductive rate of yaks seriously restricts their production and utilization. The cold season on the Tibetan Plateau lasts for eight months (October to the following May), during which time the quantity and quality of pasture decrease below the nutritional requirements of lactating yaks . The deficiency of feed intake results in a negative body energy balance and metabolic stress . On the other hand, under traditional grazing management, plateau-grazing yak calves are weaned naturally or artificially under various conditions at an age of 18-24 months , rather than the weaning age of domestic beef cattle (<6 months). The slow recovery itself and the late weaning of yak calves, which result in a poor postnatal physical condition, severely affect the onset of the next estrous cycle in the cow. Most yaks exhibit a long postpartum anestrous period and calve twice every 3 years or once every 2 years . Therefore, the early weaning of yak calves may help mitigate these adverse effects. Early weaning has become more popular in recent years for various reasons, including the better use of limited feed resources and alleviating grazing pressure on pastures by reducing the nutritional needs of cows . Weaning calves before the start of the breeding season improves the reproductive performance of cows because the cows can regain their weight faster, thus accelerating the onset of postpartum estrus. The use of milk replacer in early weaning is common in livestock production . The milk replacer has demonstrated positive benefits in animal experiments, such as improved immunity and relieved weaning stress response . Increasing evidence suggests that enhanced milk replacer feeding is beneficial for improving gut microbial development and growth performance in early-weaned lambs . Over the past few decades, probiotics have been widely used in livestock and poultry production for their ability to enhance animal disease resistance, improve feed utilization, and improve growth performance . In ruminants, yeasts and bacteria, including Lactobacillus, Bifidobacterium, Bacillus, Propionibacterium, and Enterococcus, alone or in combination, are used as additives in diets . Probiotics can decrease diarrhea, improve production and feed utilization efficiency, and strengthen the immunity system in young ruminants . Moreover, supplementation with probiotics improves the rumen and intestinal epithelial cell growth, which enhances the gastrointestinal tract development and health status of calves . Oral administration of Bacillus licheniformis can increase ruminal digestibility and total volatile fatty acid concentrations in Holstein cows and growth performance in Holstein calves . In vitro inoculation with Bacillus licheniformis also improves ruminal fermentation efficiency of forage of various qualities . However, no information is currently available on the effect of Bacillus licheniformis on the growth performance of yak calves. Compound enzyme preparations are produced from one or more preparations containing a single enzyme as the main entity, which is mixed or fermented with other single enzyme preparations to form one or more microbial products , including saccharylases, amylases, cellulases, proteases, phytases, hemicellulases, and pectinases. Depending on the differences in digestive characteristics and diet composition, specific enzyme preparations can be used for livestock . Specific enzyme complex preparations can degrade multiple feed substrates (antinutrients or nutrients), and different types of enzymes can work synergistically to maximize the nutritional value of feed . In buffalo calves, cellulase and xylanase are more effective with regard to average daily weight gain (ADG) and feed efficiency . Further, the addition of exogenous fibrolytic enzymes to wheat straw has no effect on starter feed intake and increases nutrient digestibility and recumbency, but decreases the ADG of weaned Holstein dairy calves . The effects of probiotics or compound enzyme preparations on the production performance and biochemical blood indexes of calves are not consistent . The respective discrepancies may be due to differences in the amounts of added probiotics and exogenous enzymes, the strains of probiotics, diets, and animal management strategies. Therefore, this study was conducted to compare the effects of Bacillus licheniformis and a combination of probiotics and enzymes on the growth performance and serum parameters in yak calves, so as to provide a theoretical basis for the application of probiotics in grazing yak calves. 2. Materials and Methods 2.1. Animals and Treatment This study was performed in accordance with the Chinese Animal Welfare Guidelines, and the experimental protocols were approved by the Animal Care and Ethics Committee of the Institute of Animal Husbandry and Veterinary Medicine, Tibet Academyof Agriculture and Animal Husbandry Science (No. #TAAAHS-2016-27). The feeding trial was conducted at Damxung Co., (Lhasa, China; 30.5deg N, 91.1deg E) from July to October. The average altitude was 4200 m, the average annual temperature was 1.3 degC, and the average annual precipitation was 456.8 mm. Thirty two-month-old male yaks (38.89 +- 1.45 kg body weight (BW)) were fed milk replacer solution at 3% of their BW every day and were randomly assigned to three dietary supplementation treatments (n = 10, each), according to BW and age, as follows: T1, supplemented with 0.15 g/kg Bacillus licheniformis (2 x 1010 CFU/g); T2, supplemented with a 2.4 g/kg combination of probiotics and enzymes (containing 0.4 g/kg Bacillus licheniformis, 2 x 1010 CFU/g; 1.0 g/kg yeast, 1 x 1010 CFU/g; 1.0 g/kg mixture of xylanase, cellulase, and glucanase in a 1:1:1 ratio, xylanase, 20,000 U/g, cellulase, 1500 U/g, glucanase, 6000 U/g); and a control treatment. The milk replacer, probiotics, and enzyme preparations were provided by the Chinese Academy of Agricultural Sciences (Beijing, China). All yak calves were allowed to graze on an alpine meadow during daytime for the 60-day trial, and they were individually fed milk replacer before and after grazing (0800 and 2000 h, respectively). The forage of the alpine meadow was mainly composed of Kobresia tibetica, and the nutrient composition (dry matter basis) was analyzed in our previous study , i.e., 10.4% crude protein, 2.1% ether extract, 67.8% neutral detergent fiber, 34.2% acid detergent fiber, and 4.6% ash. The powdered milk replacer was weighed and mixed with warm water (approximately 40 degC) at a ratio of 1:7 (w/v) to obtain milk replacer solution, according to our previous study . Based on preliminary assessments, the feeding amount of milk replacer was calculated so that all yak calves were able to feed without surplus . The nutrient composition of the milk replacer is shown in Table 1. 2.2. Sample Collection and Analysis The BW of each yak calf was recorded before morning feeding on d 0, 30, and 60 using a platform scale, and the ADG was calculated accordingly. The body size indexes of all yak calves were determined using a linen tape at the beginning (d 0) and end (d 60) of the experiment, as previously described . Blood samples (approximately 10 mL) were collected from the jugular vein of the yak calves using a vacuum tube before morning feeding on d 0 and 60. The blood samples were centrifuged at 1100x g for 10 min to obtain serum, which was then aliquoted in 1.5 mL centrifuge tubes and stored at -20 degC. The serum biochemical parameters, including blood urea nitrogen (BUN), globulin (GLB), blood glucose (GLU), and non-esterified fatty acids (NEFAs), were analyzed using an automatic biochemical analyzer 7020 (Hitachi, Tokyo, Japan). Metabolic hormones in the serum, including insulin-like growth factor-1 (IGF-1), epidermal growth factor (EGF), cortisol, insulin (INS), and growth hormone (GH), were determined using commercial ELISA kits (Jiahong Technology Co., Ltd., Beijing, China) according to the manufacturer's instructions. Briefly, 50 mL of each five-fold diluted serum sample was added to each well of a 96-well ELISA plate. After 30 min of incubation at 37 degC, the plate was washed five times using PBS (Servicebio, Wuhan, China) to remove unbound proteins. Then, 50 mL of HRP-conjugated antibodies was added to allow them to bind with their corresponding antigens. The 3,3',5,5'-tetramethylbenzidine working solution was added to each well, followed by stop solution. Absorbance was measured using a multi-plate reader (Varioskan LUX, Thermo Fisher Scientific, Waltham, MA, USA) at a wavelength of 450 nm. 2.3. Statistical Analysis All experimental data of this study were statistically analyzed using a one-way analysis of variance followed by Duncan's post hoc test with SPSS 26.0 software (SPSS Inc., Chicago, IL, USA). Each yak calf was considered an experimental unit. Data are expressed as means +- standard error. p < 0.05 was considered statistically significant. 3. Results 3.1. Body Weight The three treatments did not differ significantly in terms of BW on d 0, 30, and 60 (Table 2). The ADG was higher (p < 0.05) in the calves under T2 treatment than those under the control treatment, from d 0 to 30, d 30 to 60, and d 0 to 60, and higher (p < 0.05) than that of those calves under the T1 treatment from d 0 to 60, indicating that the supplementation of Bacillus licheniformis and the combination of probiotics and enzymes could improve the growth performance of early-weaned grazing yak calves. The ADG of calves under T1 treatment was higher (p < 0.05) than that of those under the control treatment from d 0 to 60. 3.2. Body Size The body size parameters did not differ significantly among the three treatments on d 0 and 60 (Table 3), indicating that the supplementation of Bacillus licheniformis and the combination of probiotics and enzymes did not affect the body size of yak calves within 60 d. 3.3. Serum Biochemical Parameters The concentrations of serum GLB, BUN, GLU, and NEFAs did not differ significantly among the three treatments on d 0 and 60 (Table 4). 3.4. Serum Hormone As shown in Table 5, the concentrations of serum IGF-1 on d 60 were higher in T2-treated calves than in the control-treated calves (p < 0.05, each). The concentrations of serum EGF and GH on d 60 were higher in the T2-treated calves than in the controls (p < 0.05). The concentration of serum COR on d 60 was higher in the control calves than those under the T1 treatment (p < 0.05). 4. Discussion Early weaning may have various benefits for cows; however, early weaned calves generally perform poorly compared to naturally weaned calves . Early weaned calves without breastfeeding grew at a lower rate and subsequently took longer to reach their target weight than breastfed calves . To improve the growth performance of early-weaned calves, several improvements were made to the composition of milk replacer or additional feeds were added . Moreover, the addition of probiotics to the diets of calves significantly improved the ADG . Dietary supplementation with compound enzyme preparations also improved growth performance in weaned piglets and growing-finishing pigs . However, previous studies also reported that supplementation with probiotics, yeast cultures or enzymes had no effect on the growth performance of calves . In the current study, the addition of Bacillus licheniformis alone or a complex of probiotics and compound enzyme preparations to the milk replacer significantly improved the performance of grazing yaks and calves compared with milk replacer alone. Further, the addition of probiotics is beneficial for the regulation of the intestinal microbiota community structure, improving intestinal health and fecal consistency, and reducing diarrhea prevalence . The supplementation of fibrolytic enzyme to the diet of crossbred calves improved their nutrient digestibility with a positive effect on daily gain . Calves typically exhibit high metabolism and fast growth; however, their growth performance is susceptible to environmental stress and nutrient absorption and digestive problems, especially in the period after weaning . Under natural grazing conditions on the Qinghai-Tibet Plateau, due to the long-term lack of pasture and harsh environmental conditions, the normal growth of yak calves is severely restricted . In the present study, none of the study animals died, which may be attributed to the supplementation with milk replacer. Therefore, the addition of probiotics and compound enzyme preparations was beneficial for the growth of grazing yak calves. In most cases, calf weight is positively correlated with body length, and body length can be used to predict calf live weight . Supplementation with Bacillus subtilis results in an increased body length and BW in Barki lambs at the third and fourth week, as observed in a four-week continuous feeding trial . In the present study, neither body size nor BW differed among the treatments, which may be due to insufficient trial duration and individual differences in animals. Therefore, more time may be required to elucidate whether the probiotic and compound enzyme preparations affected the calves' body size. To a certain extent, blood biochemical parameters reflect the metabolism and the acid-base balance of the animal body, and they vary within a certain range . The results of the current study revealed that supplementation with Bacillus licheniformis and the complex of probiotics and enzyme preparations had no effect on the blood biochemical parameters of grazing yak calves, which is consistent with previously reported results in crossbred and Holstein calves . The blood biochemical values of calves vary with the growing stage and are strongly influenced by weaning , and these possible factors may be stronger than the influence of diet on blood biochemical indicators. Insulin-like growth factors (IGFs) are small polypeptide hormones mainly synthesized and secreted from the liver, and they are structural homologs of insulin, with similar activities. These consist in binding to specific carrier proteins in the blood to form a composite factor that stimulates systemic body growth and has growth-promoting effects on almost every cell in the body . As mediators of GH action, the synthesis of IGFs is also affected by the blood level of GH . EGF is a member of the growth factor family, a single polypeptide of 53 amino acid residues that is involved in regulating cell proliferation . We found that the addition of probiotics and a combination of probiotics and enzymes significantly increased the concentration of serum IGF-1, EGF, and GH, whereas supplementation with Bacillus licheniformis alone did not achieve this effect. These results are consistent with the ADG results. GH and IGF-1 are important controllers in regulating amino acid metabolism in calves, where GH promotes the entry of amino acids in muscle tissue into cells and increases protein synthesis, and IGF-1 increases protein deposition by promoting protein synthesis . Cortisol is commonly used as a marker of stress responses (such as weanling stress) in animals, and it occurs at high serum levels for a period of time after calves are weaned . In line with our results, oral supplementation with probiotics markedly decreases the concentrations of serum cortisol in neonatal and weaned calves . Interestingly, we found that the concentrations of serum cortisol were lower in the T1 than in the T2 group, which was, however, not statistically significant. This suggested that the addition of Bacillus licheniformis alone may better alleviate weaning stress in grazing yak calves. However, the respective mechanisms remain to be resolved in more detail. A limitation of this study is that the T2 group did not strictly control a single variable compared to the T1 group, and the factors (yeast or xylanase, cellulase and glucanase) that contributed to the difference were unclear. This was due to the initial intention of this study to improve the milk replacer by adding probiotics or compound enzyme preparations, and ultimately promote the growth performance of yak calves on the Qinghai-Tibet Plateau. Further, we were unable to collect data on diarrhea and determine nutrient digestibility in grazing calves, which would have further improved our understanding of the weight gain of yaks under the various treatments. 5. Conclusions Our results suggest that supplementation with Bacillus licheniformis alone or with a complex of probiotics (Bacillus licheniformis and yeast) and compound enzyme preparations (xylanase, cellulase, and glucanase) can improve the ADG of grazing yak calves, and the complex had a better effect on the ADG. The addition of the complexes of probiotics and complex enzyme preparations also increased the concentrations of serum GH, IGF-1, and EGF, which may have led to a higher ADG. Thus, the addition of a combination of probiotics and enzymes to milk replacer may serve as an effective strategy to improve the production of yak calves. Acknowledgments We would like to thank the staff at our laboratory for their ongoing assistance. Author Contributions Conceptualization, Y.B. and D.G.; Formal analysis, J.Z.; Investigation, Y.B. and D.G.; Methodology, K.Z.; Project administration, Y.B.; Software, J.Z. and L.S.; Validation, L.S.; Writing--original draft, J.Z. and K.Z.; Writing--review and editing, Y.B., C.M. and B.X. All authors have read and agreed to the published version of the manuscript. Institutional Review Board Statement The animal study protocol was approved by the Animal Ethical and Welfare Committee (AEWC) of the Institute of Animal Husbandry and Veterinary Medicine, Tibet Academyof Agriculture and Animal Husbandry Science (No. #TAAAHS-2016-27). Informed Consent Statement Not applicable. Data Availability Statement The data presented in this study are available on request from the authors. Conflicts of Interest The authors declare no conflict of interest. animals-13-00785-t001_Table 1 Table 1 Ingredients and nutrient compositions of milk replacer powder (feed basis, %). Items Nutrient Composition 1 Dry matter, % 96.22 GE, MJ/kg 19.66 Crude protein, % 26.11 Ether extract, % 25.66 Ash, % 7.23 Calcium, % 1.17 Total phosphorus, % 0.65 1 All nutrients were measured values. animals-13-00785-t002_Table 2 Table 2 Effect of Bacillus licheniformis and the combination of probiotics and enzymes on body weight of yak calves. Items Treatments 1 SEM p-Value Control T1 T2 Body weight, kg 0 d 40.64 40.13 40.07 1.00 0.956 30 d 44.27 44.63 45.7 1.29 0.775 60 d 48.09 49.42 51.47 1.48 0.367 Average daily gain, g 0-30 d 121.21 b 150.00 ab 187.78 a 11.32 0.009 30-60 d 127.27 b 159.72 ab 192.22 a 9.18 0.006 0-60 d 124.24 c 154.86 b 190.00 a 8.88 <0.001 1 Control, yak calves supplemented with milk replacer solution of 3% BW; T1, yak calves supplemented with milk replacer solution according to BW plus 0.15 g/kg Bacillus licheniformis; T2, yak calves supplemented with milk replacer solution according to BW plus 2.4 g/kg combination of probiotics and enzymes; data with different lower-case superscript letters are significantly different (p < 0.05); n = 10; SEM, standard error of the mean. animals-13-00785-t003_Table 3 Table 3 Effect of Bacillus licheniformis and the combination of probiotics and enzymes on body size of yak calves. Items Treatments 1 SEM p-Value Control T1 T2 Body length, cm d 0 52.23 51.93 52.07 0.72 0.915 d 60 59.69 61.63 62.16 0.68 0.126 Hip height, cm d 0 60.59 59.84 60.77 0.82 0.788 d 60 64.54 63.72 64.15 0.35 0.815 Heart girth, cm d 0 69.45 69.36 68.99 0.26 0.925 d 60 78.51 78.48 78.42 0.28 0.932 1 Control, yak calves supplemented with milk replacer solution of 3% BW; T1, yak calves supplemented with milk replacer solution according to BW plus 0.15 g/kg Bacillus licheniformis; T2, yak calves supplemented with milk replacer solution according to BW plus 2.4 g/kg combination of probiotics and enzymes; n = 10; SEM, standard error of the mean. animals-13-00785-t004_Table 4 Table 4 Effect of Bacillus licheniformis and the combination of probiotics and enzymes on serum biochemical parameters of yak calves. Items Treatments 1 SEM p-Value Control T1 T2 GLB, mg/mL d 0 39.67 38.12 42.21 1.98 0.856 d 60 40.99 41.49 45.18 3.46 0.455 BUN, mmol/L d 0 24.29 26.15 24.89 1.21 0.658 d 60 27.12 24.86 25.24 1.00 0.627 GLU, mmol/L d 0 6.67 7.15 7.59 0.24 0.521 d 60 7.57 6.80 7.26 0.27 0.524 NEFAs, mmol/L d 0 566.74 591.26 543.36 16.59 0.642 d 60 667.63 583.54 599.50 25.71 0.377 1 Control, yak calves supplemented with milk replacer solution according to BW; T1, yak calves supplemented with milk replacer solution according to BW plus 0.15 g/kg Bacillus licheniformis; T2, yak calves supplemented with milk replacer solution according to BW plus 2.4 g/kg combination of probiotics and enzymes; GLB, globulin; BUN, blood urea nitrogen; GLU, blood glucose; NEFAs, non-esterified fatty acids; n = 10; SEM, standard error of the mean. animals-13-00785-t005_Table 5 Table 5 Effect of Bacillus licheniformis and the combination of probiotics and enzymes on serum hormone of yak calves. Items Treatments 1 SEM p-Value Control T1 T2 IGF-1, ng/mL d 0 273.52 295.12 287.36 18.95 0.195 d 60 156.25 b 208.69 b 319.38 a 29.55 0.001 EGF, pg/mL d 0 846.26 926.21 912.62 30.15 0.389 d 60 429.82 b 608.88 ab 729.04 a 40.71 0.007 GH, ng/mL d 0 17.59 14.25 15.77 1.68 0.625 d 60 7.94 b 11.77 ab 14.12 a 0.89 0.012 INS, mIU/L d 0 28.96 27.21 26.85 1.26 0.851 d 60 26.32 22.66 29.76 3.85 0.764 COR, ng/mL d 0 107.26 101.68 116.26 2.65 0.856 d 60 105.42 a 80.79 b 93.49 ab 4.29 0.049 1 Control, yak calves supplemented with milk replacer solution according to BW; T1, yak calves supplemented with milk replacer solution according to BW plus 0.15 g/kg Bacillus licheniformis; T2, yak calves supplemented with milk replacer solution according to BW plus 2.4 g/kg combination of probiotics and enzymes; IGF-1, insulin-like growth factors-1; EGF, epidermal growth factor; GH, growth hormone; INS, insulin; data with different lower-case superscript letters are significantly different (p < 0.05); n = 10; SEM, standard error of the mean. Disclaimer/Publisher's Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

PMC10000114

In dairy systems with Zebu breeds, calves are not immediately separated from their dams after calving; consequently, maternal care and protective behavior are important, influencing both productive performance and stockpeople's safety. Our objectives were to: (1) investigate the effects of a training protocol involving pre-calving positive stimulation, delivered prior to calving, on the maternal care of primiparous Gyr cows; and (2) evaluate the effects of this training protocol on maternal protective behavior towards handlers during the first calf handling. Primiparous dairy Gyr cows (n = 37) were allocated into two groups: training (n = 16) and control (n = 21). Animal behaviors were recorded in three periods: post-calving, first calf handling, and post-handling. Maternal protective behavior during calf handling was assessed from measures of aggressiveness, attention, displacement, and agitation. Calf latency to stand up (p < 0.01) and sex (p < 0.01) differed between the training and control groups. The training group had less touching (p = 0.03), more time not interacting with the calf (p = 0.03), tended to be less protective (p = 0.056), and moved less (p < 0.01) during the first handling of their calves. In conclusion, the primiparous dairy Gyr cows subjected to pre-calving training protocol displayed less maternal care and displacement during the first handling of their calves and tended to be less protective. aggressiveness cow-calf contact systems maternal protective behavior maternal care Zebu Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP)2015/24174-3 Coordenacao de Aperfeicoamento de Pessoal de Nivel Superior (CAPES)0.001 This research was funded by the Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP)--grant number 2015/24174-3 and in parts by Coordenacao de Aperfeicoamento de Pessoal de Nivel Superior (CAPES)--grant number 0.001. pmc1. Introduction Improving human-animal interactions addresses worldwide societal demands for better animal welfare, an impetus for improvement in diverse animal production fields involving animal handling . The quality of human-animal interaction affects the welfare of several dairy species, e.g., cattle, buffaloes, sheep, and goats (reviewed by Napolitano et al. ). On dairy farms, the importance of improving human-animal relationships is evident as animals are handled on a daily basis for milking and have direct contact with handlers . There are various ways to improve animal handling and consequently animal welfare, including a selection of handlers , improving facilities , or gentle handling procedures . Gentle handling, e.g., stroking body regions, gentle tactile stimulation, and vocal interactions in European dairy cattle (Bos taurus taurus) improved animal responses to humans and handling routines . Stroking the body regions of Brown Swiss and Austrian Simmental lactating cows, Schmied et al. reported that gentle stimulation reduced avoidance distance and increased approaches to humans. Lurzel et al. reported that gentle tactile stimulation and vocal interactions with Holstein-Friesian heifers induced behavioral responses compatible with pleasure during stroking. In Gyr (Bos taurus indicus) dairy cattle, pre-calving brushing stimulation reduced cows' reactivity score, respiratory rate and rectal temperature . Similarly, primiparous and multiparous Gyr cattle subjected to positive tactile stimulation in the prepartum period reduced reactivity during milking, e.g., stepping and kicking . The training protocol was more efficient in primiparous versus multiparous cattle to maintain adequate behaviors and milk ejection during their first milking . Paranhos da Costa et al. reported beneficial effects of a training protocol on Hostein-Gyr dairy heifers reducing reactivity during first milking and facilitating the milking routine. These results demonstrated strategies to address management challenges during milking. However, for dairy cows, the most challenging handling procedures may start immediately after calving. On tropical dairy farms, heifers are usually infrequently handled and maintained in pasture-based systems before first calving and when handling procedures occur, they are generally aversive or neutral (e.g., vaccination, hot-iron branding, artificial insemination, weighing). These less-frequent and generally aversive interactions can cause greater fear and consequently reactive responses to human presence and handling procedures . It can be aggravated in the peripartum period, which is very sensitive for both dam and calf, further complicating management. In dairy farms with European breeds, calves are usually separated from their dams within the first 24 h of life . However, on most pasture-based farms with Zebu dairy breeds, the separation of dam and calf is not performed, as it may compromise lactation persistence . Thus, the first handling of dams and calves after calving can be challenging for Zebu dairy cattle. Handling newborn calves is necessary for healthcare, including navel asepsis, weighing, and identification ; however, these procedures can be hampered by the dam's presence . Cows that perceive these interventions as a potential threat to their calf may exhibit extreme protective behaviors leading to a high risk of accidents to stockpeople and calves . Issues related to behavior and the handling of recently calved cows are relevant in the livestock industry as aggressive cows can compromise one-welfare and farm sustainability . In this context, positive handling could be used in day-to-day farm management to improve the welfare and safety of animals and handlers . Perhaps a previous positive experience (e.g., habituation to humans and gentle handling) can mitigate the aggressive reactions of recently calved cows toward humans . Additionally, Florcke et al. speculated that calmer cows would display mothering problems and perhaps reduced maternal behaviors toward offspring, but they did not provide supporting data. Furthermore, there was a low phenotypic correlation but a moderate genetic correlation between docility and maternal behavior (measured by time licking the calf) . Thus, it is plausible to expect a possible association between cow calmness and maternal performance. However, to our knowledge, no studies have evaluated effects of the gentle tactile stimulation protocols before calving on maternal behavior. The effects of a training protocol on the protective behavior of Zebu dairy cows in cow-calf contact systems are also unknown. Thus, using primiparous Gyr dairy cows, we wanted to determine the effects of a training protocol for milking, involving gentle tactile stimulation prior to calving, on: (1) maternal care during the post-calving period; and (2) maternal protective behavior towards handlers during the first handling of calves. We hypothesized that: (1) the training protocol would affect the cows' maternal behavior; and (2) cows subjected to the training protocol would have lower protective behavior against stockpeople during the first handling of their calves. 2. Materials and Methods 2.1. Animals and Handling Thirty-seven primiparous Gyr dairy cows (Bos taurus indicus), aged 46.26 +- 8.63 months, from an experimental farm station (Empresa de Pesquisa Agropecuaria de Minas Gerais-Epamig Oeste, Uberaba, Minas Gerais State, Brazil) were used. Only eutocic calvings of singleton births between August and December 2017 were included. A pasture-based system was used for all cattle. Cows were allocated into two groups: training (n = 16) and control (n = 21), based on the expected calving date (the first cow to calve was randomly allocated to one group, the next cow to calve was then allocated into the alternate group, with this pattern continuing for the remaining cows). In the training group, 40 days before the estimated calving day, cows started a training protocol for the first milking, involving gentle tactile stimulation (14 days consecutively) in a conventional tandem parlor (12 milking machines, in two rows). The protocol was performed by six trained handlers and constituted three phases, as described by Ujita et al. . Briefly, in Phase 1, cows were driven from the pasture to the milking parlor and passed through the milking stalls; in Phase 2, cows went through the milking stalls and were brushed (2 min) on the whole body (head, neck, trunk, udder, front legs, and hind legs); and in Phase 3, cows went through the milking stalls, their bodies (mainly udder and hind legs) were brushed (2 min), legs restrained and teat asepsis (pre-dipping) was performed. Additional information about the milking parlor and milking routine is described in Ujita et al. . The control group did not receive the training protocol and was subjected to typical farm management. At 30 days before expected calving, the cows of both groups were relocated from the pasture to a maternity paddock (0.55 ha; maximum stocking density: 27 cows/hectare), with both the control and training group cows kept in the same paddock. The nutritional management of both groups in the maternity consisted of corn silage and 500 g concentrate per cow, delivered to the feeder twice a day using a tractor vehicle. Additionally, water and mineral salt were offered ad libitum. Cows weighed 425.5 +- 47.3 kg (training group: 427.5 kg +- 37.1 kg; control group: 422.5 +- 37.1 kg). Details about the maternity paddock and nutritional management of cows are fully described in Vicentini et al. . During the final gestation period, cows were not handled or disturbed (except for the training group during the protocol). All routine procedures (i.e., feed delivery, calf weighing, and navel asepsis) were performed by the same six handlers trained in good practices of cattle handling. 2.2. Behavioral Observations The maternity paddock was equipped with four video monitoring cameras (GIGA, GSHDP20TB) that recorded cow behavior 24 h a day. Cows were individually identified with non-toxic paint (Koleston, Wella(r), Darmstadt, Germany) on both sides of their body. Calving was defined as complete expulsion of the calf. Thereafter, a minimum of 3 h were allowed for the cow-calf dyad to remain together without any human intervention, with the first calf's handling performed afterward. Calf handling was performed daily during handlers' working hours (8 am-5 pm); therefore, cows that calved from 5 pm to 4 am remained with their calves for 3 to 15 h. Three periods were considered to record the maternal behaviors: (1) 'post-calving period': 3 h following the complete expulsion of the calf; (2) 'first calf handling': the period of calf handling, including inspection and navel asepsis; (3) 'post-handling period': 1 h after completion of navel asepsis. After the 'post-handling period', both cow and calf were removed from the maternity pen. Based on video recordings from monitoring cameras, 189 h (4.5 h/cow) were analyzed. A single trained observer recorded the cow behaviors using focal sampling and continuous observation . During the 'post-calving' period, the latency of the first calf touch by the cow ('cow latency'), and the latency of the calf to stand on its four feet ('calf latency') were recorded in minutes (Table 1). Unsuccessful attempts by calves to stand until they were able to stand up without falling ('calf attempts') were recorded as number of occurrences (Table 1). Behaviors related to cow-calf interaction ('touching', 'not interacting' and 'suckling') in both the 'post-calving' and 'post-handling' periods were recorded as percentages of observation time (Table 1). During the 'first handling' period, cow protective maternal behavior was assessed by a single trained observer using two scoring systems, 'maternal composite score (MCS)' and 'maternal protective behavior (MPB)', as described by Vicentini et al. . The MCS was obtained by adding the scores of the four main behaviors: 'aggressiveness' (1-3), 'attention' (1-3), 'displacement' (1-5), and 'agitation' (1-4) . Full descriptions of the scores are included as the Supplementary Material (Table S1). The sums of the MCS scores ranged from 4 (minimum) to 10 (maximum) and subsequently transformed from 1 into 7. The MPB applied scoring from 1 (calm cows) to 5 (aggressive cows) . Navel disinfection was conducted by two familiar handlers that worked in pairs. The handlers' approach was standardized, aiming to be consistent and avoid influencing the cows' behavior, as described by Vicentini et al. . After the 'post-handling period', the cow and calf were moved from the maternity paddock to the corral where the colostrum milking and calf identification procedures occurred and the calf was weighed (Digitron Scale). 2.3. Statistical Analyses Descriptive statistics and normality tests were conducted for all behavioral variables using the PROC Univariate of Statistical Analysis System (SAS(r) Institute, INC., Cary, NC, USA). To evaluate the effects of the treatment (training group vs. control group) on cow and calf behaviors, and maternal protection scores, general linear models were fitted, using PROC GLIMMIX of SAS and adopting the lognormal distribution for variables with non-normal distribution ('cow latency', 'calf latency', 'calf attempts', MCS, and MPB). Cows' behaviors ('cow latency', 'touching', 'not interacting', and 'suckling') and calf behaviors ('calf latency' and 'calf attempts') at 'post-calving' and 'post-handling' periods, and maternal protection scores (MCS and MPB) were used as dependent variables. Treatment (training group vs. control group), 'calf sex' (male or female), treatment, and calf sex interaction were used as the fixed effects. The 'Calf weight' (in kilograms) and cow age (in months) were included as covariates with linear effects. Complementarily, Chi-square tests in contingency tables were used to estimate the associations between treatment (training or control groups) with scores for aggressiveness, attention, displacement, and agitation. Pearson correlation coefficients were used to investigate the relationships between cow and calf behaviors in 'Post-calving' and 'Post-handling' periods. p values <= 0.05 were considered significant and p values <= 0.10 tendencies. 3. Results There were more male (n = 20) than female (n = 17) calves, and males tended to be heavier (Table 2). There were effects of 'calf sex' (F = 9.94; p < 0.01) and 'calf sex' * 'calf weight' interaction (F = 6.97; p = 0.01) on 'calf attempts' to stand up; male calves had almost double the number of attempts than females (Table 2). Regarding cow behaviors, the 'calf weight' (F = 3.33; p = 0.07) tended to affect the 'cow latency', with heavier calves taking longer to be touched by their dams. There were relationships between the cow and calf behaviors. A positive correlation between the 'calf latency' and 'calf attempts' (r = 0.63; p < 0.01) indicated that the calves with the longest time to stand up were also those that made more attempts to do so. In addition, a positive correlation between 'calf latency' with 'not interacting' in both the 'post-calving' (r = 0.36; p = 0.03) and 'post-handling' (r = 0.41; p = 0.01) periods was detected. Finally, a tendency between 'calf attempts' and 'touching' (r = -0.30; p = 0.09), and a significant correlation between 'touching' and 'not interacting' (r = -0.94; p < 0.01), both at 'post-calving period' were identified. Regarding the effects of a training protocol on maternal behaviors in the 'post-calving period' there was a significant effect of the treatment * 'calf sex' interaction on the variables 'touching' (F = 4.79; p = 0.03) and 'not interacting' (F = 4.85; p = 0.03). Male calves were less touched than females by cows from the training group, but there was no difference in the control group (Table 2). The training group dams of male calves spent less time 'touching' and more time 'not interacting' than those from control group (Table 2). During the 'post-handling period', there was an effect of the 'calf weight' on the 'touching' behavior (F = 3.96; p = 0.05), in which heavier calves were touched longer by their dams. Cows from the training and control groups did not differ in other assessed behaviors. Distributions of the scores for 'displacement', 'agitation', 'attention' and 'aggressiveness' plus 'maternal composite score' (MCS), and 'maternal protective behavior' (MPB) are presented in Figure 1. Treatment group had a tendency on MCS (F = 3.92; p = 0.056; trained: 3.18 +- 1.75 vs. control: 3.80 +- 1.47) and an effect on 'displacement' (F = 10.05; p < 0.01; trained: 1.87 +- 0.80 vs. control: 2.33 +- 0.48) scores. Scores for 'agitation', 'attention', 'aggressiveness' and MPB did not statistically differ between treatment groups. Chi-square revealed an association between treatment with 'displacement' score, with higher percentage of score 0 for the training group and a higher percentage of score 3 for the control group (kh2 = 11.11; p < 0.01). For all other measures, there were no significant associations with treatment groups (p > 0.05). 4. Discussion Maternal behavior is an important trait in many domestic species . However, in Zebu breeds, this characteristic is even more relevant as it impacts both dam productivity and calf performance . In the present study, male calves tended to be heavier and made more attempts to stand up. Despite being heavier, the higher frequency of attempts combined with the higher latency of males to stand for the first time can be understood as a signal of lower vigor compared to female calves. Lower motility can be a reliable indicator of low vigor in Zebu calves . Indeed, at birth, male calves have greater chance of poor vigor than females . Calf vigor must be considered an important indicator of offspring survival. In Nellore cattle, Schmidek et al. reported the risks of low vigor and death were ~20% greater in males compared to females. Several factors influence the postpartum behavior of cows, with an important role of calf vigor in arousing the mother's interest . In our study, heavier calves experienced a delay in being touched by their dams. One possible explanation was exhaustion from the calving process. Although dystocia did not occur in our study, cows delivering heavier calves can experience severe pain or exhaustion during calving . This phenomenon was evinced by correlations between cows and calf behaviors (longer latency to stand up was related to less maternal touch and interactions). Edwards and Broom reported the influence of weariness on delayed standing after calving, which can also affect first maternal care (e.g., touching, cleaning, and suckling). Additionally, pain signs and weariness can be more evident in primiparous than multiparous cows , and our studied cows were all primiparous. Future studies could include calving duration as a potential indicator identifying cows with more strenuous efforts to deliver heavier calves, resulting in longer intervals of rest to recover before approaching the calf. In addition to vigor, calf sex appears to have an important role in triggering cows' nursing behavior. Female calves were touched and interacted more with their dams than males during the post-calving period. This behavior can be attributed to the exhaustion from the calving process. As discussed above, male calves tended to be heavier, likely resulting in a more exhausting calving process. Detrimental effects of difficult calving (e.g., pain and fatigue have been reported and may impair maternal care ). Stehulova et al. studying Gasconne cattle reported that cows provided more maternal care to low-weight calves, and licked and followed more female than male calves. Similarly to taurine cattle, Zebu cows of the Guzerat breed were described as spending more time and providing more maternal care to low-weight calves . Training influenced the maternal care delivered by cows with male calves, with the training group cows spending less time performing maternal care behaviors (less 'Touching' and more 'Not interacting') than cows in the control group. This was the first evidence of a possible effect of a training protocol to the first milking and/or use of gentle tactile stimulation on maternal behavior in Zebu cattle, since no previous studies have evaluated this topic. Further studies are needed to better elucidate biological reasons for the effects of training only being manifested in the dams of male calves. Previous studies reported reductions in fear, reactivity, and aversiveness during the first milking in response to gentle tactile stimulation for Gyr and Holstein-Gyr cows . Additionally, Florcke et al. speculated that very calm cows would have limited maternal performance. Unfortunately, in the present study, we did not perform a temperament assessment of these cows in the absence of their calves to confirm whether trained cows were calmer or more docile than control cows during handling. We can only presume that the gentle handling and training protocols may modulate some aspects of maternal behavior towards their calves, that can be dependent on calf sex. Regarding maternal protectiveness, control group cows were more displaced during the first handling of their calves and tended to be scored as more protective based on MCS, without significant effects on the MPB, aggressiveness, attention, or agitation scores. Lower maternal defense responses toward humans in trained cows were expected, based on previous evidence that habituation and gentle interactions decrease aversive and avoidance reactions toward people in cattle, and improve human-animal interactions . Evaluating beef Gyr, Brahman, and Gyr-Brahman cross cows, Orihuela et al. observed that more aggressive cows towards handlers were those with more intensive protective behavior reaction to separation from their calves during handling. However, the authors did not report any relationship between the aggressive behaviors and cow temperament in the peripartum period . Beef cows are recurrently described to be more protective of their calves than dairy cows . Using qualitative behavior assessment, Ceballos et al. reported that dairy Holstein-Gyr cows with aggressive behavior during their calves handling were those considered more 'frightened' and 'irritated' than the cows classified as 'loving' and 'attentive'. Although, in general, Zebu dams demonstrate strong protective behavior to their calves, we emphasize the plasticity of this phenotype, influenced by several factors . In this case, environmental effects and handling routine may have modulated maternal behavior. Repeated handling and habituation are useful tools to improve animal responses to handling . Cows with lower milking reactivity, better productivity, and calmer response when restrained were already described as a result of positive tactile stimulation protocol . Despite having a weak effect on maternal protectiveness, we attributed the reduced displacement and tendency of a lower MCS to gentle tactile stimulation received during training. Habituation to human presence and gentle handling may influence how cattle perceive humans ; cattle may regard them as something other than a potential risk for their calves. Although the effects documented herein suggest a relationship between the training protocol and maternal performance, our study had some limitations, requiring thoughtfulness to extrapolate our results. Even though we observed a tendency of the training protocol to affect maternal protectiveness (MCS score) of Gyr dairy cows, the differences between individuals were important. Some aspects of these individual traits may influence a stimuli response even in standardized protocols . Individuals' past experiences (before conception or even during gestation) may have masked or influenced cow behavior in some way, mainly toward handlers . Furthermore, we only had 16 primiparous cows in the training group and a single group per treatment (without replication). Therefore, pre-milking training effects on maternal protectiveness must be confirmed in future studies with greater sample sizes and preferably using a replicated design with both male and female offspring. Moreover, it is important to evaluate intra-observer reliability when measuring animal behavior as it gives confidence about the quality of assessment made by the same observer; unfortunately, we did not evaluate it in our study. Nevertheless, the observer has been trained in observing animal behavior with years of experience in this task. Future studies should consider including a baseline (pre-calving) reactivity assessment of both groups (trained and control) to better elucidate the potential effects of the training protocol on cows' reactivity post-calving. Finally, more studies should investigate the possible effects of training protocols for longer intervals after calving, evaluating the long-term effects on cow-calf bonds across lactation in cow-calf contact production systems. 5. Conclusions A pre-calving training protocol for the first milking involving gentle tactile stimulation on primiparous Gyr dairy cows was associated with reduced displacement and lower maternal defense, based on a maternal composite score, which can be beneficial for the handling routine of calves. However, primiparous Gyr dairy cows subjected to the training protocol spent less time touching their male calves. In addition, maternal care was also affected by calf latency to stand up, weight, and sex. The potential long-term effects of lower maternal care by trained cows on calf development need to be determined in future studies. In general, our findings bring new perspectives about using a training protocol to habituate primiparous cows to the first milking. In addition to the relevant benefits already known in productivity and animal welfare, the adoption of a training protocol could modulate the protective responses of dams to the management of their calves. Acknowledgments The authors thank Empresa de Pesquisa Agropecuaria de Minas Gerais (EPAMIG) for kindly providing the animals and infrastructure necessary for this study, and the students and staff who collaborated with this study. We are grateful to John P. Kastelic (University of Calgary) for correcting style and English grammar. Supplementary Materials The following supporting information can be downloaded at: Table S1. Scores used to assess maternal protectiveness, including the maternal protective score (MPS), displacement (DIS), agitation (AGI), attention (ATT), and aggressiveness (AGG) scores as described by Ceballos et al. Click here for additional data file. Author Contributions Conceptualization, R.R.V., L.E.F., A.U., J.A.N. and A.C.S.; methodology, R.R.V., L.E.F., A.U., J.A.N., M.C.C. and A.C.S.; formal analysis, R.R.V. and A.C.S.; writing--original draft preparation, R.R.V., M.C.C. and A.C.S.; writing--review and editing, R.R.V., M.C.C., J.A.N. and A.C.S.; supervision, L.E.F. and A.C.S.; project administration, L.E.F.; funding acquisition, L.E.F. All authors have read and agreed to the published version of the manuscript. Institutional Review Board Statement The animal study protocol was approved by the Ethics Committee of the Instituto de Zootecnia do Governo do Estado de Sao Paulo (protocol code 230-16/6 April 2016) for studies involving animals. Data Availability Statement The data presented in this study are available on request from the corresponding author. Conflicts of Interest The authors declare no conflict of interest. Figure 1 Distribution of displacement, agitation, attention, and aggressiveness scores, maternal composite score (MCS), and maternal protective behavior (MPB) of primiparous Gyr dairy cows at the first handling of their calves. animals-13-00921-t001_Table 1 Table 1 Ethogram of primiparous Gyr dairy cow and calf behaviors in Post-calving and Post-handling periods. Categories Description Post-calving period Cow latency (min a) Period from complete expulsion of the fetus (calving) to the first time the cow touches the calf with the muzzle and/or tongue. Calf latency (min.) Period from expulsion of the fetus (calving) to the calf stands on four legs without falling. Calf attempts (freq b) Number of calf unsuccessful attempts to stand. Post-calving and Post-handling periods Touching (% c) Cow's tongue or muzzle is in physical contact with any part of the calf's body. Not interacting (%) Cow is standing or lying without physical contact and/or without any type of physical interaction with the calf. Suckling (%) Cow is standing still while the calf sucks her teats, or makes contact with teats and/or udder region. a min = latency in minutes; b freq. = frequency (in number); c % = percentage of observation time. animals-13-00921-t002_Table 2 Table 2 Means (+-standard error) of primiparous Gyr dairy cow behaviors and calf sex, behavior, and weight during the post-calving and post-handling periods. Categories Mean +- SEM Treatments Training Control Calf sex (freq 1) Male () 11 9 Female () 6 11 Calf weight (kg 2) 22.70 +- 0.52 23.62 +- 0.91 22.05 +- 0.61 Male () 23.29 +- 0.72 a* 23.72 +- 1.22 22.92 +- 0.87 Female () 21.87 +- 0.73 b* 23.42 +- 1.43 A* 21.02 +- 0.76 B* Calf latency (min 3) 62.51 +- 7.51 68.87 +- 11.16 57.66 +- 10.25 Male () 68.28 +- 11.18 73.80 +- 15.77 63.27 +- 16.38 Female () 54.93 +- 9.38 60.66 +- 15.13 51.50 +- 12.44 Calf attempts (freq.) 13.54 +- 1.00 14.06 +- 1.47 13.14 +- 1.38 Male () 16.28 +- 1.38 a** 15.80 +- 1.93 16.72 +- 2.05 Female () 9.93 +- 0.82 b** 11.16 +- 1.83 9.20 +- 0.72 'Post-calving Period' Cow latency (min.) 4.60 +- 0.81 4.75 +- 1.54 4.50 +- 0.91 Male () 5.30 +- 1.24 5.40 +- 2.21 5.23 +- 1.49 Female () 3.64 +- 0.89 3.66 +- 2.01 3.63 +- 0.92 Touching (% 4) 52.95 +- 2.81 49.38 +- 4.48 55.72 +- 3.56 Male () 48.92 +- 18.36 42.96 +- 4.87 B,b** 55.63 +- 7.32 A** Female () 57.51 +- 11.59 60.95 +- 6.77 a** 55.80 +- 3.11 Not interacting (%) 42.00 +- 2.87 45.63 +- 4.66 39.17 +- 3.57 Male () 47.43 +- 4.49 53.12 +- 5.07 a,A** 41.03 +- 7.33 B** Female () 35.85 +- 2.81 32.15 +- 5.91 b** 37.69 +- 3.08 Suckling (%) 3.94 +- 0.90 4.04 +- 1.20 3.85 +- 1.32 Male () 2.90 +- 3.78 3.39 +- 1.57 2.36 +- 0.90 Female () 5.11 +- 6.18 5.22 +- 1.95 5.05 +- 2.25 'Post-handling Period' Touching (%) 38.91 +- 2.99 36.87 +- 4.10 40.53 +- 4.33 Male () 39.73 +- 4.36 38.00 +- 5.76 41.66 +- 6.91 Female () 37.98 +- 4.18 35.00 +- 5.80 39.61 +- 5.77 Not interacting (%) 56.41 +- 3.09 56.45 +- 4.27 56.37 +- 4.50 Male () 53.50 +- 4.53 54.00 +- 6.25 52.96 +- 6.98 Female () 59.66 +- 4.16 60.55 +- 4.84 59.17 +- 6.03 Suckling (%) 3.47 +- 0.94 3.95 +- 1.46 3.08 +- 1.26 Male () 4.47 +- 1.40 3.66 +- 1.69 5.37 +- 2.35 Female () 2.35 +- 1.23 4.44 +- 2.90 1.21 +- 1.15 1 freq. = frequency (in number); 2 kg: kilograms; 3 min = latency in minutes; 4 % = relative frequency; a-b In the same column of each category, means without a common lowercase superscript indicate significance (p <= 0.05) ** or tendency (p <= 0.10) *; A-B In the same row of each category, means without a common uppercase superscript indicate significance (p <= 0.05) or tendency (p <= 0.10). 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PMC10000115

This study aimed to evaluate the effect of the ovarian status and steroid hormone concentration on the day of TAI on the reproductive performance of dairy cows subjected to estrus synchronization treatment and timed artificial insemination with sexed semen. Seventy-eight cyclic Holstein cows pre-treated with PGF2a-GnRH were divided in two groups--I (Preselect-OvSynch, n = 38) and II (OvSynch+PRID-7-day+eCG, n = 40)--and inseminated with sexed semen. The presence of preovulatory follicle (PF) with or without corpus luteum (CL), the PF diameter, the estradiol (E2) and progesterone (P4) concentrations on the day of TAI, the pregnancy rate (PR) and embryo loss were determined. On the day of TAI, 78.4% of all the pregnant cows presented a PF (mean size 1.80 +- 0.12 cm) without CL, low P4 (0.59 +- 0.28 ng/mL) and high E2 (12.35 +- 2.62 pg/mg) concentrations. The positive correlation between the size of the PF and the level of E2 in the pregnant cows from group II was stronger than that of group I (R = 0.82 vs. R = 0.52, p < 0.05). The pregnancy rate on day 30 (57.5% vs. 36.8%) and day 60 (50% vs. 26.3%; p < 0.05) and the embryo losses (13% vs. 28.5%) showed better effects of treatment in group II. In conclusion, the ovarian status and the steroid hormone concentration on the day of TAI influence the pregnancy rates of dairy cows subjected to estrus synchronization and timed artificial insemination with sexed semen. cows ovarian status steroid hormones pregnancy sexed semen Ministry of Education and Science of the Republic of BulgariaD01-62/18.03.2021 The research leading to these results received funding from the Ministry of Education and Science of the Republic of Bulgaria under the National Scientific Program INTELLIGENT ANIMAL HUSBANDRY, grant agreement no. D01-62/18.03.2021. pmc1. Introduction Insemination with sexed semen is an intelligent tool to accelerate herd expansion, minimize waste production, improve animal welfare, increase profitability and reduce greenhouse gas emissions from beef and dairy farming . Semen sexing is an assisted reproductive technology that allows the sorting of spermatozoa carrying the Y-chromosome and the production of newborn animals of a precisely defined and preferred sex . Its main advantage is the production of more female offspring with valuable genetic traits in a shorter period. The use of sexed semen results in a reduction in the number of cows required for progeny testing, the fast production of replacement dairy heifers and a significant reduction in the herd-renewal period. Accelerated genetic progress and the accomplishment of in-house production of replacement heifers increase the degree of farm biosecurity. In line with modern standards of animal welfare, there is a reduction in difficult births when the sexed semen originates from bulls selected for ease of calving . Despite the aforementioned advantages, the concentration of spermatozoa in sexed semen is lower than in conventional semen, and the sorting procedure usually leads to some morphological damage to the sperm cells as well as reduced mitochondrial activity, which is related to low fertility . The standard dose of one straw with sexed sperm is approximately 2 x 106 sperm, while the straws with conventional semen contain 15-20 x 106 spermatozoa per dose . Sperm cell damage is associated with lower survival in the genital organs of female animals (12-16 h for sexed vs. 24-36 h for conventional semen) and requires excellent heat-detection management . Significantly greater conception rates in heifers than in lactating cows and animals with observed hormonally induced estrus have been determined by different authors . Because of this reduced fertility, most of these authors suggest the application of sexed semen for the insemination of heifers with observed estrus. Nevertheless, studies about the use of sexed semen in lactating dairy cows are in progress. Maicas et al. report increased pregnancy per artificial insemination (P/AI) in lactating cows when target animals were selected based on base parity, body condition score (BCS), economic breeding index (EBI) and days in milk. In this aspect, DeJarnette et al. recommend artificial insemination with sexed semen mainly in dairy cows with observed estrus, and recommend avoiding the use of a fixed-time insemination, as the success rate was highly variable. In contrast, Karakaya-Bilen et al. , comparing three TAI protocols with conventional and sexed semen, conclude that presynchronization increases ovulation and improves the P/AI in cows inseminated with both semen types. According to Darke et al. , there is no significant difference in P/AI when cows are inseminated with sexed semen at 16 or 22 h after GnRH treatment, suggesting that 16 to 22 h after GnRH injection probably encompasses the optimal window for the timing of AI with sex-sorted semen. However, Louber et al. underline that cows inseminated with sexed semen 16 h after the Double-Ovsynch program yielded more P/AI than cows inseminated 24 h after the same treatment. The lower result was associated with decreased time for sperm transport and capacitation, as well as differences in the hormonal milieu at critical time points near the end of the synchronization protocol. Guner et al. observed a higher pregnancy rate in cows that expressed estrus following synchronization with progesterone-based OvSynch than cows with no observed estrus. Different studies have reported the influence of estradiol and progesterone concentrations on follicular development, ovulation and the pregnancy rate in cows with natural or synchronized estrus and TAI . Nevertheless, questions remain open regarding the effects of animal selection in synchronization programs and TAI with sexed semen, as well as responses to different hormonal treatments and steroid hormone concentrations at the time of AI on the pregnancy rate. Inadequate preovulatory concentrations of estradiol in cows around the time of fixed-time insemination was shown to be a reason for lower pregnancy rates associated with improper uterine pH at the time of insemination . A study by Herlihy et al. demonstrated differences in periovulatory E2 concentrations between treatments to synchronize estrus and ovulation. They observed increased periovulatory E2 concentrations with a CIDR-based compared with an OvSynch protocol. Preovulatory concentrations of estradiol probably play a crucial role in pregnancy maintenance, not only directly by regulating uterine gene expression but also indirectly throughout the subsequent estrous cycle . In most mammals, progesterone production by the CL is essential for the establishment and maintenance of pregnancy and the stimulation of the uterine functions that allow early embryonic development, implantation, placentation and successful fetal and placental development to term . According to Cerri et al. reduced concentrations of progesterone before ovulation can modify endometrial gene expression beyond steroid receptors to a variety of other genes that might be essential to early embryonic survival. They determined that cows with lower plasma concentrations of progesterone and greater concentrations of estradiol had larger ovulatory follicle diameters at the end of the synchronization protocol than cows with high progesterone. Baruselli et al. reported the influence of progesterone concentrations during superovulation protocols on follicular growth, oocyte quality and embryo quality, and the need for adjustments to the protocols depending on the animal category and breed. The aim of the current study was to determine the effect of the ovarian status and steroid hormone concentration on the day of TAI on the reproductive performance of dairy cows subjected to different estrus synchronization treatments and timed artificial insemination with sexed semen. 2. Materials and Methods 2.1. Animals This study was carried out with 78 clinically healthy Holstein cows reared on a commercial dairy farm in Bulgaria, located at latitude of 42.183 N, longitude 25.567 E. The animals were from first to third lactation, weighing 680-700 kg, >=63 DIM (range 63 to 120 DIM),with an average daily milk yield >= 28 kg (range 28 to 34 kg), and located in a loose housing system with individual boxes and a self-locking feed fence. Their feed was with a total mix ration, and their daily diet comprised corn silage, triticale haylage, straw, beer mash, soybean meal, medium-coarse grain soft wheat, corn grain, mineral and vitamin supplement, natrium chloride and a toxin binder with water intake ad libitum. The investigation was conducted between October and December. All procedures were in agreement with the Bulgarian Veterinary Law (25 January 2011) regarding the living conditions and welfare of livestock animals used for experimental purposes, which is adapted to European Union regulation 86/609 (approval no. 3601/09.12.2020). 2.2. Experimental Design, Hormonal Treatments, Timed Artificial Insemination, Pregnancy Rate and Embryo-Loss Determination Animal selection was based on ultrasound examination of reproductive organs and ovarian status determination. A transrectal ultrasonography was conducted using ultrasound scanner SonoScape S2 Vet and a multifrequency (7-12 MHz) linear transducer (SonoScape Medical Corporation. Shenzhen, China). Only cows with corpus luteum (CL) and at least one large follicle (>=10 mm in diameter) in one of the ovaries were included in the study. Their follicular diameters were measured using the in-built scale provided with the ultrasound, and in calculation, only the largest was used. The animals were divided into two groups according to the hormonal treatments, as the proportions of animals that were close in terms of days in milk, average milk yield and number of lactations were almost the same. The estrus synchronization protocols were designed to start on day 6-7 of estrous cycle . After animal selection, both groups were subjected to presynchronization treatment, including single intramuscular injection of PGF2a (25 mg dinoprost tromethamine, Enzaprost T(r), CEVA Sante Animale, Libourne, France) on the day of the first ultrasound, followed by intramuscular GnRH administration (100 mg gonadorelin as diacetate, Ovarelin(r), CEVA Sante Animale, Libourne, France) on the day of heat detection (3rd or 4th day). The heat detection after the prostaglandin injection in the presynchronization treatment was registered by an electronic AfiMilk(r) system for activity monitoring (AfiMilk, Ltd., Kibbutz Afikim, Israel). Day 7 after the end of the hormonal pre-treatment was accepted as day 0 of estrus synchronization protocol. The first group (group I, n = 38) was treated with Preselect-OvSynch protocol, including 100 mg GnRH on days 0 and 9 and 25 mg PGF2a on day 7. Timed artificial insemination was carried out 20 h after the last GnRH injection. The second group (group II, n = 40) was included in OvSynch+PRID-7-day+eCG protocol. On day 0, a progesterone-releasing intravaginal device (PRID Delta(r) containing 1.55 g progesterone, CEVA Sante Animale, Libourne, France) was inserted into the vagina and 100 mg GnRH was injected intramuscularly. On day 7, the PRID was removed and each animal received 25 mg PGF2a and 500 UI eCG (equine chorionic gonadotropin; Folligon(r), Intervet International B.V., Boxmeer, The Netherlands) followed by a second injection of 100 mg GnRH 56 h later. The artificial insemination was 16 h after the second GnRH administration. The ovarian status (presence of follicles with or without CL in one of the ovaries and diameter of the preovulatory follicle) was determined by ultrasound immediately before TAI . The largest follicle located in the left or the right ovary was defined as a preovulatory follicle (PF). The timed artificial insemination of all cows was performed using frozen sexed semen in straw from the same bull with a high fertility index. The semen was deposited deep cervically by the same operator. The reproductive performance included registration of pregnancy rate and embryo loss. The pregnancy status (pregnant or non-pregnant cows) was determined by transrectal ultrasonography on days 30 and 60 after the TAI. A positive pregnancy diagnosis was recorded upon observation of an echogenic embryo located in echogenic uterine lumen . Embryo loss was calculated according to the pregnancy results on days 30 and 60 after TAI. 2.3. Measurement of Steroid Hormone Concentrations Blood samples were collected on day of TAI before the ultrasound examination. The serum from the coagulated blood samples was separated by centrifugation (3000x g for 15 min) and stored in a sterile tube at -20 degC until analysis. Steroid hormone concentrations were measured by an automated quantitative enzyme-linked fluorescent immunoassay (ELFA; VIDAS; ImmunoDiagnostic Assay System, bioMerieux, Craponne, France). For the progesterone (P4) assay, we used a VIDAS(r) Progesterone kit with an analytical sensitivity of 0.1 ng/mL and imprecision within and between runs of 5.7-3.8% and 6.2-3.8%, respectively. The level of estradiol-17b (E2) was determined using a VIDAS(r) Estradiol II kit with an analytical sensitivity of 9 pg/mL and imprecision within and between runs of 7.5-3.9% and 9.5-5.1%, respectively. The measurement range of the kit was 9-3000 pg/mL; therefore, the samples with values < 9 pg/mL were counted as zero. 2.4. Statistical Analysis The results were processed by computer program Statistica version 10.0 (Stat-Soft. Inc. Tulsa, OK, USA). The values of different parameters are presented as mean +- standard deviation (mean +- SD). An analysis of variance for main effects (ANOVA) was used to estimate the statistical significance of the influence of different factors on the likelihood of pregnancy. The strength of the relationship between parameters was assessed by Pearson's linear correlation test. The significance of the differences between the mean values for different groups and subgroups was compared using the post hoc Tukey's test or a Chi-squared test for comparison of proportions with small samples. Statistical significance was considered at p < 0.05. 3. Results At the beginning of the study, the selected animals had corpus luteum, small or medium follicles and at least one large follicle located in one of the ovaries, which was accepted as an indicator of cyclicity. All cows had a positive response after the PGF2a injection of the presychronization treatment, evidenced by the detection of estrus behavior between 3 and 4 days later. The comparative analysis of the ovarian status showed that in group I, 16.6% more non-pregnant cows had PF with CL on the day of TAI, while of the pregnant animals, 28.6% more had PF without CL. (Table 1). In group II, a significantly higher percentage of PFs without CL on the day of TAI was observed in both the non-pregnant and the pregnant animals (70.6% and 87%; p < 0.05). The mean diameters of the PFs in the non-pregnant and pregnant cows treated with Preselect-Ovsynch were close (p = 0.12). The value of this parameter in the OvSynch+PRID+eCG group was higher in the pregnant than in the non-pregnant animals (1.88 +- 0.10 mm vs. 1.68 +- 0.19 mm; p < 0.05). The mean PF diameter in the pregnant cows in group II also differed significantly (p < 0.05) from the follicular diameters in the pregnant and non-pregnant cows in group I. The blood progesterone concentrations on the day of TAI indicated significant (p < 0.05) differences between the pregnant and non-pregnant dairy cows, irrespective of the estrus synchronization protocol used. This was clearly expressed in group I, where the concentration of P4 in the non-pregnant animals exceeded that in the pregnant animals by a few times (Table 1). The lowest level of P4 on the day of TAI was recorded in the pregnant cows from group II. The mean estradiol-17b concentrations in the pregnant and non-pregnant cows in both groups also differed significantly (p < 0.05). The highest level of E2 on the day of TAI was determined in the pregnant animals that received OvSynch+PRID+eCG treatment. It was higher compared to the measured level E2 in the non-pregnant animals in this group (p < 0.05) and the E2 values in the whole group I (Table 1). The pregnancy rate on day 30 tended to be higher by about 20% in the OvSynch+PRID+ to the Preselect-OvSynch-treated cows, but the difference was not significant (p = 0.16). However, on day 60, the PR values were significantly greater in group II compared to group I (Table 1). In addition, the embryo losses were 15.5% lower in the animals treated with protocol II than in those treated with protocol I. The animals with embryo loss in pregnant subgroup I had PF only, and PF and CL on the day of TAI at rates of 25% and 75%, respectively. At the same time, 100% of the cows with embryo loss in the pregnant subgroup II exhibited PF and CL. From a total of seven animals, PF with a Cl was detected in 85.7% of cows, while in 14.3%, this was not observed. The average diameter of the PFs on the day of TAI was 1.76 +- 0.07 mm. The pregnancy reductions of 10.3% in group I and of 7.5% in group II between 30 and 60 days were not statistically significant. The investigated effects, irrespective of the estrus synchronization treatment used, are presented in Table 2. Comparative analysis showed that overall, the pregnant cows had significantly more cases of PF without CL in the ovaries on the day of TAI. The mean values for the PF diameter and E2 concentration on the day of TAI were higher in the pregnant group than in the non-pregnant group (p < 0.05), whereas the concentration of P4 was lower (Table 2. p < 0.05). A strong correlation was observed between the sizes of the preovulatory follicles and the level of E2 in all the animals (R = 0.93, p < 0.001), as well as in the pregnant animals treated with both synchronization schemes (R = 0.79, p = 0.001). Interestingly, in the pregnant cows from group II, this correlation was stronger compared to that in the pregnant cows from group I (R = 0.82 vs. R = 0.52, p < 0.05). The analysis (Table S1) showed that both the PF size and the E2 concentration were influenced by the ovarian status (p = 0.007) and by the treatment protocol (p = 0.019). The observed power of the effect of pregnancy status on these parameters was the highest (observed power 0.94, p < 0.05). 4. Discussion The success of the artificial insemination of dairy cows with sexed semen is rather variable and depends on different on-farm factors, the age, BCS and parity of the animals, sire selection, service number, estrus detection management and cow fertility potential . A reduced number of spermatozoa per dose, the limited lifespan of sorted sperm cells, the insemination of cows with inadequate development of ovulatory follicles, or failed ovulation due to a hormonal imbalance and the performance of artificial insemination at an inappropriate time are the main reasons for low conception rates . In this context, some authors recommend eliminating some of these problems by including dairy cows in estrus synchronization programs with fixed times of artificial insemination . A major advantage of these programs is the possibility of timed AI, which is less time-consuming, makes work with the animals easier and provides control over ovulation time . Starting the OvSynch treatment between days 5 and 9 of the estrus cycle is associated with a greater probability of having ovulations with a new CL and the development of a new follicular wave. This results in the synchronized ovulation of a newly formed dominant follicle at the end of the synchronization program and better conception rates . Different studies reveal a positive effect of progesterone-releasing devices on conception rates after their inclusion in estrus synchronization protocols . This study presents results regarding the effects of ovarian status and steroid hormone concentrations at the time of TAI on the pregnancy rates of dairy cows treated by Preselect-OvSynch and OvSynch+PRID+eCG estrus synchronization protocols, starting on day 6-7 of the estrous cycle and timed AI with sexed semen from a high-fertility bull. The uniform distribution of the animals in both treatment groups was proven by the fact that the same ovarian status was observed before the start of the experiment, and by the positive response of all the cows to the presynchronizing PGF2a-GnRH treatment which was evidenced by heat detection 3 to 4 days later. The strategy for presynchronization, involving the induction of ovulation 6-7 days before the start of the estrus synchronization protocol, results in more animals having ovulations after the first GnRH injection of the protocol . If cows fail to ovulate after the first GnRH injection, the lifespan of the preovulatory follicle at the end of the synchronization protocol is prolonged, which is associated with the ovulation of aged oocytes, reduced embryo quality and low pregnancy rates . Nevertheless, we considered the ovarian status and the concentration of steroid hormones on the day of TAI as determining factors in the successful ovulation and the achievement of an acceptable PR in the use of sexed semen. Most of the non-pregnant animals in group I (58.3%) had PF and CL on the day of TAI, while the majority of the animals that became pregnant (64.4%) bore PF without CL. This information and the significant (p < 0.05) difference in the ovarian status on the day of TAI between the different subgroups of cows in group II supported the aforementioned hypothesis. The absence of CL on the day of TAI in more than 70% of the pregnant animals after the OvSynch+PRID+eCG treatment was in agreement with other reports of the better synchronizing effect of progesterone-based protocols in comparison with conventional OvSynch. The phenomenon behind the reduction in the efficiency of conventional OvSynch protocols may be incomplete luteolysis after the PGF2a injection, followed by inadequate levels of progesterone and the suppression of the second follicular wave, which makes the synchronization of ovulation and TAI impossible . This is crucial for successful insemination with sexed semen because of the limited lifespan of the spermatozoa in the female genital organs, the reduced number of sperm per straw, and the potential pre-capacitation induced by the sorting procedure . Different researchers stated that progesterone supplementation before timed AI imitates P4 concentration in the late luteal phase of a regular estrous cycle, leading to an increase an ovulation synchrony and P/AI . This information is in agreement with the better pregnancy rate in group II observed in the current experiment. Our hypothesis supposes that the diameters of the preovulatory follicles and steroid hormone concentrations can affect ovulation and, consequently, pregnancy after AI with sexed semen. The large size of PFs (in the range of 1.72 cm to 1.88 cm) was associated with a positive effect on the ovulation and PR in both groups. The determination of the highest mean diameter of the PFs in the pregnant animals from the second group and the relative enhancement of the sizes of the preovulatory follicles in the non-pregnant cows from the first group may be explained by the administration of eCG during the PGF2a injection. The influence of follicular size near the time of TAI on pregnancy success is still debatable. Bello et al. determined the best conception rates after the ovulation of a 1.6 cm follicle, whereas conception rates decreased if the preovulatory follicle was either smaller or larger than 1.6 cm. According to Colazo et al. , after the second GnRH injection in synchronization programs with or without P4 supplementation, there was a variable range of ovulatory follicle sizes between lactating dairy and beef cows. They reported that follicles between 1.7 cm and 2.5 cm in diameter retain their capacity to ovulate, and cows with ovulatory follicles greater than 2.0 cm have an increased probability of pregnancy loss. Conversely, Berg et al. found that follicles smaller than 1.5 cm ovulated significantly later, while cows with medium-sized and large follicles ovulated earlier than other animals. These controversial results presume that follicular size is not the only factor responsible for successful ovulation and conception. In the current study, estrus synchronization treatments had a considerable effect on the functional status of the ovarian structures on the day of TAI. They were associated with the production of progesterone and estrogen from the CL and the preovulatory follicle, respectively. The significant (p < 0.05) difference in the mean P4 concentration between the non-pregnant and pregnant animals subjected to both treatments was probably due to the high percentage of animals bearing functional and active CL in the non-pregnant group at the time of TAI, caused by failed or incomplete luteal regression. The lower concentration of P4 determined in the pregnant animals from group II can be attributed to better luteolysis after the application of the Ovsynch+PRID+eCG protocol. In this regard, Garcia-Munoz et al. observed insignificant differences between treated and control groups after the administration of exogenous progesterone around the time of ovulation, and treatment with d-cloprostenol 120 h after ovulation. It is still not clear whether exogenous progesterone influences the sensitivity of the cow CL to PGF treatment. Although the same number of animals presented CL at the time of TAI, we observed a slight increase in P4 in non-pregnant subgroup II compared to pregnant subgroup I. One reason for this result could be the residual quantities or the delayed clearance of the supplemented progesterone. Cerri et al. also reported augmented levels of P4 observed at the time of TAI after PRID removal and the first PGF2 alfa administration. The significant difference (p < 0.05) in the mean E2 concentration between the pregnant and non-pregnant animals of the treated groups was an indicator of the increased steroid activity of the larger preovulatory follicles, which was confirmed by correlative analysis. This effect was clearly visible in group II, where the highest E2 concentration corresponded with the largest size of the preovulatory follicles. A similar increase in E2 in the larger PFs was observed in group I. On the other hand, the preovulatory follicles formed after Preselect-Ovsynch or Ovsynch+PRID+eCG treatment probably had different levels of estradiol production. Despite the insignificant difference between the mean diameters of the PFs in both subgroups subjected to Preselct-Ovsynch, a significantly (p < 0.05) higher mean concentration of estradiol-17b in the pregnant subgroup was observed. In this respect, Bridges and Fortune emphasized that extended progesterone exposition after the incomplete regression of the CL had a negative indirect effect on estradiol-17b production. Herlihy et al. also reported a positive correlation between increased preovulatory follicle size and circulating concentrations of E2 in progesterone-based synchronization protocols due to a longer period of preovulatory follicle growth. After P4 withdrawal, an increase in LH pulse frequency and mean LH concentrations promoted follicle growth and increased estrogen production. The second GnRH caused a rapid decrease in circulating estrogen concentrations, and OvSynch had less peak preovulatory E2 than the CIDR-TAI protocol. An additional factor in the increase in steroid activity in the preovulatory follicles of group II was probably the PMSG stimulation. According to Hirako et al. , the administration of 500 UI eCG in cattle lead to a significant increase in the release of estradiol-17b. Despite the lack of significant differences in PR on day 30, the PR tended to increase after the Ovsynch+PRID+eCG treatment compared to the Preselect-Ovsynch treatment (38.6% vs. 57.5%; p = 0.09). The lower percentage of pregnant cows in group I, which was probably due to incomplete luteolysis, corresponded with the high P4 level on the day of TAI, which results in reduced pulsatile LH release and negatively affects final follicle maturation, oocyte maturity, and gamete transport . Martins et al. determined that complete functional luteolysis had a strong positive relationship with the time of ovulation. The lack of complete regression of the corpus luteum resulted in a low P/AI because of the increased progesterone concentration close to the time of AI in cows subjected to conventional Ovsynch treatment . The higher pregnancy rate in group II can be explained by the inclusion of exogenous progesterone and eCG in the second protocol. In line with the current study, Colazo et al. determined that the inclusion of PRID in the conventional Ovsynch protocol had a positive effect on P/AI. In fact, progesterone-releasing devices (CIDR or PRID) provide a slow release of progesterone, which regulates the number of estrogen receptors in the medial basal hypothalamus, increasing the responsiveness to estradiol, leading to a preovulatory LH surge. The withdrawal of the progesterone device leads to decreased progesterone and increased LH pulse frequency and mean concentrations of LH, which are associated with an increased number of LH receptors in the granulosa and theca cells, estradiol production by the follicle, and the stimulation of LH surges, thus resulting in ovulation . Additional evidence for the positive influence of the Ovsynch+PRID+eCG treatment on pregnancy was the registration of a higher percentage of PR in the second group on day 60 (p < 0.05). The pregnancy rate obtained with the Preselect-Ovsynch on day 30 after TAI (36.8%) was close to the 35.5% recorded by Karakaya-Bilen et al. , but lower than the 49.0% obtained by Drake et al. . Lauber et al. recorded 50% P/AI in presynchronized dairy cows on day 34 after double Ovsynch treatment and TAI with sexed semen. At the same time, the P/AI in Ovsynch+PRID+eCG (57.5%) was greater than the values observed in primiparous (49.5%) and multiparous (47.6%) lactating cows treated with a PRID-based protocol and inseminated with sexed semen . In agreement with this study was Brozos et al. Ref. 's finding of 54.3% P/AI after the use of PGF2a-GnRH pre-treatment and PRID-based protocols during the winter season. Furthermore, the sexed semen for the AI in our study originated from a bull selected for its high fertility. Drake et al. investigated the interaction between treatment with sex-sorted semen and P/AI according to the bull fertility and detected deviations in the P/AI from 38.2% to 60.3%. Similar to the findings of previous authors, we also observed decreasing PR between both consecutive ultrasound examinations for pregnancy, which was an indicator of late embryo loss. The embryo loss after the use of both synchronization protocols (28.5% and 13%) corresponded with the data obtained by Karakaya-Bilen et al. and Guner et al. . In disagreement with the obtained data was the embryo loss (5% and 6%) in the dairy cows on days 34 +- 3 and 80 +- 17 determined by Lauber et al. ; however, that study used a different estrus synchronization treatment. The higher percentage of embryo loss in group I can be attributed to the ovulation of smaller follicles at the time of TAI and the formation of CL, releasing less P4, which is critical for embryo development through the early gestation period . In this respect, the diameter of the PFs on the day of TAI in the animals with pregnancy loss in subgroup I (<=1.7 mm) was below the calculated average size (1.8 +- 0.12 mm) of the PFs for all the pregnant animals. The assertion of the effect of the ovarian status was supported by the significantly (p < 0.05) higher percentage of animals that became pregnant when PF without CL and diameters between 1.68 cm and 1.88 cm on the day of TAI were presented. It is known that the large luteal cells of the future CL, producing 80 to 90% of the progesterone, originate from granulosa cells . Larger ovulatory follicles provided a greater number of granulosa cells, which resulted in a larger CL volume on day 7, producing enough progesterone for adequate pregnancy maintenance . The difference (p < 0.05) between the mean values of the steroid hormones on the day of TAI between the non-pregnant and pregnant cows was an indicator of the significant effect of the E2 and P4 concentrations on the pregnancy results. A low P4 concentration at TAI in pregnant animals was also recorded by Souza et al. . They observed that a blood progesterone level > 0.5 mg/mL at a time close to insemination decreases fertility by about 50%, while the P4 dropping to <0.5 ng/mL 2 days after the GnRH injection suggested a greater probability of pregnancy. Denicol et al. underline the importance of the endocrine environment during follicular growth and the maturation of viable oocytes, and accept the size of the ovulatory follicle as a vital component in the probability of pregnancy. An inadequate endocrine profile at the time of TAI was shown to decrease oocyte quality, gamete transport, fertilization and the uterus's ability to maintain pregnancy . According to Brozos et al. , even after the second PGF2 treatment in the PRID-based protocol, a considerable number of cows had high progesterone (>1 ng/mL) at the time of AI; this occurred without a decrease in P4 concentration in the period between the first PGF2 injection and the AI. All the aforementioned studies assumed that the ratio between estrogen and progesterone on the day of TAI is a very important factor in successful conception in synchronized dairy cows inseminated with sexed semen. Future detailed investigations with a large number of cyclic cows can clarify this matter. 5. Conclusions The ovarian status and steroid hormone level on the day of TAI significantly affected the reproductive performance of dairy cows subjected to estrus synchronization and artificial insemination with sexed semen. The presence of a large PF, the production of more estrogen, a lack of CL, and low progesterone levels at the time of AI are factors that ensure acceptable pregnancy rates. The application of presynchronization treatment with the Ovsynch+PRID+eCG protocol can additionally improve pregnancy results after AI with sex-sorted semen. The confirmed strong correlation between the values of the PF and E2 concentrations allows ultrasound examination of the ovarian status of cows on the day of TAI to be recommended as a reliable tool for the selection of candidates for insemination with sexed semen. This will improve the utilization of sex-sorted semen from bulls with high genetic value, and accelerate genetic progress. Supplementary Materials The following are available online at Table S1: Main factors influencing PF size and E2 concentration in dairy cows inseminated with sex-sorted semen (ANOVA main effects analysis). Figure S1: Ultrasound image of the ovarian status of dairy cows on day of TAI (A--preovulatory follicles in both ovaries without corpus luteum; B--corpus luteum located in the left ovary and preovulatory follicle in the right ovary); Figure S2: Ultrasound image of pregnancy in dairy cows on days 30 (A) and 60 (B) after TAI with sexed semen. Click here for additional data file. Author Contributions Conceptualization and methodology, S.Y. and A.A.; investigation, S.Y., I.F., A.A., B.S. and B.I.; resources, D.Y. and B.S.; data curation, S.Y., E.K. and D.Z.; writing--original draft preparation, S.Y. and E.K.; writing--review and editing, S.Y. and E.K.; visualization, S.Y. and A.A.; supervision, S.Y.; project administration, D.Y. and D.Z.; funding acquisition, all contributors. All authors have read and agreed to the published version of the manuscript. Institutional Review Board Statement The experimental work with animals was in accordance with Bulgarian Veterinary Law (25 January 2011) regarding the living conditions and welfare of livestock animals used for experimental purposes (farm license no. 3601/09.12.2020), which is adapted to European Union regulation 86/609. The use of farm animals was approved by the Ethics Committee at Trakia University (permission no. 233/11.04.2019) with the agreement of the Bulgarian National Animals Ethics Committee. Informed Consent Statement Informed consent was obtained from all subjects involved in the study. Data Availability Statement All data are included in the present article and its Supplementary Materials. Conflicts of Interest The authors declare no conflict of interest. Figure 1 Preselect-OvSynch (A) and OvSynch+PRID+eCG (B) estrus synchronization protocols in dairy cows (US--ultrasound examination; PGF2a--prostaglandin F2 alfa; GnRH--gonadotropin-releasing hormone; PRID--progesterone-releasing intravaginal device; TAI--timed artificial insemination). animals-13-00896-t001_Table 1 Table 1 Ovarian status and steroid hormone concentration on day of TAI, pregnancy rate (PR) and embryo loss according to different estrus synchronization treatments. Parameters Group I * (n = 38) Group II ** (n = 40) Non-Pregnant Subgroup (n = 24) Pregnant Subgroup (n = 14) Non-Pregnant Subgroup (n = 17) Pregnant Subgroup (n = 23) Ovarian status Preovulatory follicle with corpus luteum %, (n/n) 58.3 (14/24) 35.7 (5/14) 29.4 (5/17) 1 13 (3/23) 1 Preovulatory follicle without corpus luteum %, (n/n) 41.7 (10/24) 64.3 (9/14) 70.6 (12/17) 2 87 (20/23) 2 Diameter of preovulatory follicle (mean +- SD, cm) 1.60 +- 0.24 a 1.72 +- 0.20 a 1.68 +- 0.19 a 1.88 +- 0.10 b Steroid hormone concentrations Progesterone (mean +- SD, ng/mL) 2.35 +- 3.07 a 0.68 +- 0.26 b 0.82 +- 0.24 b 0.52 +- 0.12 c Estradiol-17b (mean +- SD, pg/mL) 7.32 +- 0.80 a 10.24 +- 0.36 b 10.04 +- 2.12 b 12.86 +- 2.26 c PR on day 30 %, (n/n) 36.8 (14/38) 57.5 (23/40) PR on day 60 %, (n/n) 26.3 (10/38) a 50 (20/40) b Pregnancy loss % 28.5 (4/14) 13 (3/23) * Preselect-OvSynch protocol, ** OvSynch+PRID-7-day+eCG protocol. Values with different superscript letters within a row are different at p < 0.05. Values with different superscript numbers within a column are different at p < 0.05. animals-13-00896-t002_Table 2 Table 2 Ovarian status and steroid hormone concentration on day of TAI according to pregnancy status of the cows. Groups Non-Pregnant (n = 41) Pregnant (n = 37) Ovarian status on day of TAI Preovulatory follicle with corpus luteum %, (n/n) 46.3 (19/41) a 21.6 (8/37) b,1 Preovulatory follicle without corpus luteum %, (n/n) 53.7 (22/41) a 78.4 (29/37) b,2 Diameter of preovulatory follicle (mean +- SD, cm) 1.64 +- 0.26 a 1.80 +- 0.12 b Steroid hormone concentrations Progesterone (mean +- SD, ng/mL) 1.58 +- 1.06 a 0.59 +- 0.28 b Estradiol-17b (mean +- SD, pg/mL) 8.68 +- 1.82 a 12.35 +- 2.62 b Values with different superscript letters within a row are different at p < 0.05. Values with different superscript numbers within a column are different at p < 0.05. 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PMC10000116

Acute myeloid leukemia (AML) comprises a group of hematologic neoplasms characterized by abnormal differentiation and proliferation of myeloid progenitor cells. AML is associated with poor outcome due to the lack of efficient therapies and early diagnostic tools. The current gold standard diagnostic tools are based on bone marrow biopsy. These biopsies, apart from being very invasive, painful, and costly, have low sensitivity. Despite the progress uncovering the molecular pathogenesis of AML, the development of novel detection strategies is still poorly explored. This is particularly important for patients that check the criteria for complete remission after treatment, since they can relapse through the persistence of some leukemic stem cells. This condition, recently named as measurable residual disease (MRD), has severe consequences for disease progression. Hence, an early and accurate diagnosis of MRD would allow an appropriate therapy to be tailored, improving a patient's prognosis. Many novel techniques with high potential in disease prevention and early detection are being explored. Among them, microfluidics has flourished in recent years due to its ability at processing complex samples as well as its demonstrated capacity to isolate rare cells from biological fluids. In parallel, surface-enhanced Raman scattering (SERS) spectroscopy has shown outstanding sensitivity and capability for multiplex quantitative detection of disease biomarkers. Together, these technologies can allow early and cost-effective disease detection as well as contribute to monitoring the efficiency of treatments. In this review, we aim to provide a comprehensive overview of AML disease, the conventional techniques currently used for its diagnosis, classification (recently updated in September 2022), and treatment selection, and we also aim to present how novel technologies can be applied to improve the detection and monitoring of MRD. acute myeloid leukemia (AML) measurable residual disease (MRD) microfluidics surface-enhanced Raman scattering (SERS) European Regional Development Fund (ERDF)030782 Foundation for Science and Technology (FCT)UIDB/50026/2020 UIDP/50026/2020 Norte Portugal Regional Operational Programme (NORTE 2020) Health From Portugal C630926586-00465198 FCT studentshipSFRH/BD/148091/2019 FCTDL 57/2016 This work was supported by European Regional Development Fund (ERDF) through COMPETE2020, under the IMPAct-L project (030782); by the Foundation for Science and Technology (FCT) projects UIDB/50026/2020 and UIDP/50026/2020; and by the project NORTE-01-0145-FEDER-000055, supported by Norte Portugal Regional Operational Programme (NORTE 2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (ERDF). This project also received funding of the project Health From Portugal (C630926586-00465198), supported by Component C5-Capitalisation and Business Innovation, under the Portuguese Resilience and Recovery Plan, through the NextGenerationEU Fund. A.T. acknowledges the FCT studentship SFRH/BD/148091/2019. B.S.-M. acknowledges funding by FCT, grant number DL 57/2016. pmc1. Introduction Hematopoiesis is a process that promotes the formation, development, and maturation of all blood constituents from hematopoietic stem cells (HSCs) . HSCs are multipotent cells that can self-renew and have the capacity to regenerate all the different cell types of the hematopoietic system, including the formation of erythroid cells, platelets, neutrophils, monocytes/macrophages, basophils, eosinophils, and lymphocytes . These cells have a key role in the continuous replenishment of blood cells throughout life . However, several intrinsic and extrinsic factors might have an impact on hematopoiesis . Disturbance in the ability of the HSCs to proliferate and differentiate in the different blood cells can cause an accumulation of immature blasts and hematopoiesis failure, consequently leading to the development of distinct hematopoietic disorders, such as leukemia. Leukemia can be grouped according to the affected cell lineage and the proliferation rate of immature cells. As such, leukemia can affect myeloblasts (myeloid) or lymphocytic precursors (lymphoid), and can be classified as acute (fast proliferation) or chronic (slow proliferation) . Consequently, leukemia is mainly divided into four types: acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphoid leukemia (ALL), and chronic lymphoid leukemia (CLL) . Depending on the type of leukemia, patients undergo tailored chemotherapy until they clinically reach complete remission (CR). However, after achieving CR and despite having no symptoms or signs of the disease, some patients may present small undetectable numbers of leukemic stem cells (LSCs) in the bone marrow . This condition is named minimal, or, more recently, measurable residual disease (MRD), and it constitutes the major cause of leukemia relapse. Until very recently, none of the conventional diagnostic tests used was sensitive enough to assess MRD in the early stages. In the following sections, we provide an overview on AML and the current tools used in the clinical setting for their diagnosis, monitoring, and therapeutics, and then focus on the new technologies and their potential to assess MRD. 2. Acute Myeloid Leukemia AML is a heterogeneous group of hematopoietic neoplasms and the most common type of acute leukemia in older individuals . AML incidence rises with age, increasing from ~1.3 per 100,000 of the population in those less than 65 years old to 12.2 cases per 100,000 of the population in those over 65 years of age, and with a median age of diagnosis of 68 years old . Prognosis also changes with age, younger individuals having a more favorable prognosis. In the majority of cases, the disease appears as a de novo malignancy in healthy individuals, but patients with underlying hematological disorders or prior chemotherapy are at higher risk of developing the disease . The estimated 5-year overall survival (OS) for older patients, those with secondary AML, or those with relapsed disease is an alarming 5-10%. The appearance and even the accumulation of mutations in HSCs and normal dividing cells are often associated with aging . A common age-related condition characterized by the acquisition of somatic mutations in leukemia-associated myeloid malignancy driver genes is clonal hematopoiesis of undetermined potential (CHIP) . However, these mutations must be present at a variable allele frequency >=2% in healthy individuals non-diagnosed with myeloid neoplasm or other obvious hematologic conditions . Taking this into account, CHIP is considered a potential pre-stage of leukemia, associated with a 0.5-1.0% risk (per year) for developing the hematologic disease . AML disease arises from continuous genetic and epigenetic alterations in HSCs and progenitor cells . It is known that somatic mutations in genes codifying epigenetic modifiers as NPM1, DNMT3A, IDH1/2, and TET2 are accumulated with age and/or during AML progression in HSCs . Shlush et al. found that there is a potential order for a mutation to happen, therefore mutations in DNMT3A and IDH2 occur in pre-leukemic HSCs, and mutations on NPM1c and FLT3 and NRAS and RUNX1 appear later in a leukemic state. In addition, even in cases of absence of any large chromosomal abnormality, genetic mutations which are identified in more than 97% of the cases are involved in the development of AML . Furthermore, emerging evidence suggests that LSCs induce molecular changes in the distinct hematopoietic and non-hematopoietic cell populations in the bone marrow (BM) niche . The latter happens via exosome secretion, facilitating the development of a malignant microenvironment while disrupting normal hematopoiesis . However, the conundrum of 'the chicken or the egg' of whether the niche favors some mutations or whether the mutations precede alterations in the niche remains an open question. It is possible that these two events are concurrent, where either niche alterations are genotoxic and oncogenic and/or that malignant cells might transform the normal niche to promote their survival and expansion. As a result of the dysregulated differentiation of normal blood cells, an accumulation of immature myeloid progenitor cells is observed not only in the BM , but also in the peripheral blood (PB). Consequently, the ability of the BM to produce normal white and red blood cells and platelets is reduced . Occasionally, clonal blasts can be found in other organs, such as the liver, spleen, and lymph nodes, resulting in anemia, bleeding, or organ infiltration and, ultimately, in hematopoietic failure . Finally, due to the highly heterogeneous genetic landscape of LSCs, early detection and personalized treatment constitute the biggest challenges in the field of AML. Furthermore, expanding the knowledge of the genetic landscape might promote a better understanding of therapeutic fragilities in AML, contributing to the definition of new potential targets, the development of new therapies, and the improvement of current strategies . 2.1. Diagnosis At the initial stages of the disease, PB analysis showing a high number of abnormal white blood cells (WBC), or even a very low blood count, can indicate the presence of AML. Hence, the confirmation of the AML diagnosis is performed by further morphologic and cytogenetic assessment in BM aspirates . The immunophenotyping by multiparameter flow cytometry (MFC) is conventionally used to accurately diagnose AML based on the identification of intracellular and extracellular markers (e.g., CD13, CD33 and CD34). On the other hand, the conventional cytogenetic analysis is mandatory in the assessment of AML disease. An alternative to detect specific abnormalities (RUNX1::RUNX1T1, CBFB::MYH11, KMT2A (MLL) and MECOM (EVI1) gene fusions or myelodysplasia-related chromosome abnormalities) is fluorescence in situ hybridization. Beyond these technical approaches, different molecular genetic tests have been used to screen all AML characteristic genetic abnormalities, and the tests are needed for differential diagnosis and targeted treatments. The final diagnosis and monitoring of AML always involves a BM tissue biopsy (BM trephine biopsy) or a BM biopsy. Those are invasive, painful, and create discomfort and harm for leukemia patients . Therefore, less invasive procedures are needed while ensuring an accurate diagnosis revealing more detailed information about the disease progression. One of the less invasive procedures that have been extensively studied and applied as a complementary method is the PB biopsy, also known as liquid biopsy . This is currently a complementary technique to the gold standard method for the detection of leukemia or MRD because of a smaller number of LSCs in the PB when compared with BM aspirates . 2.2. Classification The classification of hematological malignancies has advanced tremendously since the 1970s. The classification of AML is based on two main systems: the French-American-British (FAB) and the World Health Organization (WHO) (Table 1) . The FAB classification was introduced in 1976 and it is based on the morphology and cytochemistry of blasts, the degree of myeloid or monocytic differentiation, and their degree of maturation . As such, this classification recognizes eight distinct subtypes of AML, from M0 to M7 . This system has been widely used for the classification of hematological disorders in the last decade since characterization of the blasts is easily accessible and low cost. However, with recent advances in the genetic and molecular characteristics of AML, this classification is now becoming incomplete and outdated. Consequently, the WHO system has been increasingly used to classify AML. This system was firstly introduced in 1999 and reviewed in 2016 , combining the previous features from the FAB classification with genetic data (chromosomal and molecular), biological data, and clinical data . More recently, in September 2022, new updates about AML classification were published . This updated version of the classification provides a more genetically defined classification as it retains most of the previously defined AML types (with recurrent genetic abnormalities) and includes other genetically-related entities to define AML and changes in the blast thresholds. In summary, this revision of the WHO classification (completed by leading experts), maintains the blast threshold at 20% for the diagnosis of the majority of AML subtypes, but decreases it to >=10% blasts in BM or blood in the presence of recurrent genetic abnormalities (except for AML with t(9;22)(q34.1;q11.2)/BCR::ABL1) (Table 1) . The case of AML with BCR::ABL1 still requires >=20% blasts, due to avoiding overlap with chronic myeloid leukemia in the accelerated phase . In addition, some predisposing features (therapy-related, prior myelodysplastic syndrome (MDS) or MDS/myeloproliferative neoplasm (MPN), and germline predisposition) designed as qualifiers of the primary diagnosis have also been added. The WHO system provides a better characterization of AML patients, offering clear advantages for treatment personalization . 2.3. AML Pathogenesis and Prognosis In the new diagnosis of AML, a high percentage (50-60%) of cytogenetic abnormalities are found . These abnormalities are usually associated with non-random chromosomal translocations that result in gene arrangements implicated in a modification in the locus encoding for a transcriptional activator, leading to the expression of a fusion protein . These fusion proteins promote changes in the expression of target genes responsible for myeloid development maturation and proliferation, an abnormality that potentiates AML transformation . As a result, AML is constituted by a heterogeneous population of malignant cells. The different AML clones have distinct cytogenetic alterations and mutations that produce considerable genetic complexity. Taking this into account, a risk classification system in AML patients at diagnosis was also defined. This system was also updated this year (September, 2022), but maintained the division into three risk categories: favorable, intermediate, and adverse . However, it is important to note that after the recent revision and refinement, in addition to the baseline genetic abnormalities, the response to initial therapy and early MRD testing are now considered factors that contribute to the patient's risk classification . Thus, AML patients with a favorable prognosis present translocations of t (8; 21) (q22;q22.1)/RUNX1::RUNX1T1 with an occurrence of 10%; inversions of inv. (16) (p13.1q22) with an occurrence of 5% ; and mutated NPM1 without FLT3-internal tandem duplication (ITD) and in-frame mutations affecting the bZIP region of CEBPA . The intermediate risk group includes all AML cases with FLT3-ITD; mutated NPM1 with FLT3-ITD; wild-type NPM1 with FLT3-ITD; with translocations: t(9;11)(p21.3;q23.3) involving the MLLT3::KMT2A genes, and all the cytogenetic and/or molecular abnormalities not classified as favorable or adverse . Finally, the adverse-risk group now includes recurring cytogenetic abnormalities, such as the translocations: t(3q26.2;v) involving the MECOM gene; t(8;16)(p11;p13) associated with the KAT6A::CREBBP gene fusion; t(6;9)(p23;q34.1)/DEK::NUP214; t(v;11q23.3)/KMT2A-rearranged; t(9;22)(q34.1;q11.2) involving BCR::ABL1 genes; t(3;3)(q21.3;q26.2) involving the GATA2, MECOM(EVI1) genes or inversions: inv(3)(q21.3q26.2); -5 or del(5q); -7; -17/abn(17p). AML patients that present hyperdiploid karyotypes with multiple trisomies and complex karyotypes or monosomal karyotypes are also included in this group. Additionally, mutated ASXL1, BCOR, EZH2, RUNX1, SF3B1, SRSF2, STAG2, U2AF1, or ZRSR2 and mutated TP53 are also classified as adverse risk ; and if adverse-risk cytogenetic abnormalities are present in NPM1-mutated AML, they are also classified as adverse risk . Patients included in the adverse risk group (~40%) have reduced rates of CR being prone to develop recurrence . 2.4. Therapy and Outcomes The current AML intensive treatment protocols are the same that were established more than 50 years ago. The remission-inducing chemotherapy usually consists of a combination of high doses of two cytotoxic drugs, cytarabine and anthracycline, with or without a purine analog . After the induction therapy to clear LSCs and consequently induce CR, patients follow consolidation chemotherapy regimens (consisting of an intermediate dose of cytarabine) to deepen remission and maximize response . In some cases, maintenance therapy is also applied to patients that have achieved remission, with the main goal of decreasing the risk of relapse . In other cases, such as patients classified with adverse risk, allogeneic hematopoietic stem cell transplantation (alloSCT) is also recommended, but it is dependent on the risk of relapse (>35% to 40%), age (<60 years), and other comorbidities . Although conventional therapy has better results in younger patients, this approach presents poor results in elderly individuals or in patients that present some comorbidities . This is due to the combination of the high toxicity of the drugs and the heterogeneity of the disease . Thus, some alternative treatment options are idarubicin (FLAG-IDA), fludarabine, cytarabine, azacitidine, decitabine, granulocyte colony-stimulating factor, and also mitoxantrone-based cytarabine regimens. Current research has been devoted to the identification of molecular targets allowing the implementation of personalized treatments. In 2017 and 2018, a handful of new drug formulations were approved by the FDA: new liposomal formulation of cytarabine and daunorubicin (CPX-351), a BCL-2 inhibitor (venetoclax), the IDH inhibitors (ivosidenib and enasidenib), the FLT3 inhibitors (gilteritinib and midostaurin) the anti-CD33 monoclonal antibody gemtuzumab ozogamicin, the hedgehog signaling pathway inhibitor (glasdegib), and the oral HMA CC-486 . Some of these therapeutic options have already demonstrated very good results in clinical trials. For example, CPX-351 treatment improved the 5-year OS by 10% in patients aged 60-75 years, and with newly diagnosed high-risk or secondary AML when compared with conventional chemotherapy (cytarabine for 7 days plus daunorubicin on days 1, 2, and 3 [7 + 3 regimen] and cytarabine for 5 days and daunorubicin on days 1 and 2 for the second induction [5 + 2 regimen]) . The use of a kinase inhibitor, midostaurin, as an adjunct to conventional therapy, daunorubicin and cytarabine (induction therapy), and then to high doses of cytarabine (consolidation therapy) also promoted an increase in overall and event-free survival in AML patients with FLT3 mutation, and, more precisely, increased the 4-year OS from 44.3% to 51.4% . Currently, midostaurin is often used as first-line therapy in AML patients with FLT3 mutation . Despite these advances, the choice of the therapeutic regime (combination of drugs on doublet and triplet regimens) has always been based on the patient's eligibility or ineligibility to undergo intensive chemotherapy . It is important to note that there are still no generally accepted or validated criteria for considering a patient ineligible for intensive therapy. However, in the context of clinical trials, there are some criteria to consider a patient not suitable, which can be used as a guideline in routine practice . According to the 2022 revision completed by Dohner et al., patients not suitable for intense therapy are (1) patients >75 years old (cannot be an absolute criterion, because AML patients with a more favorable disease and without relevant comorbidities can be submitted to intensive chemotherapy); or (2) Eastern Cooperative Oncology Group (ECOG) performance status >2 and/or age-related comorbidities, such as severe cardiac disorder, severe pulmonary disorder, creatinine clearance <45 mL/min, a hepatic disorder with total bilirubin >1.5 times, or any other comorbidity that the physician assesses to be incompatible with intensive chemotherapy . Thus, for patients who are not candidates for intensive remission-induction therapy, venetoclax plus either hypomethylating agents or low doses of cytarabine have emerged as an effective treatment option based on the achievement of a high and rapid rate of response . Finally, for patients that do not want any treatment or cannot tolerate any type of anti-leukemic therapy, the best support care is proposed. More recently, immune therapies based on antibody therapies have revolutionized the treatment of leukemia. This includes chimeric antigen receptor T cells (CAR T cells), which are T cells taken from the patient and educated in the laboratory to express a specific antigen receptor target or CAR designed against a specific cell-surface antigen. These immunotherapeutic strategies have shown good results in other types of leukemia, particularly CLL and ALL , showing superior remission rates when compared with conventional therapy regimens. Despite several efforts to achieve similar success of antibody-based or cellular immunotherapies in AML , it is still a challenge to identify a good target antigen for this hematologic malignancy. In conclusion, due to the heterogeneous nature of AML, there is a pressing need for more fundamental research and biomarker identification to optimize and personalize AML therapies. Advances in this field will have the potential to improve quality of life, increase the time of at-home care, and reduce early organ damage and mortality while reducing the clinical, emotional, psychosocial, and economic burden associated with current intensive therapies. 3. Measurable Residual Disease (MRD) After the first induction of chemotherapy, CR rates reach approximately 80% and the disease burden is reduced by 99.9%. However, some residual LSCs (up to 1010 to 1012 cells) can remain, causing almost every patient to relapse if no consolidation therapy is provided . Even when consolidation therapy is performed to eradicate any persistent LSCs, above 50% of patients will still relapse or develop refractory disease . This is caused by the persistence of LSCc not detected by conventional technologies, and constitutes the termed minimal residual disease. However, more recently (in 2018), this nomenclature has been replaced by measurable disease (MRD) . The term "measurable" has been proposed to indicate contexts where levels of LSCs are detectable by modern technologies that already have a higher sensitivity . MRD monitoring is performed routinely in clinical practice since it is known that the presence of MRD is a strong prognostic indicator of relapse risk. Besides, MRD can also have implications in the planning and personalization of treatment, when assessed in conjunction with other well-established clinical, cytogenetic, and molecular data . Furthermore, it has been shown that when MRD detection is successfully performed at an early stage, it results in improved prognosis, disease management, and patient outcome . Given the importance of efficient and sensitive detection of MRD, which to date remains challenging, a large body of research has concentrated on developing new precise and sensitive detection strategies. Due to AML and the short period between CR and relapse, finding particular genetic mutations or aberrant protein expression patterns constitutes a major challenge . Additionally, the challenge of detecting a MRD condition is due to the fact that the number of leukemia cells is very low compared to other blood cells; i.e., in ratios of 1:104 to 1:106 LSCs to WBCs, which makes this type of evaluation an extremely difficult task . Thus, powerful and highly sensitive techniques are required to perform the molecular MRD assessment, reaching a limit of detection of 10-3 or lower, and PB and BM may be used . The Emerging Role of MRD in the Therapeutic Scenario Recently, the research community has demonstrated the relevance of MRD assessment towards guiding and personalizing therapy in leukemia patients . The results of studies and clinical trials carried out in this field point towards two main MRD-driven patient management strategies: (i) the use of MRD in the pre-transplant scenario (induction and before transplantation), helping to select the most appropriate consolidation therapy and risk stratification; and (ii) the evaluation of MRD in the post-transplant setting, such as taper off maintenance therapy . Thus, the stronger added value of MRD is based on its use as a (a) monitoring tool to detect imminent disease relapse, (b) a prognostic biomarker to allow the refinement of post-remission relapse risk assessment, and also (c) as a potential surrogate endpoint (still under exploration) . In agreement, the recent 'Diagnosis and management of AML in adults: 2022 recommendations from an international expert panel on behalf of the ELN', recommends MRD assessment only after two cycles of induction/consolidation treatment, at the end of treatment, and during follow-up (every 3 months for up to 24 months) . This recommendation validates not only the more frequent assessment of MRD at different and specific time points during treatment, but also the application of MRD as an independent biomarker to predict outcome, and an asset for detailed patient monitoring . Although MRD negativity has been best stablished as a potential surrogate for clinical benefit in ALL, APL, and chronic lymphocytic leukemia, for AML this is still unclear due to the heterogeneity of the disease and the complexity of the MRD assessment . In AML, MRD assessment is relevant for understanding the efficiency of induction chemotherapy and the intensity of the conditioning regimen; determination of the optimal type of post-remission therapy, such as hematopoietic stem cell transplantation in patients at high risk of relapse or maintenance therapy with hypomethylating agents; and choice of immunosuppressive agents, among others . Currently, the use of MRD analysis to guide treatment decisions in AML patients is still not widely accepted, despite being a promising strategy. Nevertheless, many clinical centers have already started to guide treatment based on MRD, especially in the early stages of disease, given its strong clinical relevance and its potential to aid therapeutic decisions . However, it is important to mention that most of the information until now is derived from clinical studies in patients submitted to intensive treatment. In the context of less intensive treatments (hypomethylating agents treatment or targeted therapies), the investigation about the predictive value of MRD assessment is still less extensive . For example, MRD detection on AML patients with CR guides the early decision to proceed with an alloSCT. Post-transplantation monitoring of MRD could also guide the prescription of immune-suppressive drugs or more intensified chemotherapy regimens or with hypomethylating agents plus venetoclax; targeted therapy combinations when indicated, usually taking into account specific molecules abnormalities (FLT3 or IDH inhibitors); and antibody therapies . While having already demonstrated the high relevance of MRD detection, one of the challenges is still to be able to detect this condition, particularly in the early stages. Thus, it is crucial to use reliable tools, and with high sensitivity. 4. Traditional Techniques for Diagnosis and Monitoring of MRD The European LeukemiaNet MRD Working Party has released comprehensive, consensus recommendations for the use of appropriate techniques having taken into consideration their technical limitations . Well-established techniques, such as reverse transcription polymerase chain reaction (RT-PCR) and multiparameter flow cytometry (MFC), are recommended by the European Leukemia Network AML Measurable Residual Disease Expert Committee to detect MRD in AML. Additionally, other strategies have also been applied as well as next-generation sequencing (NGS) . All these techniques present advantages and disadvantages regarding levels of sensitivity, applicability to the various heterogeneous types of AML, and costs and level of technical expertise needed to process the data. It is worth noting that an ideal MRD detection method should discriminate different cellular populations that could or not cause relapse. Except for the detection of acute promyelocytic leukemia by PCR, current detection methods are not sensitive enough to detect MRD in a regime with very low residual LSCs . 4.1. Multiparametric Flow Cytometry (MFC) MFC is one of the most common techniques used for the diagnosis, classification, and prognosis of AML disease. This technique identifies a signature of aberrant expression of antigens present on AML cells, through specific panels of fluorochrome-labeled monoclonal antibodies . Recent advances, based on the development of multicolor panels (>8 colors), brought the levels of sensitivity of cytometric analysis closer to those obtained by molecular biology techniques . Thus, MRD analysis based on MCF presents moderate sensitivity, with a LOD of 1:10-3 up to 1:10-4 (1 leukemic cell in 10,000 to 10,000 WBCs), which is dependent on the number of cells analyzed, the gating strategy, and the number of antibody markers used . The main advantage of MCF over other techniques is the ability to characterize millions of cells in a short period of time, while distinguishing viable cells from debris and dead cells . Furthermore, it has the advantage of being applicable to the vast majority of AML patients, unlike other techniques that focus on the genetic and/or molecular signature . MRD condition can be assessed/monitored in AML patients by MFC, based on leukemia-associated immunophenotypes (LAIPs) . Briefly, it is known as LAIPs, the abnormal expression of immunophenotypic markers that provide the distinction of the leukemic cells. Conventionally, for the detection of LAIPs, several markers can be used, including CD34, CD117, CD45, CD33, CD13, CD56, CD7, HLA-DR (for MRD), among others . Much work has been completed to improve this strategy and its application in MRD. In most of the cases, the improvements consist of the combination of several antibodies with different fluorophores (at least eight) to detect more than one LAIP at a time, improving the diagnostic performance . Several other studies were conducted to understand the ability and efficiency of cytometric analysis to detect MRD. For example, a study with 451 BM samples from adult AML patients assessed the clinical utility of MRD detection, demonstrating that MFC is useful for outcome prediction and guidance of post-remission in AML . The biggest drawback of this technique is the requirement of significant technical expertise and the intervention of an interpreting pathologist. The future use of artificial intelligence to reduce potential bias and/or human interpretive errors can bring accurate interpretations of the results. The low sensitivity and the high change of the aberrant immunophenotype during disease progression makes the assessment of MRD more difficult, leading to a high rate of false negatives and limiting the detection of the different aberrant markers. 4.2. Molecular-Based Techniques Molecular techniques are used to assess the presence of MRD by analyzing well-known AML mutations . The most common markers used to monitor MRD are NPM1 insertion, CFB-MYH11, AML1-ETO, and PML-RARA (chimeric fusion genes) . NPM1 is one of the most prevalent mutations in AML (present in 25-35% of patients) and is reported as a definitive leukemia-founder mutation that has already been validated as an optimal and reliable MRD marker . Recent studies are focused on introducing/validating new markers for MRD monitoring; for example, many studies are focused on the relevance of IDH mutations . The IDH1 and IDH2 mutations area is also prevalent in AML (occur in ~20% of cases) but there are still conflicting data, whether they represent pre-leukemic mutations or dominant clonal mutations, hence their value in monitoring MRD is not yet well established . Moreover, NPM1 is frequently one of the references for the studies involving IDH mutations . Furthermore, the prognostic significance of combined mutations, such as NPM1, FLT3, DNMT3A, and IDH is still unclear. However, recent studies state that some combinations may have an impact on the risk of relapse and overall survival of AML patients compared with other combinations . Thus, in AML disease, and even in the MRD condition, it is crucial to use reliable and sensitive molecular techniques to detect these mutations, even when present in small amounts, in order to have a better understanding of their prognostic value. 4.2.1. Reverse Transcription Polymerase Chain Reaction (RT-PCR) In AML, the development of RT-PCR has been crucial to detect residual elements conventionally characterized by molecularly cloned chromosome translocations . The use of RT-PCR enables the detection of the fusion genes RUNX1-RUNX1T1 and CBFB-MYH11 as well as PML-RARA and mutated-NPM1 , important for the sensitive detection and quantification of MRD in AML . In fact, in the case of CBF fusion transcripts (RUNX1-RUNX1T1 and CBFB-MYH11), some studies in PB or BM have demonstrated the prognostic value of detection and quantification of MRD at specific time points allowing the identification of patients with a high risk of relapse . In addition, several studies have investigated the clinical implications of monitoring NPM1 and its association with relapse and survival rates . In the study performed by Ivey et al., using BM and PB of the AML patients, the presence of NPM1-mutated transcripts after the second chemotherapy cycle was associated with a significantly higher relapse risk and poorer survival rates, independent of other known prognostic factors . Given the high specificity and sensitivity for LSCs, decreased risk of contamination and better evaluation of RNA quality, the technique of RT-PCR has been widely implemented for routine patient care . However, the detection of MRD based on RT-PCR or PCR is currently limited to around 50% of patients, since not all patients carry the fusion transcript RUNX1-RUNX1T1 (characteristic of t[8;21]), CBFB-MYH11 [inv 16] or t[16;16]), or NPM1 mutations . The optimized RT-PCR assays presents a limit of detection (LOD) of 10-4 to 10-6, and is therefore more sensitive or equal than other technologies used, such as MFC (range 1:10-3 up to 1:10-4 ) . As is the case with any other technique, there are some limitations of RT-PCR-based MRD tests, namely their dependence on specific mutations, requiring individual standard reference curves based on serial dilutions of targets, intensive labor, expertise, cost, time-consuming, and computationally demanding work . 4.2.2. Next-Generation Sequencing (NGS) Next-generation sequencing (NGS) refers to the deep, high-throughput, in-parallel DNA sequencing technologies that were developed in the decades after 1977, when the Sanger DNA sequencing method was developed. Unlike Sanger sequencing, NGS procedures provide a massive parallel and extremely high-throughput analysis (millions to billions of nucleotides) of multiple samples, bringing much faster results (due to multiplexing), and at a lower cost than individual testing . Therefore, NGS is a tool that easily assesses genomic, transcriptomic, and epigenomic features . More specifically, in the AML context, NGS has extreme relevance for diagnosis, due to the high clonal heterogeneity characteristic of this disease . It provides information about the different AML mutations present in a patient, providing more information and allowing the design of personalized therapies, which will ultimately result in a better prognosis of remission and less cases of relapse . It was already described that for patients in CR, the estimated percentage of the MRD measured by NGS was much greater than of aberrant blasts detected by MFC . This technology has also revealed a set of mutations in rare mutant cells and gene sequences, as well as the discovery of genetic alterations that occur between diagnosis and relapse times . In a study with a cohort of 482 patients, at least one mutation was detected in 430 patients (89.2%) showing the broad clinical application of targeted NGS . Additionally, NGS showed a significant additive prognostic value compared to flow cytometry for the detection of MRD . Recently, Alonso et al. tested a 19-gene AML-targeted NGS in a small cohort of 162 patients and showed that well-defined NGS panels are reliable in guiding clinical decisions by the current standards. Results had a 100% correlation with conventional molecular biology techniques, and all patients were successfully classified according to 2016 WHO classification systems (2016 WHO diagnostic categories, 2017 European LeukemiaNet, and Genomic classification) . NGS was also explored in the context of allogeneic hematopoietic cell transplantation (alloHCT). For example, Thol et al. published a clinical study with a cohort of more than one hundred patients, demonstrating that MRD detection utilizing NGS before alloHCT is highly predictive of relapse and survival, and can therefore improve patient management . It is worth noting that in this study similar results were obtained from both BM and PB samples, demonstrating the usefulness of using less invasive body fluids . Thus, through the molecular characterization of AML cells, NGS promotes a personalized and precise method for the assessment of MRD, since can reach a sensitivity of 10-4 to 10-5 . However, the widespread use of NGS has been limited due to its high cost, and the expertise required for the data analysis and interpretation . Furthermore, other drawbacks of this technique are the non-standardization of the assay, the high variance of sensitivity across different platforms, and a long waiting time for the result analysis. In summary, MRD has been shown to be an important and independent prognostic factor and predictor of relapse, and also plays a crucial role in the design of personalized treatment strategies. However, when values of cells are lower than 10-5, it is generally more challenging to be detectable by the diagnostic procedures used in clinical settings. Thus, the development of the aforementioned techniques enables a more reliable and satisfactory detection of MRD and CR as well as the stratification of patients into different subtypes. Still, the sensitivity of some of these techniques is far from ideal, and their implementation in clinical routine is still pending. Despite the technological challenges, it remains crucial to develop new technologies for MRD detection and quantification that are more affordable, faster, and offer higher accuracy and sensitivity. 5. Microfluidics for MRD Detection in AML Disease 5.1. Microfluidics in MRD Condition The major limitation of current MRD detection techniques is the inability to detect very low amounts of LSCs, considered rare tumor cells and named circulating leukemic cells (CLCs). These type of cells are present in body fluids, such as BM or PB. Hence, the pressing need of developing new tools with high sensitivity that could be easily implemented in clinical practice. Microfluidic systems are able to process fluids at high throughputs in microchannels. Due to the channel dimensions, in the order of 10 s to 100 s of micrometers, and to the laminar flow conditions, these devices exhibit many advantages to manipulate cells from a complex sample. Microfluidic devices also display a very high surface-to-volume ratio, holding extremely low amounts of fluids (10-9 to 10-18 L) . The substantial reduction of the scale of the experiments offers unique advantages compared to more conventional analytic approaches, such as reducing the necessary amounts of samples and reagents. Microfluidics is also compatible with multiplexing and automation, and allows quick and efficient cell separation and detection at high resolution, high sensitivity, and low costs . Currently, there are reports of many microfluidic devices that offer the capacity to selectively isolate cells from a mixture using several methodologies based on different cell characteristics, such as size , deformability , density , electrical and magnetic properties , and surface charge . Microfluidic strategies for cell isolation also exploit immunoaffinity, either for negative or positive enrichment of cells in body fluids, or using a combination of both . These assays are not only very efficient but also maintain cell integrity, which is crucial for downstream analyses. Negative enrichment consists of using immunoseparation methods to target specific antigens on the membrane of unwanted cells, for example targeting the CD45 surface marker for the depletion of WBCs . This method allows an unbiased separation of target cells, but it results in lower purity when compared with positive enrichment approaches . The CellSearch(r) system is an example of a positive enrichment technique that has obtained FDA clearance for the separation of Circulating Tumor Cells (CTCs) from the blood of metastatic patients. In this case, magnetic nanoparticles immunoconjugated to target EpCAM are used to separate the cells of interest from the whole blood sample. Although positive selection promotes greater purity of CTCs, this strategy fails to isolate cells with low expression or negative expression of the surface-marker used . Similarly to CTCs, microfluidics can be used to isolate CLCs from body fluids. The application of microfluidics in AML has been explored in many other ways, including a very interesting work for single-cell DNA sequence . In this review, we have focused on microfluidics as a tool to isolate CLCs in order to demonstrate the high relevance of this technique in the MRD detection as a less invasive tool for patient diagnosis and monitoring. Enriching and isolating CLCs would not only provide a way to better detect and monitor MRD (where the number of LSCs is very low), but it would also be invaluable for early diagnosis and accurate patient stratification. 5.2. Microfluidics for Isolation of Circulating Leukemic Blasts (CLCs) Recent studies have shown the capacity of microfluidics to isolate CLCs, which can be used as biomarkers to determine the occurrence of MRD in AML patients . However, the isolation of CLCs can be more complicated than that of CTCs, since they do not have any ubiquitous characteristic that can aid their identification. It is important to emphasize that CLCs and CTCs differ in both, morphological and biological aspects. The most evident parameter is the size, while CTCs have bigger sizes (~14-25 mm), CLCs are a very similar size to WBCs (~7-15 mm) . Therefore, in the case of CLCs, it is not useful to use filtration systems or other types of size-based assays. Another very common technique described to isolate CTCs is positive immunoisolation. The antibodies most commonly used for this purpose are the ones that recognize epithelial cell adhesion molecule (EpCAM). This approach renders high levels of purity because this marker is highly expressed in cancer cells from solid tumors, while WBCs do not express this glycoprotein. On the other hand, antibodies that are used in the context of AML to promote the enrichment of CLCs can also be expressed by blood cells, so the number of interfering cells would be higher, meaning low purity outcomes. Therefore, to overcome these obstacles, an additional step is necessary to distinguish cancer cells from healthy blood cells, using specific markers of aberrant leukemic cells. In this sense, and to detect MRD in patients with AML, Jackson et al., used a microfluidic assay to target several antigens, including CD33, CD34, and CD177 . The antibodies used to recognize these antigens cover about 70% to 90% of the AML patient population. They also included CD7 and CD56 to detect aberrant markers and consequently identify the CLCs fraction of the total of cells isolated . The study also demonstrated that this microfluidic assay is able to detect relapse two months earlier when compared with the technologies currently used--PCR or flow cytometry analysis of a BM biopsy . Recently, an inertial microfluidic chip based on cell stiffness (LSCs are stiffer than the non-target cells) and differential cell size to detect rare leukemic cells was developed by Khoo and collaborators . For the optimization and validation of the system to detect low amounts of cells in blood, the authors started using samples spiked with LSCs (cell count < 5%) showing a detection limit of 5 LSCs among 106 leukocytes. This efficiency exceeds the minimum detection rate obtained by the methods currently used. After preliminary validations with cell lines, the study used clinical blood samples (including ALL, MDS for various subtypes of AML patients) demonstrating that the system had the ability to promote rapid enrichment of blasts, although the blood samples had to be pre-processed before being run in the system to remove red blood cells . After isolation, the morphology and expression of the recovered cells was identified by cytopathological and/or immunocytochemistry analysis . This procedure allows the potential detection of low amounts of blasts and, subsequently, has added value in the detection of MRD. In a recent work, Lai et al. also developed a non-invasive strategy for LSCs capture designed LSC-Chip (Leukemic Stem Cell Specific Capture Chip) . This strategy consists of microfluidic herringbone chips with a CD34-antibody-modified surface. To bind the antibody to the surface of the chips, they used a reversible disulfide strategy to promote further analysis, including scRNA-Seq . The main objective of the herringbone structures is to 'force' cells to travel through streamlines, and to interact more frequently with the antibody-modified surface, with the primary aim of improving target cell capture. The capture efficiency of the chip was tested using different cell lines such as KG-1a (CD34+) and HCT116 (CD34-). The best results obtained reached a value of 84.55% using KG-1a cells in PBS and 77.75% using the same cells spiked in whole blood, and both used a flow rate of 1.5 mL/h. The LSCs chips were validated using PB samples (non-invasive sampling) from 36 AML patients diagnosed as CR and non-remission, and 10 healthy donors . The results achieved demonstrated the potential of the application of the LSCs-Chip for remission status monitoring in AML. A novel approach aimed at the isolation and detection of AML cells, more precisely drug-resistant ones, has recently been implemented based on the combination of superparamagnetic nanomaterials and microfluidics . The magnetic capture efficiency of the cells of interest using the microfluidic device, which in this case were HL-60 cells that overexpress CXC chemokine receptor 4 (CXCR4), was 82.92% . All these studies have demonstrated the relevance and potential of using microfluidics to separate/isolate cells of interest more efficiently, with lower sample volume consumption and in a shorter time compared to traditional methods. 6. Emerging Technologies for the Diagnosis of MRD 6.1. Digital PCR (dPCR) The inclusion of new techniques with the ability to identify disease-related mutations present at very low frequencies is crucial for the assessment of the MRD evaluation in the clinical and research stages. Digital PCR (dPCR) is a high-performance technology that produces an absolute quantification. It is based on the amplification of target genes without a standard reference curve, unlike its conventional version, RT-PCR . Due to its advantages when compared with conventional PCR, dPCR is starting to be explored as a replacement of the outdated version in numerous applications, and AML is no exception. Compared with NGS, it allows less multiplexing, but it is cheaper and presents a good sensitivity. In this context, Hindson et al. compared the microfluidic-based system (dPCR) with traditional Taqman probe (or similar) based quantitative PCR systems, which resulted in even greater sensitivity (up to 10 times), accuracy, and high reproducibility of the newly developed technology . Due to the fact that this technology is considered an asset in MRD monitoring, it has recently been introduced as a molecular assay, and it has been applied in patients for the detection of mutations, such as IDH1/2, DNMT3A, and NPM1 . Parkin and colleagues published a work in which they tried to optimize the dPCR to characterize MRD and to be used in AML prognosis. The authors essentially applied dPCR to measure a variety of mutated alleles of recurrently mutated genes in BM samples of CR AML patients. It was possible to detect variant alleles with a frequency as low as 0.002% . Koizumi et al. used mRNA to detect the presence of Wilms' tumor 1 (WT 1) in several hematological malignancies, including AML, by dPCR, and compared the results with quantitative real-time polymerase chain reaction (RQ-PCR). Both methods correlated strongly with each other, but dPCR was capable of detecting lower levels of WT1 . Bussaglia et al. also applied a dPCR method for the detection of low levels of WT1, but, in parallel, also performed the MFC technique for immunophenotype analysis (using markers for MRD assessment) to establish some prognostic correlations . The optimization of dPCR allowed the first quantitative measurement of frequent genetic mutations in AML and many other malignancies, using the detection of rare variants with high sensitivity, managing to detect 1 in 50,000 mutant cells. dPCR also allowed the provision of several conclusions on the biology of residual AML and pre-leukemia in early CR . In a cohort of 99 patients the occurrence of molecular MRD (NPM1mut) by dPCR strongly correlated with RQ-PCR, showing prognostic relevance . Beyond WT1 and NMP1, dPCR was also performed to IDH1/2 , the PML-RARA , BAALC , MN1 . We may conclude that dPCR is a promising tool for MRD detection since it presents good levels of sensitivity, able to achieve from 10-4 down to 10-6 . Besides, dPCR offers other advantages, such as being much faster and easier to interpret data, without requiring technical expertise, and with lower error rates. As such, dPCR is considered a powerful and feasible tool to improve the diagnosis and stratification of patients and to monitor MRD . 6.2. LNA-qPCR Another promising tool that has also been used is the incorporation of locked nucleic acids (LNA) nucleotides into PCR probes and primers. Briefly, LNA bases can be incorporated into any DNA or RNA oligonucleotide, inflicting a conformational modification in the local helix . This alteration provides a higher hybridization affinity and stronger binding strength for complementary sequences as well as better duplex formation . Moreover, the incorporation of LNAs increases melting temperatures by several degrees (per LNA monomer introduced: +1-+8 degC against DNA and +2-+10 degC against RNA ), which permits probes and primers to be shortened . All these positive aspects of the incorporation of LNAs in PCR increase the success of the amplification and, additionally, provide a higher specificity due to the elimination of undesired products , and, besides PCR, it has also been integrated into Molecular Beacon, TaqMan Beacon, microarray probes, and antisense oligonucleotides . In the AML context, the LNAs strategy has been applied with the main goal of improving the detection of AML-specific mutations. For example, Laughlin et al. developed a method for the detection of NPM1 mutations that are frequently found in AML patients with a normal karyotype . It consists in using an oligonucleotide that contains LNA nucleotides to act as a PCR clamp, thereby inhibiting amplification of the normal sequence and promoting preferential amplification of DNA with a mutation. This method provides increased analytical sensitivity of the assay . In another work, the E-ice-COLD-PCR method (enhance improved and complete enrichment co-amplification at lower denaturation temperature polymerase chain reaction) was used for the detection of low levels of NPM1 mutations . This method uses a non-extendable blocker probe that integrates LNA bases to block wild-type amplification, promoting the enrichment of the four different types of NPM1 mutations, such as A, B, D, and J . Some studies have already been conducted using the LNAs for the assessment of specific mutations during MRD monitoring . For example, the work developed by Abdelhamid and collaborators used LNA-RQ-PCR, which is a RQ-PCR-based technique incorporating LNA to modify the reverse primer in order to amplify only the mutant allele that is weakly present in the neoplastic DNA studied . This strategy allowed a rapid and sensitive quantification of IDH1 and IDH2 gene mutations in MRD AML . A similar strategy was previously used by Jeziskova et al. for the detection of IDH2 mutations . In addition, a very recent work developed by Kao et al. also demonstrated the application of LNA-qPCR in detecting IDH1/2 mutations and monitoring MRD . For this study, BM samples from 88 IDH1/2-mutated AML patients that received standard chemotherapy, azacitidine, or low-dose cytarabine as induction therapy were used. The authors then applied LNA-qPCR to quantify the IDH1/2 mutants MRD kinetics in the samples and compared the results with those obtained for NPM1 qPCR. The results demonstrated that IDH1/2 LNA-qPCR MRD was concordant with remission status or NPM1-MRD in 79.5% of patients . Finally, authors demonstrated the potential of LNAs for detecting NPM1 mutations using both PB and BM samples . The results demonstrated the potential of MRD measurement by mutant NPM1, allowing the identification of a group at high risk of recurrence and even detection of relapse. Thus, all these studies demonstrated the potential of LNAs integrated with quantitative PCR to provide an easier but more specific and sensitive way to detect low levels of specific and relevant mutations in AML and MRD conditions. 6.3. Surface-Enhanced Raman Scattering for Diagnosis of AML At present, Raman spectroscopy and its variant surface-enhanced Raman scattering (SERS) spectroscopy are among the most powerful analytical techniques available for the analysis of biochemical markers. Its application has already been demonstrated in a wide number of fields, from environmental pollution , food industry , biomedical , medicine to arts . Raman spectroscopy is a scattering technique that studies the specific vibrations of molecules by the analysis of their characteristic Raman spectrum (Raman intensity versus wavelength shift spectrum) . This technique provides a vibrational fingerprint of the studied sample that can be directly correlated to its structure, constituents, symmetry, and environment . Thus, Raman spectroscopy can be used to determine chemical composition, molecular structures/conformations, and interactions between different molecules, allowing qualitative and quantitative results . Qualitative analysis can be performed by measuring the frequency of scattered radiation, while quantitative analysis is performed by measuring the intensity of scattered radiation . However, the sensitivity of conventional Raman spectroscopy is intrinsically low, which limits its wide application in the biomedical field . One way to overcome this limitation is to use metal nanoparticles or other plasmonic nanostructures to enhance the Raman signal of molecules that are in their vicinity, known as SERS . SERS is an advanced analytical technique that can be used for the ultra-sensitive detection of various analytes, with astonishingly low detection limits, even up to a single molecular level . This technology was already explored in rare cells detection in the distinction of different cell populations or even in DNA mutation detection. For example, Nima et al. demonstrated a strategy based on SERS to detect CTCs, where four different antibodies and Raman tags were used, showing the possibility of identifying a single cancer cell, with high accuracy/precision, in the middle of millions of blood cells . Wu et al. also developed three types of active SERS nanoparticles to detect CTCs in blood samples with high sensitivity (detection limit of 1 cell/mL) and specificity, without prior enrichment . Additionally, an efficient and non-invasive method was also developed based on an active SERS platform. Briefly, this method consists of the incorporation of the platform in the microfluidic device to isolate CTCs, promoting a label-free detection and its identification through structural and biochemical analysis . This demonstrates the relevance and applicability of SERS in the difficult task of detecting rare cells. On the distinction of different cell populations, Teixeira et al. demonstrated the potential of the SERS technique to detect the profiles of different cell types (malignant and non-malignant) and to distinguish them based on spectral differences . In a recent study, Oliveira et al. demonstrated the multiplex phenotyping of single cancer cells using SERS probes . Furthermore, the possibility to detect DNA mutations using SERS was also demonstrated. More precisely, a SERS chip with the ability to detect specific KRAS mutations and differentiate between wild-type and mutated KRAS genes in different cancer cells was developed . The same SERS chip was optimized and combined with supervised machine learning allowing the intelligent cancer classification according to its mutational landscape . The application of this sensitive and precise technology, with added value in the detection of malignant cells, has already been explored in the context of leukemia or even to distinguish these cells from the healthy ones. In 2014, Gonzalez-Solis and colleagues mentioned the relevance of SERS technology associated with leukemia. They analyzed the biochemistry of blood serum from leukemia patients and healthy donors, through Raman spectroscopy, and managed to distinguish between normal and leukemia cells, and also to identify different types of leukemia . In CLL, a SERS technique was used to detect leukemic cells, using SERS probes composed by nanoparticles functionalized with PEG and antibodies (to recognize three surface proteins of interest in malignant B cells). To confirm the results of this method, flow cytometry was used . In ALL, the differentiation between normal B-lymphocytes and B-ALL cells at different maturation stages using Raman spectroscopy and Principal component analysis (PCA) was demonstrated, with important implications for the clinical practice. The high sensitivity of Raman and feature-rich Raman spectra offers the possibility of multiplex detection, and it could represent a major step in blood analysis through the realization of a non-destructive, label-free Raman-based flow cytometer . Furthermore, in the context of B-cell hematological malignancy, a SERS-based strategy was also used to simultaneously detect two MRD surface markers (CD19 and CD20) in patient blood samples. The results were compared with flow cytometry and demonstrated the great potential of the approach for MRD detection with high sensitivity . Finally, in the context of AML, a preliminary study was carried out using a sensitive and robust platform, based on nanoparticles and hollow core photonic crystal fibers, to monitor and detect leukemic cells (using HL-60 cells) . In addition, electroporation-based SERS spectroscopy using silver nanoparticles was used to detect AML cells (HL-60 and K562 cells) and also differentiate them from healthy cells (PBMCs, Peripheral blood mononuclear cells) . It is important to note that some SERS methods have been also used in AML patient samples, more precisely in APL subtype, to discriminate subtypes or molecular tests. For example, Ye et al. used Ag nanoparticles-based SERS technique to discriminate acute monocytic leukemia and the AML subtype acute promyelocytic leukemia using plasma from patient samples . Another study also demonstrated the possibility of using SERS (through Ag nanoparticles) to distinguish between AML subtypes and control samples, and also for the prognostic stratification of AML patients using characteristic SERS bands . Interestingly, for the discrimination between AML patients and control samples, the relevant specific patterns referred to nucleic acids, and also amino acids, glucose (and products), and protein (and derivatives). On the other hand, to distinguish between different subtypes of AML, different nucleic acids patterns were used. This study demonstrated the potential of SERS technique to be translated to clinical practice, then, and to aid in the differential diagnostic and prognostic stratification of AML patients . A different approach developed by Moisoiu et al. showed the application of SERS for the detection of cancer DNA, more precisely for the characteristic methylation pattern of cancer DNA without pre-amplification in AML patients. The results demonstrated that with SERS, it is possible to detect a unique methylation pattern of cancer DNA and, even more, to promote a discrimination between control samples and AML patients, with a specificity of 82% and a sensitivity of 82% . The studies described above have evidenced the high sensitivity and precision of Raman spectroscopy, and its capacity to differentiate tumor cells from healthy blood cells in a non-destructive way, and even to discriminate patient subtypes. Despite the very promising potential of this technique in cancer and also specifically in AML context, more studies are needed. For example, no specific studies using SERS for the detection of CLCs from blood samples and for the evaluation of MRD or even for specific DNA mutations in AML have been described so far. 7. Conclusions The detection of MRD has a strong clinical relevance, but it is still the greatest challenge in AML disease management. New technological advances have been focused on increasing sensitivity and specificity to create essential tools for the early diagnosis of AML and its relapse. Some of the most promising tools to improve the current landscape include microfluidics and SERS or a combination of both. Indeed, several techniques may be needed, in order to maximize the availability of clinically useful information, with the ultimate goal of promoting more personalized treatment approaches. Author Contributions A.T. and L.C.; writing--original draft preparation, A.T., S.A.-C., B.S.-M., A.C.A., P.L. and L.D.; writing--review and editing. All authors have read and agreed to the published version of the manuscript. Institutional Review Board Statement Not applicable. Informed Consent Statement Not applicable. Data Availability Statement The data can be shared up on request. Conflicts of Interest The authors declare no conflict of interest. Figure 1 Scheme summarizing the development of leukemia. Representation of normal hematopoietic development (A) versus leukemic development and (B) when normal hematopoietic development is initiated in the bone marrow by repopulation of hematopoietic stem cells (HSCs) (A). Formation of leukemic blasts and leukemia stem cells (LSC) and their accumulation in the bone marrow (B). Figure 2 The sequence of events in acute myeloid leukemia from diagnosis to relapse: AML disease diagnosis followed by chemotherapy, MRD detection and relapse. Figure 3 Whole blood is processed through three microfluidic devices modified with mAbs specific for CD33 (red), CD34 (yellow), and CD117 (blue) expressing cells. (A) SEMs of the sinusoidal channel array (50 channels in the array) and the entrance of the single channel that addresses all sinusoidal channels. (B) Schematic of the affinity isolation assay. (C) Selected cells are then immunostained against CD45 and the aberrant marker, and selected cells are released from the capture surface and carried hydrodynamically into flat-bottomed wells, where the cells are imaged. (D) CLCs are identified by positive aberrant staining (aberrant (+)) and positive CD45 and DAPI staining, whereas other blood components only show CD45 and DAPI staining (aberrant(-)). Reproduced with permission from Jackson et al. . Figure 4 Overview of blast cell isolation using inertial microfluidic chip, blast cell biochip (BCB). (A) 3D overview of BCB: (i.) distribution of unselected samples at the entry position; (ii.) distribution of samples at the bifurcation point; (iii.) biochip incorporated into the screening system; and (iv.) enrichment procedure of leukemic blast cells using BCB. (B) From top to bottom, results obtained with healthy leukocytes for cell stiffness measurements: cell stiffness of blast cell types compared to healthy leukocytes; characterization of the distribution of healthy leukocytes and target cells under optimal flow rate; and, finally, frames captured with a high-speed camera demonstrating the focused flows of target cells among the other blood cells. Reproduced with permission from Khoo et al. . cancers-15-01362-t001_Table 1 Table 1 Summary of the classification of AML with percentage of blasts required for the diagnosis . AML with Recurrent Genetic Abnormalities (Now Requiring >=10% Blasts in BM or PB) APL with a translocation between chromosomes 15 and 17-t(15;17)(q22;q12)/PML-RARA APL with other RARA rearrangements AML with a translocation between chromosomes 8 and 21-t(8;21)(q22;q22); RUNX1::RUNX1T1 AML with a inversion or translocation in chromosome 16-inv(16)(pl3.1q22) or t(16;16)(p13.1;q22)/CBFB::MYH11 AML with a translocation between chromosomes 9 and 11-t(9;11)(p21.3;q23.3); MLLT3::KMT2A AML with other KMT2A rearrangements AML with a translocation between chromosomes 6 and 9-t(6;9)(p22.3;q34.1)/DEK-NUP214 AML with a translocation or inversion in chromosome 3-inv(3)(q21.3q26.2) or t(3;3)(q21.3;q26.2)/GATA2;MECOM(EVI1) AML with other MECOM rearrangements AML with other rare recurring translocations AML with mutated NPM1 AML with in-frame bZIP CEBPA mutations AML with translocation between chromosomes 9 and 22 t(9;22)(q34.1;q11.2)/BCR::ABL1 Myeloid Sarcoma Acute leukemia of ambiguous lineage Acute undifferenciated leukemia Mixed phenotype acute leukemia (MPAL) with translocation between chromosomes 9 and 22-t(9;22)(q34.1;q11.2)/BCR::ABL1 MPAL t(v;11q23.3)/KMT2A-rearranged MPAL, B/myeloid, not otherwise specified MPAL, T/myeloid, not otherwise specified Categories designated AML (>=20% blasts in BM and PB) or MDS/AML (if >=10 to 19% blasts in BM and PB) AML with mutated TP53 AML with myelodysplasia-related gene mutations defined by: mutations in ASXL1, BCOR, EZH2, RUNX1, SF3B1, SRSF2, STAG2, U2AF1, and/or ZRSR2 AML with myelodysplasia-related cytogenetic abnormalitiesj AML not otherwise specified Myeloid neoplasms associated to Down syndrome Transient abnormal myelopoiesis associated with Down syndrome Myeloid leukemia associated with Down syndrome Blastic plasmacytoid dendritic cell neoplasm The classification is adapted from references . 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PMC10000117

This study aims to investigate the effects of partial dietary replacement of fish meal with unfermented and/or fermented soybean meal (fermented by Bacillus cereus) supplemented on the growth performance, whole-body composition, antioxidant and immunity capacity, and their related gene expression of juvenile coho salmon (Oncorhynchus kisutch). Four groups of juveniles (initial weight 159.63 +- 9.54 g) at 6 months of age in triplicate were fed for 12 weeks on four different iso-nitrogen (about 41% dietary protein) and iso-lipid (about 15% dietary lipid) experimental diets. The main results were: Compared with the control diet, the diet with replaced 10% fish meal protein with fermented soybean meal protein supplementation can significantly (p < 0.05) influence the expression of superoxide dismutase, catalase, glutathione peroxidase, glutathione S-transferase, nuclear factor erythroid 2-related factor 2, tumor necrosis factor a and interleukin-6 genes, the growth performance, the serum biochemical indices, and the activity of antioxidant and immunity enzymes. However, there was no significant effect (p > 0.05) on the survival rate (SR) and whole-body composition in the juveniles among the experimental groups. In conclusion, the diet with replaced 10% fish meal protein with fermented soybean meal protein supplementation could significantly increase the growth performance, antioxidant and immunity capacity, and their related gene expression of juveniles. fermented soybean meal coho salmon growth performance antioxidant immunity gene expression Guangxi Minzu University2018KJQD14 2018RSCXSHQN02 National Natural Science Foundation of ChinaU20A2064 Innovation-driven development special fund project of GuangxiAA17204044 Shandong Provincial Key Research and Development Programs2019JZZY020710 This work was supported by grants from the Scientific research foundation for the introduced talents of Guangxi Minzu University (2018KJQD14), the National Natural Science Foundation of China (U20A2064), Innovation-driven development special fund project of Guangxi (AA17204044), Innovation team fund project of young Xiangsi Lake scholars of Guangxi Minzu University (2018RSCXSHQN02) and Shandong Provincial Key Research and Development Programs (2019JZZY020710). pmc1. Introduction Coho salmon (Oncorhynchus kisutch) has become one of the most promising fish in China because of its fast growth rate, high economic value, rich nutrition, containing a variety of minerals, and delicious meat . At present, the feed needed by the salmon aquaculture industry is mainly fish meal, and fish meal has been the main aquatic feed protein source for aquaculture because of its high protein content, balanced amino acid composition and rich nutrition . However, due to the continuous growth of the modern aquaculture industry, global fish meal is lacking, and the price of fish meal continues to rise, which has been unable to meet the needs . Therefore, it is urgent to find a suitable protein source to replace fish meal in the aquaculture industry. Soybean meal is a plant protein with high digestive protein content, wide source, and low price, so it is currently recognized as the best choice to replace fish meal in aquatic feed . However, the soybean meal contains unbalanced amino acids and soybean antigen protein, urease, trypsin inhibitor, soybean lectin, phytic acid, saponins, phytoestrogens, anti-vitamins and allergens, and other anti-nutritional factors , which can affect the palatability, and inhibit the digestion and absorption of nutrients, and cause the damage of tissue and organ, and seriously affect the health of aquatic animals . Microbial fermentation is a commonly used biological method for treating soybean meal antigens and palatability, and soybean meal after microbial fermentation can reduce most of the anti-nutritional factors, produce carbohydrates, digestive enzymes and other nutrients, degradation of macromolecular protein, produce small active peptides, organic acids, thereby enhancing its nutritional value and enhance the digestion and absorption of nutrients . In addition, fermented soybean meal can also provide animals with probiotics, prebiotics and flavonoids and other active substances and increase the antioxidant properties of free amino acid content and the concentration of phenolic compounds . At present, there are relatively few studies on the replacement of fish meal with fermented soybean meal in coho salmon. The antibacterial substances produced by Bacillus cereus have the effects of promoting growth, regulating immune function, and treating diseases in livestock and poultry . Therefore, coho salmon was selected as the research object, and Bacillus cereus was used as a fermentation strain to explore the effects of replacing part of fish meal with fermented soybean meal on the growth performance, muscle composition, antioxidant and immunity capacity, and their related gene expression of juvenile coho salmon in this study. The results provide a theoretical basis for the development and optimization of coho salmon compound feed and the healthy development of the artificial breeding industry. 2. Materials and Methods 2.1. Experimental Diets Four different iso-nitrogen (about 41% dietary protein) and iso-lipid (about 15% dietary lipid) experimental diets were designed and based on the references , in which the soybean meal could replace 10% fish meal protein. The G0 diet contained 28% fish meal protein (control group). Three other diets (G1, G2 and G3) were replaced 10% fish meal protein with unfermented and/or fermented soybean meal: The G1 diet replaced by 10% unfermented soybean meal protein, the G2 diet replaced by 5% unfermented soybean meal protein and 5% fermented soybean meal protein, and the G3 diet replaced by 10% fermented soybean meal protein, based on per kg of dried feed, as shown in Table 1. All the feed materials were provided by Conkerun Ocean Technology Co., Ltd. in Shandong, China, and they were animal food-grade. The soybean meal was fermented by Bacillus cereus, and the bacterial strain was collected from mangrove root soil in Maowei Sea, Qinzhou, Guangxi, China (21deg81'66'' N, 108deg58'46'' E). The experimental strains and fermentation conditions were derived from preliminary experiments in our lab. The inoculation amount of Bacillus cereus was 10% (v/m), the ratio of material to water was 1:1.4, and the fermentation was cultured at 37 degC for 60 h. The fermented soybean meal was dried for 24 h in a blast drying baker at 37 degC. A hammer mill was used to grind raw all the dry materials into a fine powder (80-mm mesh), then all the dry materials were mixed in a roller mixer for 15 min and added some water to make a hard dough. Floating pellets with a diameter of 2.0 x 3.0 mm were obtained by a single screw extruder, and they were dried in the air flow at 37 degC until the water content was below 100 g/kg. Then the dry floating pellets were sealed in plastic bags and stored at -20 degC until use. 2.2. Experimental Fish and Culture Six hundred juvenile coho salmon at the age of 6 months were from a hatchery located in Benxi rainbow trout breeding farm in Liaoning, China. Outdoor feeding and breeding experiments of juvenile coho salmon were carried out at a rainbow trout breeding farm in Nanfen District, Benxi City, Liaoning, China. After being disinfected using a concentration of 1/100,000-1/50,000 potassium permanganate, the juveniles were acclimatized for 14 days, using water temperature at 10-18 degC, water intake >= 100 L/s, surface velocity >= 2 cm/s, dissolved O2 >= 6.0 mg/L, pH 7.8-8.3 and natural light. The juveniles were fed three times a day at 08:00, 12:00 and 16:00 h, using a control diet (28% fish meal protein), and the daily feeding quantity was fed until the fish was no feeding behavior at the feeding time. After being acclimatized for 14 days, 390 juvenile coho salmon (initial weight 159.63 +- 9.54 g) were selected for the formal experiment, and 30 of the selected juveniles were freely taken for initial samples. The remaining 360 of them were assigned randomly into 4 groups in triplicate, making a total of 12 net cages (1.0 x 1.0 x 0.8 m, L x W x H) with 30 fish in each net cage. The juveniles were cultured in the same breeding environment, and they were fed for 12 weeks using one of the 4 diets above (Table 1) and the daily feeding quantity was fed until the fish was no feeding behavior at the feeding time. 2.3. Sampling The juvenile coho salmon were sampled at day 0 and the end of 12 weeks, respectively, after being starved for 24 h. All sample fish were separately anesthetized using 40 mg/L of 3-aminobenzoic acid ethyl ester methane sultanate (MS-222, Adamas Reagent, China). Then, their body weight and length were individually measured. At day 0, 20 juveniles were taken for dissecting liver samples and the other 10 juveniles for the sampling of whole fish. At the end of 12 weeks, 9 fish per net cage were randomly taken for the samples, 3 of which were for whole fish samples and 6 for the samples of serum, viscera mass, and liver. A sterile syringe was used to collect blood from the tail vein of juvenile coho salmon; then, the blood was transferred to a 2 mL sterile enzyme-free centrifuge tube. At 3000x g and 4 degC, the blood was centrifuged in a centrifuge for 15 min, and the supernatant was serum. The liver weight and visceral mass weight were weighed and recorded separately for analysis of the growth performance. All the experimental samples were stored at -80 degC for subsequent analysis. 2.4. Calculations and Analytical Methods 2.4.1. Growth Performance The survival rate, weight gain rate, specific growth rate, condition factor, hepatosomatic index, viscerosomatic index, feed conversion ratio, and protein efficiency ratio are calculated according to the following formulas. Survival rate (SR, %)=100 xfinal amount of fishinital amount of fish Weight gain rate (WGR, %)=100 xfinal body weight (g) - initial body weight (g)initial body weight (g) Specific growth rate (SGR, %/d)=100 xln(final body weight (g)) - ln(initial body weight (g))days Condition factor (CF, %)=100 x body weight (g)(body length (cm))3 Hepatosomatic index (HSI, %)=100 xliver weight (g) body weight (g) Viscerosomatic index (VSI, %)=100 xviscera weight (g) body weight (g) Feed conversion ratio (FCR)=total diets weight (g) final body weight (g) - initial body weight (g) Protein efficiency ratio (PER, %)=100 xfinal body weight (g) - initial body weight (g) total intake of crude protein weight (g) 2.4.2. Determination of Feed and Whole Fish Composition The compositions of feed and whole fish were analyzed following the standard methods of the Association of Official Analytic Chemists (AOAC, 2005) . The samples were dried at 105 degC until constant weight in an oven to determine moisture content. The muffle furnace at 550 degC for 24 h was used to determine ash. Kjeldahl method was used to determine crude protein. Soxhlet method by ether extraction was used to determine crude lipid. 2.4.3. Determination of Serum Biochemical Parameters The indicators in serum were measured using the kit produced by Nanjing Jiancheng Bioengineering Institute (Nanjing, China) and referred to the instructions in the kit for specific operation steps. All the instructions can be found and downloaded at (accessed on 1 March 2023). The total protein (TP) content was determined by the Coomassie brilliant blue method. The glucose (GLU) content was determined by the glucose oxidase method. The total cholesterol (T-CHO) content was determined by the cholesterol oxidase (COD-PAP) method. The albumin (ALB) content and alkaline phosphatase (AKP) vitality were determined by the microplate method. 2.4.4. Determination of Liver Antioxidant Capacity The indicators in the liver were measured using the kit produced by Nanjing Jiancheng Bioengineering Institute (Nanjing, China) and referred to the instructions in the kit for specific operation steps. All the instructions can be found and downloaded at (accessed on 1 March 2023). The superoxide dismutase (SOD) was determined by the water-soluble tetrazole salt (WST-1) method. The catalase (CAT) was determined by the visible light method. The malondialdehyde (MDA) was determined by the thiobarbituric acid (TBA method). The total antioxidant capacity (T-AOC) was determined by the ferric-reducing ability of plasma (FRAP) method. The glutathione peroxidase (GSH-PX), glutathione S-transferase (GST), hydroxyl radical clearance ratio (OH*-CR) and superoxide radical clearance ratio (O2*-CR) were determined by the colorimetric method. The reduced glutathione (GSH) was determined by the microplate method. 2.4.5. Expression of Antioxidant and Immunity Genes The method of Ding et al. was applied to determine the expression of sod, cat, gsh-px, gst, nrf2, tnf-a and il-6 mRNA in the liver of the juvenile coho salmon. Briefly, the Steady Pure Universal RNA Extraction Kit and the Evo M-MLV reverse transcription kit (Accurate Biology Biotechnology Engineering Ltd., Changsha, China) were used to extract 500 ng of total RNA from samples and reverse-transcribe it into cDNA. The polymerase chain reaction (PCR) conditions were 50 degC for 30 min, 95 degC for 5 min, and 5 degC for 5 min. The forward and reverse primers of sod, cat, gsh-px, gst, nrf2, tnf-a and il-6 genes for reverse transcription were designed by referencing the corresponding genomic sequences of coho salmon in the National Center for Biotechnology Information (NCBI) database. The primers were synthesized by Sangon Biotech (Shanghai) Co., Ltd. (Shanghai, China). The primers were shown in Table 2, and b-actin was chosen as the nonregulated reference gene. The real-time quantitative polymerase chain reaction (RT-qPCR) was conducted using an RT-qPCR System (LightCycler(r) 96, Roche, Switzerland) and SYBR Green Pro Taq HS qPCR kit (Accurate Biology Biotechnology Engineering Ltd., Changsha, China). The RT-qPCR conditions were as follows: initial denaturation at 95 degC for 30 s, 40 cycles of denaturation at 95 degC for 5 s, annealing at 60 degC for 30 s and extension at 72 degC for 20 s. The 2-DDCT method was applied to calculate the relative expression levels of sod, cat, gsh-px, gst, nrf2, tnf-a and il-6 mRNA. 2.5. Statistical Analysis All the data were analyzed using IBM SPSS Statistics 25 (Chicago, IL, USA) and one-way analysis of variance (ANOVA) and tested for normality and homogeneity of variance. Duncan's test was used for multiple comparison analysis when it was significantly different (p < 0.05). Statistics are expressed as means +- standard deviation (SD). 3. Results 3.1. Effect of Replacing a Portion of Fish Meal with Unfermented and/or Fermented Soybean Meal on the Growth Performance of Juvenile Coho Salmon The WGR, SGR, CF, and PER of the juveniles in G3 and the HSI, VSI, and FCR of the juveniles in G1 and G2 were significantly higher (p < 0.05) than those of the juveniles in G0. The HSI, VSI, and FCR of the juveniles in G3 and the WGR, SGR, CF, and PER of the juveniles in G1 and G2 were significantly lower (p < 0.05) than those of the juveniles in G0. However, there was no significant difference in the SR of the juveniles between the groups (p > 0.05), as shown in Table 3. 3.2. Effect of Replacing a Portion of Fish Meal with Unfermented and/or Fermented Soybean Meal on the Whole-Body Composition of Juvenile Coho Salmon No significant difference (p > 0.05) was found in the moisture, crude protein, crude lipid, and ash of juvenile coho salmon fed diets of replacement of fish meal with unfermented soybean meal and/or fermented soybean meal, as shown in Table 4. 3.3. Effect of Replacing a Portion of Fish Meal with Unfermented and/or Fermented Soybean Meal on the Physiological and Biochemical Indices in Serum of Juvenile Coho Salmon The TP, GLU, ALB, AKP, and T-CHO of the juveniles in G3 were significantly higher (p < 0.05) than those of the juveniles in G0. The TP, GLU, ALB, AKP, and T-CHO of the juveniles in G1 and G2 were significantly lower (p < 0.05) than those of the juveniles in G0, as shown in Table 5. 3.4. Effect of Replacing a Portion of Fish Meal with Unfermented and/or Fermented Soybean Meal on the Antioxidant Capacity in the Liver of Juvenile Coho Salmon The SOD, CAT, GSH-PX, GSH, GST, OH*-CR, O2*-CR, and T-AOC of the juveniles in G3, and the MDA of the juveniles in G1 and G2 were significantly higher (p < 0.05) than those of the juveniles in G0. The MDA of the juveniles in G3 and the SOD, CAT, GSH-PX, GSH, GST, OH*-CR, O2*-CR, and T-AOC of the juveniles in G1 and G2 were significantly lower (p < 0.05) than those of the juveniles in G0, as shown in Table 6. 3.5. Effect of Replacing a Portion of Fish Meal with Unfermented and/or Fermented Soybean Meal on the Expression of Antioxidant and Immune Genes in the Liver of Juvenile Coho Salmon The expression of the sod, cat, gsh-px, gst, and nrf2 genes in the liver of the juveniles in G3 and the expression of the il-6 and tnf-a genes in the liver of the juveniles in G1 and G2 were significantly higher (p < 0.05) than those of the juveniles in G0. The expression of the il-6 and tnf-a genes in the liver of the juveniles in G3 and the expression of sod, cat, gsh-px, gst, and nrf2 genes in the liver of the juveniles in G1 and G2 were significantly lower (p < 0.05) than those of the juveniles in G0, as shown in Figure 1. 4. Discussion The growth performance of fish can be used to reflect growth and health status, and it is affected by many factors, such as fish species, growth stage, nutrient deficiency, metabolic disorders, anti-nutritional factors, and toxic and harmful substances . The results of this study showed that partial replacement of fish meal with fermented soybean meal could significantly increase the growth performance of juvenile coho salmon. However, partial replacement of fish meal with unfermented soybean meal could significantly decrease the growth performance of juvenile coho salmon. The reasons are supposed to be: First, unfermented soybean meal had adverse factors such as poor palatability, essential amino acid imbalance, low phosphorus utilization, high anti-nutritional factors, and easily cause lipid metabolism disorder, which will lead to decreased growth performance . Second, fermented soybean meal could reduce and even eliminate anti-nutrient factors, and the protein could be degraded into easily digestible peptides or amino acids; thus, fermented soybean meal could improve the nutritional quality of feed and the digestibility of fish . Third, the active bacteria, organic acids, and vitamins in fermented soybean meal would also play a positive role in growth performance . Similar studies had shown that feeding largemouth bass (Micropterus salmoides) and Macrobrachium nipponense (Macrobrachium nipponense) with the diet with partial replacement of fish meal with fermented soybean meal significantly improved their growth performance. Serum biochemical indexes of fish are closely related to metabolism, nutrient absorption, and health status. They are important indexes to evaluate physiology and pathology and are widely used to measure metabolism and health status . TP and ALB in the blood are synthesized by the liver, and the increase of TP and ALB content indicates that the ability of the liver to synthesize protein is enhanced. AKP is one of the important indicators of fish physiological activity and disease diagnosis, which can reflect the anti-stress ability of biological organisms . T-CHO is an important index to reflect the body's lipid metabolism . GLU is the main functional substance of the body, and its content is affected by nutrition and feed intake . The results of this study showed that partial replacement of fish meal with fermented soybean meal could significantly increase the serum biochemical indexes of juvenile coho salmon, indicating that fermented soybean meal could be used as a protein substitute for fish meal to improve the health of juvenile coho salmon. The reasons are supposed to be: First, fermented soybean meal could improve the intestinal structure and function of fish, increase the activity of digestive enzymes, and increase the absorption and utilization of dietary proteins and lipids . Second, compared with macromolecular proteins, the small peptides in fermented soybean meal are more easily absorbed by fish, which could improve the diet protein utilization rate, consequently enhancing the serum protein content of fish . Third, fermented soybean meal could decrease the content of soybean saponins, increase the activity of a-glucosidase, and improve the absorption of glucose . Fourth, fermented soybean meal could not only reduce the inhibitory effect of soy isoflavones on serum T-CHO levels but also stimulate the antioxidant system of the body, thereby inhibiting the process of lipid oxidation and increasing the content of T-CHO in the serum . In addition, bioactive peptides during fermentation can act as immune stimulants to enhance AKP activity . Nuclear factor erythroid 2-related factors (nrf2) is an important nuclear transcription factor and can be involved in a variety of cellular processes, including maintaining intracellular redox balance, cell proliferation/differentiation, metabolism, protein homeostasis and inflammation regulation, and disease development . The activation of the nrf2 signaling pathway can initiate the expression of multiple downstream target proteins, such as SOD, CAT, GPX, glutathione ligase (g-GCS), glutathione catalase (GR), glutathione S-transferase (GST) and glucose-6-phosphate kinase (G-6-PDH) . The expression of these genes is an important way for the body to resist oxidative stress damage . Nrf2 signaling pathway can negatively regulate various cytokines (TNF-a, IL-1 and IL-6), chemokines, cell adhesion factors, matrix metalloproteinases, cyclooxygenase-2, inducible nitric oxide synthase, and other inflammatory mediators, which plays a protective role in the dysfunction caused by inflammation . IL-6 and TNF-a are often used as indicators of the inflammatory response . MDA content has been used by many researchers to evaluate the effect of protein replacement sources on the antioxidant capacity of fish, which can be used as an important marker of endogenous oxidative damage in organisms . The results of this study showed that partial replacement of fish meal with fermented soybean meal could significantly increase the antioxidant capacity and the expression of their related gene in the liver and significantly decrease the expression of il-6 and tnf-a gene in the liver of juvenile coho salmon. However, partial replacement of fish meal with unfermented soybean meal could significantly decrease the antioxidant capacity and the expression of their related gene in the liver and significantly increase the expression of the il-6 and tnf-a genes in the liver of juvenile coho salmon. The reasons are supposed to be: First, the soybean globulin and b-conglycinin in soybean meal could destroy the antioxidant system of fish and cause oxidative damage . Previous studies have shown that soybean meal in feed may cause oxidative stress in fish such as gilthead sea bream (Sparus aurata) . Second, a high concentration of soybean peptides and phenols in fermented soybean meal could up-regulate nrf2 gene expression, induce the expression of the sod, cat, gsh, and gsh-px genes, and improve the antioxidant ability of the body . Lee et al. found that an appropriate proportion of fermented soybean meal in a diet can increase the activities of SOD, GSH-Px, and GSH in the liver . Third, Bacillus could stimulate the production of antioxidant enzymes and antioxidants, thereby scavenging free radicals, maintaining homeostasis, improving antioxidant capacity, and activating the Nrf2 pathway . Fourth, the replacement of fish meal protein with 10% fermented soybean meal protein was insufficient for causing a change in the body's ability to recognize foreign bodies and did not lead to an inflammatory reaction . In addition, after soybean meal fermentation, a unique fragrance could be formed, which can promote the feeding of aquatic animals and increase their immunity . However, the results of this study showed that partial replacement of fish meal with unfermented and/or fermented soybean meal had no significant effect on the survival rate and whole-body composition of juvenile coho salmon. The reasons are supposed to be: First, the energy required by fish to maintain normal life activities mainly depends on the breakdown of protein and fat, and fish meal contains a complete set of essential amino acids that meet the protein requirements of most aquatic animals . Second, the crude protein and crude fat contents of the four diets in this study were the same and were enough to satisfy the daily needs of juvenile coho salmon. Third, fish body composition is affected by external conditions such as feed nutrients, food composition, aquaculture water environment and season, but fish body composition was not affected by plant protein levels . Similar results were obtained in pompano (Trachinotus ovatus) and Florida pompano (Trachinotus carolinus) fed with fermented soybean meal partially replacing fish meal. However, studies have shown that a high proportion of fermented soybean meal instead of fish meal significantly increased the whole-body moisture and reduced crude protein and crude lipid content of Japanese seabass (Lateolabrax japonicus) . In giant grouper (Epinephelus lanceolatus), high levels of fermented soybean meal replacement also significantly increased whole-fish moisture and decreased crude protein and crude lipid content . The above inconsistent results might be related to the strains of fermented soybean meal, the basic feed formula, the substitution ratio of fermented soybean meal, the types of aquatic animals, the breeding cycle, and the growth stage. 5. Conclusions In conclusion, the diet with replaced 10% fish meal protein with fermented soybean meal protein supplementation can significantly influence the expression of superoxide dismutase, catalase, glutathione peroxidase, glutathione S-transferase, nuclear factor erythroid 2-related factor 2, tumor necrosis factor a and interleukin-6 genes, the growth performance, the serum biochemical indices, and the activity of antioxidant and immunity enzymes of juvenile coho salmon. The results provide a theoretical basis for the development and optimization of coho salmon compound feed and the healthy development of the artificial breeding industry. Author Contributions Q.Z. contributed to conceptualization, data curation, formal analysis, funding acquisition, investigation, methodology, project administration, validation, visualization, writing, reviewing, and editing. F.L. contributed to data curation, formal analysis, investigation, methodology, resources, software, validation, visualization, and writing the original draft. M.G. contributed to the investigation, methodology, validation, and visualization. M.Q. contributed to the investigation, methodology, validation, and visualization. J.W. contributed to the investigation, methodology, validation, and visualization. H.Y. contributed to the investigation, methodology, validation, and visualization. J.X. contributed to funding acquisition, resources, software, validation, and visualization. Y.L. contributed to conceptualization, data curation, formal analysis, funding acquisition, investigation, methodology, project administration, software, supervision, validation, visualization, writing, reviewing, and editing. T.T. contributed to data curation, funding acquisition, validation, writing, reviewing, and editing. All authors have read and agreed to the published version of the manuscript. Institutional Review Board Statement This study was reviewed and approved by the guidelines of Guangxi Minzu University, Nanning, China, for the care and use of laboratory animals on 18 June 2021 (approval number: No. GXUN 2021-006), and this research does not contain any studies with human participants. Informed Consent Statement Not applicable. Data Availability Statement The data that support the findings of this study are available from the corresponding author upon reasonable request. Conflicts of Interest The authors declare no conflict of interest. Figure 1 Effect of fish meal replaced by unfermented and/or fermented soybean meal on the expression levels of the antioxidant and immunity genes in the liver of juvenile coho salmon, in which (#) indicates superoxide dismutase (sod), () catalase (cat), () glutathione peroxidase (gsh-px), () glutathione S-transferase (gst), () nuclear factor erythroid 2-related factor 2 (nrf2), () tumor necrosis factor a (tnf-a), and (#) interleukin-6 (il-6). All above data are mean +- SD (n = 3 x 3 x 3), and different superscript letters indicate significant differences among the data (p < 0.05). animals-13-00945-t001_Table 1 Table 1 Experimental diet formula (g/kg of dried feed) and approximate composition (%, dry matter percentage). Ingredients G0 G1 G2 G3 Fish meal 1 401.00 258.00 258.00 258.00 Soybean meal 2 0.00 213.60 106.80 0.00 Fermented soybean meal 3 0.00 0.00 90.50 181.10 Chicken powder 4 100.00 100.00 100.00 100.00 Shrimp powder 5 100.00 100.00 100.00 100.00 Wheat middling 6 178.80 178.40 178.40 178.40 Starch 30.20 30.20 30.20 30.20 Cellulose 85.30 0.00 16.30 32.50 Fish oil 40.10 55.30 55.30 55.30 Soybean oil 40.10 40.00 40.00 40.00 Ca(H2PO4)2 10.10 10.10 10.10 10.10 Mineral premix 7 5.20 5.20 5.20 5.20 Vitamin premix 8 5.20 5.20 5.20 5.20 Choline 3.00 3.00 3.00 3.00 Vitamin C 1.00 1.00 1.00 1.00 Approximate composition Crude protein 41.78 41.42 41.38 41.26 Fish meal protein 28.00 18.00 18.00 18.00 Soybean meal protein 0.00 10.00 5.00 0.00 Fermented soybean meal protein 0.00 0.00 5.00 10.00 Crude lipid 15.21 15.22 15.21 15.20 Note: 1 Fish meal: protein content 70.00%, lipid content 8.00%. 2 Soybean meal: protein content 46.81%, lipid content 1.84%. 3 Fermented soybean meal: protein content 55.21%, lipid content 1.93%. 4 Chicken powder: protein content 62.00%, lipid content 12.00%. 5 Shrimp powder: protein content 49.00%, lipid content 8.00%. 6 Wheat middling: protein content 11.00%, lipid content 1.60%. 7 Composition (mg/kg mineral premix): AlK(SO4)2*12H2O, 123.7; CaCl2, 17,879.8; CuSO4*5H2O, 31.7; CoCl2*6H2O, 48.9; FeSO4*7H2O, 707.4; MgSO4*7H2O, 4316.8; MnSO4*4H2O, 31.1; ZnSO4*7H2O, 176.7; KCl, 1191.9; KI, 5.3; NaCl, 4934.5; Na2SeO3*H2O, 3.4; Ca(H2PO4)2*H2O, 12,457.0; KH2PO4, 9930.2. 8 Composition (IU or g/kg vitamin premix): retinal palmitate, 10,000 IU; cholecalciferol, 4000 IU; a-tocopherol, 75.0 IU; menadione, 22.0 g; thiamine HCl, 40.0 g; riboflavin, 30.0 g; D-calcium pantothenate, 150.0 g; pyridoxine HCl, 20.0 g; meso-inositol, 500.0 g; D-biotin, 1.0 g; folic acid, 15.0 g; ascorbic acid, 200.0 g; niacin, 300.0 g; cyanocobalamin, 0.3 g. animals-13-00945-t002_Table 2 Table 2 Real-time quantitative PCR primers for genes of coho salmon. Gene Primer Sequence GenBank Tm (degC) Size (bp) b-actin 1 F: CCAAAGCCAACAGGGAGAA R: AGGGACAACACTGCCTGGAT BG933897 60 91 Sod 2 F: CCGTTGGTGTTGTCTCCGAAGG R: GAGGGTGACAATGCTCCAGTGAAG XM_014198383 60 101 gsh-px 3 F: GATTCGTTCCAAACTTCCTGCTA R: GCTCCCAGAACAGCCTGTTG BG934453 60 140 gst 4 F: CGCATTGACATGATGTGTGA R: TGTCGAGGTGGTTAGGAAGG DQ367889 60 121 cat 5 F: GCGTTCGGGTACTTTGAGGTGAC R: TGGAGAAGCGGATGGCGATAGG BG935638 60 103 nrf2 6 F: TAGAGACGAGCAGCGAGCCAAG R: GTTGAAGTCATCCACAGGCAGGTC NM_001139807 60 82 il-6 7 F: GAGCTACGTAACTTCCTGGTTGAC R: GCAAGTTTCTACTCCAGGCCTGAT XM_014143031 60 129 tnf-a 8 F: GGCGAGCATACCACTCCTCT R: TCGGACTCAGCATCACCGTA AY848945 60 124 Note: 1 b-actin: Reference gene. 2 sod: Superoxide dismutase gene. 3 gsh-px: Glutathione peroxidase gene. 4 gst: Glutathione S-transferase gene. 5 cat: Catalase gene. 6 nrf2: Nuclear factor erythroid 2-related factor 2 gene. 7 il-6: Interleukin-6 gene. 8 tnf-a: Tumor necrosis factor a gene. animals-13-00945-t003_Table 3 Table 3 Effect of fish meal replaced by unfermented and/or fermented soybean meal on growth performance of the juvenile coho salmon. G0 G1 G2 G3 Initial weight (g) 159.63 +- 9.54 159.63 +- 9.54 159.63 +- 9.54 159.63 +- 9.54 Final weight (g) 583.49 +- 10.97 c 473.01 +- 12.16 a 545.08 +- 6.09 b 617.07 +- 4.28 d SR 1 (%) 93.2 +- 1.73 91.2 +- 2.11 92.2 +- 1.24 94.6 +- 1.21 WGR 2 (%) 265.53 +- 6.87 c 196.32 +- 7.68 a 241.46 +- 3.81 b 286.57 +- 2.68 d SGR 3 (%/d) 1.54 +- 0.02 c 1.29 +- 0.03 a 1.46 +- 0.01 b 1.60 +- 0.01 d CF 4 (%) 1.91 +- 0.02 c 1.42 +- 0.02 a 1.67 +- 0.01 b 2.07 +- 0.02 d HIS 5 (%) 1.58 +- 0.03 b 1.71 +- 0.03 c 1.68 +- 0.02 c 1.46 +- 0.02 a VSI 6 (%) 11.67 +- 0.31 b 12.75 +- 0.64 c 12.51 +- 0.35 c 10.24 +- 0.43 a FCR 7 1.64 +- 0.03 b 1.95 +- 0.03 d 1.86 +- 0.02 c 1.53 +- 0.02 a PER 8 (%) 231.31 +- 7.42 c 181.93 +- 3.22 a 208.44 +- 6.85 b 252.19 +- 5.31 d Note: All above data are mean +- SD (n = 3 x 3 x 3) except SR is mean +- SD (n = 30 x 3), and different superscript letters in the same row indicate significant differences among the data (p < 0.05). 1 SR: Survival rate. 2 WGR: Weight gain rate. 3 SGR: Specific growth rate. 4 CF: Condition factor. 5 HSI: Hepatosomatic index. 6 VSI: Viscera index. 7 FCR: Feed conversion ratio. 8 PER: Protein efficiency ratio. animals-13-00945-t004_Table 4 Table 4 Effect of fish meal replaced by unfermented and/or fermented soybean meal on whole body composition of the juvenile coho salmon (%/per g of wet weight). G0 G1 G2 G3 Moisture 75.22 +- 0.19 75.27 +- 0.10 75.38 +- 0.68 75.04 +- 0.36 Crude protein 18.25 +- 0.17 17.67 +- 0.57 17.89 +- 0.81 17.98 +- 0.44 Crude lipid 5.36 +- 0.39 5.28 +- 0.34 5.30 +- 0.09 5.34 +- 0.13 Ash 1.45 +- 0.07 1.50 +- 0.06 1.67 +- 0.10 1.52 +- 0.09 Note: All above data are mean +- SD (n = 3 x 3 x 3), and different superscript letters in the same row indicate significant differences among the data (p < 0.05). animals-13-00945-t005_Table 5 Table 5 Effect of fish meal replaced by unfermented and/or fermented soybean meal on serum physiological and biochemical indices of the juvenile coho salmon. G0 G1 G2 G3 TP 1 (g/L) 54.46 +- 0.17 c 36.21 +- 0.21 a 44.98 +- 0.06 b 60.28 +- 1.34 d GLU 2 (mmol/L) 5.06 +- 0.35 c 2.97 +- 0.14 a 4.08 +- 0.33 b 5.87 +- 0.12 d ALB 3 (g/L) 38.79 +- 2.37 c 19.23 +- 0.92 a 30.3 +- 2.76 b 44.27 +- 2.79 d AKP 4 (U/mL) 20.36 +- 1.25 c 8.26 +- 1.20 a 13.19 +- 1.52 b 23.69 +- 0.92 d T-CHO 5 (mmol/L) 7.77 +- 0.33 c 3.19 +- 0.07 a 4.94 +- 0.87 b 8.54 +- 0.11 d Note: All above data are mean +- SD (n = 3 x 3 x 3), and different superscript letters in the same row indicate significant differences among the data (p < 0.05). 1 TP: Total protein. 2 GLU: Glucose. 3 ALB: Albumin. 4 AKP: Alkaline phosphatase. 5 T-CHO: Total cholesterol. animals-13-00945-t006_Table 6 Table 6 Effect of fish meal replaced by unfermented and/or fermented soybean meal on the antioxidant capacity in the liver of the juvenile coho salmon. G0 G1 G2 G3 SOD 1 (U/mg) 822.5 +- 32.71 b 627.12 +- 32.84 a 642.77 +- 46.08 a 977.36 +- 54.18 c CAT 2 (U/mg) 318.75 +- 15.31 b 221.77 +- 20.15 a 257.31 +- 22.94 a 365.02 +- 16.37 c GSH-PX 3 (U/mg) 19.48 +- 2.48 c 9.09 +- 1.5 a 13.62 +- 1.44 b 23.27 +- 1.81 d GSH 4 (U/mg) 118.13 +- 9.42 c 68.32 +- 4.35 a 88.56 +- 11.77 b 153.86 +- 16.24 d GST 5 (U/mg) 41.86 +- 3.28 c 26.62 +- 2.25 a 34.71 +- 2.86 b 50.52 +- 4.96 d MDA 6 (mmol/g) 3.82 +- 0.25 b 6.13 +- 0.33 d 4.95 +- 0.27 c 3.12 +- 0.27 a OH*-CR 7 (U/g) 102.12 +- 9.15 c 42.86 +- 3.98 a 75.54 +- 6.21 b 136.96 +- 11.75 d O2*-CR 8 (U/g) 70.12 +- 3.18 c 34.84 +- 1.75 a 51.43 +- 2.37 b 84.27 +- 4.41 d T-AOC 9 (mmol/g) 2.26 +- 0.07 c 1.27 +- 0.15 a 1.67 +- 0.18 b 2.73 +- 0.12 d Note: All above data are mean +- SD (n = 3 x 3 x 3), and different superscript letters in the same row indicate significant differences among the data (p < 0.05). 1 SOD: superoxide dismutase. 2 CAT: catalase. 3 GSH-PX: glutathione peroxidase. 4 GSH: glutathione. 5 GST: glutathione S-transferase. 6 MDA: malondialdehyde. 7 OH*-CR: hydroxyl radical clearance ratio. 8 O2*-CR: superoxide radical clearance ratio. 9 T-AOC: total antioxidant capacity. 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PMC10000118

The availability of floral resources is crucial for honey bee colonies because it allows them to obtain protein from pollen and carbohydrates from nectar; typically, they consume these nutrients in the form of bee bread, which has undergone fermentation. However, the intensification of agriculture, urbanization, changes to the topography, and harsh environmental conditions are currently impacting foraging sites due to habitat loss and scarcity of food resources. Thus, this study aimed to assess honey bee preference for various pollen substitute diet compositions. Bee colonies perform poorly because of specific environmental problems, which ultimately result in pollen scarcity. Pollen substitutes located at various distance from the bee hive were also investigated in addition to determining the preferences of honey bees for various pollen substitute diets. The local honey bee (Apis mellifera jemenitica) colonies and different diets (four main treatments, namely, chickpea flour, maize flour, sorghum flour, wheat flour; each flour was further mixed with cinnamon powder, turmeric powder, flour only, flour mixed with both cinnamon and turmeric powder) were used. Bee pollen was used as a control. The best performing pollen substitutes were further placed at 10, 25, and 50 m distances from the apiary. Maximum bee visits were observed on bee pollen (210 +- 25.96) followed by chickpea flour only (205 +- 19.32). However, there was variability in the bee visits to the different diets (F (16,34) = 17.91; p < 0.01). In addition, a significant difference in diet consumption was observed in control (576 +- 58.85 g) followed by chickpea flour only (463.33 +- 42.84 g), compared to rest of the diets (F (16,34) = 29.75; p < 0.01). Similarly, foraging efforts differed significantly (p < 0.01) at the observed time of 7-8 A.M., 11-12 A.M., and 4-5 P.M. at the distance of 10, 25, and 50 m away from the apiary. Honey bees preferred to visit the food source that was closest to the hive. This study should be very helpful for beekeepers in supplementing their bee colonies when there is a shortage or unavailability of pollens, and it is much better to keep the food source near the apiary. Future research needs to highlight the effect of these diets on bee health and colony development. honey bee supplementary diet pollens foraging behavior Ministry of Education in KSAKKU-IFP2-DA-7 This research was funded by the Ministry of Education in KSA through a project number KKU-IFP2-DA-7. pmc1. Introduction Honey bees (Apis mellifera jemenitica) have a high commercial value for honey production as well as pollination in a range of agricultural crops . Honey bees, like other invertebrates, are poikilothermic; they cannot regulate their body temperature and must go into hibernation when the ambient temperature is too high . Due to restricted foraging activity, their dietary requirements and metabolic activities are decreased during this period . High summer temperatures and dry weather are the main factors contributing to honey bee mortality in Saudi Arabia. This is due to decreased plant flowering and pollen availability due to heat stress . Native populations of A. mellifera jemenitica in Saudi Arabia are much more tolerant of heat than the common races. A. mellifera jemenitica exists in central Saudi Arabia, which has the warmest summer temperatures, >45 degC . Additionally, A. mellifera jemenitica possesses a remarkable ability to hunt for pollen and possesses high fecundity . The pollen quantity was discovered to have a positive correlation with temperature and a negative correlation with rainfall, relative humidity, and wind speed. The months with the highest pollen quantity (95% of all pollen) were May through September in Abha Saudi Arabia, while in September and October, there was a spike in flight activity. August through September had seen the highest concentrations of pollen brought back to the hive, while November through December had seen the lowest pollen concentrations in the Al-Ahsa region of Saudi Arabia . In addition, other seasonal and climatic fluctuations (precipitation, hail, etc.) produce considerable losses in floral resources throughout the year . Meanwhile, flowers are essential for honey bee brood production, immunological function, and overwintering survival . While nectar is a source of carbohydrates, pollen supplies proteins, lipids, and micronutrients . When the natural flora is insufficient, the queen bee's egg-laying level decreases, resulting in a fall in the colony's population level . Malnutrition reduces individual survival rates, causes larval life to cease, renders the colony prone to disease, and drives individuals to leave the colony . Usually, a honey bee colony obtains 10-26 kg of pollen each year from flowers as a rudimentary source of protein content and amino acid composition for the well-being of their colony . Furthermore, appropriate protein and carbohydrate stores in the colony are suggested to aid honey bees in fighting or tolerating different stressors associated with modern apiculture . Although pollen remains the most desirable and appealing protein source for honey bees, pollen replacements have advantages. Pollen introduced to the colonies from the outside is costly to get in large quantities, and it also entails the danger of introducing infections or pesticides into the colonies. Thus, human intervention is needed to overcome these problems, particularly for disease management and additional feeding. To compensate for the lack of nutritive forage in the environment, hives are routinely given artificial "pollen substitute" diets . As a result, better colony health for honey production and pollination can be maintained . To compensate for insufficient pollen forage and boost colony vigor prior to pollination services, beekeepers provide different "pollen substitute" diets . In order to manage honey bee colonies during a pollen-scarce season, Pande and Karnatak utilized germinated pulses as a substitute for pollen. For the production of four distinct diets--ger horse gram, ger chickpea, ger green gram/mungbean, and ger pea--various germinated pulse flours were used. Similarly, Kumar and Agrawal made six distinct combinations of artificial food using defatted soy flour, brewer's yeast, parched gram, spirulina, skim milk powder, sugar, glucose, protein hydrolysate powder, and natural pollen. This artificial diet favored the biochemical composition and net consumption, and also had a positive effect on colony parameters such as egg laying and brood production. In addition, in another study, different supplements were used such as roasted chickpea flour, broadbean flour, maize flour, and soy flour , and these supplements enhanced brood production and longevity. A study conducted in India using four flours--soybean, wheat, maize, and gram--as pollen substitutes found that pollen substitute is crucial for the growth and development of bee colonies not only during times of scarcity but especially during foraging and pollination and to overcome pesticide exposures . Meanwhile, in another study, six protein-rich ingredients--defatted soybean flour, chickpea flour, maize flour, wheat germ, pea flour, and dried brewer's yeast--were combined in various ratios with sugar powder, bee honey, and water to create ten diets , and these diets increased biological activities including diet consumption, sealed worker brood area, and pollen and honey store area. Honey bees can visit multiple food sources at once and travel up to 11 km to obtain primary food resources such as nectar and pollen , which are stored in their colonies as honey and beebread . Their foraging is one of the most well-organized behaviors found in social insects . Honey bee foragers use information gathered from their own experience, such as recall of time and place, sugar concentration to determine whether to continue or begin foraging on certain resources . Until now, research has concentrated on commonly foraged feed elements such as pollen and nectar rather than atypically foraged materials that may be ingested under drought. The objectives of the present study were to evaluate the preference of honey bees for different diets. In addition, we assessed honey bee preferences for various diet supplements placed at various distances from the colonies. 2. Materials and Methods The study was conducted at the Unit of Bee Research and Honey Production, King Khalid University Abha Saudi Arabia. The current study used local honey bee (A. mellifera jemenitica) colonies housed in the Langstroth hives. The honey bee colonies placed at the apiary did not show any clinical illness signs . All bee colonies were subjected to regularly suggested colony management procedures . 2.1. Preparation of Diets These pollen substitution diets were high in protein, carbohydrates, minerals, and fats. These items were reasonably priced in the local market. The supplemental diets listed below were created. Each diet was tested with three replications for five pollen substitute diets, including naturally collected pollen as a control on diet preference (see Table 1). The various supplemental diets were prepared separately first, measured known quantity , and carefully blended in a dough machine (Hobart dough mixer, model A200, Offenburg, Germany). The flour was fed externally to provide bees easy access to it. Honey bees must vibrate their body to collect powdered substances, a simple process requiring little time and effort . 2.2. Bee Visits and Diet Consumption Every day in the late afternoon, the consumption rate of each diet replicate was calculated by measuring diet weight before and after feeding in grams. At the end of the trial, the total amount of food consumed in each replicate was also calculated. 2.3. Choice Powder Feeding This modified procedure used three distances from colonies: 10, 25, and 50 m. Experimental colonies received all five feeds (i.e., CPCM, CPTM, CPOY, CPBH, and PNOY) in separate plates positioned at various distances as indicated in Figure 3. 2.4. Estimation of Honey Bee Numbers The numbers of honey bees were counted at different time intervals (7-8 A.M., 11-12 A.M., and 4-5 P.M.). During each time interval, the number of honey bees were counted visually three times as indicated in Figure 3. 2.5. Diet Consumption The diet weight between before and after feeding in grams per colony were calculated to determine the net weight of pollen-supplemented diets ingested within treatments after feeding 10 times to each colony . 2.6. Statistical Analysis The total amount of preferred food consumed and distance preference was compared between treatments with an analysis of variance. The data were calculated as mean and standard error using the SPSS (version 20). Graphs were created with the GraphPad Prism software (version 7.03). Furthermore, the Tukey post hoc test was used for multiple group comparisons at the 0.05 level. 3. Results 3.1. Honey Bee Visitation Maximum honey bee visitation during the first week was on the PNOY with 143 +- 12.89/week followed by CPOY (133.67 +- 13.75/week) and CPBH (78 +- 38.07/week). The fewest visits were on the MZOY (1.33 +- 0.88/week) followed by SGOY and WTOY with 2 +- 0.57/week and 2 +- 1.52/week mean visits, respectively. In the second week, maximum honey bee visits were observed in the CPOY (71.66 +- 6.06/week) and PNOY (67 +- 13.11/week). The fewest visits were on the SGOY (0.33 +- 0.33/week) followed by WTOY (0.66 +- 0.66/week) and MZOY (1 +- 0.57/week). However, overall time periods, maximum visits were on the PNOY (210 +- 25.96) followed by CPOY (205 +- 19.32), and the fewest visits were on the MZOY and SGOY with mean visits of 2.33 +- 1.45 and 2.33 +- 0.88 followed by WTOY (2.66 +- 2.18) as indicated in Figure 4. 3.2. Diet Supplement Consumption The findings showed that honey bees ingested varied amounts of all supplements throughout the research period. Honey bees consumed the highest amount of PNOY diet (404 +- 28.15 g/week) in week 1, which was followed by CPOY (350 +- 8.21 g/week) and CPBH (235.67 +- 7.85/week). The least consumption was for the WTOY diet (5 +- 4.04 g/week) followed by MZOY (7.66 +- 4.97 g/week) and SGOY (11 +- 2 g/week) as indicated in Figure 2. During week 2, the highest diet consumption amount was observed for the CPTM diet (203.33 +- 16.41 g/week) followed by the CPBH diet (192 +- 6.42 g/week), and then PNOY (172 +- 38.15 g/week). However, the overall highest consumption was for the PNOY diet (576 +- 58.85 g) followed by the CPOY diet (463.33 +- 42.84 g), the CPBH diet (427.67 +- 6.35 g), and the CPTM diet (384.67 +- 14.72 g). The least consumption was observed for the WTOY diet (7 +- 6.02 g) followed by the MZCM (16 +- 8.32) and MTTM diets (63 +- 21.37). The remaining diet treatment consumption is indicated in Figure 5. ANOVA was performed on the honey bee visits and diet consumption, and there were significant differences between the visits to the different treatments during the first week (p < 0.01). Similarly, during the second week, there were significant visits to different diets (p < 0.01). Overall, there were significant variability in some of the treatment visits (p < 0.01) as indicated in Table 2. According to Table 2, the mean diet consumption throughout the entire sample of honey bee colonies showed notable variability during the first week (p < 0.01). The same pattern was observed in consumption for the second week (p < 0.01). Similarly, overall, there were significant differences in diet consumption due to treatment (p < 0.01). There was a strong positive correlation between the two variables: honey bee visits and diet consumption. During the first week, the Pearson's correlation was 0.957. Similarly, during the second week and in the total observation period, a strong positive correlation was also found between bee visits and consumption: 0.89 and 0.94, respectively. 3.3. Foraging Efforts The foraging effort was measured by counting the number of bees visiting the best performing diet (chickpea flour mixed with different spices) placed at different distances such as 10 m, 25 m, and 50 m and observed at different time intervals. The maximum mean number of honey bee visits during 7-8 A.M. at a 10 m distance was observed in the PNOY diet (282.5 +- 2.5), which was followed by CPOY (277.5 +- 7.5). The visitation rate was observed in CPCM, which was 75 +- 5, followed by CPBH (79.5 +- 0.5). The maximum mean number of honey bees visiting the PNOY diet (145.5 +- 3) at 25 m was less than the maximum visitation rate to CPOY (252.5 +- 2.5). The least number of visits were observed in the CPTM diet with 50 +- 5 followed by the CPBH diet (51 +- 1). Similarly, at the distance of 50 m, maximum number of honey bees were observed in the PNOY diet (237 +- 7) followed by the CPOY diet (215.5 +- 14.5). The least number of visits (34.5 +- 4.5) were recorded at the CPCM diet followed by the CPBH diet (37.5 +- 2.5) as shown in Figure 6a. As indicated in Figure 6b, when diets were placed at the distance of 10 m, the maximum number of honey bees during the time of 11-12 A.M. were observed on the PNOY (226 +- 1) followed by CPOY (220.5 +- 0.5) while the fewest number of honey bees were recorded on the CPBH diet, which was 58.5 +- 0.5 followed by CPCM (65 +- 5). Similarly, when diets were placed at a distance of 25 m, the maximum number of honey bees visited the COPY diet (202.5 +- 6.5) followed by the PNOY diet (202 +- 3), while the fewest number of visits were observed on the CPCM diet with 35 +- 5 visits followed by the CPBH diet (46.5 +- 5.5). In the case of 50 m, maximum visits were observed on the copy diet (180 +- 10) followed by the PNOY diet (176 +- 1). The least number of visits were recorded on the CPCM and CPBH diets (27.5 +- 2.5 and 40.5 +- 5.5, respectively). When the foraging activity of honey bees at the time of 4-5 P.M. was observed at a distance of 10 m, the maximum number of honey bees visiting the diets were found on the PNOY treatment (205 +- 10) followed by the copy treatment (178 +- 7). The fewest bees were observed on the CPCM (50 +- 5) and the CPBH diets (51 +- 1). At the distance of 25 m, maximum number of bee visits were observed on the PNOY diet (185 +- 10) followed by the copy diet (164 +- 6). The least number of honey bee visits were recorded on the CPCM (32.5 +- 1.5) and the CPBH diets (37 +- 8). When diets were placed at a distance of 50 m, the maximum number of honey bees visiting the treatments were found on the PNOY diet (145 +- 9), followed by the copy diet (140 +- 10). The least number of honey bees visiting a diet were found on the CPCM (22.5 +- 2.5) and the CPBH diets (26 +- 3) as indicated in Figure 6c. There was a non-statistically significant interaction between time of day, distance from the colonies, and diet types (F (16,45) = 1.017 (p = 0.458). However, a significant interaction was found between time of day and distance (F (16,45) = 3.063 (p = 0.026). Similarly, a significant interaction was found between time of day and diet types (F (16,45) = 33.349 (p = 0.001). 4. Discussion Like most other invertebrates, honey bees are poikilothermic; they are unable to control their body temperatures and become inactive when the outside temperature becomes intolerable. Due to severely constrained foraging activities during hot weather, their nutritional needs and metabolic activity are reduced . Thus, the present study was designed to offer a substitute for honey bees in the harsh conditions of KSA and to investigate the effects of distance from the bee hive on visitation rate to the diets. Overall, in our study, honey bees exhibited a significant difference in visitation rates to the different diets. However, maximum visits were observed in PNOY (pollen as a control) (210 +- 25.96) followed by CPOY (chickpea flour only) (205 +- 19.32). In terms of diet consumption, PNOY (576 +- 58.85 g) followed by CPOY (463.33 +- 42.84 g) were maximum diets consumed which are in agreement with findings of Khan and Ghramh . They concluded that honey bees ingested much more pollen (11.51 +- 2.22 mg/bee) and ajeena diet, i.e., commercially available pollen substitute (10.68 +- 1.29 mg/bee) than any other diet. This attraction towards pollen may be due to the quality of the diet; pollen attracts foragers and is considered a major source of vitamins, minerals, lipids, carbohydrates, sterols, proteins, and amino acids . The nutritional value of protein would be the primary factor in honey bees' selection of pollen for food . In a study conducted in India, four distinct germinated pulse flour diets--germinated horse gram, germinated chickpea, germinated green gram/mungbean, and germinated pea--were used and following feeding, foraging behavior was seen in all diet combinations, including germinated chickpea, germinated green gram, and germinated horse gram . While our findings were in contrast with the study conducted in India, three diet formulas using the four germinated pulses soybean, mungbean, pigeon pea, and chickpea were used, and soybean was the most preferred of the four pulses and three formulations . However, the other substitutes can also be very helpful in drought or harsh conditions when there are few flowers. The fluctuation in floral sources and bee colony population density affect the annual pollen supply for bee colonies in many regions of the world. Since the flora that honey bees need is not consistently present, artificial pollen substitutes and supplements have been utilized to sustain the strength of bee colonies by lengthening the adult lifespan and maintaining brood area . While these supplemented meals and pollen replacements may offer a temporary solution to avoid bee losses in poor foraging settings, it cannot be sustained as a long-term solution in a pollen scarce locale. In our study, there were reasonable numbers of honey bees that visited and consumed chickpea flour only (CPOY). This can be the best option when there is a pollen shortage. This is because chickpea flour has a good amount of protein (21.70-23.70%), carbohydrates (59.66-66.42%), fats (4.80-6.36%), ash (2.2-3.46%), total fiber (14.80), and moisture contents (9.35%) . There are also other flours that have high amounts of proteins and other important elements (Table 3). In the present study, when honey bee visits were compared at different timings (7-8 A.M., 11-12 A.M., and 4-5 P.M.), and also diets were placed at different distances (10, 25, and 50 m) from the bee hive, maximum activity was recorded in the morning (7-8 A.M.) and at the closest distance of 10 m from the hive. In another study, contrasting results were reported. Honey bees spent the most time foraging during the day, particularly at 12:00 P.M., followed by 14:00 P.M., and finally at 10:00 A.M. every week . Similar to these findings, Pernal and Currie found that honey bees foraged more frequently in the afternoon than in the morning. Forager bees increased their activity and pollen collecting in an onion crop between the hours of 11:00 A.M. and 12:00 P.M. over several days . In all of these studies, maximum bee foraging activity was in the afternoon. This may be due to the effect of rising temperatures and falling relative humidity on anther dehiscence, reaching their highest from 11:00 A.M. to 14:00 P.M. This time period corresponded to the peak pollen-gathering activity of the bees . However, in our case, pollens were used as a control diet detached from flowers and that may be the reason why honey bees showed maximum activity in the morning (7-8 A.M.) instead of the afternoon due to the free access to pollen early in the morning. In our study, there was a constant increase in the percentage of bees foraging for pollen in the morning, but in the afternoon, that percentage declined. It was discovered that many honey bees flew orientation flights between 12:30 and 14:00 h, especially in sunny conditions, which led to a decreased percentage of pollen foragers in colonies during the middle of the day. This was also documented by a study in China . Additionally, our findings revealed a substantial decrease in foraging activity from 12:00 h to 4-5 P.M., which was likely caused by the high air temperature (exceeding 40 degC). High ambient temperatures make foraging more energy-intensive and can lead to dehydration of foragers. Importantly, foraging distance varies with month differently for the two kinds of forage. In some months, we observe greater distance for one forage type and other months we see the opposite. Overall, this implies that the distance that foraging honey bees must travel is not greatly influenced by one type of forage over another, with summer generally being the season where bees must go farther to gather forage than spring or fall. In the present study, there was a strong positive correlation between the visits and diet consumption (p < 0.01); as the visits increased, the amount of diet consumed also increased. Hence, our results suggest that when food sources are abundant near the bee hive, consumption and foraging activity may increase. Additionally, more field research is required to ascertain how these supplemental diets affect the health and productivity of honey bee colonies. This research could assist beekeepers in creating more suitable food products that reduce waste and improve the nutritional intake of their bee colonies, especially in harsh conditions when there is a shortage of flora, particularly in the case of the KSA region. 5. Conclusions In the present study, we examined the preference of honey bees towards different diets and observed the effect of distance on foraging behavior. Overall, honey bees were more attracted to the natural pollens than the other diets. However, this does not mean that honey bees were not attracted to other diets; there were a reasonable number of honey bees that visited the alternative diets such as the chickpea flour only diet. These supplemental diets can be very helpful when there is a scarcity of pollens, and they can play a very important role in the production of honey. In terms of distance, honey bees preferred to visit the food source nearest to the bee hive (10 m), and the preferred time was in the morning (7-8 A.M.). More research is required to learn how these supplements affect different physiological parameters of honey bee races under diverse climatic situations. Acknowledgments The authors extend their appreciation to the Ministry of Education in KSA for funding this research work through project number KKU-IFP2-DA-7. The authors would like to thank their colleagues at the Unit of Bee Research and Honey Production (UBRHP) at King Khalid University Abha, Saudi Arabia. Author Contributions Conceptualization, K.A.K.; Methodology, K.A.K.; Validation, H.A.G. and K.A.K.; Formal analysis, H.A.G. and K.A.K.; Investigation, H.A.G. and K.A.K.; Data curation, K.A.K.; Writing--original draft, K.A.K.; Writing--review & editing, H.A.G. and K.A.K.; Supervision, H.A.G. and K.A.K.; Project administration, H.A.G. and K.A.K.; Funding acquisition H.A.G. and K.A.K. All authors have read and agreed to the published version of the manuscript. Institutional Review Board Statement Not applicable. Data Availability Statement Data are contained within the article. Conflicts of Interest The authors declare no conflict of interest. Figure 1 A. mellifera jemenitica apiary set-up. Figure 2 Measuring known quantity of diets before mixing. Figure 3 Different diet supplements at different distances (10, 25, and 50 m) away from the apiary and measuring the number of honey bees. Figure 4 Honey bee visits towards different diet compositions. Whereas, CPCM = Chickpea flour + Cinnamon powder, CPTM = Chickpea flour + Turmeric powder, COPY = Chickpea flour only, CPBH = Chickpea flour + Both powders, MZCM = Maize flour + Cinnamon powder, MZTM = Maize flour + Turmeric powder, MZOY = Maize flour only, MZBH = Maize flour + Both powders, SGCM = Sorghum flour + Cinnamon powder, SGTM = Sorghum flour + Turmeric powder, SGOY = Sorghum flour only, SGBH = Sorghum flour + Both powders, WTCM = Wheat flour + Cinnamon powder, WTTM = Wheat flour + Turmeric powder, WTOY = Wheat flour only, WTBH = Wheat flour + Both powders, PNOY = Pollen only as a control. Different superscript letters indicate significant differences between total diet consumption. Figure 5 Honey bee diet consumption from various pollen substitutes. Whereas, CPCM = Chickpea flour + Cinnamon powder, CPTM = Chickpea flour + Turmeric powder, CPOY = Chickpea flour only, CPBH = Chickpea flour + Both powders, MZCM = Maize flour + Cinnamon powder, MZTM = Maize flour + Turmeric powder, MZOY = Maize flour only, MZBH = Maize flour + Both powders, SGCM = Sorghum flour + Cinnamon powder, SGTM = Sorghum flour + Turmeric powder, SGOY = Sorghum flour only, SGBH = Sorghum flour + Both powders, WTCM = Wheat flour + Cinnamon powder, WTTM = Wheat flour + Turmeric powder, WTOY = Wheat flour only, WTBH = Wheat flour + Both powders, PNOY = Pollen only as a control. Different superscript letters indicate significant differences between total diet consumption. Figure 6 Honey bee visits at different times and pollen substitutes present at different distances: (a) 7-8 A.M. time (b) 11-12 A.M. time (c) 4-5 P.M. time. Whereas, CPCM = Chickpea flour + Cinnamon powder, CPTM = Chickpea flour + Turmeric powder, CPOY = Chickpea flour only, CPBH = Chickpea flour + Both powders, PNOY = Pollen only as a control. animals-13-00885-t001_Table 1 Table 1 Different supplement formulations. Sr.# Pollen Supplementary Diet Ratio Abbreviations 1 Chickpea flour + Cinnamon powder 50:1 CPCM 2 Chickpea flour + Turmeric powder 50:1 CPTM 3 Chickpea flour only - CPOY 4 Chickpea flour + Both powders 50:1 CPBH 5 Maize flour + Cinnamon powder 50:1 MZCM 6 Maize flour + Turmeric powder 50:1 MZTM 7 Maize flour only - MZOY 8 Maize flour + Both powders 50:1 MZBH 9 Sorghum flour + Cinnamon powder 50:1 SGCM 10 Sorghum flour + Turmeric powder 50:1 SGTM 11 Sorghum flour only - SGOY 12 Sorghum flour + Both powders 50:1 SGBH 13 Wheat flour + Cinnamon powder 50:1 WTCM 14 Wheat flour + Turmeric powder 50:1 WTTM 15 Wheat flour only - WTOY 16 Wheat flour + Both powders 50:1 WTBH 17 Pollen only as a control - PNOY animals-13-00885-t002_Table 2 Table 2 Honey bee visit and diet consumption (ANOVA). Honey Bee Visits Sum of Squares Degrees of Freedom Mean Square F p-Value Week 1 94,945.020 16,34 5934.064 16.883 >0.001 Week 2 26,137.17 16,34 1633.574 14.922 >0.001 Total 218,023.64 16,34 13,626.478 17.913 >0.001 Diet consumption Week 1 705,552.03 16,34 44,097.002 42.080 >0.001 Week 2 238,175.92 16,34 14,885.995 12 >0.001 Total 1,615,024.64 16,34 100,939.020 29.758 >0.001 animals-13-00885-t003_Table 3 Table 3 Nutritional content of pollen substitutes reported in different studies. Pollen Substitutes Protein (%) Carbohydrates (%) Fat (%) Ash (%) Total Dietary Fiber Moisture (%) References Wheat flour 10.55 74.88 0.94 0.94 0.36 12.67 11.85 86.04 1.06 0.52 0.54 11.97 11.60 78.70 1.70 0.97 - 14.20 Maize flour 6.00 - 2.18 0.61 - 10.63 8.90 - 5.30 1.30 15.60 - 8.55 78.77 2.61 0.52 3.68 9.55 Chickpea flour 21.85 66.42 6.36 2.92 - - 21.70 59.66 5.81 3.46 - 9.35 23.70 61.10 4.80 2.2 14.80 - Sorghum flour 12.30 73.80 3.60 2.92 - - 12.21 83.45 3.76 0.68 - - 11.50 72.00 2.70 - - - Disclaimer/Publisher's Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

PMC10000119

Yield, chemical composition, and fermentation variables were compared for amaranth silages (AMS) from five cultivars (A5, A12, A14, A28, and Maria) and corn (Zea mays; CS). In vitro methane production, organic matter disappearance, microbial protein, ammonia-N concentration, volatile fatty acid levels, cellulolytic bacteria and protozoa populations, and in situ dry matter (DM) and crude protein (CP) degradability were evaluated. All crops were harvested when the plant was at the mid-milk line stage, then chopped, placed in sealed 5 L plastic bags and stored for 60 days. Data analysis was carried out using the PROC MIXED method of SAS with a randomized complete block design. The mean DM forage yield of CS was higher than the average DM yield of the amaranth cultivars (P < 0.001). In comparison with CS, the AMS had higher CP, lignin, ether extract, ash, calcium, phosphorus, magnesium, total phenolics and metabolizable protein (P < 0.001), but had lower DM, neutral detergent fiber, non-fiber carbohydrates, organic matter disappearance, lactic acid (P < 0.01) and in vitro methane production (P = 0.001). The AMS had higher (P < 0.01) pH, ammonia-N concentration, in vitro microbial protein, in situ digestible undegradable protein, and metabolizable protein compared to CS. Overall, in comparison to CS, the amaranths produced a silage of medium-quality. ensiled amaranth forage chemical composition in vitro disappearance of organic matter degradability pmcINTRODUCTION Changes in climatic conditions leading to water scarcity in semi-arid areas may lead to a reduction in yield in conventional forage crops with subsequent shortages and price increases. The production of forage depends on soil and climate (Sotomayor-Rios and Pitman, 2001), so there is a need to investigate alternative crops such as amaranth that are more tolerant of harsher conditions (Svirskis, 2009). Amaranth species are C4 dicotyledonous plants and therefore able to photosynthesize at high temperatures in conjunction with a higher water use efficiency than many C3 plants (Assad et al., 2017). These characteristics facilitate the introduction of amaranth as a crop to dry and marginal zones (Myers, 1996; Liu and Stutzel, 2004). The yield of amaranth green mass exceeds by 20-30% the productivity of the traditional silage crop-corn, 13.2 t/ha DM vs. 12.0 t/ha DM for the Iowa farms, respectively (Mehrani et al., 2012; Langemeier and Purdy, 2019). The amaranth crop yields high levels of crude protein (CP) (up to 285 g/kg DM) and good DM digestibility (590-790 g/kg) with low levels (below toxic concentrations) of oxalic and nitric acids. This data suggests a good potential as ruminant forage (Abbasi et al., 2012). It has lower water requirement compared with corn which means that amaranth silage (AS) may be a better option where water availability is limited (Ofitserov, 2001). Constant availability of animal feed is essential for the development of continuous livestock production. However, in many regions of the World, animal production is principally influenced by the variable and increasing tendency of feed price due to adverse climatic conditions, conflicts and wars in areas of production, competition from biofuel production, a variable exchange rate, and the fluctuating price of petroleum products used in the operation of farm machinery (Msangi et al., 2014). A possible solution to strengthen the self-sufficiency of concentrates and corn forage is by using a double cropping system in a single season, thus sustainably intensifying agricultural production. Double cropping is practiced in many areas of the world, where cereals such as wheat or barley are grown as the main crop followed by corn forage as a second crop (Kato, 2011; Nikkhah et al., 2011). Consequently, corn forage is harvested in late autumn (i.e., the temperature around 15-18 oC), when there is inadequate heat and sunlight to allow for sufficient drying, and consequently a low DM silage (<250 g/kg fresh weight) is produced. In such climatic conditions, there is little information in the literature on the productive potential, nutritional characteristics, and fermentation patterns of Amaranthus hypochondriacus cultivars and their potential to replace corn forage. Therefore, this study describes amaranth and corn as the second crops in a double cropping system, using barley as the main crop followed by amaranth and corn to compare their suitability to be cultivated in this region (i.e., Malayer city, Hamedan Province, Iran) under these climactic conditions. The significant considerations for growers are to confirm yield and nutritional values by the selection of ideal cultivars under these conditions. Hence, this study was conducted to investigate how five amaranth (A. hypochondriacus) cultivars when both had low DM content (<250 g DM/kg fresh weight) responded to climate in terms of biomass production, and accumulation of nutrients when compared to corn forage. MATERIALS AND METHODS All aspects of animal care in this experiment were taken from The Guide for the Care and Use of Agricultural Animals in Research and Teaching (FASS, 2020). The protocols of the study (#9530381003, 2017) had the approval of the Animal Science Committee of Tarbiat Modares University. Forage Preparation and Sampling Methods The Seed and Plant Research Improvement Institute (Karaj) supplied corn seeds (Zea mays hybrid variety SC 704). The amaranth cultivars' seeds (A5, A12, A14, A28, and Maria) were supplied by Sven-Erik Jacobsen (Quinoa Quality, Regstrup, Denmark) and had been selected for their performance from the PROTEIN2FOOD project. The experimental location was near Malayer city (Hamadan Province) at 1751 m above sea level (Taherimoghaddam et al., 2018) with a mean yearly rainfall of 343 mm (2017) and a mean temperature of 12.4 degC (Hamadan Regional Water Authority, 2022) in soft loam-clay soil (Sand: 20%, Clay: 30%, Silt: 50%) (Naderi-Mahdei and Bahrami, 2014). There were four 25 m2 replicate plots per treatment set out as a randomized complete block design. The amaranths were sown into coir beds in culture trays (at a seeding rate of approx. 0.5 kg/ha) then transplanted when 10 cm high (industry standard). The corn was direct precision drilled (Tarashkadeh Co., Karaj, Iran). There was a 50 cm row spacing for all plants in the field. Nitrogen fertilizer in the form of urea had been applied to the plots at a rate of 53 kg N/ha. Weeds were controlled by row cultivation until the plants became established. When in the beds, the amaranth was irrigated daily but the corn and the amaranth transplants in the field were irrigated every other day with a drip irrigation system (20 cm hole spacing, bar type). The mean total irrigation applied to corn and amaranth cultivars were 350 and 250 mm (equivalent to 3,500 and 2,500 m3/ha), respectively as it has been shown that amaranth needs less water than corn to achieve the same biomass (260 vs. 370 g/g) (Ofitserov, 2001) and Shadi et al. (2020) recorded no detrimental effects on amaranth growth at this level. The crops were harvested, manually, in 85 days (October 25, 2017) (Nikkhah et al., 2011; Abbasi et al., 2018) at the mid-milk line stage (Lancashire et al., 1991) and then left to wilt for 30 hours. The DM values of corn and amaranths, Maria, A5, A12, A14, and A28, at harvest were 184, 163, 130, 158, 127, and 117 g/kg wet weight, respectively. The DM after wilting of the crops are shown in Table 2. The crops were chopped, by knife, into approximately 2 cm lengths and packed tightly into 10 L plastic bags, without a microbial inoculant, vacuum sealed and left for 60 days (Shadi et al., 2020). Fresh forage and silage samples were kept for analysis. Table 2. Dry matter (g/kg as-fed) and chemical composition (g/kg DM) of the fresh forages and silages from five amaranth varieties (Maria, A5, A12, A14, A28) and corn. Item1 Fresh forage Ensiled forage Amaranth varieties P-value Amaranth varieties P-value Corn Maria A5 A12 A14 A28 SEM2 CA3 AM4 Corn Maria A5 A12 A14 A28 SEM2 CA3 AM4 DM 202 185a 141cd 174b 149c 133d 1.85 0.003 0.01 217 187a 152b 177a 155b 152b 2.92 <0.001 0.004 CP 77 160b 158b 148c 156b 170a 1.67 <0.001 0.008 77 155b 144c 141c 142c 163a 3.09 <0.001 0.002 NDFom 493 370a 351c 378a 357b 339d 2.55 0.003 0.009 482 353a 335b 355a 341b 317c 3.22 <0.001 0.006 ADFom 308 279b 252c 290a 273b 244c 2.24 0.01 0.005 308 271a 243bc 281a 262ab 234c 2.88 0.003 0.005 ADL 48.3 63.4a 60.4ab 58.9b 65.0a 67.2a 1.96 0.003 0.007 47.6 62.4ab 60.0b 56.5c 64.2a 65.2a 1.91 <0.001 0.002 WSC 120 57a 55ab 53b 52b 50b 1.57 <0.001 0.03 11.0 12.2b 13.5b 14.0a 12.0b 11.2c 1.05 0.001 <0.001 EE 26.4 43.9a 39.2b 36.1b 38.3b 33.0b 0.54 <0.001 0.006 26.0 56.2a 52.9ab 48.0b 48.1b 43.0c 1.18 <0.001 0.008 Ash 72 177c 197b 198b 204b 222a 2.63 <0.001 0.005 71 197d 227b 207c 223b 239a 2.89 <0.001 0.004 NFC 332 249b 255a 240c 245bc 236c 3.05 0.002 0.004 344 239b 251a 249a 246a 238b 3.42 <0.001 0.002 Ca 3.2 13.3 15.5 14.0 14.7 15.1 1.01 <0.001 0.28 3.3 13.9 15.9 14.4 15.0 15.4 0.92 <0.001 0.38 P 2.4 5.3 5.5 4.9 5.1 5.2 0.59 0.001 0.33 2.5 5.3 5.6 4.9 5.2 5.2 0.45 <0.001 0.15 Mg 2.1 5.4 4.5 4.8 4.7 4.4 0.24 <0.001 0.25 2.3 5.5a 4.6 4.8 4.9 4.4 0.46 <0.001 0.11 1DM = dry matter; CP = crude protein; NDFom = ash-free NDF; ADFom = ash-free ADF; ADL = acid detergent lignin; EE = ether extract; WSC = water-soluble carbohydrates; EE = ether extract; NFC = non-fiber carbohydrates (on DM basis) calculated as: 1000 - (NDFom g/kg + CP g/kg + EE g/kg + ash g/kg); 2SEM = standard error of the means; 3CA = comparing corn vs. average value of amaranths; 4AM = comparing among amaranth varieties; a, b, c, d, e among amaranths = means in the same row with different superscripts differ (P < 0.05). Chemical Analyses Samples of silage were weighed and dried at 60 degC for 48 h to determine DM percentage then ground to pass through a 1-mm sieve. Total nitrogen (N) was determined using method 990.03 of AOAC (2012) with CP being calculated as 6.25 times the total N concentration. Total ash content was measured using the method from AOAC (2012) as were calcium, magnesium and phosphorus concentrations. Ether extract (EE) was determined using AOAC (2012) method 2003.05. Neutral detergent fiber (NDF) levels were determined using alpha amylase, without sodium sulfite, and expressed exclusive residual ash (NDFom; Van Soest et al., 1991). Ash-free acid detergent fiber (ADFom) was determined using AOAC (2012) method (973.18) and acid detergent lignin (ADL) using AOAC (2012) method (973.18). Water-soluble carbohydrates (WSC) concentration was measured by the anthrone reaction assay (MAFF, 1986). The nonfiber carbohydrates (NFC) content (on a DM basis) was calculated as 1000 - (NDFom g/kg + CP g/kg + EE g/kg + ash g/kg) (NRC, 2001). Nitrate concentration was measured using colorimetry (Singh, 1988). The oxalic acid concentration was measured by spectrophotometry using the method of Savage et al. (2000). The Folin-Ciocalteau method (Makkar, 2000) was used to determine total phenolics. Non-tannin phenolics were measured by the Folin-Ciocalteau reaction after the absorption of tannins to insoluble polyvinylpyrrolidone. (Makkar, 2000). Total tannin levels were obtained by subtracting non-tannin phenolics from total phenolics with the data expressed as tannic acid (Merck GmbH, Germany). For measuring silage pH, 50 g of fresh silage was blended with 125 mL of distilled water and allowed to stand at room temperature for 1 h (Faithfull, 2002). After decanting the silage extract into a small beaker, the pH was measured using a digital pH meter (Sartorius PT-10; Germany). Two milliliters of juice from the silages were pipetted into centrifuge tubes containing 0.2 mL of acid (25% meta-phosphoric acid and 2-ethyl butyric acid 2 g/L as the internal standard), then centrifuged at 10,000 x g for 10 min at 4 degC (Galyean, 2010). Volatile fatty acids in the supernatant were quantified using gas chromatography (UNICAM 4600; SB Analytical, Cambridge, UK) with a flame ionization detector (FID; 250 degC), split-injection port (1.0 mL injection), capillary column (Agilent J & W HP-FFAP, 10 m by 0.535 mm by 1.00 mm, 19095 F-121; Agilent, CA), and helium as the carrier gas (column head pressure of 10 psi). To determine NH3-N, an extract was obtained by squeezing the silage material, filtered using Whatman 54 filter paper, then a 9 mL of aliquot was taken, mixed with 1 mL of 7.2 N H2SO4, and stored at -20 degC. After thawing, the silage extracts were analyzed for NH3-N using a phenol-hypochlorite assay (Galyean, 2010). In vitro Ruminal Gas Production and Estimated Variables A 24-h in vitro gas production (GP) was conducted to determine the silage fermentation properties according to the method of Menke et al. (1979). Rumen fluid was used from 3 fistulated Shall sheep that had been fed twice daily on a diet containing (g/kg DM) 400 alfalfa hay, 150 corn silage (CS), 100 amaranth silages, 150 rolled barley, 140 wheat bran, 50 soybean meal, and 10 vitamin-mineral premix. Rumen fluid was collected before the sheep's morning feed. One hundred and ninety-eight syringes (six treatments x four blocks x two individual samples per block x two syringes per sample x two runs, with three blanks in each run) were used. The samples (200 mg DM) were incubated in 30-mL of diluted rumen liquor (10 mL of the rumen fluid + 20 mL buffer) under a CO2 atmosphere at 39 degC. The gas volume was measured from the change in the position of the syringe piston after 24-h. The in vitro OMD and metabolizable energy (ME) were calculated using the following equations: OMD(g/kg)=1488+(8893xGP24)+(0448xCP)+(0651xXA) ME(MJ/kgDM)=220+(01357xGP24)+(00057xCP)+(000002859xCP2) In the above equations, GP24 is net gas produced after 24 h (mL/200 mg DM), CP is crude protein (g/kg DM), XA is ash (g/kg DM). Eight syringe contents per treatment from each run (four blocks x two individual samples x one syringe per sample) after soluble product removal (Van Soest et al., 1991) were used to determine truly degraded substrate (TDS) levels (Blummel et al., 1997). The partitioning factor after 24 h (PF24) was calculated from Blummel et al. (1997)PF24(mg/mL)=TDS(mg)/GP24(mL), where GP24 is 24-h gas production. Microbial CP (MCP, mg/g DM) was estimated from the equation TDS(mg)-(mLGP24x2.2mg/mL), 2.2 mg/mL being a stoichiometric factor expressing mg of carbon, hydrogen, and oxygen required for the volatile fatty acids-gas complex production needed for 1 mL of gas produced at 24 h". Eight syringes per treatment per run (four blocks x two samples) were used to determine in vitro pH, NH3-N, VFA and numbers of cellulolytic bacteria, and protozoa. Ammonia-N was measured from a strained sample of 2.5 mL by the method of Galyean (2010). The VFA levels were measured in 2mL of supernatant using gas chromatography by the method of Galyean (2010). Total protozoa numbers and subfamily counts were enumerated using the method of Dehority (2003). The cellulolytic bacteria population was determined using Hungate tubes containing media according to Bryant (1972). After 21 days the Most Probable Number method of Dehority (2003) was used to ascertain bacteria numbers. Gas production (GP) kinetics were evaluated from a 96-h in vitro experiment with gas being recorded at 2, 4, 6, 8, 12, 24, 48, 72, and 96 h of the incubation. Kinetic variables were predicted according to Carro et al. (1999):- "y = B (1 - (t-L)). In this model, y, B, c, and L are the gas volume detected at time t, asymptotic value of produced gas (mL/200 mg diet DM), first-order fractional rate constant of produced gas (/h), and lag time (h), respectively". The in vitro methane produced from each sample was measured after 24-h incubation (Menke et al., 1979). After the total gas volume had been recorded, four mL of 10 M NaOH was introduced into the syringe to absorb CO2 leaving the remainder to be measured as CH4 (Demeyer et al., 1988). In situ Degradability of DM and CP The silage DM and CP degradability was determined by the in situ method (AFRC, 1992) from bags suspended in the rumens of four fistulated Shall sheep. The sheep were fed the diet (as above) for 2 weeks to allow for adaptation. The bags were 10 x 21 cm having a 45 mm pore size (Bucksburn, Aberdeen, UK). Dried silage samples were ground to pass through a 4 mm sieve using a Cyclotec TM 1093 Sample Mill (Foss Company, Denmark). The bags each contained 5 g DM and were incubated for 2, 4, 8, 12, 24, 48, 72, and 96 h. One bag per sample was used for each time in each sheep. When the bags were removed, they were cold water washed (Hoover OPHS 612; Hoover, London, UK) for 1 h, then dried to a constant weight at 55 oC. The degradability value at time zero was determined from 3 bags per sample washed and dried as above. Bags and contents were weighed to estimate degraded DM and residues were analyzed for CP according to AOAC (2012) method (942.05). The degradation kinetics parameters of DM and CP were determined by the method of Orskov and McDonald (1979): An exponential curve as Y = a + b (1 - e-ct) fitted to the experimental data by iterative regression analysis. The effective degradability (ED) of DM and CP was then estimated as ED(%)=a+[(b+c)/(c+k)]. In these equations, 'Y' is disappearance from the bag, 'a' is the soluble and very rapidly degradable fraction, 'b' is the insoluble but potentially degradable fraction degrading at a constant fractional rate (c) per unit time (t), 'e' is the base of natural logarithms, ED is the effective degradability, and 'k' refers to the fractional outflow rate of small particles from the rumen. An assumed value for 'k' was 0.02 fraction/h (Orskov and McDonald, 1979). Effective RDP (ERDP) was calculated from the equation recommended by AFRC (1992) as: ERDP=CPx[0.8a+bc/(c+r)] where a, b, and c are the fitted parameters derived from in situ determination of feed degradability, 0.8 is the efficiency of capture of the nitrogen of the readily degradable fraction, and r is the outflow rate (0.02 fraction/h). The DUP was calculated as 0.9 (UDP - 6.25 ADIN), where UDP is dietary undegraded protein, and ADIN is acid detergent insoluble nitrogen (which is undegradable and indigestible in the rumen and abomasum). The proportion of the microbial CP (i.e., ERDP) present as true protein is 0.75 (amino acids) and its true digestibility is 0.85, therefore, the MP is calculated as: MP (g/kg DM) = [ERDP x 0.75 x 0.85] + UDP Statistical Analysis The SAS, PROC MIXED program (SAS Inst. Inc., Cary, NC) was used for the analysis of data from yield and chemical composition of the forage and silage treatments, as well as fermentation quality of the silages, using a randomized complete block design with treatment being a fixed effect and random effect of block. The model was Yijk = m + Ti + Bj + eij + eijk, where Yijk is the observation, m is the general mean, Ti is the treatment effect, Bj is the block effect and eij and eijk are the experimental and sampling errors respectively. Gas production data of the silage treatments was analyzed using a randomized complete block design as described above, including the random effect of run. Moreover, the SAS, PROC MIXED, program was used for the in situ study data analysis. The model included the fixed effect of treatment and the random effects of animal and block. The PROC UNIVARIATE was used to test the residual normality of the data. Least squares means (five means) of the amaranths were separated using the DIFF option. Moreover, the single contrast test was used to compare corn (as a treatment) vs. average amaranth value (the mean of five amaranths). The P-values <= 0.05 were accepted to define statistical significance. RESULTS Yield, Chemical Analyses, and Antinutritional Factors In Table 1, results of fresh forage illustrated that dry forage yield/ha of the corn was higher (P = 0.002) than the mean amaranth yield (6.5 vs. 5.4). Amaranth forages had lower (P < 0.01) overall mean levels of DM (156 vs. 202), NDFom (359 vs. 493), and NFC (245 vs. 332), but higher (P < 0.01) ADL (63 vs. 48), CP (158.4 vs. 77), EE (39 vs. 26), ash (199 vs. 72), calcium (14.5 vs. 3.2), phosphorus (5.2 vs. 2.4), and magnesium (4.7 vs. 2.1) levels (Table 2). Among amaranth cultivars, Maria had the highest (P = 0.01) DM value. The A28 forage had the highest (P = 0.008) CP value, followed by Maria, A5, A14, with A12 being the lowest. The NDFom values for Maria and A12 were higher (P = 0.003) than the rest. In comparison to corn, amaranth fresh forages had lower (P < 0.001) overall mean levels of WSC (53 vs. 120). Among amaranths, Maria had a higher (P = 0.03) WSC value in comparison with A12, A14, and A28. Compared to amaranth forage, corn forage had lower (P < 0.001) EE (26.4 vs. 38.1). Maria forage showed the highest (P = 0.006) EE concentration among amaranth cultivars. The ash content of corn forage was lower (P < 0.001) than the mean value of amaranths (72 vs. 200). Among amaranth forages, A28 had higher ash level (P = 0.005) than the rest. The highest and lowest (P = 0.004) NFC values for amaranth forages were in A5 and A28 (255 vs. 236). In comparison to corn forage, amaranth cultivars had higher (P < 0.01) calcium (3.2 vs. 14.5), phosphorus (2.4 vs. 5.2), and magnesium (2.1 vs. 4.7) concentrations. There were no differences in calcium, phosphorus and magnesium concentrations among the amaranth forages. In Table 3, results of fresh forages show that the corn forage showed lower levels of nitrate (0.9 vs. 2.5), total phenolics (3.6 vs. 8.9), and total tannins (2.2 vs. 4.6), but higher oxalate (8.2 vs. 6.6) than the mean amaranth values (P < 0.01). In terms of amaranth forage, Maria had the highest (P < 0.001) nitrate and total phenolics values. Table 1. Yields of the fresh forages and silages from corn and five amaranth varieties. Yield Amaranth varieties P-value Corn Maria A5 A12 A14 A28 SEM2 CA3 AM4 Fresh forage, t/ha 30.0 32.5c 29.2f 35.8a 34.3b 31.6d 2.19 0.001 0.002 Dry forage, t/ha 6.5 6.1a 4.4c 6.4a 5.3b 4.8b 0.47 0.002 0.002 Silage Dry silage, t/ha 6.50 6.10b 4.43e 6.35a 5.33c 4.80d 0.47 <0.001 0.002 CP, t of DM/ha 0.50 0.94a 0.64e 0.90b 0.76d 0.78c 0.032 0.005 0.007 MP,S t of DM/ha 0.28 0.60a 0.38e 0.57b 0.46d 0.46c 0.013 0.01 0.008 OM, t of DM/ha 5.10 4.94b 3.76e 5.23a 4.50c 4.06d 0.30 <0.001 0.003 OMD, t of DM/ha 3.28 2.57c 2.25e 3.22a 2.36d 2.16f 0.57 0.003 <0.001 1DM = dry matter; CP = crude protein estimated from total nitrogen content x 6.25; MP = metabolizable protein; OM = organic matter; OMD = organic matter disappearance calculated as 148.8 + (8.893 x GP24) + (0.448 x CP) + (0.651 x XA), (Menke et al., 1979). 2SEM = standard error of the means; 3CA = comparing corn vs. average value of amaranths; 4AM = comparing among amaranth varieties; a,b, c, d, e, f among amaranths = means in the same row with different superscripts differ (P < 0.05). Table 3. Anti-nutritional factors content (g/kg DM) of the fresh forages and silages of five amaranth varieties (Maria, A5, A12, A14, A28) and corn Item Fresh forage Ensiled forage Amaranth varieties P-value Amaranth varieties P-value Corn Maria A5 A12 A14 A28 SEM3 CA4 AM5 Corn Maria A5 A12 A14 A28 SEM3 CA4 AM5 Nitrate 0.9 3.2a 2.9b 2.6bc 2.2c 1.7d 0.24 <0.001 0.001 0.8 1.9a 1.6b 1.7a 1.7a 1.2c 0.19 <0.001 0.001 Oxalate 8.2 4.4c 7.3b 4.7c 9.3a 7.4b 0.31 0.004 <0.001 7.4 4.1c 7.0b 4.5c 8.9a 7.1b 0.42 0.006 <0.001 TP1 3.6 10.6a 8.9b 9.5b 7.7c 7.7c 0.31 <0.001 0.006 2.2 7.6a 6.4b 6.1c 4.8cd 4.1d 0.14 <0.001 <0.001 TT2 2.2 6.7a 4.5b 4.7b 3.4c 3.7c 0.19 <0.001 <0.001 1.2 4.2a 3.2b 2.8b 1.8c 1.7c 0.15 <0.001 <0.001 1TP = total phenolics; TT2 = total tannins; 3SEM = standard error of the means; 4CA = comparing corn vs. average value of amaranths; 5AM = comparing among amaranth varieties; a, b, c, d among amaranths = means in the same row with different superscripts differ (P < 0.05). According to Table 1, CS had higher (P < 0.01) yields of DM (6.5 vs. 5.4), OM (5.1 vs. 4.5), and OMD (3.28 vs. 2.5), but lower (P < 0.01) CP (0.5 vs. 0.8) and MP (0.28 vs. 0.5) yields in comparison with the mean values of AMS. Silage of A12 cultivar had the highest (P < 0.001) DM yield/ha, and A5 had the lowest among AMS (6.35 vs. 4.43). In terms of OM and OMD yields, A12 silage had the highest (P < 0.01) followed by Maria, A14, A28, and A5 having the least (P < 0.01). The A12 had the highest silage DM yield (6.35) whereas Maria had the highest CP yield (0.94) among all the AMS (P < 0.01). In comparison to CS, amaranth silages had lower (P < 0.001) overall mean levels of DM (165 vs. 217), NDFom (340 vs. 482), and NFC (245 vs. 344), but higher (P < 0.01) CP (149 vs. 77), ADL (61.7 vs. 47.6), WSC (12.6 vs. 11), EE (49.6 vs. 26.0), ash (219 vs. 71), calcium (14.9 vs. 3.3), phosphorus (5.2 vs. 2.5), and magnesium (4.8 vs. 2.3) levels (Table 2). Among the AMS, A12 and Maria had higher (P < 0.01) NDFom values than A14, A28 and A5. The A12 silage had the lowest (P = 0.002) ADL value. In amaranth silages, A28 had the lowest (P < 0.001) WSC value and A12 the highest (P < 0.01) (11.2 vs. 14.0). Maria silage showed the highest (P < 0.001) EE concentration and A28 had lowest ash level (P = 0.004) in comparison with the other cultivars. Among amaranth silages, A12 and A5 had the highest (P = 0.002) NFC values, followed by A14, Maria, and A28. There were no differences in calcium, phosphorus and magnesium concentrations among the ensiled amaranth. Table 3 shows the results of silages which illustrated that corn silage showed lower (P < 0.001) levels of nitrate (0.83 vs. 1.6), total phenolics (2.2 vs. 5.8), and total tannins (1.2 vs. 2.7), but higher (P = 0.006) oxalate (7.4 vs. 6.3) than the mean of AMS values. Among AMS, Maria, A12, and A14 had higher (P = 0.001) nitrate values in comparison to A5 and A28. Moreover, A14 had the highest (P < 0.001) oxalate content and A12 had the lowest. Silage Fermentation Characteristics The mean pH values and ammonia-N levels in AMS were higher (P < 0.001) than in CS, but CS had higher values (P < 0.001) of acetate and lactate (Table 4). Propionic acid levels showed no difference between CS and AMS (P = 0.25) and among ensiled amaranth (P = 0.08). Among AMS, Maria had the lowest (P = 0.002) butyric acid level and A28 the highest. In terms of AMS, A28 had higher (P = 0.007) pH and ammonia-N values but a lower (P < 0.001) lactic acid level. Maria and A12 had the highest acetic acid levels followed by A5 and A14 with A28 having the lowest (P < 0.001). Table 4. Silage fermentation characteristics of five amaranth varieties (Maria, A5, A12, A14, A28) and corn. Item Amaranth varieties P-value Corn Maria A5 A12 A14 A28 SEM1 CA2 AM3 pH 3.90 4.28d 4.78b 4.36c 4.60d 4.96a 0.03 <0.001 0.007 Ammonia-N, g/kg total N 42.3 53.5c 64.0b 57.0c 56.5c 66.6a 1.86 <0.001 0.002 Lactic acid, g/kg DM 70.2 62.1a 55.8b 60.1a 60.0a 51.0c 1.94 <0.001 <0.001 Volatile fatty acids Acetic acid, g/kg DM 23.3 19.4a 17.1b 18.9a 16.7b 15.9c 0.86 <0.001 <0.001 Propionic acid, g/kg DM 2.8 2.1 2.5 2.6 3.1 3.2 0.31 0.25 0.08 Butyric acid, g/kg DM 0.68 0.64c 0.92b 0.67c 0.90b 1.06a 0.09 0.05 0.002 1SEM = standard error of the means; 2CA = comparing corn vs. average value of amaranths; 3AM = comparing among amaranth varieties; a, b, c, d among amaranths = means in the same row with different superscripts differ (P < 0.05). In vitro Gas Production and Estimated Variables There was no difference between the in vitro ruminal pH of CS and that of the mean AMS value (Table 5) and neither was there among the AMS in vitro pHs. The ammonia-N value in CS was lower (P < 0.001) than the AMS mean value. The in vitro total VFA level, the molar proportion of acetate, and the acetate-to-propionate ratio were higher (P < 0.01) in CS than the AMS mean value, but CS had a lower (P < 0.001) in vitro butyric acid level. There was no difference in the in vitro isovaleric acid molar proportion between CS and the mean value from the amaranths. There were no differences in the in vitro ruminal proportions of acetic, propionic, butyric and isovaleric acids, or acetate to-propionate ratio among the AMS, but in vitro total VFA levels in A5, A14, and A28 were lower (P = 0.02). Table 5. In vitro ruminal fermentation characteristics, protozoa count (x 105/mL digesta) and cellulolytic bacteria enumeration (log10/mL digesta) of five amaranth varieties (Maria, A5, A12, A14, A28) and corn. Item Amaranth P-value Corn Maria A5 A12 A14 A28 SEM1 CA2 AM3 pH 6.70 6.67 6.71 6.76 6.80 6.82 0.13 0.19 0.11 Ammonia-N, mg/dL 10.7 22.1c 22.8b 23.7ab 24.3a 24.4a 0.39 <0.001 0.002 Total VFA4, mmol/L 60.9 58.1a 55.0b 57.6a 55.5b 55.2b 1.36 <0.001 0.02 VFA (mol/100 mol) Acetic acid 70.80 66.12 66.9 66.1 65.9 65.9 0.85 0.005 0.38 Propionic acid 21.0 22.9 22.6 22.5 221.0 22.6 0.32 0.45 0.19 Butyric acid 4.55 6.36 6.48 7.21 7.46 7.60 0.83 <0.001 0.12 Isovaleric acid 3.67 4.59 4.00 4.15 4.65 3.93 0.73 0.56 0.31 A:P5 3.37 2.90 2.91 2.93 2.99 3.03 0.25 0.009 0.37 Total protozoa 7.45 6.07 6.20 6.14 6.17 6.09 0.11 0.022 0.26 Isotrichidae 1.83 1.55 1.61 1.70 1.70 1.60 0.068 0.22 0.21 Entodiniinae 3.43 2.62 2.85 2.63 2.54 2.60 0.090 <0.001 0.18 Diplodiniinae 1.46 1.09 1.01 1.06 1.10 1.13 0.057 0.040 0.54 Ophryoscolecina 0.73 0.71 0.73 0.75 0.83 0.76 0.063 0.35 0.25 Cellulolytic bacteria 8.23 8.01 8.01 8.08 7.96 7.90 0.10 0.005 0.66 1SEM = standard error of the means; 2CA = comparing corn vs. average value of amaranths; 3AM = comparing among amaranth varieties; 4VFA = volatile fatty acids; 5A:P = Acetic acid: Propionic acid ratio; a, b, c among amaranths = means in the same row with different superscripts differ (P < 0.05). The in vitro ruminal total protozoa population of CS was higher (P = 0.022) than the mean value from AMS (Table 5). The cellulolytic bacteria numbers in CS were higher (P = 0.005) than the mean value from AMS. The in vitro 24-h CS incubation had higher (P < 0.001) GP24, OMD, ME, TDS, and CH4, but lower PF24 and MCP (Table 6) in comparison with the mean value from the AMS. Concerning the CS 96-h incubation, the 'b' value was higher and the 'c' value lower (P < 0.001) than the mean AMS value. The A12 cultivar had the highest (P < 0.01) in vitro GP24, OMD, ME, and TDS and the lowest (P = 0.001) PF24 of the AMS. The A28 cultivar had lower in vitro CH4 production, but there was no difference among other AMS. Table 6. In vitro ruminal gas production and estimated variables, truly degraded substrate and microbial protein of the corn and five amaranth varieties silages. Item1 Amaranth varieties P-value Corn Maria A5 A12 A14 A28 SEM2 CA3 AM4 24-h incubation GP, mL/200 mg DM 46.5 19.5c 27.3b 30.5a 18.7c 17.5c 0.81 <0.001 <0.001 OMD, g/kg 644 520c 598b 617a 524c 531c 2.89 <0.001 <0.001 ME, MJ/kg DM 9.13 6.41b 7.32a 7.72a 6.13b 6.27b 0.12 <0.001 0.004 TDS, g/kg DM 682 582a 580a 620a 487b 451c 3.43 <0.001 <0.001 PF24, g TDS/mL GP per g DM 2.93 5.97a 4.24c 4.06d 5.20b 5.15b 0.16 <0.001 0.001 MCP, g/kg DM 133 368a 279b 285b 281b 259c 3.53 <0.001 <0.001 CH4, % of total GP 22.8 20.5a 19.5a 20.5a 20.0a 18.0b 0.66 0.001 0.004 96-h incubation b, mL/200 mg DM 60.78 42.53b 44.58ab 46.81a 41.80b 38.34c 0.95 <0.001 <0.001 Lag time, h 0.059 0.066 0.067 0.063 0.068 0.067 0.010 0.34 0.70 c, /h 0.037 0.046b 0.044b 0.041c 0.049ab 0.052a 0.002 <0.001 <0.001 1GP24 = in vitro ruminal gas production at 24 h; OMD = estimated organic matter disappearance; ME = metabolizable energy; TDS = truly degraded substrate; PF = partitioning factor at 24 h of incubation; MCP = microbial crude protein; B = the asymptotic value of gas production; C = constant rate of gas production; 2SEM = standard error of the means; 3CA = comparing corn vs. average value of amaranths; 4AM = comparing among amaranths; a, b, c among amaranths = means in the same row with different superscripts differ (P < 0.05). In regard to the AMS in vitro 96-h incubation, the lowest 'b' value (P < 0.001) was recorded in A14 with A5 having the highest value. The highest 'c' value (P < 0.001) was recorded in A28 with the lowest value in A12. In situ Degradability of DM and CP The CS had more (P < 0.01) insoluble-degradable DM, 'c' and ED of DM, but less (P < 0.001) soluble DM compared to the AMS mean values (Table 7). Among the AMS, A12 had a higher (P < 0.001) 'b' fraction and ED of DM whereas A5 had the lowest 'b' and ED of DM. The highest and lowest (P < 0.001) values of 'c' among AMS were recorded in A28 and A12, respectively. Table 7. In situ degradability of dry matter and crude protein of the silages of corn and five amaranth varieties silages. Item1 Amaranth varieties P-value Corn Maria A5 A12 A14 A28 SEM2 CA3 AM4 DM degradation a, g/kg DM 344 351b 370a 368a 371a 357b 2.95 <0.001 <0.001 b, g/kg DM 479 439bc 427c 464a 444b 428c 2.76 <0.001 <0.001 c, /h 0.051 0.043b 0.044b 0.041b 0.043b 0.056a 0.004 <0.001 <0.001 ED, g/kg 607 554c 542d 586a 574b 578b 3.15 <0.001 <0.001 CP degradation a, g/kg CP 488 460b 476a 450c 466b 468b 2.38 <0.001 <0.001 b, g/kg CP 412 498a 460cd 468c 455d 482b 3.88 <0.001 <0.001 ADICP, g/kg CP 100 42c 64b 82a 79a 50c 1.23 <0.001 <0.001 c, /h 0.027 0.025c 0.031b 0.018d 0.027c 0.045a 0.003 <0.001 <0.001 ERDP, g/kg DM 48.0 99.9b 95.1b 82.0d 90.1c 115.0a 2.22 <0.001 <0.001 DUP, g/kg DM 12.09 30.87a 23.38bc 31.26a 24.74b 21.74c 1.75 <0.001 <0.001 MP, g/kg DM 42.83 94.83a 84.24b 83.75b 82.38b 95.62a 1.62 <0.001 <0.001 1a = soluble and very rapidly degradable fraction; b = insoluble but potentially fermentable fraction; c = fractional degradation rate of b; ED = effective degradability calculated for an outflow rate of 0.02/h; ADICP = Acid detergent insoluble crude protein; ERDP = effective rumen degradable protein; DUP = digestible undegradable protein; MP = metabolizable protein. 2SEM = standard error of the means; 3CA = comparing corn vs. average value of amaranths; 4AM = comparing among amaranths; a, b, c, d among amaranths = means in the same row with different superscripts differ (P < 0.05). In regard to CP degradability, CS had a higher (P < 0.001) level of 'a' and ADICP, but lower (P < 0.001) 'b', ERDP, DUP, and MP, compared to the AMS mean values. The A5 silage had the highest soluble CP fraction 'a' (P < 0.001). The Maria silage had the highest (P < 0.001) insoluble-degradable CP fraction. The highest level of ADICP was recorded in A12 and the lowest in Maria. Maximum and minimum (P < 0.001) concentrations of ADICP were obtained in A12 and Maria, respectively. The A28 silage had the highest (P < 0.01) ERDP and MP. The DUP of A12 and Maria silages were higher (P < 0.001) than the other AMS. DISCUSSION Yield, Chemical Analyses, and Antinutritional Factors Although corn forage and silage had higher DM, OM, and ME yields than the mean amaranth value, its yield was less than optimum, probably due to the late cultivation time (25th of August), when there is a decline in daylight hours. Studies have shown that declining exposure to sunlight reduces crop yield by weakening the photosynthesis process (Zhang et al., 2013). Amaranth can adapt to medium temperatures (Ofitserov, 2001), but lower exposure to sunlight is unfavorable to growth and subsequent yield (Modi, 2007). The A12 cultivar appeared to be better adapted to the climate conditions in this study. However, Abbasi et al. (2012) reported higher amaranth yields (up to 16.6 t DM/ha) compared to the current study, but levels of N fertilizer were higher (240 vs. 53 kg urea-N/ha) and the amaranth was planted a month earlier (warmer climate) and these factors would have contributed to a higher yield. Nevertheless, this study only measured yield from one year so further research is warranted. In many parts of the World, these forages are sown as second crops in the middle of summer after grain crops (barley or wheat) and harvested in the autumn. This leads to a low DM value (< 300 g/kg fresh weight) in both corn and amaranth because lower heat and sunlight levels in this season prevent optimum plant dry matter for silage (Nikkhah et al., 2011). A low forage DM increases the difficulty in making good quality silage (McDonald et al., 1991), which was seen when comparing AMS to CS. The A5 and A28 cultivars had the lowest DM and consequently produced the lowest quality silage (low lactic acid levels, high pH and ammonia-N). Late harvesting (last week of October) of AMS has resulted in insufficient heat and sunshine for obtaining optimum DM to enhance the ensilability characteristics (i.e., most AMS were ensiled with <250 g/kg DM). Excessive moisture interferes with the rapid establishment of lactic acid-producing bacteria and delays a rapidly needed decline in pH (McDonald et al., 1991), thus contributed to lower silage quality of AMS. High CP levels in amaranth silages (average 149 g/kg DM) and relatively low levels of NDFom (340 g/kg DM) and ADL (i.e., 61.7g/kg DM, comparable to high-quality alfalfa at 60 g/kg DM, MAFF, 1992) shows its suitability as a ruminant feed, as high level of the latter compound can reduce feed intake and digestibility (Van Soest, 1994). These different levels of CP and NDFom, WSC, and NFC in amaranth compared to corn may be partially accounted for by differences in genetics, metabolism, and carbon uptake per unit area (Edwards and Walker, 1983). The silage process reduces CP levels as plant and microbial proteolysis converts these compounds into NH3-N (McDonald et al., 1991), and this may affect the amaranth cultivars more because of their higher moisture content. Shadi et al. (2020) reported higher NDFom in AMS (i.e., 381 g/kg DM), which may be due to the growth stage at harvest (i.e.,Shadi et al. (2020) harvested at 93 days compared to 60 days in this study). The Maria and A12 silages had higher CP values than the other AMS which is likely a result of less dilution by ash in some varieties. Karimi Rahjerdi et al. (2015) recorded differences in chemistry among amaranth silage cultivars. The corn forage had the optimal WSC values (60-80 g/kg DM; Wang et al. (2018) for good quality silage whereas amaranth forages had WSC values below 60 g/kg DM leading to medium silage quality. Different amounts of residual WSC among AMS was an indication of differences in silage microbe fermentation (McDonald et al., 1991). The higher EE levels in AMS in comparison with forages were caused by changes in proportions of WSC, protein, and NDF. The nature of amaranth being a halophyte means that it has higher ash levels compared to corn (Edwards and Walker, 1983). Also, wilting amaranth may also increase ash concentration through soil contamination. High ash levels in the diet from amaranth can raise concerns about dietary energy and mineral imbalance. The CS Ca:P ratio at 2:1 is recommended by NRC (2005) for the prevention of the production of urinary calculi in sheep, whereas the AMS Ca:P ratio varied between 2.5:1 and 3.0:1, so an adjustment is needed if AMS is part of the diet. This ash level and high Ca:P ratio in AMS mean that it can be used to balance dietary Ca. Amaranth ash values in this study were similar to values recorded by Shadi et al. (2020); 182 to 215 g/kg DM) but higher than values recorded by Rezaei et al. (2014) and Karimi Rahjerdi et al. (2015), at 110 and 138 g/kg DM, respectively. These differences relate to cultivar, growth stage when harvested and local soil and climate conditions (Viglasky et al., 2009). Amaranth forage nitrate values were higher than those in corn but below values considered toxic to ruminants (i.e., < 6 g/kg DM; Radostits et al., 2007). A28 forage had higher CP and lower nitrate levels than other amaranths suggesting higher levels of photosynthesis and nitrate to protein conversion (Hopkins and Huner, 2008). Differences in nitrate levels among AMS (0.36) were smaller in comparison with their forages (1.5), as the silage fermentation process converts some nitrate to ammonia (Van Soest, 1994). Shadi et al. (2020) recorded lower nitrate values (i.e., 0.3-0.69 g/kg DM) at a 93 day harvest than in this study's 60-day harvest suggesting a lowering of nitrate with plant maturity (Abbasi et al., 2012). There was a difference in oxalate levels between corn and mean of the amaranths, and also among the amaranths, both in terms of forage and silage but the levels were all below 20 g/kg DM, the level considered to be toxic for ruminants (Rahman et al., 2013). Other studies have reported a wide variation of oxalic acid levels in amaranths (i.e., 2.0-114 g/kg DM; Teutonico and Knorr, 1985), relating to soil conditions, plant growth stage, and plant variety (Bressani, 1993). Bacterial degradation of oxalic acid in the silage fermentation process tends to reduce the levels (Rahman et al., 2021). Total phenolic and total tannin levels in amaranth and corn (forage and silage) were below 10 g/kg DM and had no detrimental effect (Benchaar et al., 2008) on in vitro fermentation. Shadi et al. (2020) reported similar levels of total phenolics (4.1-7.0 g/kg DM) and total tannins (1.7-4.9 g/kg DM). Differences in total phenolic levels among AMS (3.5) were higher than among the forages (2.9), indicating differences in silage pH, bacterial activity and extent of fermentation (Makkar and Singh, 1993). Silage Fermentation Characteristics The lower pH of CS in comparison with the AMS mean pH (Table 4) relates to the higher WSC level in the corn forage (Table 2) as WSC is converted into lactic acid (Kaiser et al., 2004). The amaranths produced medium quality silage in line with their levels of DM and WSC (Faithfull, 2002). The A28 silage had the highest pH which reflected it having the lowest WSC level in the forage and the lowest lactic acid and highest ammonia-N levels in the made silage. AMS in this study had higher pH values than those recorded by Rezaei (pH = 3.9) et al. (2014), but, compare well to the mean pH (4.6) recorded by Seguin et al. (2013) of amaranth silages harvested at different growth stages. Silage pH is affected by forage WSC level, forage buffering capacity and lactic acid and ammonia-N levels accumulated in the made silage (McDonald et al., 1991; Kaiser et al., 2004). Lower levels of ammonia-N in CS in comparison with AMS are due to the lower CP level and pH resulting in less protein being degraded in the fermentation process (Kaiser et al., 2004). The AMS were low DM silages and as such had relatively low ammonia-N levels (Chamberlain and Wilkinson, 2000) indicating reasonable quality. Ammonia-N levels in the AMS were higher than that recorded by Karimi Rahjerdi et al. (2015) (40 g/kg total N), but in line with those recorded by Rezaei et al. (2014, 2015) (60.2-69.3 g/kg total N, respectively). The AMS lactic acid levels were within the 55-63 g/kg DM range accepted as adequate for medium quality silage (ZoBell et al. 2004) and in line with levels recorded by Shadi et al. (2020). Rezaei et al. (2015) reported higher lactic acid concentration (69.1 g/kg DM), relating to higher WSC values in the forage (76.9 g/kg DM) that produced lactic acid from homolactic fermentation (McDonald et al., 1991). In CS, acetic acid levels were higher than the AMS mean value which indicates more heterolactic fermentation (Kaiser et al., 2004). These higher acetic acid concentrations in CS were also reported by Rezaei et al. (2014) and Shadi et al. (2020). The concentration of butyric acid was low in all silages and is an indication of minimal clostridial fermentation (Kaiser et al., 2004). Nitrate levels are negatively correlated with clostridia numbers (Spoelstra, 1983) as during fermentation nitrate is converted into nitrite and nitric oxide which are toxic to clostridia (Weissbach, 1996). In vitro Gas Production and Fermentation Variables Table 5 shows that both the CS and all the AMS have values of in vitro pH between 5.9 and 7 that are considered to be acceptable physiological levels (Wales et al. 2004), and all ammonia-N values were higher than that needed for optimum ruminal microbial growth (>50 mg/L) (Satter and Slyter, 1974). The ammonia-N levels in AMS were higher than those in CS, and CP levels were higher in AMS, likely contributing to this difference (Table 2). The CS had higher levels of VFA and acetic acid in comparison with the mean values of the AMS which is related to its higher levels of OM (Table 2) and TDS (Table 6) leading to an increase in microbial activity (Van Soest, 1994). The Maria and A12 silages had higher VFA levels than the other cultivars and also higher OM levels. The higher cellulolytic bacteria numbers in CS would explain the higher acetic acid levels in comparison with AMS as these bacteria produce acetic acid as an endpoint of OM fermentation (Newbold et al., 2015). The similar acetic acid levels among the AMS could be correlated to their similar cellulolytic bacteria numbers (Newbold et al., 2015). The higher protozoa numbers from CS in comparison with the mean value from AMS relate to its lower level of phenolics and the higher acetic acid level as there is a correlation between acetic acid level and protozoa population, due to the role of protozoa in the synthesis of VFA and feed degradation (Newbold et al., 2015). Results for all silage samples identify four genera of protozoa with Entodinium being the dominant genus (Newbold et al., 2015). The higher values of GP24, OMD, ME, and TDS from CS compared to AMS were similar to the results of Lotfi et al. (2022) and Shadi et al. (2020) and may be due to higher levels of NFC, cellulolytic bacteria and OM in CS (Van Soest, 1994). The higher mean PF24 value of AMS compared to CS could have been due to its higher ash level as ash does not add to GP (Makkar, 2010) The lower MCP of CS in comparison with AMS relates to its lower CP level and higher numbers of protozoa (Dehority, 2003). Of the AMS varieties, A12 had the highest in vitro GP24, OMD, ME, and TDS and the lowest ash level so in proportion had a higher level of fermentable substrate per unit of DM (Van Soest, 1994). Lotfi et al. (2022) and Shadi et al. (2020) reported a high GP24 (145 mL/g DM) from A5 as it had the highest OM level of the amaranth silages tested. The AMS, A12 had a PF24 in the range for conventional feedstuffs (2.75-4.41) (Makkar, 2010). The mean value of in vitro methane produced from AMS was lower than CS and that is related to the lower protozoa numbers which are likely a result of the lower hydrogen production related to different fermentation pathways producing less hydrogen in fermentation with AMS (Newbold et al., 2015). Also, the lower acetic acid levels in AMS correlate with a lower methane output (Newbold et al., 2015). Lotfi et al. (2022) and Shadi et al. (2020) recorded similar results. In situ Degradability of DM and CP In terms of DM degradability, the mean of fraction 'a' from the AMS was higher in comparison with CS may relate to its higher soluble and very rapidly CP degradable fraction (g/kg DM) in AMS compared to CS (Table 7), and this may have led to higher soluble and very rapidly DM degradable fraction in AMS compared to CS. In CS, the higher fraction 'b' relates to its higher level of NDFom as this is known to slowly degrade in the rumen (NRC, 2001). The higher ED of DM of CS compared to the mean AMS value relates to its higher NFC value. In terms of differences among the AMS, the lower 'a' fraction of Maria and A28 in comparison with A5, A12, and A14 relates to its lower NFC levels (Table 1). Whereas, A12 had a higher 'b' fraction and ED compared to the other AMS which may be due to its higher NDF level (Table 2). Another study where amaranths were grown in drier conditions recorded A5 having the highest ED of DM (632 g/kg DM) and A28 the lowest (509 g/kg DM) (Shadi et al., 2020). However, when Lotfi et al (2022) grew amaranth in hot humid conditions, the highest ED of DM was recorded for A14 (619 g/kg DM) with A5 having the lowest (593 g/kg DM). Karimi-Rahjerdi et al. (2015) recorded higher DM degradability (808-812 g/kg DM) for the two varieties, Kharkovskiy and Sem which may reflect differences in plant chemistry, climate, soil management and silage production methods (Van Soest, 1994). In terms of the degradability of CP, the higher fractions 'a', 'b', ED, ERDP, and DUP in AMS in comparison with CS may relate to their higher CP levels as this was reported by Lotfi et al., (2022) and Shadi et al. (2020). The AMS also had a higher DUP in comparison with CS, indicating that higher amounts of CP being digested post-rumen resulted in more efficiency in CP use (Van Soest, 1994). Among AMS, A28 had higher CP quality indices (ED, ERDP, and MP), which relates to its higher CP and low ADICP. However, synchronization between energy and protein in the rumen is needed to improve ERDP utilization (NRC, 2001). Maria silage had a lower ED of CP in comparison with A28, but its MP compared well to A28 as it had the highest OM and NFC and a high level of CP. The higher DUP of Maria in comparison with the other AMS, would likely cause more efficient CP utilization as more protein was digested post-ruminally (Van Soest, 1994). Differences in DUP in AMS relate to CP levels and increases in protein breakdown in the rumen (Do et al., 2013). This means that CP degradability and silage MP are affected by different silage components and they should all be taken into account in the results (McDonald et al., 1991). CONCLUSIONS The chemistry, particularly the CP and MP, and OMD of AMS suggests that this silage has potential as a feed for ruminants. The anti-nutrient levels in the tested varieties were below the toxic levels for ruminants. In terms of the forage yield, CP and MP, Maria and A28 had the highest levels but regarding OMD content, A5 and A12 had the highest levels. Overall, it can be concluded that A12 silage has the best balance of nutritive value and digestibility compared to other varieties. However, more in vivo study is required to confirm these nutritional characteristics. ACKNOWLEDGMENT The authors would like to thank the National Science Foundation (Tehran, Iran) (grant ID is 95842798) for the financial support given to carry out this trial, and gratefully acknowledge Mr. Gary Easton for his English language correction of the manuscript. Conflict of interest The authors declare that there were no conflicts of interest.

PMC10000120

Primary mucosal melanomas (MMs) are uncommon tumors originating from melanocytes located in the mucous membranes at various anatomic sites within the body. MM significantly differs from cutaneous melanoma (CM) regarding epidemiology, genetic profile, clinical presentation, and response to therapies. Despite these differences, that have important implications for both disease diagnosis and prognosis, MMs are usually treated in the same way as CM but exhibit a lower response rate to immunotherapy leading to a poorer survival rate. Furthermore, a high inter-patient variability can be observed in relation to therapeutic response. Recently, novel "omics" techniques have evidenced that MM lesions have different genomic, molecular, and metabolic landscapes as compared with CM lesions, thus explaining the heterogeneity of the response. Such specific molecular aspects might be useful to identify new biomarkers aimed at improving the diagnosis and selection of MM patients who could benefit from immunotherapy or targeted therapy. In this review, we have focused on relevant molecular and clinical advancements for the different MM subtypes in order to describe the updated knowledge relating to main diagnostic, clinical, and therapeutic implications as well as to provide hints on likely future directions. cancer mucosal melanomas diagnosis prognosis genetic metabolism immunotherapy Italian Ministry of Health--Ricerca Corrente2021-1309 This research was funded by Italian Ministry of Health--Ricerca Corrente (ndeg 2021-1309). pmc1. Introduction Primary Mucosal Melanoma (MM) is a rare malignancy originating from melanocytes located in the mucosae at various anatomic sites within the body . Estimation of cancer incidence in United States for 2023 indicates that melanomas will represent 5% of new cancer cases, of which MM will constitute <=2% diagnoses . Melanocytes' primary role is the synthesis of the melanin pigment that, in the skin, is transferred to keratinocytes and serves as a shield to protect the human body surface from sunlight UV-radiation. Despite the fact that these pigmented cells are markedly distributed into the skin and the vast majority of melanomas are of cutaneous origin , they can also be found in other tissues. Originating from pluripotent embryonic cells (namely, the neural crest cells), melanocytes have the property to migrate not only under the epidermis to populate all parts of the skin but also within the basal cell layer of the epithelium at different anatomic districts. Melanocytes are mainly present in the inner ear, nervous system, and heart; in addition, generally they can be found in nearly all tissues to a differing degree . Melanin-containing melanocytes are present in the basal cell layer of the epithelium even at mucosal sites with no visible signs of melanin pigmentation . At the oral level, melanocytes may or may not produce melanin and, analogously to what happens into the skin, the amount of synthetized melanin is constitutionally determined . As is commonly known, there are substantial variations in rates of melanin pigmentation among people from different racial/ethnic groups as well as among those belonging to the same racial/ethnic group . Furthermore, when considering the oral site, melanin pigmentation is common in black people , being even more frequent in darker skinned whites (Caucasians) than in lighter skinned whites . Additionally, melanin distribution into the oral mucosae may be heterogeneous, with the pigmentation being patchy or uniform and more frequently affecting the gingiva . Smaller amounts of melanin-containing melanocytes can be also found in other distinct mucosal membranes, determining that melanomas can arise in any site where such cells are present: the respiratory, gastrointestinal, and urogenital tracts as well as in the eye . MM significantly differs from cutaneous melanoma (CM) regarding epidemiology, genetic profile, clinical presentation, and response to therapies . Despite these differences that have important implications for disease management, the MMs are usually treated in the same way as CMs . However, MM patients exhibit a lower response rate to immunotherapy than CM patients, with a poorer survival rate . Furthermore, a high inter-patient variability can be observed in relation to treatment response. Recently, novel omics' techniques have revealed that MMs have a different molecular landscape and genomic profiling as compared with CM that can explain their different response . Such specific molecular aspects are useful for identifying new biomarkers for improving diagnosis as well as the selection of MM patient candidates to achieve the best benefit from immunotherapy or targeted therapy. Here, we will we provide an updated overview of the relevant molecular and clinical advancements on MM subtypes in order to describe the state of the art on major diagnostic, clinical, and therapeutic implications as well as likely future directions. 2. Mucosal Melanomas 2.1. Epidemiology MMs are very rare tumors representing less than 2% of all melanomas , with about 850 new cases reported in Europe in 2013 . Unlike CM, whose incidence is increasing, the MM incidence is estimated to remain stable . Usually, MMs are diagnosed in people aged over 65 years, with a rate of 6.3 per million, and they are more frequent in women than men . MMs occur with about equal frequency in all races, therefore accounting of a substantial fraction of all melanomas diagnosed in black people, Asian people, and Hispanic people when compared with melanomas occurring amongst Caucasian people . This reflects the incidence of CM in these populations . Defined as melanomas occurring in mucosal membranes at various anatomic sites, these lesions are most frequently observed in the head and neck regions (approximately 50% of cases) followed by gastrointestinal (37%), female genital (6%), and urinary tracts (4%) rather than other locations such as pharynx, larynx, urinary tract, cervix, esophagus, and gallbladder . Furthermore, lung site involvement is mostly secondary . 2.2. Risk Factors Unlike CM, where association with UV sunlight exposure represents a well-defined risk factor, genomic studies have suggested that UV light plays a nearly null role in MM carcinogenesis . In fact, MMs develop on sun-shielded surfaces. Risk factors for this rare melanoma subtype remain poorly understood. Currently, there is no clear evidence to support the pathogenic role of chemical carcinogens or viruses despite their suggestion as etiologic factors . Interestingly, some authors have reported that oral melanosis, which precede about one third of MMs in the oral cavity, may be associated with cigarette smoking . 2.3. Molecular Features Globally, MMs are characterized by the presence of distinct molecular features, frequently characterized by DNA structural changes and mutation signatures of unknown etiology, with respect to CM . MMs are characterized by a relatively low tumor mutational burden (TMB) compared to CM . In particular, MM has an average of about 8000 non-synonymous single nucleotide variants (nsSNVs) per tumor, a more than 10-fold lower mutation rate than CM (which, with an average of more than 80,000 nsSNVs, exhibits one of the highest somatic neoplastic mutation rates) . This difference may explain the molecular reason for discordant clinical responses to immunotherapy that are observed in Asian people, where MM represents a substantial fraction of all diagnosed melanomas, versus white Caucasian people . From the qualitative point of view, mutational profiling of MM as inferred by whole genome/exome sequencing approaches is different from that observed in CM . According to recent NGS-based studies as well as meta-analysis and systematic review, MMs are characterized by a lower load of somatic mutations and lack of either UV-induced mutational signatures or oncogenic fusion gene transcripts frequently reported in CM . In Figure 1, the main genes and their functional pathways involved in MM pathogenesis are represented. Overall, the genes mostly mutated (>10%) are neurofibromin 1 (NF1), KIT proto-oncogene receptor tyrosine kinase (KIT), and splicing factor 3b subunit 1 (SF3B1); the remaining genes commonly mutated but at lower rates (5% to 10%) are neuroblastoma RAS viral oncogene homolog (NRAS), sprout-related EVH1 domain containing protein 1 (SPRED1), ATP dependent helicase (ATRX), and B-Raf proto-oncogene (BRAF) . The MITF gene, involved in melanocyte development and differentiation, has been found to either rarely carry the germline variant E318K--widely demonstrated to confer an increased CM risk--in vulvar melanoma either to be commonly amplified in several MMs . Despite the fact that the BRAF and NRAS genes are preponderantly mutated in CM lesions (together accounting for about 75% of cases), their role in MM pathogenesis is not secondary. Activating mutations of the BRAF oncogene are detected in approximately 50% of CM , but at much lower frequency in MMs . Considering NGS-based data, the frequency of mutations for BRAF and NRAS oncogenes has been reported at even higher rates (up to more than 20%) in different types of MM, with the most prevalent incidence in the mucosae of the head and neck district . In sinonasal MM, a high prevalence of NRAS mutations has been reported ; however, a lack of KIT alterations with a high prevalence of BRAF mutations has been reported in some populations, such as those from South Italy or from Germany , but not in the majority of the others (as those from Japan, Poland, Spain, and the United States ), further suggesting that patients' origin may account for different mutation rates in candidate cancer genes. In some MM cases with a lower rate of BRAF mutations, an increased activity of the Cyclin D1-Cyclin-dependent kinase 4/6 (CCDN1-CDK4/6) complex, mostly through gene amplification, has been demonstrated as contributing to MM pathogenesis . Finally, in BRAF/NRAS-wild-type MMs, a higher rate of mutation into the Telomerase Reverse Transcriptase (TERT) promoter has been described, specifically for sinonasal melanomas . Not having UV radiation as the most preponderant carcinogenic effect--usually occurring in the CM etiology--the pathogenic sequence variations found in the BRAF and NRAS genes appear to be more heterogeneous in MMs . In fact, a markedly high prevalence of mutations at the V600 codon of BRAF (nearly all BRAF-mutated CM lesions present V600E/K variants, with particular reference to melanomas arising on skin areas intermittently exposed to UV) is lacking among MM cases; conversely, sequence variations mostly affect other regions of the BRAF gene than codon 600, though most of them in codons of the exons 11 and 15 are deputed to encode the protein domains where the kinase activity resides (among others, codons D594, G469, and K601 are frequently altered) . This situation closely resembles what occurs in the adenocarcinoma subtype in the non-small cell lung cancer (NSCLC), in which the V600E mutations account for a fraction of BRAF-mutated NSCLC cases (about 40%, mainly females, and never-smoker patients), whereas the majority of them (about two thirds, regardless of the smoking habitus) carry non-V600 mutations . Similarly, a poor prevalence of mutations into the Q61 codon of the NRAS exon 3, which is found in nearly all RAS-mutated CM cases, has been observed in MM; conversely, the G12 and G13 codons within the exon 2 of the gene are maximally involved in NRAS-mutated MM cases . The latter codons are also prevalently mutated in the KRAS gene among lung and colorectal adenocarcinomas . Such evidence strongly supports the hypothesis that causative factors promoting mutations in BRAF and NRAS genes in MM can be more similar to those involved in the onset and progression of lung and colorectal carcinomas than to those involved in cutaneous melanocytic transformation. Unfortunately, such causative factors remain to be determined. As already depicted above, the anatomical sites where MMs originate deeply affect the spectrum and distribution of the mutations in driver genes . Considering the main one, head and neck melanomas present a lower prevalence of KIT mutations as compared to the genitourinary tract and anorectal melanomas in which mutations and amplifications of this gene are common; among genital MMs, vaginal but not vulvar melanomas present very low rates of KIT mutations . Furthermore, the frequency of different mutations may vary across different studies and reports on patients of different geographical origin and different genetically driven constitutional features. For other genes, a different anatomical distribution of alterations has also been reported: ATRX and SF3B1 mutations occurred more commonly in lower anatomy MMs and TERT or CTNNB1 in the upper anatomy . Overall, MMs show specific genetic signatures depending on the site of origin. A higher percentage of mutations involving the SF3B1 gene was observed in patients with MM of anorectal origin. In patients with vulvovaginal melanoma, the involvement of the TP53 gene is common, while mutations involving the MYC gene are frequently detected in patients with sinus or nasopharyngeal melanoma . In summary, the mitogen-activated protein kinase (MAPK) pathway--including the RAS-RAF-MEK-ERK signal transduction cascade--regulates the main processes of cell proliferation and cell survival in both MM and CM lesions. However, while in CM the MAPK cascade is directly activated by a constitutive alteration of one of its component (BRAF or NRAS), in MM the oncogenic MAPK activation is indirectly promoted by effectors external to the pathway (KIT and/or NF1) . In particular, KIT encodes a tyrosine kinase receptor of the cell membrane and its activation through mutation and/or amplification result in a continuous RAS-driven induction of cell proliferation, whereas NF1 encodes for a negative regulator of RAS signaling and its genetic or functional silencing results in RAS activation and enhancement of the malignant transformation and progression . Inactivation or functional loss of NF1 may also cooperate with other so-called RASopathy genes--such as SPRED1, which is involved in melanoma genesis --in sustaining constitutive RAS activation . Tumors with mutations in NF1 and constitutive activation of RASopathy genes are expected to be associated with a higher mutational load and, consequently, a greater probability of generating neoantigens . Unfortunately, MMs are poorly immunogenic, regardless of their intracellular mutational status, probably due to their lack of ability to induce a strong adaptive immune response in the tumor microenvironment; the reduced susceptibility to PD-1/PD-L1 and/or CTLA-4 blockade is highlighted by the extremely poor clinical outcomes of patients with advanced MM using immune checkpoint inhibitors . 2.4. Clinical Behavior Globally, MMs are characterized by an aggressive clinical behavior and a prognosis worse than CM . Early diagnosis of the disease can be hindered by both its rarity and its mostly occult anatomical origin site; a possible multifocality; and/or the fact that about half of MMs are amelanotic may contribute to delaying diagnosis . MMs patients are most likely to be diagnosed with regional or distant metastases, with a reported 5-year overall survival rate of approximately 25% . In the large series reported by Lian et al. , predominant metastatic sites included regional lymph nodes (21.5%), lung (21%), liver (18.5%), and distant nodes (9%). Interestingly, when matched for staging, prognostic, and molecular factors, no significant survival difference among MMs arising at different sites was found by Cui et al. in a series of 706 patients . 3. Clinical Presentation MM clinical presentation is extremely variable. The site of origin and the size of the tumor, along with the possible metastatic involvement of other locations within the human body, can clearly determine different clinical scenarios. 3.1. Head and Neck Although almost all malignant melanomas of the head and neck district are constituted by cutaneous lesions, mostly arising on the face skin, this region represents the most common site of MM origin and it accounts for more than half of all diagnosed MMs . MMs may involve the nose and paranasal sinuses, oral cavity, pharynx, larynx, and esophagus . The nose and paranasal sinuses followed by the oral cavity are the most common sites of origin , whereas it is uncommon to find it in the primitive mucosa of the larynx, pharynx, esophagus, or tracheobronchial tree . MMs arising from the respiratory mucosa (such as the nasal cavity) have different clinicopathologic characteristics than those involving the oral mucosa, though they are characterized by similar adverse prognosis and outcome . Within the nasal cavity, the origin of the disease from either the lateral nasal wall or nasal septum is preferential, whereas, considering the sinuses, the maxillary sinus, followed by the ethmoid, the frontal, and the sphenoid sinuses, these represent the anatomic privileged sites of its origin . Typically, most of patients developing a MM in the nasal cavity presents a clinically localized disease and tend to exhibit a more favorable prognosis than those affected by MMs of paranasal sinuses or of other head and neck sites, which are often diagnosed at a more advanced disease stage with subsequent poorer outcome . Sinus melanomas are asymptomatic in this anatomically silent area until the neoplastic growth invasion of adjacent structures is evident. In a pooled data analysis of approximately 200 patients from five series, the five-year survival for patients with nasal melanoma was 31% compared to approximately 0% for those with sinus melanoma . Sinonasal MM commonly metastasizes to the lung and liver. At the time of the diagnosis, nodal secondary involvement by the tumor is present in about 20% of patients . Clinical symptoms--such as epistaxis, unilateral nasal obstruction, or persistent rhinorrhea, loss of smell, and facial pain--are nonspecific, often indistinguishable from those of benign sinonasal disease; therefore, early diagnosis can be delayed in several cases . Moreover, proptosis, diplopia, or neurological symptoms are symptomatic of a more advanced disease stage . Most of such MMs is likely to show features endoscopically as polypoid, fleshy lesions with strict unilateral involvement with different degrees of pigmentation while they are rarely amelanotic . Oral cavity MMs represent almost 30% of MMs of the head and neck, mostly involving the palate and maxillary gingiva . Again, they are asymptomatic in the early stages, usually presenting with a pigmented patch or a mass characterized by a rapid growth rate . Diagnosis may be easier due to greater accessibility of the oral cavity for clinical examination. Those lesions can be amelanocitic or characterized by a wide range of colors varying from black, brown, grey, to reddish or white . At the time of the diagnosis, the probability of detecting distant metastases is low (5-10%) with no significant difference between oral and sinonasal MMs; however, the incidence of nodal metastases at this level is highest than in sinonasal lesions, especially in presence of a depth of infiltration > 5 mm . 3.2. Gastrointestinal MMs Approximately, 37% of MMs arise from the gastrointestinal tract, which represents the second most privileged site of origin for those malignancies . Their preferred location is the anorectal tract (>50%), followed by the stomach, small intestine, and colon . However, anorectal MMs are very rare accounting for approximately 0.5% of all colorectal and anal cancers . MM rectal origin is more frequent than anal primary involvement and notably, given their infrequent occurrence, those tumors are studied together as a unique entity . They are most frequent in women aged over 50 years and are characterized by a very poor prognosis . It is also interesting to report that there is an unclear association between HIV infection and anorectal MM, though we think it noteworthy to take this information into account in the management of such patients . Because of the hidden location and the lack of specific symptoms, patients are likely to present with disseminated disease . Typical, early symptoms such as itching or rectal pain are often mistakenly attributed to a benign pathology, such as hemorrhoids, polyps, or anal fissures, and so the diagnosis is delayed many times. Moreover, changes in bowel habits, bowel obstruction, rectal bleeding, anal pain, and/or rectal tenesmus occur in a more advanced disease stage. 3.3. Genitourinary MMs MM arising from the genitourinary (GU) tract is an extremely rare disease, characterized by a clinically aggressive behavior, that comprises 0.2-1% of all melanoma cases . GU MMs include tumors arising from the female GU tract (vulva, vagina, and cervix), male GU tract (penis and scrotum), and urinary tract (urethral and bladder) . In a series of 817 primary GU melanoma cases from the Surveillance, Epidemiology, and End Results (SEER) database, the female GU tract was the most commonly involved site (89.4%), followed by the male GU tract (6.6%), and the urinary tract (4.3%) . Most cases of GU MMs occurred in the vulva, with the highest age-specific incidence rates in patients aged 85 years and older for both women and men . The labia minora followed by the labia majora and clitoris represent the most common sites of disease occurrence, whereas urethra and cervix locations are less frequently involved . Symptoms of vulvovaginal melanoma include itching, vaginal discharge, bleeding, dyspareunia, mass as well as occasional dysuria and voiding dysfunction if urethral involvement occurs . Vulvovaginal melanomas present frequently with regional lymph node or distant metastatic disease, probably due to the richly innervated lymphatic system that facilitates nodal spreading; again, their prognosis is very poor . GU melanoma usually presents in males as a pigmented macule, papule, plaque, or an irregularly demarcated, ulcerated lesion on the glans penis at an early disease stage. Moreover, patients may present obstructive symptoms, hematuria, urethral discharge, and urinary fistula in more advanced disease stages . Prognosis is also very poor. Hematuria, complaints of a protruding mass, obstructive urinary symptoms, including a weak urinary stream, urethral discharge, flank pain, and hydronephrosis are symptoms related to urinary tract MMs . 4. Diagnosis MMs presenting ''ABCDE'' features can be clinically recognized in visible regions such as the vulva, the penis, and the oral cavity . However, it is important to take into account that amelanocitic lesions may be difficult to diagnose. A careful clinical examination as well as palpation for the detection of suspicious regional or distant lymphadenopathies along with a visual inspection for detection of occult mass must be carried out in such patients. Particular attention should be paid to cutaneous examination and careful exploration of visible mucosae due to the possibility of metastatic CM. Specialist consultations by an otolaryngologist, urologist, gynecologist, and gastroenterologist are often necessary for MM diagnosis because of the necessity to perform biopsies of suspicious lesions. Pathology examination of biopsy specimen represents the gold standard for diagnosis. Macroscopically pigmented lesions are highly suspect for melanoma. If pigment is absent, then diagnosis may be less simple. Immunohistochemical staining positive for protein S-100, HMB-45, Melan-A, Mart-1, and tyrosinase support the diagnosis of melanoma . 5. Staging Once diagnosis of malignancy is performed an accurate staging of the disease is mandatory in order to obtain prognostic information and to determine the further management of the patient. Usually, laboratory analyses along with instrumental tests including endoscopic studies of the head and neck region or gastrointestinal tracts, a total body computed tomography (CT) scan, positron emission tomography (PET), ultrasonography of various areas within the human body, as well as magnetic resonance imaging (MRI) are useful to complete the diagnostic path and to detect putative loco-regional and systemic extension of the disease. However, no consensus on staging MMs from various anatomic sites exists . Due to the rarity of disease, the adequacy of staging criteria for providing prognostic information is not clear and many oncologists use different systems to stage MM . Globally, MMs of the aero-digestive tract are staged in Italy according to AJCC staging (8deg ed) and vulvar melanomas are staged following the AJCC staging criteria for CM; for the urethral, vaginal, rectal, and anal MMs, there is no consensus for staging . Some clinicians prefer to utilize a simple and practice system for staging MM patients, which was proposed by Ballantine for Head and Neck MMs in 1970 . This system is characterized by three stages, depending on disease extension: (I) local, (II) regional (nodal metastases), or (III) disseminated. 6. Management 6.1. Surgery Surgery represents the mainstay for treatment of localized disease. Complete tumor removal with the pathological finding of clear margins seems to be associated with better outcomes , though with no clear benefit on overall survival (OS) . Despite aggressive surgery, the occurrence of local or distant recurrences are very common with the majority of patients ultimately dying of disseminated disease. Factors associated with local recurrence include tumor size, vascular invasion, and not radical tumor resections. Furthermore, the surgical strategy may be challenging and it should be tailored individually taking into account the tumor stage or its anatomic site, because of its potential morbidity. Overall, the poor prognosis related to the disease induces the consideration of the residual patient quality of life in any clinical decision-making in order to avoid unnecessary and harmful surgical efforts. For instance, endoscopic resections can be offered to selected patients with lower morbidity and similar local control . 6.2. Radiotherapy Radiotherapy may be helpful for increasing local control and reducing the rates of local failure, especially when surgery achieves no negative margins or is aimed at obtaining conservative surgical management of the disease such as in anorectal MMs . It is also a treatment worth considering for bulky mass or symptomatic lesions with palliative scope. 6.3. Systemic Therapy Before 2010, the prognosis of patients affected by advanced CM was very poor irrespective of melanoma subtype, as few effective systemic therapies were available. In their series, Kuck et al. reported a median survival ranging from 10 to 13 months in patients affected by metastatic melanoma from cutaneous, acral, uveal and unknown origin . In the same report, patients affected by MM exhibited a poorer outcome with a median survival of approximately 9 months . Whilst the introduction of new systemic treatment options for the management of patients affected by CM, such as immunotherapies (anti-CTLA-4 and anti-PD-1 antibodies ) or targeted therapies (BRAF, MEK inhibitors ) either alone or in combination, represented a significant breakthrough in clinical practice, the development of systemic therapies for MMs has been much slower . Actually, 3 immune checkpoint inhibitors (ICIs) have been approved for treatment advanced melanoma patients: ipilimumab, a monoclonal antibody against cytotoxic T-lymphocyte-associated protein 4 (CTLA-4); the anti-programmed cell death receptor 1 (PD-1) agents pembrolizumab and nivolumab; as well as the combination of ipilimumab plus nivolumab . Recently, based on results of the phase 2/3 Relativity-047 randomized trial , a new drug that combines nivolumab with relatlimab, opdualag, has been approved in an attempt to block both the immune checkpoint protein PD-1 and LAG-3. Across different trials, PD-1 blockade with either nivolumab or pembrolizumab resulted in response rates ranging from 26% to 44% when used as single agents and significantly improved OS when compared with ipilimumab and dacarbazine . Furthermore, a durable, sustained survival benefit can be achieved with first-line nivolumab plus ipilimumab or nivolumab alone . However, CM and MMs are molecularly different tumors and most of these studies have been performed on patients affected by CM, limiting data extrapolation to MMs because of their rarity. It was also suggested that the efficacy outcome of ICIs was poorer in MM than CM patients, but no formal perspective comparisons, to the best of our knowledge, were made between such melanoma subtypes . Moreover, due to the rarity of MMs, knowledge about disease treatment with ICIs are limited, mostly deriving from small population studies, retrospective analyses, and case reports . A multicenter retrospective study comparing immunotherapy (anti-PD-1 and anti-CTLA-4) versus chemotherapy (mostly dacarbazine) showed a longer median OS for patients affected by advanced MM who received ICIs (approximately 16 months) respect to those treated with chemotherapy (8.8 months) . Furthermore, Hamid et al. reported an overall response rate (ORR) of 22% and 15% with a median progression-free survival (PFS) of approximately 2.8 months (in ipilimumab-naive or ipilimumab pre-treated patients, respectively) in a post-hoc analysis of pembrolizumab of KEYNOTE-001, -002, and -006 trials . A large report of the efficacy and safety of an ICI in MM was reported by D'Angelo et al. in their pooled analysis, where the combination regimen nivolumab plus ipilimumab was compared with either nivolumab or ipilimumab alone . This combo immunotherapy achieved a better ORR (37.1%) as compared with both single agents (23.3% and 8.3%, respectively), but showed a higher toxicity. Despite this positive result, ORR by this combination was found to be markedly inferior as compared with the ORR of 60% observed in CM patients. Furthermore, the same combo achieved 43% of ORR with a PFS of 5.8 months in a naive subgroup of MM patients from Check-Mate 067 as compared with nivolumab (ORR 30% and PFS 3 months) and ipilimumab (ORR 7% and PFS 2.6 months) . The combo ipilimumab-nivolumab regimen appears to be a rational systemic approach for treating fit patients affected by advanced MM, though this type of treatment is burdened by important grade 3-4 toxicities (approximately 55%) . Alternatively, a less toxic monotherapy with anti PDl-1 agents, such as nivolumab or pembrolizumab, may be considered for unfit patients in order to implement their treatment compliance. A retrospective cohort study evaluated treatment efficacy of anti-PD-1 +- ipilimumab in 545 MM patients. Three hundred and forty-eight (64%) patients received anti-PD-1 whereas the remaining 197 (36%) received the anti-PD-1/ipilimumab combination. The ORR, PFS, and OS did not significantly differ by primary site (naso-oral, urogenital, anorectal, other), ethnicity/race, and treatment. Only the ORR for naso-oral primary site was numerically higher for the anti-PD-1/ipilimumab combination compared with anti-PD-1 without survival benefit. These data suggest that in MMs, the addition of ipilimumab has a poor clinical benefit over anti-PD-1 . In phase 2/3 Relativity-047, 51 (7.1%) of the 714 melanoma patients were MM patients. The subgroup analysis comparing 23 cases undergoing the relatlimab-nivolumab combo toward the 28 cases undergoing nivolumab alone showed no significant differences in PFS. However, the small sample size and the rarity of MMs show that additional studies are needed to understand the efficacy of the relatlimab-nivolumab combo in patient populations that are often excluded from clinical trials . Overall, it is not clear why the response rates observed to ICIs are lower in MMs with respect to CM. It has been hypothesized that this may be a result of the lower proportion of MM patients carrying high levels of PD-L1 expression (>5%), which was found in the pooled analyses, although no evidence fully supports the role of PD-L1 in predicting response to ICIs in such patients . On the other hand, the lower MM mutation burden as compared to that observed in CM lesions may justify this clinical observation, whereas the presence of tumor-infiltrating lymphocytes (TILs) could be a predictor of anti PD-L1 response . Recently, a phase II trial aimed to compare toripalimab versus high-dose interferon-a2b (HDI) as an adjuvant therapy for resected MM. A total of 145 patients with resected MM were randomized (1:1) to receive HDI (n = 72) or toripalimab (n = 73) for 1 year. Toripalimab showed a similar relapse-free survival and a more favorable safety profile than HDI, suggesting that toripalimab might become the better treatment option . To date, limited data regarding the efficacy of neoadjuvant checkpoint inhibition for resectable MMs are available. However, the Ho et al. study, despite the small sample size (n = 36) demonstrated that the neoadjuvant +/- anti-CTLA4 immunotherapy can be a feasible approach. Indeed, median event-free survival (EFS) was 9.2 months, ORR was 47%, PRR was 35%, whereas the 3-year OS rate was 55%. Interestingly, the pathologic response achievement was associated with a significant improvement in OS and EFS suggesting the need for further investigation . Unlike CM, BRAF mutations are infrequent in MMs and therefore treatment with BRAF kinase inhibitors (i.e., dabrafenib or vemurafenib), mostly in combination with a MEK inhibitor (i.e., trametinib), rarely represents a therapeutic option for these patients . However, all MM patients should be tested for BRAF mutations and the treatment with the combination of a BRAF inhibitor and a MEK inhibitor may be considered when activating BRAF mutations are detected . Treatment with BRAF inhibitors achieved a 20% of ORR and 70% disease control rate (DCR) in a small cohort of 12 MM patients with metastatic or unresectable BRAF V600E-mutant melanoma . MMs may present activating mutations in KIT. These mutations, mainly detected in exons 11 and 13, are most common in vulvovaginal and anorectal disease sites . Tyrosine kinase inhibitors, such as imatinib or nilotinib, targeting the aberrant protein gene product were found to be able to induce a clinical response. In a multicenter phase 2 trial involving 17 MM patients harboring mutationally activated or amplified KIT, Hody et al. reported a 64% of ORR among those with KIT mutations (in exon 11, 13, and 17) treated with imatinib mesylate, whereas the drug was ineffective in patients only presenting KIT amplifications. Furthermore, nilotinib exhibited similar responses as imatinib, demonstrating a clinical effect in circumstances of disease progression after imatinib . Interestingly, new strategies for the systemic treatment of MMs have been recently reported . These systemic approaches were developed considering the important role played by the vascular endothelial growth factor (VEGF), which is strongly expressed in MMs, on either disease progression or immunosuppression . Sheng et al. investigated a combination of a VEGF inhibition (axitinib) with PD-1 blockade (toripalimab) in a phase Ib trial of 33 patients suffering from metastatic MM. Among 29 treatment naive patients, an ORR of 48.3% with a disease control rate (DCR) of 86.2% was reported. Median PFS was 7.5 months while median OS was 20.7 months. In their report, PD-L1 expression or TMB value presented no significant differences in responder versus non-responder patients, whereas gene expression profile (GEP) scores of eight selected immune-related and four angiogenesis-related genes, showed a strong correlation with clinical response . Notably, 39.4% of patients experienced Grade 3-4 toxicities but only one patient discontinued treatment. Most common side effects were diarrhea, proteinuria, hand-foot syndrome, and hypothyroidism . On the other hand, Yan et al. conducted a multicenter 2:1 randomized, open-label, phase II study involving 114 naive advanced MM patients receiving chemotherapy (carboplatin AUC 5 plus paclitaxel 175 mg/m2 every 4 weeks) with or without VEGF inhibition (bevacizumab 5 mg/kg every 2 weeks). The primary end point of the trial was PFS while OS was a key secondary end point . The combination of carboplatin plus paclitaxel and bevacizumab was found to be more active than carboplatin plus paclitaxel. A combination with the antiangiogenic drug bevacizumab significantly increased median PFS from 3.0 to 4.8 months and median OS from 9.0 to 13.6 months . Treatment appeared to be well tolerated and no significant safety signals (including no gastrointestinal perforation or hemorrhage) were reported . This regimen could represent an alternative for the treatment of advanced MMs in an immunotherapy-ineligible or immunotherapy-failure population and further studies are warranted as suggested by the authors . 6.4. Future Therapeutic Perspectives The different sites of origin associated with a remarkable genetic heterogeneity make MM a straightforward model to evaluate the cell biochemical changes that enhance tumor spreading. Indeed, mucosal melanoma cell lines capable of maintaining their heterogeneity following long-term cell culture and allowed to establish as two different metabolic profiles, the first characterized by the production and excretion of lactate in the extracellular microenvironment while the second, oxidative type was characterized by the uptake of lactate from the microenvironment and its use as primary carbon source, showed different phenotypic profiles in promoting cancer metastasis spreading . Thus, a deep knowledge of metabolic reprogramming processes may highlight potential targets useful in interfering with the dynamic cancer progression. Another targetable metabolic pathway for MM is represented by the degradation of tryptophan to kynurenine, which plays a key role in regulating the immune response. In particular, it has been found that in tumor tissue an increase in the enzyme indoleamine 2,3-dioxygenase (IDO) that regulates the degradation of tryptophan is able to promote the development of an immunosuppressive microenvironment that can inhibit effective antitumor immunological responses . To date, it is unknown whether in MM the IDO activity changes reflect or are able to modify the metabolism of the host, leading to serum variations in the concentration of tryptophan and kynurenine. Interestingly, it has been observed that the surgical treatment of non-cutaneous melanoma is able to profoundly change the metabolic profile of the host. The removal of a primary melanoma of the lung re-established the level of 5-S-Cysteinyldopa within the normal range; thus, indicating how melanoma is able to condition not only the microenvironment surrounding the tumor but also to create important metabolic changes in different body matrices . Considering that the metabolic profile tended to change according to the recurrence of the disease and to the treatment, the achievement of a homeostatic balance similar to normal range or its further imbalance could be considered as two different trajectories associated to different prognoses. The cell-free microRNas (miRNAs) may play a significant role in the development and progression of MM since in MM cells it has been observed that overexpression of some miRNa, such as miR-let-7b or miR-let-7c, is able to inhibit cell growth, migration, invasion, and metastatization whereas, conversely, it seems to induce apoptosis and cell cycle shutdown after chemotherapy treatment . The overexpression of these miRNAs was therefore able to reduce the recurrence of MM increasing the sensitivity to chemotherapy treatment thus leading to a lengthening DFS. In agreement with such evidence, a low expression of microRNA-23a-3p has been significantly associated with poor outcomes. Indeed, the Kaplan-Meier survival analysis of 117 MM patients showed that low mir-23a-3p expression was significantly associated with poor outcomes, with both the DFS and OS being markedly reduced. In this study, even after a multivariate analysis, the low expression of miR-23a-3p was confirmed as an independent prognostic indicator of reduced OS . Finally, the recent SARS-CoV-2 pandemic allowed for the introduction of new technologies based on mRNA vaccines. This methodology has proven to be safe and effective and could be translated into the oncology field. The building of mRNAs that go inside the immune cells favoring the production of individual, specific tumor proteins able to enhance immunological responses would definitively pave the way for highly personalized therapies. Briefly, mRNA vaccines represent an attractive vector to deliver tumor antigens into dendritic cells (DCs) that are the antigen-presenting cells of our immune system . The encoded proteins can be translated and processed into peptides, which can be subsequently exhibited on the DCs' surface; thus, enhancing the immunological effects mainly through adaptive immune responses. In this context, the peptides sharing with the CD8+ T cells trigger the cytotoxic reaction while exposure to CD4+ T cells determines antibody production against the tumor cells . Underlying the immune response processes turns out to be the relevant modulation of the interferon type I (IFNs) capable of initiating or adjuvating the cytotoxic activity of CD8 cells . However, to date it remains controversial whether IFNs have a stimulatory or inhibitory action on CD8+ T-cell immunity since the mechanisms behind this duality are unclear. Strategies to optimize the intracellular delivery of mRNA vaccines, allowing a more efficient antigen translation and providing potent and specific immune activations, are being investigated. Among others, lipid formulations seem to induce antigen-presenting cell maturation via the intracellular stimulator of interferon genes (STING) pathway and result in systemic cytokine expression and enhanced anti-tumor efficacy . To further support such a therapeutic strategy to a wider extent, preliminary results from a study on adjuvant treatment with the personalized cancer vaccine mRNA-4157 and pembrolizumab in stage III/IV melanoma patients undergoing complete resection of cutaneous melanoma have just demonstrated a statistically significant and clinically meaningful reduction in the risk of disease recurrence or death compared to pembrolizumab monotherapy (KEYNOTE-942) . 7. Conclusions The life expectancy of patients with MMs remains lower than that of patients with CMs since they are diagnosed at a more advanced stage and have a lower burden of point mutations and a wider number of structural chromosomal variants that make them less responsive to immunotherapy treatments. These biochemical features impose the need to identify new specific and selective druggable transcriptomics and metabolic pathways for future more efficient therapeutic applications. In this context, the application of omics' technologies could represent an innovative impulse to better understand the dynamic cell metabolic reprogramming that leads to cancer development and progression explaining the phenotypic heterogeneity and its biological and biochemical behavior of MMs that can contribute to improve their diagnosis and treatment. Author Contributions Conceptualization, D.A.S., G.P. and G.C. (Giuseppe Corona); methodology, D.A.S., G.P. and G.C. (Giuseppe Corona); writing--original draft preparation, D.A.S., G.P., G.M., M.C., M.G.D., L.F., P.P., G.C. (Giuseppe Corona), M.M., A.C., G.L.R. and G.C. (Giuseppe Corona); writing--review and editing, D.A.S., G.P., G.M., M.C., M.G.D., L.F., P.P., G.C. (Giampiero Capobianco), M.M., A.C., G.L.R. and G.C. (Giuseppe Corona); funding acquisition, G.C. (Giuseppe Corona). All authors have read and agreed to the published version of the manuscript. Data Availability Statement Not applicable. Conflicts of Interest The authors declare no conflict of interest. Figure 1 Main genes and their functional role in MM. ATRX, ATP-dependent helicase; BRAF, B-Raf proto-oncogene; CDK4/6, Cyclin-dependent kinase 4/6; CCND1, Cyclin D1; CDKN2A/B, Cyclin-dependent kinase inhibitor of kinase 2A/B; E2F, E2F transcription factor 2; ERK, Extracellular-related kinase; KIT, receptor tyrosine kinase; MDM2, mouse double minute 2 p53 binding protein homolog; MITF, Microphthalmia-associated transcription factor; MEK, Mitogen-activated protein kinase-extracellular related kinase; mTOR, mammalian target of rapamycin; NF1, neurofibromin 1; NRAS, neuroblastoma RAS viral oncogene homolog; PI3K, Phosphatidylinositol 3 kinase; PTEN, Phosphatase and tensin homologue; RB1, retinoblastoma 1; SF3B1, splicing factor 3b subunit 1; SPRED1, sprout-related EVH1 domain containing protein 1; TERT, telomerase reverse transcriptase; TP53, tumor protein p53. Figure 2 Distribution of MM anatomical locations (head and neck, gastrointestinal, urinary, and genital) and correspondent mutation frequencies for the main driver oncogenes (BRAF, NRAS, and KIT). Mutation prevalence values were mainly derived by NGS-based approaches (for references, see text). Disclaimer/Publisher's Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

PMC10000121

A high intramuscular fat content characterizes Wagyu (WY) cattle breed. Our objective was to compare beef from WY, WY-by-Angus, or Wangus (WN) steers with European, Angus-by-Charolais-Limousine crossbred steers (ACL), considering metabolic biomarkers pre-slaughtering and nutritional characteristics, including health-related indexes of the lipid fraction. The fattening system with olein-rich diets and no exercise restriction included 82 steers, 24 WY, 29 WN, and 29 ACL. The slaughter ages and weights were (median and interquartile range) 38.4 mo.-old (34.9-40.3 mo.) and 840 kg (785-895 kg) for WY; for WN, 30.6 mo. (26.9-36.5 mo.) and 832 kg (802-875 kg), and for ACL steers, 20.3 mo.-old (19.0-22.7 mo.) and 780 kg (715-852 kg). Blood lipid-related metabolites, except for non-esterified fatty acids (NEFA) and low-density level cholesterol (LDL), were higher in WY and WN than in ACL, while glucose was lower in WY and WN. Leptin was higher in WN than in ACL. Pre-slaughtering values of plasma HDL underscored as a possible metabolic biomarker directly related to beef quality. The amino-acid content in beef did not differ among experimental groups, except for more crude protein in ACL. Compared to ACL, WY steers showed higher intramuscular fat in sirloin (51.5 vs. 21.9%) and entrecote (59.6 vs. 27.6%), more unsaturated fatty acids in entrecote (55.8 vs. 53.0%), and more oleic acid in sirloin (46 vs. 41.3%) and entrecote (47.5 vs. 43.3%). Compared to ACL entrecote, WY and WN showed better atherogenic (0.6 and 0.55 vs. 0.69), thrombogenicity (0.82 and 0.92 vs. 1.1), and hypocholesterolemic/hypercholesterolemic index (1.9 and 2.1 vs. 1.7). Therefore, beef's nutritional characteristics depend on breed/crossbred, slaughtering age and cut, with WY and WN entrecote samples showing a healthier lipid fraction. Black-Japanese angus oleins MUFA PUFA amino acids health-related indexes Centre for the Development of Industrial TechnologyCDTI-IDI-20180254 This research was funded by the Centre for the Development of Industrial Technology from the Spanish Ministry of Science and Innovation, grant number CDTI-IDI-20180254. pmc1. Introduction The Black-Japanese breed, also called Wagyu, is known worldwide because of the outstanding quality and organoleptic characteristics of its meat due to its extensive fat infiltration of muscle . Gotoh et al. determined a very high level of intramuscular fat content (IMF) in the Longissimus thoracis et lumborum muscle of 24 months old Wagyu steers (23%), while German Angus, Belgian Blue, and Holstein Friesian bulls showed lower IMF (<5%). Wagyu cattle have not only higher IMF but also a different fatty acid composition (FA) with a larger oleic acid (OA) concentration (52.9% of total fatty acids) when compared to Hanwoo steers (47.3%) or corn-fed Angus (39.8%) . Meat chemical composition and the FA profile are directly linked to beef quality , with an increased crude fat content (range 23.8-48.6%) improving tenderness, juiciness, and fattiness and thereby its acceptability by the consumers in Japan . Consumer concerns are also linked to issues related to the health and convenience of beef intake and the advantages and disadvantages of red beef consumption . Although beef is a nutrient-dense protein source, the saturated fat content has been associated with an increased risk of cardiovascular disease , diabetes type 2 , and mortality due to non-communicable diseases . Contrary to these associations, clinical trials have demonstrated that the inclusion of beef in a healthy and balanced diet does not negatively influence disease risk factors . Furthermore, a recent meta-analysis shows that out of the top 15 dietary components related to non-communicable disease risk, a diet high in red meat was ranked as the lowest dietary factor related to increased risk . Due to the high OA content, Wagyu beef has even been related to a reduction in the blood cholesterol level in human consumers . Therefore, besides the quantity, the lipid composition of meat is of increasing interest not only due to its effect on the organoleptic characteristics of the meat but to rate how healthy beef is . In fact, cardiovascular disease and coronary heart disease risk are reduced when saturated fatty acids (SFA) are replaced by monounsaturated fatty acids (MUFA) and polyunsaturated fatty acids (PUFA) . These effects can be achieved by modifying the profile of the ingested fat and the kind of beef consumed . The effects of the diets may vary depending on the bovine breed . Key fatty acid metabolism-associated genes with upstream regulators (SREBF1) are related to a higher FA index in beef . In the Wagyu breed, condensed barley distillers soluble promoted growth, a higher glycolytic and lower oxidative muscle metabolism , with this breed steers producing higher MUFA percentage in carcasses than Angus steers when fed high-roughage diets . Increasing the concentration of oleic acid in diet, Wagyu beef increased the OA level in IMF , but there is no evidence about Angus-crossbreed managed under Spanish farming conditions. The evidence suggests that depending on the breed, the nutritional system can induce relevant improvement in the beef fatty acid profile. Nutritional and management systems trigger changes in the homeostasis of the fattened animals, inducing blood metabolite changes . These changes may be the link between management and final beef characteristics. Indeed, metabolite concentrations are commonly used to assess health and nutritional status in cattle . In Wagyu cattle, metabolic markers show different patterns than in another leaner breeds . The main metabolic parameters related to marbling are cholesterol, glucose, and urea, among others . Although these parameters may change with fed diets, the relationship among metabolic parameters, beef composition, and fatty acid profile remains unclear. A previous study of our group described healthy growing purebred Wagyu, crossbred Wagyu-by-Angus (Wangus), and Angus-by-European crossbreeds, such as Charolais and Limousine (ACL) steers, in a Spanish fattening system involving olein-rich diets, no exercise restriction and high animal welfare . We hypothesize that this production system results in different beef products depending on the experimental group, in fact, on the breed/crossbred. Furthermore, the aim of the study was to describe the beef characteristics of these groups raised in this Spanish fattening system and to explore the relationship between blood metabolic parameters prior to slaughtering and beef characteristics, possibly associated with the group and age at slaughtering. This information is highly relevant to choose the optimal system to produce a more appreciated, high-marbled beef with Wagyu animals efficiently in other world regions outside Japan. 2. Materials and Methods 2.1. Animals and Farm Management This study was conducted under commercial farming practices on the farm "Mudejar-Wagyu", located in the North-Centre of Spain, with the appropriate farming measures and clinical activities . All available, healthy steers slaughtered during the study time were included in the study. A total of 82 steers were studied: 24 purebred Black-Japanese or Wagyu, (WY); 29 crossbred Wagyu-by-Angus or Wangus (WN); and 29 Angus-by-European Charolais and Limousine crossbred steers (ACL). Data were recovered at once, at the end of the fattening period, without repeated measurements. A different objective-slaughtering age was intended by experimental group, following the usual slaughtering times. This guaranteed an adequate carcass quality, with an earlier expected slaughtering age in European crossbreds, to avoid excessive fattening (WY 37 mo., WN 31 mo., and ACL 22 mo.-old), assuring a comparable and marketable beef after slaughtering the steers from the three experimental groups. The actual slaughter ages, expressed as median and interquartile range, were 38.4 mo.-old (34.9-40.3 mo.) for WY; for WN, 30.6 mo. (26.9-36.5 mo.) and for ACL steers 20.3 mo.-old (19.0-22.7 mo.). The animals were raised as described previously . Castrated male calves were housed during the last phase of fattening in groups of 10 animals/pen (20 m2/animal). All barns were open (natural ventilation), with anti-slip, concrete or soil floors and chopped straw bedding, and access to open areas. Brushers and sprinklers were available in the pens assuring a high level of animal welfare. The nutritional management of the animals included ad libitum water and diets adjusted to their requirements . From 10 to 22 mo. of age, the diet was a wet total mixed ration (wet TMR), and afterward, the diet was a dry TMR enriched in oleins. ACL animals received the finalization diet at least 2 months before slaughtering, independently of age. Further details on diets and management are described in the previous study on productive and health parameters , and the detailed composition of this finalization diet, enriched in oleins, is resumed in Table 1 and Table 2. Animals were slaughtered according to European Union regulation 1099/2009. Carcasses were aired for temperature decrease up to 4 degC in less than 24 h and kept at 2 degC for 72 h. At the meat processing facility, 200 g fresh meat samples of the Longissimus lumborum muscle, between the lumbar ribs (sirloin) and Longissimus thoracis between the 6 and 7th ribs (entrecote or ribeye steak) were taken from each carcass on the 5th day after slaughtering. Samples were adequately identified, vacuum-packed, and sent under a stable temperature of 4 degC to the diagnostic laboratory (Labocor Analitica, Colmenar Viejo, Madrid, Spain), operating under the Spanish National Accreditation Body (ENAC; complying the applicable requirements and conditions of the standard GMP+ B10 Laboratory Testing of the GMP+ FC scheme (based on GMP+ C6) of GMP+ International and the UNE-EN ISO/IEC 17025:2017). 2.2. Description of the Slaughtered Animals: Weight, Height and In Vivo Fatness Animals that could enter the weighing booth during the last week before slaughtering (n = 15/24 WY, 14/29 WN, and 29/29 ACL) were weighted (BW, Kg) at the farm before slaughtering using an electronic balance (TRU-TEST, Auckland, New Zealand; accuracy of +-0.5 Kg), while its height (cm) was determined with the animal standing, using a measuring stick, from a point directly over the hip bones (hocks) to the floor. In vivo evaluation of the body-fattening stage was performed with ultrasound using the PieQuip technology and a MyLab One(r) ultrasound system (Esaote, Barcelona, Spain) with an animal science probe (ASP) of 18 cm length and a frequency of 3.5 Mhz linear transducer . Measurements were made at the closest moment before slaughtering when images could be processed. As previously determined , in Wagyu and Wangus animals older > 22 mo. of age, the ultrasound images could not be recovered due to excessive dorsal fat layer (>20 mm). Therefore, the total of animals scanned near slaughtering with valid figures was 9 purebred Wagyu, 19 Wangus, and 29 ACL steers (Table 3). Ultrasound scanning traits related to growth and fat deposition were:Rump fat thickness (RF, mm), at the "P8" rump site. Measured at the level of the rump, at the intersection of the gluteus medius and biceps femoris muscles. Depth of the gluteus medius muscle (GMD, in mm). Measured at point P8. Ribeye area (REA, in cm2) of the Longissimus thoracis muscle measured between the 12-13th ribs. Back fat (BF, mm). It was 12-13th rib fat thickness. Percentage of intramuscular fat estimation (marbling; IMF) of the Longissimus lumborum muscle was measured between the 12-13th ribs as a numerical value given by the ultrasound equipment software and positively correlated to intramuscular fat. 2.3. Metabolic Status of the Animals Before slaughtering, blood samples were extracted for sanitary reasons by the official veterinarians, and the authors received an aliquot for metabolic assessment. Once obtained, plasma was separated by centrifugation at 4500x g for 15 min and stored in polypropylene vials at -80 degC until later analysis. Parameters of plasma total cholesterol (TC, mg/dL), triglycerides (TG, mg/dL), high- (HDL, mg/dL) and low-density lipoprotein cholesterol (LDL, mg/dL), glucose (GLU, mg/dL), fructosamine (FRU, mg/dL), lactate (LAC, mg/dL), b-hydroxy butyrate (BHB, mmol/L), non-esterified-fatty acid (NEFA mmol/L) and urea (UR, mg/dL) were assessed with clinical chemistry analyzer (Konelab 20; Thermo Fisher Scientific, Waltham, MA, USA), according to the manufacturer's instructions. Plasma leptin concentration (LEP, mg/mL) was determined with a multispecies ELISA kit (Demeditec Diagnostics GmbH, Kiel, Germany; assay sensitivity 0.25 mg/mL and intra-assay variation coefficient < 15%). 2.4. Meat Nutritional Analyses Meat analyses were performed immediately after reception at the laboratory Labocor Analitica on fresh samples. Samples were minced and homogenized. Crude protein content was determined by Kjeldahl with a conversion factor of 6.25, and total fat was determined by ethyl ether extraction with previously acid-hydrolyzed samples (Soxhlet technique ). Moisture was gravimetrically measured by drying at 103 +- 2 degC , and ash was gravimetrically assessed after combustion at 550 degC in a muffle furnace to a constant weight. Energy content (kcal) was calculated based on 100 g portion using according to the equation of Merrill and Watt as the following: energy value (kcal/100 g) = (Protein% x 4) + (Fat% x 9) + (Carbohydrate% x 4). Cholesterol in meat was assessed with the enzymatic method (Kit Boehringer Mannheim) and read by spectrophotometry. quantified with a glass electrode (FC2323) specifically designed for meat, introduced 1 cm into the meat, performing 3 measurements/piece, and recalibrated by each sample. Fatty acid content of beef was determined by extracting the fat fraction, following the method of ISO for determining FA methyl esters by gas chromatography, set up, and verified. Only cis-isomers of fatty acids were detected. Fatty acids were expressed as a concentration in fresh meat and/or as a percentage of the total amount of the identified fatty acids. The total contents of saturated fatty acids (SFAs), unsaturated fatty acids (UFAs), monounsaturated fatty acids (MUFAs), polyunsaturated fatty acids (PUFAs), and o-3 and o-6 PUFAs were calculated. In addition, the health-related indexes of the lipid fraction were assessed by the following calculations:o-6/o-3 ratio: o-6 to o-3 fatty acids ratio (optimum value < 4; although it is a discussed cut-off) . So-6 PUFA/So-3 PUFA Atherogenic index (AI ; the lower the healthier). AI = [12:0 + (4x14:0) + 16:0]/(So-3 PUFA + So-6 PUFA + SMUFA) Thrombogenicity index (TI ; the lower the healthier). TI = (14:0 + 16:0 + 18:0)/[(0.5 x SMUFA) + (0.5 x So-6 PUFA) +(3 x So-3 PUFA) + (So-3 PUFA/So-6 PUFA)] Hypo-/hypercholesterolemic ratio (h/H; modified from Fernandez et al. ; the higher the healthier). h/H = [(C18:1 + C18:2n-6 + C18:3n-6 + C18:3n-3 + C20:3n-6 + C20:4n-6 + C20:5n-3 + C22:4n-6 + C22:5n-3 + C22:6n-3)/(C14:0 + C16:0)]. 2.5. Amino Acids Assessment A representative subset of the entrecote samples of each breed group was randomly selected (n = 13/24 WY, 12/29 WN and 8/29 ACL). Samples were hydrolyzed with HCL 6N, at the Laboratory of the Veterinary Faculty at the Universidad Catolica of Valencia (Valencia, Spain), by high-performance liquid chromatography (HPLC), with the derivatization method (Waters AccQ-Tag reagent kit). Then, the amino acids were separated on a reverse phase C18 column (AccQ Tag, 3.9 x 150 mm, Waters Co, Milford, CT, USA) using a gradient of two eluents from the kit as mobile phase. Three mL of sample were injected for each analysis carried out in 50 min. The detection of the amino acids was carried out with a fluorescence detector set up to work at 250 and 395 nm (excitation and emission wavelengths, respectively). Amino acids were identified by retention times and quantified by the external standard technique using an amino acid standard [a-aminobutyric acid (aAba)] and processed with the EZChromeElite program (Agilent, Santa Clara, CA, USA). Results were expressed in g/100 g of dry matter. Tryptophan content was determined following the protocol described by Yust et al. , in which alkaline hydrolysis of samples of meat and ashes was performed. In brief, tryptophan was extracted from other amino acids and assessed by reverse phase HPLC and its detection by absorbance set up with 280 nm wavelength. The same reverse phase C18 column was used for separating derivatized amino acids according to the Waters AccQ-Tag method described above. Tryptophan was quantified using a standard curve with different concentrations of tryptophan standard. Results were expressed in g/100 g of dry matter. 2.6. Statistical Analyses Data were analyzed using SPSS(r) v.25 (IBM, Armonk, NY, USA). Normality of variables was assessed with Kolmogorov-Smirnov and Shapiro-Wilk tests. Most of the variables showed a skewed distribution, and all the variables were reported as median and range instead of average and standard deviation as best measures of centrality and variability for skewed distributions; therefore, non-parametric tests were used for assessing intergroup differences such as Kruskal-Wallis test for independent samples. The comparisons were made among experimental groups (three groups, one full breed: WY and two crossbreeds: WN and ACL), separated by beef cut (sirloin or entrecote) or between cuts (sirloin or entrecote) separated by experimental groups. Potential pairwise relationships among numerical variables of steers characteristics prior slaughtering, blood metabolites, meat nutritional parameters, and amino acid values were assessed with Spearman tests. Differences associated with p-values <= 0.05 were considered significant. 3. Results 3.1. Pre-Slaughter Metabolic Data WY and WN steers showed levels of metabolites in plasma that were not statistically different (Table 4). Concentrations of all lipid-related metabolites, except NEFA and LDL, were higher in WY and WN steers than in ACL animals. Conversely, glucose values were lower in WY and WN steers. Levels of the hormone leptin, which are related to the lipid profile, were higher in WN than in ACL steers. 3.2. Nutritional, Fatty Acid, and Amino Acid Profiles in Beef Samples Nutritional analyses showed that for both meat cuts, WY and WN steers showed higher fat infiltration than ACL animals, including 2 to 2.5-fold higher IMF (in the fresh matter; Table 5). However, ACL meat had a higher content of protein and lower content of saturated fatty acids in both cuts of meat. Entrecote samples showed more IMF and less SFA in all breeds (Table 5). The relative content of saturated fatty acids, expressed as the percentage of the total amount of identified fatty acids, varied depending on the cut of meat. Only in ACL animals, the amount of PUFA and the ratio o-6/o-3 were significantly higher in sirloin than in entrecote (Table 6). Entrecote samples from ACL steers contained more saturated fatty acids than the corresponding cut from the other two breeds, with the exception of stearic acid (C18:0, Table 7). However, this pattern was absent in sirloin samples, where only stearic acid values differed, with WN steers presenting the lowest level. The relative content of unsaturated fatty acids was higher in sirloin from WN steers and lower in entrecote from ACL steers (Table 6), with WY and WN samples showing the highest concentrations of oleic acid (C18:1; Table 7) and linoleic acid (C18:2n-6). Conversely, the content of linoleic acid (C18:2n-6) was highest in ACL steers. C17:1 was the most abundant fatty acid in sirloin from WN animals. Next, we correlated the fat characteristics of meat with healthiness indexes for humans. The ratio of monounsaturated to saturated fatty acids (MUFA/SFA) was higher in WY and WN than in ACL meat (Table 6). The ACL entrecote showed the lowest hypocholesterolemic/hypercholesterolemic index, indicating less healthy meat, while sirloin did not differ substantially in this index across the breeds. Moreover, for both cuts of meat, ACL steers showed the highest thrombogenicity index, indicating less healthy meat, and their entrecote showed the highest atherogenic index. Consistently, ACL meat showed the highest ratio of o-6 to o-3 than Wagyu in both cuts, and Wangus in sirloin, which ideally should be <4. The amino acid content of entrecote samples did not differ among breed/crossbreed (Table 8), except that ACL steers showed higher absolute levels of arginine and threonine than the other two breeds and higher leucine content than Wagyu due to the higher crude protein content. 3.3. Relationships between Variables In sirloin, the IMF content samples correlated positively with the total content of monounsaturated fatty acids due to the oleic acid content and inversely with the content of polyunsaturated fatty acids or PUFAs . In entrecote and sirloin, the human health-related indexes AI and TI correlated negatively with unsaturated fatty acids but positively with saturated fatty acids, while the opposite was true for the h/H ratio. Among blood metabolites, IMF correlated positively with total cholesterol and high-density lipoprotein cholesterol, regardless of the meat cut (Table 9). b-hydroxybutyrate correlated with moisture (negatively) and IMF (positively), but only in sirloin, while the negative correlation between the total content of PUFAs and urea was found only in entrecote. Plasma total cholesterol and HDL were revealed to be positively associated with the IMF in beef in both cuts and negatively with the amount of o-6 PUFAs in entrecote and sirloin, independently of breed/crossbreed. Figure 2 summarizes the significant pairwise correlations between variables by breed/crossbreed. In WY meat, moisture and IMF did not correlate with health-related indexes. In WN or ACL meat, increased moisture and decreased IMF correlated with better health-related indexes. In meat from WN and ACL steers, but not WY animals, moisture and IFM correlated negatively with the atherogenic index and positively with the ratio h/H. Content of specific fatty acids correlated with the corresponding total amounts. In all breeds, higher content of palmitic acid (C16:0), the most frequent saturated fatty acid, was associated with worse health-related indexes. The content of other fatty acids correlated differently with health-related indexes depending on the experimental group. In WY and ACL meat, the content of stearic fatty acid (C18:0) was positively correlated with the TI index. In WY meat, the content of linoleic acid [C18:2n-6] and linolenic acid [C18:3n-3] did not correlate with h/H, TI, or AI. However, a robust positive correlation with health-related indexes was observed in WN and ACL steers for the linoleic acid, while such a relationship was observed for linolenic acid, but only in WN meat. The stearidonic fatty acid [C18:4n-3] correlated negatively with TI in WY and WN steers, while it was positively related to h/H and AI in ACL steers. More pre-slaughter metabolic parameters correlated with fatty acid composition and leptin content in meat from WN animals than in meat from WY or ACL steers (UREA, TC, and HDL positively correlated to saturated fatty acids). Leptin levels, although highest in WN animals, significantly correlated with NEFA and glucose metabolic parameters in purebred WY steers, while leptin correlated positively in ACL animals only with triglycerides . 4. Discussion Wagyu and Wangus steers are of high commercial interest worldwide because of their high beef quality, due in part to high IMF content. The impact of different fattening systems on beef quality and fat profile is poorly understood. Therefore, we aimed to describe the pre-slaughter metabolic parameters, beef quality, and fat profile of the final marketable meat samples from three high-marbling bovine breeds/crossbreeds raised under the same Spanish fattening system but slaughtered at different ages. We also analyzed pairwise relationships among variables. As we hypothesized, pure and crossbred WY steers presented higher IMF content, lower crude protein content, and a healthier fatty acid profile than the European ACL crossbred steers. Regarding the pre-slaughter metabolic biomarkers, ACL animals showed lower levels of blood plasma metabolites related to lipid metabolism, including leptin, than WY and WN animals. The exception was LDL. These differences may be due to the experimental group but also to the different slaughter ages (ACL animals were slaughtered with an age of at least 10 months younger than WY and WN steers), a factor demonstrated to alter the metabolic status of cattle . The only metabolic parameter that was higher in ACL animals than in the other was plasma glucose concentration, which supports previous findings demonstrating higher insulin levels and lower plasma glucose in WY steers than in European crossbreeds, and a positive correlation between IMF and insulin in cattle . We found that pre-slaughtering blood plasma BHB correlated negatively with beef moisture and positively with IMF, independently of breed/crossbreed. The production of BHB inhibits some class I histone deacetylases , reducing lipolysis in adipocytes. The availability of fatty acids in the liver, which is strongly regulated by insulin, is a critical determinant of ketogenesis, so the reduction of lipolysis may act as a feedback mechanism to limit BHB production . Such feedback may explain the positive correlation between BHB and IMF: increased BHB reduces lipolysis and therefore increases IMF. It may also explain the negative correlation between BHB and moisture: moisture correlates inversely with IMF. Pre-slaughtering values of HDL related to lipid beef characteristics, underscoring a possible metabolic biomarker directly related to beef quality, i.e., foreseeing a high level of IMF and o-6 PUFA in beef, result highly interesting. Moreover, the correlations between metabolites and fat profiles differed clearly among experimental groups. In general, WN meat showed stronger correlations between metabolic parameters and fatty acids than WY meat . This may indicate that the metabolism of WY animals is better adapted to high body fat than the metabolism of WN animals. As expected, WY and WN steers showed more fat infiltration in both meat cuts due to their typical Wagyu beef characteristics, and accordingly, the energy content of Wagyu and Wangu beef was significantly higher than that of ACL steers, and the absolute amount of SFA per 100 g of meat was the lowest in the ACL beef samples. We did not observe differences in IMF between purebred WY and crossbred WN animals. Previous work confirmed that crossbreds with WY show stronger IMF content and greater tenderness and marbling score , which would explain the high IMF values in our WN steers. Therefore, our work illustrates that the relationships among the meat characteristics that we examined depend on the experimental group (i.e., on breed/crossbreed), even when diet and management are the same. These differences could also relate to the different slaughtering age (linked, in turn, to the group) effects that we cannot separate in this study. Regarding the assessment of the lipid fraction in beef, among the three experimental groups in our study, ACL steers showed the lowest levels of MUFAs. Previous work found higher MUFA values in subcutaneous fat from WY steers (60.7%) and crossbred WN steers (60.1%) than from Holstein animals (57.6%) . This result reflects the higher ratio of C18:1 to C18:0 desaturases in the WY breed, which leads to higher oleic acid content . Interestingly, our WN crossbred showed even higher MUFA values than WY in sirloin; the WY values were similar to those in a previous study . On the other hand, other authors did not observe such differences when they compared WY animals with the genetically similar Hanwoo and Jeju Black breeds . In our work, this specific high-olein diet may have promoted a higher content of MUFAs in WY and WN steers. Similar results have been reported in steers fed with diets rich in oleic acid, which increased the oleic acid content in meat fat from 1.72 to 4.22% . This may have important implications for the marketability of this meat since consumption of Hanwoo beef from animals with high oleic acid content might reduce the risk of cardiovascular disease . The link between a grain-fed diet and higher, healthier levels of MUFAs in beef was also observed previously in various breeds, such as Angus, Hereford, and Hanwoo, among others . Angus-by-Charolais or Limousine beef in our study showed the highest content in PUFAs and the highest o-6/o-3 ratio across all three breed/crossbreds, with nearly doubling the content of linoleic acid (C18:2n-6). In contrast, the entrecote pieces of ACL showed the highest content of saturated fatty acids, similar to that reported in former reports . In the current study, a high-olein diet appeared to induce high levels of PUFAs in all three breeds/crossbreds. These values were like those previously described in beef cattle of other breeds (4-5%) and higher than those reported in grain-fed WY steers (2.6-3.5%) . It seems, therefore, that a high-olein diet improves beef quality more intensively, leading to levels of PUFAs and o-6/o-3 ratio healthier for humans in WY steers than in European crossbred animals. The health-related indexes AI and TI in our work correlated negatively with the content of unsaturated fatty acids (improving the effect for healthiness in humans) and positively with the content of saturated fatty acids (worsening the effect for healthiness in humans). These relationships seem logical given that the indexes are calculated in a way that saturated fatty acid content is penalized due to its assumed adverse effects on human health. One study has linked worse AI and TI indexes to higher saturated fatty acid content in beef as well as a greater risk of cardiovascular disease in humans who consume that beef . However, the inclusion of beef in a healthy, balanced diet does not necessarily increase the risk of cardiovascular disease , especially if the fatty acids in the meat are predominantly unsaturated. Although the two cuts of meat in our study are similarly highly regarded on the market, we found interesting differences in nutritional parameters and in the lipid fraction between cuts from the same breed/crossbreed, some of them previously found , as well as in correlations between those variables and health-related indexes. Independently of the experimental group, entrecote showed more IMF than sirloin pieces and, accordingly, more energy content and SFA in g/100 g of meat. In the lipid fraction, the main difference between sirloin and entrecote differed by group, with ACL animals showing higher PUFA and lower SFA contents in sirloin than in entrecote. The relationship among fatty acids also varied by meat cut. The higher content of total fat and total saturated fatty acids in entrecote probably weakened some relative relationships among specific MUFAs, PUFAS, and health-related indexes, indeed observed in sirloin. For example, content in o-6 fatty acids correlated negatively with palmitic acid and positively with linolenic acid in sirloin but not in entrecote. Only in entrecote did we find correlations of lower values of AI with higher values of linoleic acid and higher values of h/H, indicating healthier beef, with linoleic acid being a potential marker. In fact, linoleic acid is known to be beneficial to human health , and it is valued by beef consumers . Therefore, the cut should always be taken into account when analyzing the beef quality and healthiness of humans. Congruently with a lower absolute content in intramuscular fat, Angus-by-Charolais or Limousine beef samples had more crude protein per 100 g of entrecote and a higher absolute content of several amino acids. Unfortunately, none of the three types of beef showed higher content of essential amino acids. Moreover, a higher protein level may not always be desirable: for example, higher levels of leucine and methionine in ACL beef can lead to a bitter taste in consumers . Wagyu and Wangus beef may be superior to ACL beef in this regard. In addition, WY and WN beef showed a higher ratio of MUFAs to saturated fatty acids, indicating better organoleptic qualities . The three kinds of beef should be compared in taster panel studies. Finally, the health-promoting value of meat can be assessed based on indexes of its various long-chain fatty acids . As we hypothesized, the different groups in our study showed different indexes linked to different intramuscular lipid fractions. Congruently with the higher content in PUFAs and higher o-6/o-3 ratio in beef, ACL entrecote showed the lowest h/H index and the highest atherogenic index, and both meat cuts from ACL steers showed the highest thrombogenicity index. These results support the idea that beef quality strongly depends on the breed/crossbreed . In fact, specific genes in WY steers have been positively linked to high fat deposition , and the maternal gametic effect may also contribute to fat deposition . This study implies that the fat composition of beef influences many aspects of its quality and health value . In a previous study, adding olive oil to the diet of Blonde d'Aquitaine steers led to a similar atherogenic index as that in our ACL bulls (0.6), which further improved when soy oil was used instead of olive oil . In the current study, a high-olein diet led to better entrecote fat in WY steers than in other groups, as well as lower AI and TI (Table 6). In general, lower atherogenic and thrombogenicity indexes, a higher h/H ratio, and a o-6/o-3 ratio lower than four are desirable . This kind of fat profile lowers LDL , increases HDL, and decreases triglycerides in humans , although the magnitude of the health benefit requires further study . Moreover, based on the lipid fraction, beef fat from WY may be healthier for humans than beef fat from European crossbreeds raised under the same conditions. These considerations also suggest that raising WY steers on pasture or feeding them forage may further improve the health-related indexes of WY beef, as suggested before , which is an interesting question for further research . The information about the influence of breed/crossbreed and management systems on the lipid fraction of meat is further scarce, and although recent efforts in "foodomics" have brought some light to this topic , further research is required. 5. Conclusions Differences among experimental groups in certain metabolic parameters were found, probably linked not only to the breed or crossbreed but also to slaughtering age. Pre-slaughtering values of plasma HDL underscored as a possible metabolic biomarker directly related to beef quality and concretely to intramuscular fat. Although the fat profile and nutritional content of beef depend on the cut, on the breed/crossbreed, and on the slaughtering age, purebred WY and their crossbred WN, raised in Spain under fattening conditions characterized by olein-rich diets, no exercise restriction, and high animal welfare, achieve high IMF content and quality that are similar to those of animals fattened in Japan. Acknowledgments We thank Pedro Cuesta and Iagoba Cano (Department of Research Support, Complutense University of Madrid) for their help with statistical analyses. We thank all the farmers and their staff for their contributions. We are particularly grateful to Antonio Valero, Monica Gejo and their colleagues at Fincas del Turia-Mudejar-Wagyu. Author Contributions Conceptualization, J.M.V.-M., A.F.-N., E.d.M., A.V., D.M., F.S., S.S.P.-G. and S.A.; methodology, J.M.V.-M., J.C.G., R.P.-C., A.R.-R. and M.L.P.-S.; software, J.M.V.-M., S.S.P.-G. and S.A.; validation, A.F.-N., M.V.-G., S.S.P.-G. and S.A.; formal analysis, A.F.-N., E.d.M., S.S.P.-G. and S.A.; investigation, A.F.-N., M.V.-G., J.C.G., R.P.-C., A.R.-R., F.S., S.S.P.-G. and S.A.; resources, D.M., F.S., S.S.P.-G. and S.A.; data curation, J.M.V.-M., E.d.M., J.L.P.-P., M.L.P.-S., S.S.P.-G. and S.A.; writing--original draft preparation, J.M.V.-M., A.F.-N., E.d.M. and M.V.-G.; writing--review and editing, A.F.-N., M.V.-G., J.L.P.-P., R.P.-C., A.R.-R., A.V., D.M., F.S., S.S.P.-G. and S.A.; visualization, D.M., F.S., S.S.P.-G. and S.A.; supervision, F.S., S.S.P.-G. and S.A; project administration, S.S.P.-G. and S.A; funding acquisition, S.S.P.-G. and S.A. All authors have read and agreed to the published version of the manuscript. Institutional Review Board Statement All procedures involving the animals were following the guidelines of the European Communities Council (2010/63/UE). Informed Consent Statement Not applicable. Data Availability Statement The data presented in this study are available on request from the corresponding author. The data are not publicly available due to farm privacy. Conflicts of Interest The authors declare no conflict of interest. Figure 1 Spearman coefficients for statistically significant pairwise correlations (p < 0.001, with absolute value > 0.4) between human health-related indexes, total content of fatty acid types, and specific fatty acids, analyzed separately by meat cut, entrecote and sirloin samples, regardless of experimental group. The "+" symbol represents a positive correlation and "-", a negative correlation. AI: atherogenic index; h/H: hypocholesterolemic/hypercholesterolemic index; IMF: intramuscular fat in fresh matter; MUFA: monounsaturated fatty acid; PUFA: polyunsaturated fatty acid; SFA: saturated fatty acid; TI: thrombogenicity index; UFA: unsaturated fatty acid; SH: total sum of; o-3: omega-3 fatty acids; o-6: omega-6 fatty acids; o-6/o-3: ratio of o-6 to o-3 fatty acids. Figure 2 Pairwise correlations among health-related indexes, total content of different fatty acid types, specific fatty acids, and blood metabolites analyzed separately by experimental group, in Wagyu, Wangus, and Angus-by-Charolais or Limousine steers, regardless of meat cut. Only correlations for which Spearman's coefficient had an absolute value > 0.4 and p < 0.001 are shown. "+" symbol indicates a positive correlation; "-" indicates a negative correlation. AI: atherogenic index; BHB: b-hydroxybutyrate; FRU: fructosamine; GLU: glucose; HDL: high-density lipoprotein cholesterol; h/H: hypocholesterolemic/hypercholesterolemic index; IMF: intramuscular fat in fresh matter; LACT: lactate; LDL: low-density lipoprotein cholesterol; MUFA: monounsaturated fatty acid; NEFA: non-esterified fatty acid; PUFA: polyunsaturated fatty acid; SFA: saturated fatty acid; TC: serum total cholesterol; TG: triglycerides; TI: thrombogenicity index; UFA: unsaturated fatty acid; : total content; o-3: omega-3 fatty acids; o-6: omega-6 fatty acids; o-6/o-3: ratio of o-6 to o-3 fatty acid. animals-13-00864-t001_Table 1 Table 1 Diet composition (percentage of dry matter) given to all steers in the study during the final phase of the fattening period. Nutrient Finishing Dry-TMR Humidity (%) 11.35 Dry matter (%) 88.65 Crude fiber (%) 11.15 Ash (%) 5.58 NDF (%) 26.43 Crude protein (%) 13.19 Crude fat (%) 6.98 ADF (%) 14.16 Ca (%) 0.628 P (%) 0.292 Na (%) 0.219 Cl (%) 0.419 Mg (%) 0.219 K (%) 0.778 S (%) 0.172 Vitamin E (mg/kg) 41.62 Vitamin A (KUI/kg) 4.23 Vitamin D (KUI/kg) 1.18 NFC 47.83 Starch (%) 40.46 ME (kcal/kg) 3026.4 TDN (%) 80.93 ADF: acid detergent fiber; Ca: calcium; Cl: chlorine; K: potassium; ME: metabolizable energy; Mg: magnesium; Na: sodium; NDF: neutral detergent fiber; P: phosphorus; S: sulfur; NFC: non-fibrous carbohydrates, calculated according to Institut National de la Recherche Agronomique (INRA) guidelines; TDN: total digestive nutrients; TMR: total mixed ration. animals-13-00864-t002_Table 2 Table 2 Fatty acid profile (expressed in percentage of total fatty acids) of the diet used to feed all steers in the study during the final phase of the fattening period. Fatty Acid Finishing Dry-TMR Saturated fatty acids (SFA, %) C14:0 0.19 C16:0 15.71 C17:0 0.16 C18:0 5.16 C20:0 0.55 Unsaturated fatty acids (UFA, %) C16:1 0.76 C17:1 0.11 C18:1 41.83 C18:2n-6 32.55 C18:3n-3 2.69 C20:1 0.29 Fatty acid profile SHSFA (%) 21.77 SHMUFA (%) 42.99 SHPUFA (%) 35.24 MUFA/SFA 1.97 o-3 (%) 2.69 o-6 (%) 32.55 o-6/o-3 (%) 12.10 MUFA: monounsaturated fatty acids; PUFA: polyunsaturated fatty acids; SFA: saturated fatty acids; TMR: total mixed ration; UFA: unsaturated fatty acids; SH; total sum of; o-3: omega-3 fatty acids; o-6: omega-6 fatty acids; o-6/o-3 ratio: o-6 to o-3 fatty acids ratio. animals-13-00864-t003_Table 3 Table 3 Characteristics of steers measured prior slaughtering, including last in vivo ultrasound evaluation of muscle and fat deposition (median and interquartile range). Characteristic WY WN ACL p-Value n Median (IQR) n Median (IQR) n Median (IQR) Age (mo.) 24 38.4 (34.9-40.3) a 29 30.6 (26.9-36.5) b 29 20.3 (19.0-22.7) c <0.001 Weight (kg) 15 840 (785-895) 14 832 (802-875) 29 780 (715-852) 0.070 Height (cm) 15 147.0 (145.0-150.0) 14 146.0 (141.5-147.5) 29 143.0 (140.0-146.0) 0.100 BF (mm) 9 18.6 (16.9-23.5) 16 22.5 (19.3-23.6) 29 19.8 (15.3-24.8) 0.500 REA (cm2) 9 91.7 (84.9-104.8) a 16 100.0 (88.8-111.4) a 29 110.3 (101.7-118.9) b 0.005 RF (mm) 9 14.1 (13.1-20.1) 16 14.1 (9.9-19.0) 29 15.9 (13.4-18.1) 0.600 GMD (mm) 9 92.0 (87.1-98.4) a 16 101.4 (95.4-107.6) a 29 116.4 (107.5-123.4) b <0.001 IMF 9 7.9 (7.2-8.1) a 16 6.8 (6.4-7.8) b 29 6.6 (6.1-7.1)b 0.006 ACL: Angus-by-Charolais or Limousine animals; BF: back fat thickness measured between the 12-13th ribs; GMD: depth of the gluteus medius muscle measured at point "P8" on the rump; IMF: in vivo intramuscular fat estimation (numerical values provided by the software); IQR: interquartile range; REA: ribeye area of the Longissimus thoracis measured between the 12-13th ribs; RF: rump fat thickness at point "P8"; WY: Wagyu; WN: Wagyu-by-Angus (Wangus). Different superscripts in values of the same row indicate significant differences with the p-value of the last column. animals-13-00864-t004_Table 4 Table 4 Plasma metabolites determined one week prior to slaughter in steers of different breed/crossbreed (median and interquartile range). Metabolite WY WN ACL p-Value TC (mg/dL) 163.3 (150.0-194.0) a 145.5 (124.5-162.7) ab 111.5 (98.0-137.0) b <0.001 HDL (mg/dL) 71.0 (61.1-90.0) a 54.2 (47.2-80.5) a 42.5 (39.0-53.5) b <0.001 LDL (mg/dL) 17.3 (12.2-20.0) 19.8 (17.0-22.3) 17.5 (15.3-24.2) 0.300 TG (mg/dL) 29.0 (26.1-37.2) a 28.3 (25.5-43.0) a 19.1 (16.4-35.2) b 0.040 BHB (mmol/L) 0.45 (0.38-0.58) a 0.43 (0.35-0.53) a 0.35 (0.27-0.41) b <0.001 NEFA (mmol/L) 0.16 (0.11-0.39) 0.21 (0.15-0.38) 0.26 (0.2-0.3) 0.500 GLU (mg/dL) 76.3 (73.1-91.2) a 81.8 (76.8-91.5) a 92.4 (85.0-97.4) b 0.010 FRU (mg/dL) 294.0 (274.7-310.7) 289.0 (270.5-315.5) 300 (278.5-329.0) 0.400 LAC (mg/dL) 20.2 (16.0-39.5) 19.6 (12.8-35.3) 20.0 (13.2-32.1) 0.700 UR (mg/L) 27.2 (23.0-34.2) ab 29.5 (23-42.1) a 23.5 (20.3-28.1) b 0.040 Leptin (mg/mL) 12.1 (0.36-14.9) ab 17.9 (11.0-26.2) a 8.9 (5.5-11.7) b 0.022 ACL: Angus-by-Charolais or Limousine animals; BHB: b-hydroxybutyrate; FRU: fructosamine; GLU: glucose; HDL: high-density lipoprotein cholesterol; LAC: lactate; LDL: low-density lipoprotein cholesterol; NEFA: non-esterified fatty acid; TC: total cholesterol; TG: triglycerides; UR: urea; WN: Wangus; WY: Wagyu. Values are expressed as median (interquartile range). Different superscripts in values of the same row indicate significant differences with the p-value in the last column. animals-13-00864-t005_Table 5 Table 5 Nutritional composition of fresh sirloin and entrecote samples from Wagyu (WY), Wangus (WN), and Angus-by-Charolais or Limousine (ACL) steers (median and interquartile range). Sirloin Entrecote Parameter WY WN ACL p-Value WY WN ACL p-Value pH 5.4 (5.3-5.56) a 5.6 (5.5-5.8) b 5.7 (5.4-5.8) b 0.014 5.5 (5.4-5.6) a 5.6 (5.5-5.8) b 5.7 (5.6-5.8) b 0.003 Moisture (%) 59.4 (56.5-62.6) a 61.2 (56.9-66.6) a 72.2 (70.1-74.2) b <0.001 54.9 (50.4-60.0) a 59.5 (55.4-63.5) a 69.1 (67.3-71.3) b <0.001 IMF (%) 20.5 (19.0-26.1) a 18.0 (11.6-24.7) a 6.1 (3.7-8.3) b <0.001 26.9 (21.1-32.6) a 20.9 (16-26.6) a 8.6 (5.6-11.6) b <0.001 P (g/100 g) 18.4 (17.1-19.4) a 18.6 (18.1-20.5) a 20.5 (19.6-21.4) b <0.001 16.7 (15.5-18.3) a 18.2 (17.2-20) b 21.05 (20.5-21.8) a <0.001 Ash (%) 0.9 (0.8-1.0) a 1.0 (0.9-1.1) a 1.2 (1.0-1.3) b <0.001 0.8 (0.7-1.0) a 0.9 (0.8-1.0) a 1.1 (1.0-1.2) b <0.001 E (kcal/100 g) 259 (227-309) a 241 (185-284) a 137 (113-157) b <0.001 312 (262-361) a 269 (228-308) a 162 (141-190) b <0.001 E (kJ/100 g) 1074 (944-1282) a 1002 (772-1175) a 574 (478-656) b <0.001 1291 (1085-1494) a 1115 (947-1275) a 681 (593-791) b <0.001 SFA (g/100 g) 9.3 (6.9-11.4) a 7.3 (4.4-10.3) a 2.3 (1.6-3.8) b <0.001 11.5 (8.3-13.9) a 8.7 (6.8-11.4) a 3.9 (2.5-5.3) b <0.001 Cholesterol (%) 0.65 (0.44-1.09) ab 0.52 (0.37-0.64) a 0.66 (0.51-0.87) b 0.030 0.81 (0.59-1.02) 0.70 (0.49-0.94) 0.69 (0.55-1.10) 0.500 E: energy content in 100 g of meat; IMF: intramuscular fat in fresh matter (%); P: crude protein content (g/100 g); SFA: saturated fatty acids (g/100 g). Values are expressed as median (interquartile range). Different superscripts in values of the same row indicate significant differences with the p-value of the corresponding column. animals-13-00864-t006_Table 6 Table 6 Fatty acid profile (percentage of total fatty acids) and health-related indexes of sirloin and entrecote meat from Wagyu (WY), Wangus (WN), and Angus-by-Charolais or Limousine (ACL) steers (median and interquartile range). Parameter * Sirloin Entrecote WY WN ACL p-Value WY WN ACL p-Value SHSFA (%) 44.4 (40.9-46.2) a 42.1 (37.2-44.) b 43.8 (41.8-47.2) a 0.02 43.2 (40.6-44.9) a 41.67 (36.5-43.0) a 45.6 (43.0-47.5) b <0.001 SHMUFA (%) 50.3 (48.7-53.3) a 52.5 (50.1-54.9) a 46.0 (44.8-48.1) b <0.001 52.1 (49.0-55.3) a 53.1 (50.8-54.9) a 48.36 (45.5-50.0) b <0.001 SHPUFA (%) 4.7 (4.1-6.2) a 4.4 (3.8-6.6) a 7.11 (6.2-10.1) b 0.001 4.13 (3.5-5.2) a 4.3 (3.8-5.1) a 5.76 (5.0-6.3) b <0.001 MUFA/SFA 1.2 (1.1-1.3) a 1.3 (1.1-1.5) b 1.1 (1.0-1.1) c <0.001 1.2 (1.1-1.4) a 1.3 (1.2-1.5) a 1.1 (1.0-1.2) b <0.001 o-3 (%) 0.67 (0.55-0.78) 0.59 (0.43-0.68) 0.58 (0.49-0.77) 0.410 0.66 (0.49-0.77) 0.55 (0.48-0.63) 0.56 (0.45-0.66) 0.200 o-6 (%) 3.6 (3.1-5.1) a 3.5 (3.1-5.5) a 6.1 (5.4-9.4) b <0.001 3.2 (2.6-4.3) a 3.4 (3.1-4.2) a 4.8 (4.3-5.5) b <0.001 o-6/o-3 6.5 (4.7-9.3) a 8.1 (5.5-9.6) a 11.5 (8.2-16.8) b 0.002 5.1 (4.1-7.1) a 7.0 (5.5-9.5) ab 8.9 (6.3-10.6) b 0.004 AI 0.60 (0.54-0.65) 0.54 (0.47-0.66) 0.61 (0.53-0.68) 0.180 0.60 (0.51-0.65) a 0.55 (0.47-0.63) a 0.69 (0.61-0.75) b <0.001 TI 0.88 (0.77-1.14) a 0.92 (0.79-0.99) a 1.1 (1.0-1.2) b <0.001 0.82 (0.71-1.0) a 0.92 (0.82-1.0) a 1.1 (1.0-1.2) b <0.001 h/H 1.9 (1.8-2.1) 2.1 (1.8-2.4) 1.9 (1.7-2.1) 0.140 1.9 (1.8-2.2) a 2.1 (1.8-2.3) a 1.7 (1.6-1.9) b <0.001 * Only cis isomers of the FA were detected. AI: atherogenic index; h/H: hypocholesterolemic/hypercholesterolemic index; IMF: intramuscular fat; MUFA: monounsaturated fatty acid; PUFA: polyunsaturated fatty acid; SFA: saturated fatty acid; TI: thrombogenicity index; UFA: unsaturated fatty acid; SH; total sum of; o-3: omega-3 fatty acids; o-6: omega-6 fatty acids; o-6/o-3 ratio: o-6 to o-3 fatty acids ratio. Values are expressed as median (interquartile range). Fatty acids values are relative percentages of the total amount of identified fatty acids. Different superscripts in values of the same row indicate significant differences with the p-value of the last column. animals-13-00864-t007_Table 7 Table 7 Percentage of saturated and unsaturated fatty acids in sirloin and entrecote meat from Wagyu (WY), Wangus (WN), and Angus-by-Charolais or Limousine (ACL) steers, expressed as a percentage of the total amount of fatty acids detected (median and interquartile range). Sirloin Entrecote Fatty Acid * WY WN ACL p-Value WY WN ACL p-Value Saturated fatty acids (SFA, %) C14:0 2.2 (1.9-2.4) 2.1 (1.7-2.5) 2.1 (1.8-2.5) 0.860 2.2 (2.0-2.4) a 2.1 (1.7-2.3) a 2.4 (2.0-2.9) b 0.003 C15:0 0.32 (0.26-0.28) 0.35 (0.28-0.41) 0.33 (0.30-0.41) 0.410 0.28 (0.22-0.33) a 0.31 (0.27-0.37) a 0.36 (0.31-0.44) b 0.001 C16:0 24.1 (22.5-25.0) 23.6 (21.2-25.1) 23.8 (22.9-25.4) 0.590 24.1 (22.2-25.0) a 23.0 (21.7-25.0) a 25.7 (24.8-26.7) b 0.001 C17:0 0.74 (0.71-1.03) 0.92 (0.81-1.02) 0.94 (0.84-1.08) 0.210 0.74 (0.64-0.89) a 0.78 (0.67-0.93) a 0.91 (0.82-1.06) b 0.001 C18:0 16.6 (14.7-18.5) a 14.5 (12.5-15.8) b 16.6 (15.3-17.9) a 0.002 15.2 (12.6-16.6) 14.4 (12.3-16.4) 16.0 (12.7-18.8) 0.300 Unsaturated fatty acids (UFA, %) C14:1 0.46 (0.39-0.56) 0.56 (0.4-0.66) 0.43 (0.32-0.55) 0.090 0.57 (0.44-0.7) 0.56 (0.50-0.71) 0.61 (0.43-0.72) 0.800 C16:1 2.7 (2.5-3.2) 3.0 (2.6-3.7) 2.9 (2.5-3.4) 0.330 3.48 (2.95-3.69) 3.3 (2.9-3.8) 3.5 (2.8-4.1) 0.900 C16:2 0.32 (0.25-0.47) 0.40 (0.25-0.49) 0.31 (0.22-0.37) 0.300 0.29 (0.26-0.47) 0.27 (0.23-0.36) 0.27 (0.19-0.40) 0.300 C17:1 0.65 (0.55-0.74) a 0.81 (0.68-0.89) b 0.71 (0.65-0.81) a 0.001 0.64 (0.54-0.74) 0.82 (0.62-0.90) 0.71 (0.62-0.87) 0.060 C18:1 46.1 (44.8-48.4) a 47.8 (46.1-50.1) a 41.3 (40.6-43.7) b <0.001 47.47 (44.33-50.25) a 48.4 (46.1-50.2) a 43.3 (40.1-45.0) b <0.001 C18:2n-6 4.0 (3.1-5.1) a 3.5 (3.1-5.5) a 6.1 (5.1-8.0) b <0.001 3.17 (2.64-4.32) a 3.4 (3.2-4.3) a 4.8 (4.1-5.5) b 0.001 C18:3n-3 0.27 (0.23-0.33) 0.30 (0.23-0.39) 0.33 (0.29-0.38) 0.110 0.27 (0.24-0.31) 0.26 (0.23-0.33) 0.30 (0.24-0.35) 0.300 C18:4n-3 0.37 (0.31-0.45) a 0.30 (0.22-0.37) b 0.26 (0.22-0.31) b 0.010 0.39 (0.34-0.44) a 0.28 (0.20-0.34) b 0.29 (0.21-0.36) b <0.001 C20:1 0.25 (0.19-0.33) 0.24 (0.16-0.27) 0.20 (0.16-0.26) 0.250 0.27 (0.19-0.42) a 0.29 (0.16-0.27) a 0.16 (0.14-0.24) b 0.02 * Only cis isomers of the FA were detected. Values are expressed as median (interquartile range). Fatty acids values are relative percentages of the total amount of identified fatty acids. Different superscripts in values of the same row indicate significant differences with the p-value of the last column. animals-13-00864-t008_Table 8 Table 8 Amino acid profiles (g/100 g) in entrecote from Wagyu (WY), Wangus (WN), and Angus-by-Charolais or Limousine (ACL) steers (median and interquartile range). Amino Acid WY WN ACL p-Value Alanine 1.0 (0.87-1.4) 1.2 (1.0-1.3) 1.3 (0.96-1.4) 0.720 Arginine 1.2 (0.92-1.5) a 1.2 (1.0-1.3) a 1.7 (1.5-1.9) b 0.022 Aspartame 2.4 (2.0-2.8) 2.6 (2.3-3.1) 2.9 (2.4-3.1) 0.370 Glutamate 4.2 (3.3-4.6) 4.2 (6.8-5.1) 4.8 (4.0-5.2) 0.330 Glycine 1.4 (1.1-1.5) 1.3 (1.2-1.5) 1.5 (1.3-2.2) 0.470 Histidine 0.88 (0.75-1.0) 0.89 (0.79-0.96) 1.1 (0.82-1.2) 0.420 Isoleucine + 1.1 (1.0-1.3) 1.3 (1.1-1.5) 1.5 (1.0-1.7) 0.290 Leucine + 1.7 (1.5-1.8) a 1.9 (1.8-2.0) ab 2.1 (1.9-2.2) b 0.046 Lysine + 2.2 (1.8-2.5) 2.3 (2.0-2.7) 2.6 (1.7-2.8) 0.580 Methionine + 0.61 (0.52-0.67) 0.68 (0.58-0.72) 0.83 (0.66-0.95) 0.062 Phenylalanine + 1.1 (0.91-1.2) 1.2 (1.1-1.4) 1.3 (1.1-1.4) 0.200 Proline 1.3 (0.96-1.6) 1.2 (1.1-1.4) 1.4 (1.1-1.9) 0.790 Serine 1.1 (0.92-1.3) 1.1 (0.99-1.3) 1.3 (1.1-1.3) 0.450 Threonine + 0.82 (0.75-1.1) a 0.96 (0.70-1.0) a 1.6 (1.1-1.8) b 0.012 Tryptophan + 0.19 (0.16-0.20) 0.20 (0.18-0.23) 0.19 (0.15-0.22) 0.410 Tyrosine 0.94 (0.77-1.1) 1.2 (0.97-1.3) 1.3 (0.74-1.4) 0.100 Valine + 1.4 (1.2-1.5) 1.4 (1.3-1.6) 1.6 (1.3-1.8) 0.400 Total essential amino acids 9.0 (7.8-10.1) 10.1 (8.7-11.1) 11.4 (8.9-12.5) 0.210 + Essential amino acids. Values are expressed as median (interquartile range). Different superscripts in values of the same row indicate significant differences with p-value in the last column. animals-13-00864-t009_Table 9 Table 9 Spearman coefficients for statistically significant pairwise correlations (p < 0.001) between human health-related indexes and blood metabolites in entrecote and sirloin samples. Entrecote BHB TC HDL Urea Moisture -0.481 -0.564 IMF (fresh) 0.465 0.537 SHPUFA -0.478 -0.404 o-6 -0.409 -0.486 Sirloin Moisture -0.541 -0.413 -0.504 IMF (fresh) 0.556 0.412 0.500 SHPUFA -0.485 o-6 -0.473 o-6/o-3 -0.453 Only significant coefficients with absolute values > 0.4 are shown. BHB: b-hydroxybutyrate; HDL: high-density lipoprotein cholesterol; IMF: intramuscular fat in fresh matter; PUFA: polyunsaturated fatty acids. TC: serum total cholesterol; SH: total sum of; o-6: omega-6 fatty acids; o-6/o-3: ratio of o-6 to o-3 fatty acids. 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PMC10000122

The objectives were to determine the effects of graded inclusion rates of wheat bran (WB) on apparent ileal (AID), apparent total tract (ATTD), and hindgut digestibility of nutrients and tested the influence of ileal digesta collection on proceeding fecal nutrient digestibility in pigs. Six barrows with an initial mean body weight of 70.7 +- 5.7 kg fitted with an ileal T-cannula were used. The animals were assigned to a replicated 3 x 3 Latin square design with three diets and three periods. A basal diet was based mainly on wheat, soybean meal, and cornstarch. Two additional diets were formulated to contain 20 or 40% of WB at the expense of cornstarch. Each experimental period consisted of a seven-day adaptation period and a four-day collection period. After the adaptation period, fecal samples were collected on day 8, and ileal digesta were collected on days 9 and 10. Another set of fecal samples was collected on day 11 to determine the influence of ileal digesta collection on proceeding total tract nutrient digestibility. The AID of energy, dry matter (DM), organic matter (OM), crude protein, and phosphorus linearly decreased (p < 0.05) with an increasing inclusion rate of WB from 0 to 40%. The ATTD of energy, DM, OM, crude protein, ether extract, and phosphorus linearly decreased (p < 0.01) as the inclusion rate of WB increased. Hindgut digestibility of DM, OM, and ether extract linearly increased (p < 0.05) with an increasing inclusion rate of WB. The ATTD of GE and most nutrients did not differ between the two fecal collection periods of before and after ileal digesta collection. Taken together, the inclusion of a fiber-rich ingredient reduced ileal and fecal digestibility of nutrients but increased hindgut digestibility of some nutrients, and total tract digestibility of nutrients did not differ whether the fecal samples were collected before or after two days of ileal digesta collection in pigs. digestibility fiber hindgut fermentation swine Konkuk UniversityThe present research was supported by Konkuk University in 2020. pmc1. Introduction Dietary fiber in swine diets increases endogenous losses of nutrients and passage rate of digesta in the gastrointestinal tract of pigs, therefore, decreases the digestibility of energy and nutrients . As high-fiber ingredients are increasingly used in pig diets due to the fluctuation in the price of conventional feed ingredients , research on the effects of dietary fiber on the digestive physiology of pigs becomes more important for precise swine nutrition. While most nutrients are digested and absorbed in the stomach and the small intestine of pigs, a large portion of fibers is not digested and absorbed until reaching the hindgut due to the lack of endogenous fiber-degrading enzymes . Consequently, the ileal digestibility of fibers is much less than that of nitrogen-free extract, protein, and fat. The influences of dietary fiber on total tract digestibility of nutrients have been investigated in many studies , whereas information for the hindgut digestibility (HD) of energy and nutrients is scarce due to the difficulties in determining the HD of nutrients . Therefore, the first objective of the present study was to determine the effects of graded inclusion rates of wheat bran (WB), a high-fiber ingredient, on apparent ileal digestibility (AID), apparent total tract digestibility (ATTD), and HD of energy and nutrients in pigs. To simultaneously determine both ileal and total tract digestibility of nutrients in pigs cannulated with a T-cannula at the distal ileum, fecal samples were collected before collecting ileal digesta to avoid any potential influence of ileal digesta collection on proceeding total tract nutrient digestibility. To our knowledge, however, total tract digestibility values determined using feces collected before and after ileal digesta collection have not been compared. Thus, the second objective was to investigate the influence of ileal digesta collection before fecal collection on the ATTD of energy and nutrients in pigs. 2. Materials and Methods All protocols for the experiment were reviewed and approved by the Institutional Animal Care and Use Committee of Konkuk University (KU15028; Seoul, Republic of Korea). The animal experiment was conducted in an environmentally controlled room at Konkuk University. 2.1. Animals, Diets, and Feeding Six crossbred barrows with an initial body weight of 70.7 kg (standard deviation = 5.7) equipped with a T-cannula at the distal ileum were used . The animals were individually housed in pens (1.2 m x 1.6 m) that were equipped with a feeder and a nipple drinker. The animals were allotted to a replicated 3 x 3 Latin square design with 3 dietary treatments and 3 periods. Potential first-order carryover effects in the Latin square design were minimized using a systemic Microsoft Excel macro developed by Kim and Stein . A basal diet was prepared mainly based on wheat, soybean meal [49% crude protein (CP)], and cornstarch, and two additional diets were formulated to contain 20% or 40% of WB at the expense of cornstarch in the basal diet (Table 1 and Table 2). Soybean oil was used to maintain similar ether extract (EE) concentrations in all diets. All experimental diets contained 0.5% of chromic oxide as an indigestible index. Vitamins and minerals were included to meet or exceed the requirement estimates . Daily feed allowance per pig was calculated as approximately 3% of body weight. The quantity of daily feed allowance was adjusted at the beginning of each period based on the body weight. The daily feed allowance was divided into 2 equal meals and fed to pigs at 0900 and 1700 h. Water was available at all times. 2.2. Sample Collection An experimental period consisted of a seven-day adaptation period and a four-day collection period. After the adaptation period, the fecal samples were collected for 24 h from 0900 h on day 8 . The ileal digesta samples were collected from 0930 to 1700 h on days 9 and 10 . A plastic sample bag with wire was fixed to the T-cannula to collect the ileal digesta . The plastic sample bag was changed every 30 min or when the sample bag was filled with ileal digesta. On day 11, the fecal samples were collected again for 24 h from 0900 h. Collected ileal digesta and fecal samples were immediately stored at -20 degC. 2.3. Chemical Analyses The frozen ileal digesta and fecal samples were dried in a freeze drier. Samples of ingredients, diet, ileal digesta, and feces were analyzed for gross energy (GE; Parr 1261 bomb calorimeter; Parr Instruments Co., Moline, IL, USA), dry matter (DM; method 930.15, ), CP (method 990.03), EE (method 920.39), and amylase-treated neutral detergent fiber (aNDF; method 2002.04). Those samples were also analyzed for ash (method 942.05) to calculate organic matter (OM) and were analyzed for Ca (method 978.02) and P (method 946.06) following the AOAC procedures. Chromium (Cr) concentrations in the diet, ileal digesta, and fecal samples were determined using a UV/Vis spectrophotometer (Optizen 2120UV, Mecasys Inc., Daejeon, Republic of Korea). 2.4. Calculations The AID and ATTD of GE and nutrients were calculated using the following equation :AID or ATTD (%) = 100% - (Nutrdigesta / Nutrdiet) x (Crdiet / Crdigesta) x 100% where Nutrdigesta is the concentration of GE or nutrients in the ileal or fecal digesta, Nutrdiet is the concentration of GE or nutrients in the experimental diet, Crdiet (%) is the Cr concentration in the experimental diet, and Crdigesta (%) is the Cr concentration in the ileal or fecal digesta. Units for GE and nutrients are expressed as kcal/kg and %, respectively. The HD of GE and nutrients were calculated using the following equation :HD (%) = ATTD (%) - AID (%) 2.5. Statistical Analyses Data were analyzed using the GLM procedure of SAS 9.4 (SAS Inst. Inc., Cary, NC, USA). The initial model included dietary treatment, replication, the animal within replication, and the period within replication, but only dietary treatment was used in the final model. Least squares means of each dietary treatment were calculated. Polynomial contrasts were used to test linear and quadratic effects of dietary WB on AID, ATTD, and HD of GE and nutrients. Orthogonal polynomial contrasts were used to test the effect of the fecal collection period, linear and quadratic effects of dietary WB, and interactions between the fecal collection period effect and linear and quadratic effects of dietary WB based on the 2 x 3 factorial treatment arrangement. The experimental unit was a pig. The statistical significance was declared at p-values less than 0.05, and the tendency at p-values between 0.05 and 0.10. 3. Results During the experimental period, all pigs were healthy and readily consumed their feed allotments. Two observations were missing as two pigs lost their cannula during the last period. The AID of GE, DM, OM, CP, and P linearly decreased (p < 0.05) with an increasing inclusion rate of WB from 0 to 40% (Table 3). The ATTD of GE, DM, OM, CP, EE, and P also linearly decreased (p < 0.01) as the inclusion rate of WB increased, but the ATTD of aNDF was linearly increased (p < 0.05). The HD of DM and OM linearly increased (p < 0.05) with an increasing inclusion rate of WB. There was no interaction between the fecal collection period and dietary treatments in the ATTD of GE and nutrients (Table 4). The ATTD of GE and most nutrients did not differ between the two fecal collection periods of before and after ileal digesta collection, except that the ATTD of OM tended to be less (p = 0.076) when fecal samples were collected after ileal digesta collection. The ATTD of GE, DM, OM, CP, EE, and P decreased (p < 0.01) with increasing the inclusion rate of WB from 0 to 40% regardless of the fecal collection period. 4. Discussion The chemical composition of the ingredients used in the present work (Table 1) was within a range of literature values . The analyzed composition of experimental diets was reasonably similar to the expected values (Table 2). As expected, CP and aNDF concentrations in the experimental diets increased as the inclusion rate of WB increased at the expense of cornstarch. As the experimental diets were formulated by using WB at the expense of cornstarch in the present work, the changes in energy and nutrient digestibility by the inclusion of WB would be the reflection of the digestibility differences between WB and cornstarch. Decreased AID of GE, DM, and OM is likely due to the greater nutrient digestibility in cornstarch compared with WB which contains a relatively large quantity of fibers. The AID of starch has been reported to be greater than 90% when fed to growing pigs , whereas the AID of aNDF in WB ranged from 12.5% to 33.3% in the present work. The negative effects of dietary fiber concentrations on the ileal digestibility of GE and nutrients in the present work are in good agreement with a previous study by Huang et al. , who reported reduced AID of GE, DM, and OM by the inclusion of WB at 0%, 9.7%, and 48.3% as a fiber source in a corn-soybean meal-based diet fed to pigs. This observation indicates that the GE, DM, and OM digestibility of WB were less than that of a corn-soybean mixed diet. The AID of GE in the basal diet observed in the present work is greater than that in the previous study , which is more likely due to the different basal diet formulations. Highly digestible ingredients, cornstarch and soybean oil were used at 40% and 3%, respectively, for the basal diet in the present work whereas Huang et al. used corn and soybean meal that are less digestible compared with cornstarch. The larger reduction of AID of GE (84.5% to 62.5%) by increasing the WB inclusion rate in the present work compared with the data in Huang et al. (66.6% to 54.7% with a WB inclusion rate of 48.3%) can be explained by the replacement of cornstarch with WB for other two diets in the present work. Similarly to the AID of GE, the reductions of AID of DM and OM by WB inclusion in the present work were also larger than those in the study by Huang et al. . These observations can also be explained by the use of cornstarch in the present study whereas corn and soybean meal fed to pigs by Huang et al. . The reduction of ATTD of GE, DM, and OM by increasing the inclusion rate of WB is consistent with the observations in previous experiments . In the study by Huang et al. , the ATTD of GE was reduced from 83.6% to 74.1%, which is less reduction of ATTD of GE compared with the present work. Similarly to the AID, the magnitude of energy ATTD reduction is highly dependent on the differences between ATTD of WB and ATTD of ingredients used in the basal diet. The reduction of ATTD of DM and OM was more apparent compared with the previous work . Jaworski et al. also measured the effects of dietary WB on the ATTD of energy and nutrients, in which WB was included at 0%, 15%, and 30% in a corn-soybean meal basal diet. The ATTD of GE was reduced from 91.5% to 82.2%, which is less reduction of ATTD of GE compared with the present work. This is likely due to the different basal diets between the work by Jaworski et al. and the present work and partially due to the greater inclusion rate of WB in the present work. It may be interesting to note that the aNDF concentrations in the basal diets in the present work, the study by Jaworski et al. , and the study by Huang et al. were 4%, 9%, and 12%, respectively, which potentially explains the ATTD of GE in the basal diets. The negative correlation between aNDF and ATTD of GE has been previously reported . The reason for the reduction of the ATTD of GE by increasing the inclusion rate of WB is likely due to the less ATTD of GE in WB compared with cornstarch. Apparently, over 98% of starch is digested and absorbed at the ileal level, but a large quantity of nutrients in WB is not digestible at the total tract level. The reduction of ATTD of DM and OM by the inclusion of WB would also be due to similar reasons as for the ATTD of GE. While most ingested starch is digested and absorbed before the large intestine , the portion of undigested fibers at the ileal level becomes the substrate for microbial fermentation in the hindgut of pigs. The increased HD of DM and OM by the inclusion of WB in the present work suggests that some fibers in WB undigested at the ileal level were fermented in the hindgut and absorbed, possibly as volatile fatty acids. However, Huang et al. failed to find the effects of WB inclusion on HD of DM or OM, which is likely due to the relatively high aNDF concentration (12%) of the basal corn-soybean meal diet. The fibers in corn and soybean meal may have been sufficient for microbial fermentation in the hindgut. Additionally, digesta retention time in the hindgut for microbial fermentation may have been longer in the present work, potentially due to the less fiber concentration compared with the study by Huang et al. , which may also be one of the reasons for the differences between the studies . The linearly decreased AID and ATTD of CP by the inclusion of WB in the present work is likely due to the less CP digestibility of WB compared with wheat and soybean meal that were the sources of CP in the basal diet. The AID of CP in WB has been reported to be less than that in wheat and soybean meal . Huang et al. and Jaworski et al. also observed decreased AID and ATTD of CP when WB replaced corn and soybean meal in the diets fed to pigs. However, the HD of CP was not affected by the inclusion of WB in the present study. Although the possibility of nitrogen disappearance from WB still exists, the present results indicate that the reductions of ATTD of CP by the inclusion of WB were mainly due to the changes in CP digestibility at the ileal level but not in the hindgut of pigs. The present observations are supported well by the results of Huang et al. . In this study, to minimize potential confounding effects of EE with WB inclusion rates on nutrient digestibility, the EE concentrations were equalized in all experimental diets by lowering soybean oil inclusion rates with an increasing inclusion rate of WB as the EE concentration in the WB is greater than that in cornstarch. The reduced AID and ATTD of EE by the inclusion of WB are due to the less digestibility of EE in WB compared with soybean oil. It has been reported that free-form oils are more digestible compared with intact-form oils when fed to pigs . The ATTD of EE in WB was reported to be approximately 60% . The higher AID of EE than ATTD of EE resulted in negative values for the HD of EE in all experimental diets regardless of the inclusion rate of the WB. The present observation is consistent with Huang et al. and Chen et al. who suggested that the negative HD of EE resulted potentially from the synthesis of short-chain fatty acid by microbes in the large intestine of pigs. Additionally, it was reported that the concentration of short-chain fatty acid in the feces increased with the inclusion of WB in pig diets . The lack of effects of WB on the AID of aNDF is likely due to the lack of fiber-degrading enzymes secreted in the small intestine of pigs and limited concentrations of microbes in the small intestine, and the present results agree with a previous study by Huang et al. . In contrast to the AID of aNDF, the ATTD of aNDF was linearly increased as the dietary WB concentrations in the present work. The reason for the increased ATTD of aNDF by the inclusion of WB is unclear. In the previous experiments , the ATTD of aNDF decreased by the inclusion of WB, which indicates that the ATTD of aNDF in WB was less than the ATTD of the basal diets mainly consisted of corn and soybean meal. However, the aNDF concentration of the basal diet in the present work was much less than those in previous studies , and thus, the contribution of microbial cell wall components to the fecal output from the pigs fed the basal diet may have been relatively large in the present work, resulting in low ATTD of aNDF. Microbial cell walls composed of peptidoglycan have been suggested to be analyzed as fiber , mostly aNDF . Cervantes-Pahm reported that pigs fed a fiber-free diet had negative values for ATTD of total dietary fiber, which indicates microbial cell wall components mostly from the hindgut of pigs are analyzed as aNDF. The contribution of microbial cell wall components to the fecal output from the pigs fed the experimental diets containing WB at 20%, or 40%, may have been reduced compared with that from the pigs fed the basal diet, resulting in increased ATTD of aNDF in the present work. The lack of effects of dietary treatments on the AID and ATTD of Ca would be mainly due to the low Ca concentration in WB, assuming similar Ca digestibility values in limestone and dicalcium phosphate. To meet the requirement for standardized total tract digestible P concentrations among the experimental diets, the supplemental level of dicalcium phosphate decreased with an increasing inclusion rate of WB. The decreased AID and ATTD of P with an increasing inclusion rate of WB in the present work can be explained by the less digestibility of P in WB compared with dicalcium phosphate . Although statistical analyses were not performed, the greater ATTD of Ca and P compared with AID values are consistent in the present work, which indicates hindgut absorption of Ca and P in pigs. In agreement, Mok et al. also reported greater ATTD of P compared with AID of P. However, Zhang et al. failed to find the significant hindgut digestibility of Ca and P in pigs. Potential reasons for this consistency include pig breeds, forms of dietary Ca and P, and experimental procedures. Further research is warranted to address the inconsistency in the hindgut digestibility of Ca and P. The decreased AID and ATTD of nutrients by the inclusion of WB in the present work can be at least partially explained by the contribution of ingredient-specific endogenous losses of nutrients and the passage rate of digesta. Endogenous losses of nutrients are divided into basal endogenous losses and ingredient-specific endogenous losses . In the present work, WB-specific endogenous losses are assumed to increase with the WB inclusion rate. As the quantity of the specific endogenous losses of nutrients increases, apparent nutrient digestibility values decrease due to the increased nutrient excretions. Additionally, the passage rate of digesta, although not measured in the present experiment, is also a critical factor that can affect nutrient digestibility in pigs . An increased dietary fiber content makes the passage rate of digesta faster, and eventually, lowers the time for digestion and absorption of nutrients. One of the hypotheses of the present work was that the nutrient digestibility values from fecal samples before ileal digesta collection and after ileal digesta collection would be different potentially due to the influence of ileal digesta collection on the hindgut environment and digesta passage. Fecal collection before ileal digesta collection is a general practice in experiments for determining both ileal and total tract digestibility in pigs . In a study by Wilfart et al. , the ileal digesta samples were collected prior to the collecting of fecal samples, but a three-day blank period was used to minimize the potential influence of ileal digesta collection on proceeding total tract nutrient digestibility. Ileal digesta collection was assumed to lower the amount of digesta that enters the hindgut of pigs, and thus, to increase the digesta retention time for hindgut fermentation. Additionally, ileal digesta collection may also change the microbial populations in the hindgut. Thus, the potential influence of ileal digesta collection on the proceeding fecal digestibility may be dependent upon the amount of fibers that enters the hindgut of pigs . Therefore, to address the hypothesis, fecal samples were collected before and after ileal digesta collection from pigs fed varying fiber concentrations in the present work. Unexpectedly, however, two days of ileal digesta collection did not affect total tract nutrient digestibility on the next day. The lack of influence of ileal digesta collection on proceeding total tract nutrient digestibility is likely due to the ileal digesta collection procedure. In the pigs equipped with an ileal T-cannula, the collected ileal digesta were not the total quantity of ileal digesta but some portion of digesta that came out of the ileum through the T-cannula. Moreover, ileal digesta were collected only for 7.5 h per day. Thus, the quantity of ileal digest that entered the hindgut may have been sufficient to maintain a general digesta retention time and microbial populations in the hindgut of pigs. 5. Conclusions In conclusion, increasing the dietary aNDF concentrations from 4% to 20% reduced the digestibility of energy and nutrients but increased the hindgut digestibility of DM and OM. The total tract digestibility of nutrients did not differ whether the fecal samples were collected before or after two days of ileal digesta collection in pigs. Author Contributions Conceptualization, A.R.S. and B.G.K.; Methodology, A.R.S. and B.G.K.; Formal Analysis, A.R.S. and J.S.; Investigation, A.R.S.; Data Curation, A.R.S. and J.S.; Writing--Original Draft Preparation, A.R.S.; Writing--Review and Editing, J.S. and B.G.K.; Supervision, B.G.K. All authors have read and agreed to the published version of the manuscript. Institutional Review Board Statement Not applicable. Informed Consent Statement Not applicable. Data Availability Statement The data presented in the current work are available. Conflicts of Interest We certify that there is no conflict of interest with any financial organization regarding the material discussed in the manuscript. animals-13-00799-t001_Table 1 Table 1 Analyzed energy and nutrient composition of ingredients (as-is basis) 1. Item, % Ingredient Wheat Soybean Meal Wheat Bran Gross energy, kcal/kg 4009 4413 4234 Dry matter 90.2 91.6 90.4 Crude protein 13.0 48.9 16.3 Ether extract 2.04 2.39 4.17 Amylase-treated neutral detergent fiber 8.27 5.40 39.64 Ash 1.64 5.46 4.42 Calcium 0.10 0.28 0.08 Phosphorus 0.33 0.66 0.95 1 Data are the mean of duplicate analyses of each ingredient. animals-13-00799-t002_Table 2 Table 2 Ingredient, calculated, and analyzed composition of experimental diets (as-fed basis). Item Wheat Bran (%) 0 20 40 Ingredient, % Ground wheat 31.1 32.4 33.5 Soybean meal, 49% crude protein 23.0 23.0 23.0 Cornstarch 40.0 20.0 - Wheat bran - 20.0 40.0 Soybean oil 3.0 2.0 1.0 Ground limestone 0.8 1.1 1.1 Dicalcium phosphate 0.7 0.1 - Vitamin-mineral premix 1 0.5 0.5 0.5 Chromic oxide 0.5 0.5 0.5 Salt 0.4 0.4 0.4 Calculated composition 2, % Crude protein 15.41 18.78 22.12 Ether extract 4.09 3.98 3.87 Amylase-treated neutral detergent fiber 3.81 11.85 19.87 Calcium 0.56 0.53 0.53 Total phosphorus 0.39 0.47 0.64 Standardized total tract digestible phosphorus 0.25 0.28 0.37 Analyzed composition 3, % Gross energy, kcal/kg 4050 4118 4080 Dry matter 92.1 90.6 90.0 Crude protein 16.1 20.1 22.0 Ether extract 4.17 4.23 4.01 Amylase-treated neutral detergent fiber 3.97 11.44 19.72 Ash 4.13 4.69 5.40 Calcium 0.69 0.63 0.68 Phosphorus 0.44 0.48 0.63 1 Provided the following quantities per kg of complete diet: vitamin A, 25,000 IU; vitamin D3, 4000 IU; vitamin E, 50 IU; vitamin K, 5.0 mg; thiamin, 4.9 mg; riboflavin, 10.0 mg; pyridoxine, 4.9 mg; vitamin B12, 0.06 mg; pantothenic acid, 37.5 mg; folic acid, 1.10 mg; niacin, 62 mg; biotin, 0.06 mg; Cu, 25 mg as copper sulfate; Fe, 268 mg as iron sulfate; I, 5.0 mg as potassium iodate; Mn, 125 mg as manganese sulfate; Se, 0.38 mg as sodium selenite; Zn, 313 mg as zinc oxide; butylated hydroxytoluene, 50 mg. 2 Calculated based on the analyzed nutrient concentrations of the ingredients and NRC (2012) values for standardized total tract digestibility of phosphorus. 3 Data are the mean of the duplicate analyses of each experimental diet. animals-13-00799-t003_Table 3 Table 3 Apparent ileal digestibility (AID), apparent total tract digestibility (ATTD), and hindgut digestibility (HD) of gross energy and nutrients in pigs fed diets containing graded concentrations of wheat bran 1. Item, % Wheat Bran, % SEM p-Value 0 20 40 Linear Quadratic Number of observations 5 5 6 Gross energy AID 84.5 75.0 62.5 1.9 <0.001 0.542 ATTD 92.1 84.8 75.7 0.6 <0.001 0.267 HD 7.6 9.8 13.2 2.3 0.108 0.842 Dry matter AID 82.5 70.4 56.8 1.9 <0.001 0.747 ATTD 91.1 83.4 74.2 0.7 <0.001 0.398 HD 8.6 13.0 17.4 2.4 0.021 0.988 Organic matter AID 85.4 75.2 62.4 1.8 <0.001 0.574 ATTD 93.7 87.0 78.8 0.5 <0.001 0.252 HD 8.3 11.8 16.4 2.1 0.016 0.844 Crude protein AID 78.3 77.3 68.9 2.4 0.013 0.232 ATTD 89.5 88.3 84.1 0.6 <0.001 0.082 HD 11.2 11.0 15.2 2.7 0.304 0.519 Ether extract AID 90.6 87.9 83.4 2.5 0.061 0.780 ATTD 80.5 68.5 53.6 6.1 0.007 0.848 HD -10.1 -19.4 -29.8 8.2 0.108 0.956 Amylase-treated neutral detergent fiber AID 12.5 26.1 33.3 9.5 0.151 0.788 ATTD 32.6 40.4 42.5 2.8 0.027 0.444 HD 19.4 14.3 9.2 9.5 0.467 0.999 Calcium AID 25.7 22.0 27.5 5.3 0.812 0.515 ATTD 45.3 40.4 37.5 4.8 0.249 0.874 HD 19.5 18.4 10.1 3.8 0.085 0.478 Phosphorus AID 41.4 21.2 18.5 4.9 0.005 0.175 ATTD 45.3 31.7 21.3 4.9 0.004 0.795 HD 3.9 10.5 2.8 5.9 0.900 0.346 SEM = standard error of the mean. 1 Values for the ATTD and HD were calculated based on fecal samples collected before the digesta collection period (day 8). animals-13-00799-t004_Table 4 Table 4 Apparent total tract digestibility of gross energy and nutrients based on fecal collection period and inclusion rates of wheat bran. Item, % Fecal Collection Period: Before Ileal Collection After Ileal Collection RMSE p-Value 1 Wheat Bran (%): 0 20 40 0 20 40 Time Lin Quad T x L T x Q Number of observations 5 5 6 5 5 6 Gross energy 92.1 84.8 75.7 92.1 83.3 75.3 1.5 0.270 <0.001 0.660 0.754 0.287 Dry matter 91.1 83.4 74.2 90.9 81.8 73.7 1.5 0.167 <0.001 0.864 0.849 0.271 Organic matter 93.7 87.0 78.8 93.7 85.6 78.1 1.2 0.076 <0.001 0.602 0.479 0.269 Crude protein 89.5 88.3 84.1 89.0 86.6 84.2 1.4 0.196 <0.001 0.191 0.631 0.201 Ether extract 80.5 68.5 53.6 78.0 62.1 49.7 10.3 0.253 <0.001 0.969 0.874 0.684 aNDF 32.6 40.4 42.5 38.6 32.1 38.4 6.3 0.405 0.125 0.521 0.111 0.104 Calcium 45.3 40.4 37.5 37.3 34.3 40.1 10.4 0.329 0.587 0.537 0.247 0.697 Phosphorus 45.3 31.7 21.3 37.5 26.7 26.0 10.5 0.475 <0.001 0.410 0.175 0.671 RMSE = root mean square error; aNDF = amylase-treated neutral detergent fiber. 1 Time = time points for fecal grab sampling; Lin, linear effect of dietary treatment; Quad, quadratic effect of dietary treatment; T x L, interaction between time points for fecal grab sampling and linear effect of dietary treatment; T x Q, interaction between time points for fecal grab sampling and quadratic effect of dietary treatment. 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PMC10000123

The dietary cation-anion difference (DCAD) has gotten much attention recently; however, there is not much evidence on organic matter digestibility, blood parameters, dry matter intake, body weight, and carcass features of male sheep fed with different DCAD diets. The effects of dietary cation-anion difference (DCAD) on these traits in male lambs under the environmental high temperatures were investigated in this study. Forty male lambs (average body weight of 39 kg) were randomly assigned to one of five treatments with eight replicates. Lambs were fed diets with DCAD levels ranging from 150 (control group) to 300, 450, 600, and 750 mEq/kg dry matter. This study lasted 100 d and used a 21-d adaptation. The results showed that the control group had the highest dry matter intake, dry matter digestibility, and crude protein digestibility (P = 0.02). Also, the lowest amount of average body weight was observed in the control group (P = 0.01). The results showed the different DCAD levels affected the statistical significance in terms of live weight, carcass weight, length and width of muscle cross section, lung weight, spleen weight, and abdominal fat (P = 0.04). As well, the highest ruminal pH was observed in the control group (P = 0.4). The results of the blood glucose parameter showed that control group had a significant effect on the blood glucose level (P = 0.04). Furthermore, the highest abdominal fat weight was observed in the control group (P = 0.04). There was no statistically significant difference between other traits, including skin weight, head weight, leg weight, carcass length, liver weight, kidney weight, heart weight, testicle weight, tail weight, rumen weight, and lactation weight. In summary, increasing DCAD in the diet could improve the production and carcass quality in lambs under environmental high temperatures. carcass quality dietary cation-anion difference digestion heat stress weight gain Zandi sheep pmcINTRODUCTION The cation-anion difference (DCAD) is defined in its most basic form as the difference in concentrations of the major anions ( S2-) and cations (Na+ and K+) per kilogram of diet dry matter (Block, 1984). Tucker et al. (1988) define the cation-anion difference (DCAD) as the potential positive or negative change caused by nonmetabolizable dietary ion mixtures. Block (1984) demonstrated the initial benefit of feeding a different DCAD by observing that treatment with -172.3 (mEq/kg DM) DCAD could prevent hypocalcemia compared to a control DCAD of +448.6 (mEq/kg DM). Investigations have revealed that diets with a lower DCAD could improve health and develop the economic life of livestock animals (Leno et al., 2017; Melendez et al., 2017; Al-Rabadi et al., 2018). Lately, attention has been given to the interaction of DCAD with vitamin D (Rodney et al., 2018a; Martinez et al. 2018a, 2018b), 5-hydroxyl-tryptophan (Slater et al., 2018), cholecalciferol/calcidiol (Rodney et al., 2018b), calcium (Ca) (Diehl et al., 2018), and the blood Ca level (Collazos et al., 2018; Lopera et al., 2018). Heat stress raises respiratory CO2 loss (respiratory alkalosis) and Na and K loss (coupled with bicarbonate ions) via elevated urine and sweat production. In principle, corrections in blood acid-base balance and associated electrolyte losses may be performed through modification of the DCAD. Diets formulated with DCAD of 200 to 370 mEq/kg DM have been recommended for optimal milk yield in lactating dairy cattle (West et al., 1991), 250 mEq/kg DM for optimal growth in chickens (Mongin, 1981) and pigs (Patience et al. 1987). In ruminants, dietary changes of this nature may directly modify ruminal pH, with associated effects on digestion, DMI, and ruminal microbial efficiency. Modifications in dietary salt concentrations to change DCAD may also directly affect diet digestibility and DMI. The effect of DCAD changes on the performance of sheep has received limited attention. Therefore, the role of DCAD on organic matter digestibility, DMI, digestibility, body weight, blood metabolites, and carcass traits of male lambs has not been directly assessed. Therefore, this study aimed to examine the effects of increasing DCAD from +150 to +750 mEq/kg of DM on organic matter digestibility, DMI, body weight, blood metabolites, and carcass traits in producing male lambs. Furthermore, we conducted this study to explore whether positive DCAD in a high-temperature environment can improve the DMI, production, and carcass traits of lambs. MATERIALS AND METHODS Animals and Management Animals were bred at Islamic Azad University scientific farm from June 2020 to October 2021 and approved by the Islamic Azad University Committee of Experimental Animal Ethics with the number code IU-2020-P016. Using a completely randomized block design, 40 Zandi male lambs (a native sheep breed in the central regions of Iran) with similar body weights (BW; 39.06 kg, SD = 0.55) were blocked to five treatments of eight replicates with 1 lamb per replicate. Lambs were fed in their individual cages during the whole experiment. Animals were fed with varying DCAD levels (mEq/kg DM): +150 (group 1 or Control group), +300 (group 2), +450 (group 3), +600 (group 4), and +700 (group 5). The diet was pelleted as TMR (total mixture ration) with a ratio of concentrate to roughage at 40:60. The sodium bicarbonate (NaHCO3) and sodium carbonate (Na+CO+) were included to increase DCAD. The experiment duration was 100 d, including a 21-d adaption period and a 79-d trial period. After that, in the trial period, lambs were fed the treatment diets at 0900 and 1800 hours. All lambs had free access to water during the experimental process. Ingredients and chemical components of diets are shown in Table 1. Table 1. Ingredients and chemical components of diet for lambs (diets were formulated according to the recommendations of NRC (2001) and gradually provided to lambs. This experiment included five diets with the same energy and protein levels) Items, % Group 1, +150 mEq/kg DM Group 2, +300 mEq/kg DM Group 3, +450 mEq/kg DM Group 4, +600 mEq/kg DM Group 5, +750 mEq/kg DM 40.00 40.00 40.00 40.00 40.00 Wheat straw 3.87 3.66 3.45 3.24 3.03 Soybean meal 19.62 19.62 19.62 19.62 19.62 Barley 7.69 7.69 7.69 7.69 7.69 Corn meal 25.46 25.46 25.46 25.46 25.46 Rice bran 2.25 2.25 2.25 2.25 2.25 Salt 0.90 0.90 0.90 0.90 0.90 NaHCO3 0.07 0.14 0.21 0.28 0.35 K2CO3 0.14 0.28 0.42 0.56 0.70 Analyzed nutrient composition, % DM 45.69 45.49 45.57 44.88 44.45 CP 16.68 16.84 16.91 16.94 16.74 Ash 6.36 6.42 6.55 6.64 6.75 OM 93.64 93.58 93.45 93.36 93.24 ADF 30.75 30.64 30.52 30.44 30.41 NDF 39.94 39.75 39.52 39.44 39.32 Na, mEq/100g DM 4.48 5.00 5.64 6.01 7.00 K, mEq/100g DM 36.78 37.10 42.10 44.10 47.10 Cl, mEq/100g DM 10.57 8.71 8.01 7.72 6.88 S, mEq/100g DM 7.87 8.31 8.73 8.11 9.12 DCAD, dietary cation-anion difference; CHOL, cholesterol; Na, sodium, K, potassium; Mg, magnesium; P, phosphorus. K, potassium; Mg, magnesium; P, phosphorus. Temperature-Humidity Index (THI) monitoring and measurement of rectal temperature Two wet and dry-bulb thermometers were hung on the feeding barns at 1.5 m above the floor to ensure necessary ventilation and be away from sunlight and rain. The National Research Council (1971) then used the following formula to determine the THI: THI=(Td+Tw)x0.72+40.6 Td and Tw are the temperature readings on the dry-bulb and wet-bulb thermometers. The daily averages of THI, Td and Tw were calculated. The rectum (about 4 to 6 cm from the annas) temperature (RT) of each lamb was recorded using an electronic thermometer at 0800, 1400, and 1800 hours every day during the experimental period. Data Collection and Determination of Digestibility Diet samples were collected daily for 21 to 100 d and composited, dried at 65 degC, and pulverized to pass a 1-mm screen for proximate chemical composition measurement of DM, crude protein (CP), crude ash, and total fat (AOAC, 1990), neutral detergent fiber (NDF), and acid detergent fiber (ADF) (Van Soest et al., 1991). Determine dry matter intake (DMI) was done according to National Research Council (1971). An atomic absorption spectrophotometer (iCE3000 SERIES, Thermo Fisher Scientific, USA) measured the contents of Na, and K. Cl content was determined using silver nitrate titration. As previously described, the magnesium nitrate method determined the S level (Wang & Beede, 1990). According to Block (1984), the DCAD was determined using the following equation: DCAD=Na(%)/0.0023+K(%)/0.0039 -Cl(%)/0.00355-S(%)/0.0016 All lambs were weighed individually every 14 d for 100 d. The DMI was recorded daily for each lamb calculated by the allowance of refusals. Carcass Composition At the end of the experiment, after 16 h of food deprivation, the lambs were weighed and slaughtered, the weight of the warm carcass and collected blood were weighed, and then the scalp, legs, lungs, liver, spleen, kidneys, heart, testicles, and fat were weighed. The inside of the intestine and stomach (full and empty) were weighed. Carcass length was measured from the inner edge of the hip bone to the front part of the breastbone with a tape measure. The cross-sectional area of the rectus muscle (between the 12th and 13th ribs) was first drawn using oil paper and then measured with a digital area meter. A caliper measured back fat thickness between the 12th and 13th ribs. The carcasses of the lambs were divided into different parts of the neck, head, chest, throat, thigh, and tail, and the weight of each part was determined. The right half of the carcass of all the lambs was taken to the cold house and kept for 24 h at a temperature of 2 to 4 degC, then each piece was separated by tissue, and the meat, fat (subcutaneous and intermuscular), and bone were separated from each piece. Separated and weighed (AOAC, 1999). In order to chemically analyze the components of the carcass (total fat and meat) obtained from the analysis of different parts of the right side of the carcass, they were mixed and ground, and the samples were analyzed according to the method suggested by AOAC. Rumen Fluid Collection and Volatile Fatty Acids Analysis Rumen fluid samples were collected from each lamb using a stomach tube connected to a syringe. The rumen fluid pool used the double tubes method to avoid excessive saliva contamination. The outer rubber tube (i.d. = 2.5 cm) was specified to the mouth gag. The inner rubber tube (o.d. = 1.2 cm) was the collecting tube (110 cm) handed into the ruminal cavity. Approximately 25 mL of fluid samples were taken 2.5 h after morning feeding every day. The pH was immediately determined with a pH meter (pH221, Lutron, Taipei, Taiwan). After that, the ruminal fluid samples were filtered through two layers of cheesecloth, and 1 mL 6 N HCl was added for preservation. Then, samples were frozen at -20 degC for later analysis of osmolality, volatile fatty acids (VFAs), and NH3-N. The ruminal fluid osmolality was measured with an osmometer (Osmometer 3D3; Advanced Instruments Inc., Boston, MA, USA). The VFAs were prepared and analyzed as described by Thammacharoen et al. (2001). NH3-N was determined with a salicylate-hypochlorite method (Bower and Holm-Hansen, 1980). Measurement of Plasma Metabolites Blood samples were collected monthly for analysis of biochemical parameters of blood serum. The samples were collected from the jugular vein and placed in EDTA vacuum tubes containing EDTA and intravenous vein. The pH of the samples was measured, and then the blood plasma was separated by centrifugation and frozen for further analysis. All samples were determined in terms of minerals such as sodium, potassium and magnesium, phosphorus, glucose, and blood cholesterol through the proposed AOAC methods. Statistical Analysis Balances of energy and protein digestible in the small intestine were calculated for each lamb during each period according to Institut National de la Recherche Agronomique (1989). Intake, weight gain, blood metabolite, and composition, BW, digestibility, and ruminal fluid parameters, were analyzed using the mixed model procedure of SAS Institute (1990) according to a randomized complete block design was used for data analysis. This experiment is performed according to the following statistical model. Yij=m+Ti+Eij In this regard, Yij is the value of each variable; m is the mean of the related trait, Ti effect of treatment, and Eij error rate. And then LSD test method was used to compare the mean of each trait. Statistical significance was defined as P < 0.05. RESULTS THI and Rectal Temperature of Lambs The dry-bulb thermometer readings varied from 29.03 to 39.25 degC, and the mean for the period was 33.94 +- 2.5 degC (SD). The wet-bulb thermometer readings varied from 20.16 to 30.01, and the mean was 25.61 +- 2.24 degC. The calculated THI varied from 75.13 to 86.55, and the mean was 81.23 +- 3.35. During the entire experiment period, the THI was consistently higher than 75. There were no significant differences in rectal temperature between the control and other groups . There were no significant differences in the rectal temperature between DCAD groups (Table 2). Table 2. Effects of dietary cation-anion difference (DCAD) on the rectal temperatures in male lambs Items Treatment Group 1, +150 mEq/kg DM Group 2, +300 mEq/kg DM Group 3, +450 mEq/kg DM Group 4, +600 mEq/kg DM Group 5, +750 mEq/kg DM P-value Rectal temperature, degC 37.8 37.9 37.8 37.6 37.8 0.85 Figure 1. The daily average temperature-humidity index (THI), dry-bulb temperatures (Td) and wet-bulb temperatures (Tw) during the experimental period. The horizontal line at 75 indicates the threshold for heat stress. DCAD Effect on Average Body Weight The analysis of variance showed a statistically significant between the studied treatments in terms of average body weight of lambs in 28 to 42 d (P = 0.01). Comparing the weighting trait showed that the highest weighting was observed in group 4 (P = 0.02) (Table 3). However, this treatment did not have a statistically significant from other treatments in terms of increasing the average body weight of the lambs (groups 3 and 5). As well, the average body weight of the lambs receiving the treatments of the group 4 was 50.37 kg in the whole period; it was statistically higher than the other treatments. Table 3. Effects of DCAD and average body weight of male lambs during each period (kg) Period Treatment Group 1, +150 mEq/kg DM Group 2, +300 mEq/kg DM Group 3, +450 mEq/kg DM Group 4, +600 mEq/kg DM Group 5, +750 mEq/kg DM P-value 0-14 36.32b 38.37a,b 38.06a,b 42.12a 41.12a 0.05 14-28 39.10b 41.01a,b 40.63a,b 45.00a 41.15a,b 0.02 28-42 40.31 42.29 42.79 47.07 42.56 0.24 42-56 45.82 45.66 46.46 49.15 47.60 0.75 56-70 48.75 49.37 48.68 51.61 51.02 0.84 70-84 50.21 55.50 50.91 54.45 53.06 0.06 84-98 51.36 55.50 53.81 55.95 55.26 0.86 98-112 52.36 58.00 56.08 57.66 55.26 0.66 Average 45.90b 47.81a,b 46.83a,b 50.37a 48.64a,b 0.00 DCAD Effect on Carcass Parts The results of analyzing the variance of the relative weight of the carcass and internal organs in the studied lambs are shown in Table 4. The results showed a statistical significance between the treatments in terms of live weight, carcass weight, carcass yield (P = 0.00), length and width of muscle cross section, lung weight, and abdominal fat (P = 0.05). Table 4. Effect of varying dietary cation-anion difference on different parts of male lambs Items Treatment Group 1, +150 mEq/kg DM Group 2, +300 mEq/kg DM Group 3, +450 mEq/kg DM Group 4, +600 mEq/kg DM Group 5, +750 mEq/kg DM P-Value Live weight, kg 61.54b 65.81a 66.90a 68.00a 66.51a 0.00 Carcass weight, kg 29.90b 33.63a 34.11a 35.98a 35.04a 0.00 Carcass yield, % 48.58b 51.10a 50.98a 52.91a 52.68a 0.00 Blood weight, kg 2.46 2.55 2.22 2.13 2.12 0.44 Skin weight, kg 3.44 3.10 3.53 3.01 3.83 0.24 Head weight, kg 2.60 2.31 2.10 2.04 2.04 0.75 Leg weight, kg 0.34 0.33 0.33 0.31 0.30 0.84 Cross-sectional length of the muscle, cm 13.24a 13.87b 13.24c 14.91b 14.41b 0.05 Cross-sectional width of the muscle, cm 5.51ab 5.42b 5.21b 5.95a 5.65ab 0.05 Carcass length, cm 112.24 112.00 111.50 112.23 112.00 0.32 Lung weight, kg 0.82a 0.61c 0.69bc 0.74b 0.84a 0.05 Liver weight, kg 0.36 0.38 0.39 0.40 0.41 0.77 Spleen weight, kg 0.16 0.18 0.19 0.19 0.20 0.07 Kidney weight, kg 0.15 0.16 0.16 0.17 0.17 0.82 Heart weight, kg 0.27 0.28 0.28 0.30 0.30 0.78 Testis weight, kg 0.10 0.11 0.11 0.12 0.11 0.89 Tail weight, kg 3.12 2.88 2.94 3.02 3.08 0.56 Abdominal fat weight, kg 2.77a 1.65c 1.80c 2.11bc 2.39ab 0.05 Muscle The effect of treatments on the cross-sectional area of the muscle in lambs is shown in Table 4. The results showed that the control group had the most significant effect on the cross-sectional length of lambs' muscles. The effects of treatments on the width of the cross-sectional area are also shown in Table 4. The results showed that the maximum cross-sectional width of the muscle was observed in group 4. Also, the lowest muscle cross-sectional width in the studied lambs in group 3 (P = 0.05). Lung The effect of treatments on the weight of the internal organs (lung) is shown in Table 4. The results showed that the highest weight of the lung was observed in group 5 (P = 0.05) and the control group. Also, the lowest lung weight was observed in group 2 (P = 0.05). Spleen The effect of treatments on the weight of the internal organs (spleen) is shown in Table 4. The results showed that the highest spleen weight was observed in group 5. Also, the lowest spleen weight was observed in the control group. Although, there was no statistical differences between different treatments. Abdominal Fat The effect of treatments on fat weight is shown in Table 4. The results showed that the highest fat weight was observed in the control group; however, this amount was not statistically significant from group 5. Also, the lowest weight of the fat was observed in group 2 (P = 0.05). Other Parts of the Carcass The effect of treatments on the weight of the carcass and internal organs are shown in Table 4. The results showed no statistically significance between the studied groups in terms of blood weight, skin weight, head weight, leg weight, carcass length, liver weight, kidney weight, heart weight, testicle weight, tail weight, and rumen weight. However, numerically, the highest blood weight, skin weight, leg weight, carcass length, liver weight, testicle weight, tail weight, and rumen weight were observed in the control group. Also, the highest weight of kidney, heart, tail, and carcass length was observed in group 4. DCAD Effect on Dry Matter Intake and Digestibility The mean comparison of treatments on DMI is shown in Table 5. The lowest DMI and CP digestibility were observed in the control group (P = 0.02). Furthermore, the highest DMI was observed in groups 5 and 4 (P = 0.01). Furthermore, the highest apparent digestibility of DMI, CP, and organic matter were observed in group 4 (P = 0.02). The lowest CP digestibility was observed in group 1 (control) (P = 0.02). Table 5. Effects of dietary cation and anion difference on dry matter intake, organic matter digestibility and crude protein digestibility in lambs Items Treatment Group 1, +150 mEq/kg DM Group 2, +300 mEq/kg DM Group 3, +450 mEq/kg DM Group 4, +600 mEq/kg DM Group 5, +750 mEq/kg DM P-value DMI, g/kg BW 31.60b 36.75a 38.11a 38.21a 37.11a 0.01 Nutrient intake, g/kg BW/d Organic matter 29.59b 34.39a 35.61a 35.67a 34.60a 0.04 Crude protein 4.44b 5.46a 6.53a 6.42a 6.83a 0.04 Neutral detergent fiber 16.60 16.90 19.10 19.07 19.04 0.09 Acid detergent fiber 8.34 8.56 9.33 9.59 9.30 0.84 Apparent digestibility, % Dry matter 73.74b 74.40a 77.10a 78.75a 80.01a 0.02 Organic matter 61.50b 68.05a 73.50a 74.86a 76.23a 0.02 Crude protein 65.87b 73.03a 77.05a 78.08a 78.22a 0.02 DCAD, dietary cation and anion difference; DMI, dry matter intake; BW, body weight; DM, dry matter. DCAD Effect on Rumen Fermentation Parameters The highest ruminal pH was observed in the control group. As well, the lowest ruminal pH was observed in group 5 (P = 0.04). The variation of DCAD did not affect total VFAs, acetate, propionate, butyrate, valeric, isovaleric, acetate/propionate, urea nitrogen, and ammonia (Table 6). Nevertheless, numerically, the highest total VFAs, acetate, propionate, butyrate, valeric, isovaleric, acetate/propionate, urea nitrogen, and ammonia, were observed in the control group. Moreover, the lowest total VFAs, acetate, propionate, butyrate, valeric, isovaleric, acetate/propionate, urea nitrogen, and ammonia, were observed in group 5 (Table 6). Table 6. Effects of DCAD on the rumen fermentation parameters of male lambs Items Treatment Group 1, +150 mEq/kg DM Group 2, +300 mEq/kg DM Group 3, +450 mEq/kg DM Group 4, +600 mEq/kg DM Group 5, +750 mEq/kg DM P-value pH 6.54b 6.68ab 6.64a,b 6.71a,b 6.82a 0.04 Total VFA, mEq/L 75.15 72.21 71.36 72.65 73.40 0.33 Acetate, % 61.46 56.55 58.22 55.13 54.12 0.44 Propionate, % 19.44 19.10 18.53 18.01 17.83 0.24 Butyrate, % 13.60 13.31 13.10 13.04 13.04 0.75 Valeric, % 0.34 0.33 0.33 0.31 0.30 0.84 Isovaleric, % 1.14 0.96 0.98 0.84 1.01 0.06 A/P 3.74 3.20 3.10 3.08 3.01 0.86 Ammonia, mEq/L 112.50 112.00 111.50 111.00 110.23 0.32 A/P, acetic acid/propionic acid. DCAD Effect on Plasma Metabolites The variance analysis results of blood biochemical parameters showed a significant difference between treatments in glucose, cholesterol, and sodium parameters in lambs (P = 0.04, Table 7). The results demonstrated that the control group had the highest effect on blood glucose levels in lambs (P = 0.04). Groups 3, 4, and 5 did not differ significantly in blood glucose levels. There was a significant difference between group 5 comparisons of the other group in blood cholesterol, with the highest effect on blood cholesterol. There were no significant differences between blood phosphorus, magnesium, and potassium levels (P = 0.05). Regardless, the highest amount of phosphorus, magnesium, and potassium in the blood was observed in group 5. The lowest levels of phosphorus, magnesium, and potassium in the blood were observed in the control group (Table 7). The highest amount of sodium in the blood was observed in group 1 (P = 04). Table 7. Effect of dietary cation-anion difference on the plasma metabolites and nitrogen balance of male lambs Items Treatment Group 1, +150 mEq/kg DM Group 2, +300 mEq/kg DM Group 3, +450 mEq/kg DM Group 4, +600 mEq/kg DM Group 5, +750 mEq/kg DM P-value Ca, mmol/L 2.82a 2.71a,b 2.64a,b 2.61a,b 2.42b 0.05 Glc, mmol/L 105.15a 104.21b 103.36c 103.55c 103.40c 0.04 CHOL, mmol/L 116.46b 116.55b 115.22b 115.13b 118.12a 0.05 P, mmol/L 1.45 1.56 1.57 2.05 2.17 0.75 Mg, mmol/L 1.13 1.00 1.76 1.30 1.76 0.82 K, mmol/L 113.39 113.30 113.44 113.50 113.52 0.84 Na, mmol/L 124a 108a,b,c 111a,b 94b,c 84c 0.04 Cl, mmol/L 112.50 112.00 111.50 111.00 110.23 0.32 Nitrogen balance, g/d 9.65 9.40 11.80 12.39 12.06 0.83 DCAD, dietary cation-anion difference; CHOL, cholesterol; Na, sodium, K, potassium; Mg, magnesium; P, phosphorus. K, potassium; Mg, magnesium; P, phosphorus. DISCUSSION In the present study, we investigated the effect of the DCAD diet on the carcass traits, production performance, and digestibility in lambs under the environmental high temperatures. In this regard, Santos et al. (2019) and Lean et al. (2019) used Meta-analyzes to determine the variable effects of DCAD on cattle performance, production, and health. They concluded that prenatal DCAD negatively increases total blood calcium levels during labor and leads to fewer diseases. Our results revealed a statistical significance between treatments in ruminal pH. Based on the findings of this investigation, the kind of cation source used in the diet of lambs has a direct effect on the anion-cation balance of the diet; it can affect ruminal fermentation parameters. The concentration of ammonia nitrogen produced in the rumen is one of the indicators to study the conditions of rumen fermentation. A good diet provides the nitrogen needed for maximum microbial protein, prevents excess ammonia-related waste, and provides sufficient nondegradable protein. Ammonia is one of the nitrogenous compounds used by microbes in rumen to synthesize protein. Rumen ammonia is produced from nitrogenous substances in the diet, urea in saliva, and penetrating urea through the rumen wall (Mathieson and Milligan 1971). Since the concentration of ammonia decreases with increasing DCAD level, it can be stated that increasing DCAD probably increases the ruminal acidity. The decomposition of CP strongly influences the production of ammonia in the diet by micro-organisms and the decomposition of the microbial population due to nitrogen recycling under adverse conditions; it can be concluded that this increase in acidity balances the culture medium in favor of further synthesis. Dudareva et al. (2004) displayed that reducing rumen ammonia production is beneficial in increasing microbial protein production or reducing rumen protein breakdown. Doepel and Hayirli (2011) reported that adding sodium bicarbonate to the diet did not affect ruminal ammonia levels, which was consistent with the results of this study. Reducing or eliminating protozoa from the rumen prevents the energy cycle between bacteria and protozoa, which reduces the breakdown of bacterial proteins. As a result, the flow of microbial nitrogen from the rumen increases, and consequently, the concentration of ammonia decreases (Thanh et al. 2020). Harrison et al. (2012) reported that potassium carbonate could be used as an effective buffer to stabilize ruminal pH, increase DMI, increase volatile fatty acid production, and increase the acetate to propionate ratio, consistent with the results of this research. Shahzad et al. (2008) displayed that increasing the level of anion-cation difference in the diet maintains the fermentation pattern to produce balanced acetate and butyrate, which in turn increases the synthesis of fatty acids of domestic origin, which provides up to 25% of milk fat. Therefore, it is observed that increasing DCAD has a positive effect on reducing environmental pollution related to methane production, which is an important point in modern practical nutrition. Previous studies have shown that increasing DCAD via potassium carbonate increases DMI (Apper-Bossard et al. 2010). As well, the highest apparent dry matter digestibility and the area below the digestibility curve (fluctuations of dry matter digestibility at different times) are related to the treatment containing potassium carbonate and magnesium carbonate with DCAD 905 + mEq level; it can be concluded that the addition of potassium carbonate to dairy cows' diet improves the rumen fermentation and provides potassium to the microbial population, increasing dry matter digestibility (West et al., 1987). Iwaniuk and Erdman (2015) also reported increased dry matter digestibility with increasing DCAD. Funk et al. (1986) investigated the effect of adding potassium with lasalocid on the digestibility of insoluble fiber in neutral detergent and concluded that the simultaneous use of the two increases the digestibility of insoluble fiber in neutral detergent. We showed that with increasing DCAD, ruminal acidity increased, which increased the digestion of insoluble fiber in a neutral detergent, followed by an increase in dry matter digestibility, which was supported by the previous study (Martinez et al. 2018). Furthermore, blood measurements are useful to reflect the metabolic status of animals. Because blood glucose concentration in ruminants is more affected by the amount of ruminal fluid propionate (McDonald et al. 2010). Therefore, various factors such as diet, source, and amount of supplementation that affect ruminal propionate production (Spears et al. 2004) can change the blood parameters. Minor changes in plasma sodium and potassium may be attributed to dietary changes in these minerals because excess sodium and potassium are excreted by the kidneys (Hu and Murphy 2004). Similar results were reported by West et al. (1991), who stated that increasing the level of DCAD (-116 to + 312 mA/kg DM) had no significant effect on plasma sodium and potassium levels. Furthermore, this study showed a significant difference between the treatments in terms of average body weight of lambs in 28 to 42 d. As well, the average body weight of the lambs receiving the treatments of the group 4 was 50.37 kg in the whole period; it was significantly higher than the other treatments. In agreement with the results of the present study, the use of different levels of organic and inorganic supplements in fattening calves caused an increase in BW and a difference in feed intake (Malcolm-Callis et al., 2000). Also, in another experiment, feeding fattening lambs with 20 mg of organic and mineral supplements reduced feed consumption (Garg et al., 2008). Despite this, some other studies reported the lack of effect of mineral and organic supplements on feed consumption. The decrease in feed consumption in lambs receiving supplements in the present study can be caused by various factors. It seems that other factors, such as the decrease in some mineral of the diet, caused the decrease in feed consumption of lambs receiving sodium bicarbonate and carbonate supplements. The use of supplements improved the efficiency of using nutrients to increase the weight of lambs in the present study. Also, the lambs consumed more than three percent of the average live BW of the feed. Therefore, saving in the use of nutrients and improving the efficiency of their use can be one of the possible factors for reducing feed consumption in this research. On the other hand, other factors, such as improving the absorption of nutrients (Yari et al., 2010) and increasing the concentration of VFAs in the blood (Malcolm-Callis et al., 2000), can also reduce feed consumption in ruminant animals. This study also showed a significant difference between the related treatments, including dry matter intake (DMI), organic matter digestibility, and CP. The increase in digestibility may refer to the lack of the rumen microbial population requirements in the basic diet (Mallaki et al., 2015). Some factors such as supplement concentration, supplement source (organic or mineral), diet balance (ratio of forage to concentrate), and base diet supplement concentration were the factors that differentiated nutrient digestibility in different studies (Garg et al., 2008; Kun et al., 2015). Our results also showed that different DCAD treatments affected some carcass parts' weight. These results were consistent with the studies by Luebbe et al. (2011) and Schoonmaker et al. (2013), in which they studied 16 mEq/100 g DM DCAD diets for a long (145 and 196 d) and a short (14 d) period, respectively. As well, Ross et al. (1994) reported that carcass marbling score increased by consuming cationic diets for 84 d. Schoonmaker et al. (2013) suggested that an increase in marbling of cattle fed positive DCAD could be a result of increased production of volatile fatty acid in the rumen due to the ability of sodium bicarbonate to increase ruminal pH and enhance fiber digestibility. These might have been contra-indicative to overall productivity such as carcass weight or marbling deposition if the steers had been fed a negative DCAD diet for a longer period. Therefore, various factors such as diet, source, and supplementation affect product performance. However, further studies and experiments on the effect of DCAD on carcass traits will be needed. In conclusion, we present the associated carcass traits, production performance, and digestibility mechanisms that contribute to the effect of the DCAD diet on lambs under heat-stress conditions. The results showed a significant difference between treatments regarding DMI and protein digestibility, ruminal pH, some blood metabolites, and carcass parts of lambs during 100 d. These results may provide the feasibility of feeding various DCADs to male lambs. Acknowledgments We thank all the teams who worked and who provided technical assistance during this study. We also thank reviewers for their valuable suggestions during manuscript reviewing. Conflict of Interest Statement None declared.

PMC10000124

In the multimodal management of osteoarthritis (OA) in recent decades, the use of feed supplements to maintain joint cartilage has been advocated. The aim of this scoping review is to present the results found in the veterinary literature on the use of undenatured type II collagen and Boswellia serrata in dogs, specifically its use in dogs with clinical signs of OA, healthy dogs after intense exercise or dogs with diseases that predispose the individual to OA. For this purpose, a literature review was carried out using the electronic databases PubMed, Web of Science and Google Scholar, from which a total of 26 records were included in this review: fourteen evaluating undenatured type II collagen, ten evaluating Boswellia serrata and two evaluating the combination of undenatured type II collagen and Boswellia serrata. The review of the records showed that undenatured type II collagen decreases the clinical signs associated with OA, improving the general clinical state with a reduction in the degree of lameness and increase in physical activity or mobility. Evaluating the response to supplementation with Boswellia serrata alone is complicated due to the limited publication of studies and variations in the purity and compositions of the products used, but in general terms, its combination with other feed supplements produces benefits by relieving pain and reducing the clinical signs of OA in dogs. The combination of both in the same product provides results similar to those obtained in undenatured type II collagen studies. In conclusion, undenatured type II collagen and Boswellia serrata are considered a valid option for the multimodal approach to the management of OA and for improving activity during intense exercise, but more studies are needed to conclude whether or not it prevents OA in dogs. osteoarthritis Undenatured type II collagen UC-II(r) Boswellia serrata feed supplements joint inflammation joint degeneration Vetoquinol Especialidades Veterinarias SAUThis review was partly funded by Vetoquinol Especialidades Veterinarias SAU. pmc1. Introduction Osteoarthritis (OA) is a slowly progressive and dynamic degenerative disease that affects mobile joints. The pathophysiology of OA has been considered a process of prolonged and repeated wear and tear of the articular cartilage with alterations in subchondral bone metabolism (sclerosis) and new bone formation at the joint margins (osteophytes) . Today, it is considered a more complex process, with metabolic and inflammatory factors. It is known that the accumulation of articular cartilage matrix degradation products stimulates the release of inflammatory mediators, such as cytokines (IL-1b, IL-6 and TNF-a) and prostaglandin E2 (PGE-2), from the synovium and subchondral bones. These factors act on cartilage by inhibiting proteoglycan synthesis and stimulating its destruction, creating a feedback loop . On the other hand, the endocrine system regulates chondrocyte activity, meaning that many growth factors are responsible for regulating chondrogenesis, such as insulin growth factor (IGF-I) and transforming growth factor b (TGF-b), which play key roles in chondrocyte maturation and survival and matrix formation, limiting catabolism by antagonising pro-inflammatory substances . The means by which such biochemical and mechanical processes contribute to the progressive failure that characterises OA are closely related to the interaction between joint damage, the immune response to perceived damage and subsequent chronic inflammation . The presence of OA in dogs is mostly secondary to a developmental or acquired pathology . The prevalence of OA differs in the veterinary literature, ranging from 2.5% to 20% for dogs older than one year and increasing to more than 80% for dogs older than eight years . Even though OA has been found to have a highly negative impact on patient well-being when compared to other common diseases , a definitive curative treatment has not been identified. For this reason, the strategies used for managing OA are broad, and multimodal management of the disease is generally carried out. Once OA is established, owners must be aware that it is a lifelong disease. It cannot be cured, and the clinician's main objective is to relieve pain, protect the joint from the disease's progression, and establish proper nutritional support, which will improve mobility and allow normal activity to be maintained or regained . For this reason, the management of OA combines pharmacological treatments with non-pharmacological treatments. Non-steroidal anti-inflammatory drugs (NSAIDs) are the usual treatment par excellence for OA and can be used in the long term. However, their use is associated with gastrointestinal effects and hepatic and renal injury . Other drugs that can be combined in instances of acute pain and are synergistic with NSAIDs are paracetamol (acetaminophen) and tramadol, although the bioavailability and half-life of the latter varies between individuals and is therefore not recommended for solo use . Tapentadol appears to have a superior analgesic profile, but further studies are required to assess its efficacy in patients with OA . Chronic OA pain has a maladaptive component caused by central sensitisation; hence, drugs such as gabapentin , amantadine and tricyclic antidepressants (amitriptyline and nortriptyline) may be chosen for neuropathic pain control. In recent years, cannabinoid therapy has been considered an option, given that some studies have associated it with an improvement in quality of life . Recently, species-specific anti-nerve growth factor monoclonal antibodies have been used as a novel therapy for chronic pain associated with OA in dogs, with positive results obtained for at least three months, although more studies are needed to evaluate the duration and analgesic effects . In addition to pain control, feed supplement products are recommended with the aim of maintaining joint cartilage. These feed supplements may reduce the inflammatory state and oxidative stress on the cellular level, acting through different mechanisms of action . The most common oral administration products used for OA in veterinary care are undenatured type II collagen , glucosamine (GLU), chondroitin (CHO) , green-lipped mussels and omega-3 fatty acids . Other feed supplements based on medicinal plants have been described, such as Boswellia serrata , Ribes nigrum, Shiitake (Lentinus edodes), Harpagophytum procumbens and Curcuminoid extracts . An innovative therapeutic approach that combines undenatured type II collagen with Boswellia serrata has provided positive results in patients with degenerative joint diseases in the case of both humans and animals . Undenatured type II collagen is a non-hydrolysed collagen which is extracted from the chicken sternum through a very limited mechanism that allows its native triple helix conformation to be preserved, presenting with a molecular weight of 300 kDa . It must be differentiated from hydrolysed (denatured) collagen, which is broken down into different peptide components by means of enzymes, heat or pH and has a molecular weight of 2 to 9 kDa . In humans, preclinical studies and in vivo clinical trials arrived at conclusions on the beneficial effects of collagen derivatives for OA and cartilage repair used as a nutritional supplement. However, in a large portion of the available in vitro studies, hydrolysed collagen from different sources and of different molecular weights was ineffective for, or even detrimental to, OA cartilage . Undenatured type II collagen is believed to act through an oral tolerance mechanism, thus initiating anti-inflammatory and protective cartilage pathways that prevent the immune system from damaging its own articular cartilage . Oral tolerance is an immune process that the body uses to distinguish harmless material in the gut, such as dietary proteins and the commensal organisms that comprise the microbiome, from potentially harmful foreign invaders . This detection is performed by the gut-associated lymphatic tissue (GALT), which is composed of mesenteric lymph nodes and patches of lymphoid tissue that surround the small intestine, called Peyer's patches, which absorb and select compounds from the intestinal lumen and, depending on the compound, activate or deactivate the body's immune response through the gastrointestinal tract . Laboratory studies on rats have indicated that Undenatured Type II Collagen orally ingested at low doses is transported through intestinal epithelial cells to underlying immune cells in Peyer's patches, where naive T cells transform into regulatory T (Treg) cells that are specifically activated by type II collagen. The activated Treg cells then migrate from the GALT through the lymphatic system and enter the bloodstream . After recognising their target (type II collagen) in the articular cartilage, Treg cells secrete anti-inflammatory cytokines such as TGF-beta, interleukin (IL)-4 and IL-10. This action suppresses the actions of cells involved in the normal degradation of collagen and other extracellular matrix proteins, resulting in a reduction in joint inflammation and associated discomfort . The mechanism by which oral tolerance is activated takes at least two to three weeks and may take up to three months in order to be fully effective . Boswellia serrata is a tree that is widely distributed in India, and its oily gum resin has traditionally been used for centuries in humans as a remedy to treat inflammatory diseases . The resinous part of Boswellia serrata possesses monoterpenes, diterpenes, triterpenes, tetracyclic triterpenic acids and four major pentacyclic triterpenic acids, i.e., b-boswellic acid, acetyl-b-boswellic acid (AbBA), 11-keto-b-boswellic acid and acetyl-11-keto-b-boswellic acid (AKBA) . Boswellic acids with the characteristic pentacyclic triterpene ring can exhibit actions related to the inflammatory cascade, with AKBA being more active, selectively inhibiting a branch of the arachidonic acid cascade (5 LOX) related to the production of leukotrienes without affecting other activities of LOX and COX. The inhibition of 5 LOX and leukotriene synthesis reduces the levels of pro-inflammatory cytokines, leading to decreased cartilage destruction and inflammatory cell chemotaxis and producing a balance in favour of cartilage regeneration . A review of UC-II(r) for the management of OA in companion animals has been published in the veterinary literature. It refers to an undenatured type II collagen patented under a registered trademark . However, it is not clear what type of information is available in the literature on the use of undenatured type II collagen or whether the results are similar to those of UC-II(r). In addition, the authors are currently unaware of any reviews in the literature on the use of Boswellia Serrata alone or in combination with undenatured type II collagen for the management of OA or for keep joint healthy in dogs. For these reasons, a scoping review was carried out regarding the use of undenatured type II collagen and Boswellia serrata as feed supplements in the management of OA and for helps support joint health in dogs. The following research question was formulated: What is known from the literature on the use of products containing Undenatured Type II Collagen and/or Boswellia serrata for keep joint healthy or amelioration of clinical signs of OA in dogs? 2. Materials and Methods A keyword search of the literature was performed to identify all the available existing publications related to the use of Undenatured Type II Collagen and Boswellia serrata for managing clinical signs of OA and helping support joint health in dogs in electronic databases commonly used in veterinary medicine: PubMed/MEDLINE (accessed on 29 January 2023)) and Web of Science (Web of Science Core Collection, MEDLINE, Current Contents Connect and Derwent Innovations Index) (accessed on 29 January 2023)). The research strategy was performed with keywords linked through Boolean terms. Similar terms for "Undenatured type II collagen and Boswellia serrata" were included by combining them with synonyms for "osteoarthritis" in dogs, as follows: ("undenatured type II collagen" OR "type II chicken collagen" OR CCII OR UCII OR "undenatured collagen type II" OR "Boswellia serrata" OR "Boswellia serrata gum resin extracts" OR "Boswellia serrata extract" OR "boswellia extract" OR "boswellic acid" OR "boswellia resin") AND (Osteoarthritis OR osteoarthrosis OR osteoarthroses OR osteoarthrosic OR arthrosis OR arthritis OR arthritic OR "degenerative arthritis" OR "degenerative joint disease" OR "degenerative joint disease djd" OR "cartilage degeneration" OR "inflammatory joint" OR "joint diseases") AND (dog OR canine). Whenever possible, both the singular and the plural forms were used. A language filter was applied, including only articles in English and Spanish. PubMed was searched by title/abstract, and the Web of Science search was performed by topic. In addition, a search was carried out in Google Scholar for the first 100 records that were obtained. Due to the maximum number of keywords allowed by the search engine, the following search strategy was used: ("undenatured type II collagen" OR UCII OR "undenatured collagen type II" OR "Boswellia serrata" OR "boswellic acid") (osteoarthritis OR osteoarthrosis OR arthrosis OR "degenerative joint disease" OR "cartilage degeneration") (dog OR canine). The inclusion criteria for each publication were as follows: (1) the use of undenatured type II collagen, Boswellia serrata or a commercial product containing these active ingredients in the study; (2) evaluation of their effects in managing clinical signs of OA or for helps support joint health using the following methods: owner and veterinarian questionnaires, chronic pain assessment scales, general clinical assessment scales (appetite, mobility and lameness) and quantitative methods using force plate gait and inflammatory biomarkers in the blood or synovial fluid; (3) oral application; and (4) studies conducted on dogs. The exclusion criteria were as follows: (1) reviews, book chapters, expert opinions of clinicians, authorities and/or reports of expert committees; (2) studies evaluating only alterations on the cellular, microbiological or histological level; and (3) studies measuring only plasma concentrations of undenatured type II collagen or Boswellia serrata. The titles and abstracts of all the records retrieved from the search were manually reviewed, and for records that used the trade name of the product or did not specify the feed supplement used for the study, we read the entire record to determine whether it met the inclusion criteria. A double-screened title and abstract analysis was performed using the results obtained from the search in PubMed and Web of Science, and the level of agreement between authors was 95.1%. The principal investigator searched and reviewed all the records. The data extracted from the studies included the date of publication, type of study design, whether or not the study was blind (owner or veterinarian), double-blind (veterinarian and owner) or not blind, the feed supplements used, adverse effects and results obtained through owner or veterinarian questionnaires, chronic pain assessment scales, general clinical assessment scales (appetite, mobility and lameness), quantitative methods (using force plate gait, an accelerometer and a GPS collar), radiographic or ultrasonographic examination and inflammatory biomarkers in the blood or synovial fluid. The records were classified according to the design of the scientific study, as described in Table 1. The scoping review is organised into three different blocks in the Results and Discussion sections. The first part compiles information on the use of undenatured type II collagen, the second describes the use of Boswellia serrata, and finally, the review focuses on the use of the combination of undenatured type II collagen and Boswellia serrata. 3. Results The search strategy resulted in a total of 49 records published up to 29 January 2023. The PubMed search showed 18 articles, and Web of Science yielded 31 articles. After eliminating duplicate articles (n = 16) and two articles that were leaflets or patents, the titles and abstracts of the rest of the records were reviewed, excluding those that did not meet the inclusion and exclusion criteria from the review (n = 8). In the search carried out with Google Scholar, four new records were found (three articles and a congress proceeding) . An article cited in the bibliographical references of the records was added to the review . In total, 28 bibliographic references were obtained for the complete review of the full text. There were three records from conference proceedings; hence, only the abstract was taken into account for the results and discussion. One congress proceeding was subsequently excluded after reading, since it provided the same information as one of the articles already included in the review, and one study was conducted in humans; hence, it was excluded. A flow diagram of the literature search and study selection process is presented in Figure 1. From the data collected on the feed supplements used in the studies, it was recorded that fourteen of them evaluated undenatured type II collagen alone, one of them evaluated Boswellia serrata alone, nine of them evaluated Boswellia serrata together with other feed supplements, and two evaluated the combination of undenatured type II collagen and Boswellia serrata (Supplementary Table S1). The data collected on the year of publication, the type of study design and whether the study was blinded or not are collected in Table 2, Table 3 and Table 4 for undenatured type II collagen, Boswellia serrata and undenatured type II collagen combined with Boswellia serrata, respectively. In total, 73% of the articles were prospective randomised placebo-controlled clinical trials, 12% were prospective randomised controlled clinical trials, and 15% were prospective clinical trials. Of the fourteen studies that evaluated the results after supplementation with undenatured type II collagen, 79% of them were prospective randomised placebo-controlled clinical trials, 14% were prospective randomised controlled clinical trials, and 7% were prospective clinical trials. Of the ten studies evaluating the use of feed supplements with Boswellia serrata, 70% were prospective randomised placebo-controlled clinical trials, 20% were prospective randomised controlled clinical trials, and 10% were prospective clinical trials. Of the two studies evaluating the combination of undenatured type II collagen and Boswellia serrata, only one study was a prospective randomised placebo-controlled trial , while the other one was a clinical trial and did not have a control group . Of the total articles reviewed, 21 of the studies used undenatured type II collagen and Boswellia serrata for the management of OA, and 5 of them used this feed supplements for helps support joint health after intensive exercise or in patients with diseases that predispose to OA, such as hip and elbow dysplasia or medial patellar luxation . 3.1. Undenatured Type II Collagen The first study to evaluate the effects of UC-II(r) in managing OA in dogs was conducted in 2005, comparing two groups of dogs supplemented with doses of 1 mg/day and 10 mg/day of active ingredient UC-II(r) with a placebo group. The results showed a decrease in overall patient pain, pain after manipulation and the degree of lameness after exercise in the two supplemented groups after 90 days . Subsequent studies conducted by the same research group evaluated the effects of active ingredient UC-II(r) at a dose of 10 mg/day, either alone or in combination with other feed supplements, over longer periods (120-150 days) on dogs with OA . They used observational numerical scales from 0 to 10 (0 no pain, 5 moderate and 10 severe and constant pain) to assess overall pain and scales from 0 to 4 (0 no pain, 1 mild, 2 moderate, 3 severe, 4 severe and constant) to assess the pain response after the manipulation of the limbs in flexion and extension, as well as lameness after exercise . The results showed that daily use of UC-II(r) reduced the level of generalised pain by 33% within the first month, with a 44% reduction in exercise-associated lameness and a 66% reduction in pain on manipulation within the first two months . They observed that the maximum benefit occurred 4-5 months after the start of supplementation, with a 62-81% reduction in generalised pain, 83-91% reduction in pain on limb manipulation and 78-90% reduction in exercise-associated lameness . The results obtained with NEXT-II were similar , although the reduction in generalized pain was not as evident as that observed for UC-II(r). The combination of GLU and CHO with UC-II(r) did not provide a greater reduction in generalised pain, pain upon limb manipulation or exercise-associated lameness compared to the reduction obtained with the single administration of UC-II(r) . The combination of UC-II(r) with HCA and CM seemed to show improved benefits through the reduction in overall pain, pain upon limb manipulation and exercise-associated lameness . The management of OA with UC-II(r) was analysed in two studies comparing the use of Robenacoxib and Cimicoxib, and there were no significant differences . The pain assessment questionnaires completed by the owners and veterinarians showed favourable results after the use UC-II(r) . The results obtained using quantitative methods for the gait analysis showed an improvement in step force . In healthy dogs subjected to intense exercise, supplementation with UC-II(r) led to increased speed of movement and activity per kilometre . On the blood level, researchers observed increased lymphocytes; decreased neutrophils, eosinophils and basophils; and decreased biomarker IL-6 and cartilage oligomeric matrix protein . Long-term supplementation with UC-II(r) showed no adverse effects or significant changes in hepatic or renal function , except for blood urea nitrogen in one study . The results are summarized in Table 5. 3.2. Boswellia serrata The only study that explicitly evaluated the use of Boswellia serrata was performed on dogs with chronic inflammatory and degenerative joint and spine disease. In this study, the authors administered a daily dose of Boswellia serrata (the extract contained >=50% triterpenic acids) at 400 mg per 10 kg and observed a beneficial response in 71% of patients. They showed a reduction in clinical signs after two weeks, such as intermittent lameness, lameness after a prolonged rest, local pain and stiffness when walking . The use of Boswellia serrata extracts has been combined with other natural herbal products (Harpagophytum procumbens, Ribes nigrum, Ananas comosus and Curcuma longa) and feed supplements (omega-3 fatty acid, GLU, methylsulfonylmethane, CHO and L-glutamine) showing improvements in all clinical sings (lameness, pain on manipulation, range of motion and joint swelling) . The results obtained using quantitative methods for gait analysis show an improvement in step force . Conflicting results between studies have been observed using assessment questionnaires completed by the owners and veterinarians . The use of feed supplements with Boswellia serrata has been described in growing puppies, and the results seem to show that although it does not prevent OA induced by these pathologies, a lesser degree of OA is evident at 12 months of age . It has been observed that the use of Boswellia serrata together with other feed supplements for patients with OA can result in a decrease in alkaline phosphatase, cholesterol and triglycerides , but no significant change was observed for haematological samples . In addition, changes in OA biomarkers have been observed, including increased IL-10 and Glutathione and decreased IL-6, IL-2 and C-reactive protein . Gastrointestinal disturbances were reported as adverse effects in two studies . The results are summarized in Table 6. 3.3. Undenatured Type II Collagen and Boswellia serrata One product is described in the literature that combines undenatured type II collagen and Boswellia serrata together: Confis Ultra (Candioli Pharma S.r.l., Beinasco, Italy). In the studies published by Martello and colleagues, the authors used one tablet (2 g) of Confis Ultra (Candioli Pharma Srl.) for every 10 kg of weight . The tablets contain 4 mg of undenatured type II collagen (Producer, city, state abbr. if Canada or USA, country), 31.5 mg of Boswellia serrata, Camellia sinensis, green tea extract, copper complexes of chlorophylls (Producer, city, state abbr. if Canada or USA, country), CHO, GLU and Hyaluronic acid. The combined supplementation, after 60 days, showed an improvement in the clinical signs of the patients associated with pain, with lameness being reduced in 69% of patients. Its use seemed to be more effective for more severe degrees of OA, as the owners reaffirmed that after 60 days of supplementation, their pets' mood, lameness and mobility improved, but no such significant difference was observed at 30 days. No adverse effects or analytical alterations were observed . The results are summarized in the Table 7. animals-13-00870-t005_Table 5 Table 5 Literature overview of evidence regarding undenatured type II collagen for the management of osteoarthritis (OA) and joint support in dogs. Reference No Animals, Groups, and Duration Objectives Main Results y Conclusion Deparle et al. 2005 Fifteen client-owned dogs with (Group I) - UC-II(r) (Group II) - UC-II(r) (Group III) 90 days Evaluate overall pain, pain during limb manipulation and exercise-associated lameness Groups II and III showed a decline overall pain, pain during limb manipulation and lameness after physical exercise (p < 0.05) D'Altilio et al. 2006 Twenty client-owned dogs with (Group I) - UC-II(r) (Group II) - GLU + CHO (Group III) - UC-II(r) 10 + GLU + CHO (Group IV) 120 days Evaluate overall pain, pain during limb manipulation and exercise-associated lameness Group II showed a reduction in overall pain (62%), pain upon limb manipulation (91%) and exercise-associated lameness (78%) (p < 0.05) Group IV showed a maximum reduction in overall pain (57%), pain upon limb manipulation (53%) and exercise-associated lameness (53%) (p < 0.05) after 120 days Peal et al. 2007 Twenty-five client-owned dogs with (Group I) - UC-II(r) (Group II) - HCA (Group III) - HCA + CM (Group IV) - UC-II(r) + HCA + CM (Group V) 120 days Evaluate overall pain, pain during limb manipulation and exercise-associated lameness Groups II and V showed reductions in overall pain (62-70%), pain upon limb manipulation (67-91%) and exercise-associated lameness (69-78%) (p < 0.05 Group IV showed a significant reduction in pain (p < 0.05) Bagchi et al. 2009 Dogs with OA (no information about sample size)- Placebo (Group I) - UC-II(r) (Group II) 120 days Evaluate overall pain, pain during limb manipulation, exercise-associated lameness and ground force plate Group II showed a reduction in overall pain (77%), pain upon limb manipulation (83%) and exercise-associated lameness (84%) (p < 0.05) Group II peak vertical force (PVF) was elevated from 7.467 +- 0.419 to 8.818 +- 0.290 N/kg b.w., and the impulse area was elevated from 1.154 +- 0.098 to 1.670 +- 0.278 Ns/kg b.w Gupta et al. 2012 Seven to ten client-owned dogs with OA per (Group I) - UC-II(r) (Group II) - GLU + CHO (Group III) - UC-II(r) + GLU + CHO (Group IV) 150 days Evaluate overall pain, pain during limb manipulation, exercise-associated lameness and ground force plate Group II showed reductions in overall pain (81%), pain upon limb manipulation (87%) and exercise-associated lameness (90%) (p < 0.05) Group III exhibited a reduction in overall pain (51%), pain upon limb manipulation (48%) and exercise-associated lameness (43%) (p < 0.05) Group IV exhibited a reduction in overall pain (36%), pain upon limb manipulation (34%) and exercise-associated lameness (40%) (p < 0.05) Increase in PVF and impulse area in Group II (p < 0.05) Yoshinari et al. 2015 Twenty client-owned dogs with (Group I) - NEXT-II (Group II) 150 days Evaluate overall pain, pain during limb manipulation and pain from physical exertion Group II showed overall pain reduced by 54.3%, pain upon limb manipulation decreased by 65.2% and pain after physical exertion reduced by 62.5% (p < 0.05) Stabile et al. 2019 Forty-six client-owned dogs with (Group I) - UC-II(r) (Group II) 30 days Evaluate the clinical scores and mobility based on the Liverpool Osteoarthritis in Dogs (LOAD) Owner-assessed data showed a similar reduction in the LOAD and mobility scores (p < 0.05) of the two groups There were no significant differences between treatments (p > 0.05) Kunsuwannachai and Soontornvipart 2020 Nine client-owned dogs (13 stifle joints) with type II collagen (Group I) 112 days Evaluate lameness score, radiographic examination scale, ultrasonographic and owner questionnaires: Canine Pain Inventory (CBPI) before and after treatment Lameness score and radiographic examination showed no difference (p > 0.05) Ultrasonographic examination showed differences at 8 and 16 weeks (p < 0.05) The CBPI was different (p < 0.05) Varney et al. 2020 Forty healthy Labrador (Group I) - UC-II(r) (Group II) 14 days Evaluated interleukin-6 (IL-6) and cartilage oligomeric matrix protein (COMP) pre and post run Group II had lower IL-6 and COMP (p < 0.05) Varney et al. 2021 Forty healthy Labrador (Group I) - UC-II(r) (Group II) 91 days Evaluate activity per kilometre and average moving speed Evaluate biomarkers of IL-6, creatine kinase-MM (CKM) and COMP post-run Activity per kilometre was greater in Group II vs. group I among males over all runs (p < 0.05) Average moving speed was greater in Group II (p < 0.05) Group II had significantly lower IL-6 and COMP (p < 0.05) No differences found between groups for CKM (p > 0.05) Cabezas et al. 2022 110 client-owned dogs with -II(r) 10 (Group I) 182 days Evaluate pain, general condition, appetite, mobility and lameness Parameters assessed were significantly lower at four, five and six months (p < 0.05) Stabile et al. 2022 Eighty-six client-owned dogs with OA Four groups:- Placebo (Group I) - Cimicoxib (Group II) - UC-II(r) (Group III) - Cimicoxib + UC-II(r) (Group IV) 30 days Veterinarian evaluation (posture, gait analysis, articular pain, and range of motion), Canine Osteoarthritis Staging Tool (COAST) and owner evaluation LOAD Reduction in LOAD, mobility scores and clinical scores was recorded in Groups II, III and IV (p < 0.05) OAD decreased 29.5% in Group II, 31.4% in Group III and 21.1% in Group IV Reduction in COAST was recorded in Groups II, III and IV (p < 0.05) Varney et al. 2022 Forty healthy Labrador (Group I) - UC-II(r) (Group II) 91 days Evaluated CBPI, LOAD and Gait Analysis Four Rivers Kennel (FRK) inflammatory index score pre-and post-run Group II had lower points for CBPI and LOAD (p < 0.05) Group II had an improved FRK inflammation index score (p < 0.05) Stabile et al. 2022 Twelve client-owned dogs with OA and 10 without free (Group I) - UC-II(r) (Group II) 30 days Evaluation of clinical score, mobility score and owner LOAD. Compare the metabolomic synovial fluid The clinical score, mobility score and LOAD were lower in Group II at 30 days (p < 0.05) The values of b-hydroxyisobutyrate, glutamine, creatine and trimethylamine-N-oxide were decreased in Group II at 30 days (p < 0.05) Citrate increased in Group II at 30 days (p < 0.05) Abbreviations: Glucosamine (GLU), Chondroitin (CHO), (-)-hydroxycitric acid (HCA) and chromemate (CM). animals-13-00870-t006_Table 6 Table 6 Literature overview of the evidence regarding Boswellia serrata for the management of osteoarthritis (OA) and joint support in dogs. Reference No Animals, Groups and Duration Objective Main Results Reichling et al. 2004 Twenty-four dogs with OA or spinal degenerative serrata (Group I) 42 days Evaluate overall efficacy (very good, good, moderate and insufficient) and clinical signs Overall efficacy was very good and good in 71% at two weeks, 67% at four weeks and 71% at six weeks (p < 0.05) Reduction in the severity and resolution of typical clinical signs such as intermittent lameness, local pain and stiff gait were reported at six weeks (p < 0.05) Moreau et al. 2014 Thirty-two owned dogs with OA Two groups:- Placebo (Group I) - Boswellia serrata (Group II-A) - Boswellia serrata Group II-B 61 days Evaluate peak vertical force (PVF) and case-specific outcome measure of disability (CSOM); locomotor activity was recorded using an accelerometer Groups II-A and II-B had higher a PVF at week four and week eight (p < 0.05) No change was observed in CSOM Groups II-A and II-B had higher locomotor activity at week eight (p < 0.05) Manfredi et al. 2018 Forty-two dogs with hip or elbow (Group I) - Boswellia serrata + GLU + CHO (Group II) +/- 300 days Evaluate orthopaedic variables (lameness, range of motion, swelling, pain) Group II had a less severe degree of osteoarthritis at 12 months Musco et al. 2019 Twenty dogs with (Group I) - Boswellia serrata + GLU + CHO (Group II) 90 days Evaluate scores for lameness, pain on manipulation and palpation, range of motion and joint swelling Evaluate metabolic effects All clinical signs (lameness, pain on manipulation and palpation, range of motion and joint swelling) improved in Group II (p < 0.05) Decreased reactive oxygen metabolites and increased biological antioxidants Decreased IL-6 and increased IL-10 (p < 0.05) Muller et al. 2019 Twenty-two owned dogs with (Group I) - Boswellia serrata + Hyaluronic acid (Group II) 84 days Evaluate owner questionnaires: Canine Pain Inventory (CBPI) and Liverpool Osteoarthritis in Dogs (LOAD) Evaluate inflammatory biomarkers CBPI were lower in Group II on days 0 and 84 (p < 0.05) LOAD scores were lower in Group II on day 84 (p < 0.05) Inflammatory biomarker IL-2 decreased in Group II (p < 0.05) Martello et al. 2019 Eight owned dogs with serrata + Cannabidiol (Group I) 30 days Evaluate efficacy using veterinary and owner questionnaires: Helsinki Chronic Pain Index (HCPI) The veterinarian and owner agreed on the high effectivity HCPI scores were lower at 30 days (p < 0.05) Caterino et al. 2021 Twenty owned dogs with + CHO (Group I) - Boswellia serrata + GLU + CHO (Group II) 90 days Evaluate orthopaedic and neurologic signs and force plate gait Improvement in PVF in 80% of patients, despite no statistical significance between groups Martello et al. 2021 Twenty-seven owned dogs with OA - Placebo (Group I) - Boswellia serrata (Group II) 60 days Evaluate C-reactive protein (CRP) and Glutathione (GSH) Owner questionnaire: HCPI GSH was higher in Group II (p < 0.05) CRP was lower in Group II (p < 0.05) No change was observed in HCPI (p > 0.05) Gabriele et al. 2022 Twenty-seven owned dogs with (Group I) - Boswellia serrata (Group II) 170 days Evaluate CRP and GSH Owner questionnaire: HCPI HCPI decreased in Group II (p < 0.05) GSH was higher in Group II (p < 0.05) CRP was lower in Group II (p < 0.05) Cardeccia et al. 2022 Twenty-four owned dogs with (Group I) - Boswellia gum (Group II) 60 days Evaluate CBPI and Hudson activity scale (HAS) No differences were observed in CBPI or HAS between groups (p > 0.05) Abbreviations: Glucosamine (GLU), Chondroitin (CHO). animals-13-00870-t007_Table 7 Table 7 Literature overview of evidence regarding the combination of Undenatured Type II Collagen and Boswellia serrata for the management of osteoarthritis (OA) and joint support in dogs. Reference Study Design and Duration Objective Main Results Martello et al. 2018 Thirteen client-owned dogs with Type II Collagen + Boswellia serrata + GLU + CHO (Group I) 60 days Conduct a general examination and orthopaedic examination and evaluate degree of lameness, overall classification (failure, improved, success) and Helsinki Chronic Pain Index (HCPI) In 84% of patients, the clinical status was "improved", in 8% it was "successful" and in 8% it "failed". In 69% of patients, the degree of lameness improved Dogs with an HCPI between 11 and 19 had a final score similar to their initial score The three cases with the highest index (HCPI > 20) showed a final score that was reduced significantly after 60 days of treatment Martello et al. 2022 Forty client-owned dogs with (Group I) - Undenatured Type II Collagen + Boswellia serrata + GLU + CHO (Group II) 60 days Evaluation of clinical signs of OA by the veterinarian and chronic pain questionnaires using HCPI by the owner An improvement in signs of OA was recorded in Group II (p < 0.05) Lower scores for HCPI were found in Group II (p < 0.05) Abbreviations: Glucosamine (GLU), Chondroitin (CHO). 4. Discussion This article is the first scoping review that compiles the results obtained in studies that used undenatured type II collagen, Boswellia serrata or both as supplementary feeds for managing OA and in dogs. After reviewing the currently available literature, it was observed that there are a limited number of studies focused on this topic, although OA has been managed for almost two decades with both undenatured type II collagen and Boswellia serrata. However, their combined use for managing OA is novel and has become very popular in recent years, with a total of two articles appearing in the last four years . All the studies obtained for the review are prospective, and more than two-thirds of them have a high level of evidence (LoE) . From an ethical point of view, it is not reasonable to leave a patient who has been diagnosed with OA without adequate pain management because they belong to the placebo control group. For this reason, some studies either did not have a control group, or the control group were treated with other food supplements or drugs. As for the Discussion, it is organised into three parts, each with a subheading, referring to the tables previously described in the Results section. 4.1. Undenatured Type II Collagen After reviewing the articles, we observed that not all the studies used the same undenatured type II collagen product in their clinical trials. In most studies, it was used under a registered trademark that refers to a patented undenatured type II collagen known as UC-II(r) (InterHealth Nutraceuticals Inc., Benicia, CA, USA, Flexadin Advanced(r), Vetoquinol SA, Tarare, France and Lonza Consumer Health Inc., Morristown, NJ, USA) . The products on the market that contain this undenatured type II collagen include a daily dose of 40 mg that provides 10 mg of active ingredient UC-II(r). Other studies used undenatured type II collagen referred to as native type II collagen (Confis Ultra, Candioli Pharma S.r.l.) or water-soluble Undenatured Type II Collagen (NEXT-II) (Ryusendo Co. Ltd., Tokyo, Japan) . One article does not specify the undenatured type II collagen product that the authors used . In a recent study, the authors found significant differences when comparing the analytical and physicochemical characteristics of feed supplements currently available on the market that contain undenatured type II collagen . However, these differences could be due to the sample analysis methods used, since there is some discrepancy between the authors' findings . Regardless, the authors recommended that this fact be taken into account when interpreting the results in a review, as it may not be possible to extrapolate the benefits obtained with a specific product in a given study to all products labelled with undenatured type II collagen . Of the fourteen published studies that evaluated the effects of undenatured type II collagen administration alone, we found that there are mainly two research groups who have evaluated this feed supplement for the management of OA: five studies were conducted at Murray State University and three were conducted at the University of Bari . This could explain why the methodologies and results obtained by the first research group are so similar to each other. In the study published by Deparle and colleagues , the authors obtained a more beneficial management of OA with the use of active ingredient UC-II(r) at 10 mg/day rather than 1 mg/day, which justifies the use of this dose in subsequent research and commercial products. However, the results of this study should be interpreted with caution, because the owners were blinded to the product assigned to these groups but not the placebo group. Studies comparing the use of UC-II(r) with other feed supplements, such as GLU, CHO, (-)-hydroxycitric acid (HCA) and chromemate (CM), showed that the former's use for multimodal pain management may be one of the most suitable options . The use of UC-II(r) for managing OA in dogs can provide sufficient clinical benefits on its own, without combinations with other complementary feeds being necessary, as there is no apparent improvement in the pain scales or ground force plate scores . UC-II(r) was compared with the use of the drug par excellence for the management of OA, NSAIDs, by Stabile and colleagues in two studies. Although equipotency with NSAIDs was not demonstrated, when comparing by subgroup and using the Liverpool Osteoarthritis in Dogs (LOAD) scale, the owners observed a reduction in pain similar to that obtained by Robenacoxib or Cimicoxib in the initial stages of OA . However, the results of the Robenacoxib study should be viewed with caution due to the lack of a control group and the fact that the owners were aware of their pets' treatment, which may have caused a placebo effect among the owners . In patients with OA that is secondary to patellar luxation, the use of undenatured type II collagen does not produce an evident reduction in pain-associated lameness, but an improvement in quality of life is observed based on the Canine Brief Pain Inventory scale; hence, it should be considered for cases where surgical intervention is not an option . These results differ from those obtained by the Murray State University research group , which may be due to the use of different scales to assess pain-associated lameness or the fact that the margin of improvement is narrower in patients with OA associated with patellar luxation. Other techniques have been used to evaluate the effects of undenatured type II collagen on patients with OA, such as radiographic examination, with no differences observed post-supplementation, because in its early stages, the diagnosis of OA by radiography is complicated. In contrast, ultrasound examination is a tool for evaluating synovial fluid, which has a better appearance after undenatured type II collagen treatment and may suggest an improvement in inflammation as a result of joint effusion. These results agree with those published by Stabile and colleagues , who evaluated inflammatory biomarkers in the synovial fluid. A recurrence of signs of OA was documented after the cessation of supplementation with UC-II(r) , which reaffirms the benefits of managing patients with this feed supplement. Long-term supplementation with UC-II(r) showed no adverse effects on, or significant changes in, biochemical parameters, suggesting that its administration is well tolerated and safe . Even in response to increased pain-reducing activity, patients on the UC-II(r) supplement showed a more adequate body weight . Varney and colleagues investigated the use of UC-II(r) with the aim of decrease joint inflammation after exercise, and they observed a higher activity per kilometre and less response from inflammatory biomarkers in dogs . 4.2. Boswellia serrata Boswellia serrata, as a feed supplement, has begun to be used in veterinary medicine for patients with OA due to the good results obtained for humans, in whom it has been observed to act as an anti-inflammatory, anti-arthritic and analgesic agent . In the present review, all the articles, with the exception of one , evaluated the use of Boswellia serrata together with other food supplements to manage OA in dogs. This makes it difficult to evaluate the specific benefits that Boswellia serrata can provide in managing OA, since we cannot discern the influences that other feed supplements have on the results obtained in these studies. In addition, it has been observed that some of the articles do not explicitly describe the dose or the percentage of Boswellia serrata used for the study; thus, the comparison between the different studies that used Boswellia serrata extracts (boswellic acids, resin, AKBA) is controversial due to variations in the purity and compositions of the products used. What the studies all seem to have in common, however, is the combined use of the product with other feed supplements or natural herbs, showing a reduction in chronic pain, improving clinical signs, especially in severe cases, and reducing inflammatory biomarkers . The decreases in alkaline phosphatase, cholesterol and triglycerides can be explained by taking into account the fact that triglycerides and cholesterol can be increased due to the inflammatory process that results in OA and reduced mobility , thus demonstrating the beneficial effect of using these feed supplements for pain management and increased physical activity. 4.3. Undenatured Type II Collagen and Boswellia serrata The authors found two articles describing studies in which undenatured type II collagen and Boswellia serrata were used together with other feed supplements (GLU, CHO and Hyaluronic acid) but did not find any studies that used the two alone. One study was a randomised controlled trial , while the other one did not have a control group was not randomised or blinded , and the bias that this may imply for the interpretation of the results must be considered. These studies focused on the management of OA and, to date, there is nothing published on the treatments' combined use for to decrease inflammation or degeneration of the cartilage joints. The fact that they have been combined with other food supplements implies that a conclusion cannot be drawn about the effects obtained using undenatured type II collagen and Boswellia serrata alone, since the authors did not know how GLU, CHO and Hyaluronic acid influenced the results. However, the results obtained are similar to those observed after the use of UC-II(r) for patients with OA in cases where CHO, GLU and Hyaluronic acid were not used. The use of UC-II(r) seems to be more effective for more severe degrees of OA, which may be due to a greater margin for improvement . It is known that it can take up to three months for the oral tolerance mechanism to be fully activated ; thus, the maximum efficacy of undenatured type II collagen may not yet be reached at 60 days. This hypothesis is consistent with the results obtained by the Murray State University research group, who obtained higher percentages for the reduction in OA-associated pain when they treated patients for longer periods of time . 5. Conclusions In conclusion, most of the records published in the veterinary literature on the use of undenatured type II collagen for the management of patients with clinical signs of OA used a study design with a high LoE, which means that the results obtained are reliable. The treatment improves the general condition and appetite, reduces the degree of lameness, and increases physical activity or mobility at the same time that it produces changes on the metabolic level and in the synovial fluid, enabling practitioners to consider it as an option for decrease joint inflammation during intense exercise and upon the appearance of clinical signs of diseases that predispose an individual to OA. On the other hand, the evaluation of the effects produced by Boswellia serrata is more complicated, because it is always combined with other feed supplements, and the compositions of the products are different, but in general terms, it produces benefits in relieving pain and reducing the clinical signs of OA in dogs. Finally, the combination of both with other feed supplements in the same product provides results similar to those obtained in studies of UC-II(r). The combination of undenatured type II collagen and Boswellia serrata alone has not been investigated for the management of OA and helps support joint health in dogs. Its tolerance and low adverse effects render it a feed supplement that can control pain associated with OA in the long term. Due to the slow onset of action of undenatured type II collagen, the use of this feed supplement should not be considered as the first-line option when an immediate analgesic effect is sought. Acknowledgments The authors thank Alvaro Ortega and Elena Garcia-Pedraza for the information provided in relation to the products mentioned in the article. Supplementary Materials The following supporting information can be downloaded at: Supplementary Table S1: Description of the feed supplements used in each of the studies. Click here for additional data file. Author Contributions Writing and edition, A.Z.; review, R.F.-P. Both authors have read and agreed to the published version of the manuscript. All authors have read and agreed to the published version of the manuscript. Institutional Review Board Statement No ethical approval was required. Informed Consent Statement Not applicable. Data Availability Statement The data presented in this study are available in the studies included in this systematic review. Conflicts of Interest The authors declare no conflict of interest. Figure 1 Flowchart of the selection process for scientific records to be analysed in this review. animals-13-00870-t001_Table 1 Table 1 Records were categorised according to the scientific study design. Type Description Study Design I Prospective Randomised Placebo-Controlled Clinical Trial II Prospective Randomised Controlled Clinical Trial III Prospective Clinical Trial animals-13-00870-t002_Table 2 Table 2 Records included in the review based on the search performed regarding undenatured type II collagen for managing clinical signs of osteoarthritis and joint support in dogs. Records of UC-II Year of Publication Study Design Blind Ref. 1 Efficacy and safety of glycosylated undenatured type-II collagen (UC-II) in therapy of arthritic dogs 2005 Type I Yes 2 Therapeutic Efficacy and Safety of Undenatured Type II Collagen Singly or in Combination with Glucosamine and Chondroitin in Arthritic Dogs 2007 Type I Double-blind 3 Therapeutic efficacy and safety of undenatured type-II collagen (UC-II) alone or in combination with (-)-hydroxycitric acid and chromemate in arthritic dogs 2007 Type I No information 4 Suppression of arthritic pain in dogs by undenatured type-II collagen (UC-II) treatment quantitatively assessed by ground force plate 2009 Abstract congress Type I No information 5 Comparative therapeutic efficacy and safety of type-II collagen (UC-II), glucosamine and chondroitin in arthritic dogs: pain evaluation by ground force plate 2012 Type I Double-blind 6 An Overview of a Novel, Water-Soluble Undenatured Type II Collagen (NEXT-II) 2015 Type I Yes 7 Evaluation of the Effects of Undenatured Type II Collagen (UC-II) as Compared to Robenacoxib on the Mobility Impairment Induced by Osteoarthritis in Dogs 2019 Type I Yes 8 Chondroprotective efficacy of undenatured collagen type II on canine osteoarthritis secondary to medial patellar luxation 2020 Type II No 9 Undenatured type II collagen mitigates inflammation and cartilage degeneration in healthy untrained Labrador retrievers after exercise 2020 Abstract congress Type I No information 10 Undenatured type II collagen mitigates inflammation and cartilage degeneration in healthy Labrador Retrievers during an exercise regimen 2021 Type I No information 11 Long-term supplementation with an undenatured type-II collagen (UC-II) formulation in dogs with degenerative joint disease: Exploratory study 2022 Type III No 12 Evaluation of clinical efficacy of undenatured type II collagen supplementation compared to cimicoxib and their association in dogs affected by natural occurring osteoarthritis 2022 Type I Yes 13 Impact of supplemented undenatured type II collagen on pain and mobility in healthy Labrador Retrievers during an exercise regimen 2022 Type I No information 14 1H-NMR metabolomic profile of healthy and osteoarthritic canine synovial fluid before and after UC-II supplementation 2022 Type II No animals-13-00870-t003_Table 3 Table 3 Records included in the review based on the search performed regarding Boswellia serrata for managing clinical signs of osteoarthritis and joint support in dogs. Records of Boswellia serrata Year of Publication Study Design Blind Ref. 1 Dietary support with Boswellia resin in canine inflammatory joint and spinal disease 2004 Prospective multicentre Type III No 2 A medicinal herb-based natural health product improves the condition of a canine natural osteoarthritis model: a randomized placebo-controlled trial 2014 Type I Double-blind 3 Effect of a commercially available fish-based dog food enriched with nutraceuticals on hip and elbow dysplasia in growing Labrador retrievers 2018 Type I Yes 4 Effects of a nutritional supplement in dogs affected by osteoarthritis 2019 Type I Double-blind 5 Placebo-controlled pilot study of the effects of an eggshell membrane-based supplement on mobility and serum biomarkers in dogs with osteoarthritis 2019 Type I Double-blind 6 Effects on Pain and Mobility of a New Diet Supplement in Dogs with Osteoarthritis: A Pilot Study 2019 Type III No 7 Clinical efficacy of Curcuvet and Boswellic acid combined with conventional nutraceutical product: An aid to canine osteoarthritis 2021 Type II Double-blind 8 Preliminary results on the efficacy of a dietary supplement combined with physiotherapy in dogs with osteoarthritis on biomarkers of oxidative stress and inflammation 2021 Type I Double-blind 9 Long-term effects of a diet supplement containing Cannabis sativa oil and Boswellia serrata in dogs with osteoarthritis following physiotherapy treatments: a randomised, placebo-controlled and double-blind clinical trial 2022 Type I Double-blind 10 A pilot study examining a proprietary herbal blend for the treatment of canine osteoarthritis pain 2022 Type I Double-blind animals-13-00870-t004_Table 4 Table 4 Records included in the review based on the search performed regarding the combination of undenatured type II collagen and Boswellia serrata for managing clinical signs of osteoarthritis and joint support in dogs. Records of Combined UC-II and Boswellia serrata Year of Publication Type of Record Blind Ref. 1 Evaluation of The Efficacy of a Dietary Supplement in Alleviating Symptoms in Dogs with Osteoarthritis 2018 Type III No 2 Efficacy of a dietary supplement in dogs with osteoarthritis: A randomized placebo-controlled, double-blind clinical trial 2022 Type I Double-blind Disclaimer/Publisher's Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

PMC10000125

Precision Livestock Farming (PLF) describes the combined use of sensor technology, the related algorithms, interfaces, and applications in animal husbandry. PLF technology is used in all animal production systems and most extensively described in dairy farming. PLF is developing rapidly and is moving beyond health alarms towards an integrated decision-making system. It includes animal sensor and production data but also external data. Various applications have been proposed or are available commercially, only a part of which has been evaluated scientifically; the actual impact on animal health, production and welfare therefore remains largely unknown. Although some technology has been widely implemented (e.g., estrus detection and calving detection), other systems are adopted more slowly. PLF offers opportunities for the dairy sector through early disease detection, capturing animal-related information more objectively and consistently, predicting risks for animal health and welfare, increasing the efficiency of animal production and objectively determining animal affective states. Risks of increasing PLF usage include the dependency on the technology, changes in the human-animal relationship and changes in the public perception of dairy farming. Veterinarians will be highly affected by PLF in their professional life; they nevertheless must adapt to this and play an active role in further development of technology. dairy cattle sensor health welfare veterinarian precision livestock farming This research received no external funding. pmc1. Introduction Digitalization, the Internet of Things (IoT), Big Data--Buzzwords which are increasingly associated with dairy farming. Not only agricultural and veterinary publications are embracing this topic, the general media also cover it: while a headline like "The Connected Cow: Optimizing Dairy Cow Health And Productivity With Technology" appears to make huge promises, another reads "Big Brother in the Cowshed" and may be understood as displaying a certain caution towards technical monitoring of animals and possibly a deterioration of the human-animal relationship. It is unclear how widespread the adoption of digital technology already is. For Germany, the professional association Bitkom stated in 2020 that 80% of farmers are using some kind of digital tool in animal husbandry . The market for precision farming, including all sectors, is expected to grow at an annual rate of 13% to reach a volume of USD 12.9 billion in 2027 . It is therefore to be expected that digitalization is becoming ever more relevant for all aspects of dairy farming, including veterinary services. This paper tries to give an overview of digitalization in the dairy cattle sector and to exemplify the current technology. It will also give a critical assessment of the impact of new technology on the veterinary profession and the animal-human relationship. It aims to provide the reader with an overview of the state-of-the-art components of PLF, the principles of its functioning and a comprehensive analysis of perspectives, limitations, opportunities and risks of PLF. 2. Subject Definition Digitalization refers to various trends and innovations; it has been proposed that it refers to "changing business models" and "new value producing opportunities" by using digital tools. It has to be differentiated from the term "digitization", which only describes putting analogue processes into a digital format . In production animal husbandry, the term digitalization often refers to sensor technology, electronic data processing or automated systems, e.g., in milking. Increasingly, the term "Precision Livestock Farming" (PLF; ) is being used. PLF includes robots or sensors collecting and producing data which are computed and analyzed by standardized operations (algorithms) to produce relevant information. The algorithms analyze for points of interest (e.g., "alarms") that serve as support or basis for decision making, mainly by the farmer himself. The main difference compared with traditional, more retrospective decision making is (1) the immediate availability and processing of data; (2) the integration of data from various different sources; and (3) the rapid decision-making process, implementing changes instantly . PLF therefore implies a system with components sensors, algorithms and interfaces for making practical use of data from livestock farming; the decision-making process, either automatic or supported by applications, is an integral part of PLF itself . In animal production, the implementation of PLF seems best documented in dairy farming . The development of PLF is fast; in 2008, Wathes et al. referred to PLF as being an "embryonic technology", only measuring certain parameters in animals. Twelve years later, in 2020, Cabrera et al. presented the "Dairy Brain", an integrated system automatically optimizing group feeding and providing early recognition of cases of clinical mastitis. Thus, PLF appears to be developing away from only measuring parameters towards integration of different components and decision support or itself making decisions . 3. Elements of PLF 3.1. Sensors A "sensor" is commonly defined as "a device that is used to record that something is present or that there are changes in something" . There are numerous articles reviewing available sensor technology, reflecting the current state of knowledge at the time of publication . In dairy cattle husbandry, "sensor" still seems to be associated primarily with automated heat detection based on activity data. This application is widely being used and is well researched; its practicability had already been documented more than a decade ago . Kempf reported a regular estrus detection rate of 95% using activity monitoring for estrus detection and generally higher effectivity compared to visual heat detection. This study is just one of many examples for estrus detection based on activity. The use of these sensors seems to be economically advantageous and may in the future be integrated with physiological data to target cows more individually for even higher reproductive performance . Most parameters around an individual dairy cow or the herd can be monitored by sensors. One biological parameter may be measured by different sensor types. Rumination monitoring may serve as an example: it may be captured using accelerometers , pressure sensors at the head or microphones . Scheurwater et al. showed the possibility to assess rumination or other activities using an intrareticular pressure sensor. The device was able to identify rumination with an accuracy of 0.92, a sensitivity of 0.97 and a specificity of 0.90. Various sensors are currently available and regularly connected to data systems--they can be understood as the "Internet of Things" (IoT) application to animal husbandry . Knight categorized sensors according to their relative position to the animal into "at cow" sensors (e.g., accelerometers and ruminal pH sensors), "near cow" sensors (e.g., cameras or microphones) and "from cow" sensors (e.g., real-time in-line analytic systems for milk; sensors covering "not-cow data" such as room thermometers also have to be included here). The author further states that while a lot of systems are commercially available, they may not all have been assessed for their viability and validity in a scientifically sound manner. Inversely, various technical options have scientifically proven potential but are not commercially available. Rutten et al. reviewed the development of sensor usage and the changes in their practical application. They describe sensors developing away from being isolated tools, instead becoming integrated into a more complex system on the farm . The phases described are (1) the sensor as tool measuring a biological or physical parameter and delivering this information; (2) interpretation of this data, e.g., an estrus alarm on the basis of changed activity or a ruminal fermentation disorder on the basis of intraruminal pH value patterns; this "alarm function" rests on a standard derived from already retrieved data, the validation of sensor data and finally an algorithm that translates measurements into an alarm. The authors describe as next step (3) data integration, combining various sensor and other information. An example would be the combination of activity and rumination data with farm data such as reproduction information or health events. Those data are generally provided externally by farmers themselves, veterinarians or milk-recording organizations. This enrichment of data leads to a more precise identification of relevant events, e.g., estrus or disease. The algorithms (see below) achieving this identification are trained to recognize data patterns, make predictions on the basis of these patterns and forecast relevant events. Animals at risk of a certain disease can thus be identified early and receive special attention . It is not only disease that can be predicted; another example would be the expected reproductive outcome of an individual animal or the herd as a whole . While this information is so far used only for decision support, the next step according to Rutten et al. would be (4) decision making. PLF information is so far mainly being used to make qualified decisions. Decision making by PLF components would suggest decisions to farmers; it would "provide fast, concrete and simple answers to complex farmers' questions" . Decision making applies to various aspects of herd management; most relevant today are decisions in the field of reproduction and animal health ("alarms") . Herd dynamics and replacement (culling), which appear difficult to model, will certainly be aided by the information provided in a PLF environment . An integrated system can therefore serve as a central hub to collect information from various sources and provide informed decisions covering all areas of herd management including feeding, culling, treatment options and alike . This appears to be the most inclusive development of PLF in a true sense. Sensors are mere providers of information, which are linked with other data from various sources. It has to be stated that so far there is no commercially available system that offers a "complete" monitoring of herds or individuals. Systems tend to be "stand alone" solutions, monitoring only one or two parameters; moreover, sensor systems are rarely communicating with each other, thus limiting the possible insight. As a consequence, farmers will have to rely on a combination of different PLF components if they want to realize full monitoring of their herd . It is therefore important for the whole industry to promote collaboration and communication within PLF . Sensors largely represent the "hardware" component of PLF and often closely interact with the animal. As they are at least partly used for medical purposes, they would, if used in humans, have the status of medical devices. These require specific regulations to ensure health and safety. As surprising as it may seem, there is hardly any regulatory framework concerning sensors in terms of both expected efficacy and safety. Some sensors are attached directly to the animal or even inserted into it, e.g., the digestive system. The disposal of sensors still present after slaughter and the potential of endangering food safety after culling may be seen as risks for human health and the environment. Alarms produced by PLF components may lead to some form of treatment or even culling of an animal; hence, there is a certain risk in system malfunctioning or misinterpretation leading to issues in animal health and welfare. The implementation of a harmonized methodology within one market (e.g., the EU) or across markets (e.g., by an international body) for evaluating these tools and a material vigilance system would undoubtedly be necessary. A licensing system, approval or guarantee scheme safeguarded independently appears to be desirable. 3.2. Algorithms An "algorithm" is being defined as a "set of mathematical instructions or rules that [...] will help to calculate an answer to a problem" . It is therefore a standardized, iterative process which produces information into "specs" needed for decision making. As previously mentioned, algorithms are at the core of PLF as they create meaningful and applicable information out of the pool of available sensor data. They may include data from other sources, e.g., milk recording. The algorithms applied have to cope with numerous challenges: (1) While the current amount of data produced by most systems is generally moderate in size , an increase is to be expected; for example, the more common use of photography-based sensors could lead to huge volumes of information. (2) If systems are interconnected, the data produced is multi-dimensional in nature, including rather simple information, e.g., animal ID, but also physical measurements, optical information and data entries from outside sources. (3) It is also necessary to account for missing, wrong or redundant data, often repetitive in nature or only slightly changing over time. Nevertheless, this less-than-perfect information needs to be processed and relevant patterns recognized. (4) In order to be useful and allow for proactive decision making, the algorithms generally need to involve a prediction part that identified events of interest likely to happen in the future . These challenges can be mastered by application of "machine learning" in which algorithms are trained to recognize relevant parts in a pool of information. This has been extensively reviewed elsewhere . Development of an algorithm can occur in two ways, depending on whether or not the point of interest is known: (1) Information or events that are already known can be analyzed for their informational characteristics and used by an algorithm to be recognized in another data set. (2) The algorithm itself identifies relevant characteristics in sensor data and defines the event; the algorithm thus identifies conspicuous patterns in the data that can retrospectively be linked to an event of interest. This process can therefore be defined as either "supervised" or "unsupervised" learning . Dittrich et al. describe the structure of algorithms for recognizing events. Numerous clinical studies have identified measurable behavioral changes in animals with certain diseases. For example, cows with clinical mastitis show different patterns in lying and standing times, and their patterns of activity and feeding behavior change. These changes are captured by sensors and will be analyzed by algorithms using the recorded patterns of healthy animals as a standard. Deviations from this standard point to the occurrence of clinical mastitis. As behavioral changes usually occur prior to the onset of clinical symptoms, the algorithm does not detect the clinical disease itself but rather predicts its occurrence. Reiter et al. describe the validation of an algorithm belonging to an accelerometer, giving an example of supervised learning. The sensor is attached to the ears of cows and measures motions in a three-dimensional way. The study does not validate the accuracy of the sensor itself in capturing motions; it rather shows the approach to train and validate the ability of the corresponding algorithm to recognize rumination in the motion data set produced by the sensor. The sensor was attached to ten cows, and observers visually identified time segments with rumination. The time segments were then marked in the data set and processed into the algorithm. In a second step, the thus trained algorithm was tested for repeatability of motion pattern recognition in a new data set by comparing sensor labels with visual observation. The study shows a highly correlated agreement between visual observation and recognition by the sensor. For example, the sensor identified an average of 1508 s spent ruminating per hour of resting phases; observation by a human recorded 1492 s of rumination, a mere difference of 16 s per hour spent by the animal resting. It is usually not known how exactly an algorithm in a commercially available system is built. The algorithms are typically not accessible in publications as this is commercially used intellectual property and therefore sensitive information. Some studies, however, illustrate the development of an algorithm. An example is the study by Carslake et al. , demonstrating the practical implementation of algorithms on the example of the behavior of calves. The authors describe the "training" of a sensor from mere measuring activity up to the interpretation of captured data. In this study, the activity and behavior of 13 calves were captured by an activity sensor and at the same time recorded on video. The aim was to correlate both sensor and visual information. The incoherent raw sensor information on activity was therefore "mapped" with labels derived from the visual information captured on video. As a result, it was possible to identify activities such as licking, suckling or playing in the sensor data set. This would allow for recognition of these activities in the field; changes in the behavior could then be identified, this possibly indicating disease. 3.3. Applications Sensors and their respective algorithms are an important element of PLF. These components produce data, either raw or processed, and this data has to be made accessible and meaningful. Applications achieving this are often computer systems installed on the farm; they form a database that contains farm-related information, e.g., production and fertility data or milk production. They can also serve as a platform for sensor-related applications and directly provide communication between these components, serving as an interface between farm production data and sensor-related information. Herd management programs, originally designed for storage and analysis of farm production data, are increasingly serving as exchange platforms combining data originating from the farm. This is developing into a two-way process because alarms from a sensor system are useful to be recorded and, inversely, a sensor-related application will benefit from farm records such as insemination, calving dates and alike. In recent years, a shift towards cloud-based solutions is occurring, in which farm data including sensor information is transmitted towards an external data storage and processed centrally . Relevant information is available to the farm by specially designed applications, through websites or via alarms, e.g., on a mobile phone . There is hardly any scientific literature on the nature and effect of these applications. Rarely, the analyses of herd management programs are used for research, an example being the study by Sorge et al. , in which the economic evaluation of dairy cows in the DairyComp(r) program suite was thematized. In any case, the amount of available data on and from dairy farms appears to be increasing, so too does the need to apply this information into practical decision making. It appears useful to make the generated information simple to grasp by proper design, e.g., by means of graphic illustration . 3.4. Interfaces Connecting data originating from different sources needs interfaces; they link electronic farm-specific data (e.g., insemination dates) with external data (e.g., milk recording data) and data from sensor systems, which may originate from different manufacturers. However, there is a tendency towards closed "ecosystems", in which components from a single manufacturer are combined. The exchange of information between components of different manufacturers may be difficult to establish. Bypassing the ecosystem barrier can be achieved externally by a so-called "warehouse". Relevant data from various sources on one farm is uploaded onto a server; there, it is collected, cleaned and sorted. Relevant information for certain analyses is then extracted from the warehouse and transformed to answer the questions regarding an individual or a group of animals, an individual farm or a cluster of farms . In the long term, linking farm-specific data with data from other sources will facilitate automatic decision making at the farm level. Ferris et al. introduced an integrated system which not only uses all available information at the farm level but also information such as weather data and pricing information. A system like this is no longer designed to measure processes or raise alarms; it predicts production-related processes at the herd level and proposes management decisions or implements these decisions autonomously. Cabrera et al. illustrate the use of such a system on the example of udder health. Animals can be identified as being at high risk for developing mastitis as early as in their first lactation. This classification is achieved by using genetic information , animal behavior and production data from the milking parlor or milk recording. The system gives recommendations for this animal such as exclusion from breeding or early culling due to low potential. Another concept integrating data is the "digital twin" , in which available information is used to create a virtual image, a doubling of a certain entity. In the case of dairy farming this could mean the 'twinning' of an individual animal, a group of animals or a herd as whole. The digital twin can then serve as a benchmark which points to risks in the future as it anticipates certain developments. This may also help in objectively assess animal welfare. The principle of this is already used in teaching so that the effect of certain decisions on a dairy herd can be modeled and studied . 4. PLF Influencing Animal Husbandry The rapid development of PLF and the possibilities it creates are evaluated differently in the literature. There are few evidence-based studies demonstrating a measurable effect of PLF usage on animal health or productivity. Knight states that while sensor technology can measure a lot of biological parameters and has become very effective in recognizing disease or disease risk, it remains unknown what the effect of their presence on the production and health parameters really is. Determining this effect faces the problem that this can only be done indirectly. As long as a PLF system on a farm is not acting and deciding fully automatically and autonomously, it may only influence human behavior. Individual farmers are likely to respond differently to automatically generated information, and their decisions may be affected by PLF to a varying degree. Eckelkamp and Bewley evaluated the behavior of dairy farmers using PLF technology. The study assessed the intensity with which farmers evaluated the health alarms generated by PLF systems and how they responded to this information. It was shown that farmers generally considered the alarms to be "true"; this did, however, not automatically trigger a certain behavioral response, e.g., examining an animal. Especially if farmers themselves did not expect animals to have problems (e.g., cows not in the transition phase), alarms tended to be ignored. The number of alarms generated by the system affected the probability of farmers acting: at 20 or fewer alarms per day, this probability was higher. Furthermore, the authors showed a "habituation": the longer the system was present on the farm, the more likely alarms were to be disregarded. This points to a risk of PLF application: the presence of PLF systems is no reliable indicator for a better or improved situation concerning animal health and welfare on a dairy farm. As PLF systems continuously produce data (and sometimes alerts in the case of deviation of observed data from expected data), stress can be put on the user, especially when the tools lack specificity (alerts generated on non-diseased animals). Therefore, a difficult compromise needs to be made between sensitivity to detect alterations and acceptable specificity, as a specificity that is too high will lead to false positive alerts for the user. This is illustrated by a study focusing on the prediction accuracy of a system monitoring rumination and activity; the sensitivity was programed to produce less than four false positive alerts/100 cows/day. As a result, however, only 30% of disease events were detected, based on rumination and activity drops . Faced with this incessant flow of data and alerts, the owner may lose confidence in the tool and even may altogether stop using information at the risk of missing sick animals. There is also a risk that alarms may be "over-used" with no critical review or examination of the animal. The possible advantages of PLF usage therefore largely rely on the performance of the tools and their operating conditions. These behavioral aspects of PLF application need to be considered in development of future applications. In summary, the effect of PLF applied on dairy farms remains unclear. In a study on Italian dairy farms , no higher milk production was found on farms using sensor technology. A Dutch study evaluated the effect of sensors being used for udder health and fertility monitoring on related parameters. Only small effects were found, and milk production was not better on farms using the sensor systems. The sensors at that time were, however, able to generate only very few and selective health alarms. Various sensor-based systems have been proposed for detection of lameness in cattle; however, their usefulness and cost-benefit ratio are often not acknowledged by farmers despite lameness being a major animal health issue . It therefore appears necessary to demonstrate the benefit of this technology under farm conditions. As mentioned before, a scheme to objectively assess the impact and risks of a PLF system appears to be necessary. 5. Animal Welfare and Animal Health Animal welfare concerns numerous aspects going well beyond animal health alone; while health is important, animal welfare also involves aspects of animal behavior and emotion . Sensor technology can capture various aspects: behavior of the animal (movement and activity), time budget, but also vocalization and social interaction can be monitored. Furthermore, physical parameters affecting behavior, e.g., ambient temperature, are monitored easily. Combining this information may very well contribute to a general improvement in animal welfare on dairy farms . Due to the constant nature of the systems, PLF may be able to monitor animals in a more intensive, more reliable and more consistent manner than traditional animal monitoring by humans alone is able to achieve . Some examples below illustrate the possibilities PLF is opening for the management of dairy farms with special respect to animal health and welfare. This list can by no means be exhaustive and covers both technology that is already available and also possibilities that so far are merely a concept. The previously mentioned monitoring of rumination is possible using different technological approaches and may serve in the early detection of disease, especially metabolic conditions. In a study using 312 dairy cows, Gusterer et al. showed that changes in rumination activity detected by a sensor system markedly preceded clinical diagnosis of a disease. The study used the monitoring of rumination frequency and length of rumination bouts and compared this information with the results of daily standardized clinical examination with ss-hydroxybutyrate measuring, all in the period from calving until day 8 post-partum. It was shown that rumination monitoring identified clinical disease up to five days before a clinical diagnosis could be made. Optical systems to determine the body condition score (BCS) of cattle have become commercially available in recent years. The BCS is generally understood as the main risk factor for metabolic disease in the transition period of dairy cows and is mainly monitored using traditional adspection methods. Optical monitoring using a three-dimensional capture of body points is more sensitive to changes and describes the BCS better and more consistently than the traditional system using half or quarter scoring points . As the BCS is a parameter intricately linked to metabolic processes, these systems open the door for high-throughput phenotyping to finally improve genetic selection on resilience parameters. Monitoring locomotion health in dairy cattle may benefit from PLF technologies. Digital systems that are collecting and sorting diagnoses from hoof care have been available for some years; they are helpful to give a more systematic, dynamic and more objective picture of diseases of the feet. . In particular, control of digital dermatitis may be improved by digital documentation as the associated algorithms help to describe the development on herd level better and aid in identification of herd-specific risk factors . Animals experiencing lameness show a different locomotive behavior , and the transition from soundly walking to mildly lame is regularly missed by farmers, the identification of which would help in controlling lameness at the herd level . The use of accelerometers has been described for lameness detection and appears to be a reliable method . Early detection of locomotion disease is also possible by use of optical monitoring systems, which, similarly to the abovementioned principle of metabolic disease detection, may recognize clinical alterations earlier than traditional methods and thus help to improve locomotion health in a herd . Heat stress is regularly identified as a challenge to animal wellbeing and production. So far, most farms operate their cooling regime on fixed schedules or according to ambient temperature. The actual level of heat stress can be predicted using environmental and animal production data and hence provide a more exact description of the challenge . The inclusion of actual body temperature data may refine strategies, making heat abatement more effective The control of feeding dairy cows is regularly impaired by the challenge to correctly assess the actual dry matter intake of the animals. Optical monitoring using the technique of "photogrammetry" may provide a more reliable and constant information flow. Photogrammetry converts optical data, i.e., the volume of feed, into mass. The monitoring of changes in volume and consequently in mass allows for the calculation of feed intake by the animals . Generally, the connection of various technical components related to the feeding process offers the perspective to make feeding more animal-oriented and efficient . Capture and analysis of livestock vocalization is so far merely a concept. It has been shown, however, that different animal vocalizations may be sorted and classified . In the long term, this may help to assess animal affective states in a quantitative and thus measurable way, allowing for an objective evaluation of animal welfare in dairy herds. Thus, the assessment of biological data from various sources may enable the analysis of "affective states" , which so far form a rather inaccessible part of animal welfare . Figure 1 Overview of a PLF system of various components on a dairy farm. The automated milking system (AMS) in this case serves as a complex of sensors capturing data from an individual animal. The attached algorithm interprets the data and creates predictions that are transmitted to the farmer to aid decision making . Figure 2 Overview of currently used devices to capture biological data from animals. All these sensors are connected and can hence be understood as the application of the "Internet of Things" to the dairy cattle sector. Figure 3 Elements of Precision Livestock Farming, adapted from Rutten et al. . While sensors are measuring parameters, algorithms are recognizing relevant changes and interpreting them (here with the example of estrus detection). Additional information from other sources helps to classify the event. The final stage is a system that autonomously acts on the basis of the information--in this example, it decides on inseminating the animal. With respect to animal breeding, the large-scale and continuous collection of phenotypic data (e.g., milk production, growth rate, behaviors and disease resistance for example) opens the way to what is known as "high-throughput phenotyping", i.e., the characterization of all the apparent characteristics of an individual. This can happen continuously and almost in real time using connected sensors and tools. This phenotyping allows for genomic studies that rapidly select animals carrying the characteristics of interest (such as disease resistance, a phenotype that is very difficult to characterize classically). It may also open up perspectives for phenotyping on traits not currently available and hence creating new possibilities for selection. To do this, it is fundamental to associate different people from different backgrounds to develop the tools, not forgetting to associate the end user in particular . Connected tools could make it possible to bring out the individual in the group and thus give visibility for the breeder to isolated individuals, especially in large numbers. However, the opposite effect is also realistic; an extreme standardization of the animals could lead to genetic impoverishment by eliminating individuals that go beyond the hoped or expected standards. 6. Human-Animal Relationship The increasing presence of PLF technology on dairy farms is likely to change the interaction between man and animal. An "alienation" further separating the animals from the people responsible for them appears possible. The animal would become a mere functional component in an increasingly efficient production system. Inversely, PLF seems to offer huge potential to better recognize and respond to animals' needs and prevent factors negatively influencing their wellbeing. This spectrum of possibilities is laid down in a report by a Dutch study that gives a comprehensive overview on the respective risks and opportunities . Covering the different perspectives of the animal, its owner and society in general, the study tries to gauge positive or negative consequences digitalization, electronic monitoring and automated decision making might bring to animal husbandry. Opportunities are generally seen in a more exact provision and care for the animals' needs, an early detection and prevention of disease; an incorrect usage of the systems and the information received from them or malfunctioning of systems themselves are identified as major risks. The authors recognize the danger of reducing the animal to a mere function and "thing", i.e., that animals may be understood as a pure functional part of the production process, discarding its properties a sentient being. From the producer's perspective, PLF opens perspectives to a more efficient production process and reduction of workload; on the other hand, the digitalization makes a farm vulnerable to data theft or cyber-attacks. Moreover, a certain dependency on the manufacturers of the systems is created. From the perspective of society, there is the chance to create more transparency of the production process, as animal husbandry may better be understood, and its principles more effectively communicated. However, extensive application of PLF technology may alter the image of animal husbandry towards being "over-engineered", this conflicting with the traditional perception of the farming sector. This conception may also be shared by farmers themselves. However, individual preferences are to be considered. For example, PLF-associated technology can be perceived merely as a tool not changing the production system itself at all. Interestingly, farmers that have a strong emotional relationship with their animals seem to recognize the potential of technology more positively . An ambivalent attitude in society is reported by Krampe et al. . The study evaluated the perception of PLF in consumers of three European countries; the already mentioned "over-engineering" of animal husbandry was identified as a concern. However, opportunities were recognized by consumers nonetheless: improvement of animal health and welfare, a positive impact on sustainability and transparency of the market-chain were mentioned. The information provided by PLF technology can very much alter the perception that the breeder or owner has of his animal. PLF allows for a farmer to go beyond the basic, production-related information of his animals: now, movement, behavior (feeding, sleeping) and exact location can be monitored and assessed. This may allow for a better understanding of the animal and a more personalized approach, bringing back the "individual animals perspective" even to very large herds. The absence of a specific regulatory framework for hardware components on animals means that the choice to equip a group of specific animals with a device is often made by the owner exclusively. While it is obvious that there can be no question of obtaining the direct consent of the animal, it is legitimate to question the circumstances in which one can freely decide whether or not to equip an animal, particularly when the objects are of an invasive character. Expertise from a specialist in animal health or behavior seems useful, especially when validating the equipment and, if necessary, the choice of technical solution. It is also essential that users be properly trained in all the risks implied by the tool. 7. Summary--PLF from the Veterinarian's Perspective Precision Livestock Farming is a collective term that describes a rapidly expanding complex of sensor technology, algorithms and applications. PLF aims to optimize animal production, health and welfare. It has become impossible to fully describe and evaluate all developments, especially as only a few have been evaluated scientifically. As has been shown on the example of dairy husbandry, the interaction of sensors and information technology creates new possibilities such as early disease recognition, monitoring of animal behavior and possibly improved animal welfare through optimized management. However, neither alarms nor information seem to be the objective of PLF; it is rather an integrated view on the farm, and possibly the whole industry, being interconnected. Decision making, not decision support, is the perspective. The information available increases in accuracy, complexity and volume. The data available may allow for a closer observation of farm events and development, better preparation of clinical activities or advice due to better data at hand and a more effective follow-up after changes have been implemented. This creates opportunities but also risks: the dependency on algorithms and their correct function and also the impossibility of comprehending all information collected and created may be hazardous. The veterinarian may be reduced to a component within the decision-making process which he or she may not be able to control or influence. Furthermore, the human-animal relationship may be affected, with the individual animal being viewed as a technical component. The same may apply to veterinary practice with treatments automatically evaluated and suggested beforehand and their success already being predicted based on historic data. This would clearly conflict with the ethical commitment of the veterinary profession. While PLF systems can provide early detection or even prediction of disease, it is unclear how the veterinarian can deal with this. An animal that has been predicted to develop clinical disease can nevertheless still be clinically normal; a treatment using pharmaceuticals is hardly permissible if the indication is not stated by a veterinarian. This touches the fundamentals of the veterinary profession as it challenges the clinical examination and diagnosis. The opportunities are there, nevertheless. The systematic collection and evaluation of animal records of all kinds opens new perspectives in herd health. Continuous and possibly remote access by the veterinarian to the data generated by PLF systems opens interesting perspectives in terms of teleconsultation or tele-expertise, creating new perspectives in optimizing health and wellbeing of the animals. This may especially be interesting for farms in remote areas, away from centers of veterinary expertise. However, the help that these systems could bring cannot hide the need to address the issue of planning resources and permanent health monitoring. The mass of data generated may make it possible to rethink the client-veterinary relationship, developing the field of telemonitoring and an "increased" clinical examination for the veterinarian, who would thus have access to data otherwise not available. Conversely, the farmer should not be overwhelmed with information and should contact the veterinarian in a manner that is agreed between both parties. It is indeed the complementarity of the approaches that should benefit the animal. The challenge is then to explain to customers what attitude to adopt when faced with these tools, which cannot entirely replace cow-side care. The use of PLF technologies will also require veterinarians and owners to be adequately trained in the use of these tools and the data and alerts they generate. Early detection or prediction of disease may very well improve animal health on dairy farms; monitoring lameness in particular could benefit from kinetic or optical sensor technology. This might also help to raise acceptance of the dairy sector in the consumers' perspective. In any case, PLF is going to alter the working routine of veterinarians. It is not up to the veterinary profession whether to accept these changes--they must be recognized, embraced and used. It is necessary for veterinarians to understand the functioning of PLF and to actively take part in its development. Author Contributions Conceptualization, J.L.K.; methodology, J.L.K. and R.G.; writing--original draft preparation, J.L.K.; writing--review and editing, J.L.K. and R.G.; visualization, J.L.K. and R.G.; supervision, R.G. All authors have read and agreed to the published version of the manuscript. Institutional Review Board Statement Not applicable. Informed Consent Statement Not applicable. Data Availability Statement Not applicable. Conflicts of Interest The authors declare no conflict of interest. Figure 4 Evaluation of opportunities and risks of PLF. Summary based on (Raad voor Dierenaangelegenheiden ). From the different perspectives (animal, producer, public), different options arise. Disclaimer/Publisher's Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

PMC10000126

Dairy processing is one of the most polluting sectors of the food industry as it causes water pollution. Given considerable whey quantities obtained via traditional cheese and curd production methods, manufacturers worldwide are encountering challenges for its rational use. However, with the advancement in biotechnology, the sustainability of whey management can be fostered by applying microbial cultures for the bioconversion of whey components such as lactose to functional molecules. The present work was undertaken to demonstrate the potential utilization of whey for producing a fraction rich in lactobionic acid (Lba), which was further used in the dietary treatment of lactating dairy cows. The analysis utilizing high-performance liquid chromatography with refractive index (HPLC-RID) detection confirmed the abundance of Lba in biotechnologically processed whey, corresponding to 11.3 g L-1. The basic diet of two dairy cow groups involving nine animals, Holstein Black and White or Red breeds in each, was supplemented either with 1.0 kg sugar beet molasses (Group A) or 5.0 kg of the liquid fraction containing 56.5 g Lba (Group B). Overall, the use of Lba in the diet of dairy cows during the lactation period equal to molasses affected cows' performances and quality traits, especially fat composition. The observed values of urea content revealed that animals of Group B and, to a lesser extent, Group A received a sufficient amount of proteins, as the amount of urea in the milk decreased by 21.7% and 35.1%, respectively. After six months of the feeding trial, a significantly higher concentration of essential amino acids (AAs), i.e., isoleucine and valine, was observed in Group B. The percentage increase corresponded to 5.8% and 3.3%, respectively. A similar trend of increase was found for branched-chain AAs, indicating an increase of 2.4% compared with the initial value. Overall, the content of fatty acids (FAs) in milk samples was affected by feeding. Without reference to the decrease in individual FAs, the higher values of monounsaturated FAs (MUFAs) were achieved via the supplementation of lactating cows' diets with molasses. In contrast, the dietary inclusion of Lba in the diet promoted an increase in saturated FA (SFA) and polyunsaturated FA (PUFA) content in the milk after six months of the feeding trial. amino acids fatty acids feed lactating cows lactobionic acid milk quality traits milk yield whey Ministry of Agriculture and Rural Support Service of the Republic of Latvia19-00-A01612-000007 This research was funded by the project No. 19-00-A01612-000007 "Economically justified processing of whey for new food and feed" supported by the Ministry of Agriculture and Rural Support Service of the Republic of Latvia. pmc1. Introduction Data published by the EUROSTAT disclose that in 2020 European union farms produced 160.1 million tons (Mt) of raw milk, 1.1% more than in 2019 . Of the total milk obtained, 96.3% was used to produce a range of processed dairy products and fresh products. Among other products, the dairy industry generates a substantial amount of whey which in 2020 corresponded to 55.5 Mt . The relative abundance of water and the high ratio of lactose to protein in dairy whey makes further processing challenging. The report of Ozel et al. reveals that from the total amount of whey generated, only 50% is being processed for the production of high added value products. Whey drying technologies using conventional approaches such as spray dryers are sometimes risky to manufacturers due to extensive fouling and blocking of the production equipment. These challenges increase with higher whey protein levels and temperatures, leading to protein denaturation . Manufacturers are forced to use ultrafiltration and/or reverse osmosis to reduce the risk of equipment clogging along with making whey more solid . However, these approaches challenge small enterprises and are not economically feasible , explaining the relatively high price for whey protein isolates. While considering the high biochemical oxygen consumption (BOD) of 50 g L-1 and chemical oxygen (COD) of 65 g L-1 values, the direct disposal of raw whey is strongly prohibited in many EU countries as it creates serious environmental problems, leading to changes in soil's physical condition, chemical indicators, and microbiota, thus affecting the yield of crops to be planted . Therefore, it is paramount to find new and cost-effective processing technologies that could stimulate the reuse of whey in many economic sectors, including animal husbandry and food production, and foster a circular economy toward the sustainable development of high added value products from by-products. The chemical composition of whey is discussed as it varies considerably depending on the milk source and the production process used. However, per 100 g-1, it contains an average of 6.5 g of total solids, which includes 5.0 g of lactose, 0.6 g of protein, 0.6 g ash, 0.2 g of non-protein nitrogenous substances, and 0.1 g of fat . Given the composition of whey, especially the high content of lactose, whey represents interest to biotechnologists as a potential source of carbon-containing molecules suitable for being used as nutrients for microbiological cultures. Cutting-edge biotechnology research came with the discovery of functional compounds derived from dairy whey with antioxidant, antimicrobial, antiaging, and immunomodulation activities, such as a-lactalbumin and b-lactoglobulin , glycomacropeptide , and lactoferrin . Lactobionic acid (Lba) is another functional product that, for the first time, was synthesized chemically by Fischer and Meyer by oxidizing lactose with bromine . To date, the production of Lba has been accomplished via enzymatic biocatalytic , electrocatalytic , or heterogeneous oxidation and is widely used in some pharmaceutical products as an excipient agent . More recently, a microbially synthesized Lba was obtained under optimized fermentation conditions of cheese whey with Pseudomonas fragi . Meanwhile, the ability of P. taetrolens to produce enzymes involved in the oxidation of acid whey lactose to Lba was highlighted by Sarenkova et al. . Due to intrinsic properties, e.g., the calcium delivery vehicle, acidity regulator, and free radical chelating agent, Lba may represent interest to stockbreeders. Recently, our group achieved encouraging results on the elaboration of Lba from acid whey through a biotechnological approach via lactose oxidation enzymes produced by P. taetrolens . Furthermore, the potential utilization of synthesized, isolated, and purified Lba has been demonstrated by Zagorska et al. , indicating the ability of Lba to contribute to pig growth performance and enhance the nutritional value of meat proteins. Furthermore, the ability of fermented acid whey permeate to act as a prebiotic while positively affecting the growth and development of normal intestinal microflora of lactating cows was highlighted in an in vivo study performed by Lakstina et al. . Moreover, the inclusion of Lba in laying hens' diets contributed to the eggshell thickness and their strengthening, as reported in the patent application . These observations, along with limited information regarding the influence of Lba on the productivity of lactating cows and the quality traits of milk, have promoted the design of this study which focuses on the evaluation of the influence of biotechnologically obtained Lba used as a feed supplement in the diet of lactating cows on their performance and the quality traits of milk. 2. Materials and Methods 2.1. Experimental Design The study was conducted at the Farm Ruki (Latvia, Vidzeme) and lasted from November 2020 to April 2021 (six months). Two groups of lactating dairy cows were included, i.e., control (Group A) and experimental (Group B). Each group included in the current study was composed of nine animals of two breeds, i.e., Holstein Black and White and Red dairy cows. The animals were up to 60 days in lactation (DIM), representing different lactations (from 1 to 8) divided proportionally between the groups. The basic feed for both groups was prepared directly on the farm as a partial mixed ration (PMR). The composition for one animal included grass silage 41 kg, hay 0.5 kg, rapeseed cakes 3.6 kg, grain flour 8.3 kg, and premix 0.6 kg (JOSERA Cami 0.25 kg, sodium bicarbonate 0.15 kg, DairyPilotFlavoVital(r) 0.1 kg, lime 0.1 kg, and salt 0.02 kg). Group A was additionally fed 1.0 kg sugar beet molasses, while the diet of Group B was supplemented with 5.0 kg Lba-rich whey. The amount of Lba supplemented into the basic diet was estimated based on the carbohydrate content of molasses. The proximate composition of the liquid fraction rich in Lba and molasses is given in Table 1. Dairy cows were housed under tie stalls and individually fed and watered ad libitum. Cows were milked twice daily. 2.2. Dairy Lactating Cows' Performances and Quality Traits of Milk Samples Milk productivity traits: milk yield kg d-1 and sampling for testing were conducted twice per month during the experiment. Milk composition and quality indices were determined at the beginning and end of the experiment. Raw milk samples were taken during morning milking and divided into two parts: one part was preserved using Broad Spectrum MicroTabs II (BSM II) and immediately delivered to the Dairy Laboratory, Ltd. The analysis of raw milk quality traits, i.e., fat, protein, casein, and urea content, was performed using the MilkoScan FT6000 (FOSS, Hilleroed, Denmark) mid-infrared spectroscopic approach following the guidelines outlined in the ISO 9622|IDF 141:2013. The estimation of somatic cells was performed using an instrumental flow cytometry method by FossomaticTM (FOSS, Hilleroed, Denmark) according to the ISO standard 13366-2|IDF 148-2:2006. The second part of the raw milk samples devoted to the analysis of fatty acids (FAs) and amino acids (AAs) was kept at a temperature of -20 +- 1 degC until further processing and analysis, a maximum of two weeks. The somatic cell count (SCC) per 1 mL of milk was converted to standardized units, i.e., somatic cell score (SCS), by using the following equation :(1) SCS=log2SCC100+3 where SCC is somatic cells per mL of milk. The SCS in milk was used as a quality and indirect animal health indicator. In order to evaluate results among groups and study phases (beginning and end of the experiment), the milk yield and its composition were transformed to energy-corrected milk (ECM), which indicates the amount of energy in the milk considering the values of milk, fat, and protein yield (ICAR, 2017). The ECM was determined following the equation (2) ECM=FYx38.3+PYx24.2+MYx0.78323.14 and was expressed in kg d-1, where ECM is energy-corrected milk, FY is fat yield in kg, PY is protein yield in kg, and MY is milk yield in kg. The predicted milk protein efficiency ratio (PER) was calculated according to three equations proposed by Alsmeyer, Cunningham, and Happich (1974), taking into account the values of AAs:(3) PER1=-0.684+0.456LEU-0.047PRO where LEU and PRO are the content of leucine and proline, respectively. (4) PER2=-0.468+0.454LEU-0.105TYR where LEU and TYR are the content of leucine and tyrosine, respectively. (5) PER3=-1.816+0.435MET+0.780LEU+0.211HIS-0.944TYR where MET, LEU, HIS, and TYR is the content of methionine, leucine, histidine, and tyrosine, respectively. The ratio of essential AAs (E) to the total AAs (T) of the protein was calculated based on the equation provided by Chavan et al. :(6) ET=EAATAAx100% where E/T is a ratio of essential amino acids (E) to the total amino acids (T); EAA is the sum of essential amino acids; and EAA is the sum of total amino acids. The index of atherogenicity (IA) which characterizes the atherogenic potential of fatty acids, the index of thrombogenicity (IT), a ratio of hypocholesterolemic to hypercholesterolemic (HH) values, and the health-promoting index (HPI) were calculated according to Equations (7-10) proposed by Chen and Liu (2020). (7) IA=C12:0+4xC14:0+C16:0UFA where IA is the index of atherogenicity and UFA is the sum of unsaturated fatty acids. (8) IT=C14:0+C16:0+C18:00.5xMUFA+0.5xn-6PUFA+3xn-6PUFA+n-3n-6 where IT is the index of thrombogenicity; MUFA is the sum of monounsaturated fatty acids; and PUFA is the sum of polyunsaturated fatty acids. (9) HH=cis-C18:1+PUFAC12:0+C14:0+C16:0 where HH is a ratio of hypocholesterolemic to hypercholesterolemic values; cis implies the isomeric form of C18:1 fatty acid; and PUFA is the sum of polyunsaturated fatty acids. (10) HPI=UFAC12:0+4xC14:0+C16:0 where HPI is the health-promoting index and UFA is the sum of unsaturated fatty acids. 2.3. Chemicals, Standards, and Reagents A mixture of C4-C24 fatty acid methyl esters (FAMEs) and amino acids (AAs) with a purity of >=99.0% were acquired from Sigma-Aldrich Chemie Ltd. (St. Louis, MO, USA). Acetonitrile, methanol, n-hexane, and formic acid (puriss p.a., >=98.0%) of liquid chromatography-mass spectrometry (LC-MS) grade were purchased from the same producer. Lactobionic acid (Lba) with purity >=97.0% was obtained from Acros Organics (Geel, Belgium). Ammonium hydroxide solution (25% v/v) and diethyl ether (puriss p.a., >=99.5%) were obtained from Chempur (Piekary Slaskie, Silesia, Poland). Hydrochloric acid (37% v/v) was purchased from VWRTM International, GmbH (Darmstadt, Germany). Sodium hydroxide, potassium hydroxide, phenolphthalein, and 0.5 M trimethylphenylammonium hydroxide solution (TMPAH) in methanol for GC derivatization were of reagent grade and were obtained from Sigma-Aldrich Chemie Ltd. 2.4. Production of Lactobionic Acid from Pre-Concentrated Whey In this study, pre-concentrated whey obtained from a local producer Jaunpils Ltd. (Jelgava, Latvia), with a proximate composition depicted in Table 2, was used as a carbon source for P. taetrolens DSM 21104 during the production of Lba. The description of the operational and process conditions for the production of Lba was provided in detail in our previous study . 2.5. The HPLC-RID-DAD Analytical Conditions for Lactobionic Acid and Lactose Determination Lba was analyzed using a Shimadzu series LC-20 high-performance liquid chromatography system equipped with the SPD-M20A photodiode-array detector (Shimadzu Corporation, Tokyo, Japan). All samples before HPLC analyses were centrifuged at 14,200x g for 10 min to remove cell debris and other water-insoluble substances. The LBA was determined using a refractive index detector RID-10A (Shimadzu Corporation, Tokyo, Japan). Chromatographic separation of Lba was carried out using a hybrid silica-based YMC-C18 column (4.6 mm x 250 mm, 5 mm; YMC, Kyoto, Japan) operating at 40 degC and a flow rate of 1.0 mL min-1. The separation of Lba was conducted using an isocratic mobile phase with 2 L elution containing 1.15 mL H3PO4, 14.36 g KH2PO4, and 20 mL acetonitrile. The detection wavelength was set at 210 nm. The injection volume was 10 mL. The quantitative analysis of lactose was performed using the same system while utilizing a refractive index detector RID-10A (Shimadzu Corporation, Tokyo, Japan). Chromatographic separation was conducted using an Altima Amino (4.6 x 250 mm; 5 mm; GraceTM, Columbia, MD, USA) column. The temperature of the column and flow cell was maintained at 30 degC. A mixture of H2O and MeCN (75:25, v/v) was used as the mobile phase in the isocratic mode. The flow rate of the mobile phase was 1.0 mL min-1. The injection volume was 10 mL. System control, data acquisition, analysis, and processing were performed using Empower 3 Chromatography Data Software version (build 3471) (Waters Corporation, Milford, MA, USA). 2.6. Acid Hydrolysis of Milk for Amino Acid Determination Before acid hydrolysis, each milk sample was defatted and freeze-dried to obtain protein isolates. Then, the prepared isolates 200.0 mg +- 0.1 were subjected to acid hydrolysis with 5.0 mL of 6M HCl solution according to the ISO 13903:2005 standard with modifications. The hydrolysis was undertaken in 22.0 mL glass Headspace chromatography bottles (PerkinElmer, Inc., Waltham, Massachusetts, USA) with screw caps and silicone seals in a drying cabinet of Pol-Eko Aparatura SP.J. (Wodzislava Slonska, Poland) at a temperature of 110 degC for 24 h. Before hydrolysis, to slow down the oxidation-reduction reaction of the compounds of interest, the stabilizing reagent phenol was added directly to the sample in the amount of 0.02% (w/w). After hydrolysis, the volume of hydrolysate was adjusted to 7.0 mL with H2O, and it was normalized to 6.5-6.8 using 2.18 mL of 25% NH4OH solution. The final volume was 10.0 mL. The obtained hydrolysate was subjected to 1 min intensive Vortexing using the "ZX3" vortex mixer (Velp(r) Scientifica, Usmate Velate, Italy), followed by centrifugation at 16,070x g for 10 min at 19.0 +- 1 degC in a "Hermle Z 36 HK" centrifuge (Hermle Labortechnik, GmbH, Wehingen, Germany). Before LC-MS analysis, the collected supernatant was filtered using a 0.22 mm hydrophilized polytetrafluoroethylene (H-PTFE) membrane filter (Macherey-Nagel GmbH & Co. KG, Dueren, Germany). 2.7. The HPLC-ESI-TQ-MS/MS Analytical Conditions for Amino Acids The chromatography analysis of AA was conducted using a "Shimadzu Nexera UC" series liquid chromatography (LC) system (Shimadzu Corporation, Tokyo, Japan) coupled to a triple quadrupole mass-selective detector (TQ-MS-8050, Shimadzu Corporation, Tokyo, Japan) with an electrospray ionization interface (ESI). A sample of 3 mL was injected onto a reversed-phase "Discovery(r) HS F5-3" column (3.0 mm, 150 x 2.1 mm, Merck KGaA, Darmstadt, Germany) operating at 40 degC with a flow rate of 0.25 mL min-1. The mobile phases used were acidified H2O (1.0% HCOOH v/v) (A) and acidified MeCN (1.0% HCOOH v/v) (B). The program of stepwise gradient elution of the mobile phase B for 20 min was implemented as follows: T0 min = 5.0%, T5.0 min = 30.0%, T11.0 min = 60.0%, T12.0 min = 80.0%, and T12.1 min = 5.0%. Finally, re-equilibration for 3 min was conducted after each analysis following the initial gradient conditions. The MeCN injections were included as a blank run after each sample to avoid the carry-over effect. Data were acquired using "LabSolutions Insight LC-MS" version 3.7 SP3, which was also used for instrument control and processing. Ionization in the positive ion polarity mode was applied in this study. At the same time, data were collected in profile and centroid modes, with a data storage threshold of 5000 absorbance for MS. The operating conditions were as follows: detector voltage 1.98 kV, conversion dynode voltage 10.0 kV, interface voltage 4.0 kV, interface temperature 300 degC, desolvation line temperature 250 degC, heat block temperature 400 degC, nebulizing gas argon (Ar, purity 99.9%) at a flow rate of 3.0 L min-1, heating gas carbon dioxide (CO2, purity 99.0%) set low at 10.0 L min-1, and drying gas nitrogen (N2, separated from air using a nitrogen generator system from "Peak Scientific Instruments Ltd." (Inchinnan, Scotland, UK), purity 99.0%) at flow 10.0 L min-1. All AAs were observed in the programmed and optimized multiple reaction monitoring (MRM) mode. Quantitative analysis of AAs was performed by injecting 3.0 mL of calibration solution at 15 degC with the range of 0.075-2.5 mM L-1. The working solution was prepared immediately before being used. Representative chromatographic separation of 18 AAs is given in Figure 1. 2.8. Preparation of Lipid Fraction via Alkaline-Assisted Hydrolysis with Subsequent Liquid-Liquid Extraction The isolation of lipophilic fraction from dry milk cream (please refer to Section 2.6. Acid Hydrolysis of Milk for Amino Acid Determination) was performed following the procedure described by Radenkovs et al. with minor modifications. For the release of bound forms of fatty acids (FAs), 10% (w/v) KOH dissolved in 80% MeOH (MeOH:H2O ratio 80:20 v/v) was applied. Briefly, duplicate samples of 3.0 +- 0.1 g of freeze-dried cream obtained after milk separation were weighed in 50 mL reagent bottles with screw caps. For the hydrolysis of FAs, 30 mL of freshly prepared methanolic KOH was added to each cream sample, and the mixture underwent incubation in a water bath "TW8" (Julabo(r), Saalbach-Hinterglemm, Germany) at 65 degC temperature for 3 h. After hydrolysis, the cleavage of bonds present in the potassium salts of FAs was obtained by adjusting the pH of the solution to pH 2.0 +- 0.2 by adding 3.3 mL HCl (6M). The extraction of FAs was implemented via liquid-liquid phase separation using n-hexane as the sole solvent. After hydrolysis, samples were cooled to room temperature (22 +- 1 degC) and quantitatively transferred to Falcon 50 mL conical centrifuge tubes (Sarstedt AG & Co. KG, Numbrecht, Germany). Afterwards, 10 mL of n-hexane was added to each tube, followed by vortex-mixing for 1 min. Finally, the layers were separated via centrifugation at 3169x g for 10 min in a "Sigma, 2-16KC" centrifuge (Osterode near Harz, Germany). The top n-hexane layer was decanted and collected. The extraction procedure was repeated three times. First, the resulting lipid fraction (30 mL) was evaporated using a "Laborota 4002" rotary evaporator (Heidolph, Swabia, Germany) at 65 degC, and the dry residues were then reconstituted in 5 mL of n-hexane and filtered through a polytetrafluoroethylene hydrophobic (PTFE) membrane filter with a pore size of 0.45 mm. The filtrates were quantitatively transferred to 20 mL scintillation glass vials and subjected to drying under a gentle stream of N2 to complete dryness. Prepared samples were kept at a temperature of -18 +- 1 degC until further analysis and were used within a maximum of two weeks. Before GC-MS analysis, obtained dry lipid fractions were reconstituted in 2 mL pyridine. 2.9. Preparation of Fatty Acids for GC-MS Analysis The TMPAH reagent was used as a methylation agent of the functional groups to obtain volatile FAMEs derivatives. The methylation procedure was performed following the methodology ensured by Radenkovs et al. . 2.10. The GC Conditions for FAMEs Analysis The analysis of FAMEs was performed using a "Clarus 600" system PerkinElmer, Inc. (Waltham, MA, USA) coupled with a quadrupole "Clarus 600 C" mass-selective detector (Waltham, MA, USA). The conditions were adopted from Radenkovs et al. . 2.11. Statistical Analysis The obtained data were analyzed using descriptive statistics, and the differences between the study groups and phases of the study were assessed using ANOVA with Student's t-test correction, setting the confidence level at p <= 0.05. Statistical processing of the data was carried out using the MS Office program Excel version 2016 (Microsoft Corporation, Redmond, Washington, USA). 3. Results and Discussion 3.1. Animals' Performances and Quality Traits of Milk Balanced feeding for lactating cows, especially at the beginning of lactation, is crucial as it influences cows' performances, overall health, and milk quality traits. The selection of a supplement to compensate for the lack of energy in feed depends on factors such as feeding technology, supplement availability in the market, and costs. In the current study, sugar beet molasses for Group A and Lba for Group B were selected as supplements to compare the effect on cows' performances and milk quality traits. The productivity results were analyzed for each group at the study's beginning and end (see Table 3). As seen at the beginning of the experiment, the milk yield values were not significantly different (p >= 0.05) between the study groups. However, a substantial difference (p <= 0.05) was observed comparing milk yield within the study phase between initial and final values. It was observed that at the end of the experiment passing six months, the milk yield in Group A decreased by 19.3%, while in Group B, the decrease amounted to 23.0%, which was 3.7% higher in Group A (Table 3). No apparent influence of dietary treatment on lactation performance was found; this observation is in line with Penner and Oba . The decrease in milk yield is explained by the lactation phase, which directly influences the milk yield as the number of lactation days increased during the study . A similar observation was made by Vijayakumar et al. , indicating that cows with the second lactation produced 24.18% greater milk than the first lactation cows, while the fourth lactation cows showed a decreased milk yield by 16.04% from the third lactation. According to a study reported by the National Research Council , fat content in milk is the most varying value, while lactose is the least, and this observation was also reinforced in this study. As seen, the fat content between the groups within the beginning phase of the experiment varied significantly (p <= 0.05), corresponding to 23.5% (Table 3). The percentage difference between the groups at the end of the experiment amounted to 15.4%, which was 8.1% lower than at the beginning. Such a difference could be the case of the animals' physiological states, e.g., the availability of hormones such as adrenaline and noradrenaline that are reported to be responsible for lipolytic activity in adipose tissue . At the end of the experiment, the most apparent increase in fat content was found in Group A, corresponding to a 14.7% increase compared with the initial value. A similar increase in fat content was also observed in Group B, though this value corresponded to 7.1%. It was reported that the supplementation of molasses in dairy cows' diets substantially contributes to a higher fat yield and concentration of fatty acids in primiparous cows . The increase in fat content of Group A fed with a basic diet supplemented with molasses can be explained by an enhanced rumen fermentation process moderated by pH, thus promoting mammary de novo fatty acid synthesis . This statement was further reinforced by , indicating the increase in effective ruminal degradability (ERD) of dry matter in an in situ ruminal study. It is worth noting that the supplementation of lactating cows' diets with Lba could be considered a promising carbon-containing alternative to molasses, ensuring an increase rather than a decrease in fat in milk without affecting acidosis. Multiple pieces of scientific evidence have revealed that milk composition, especially crude protein and fat content, strongly depends on the milk yield . Hence, highly productive dairy cows will provide a lower protein yield in milk than those with low productivity . However, one of the critical factors determining the amount of protein in milk is the availability of nutrients, especially those rich in proteins, that the animal ingests in the feed . The results of this study imply that the content of protein in milk from the dairy cows of Group A who received a high feed diet rich both in carbohydrates and protein (Table 1) was found to be significantly (p <= 0.05) higher compared with the initial value, corresponding to a 9.1% increase. Previous studies also observed an increase in protein content, reflecting a higher nutritional value of milk from low-productivity dairy cows . However, in Group B, in which the feed of animals was supplemented with biotechnologically produced Lba, the protein content, considering the lower milk yield at the end of the experiment, remained intact, corresponding to 3.8%. The observed values are consistent with those reported by Murphy . A plausible explanation for obtaining higher values of protein in milk from Group A relies upon the availability of readily digestible compounds present in molasses, such as sucrose, fructose, and glucose , while in Lba-rich whey solution, the main representative is lactose. Casein and whey protein are milk's major proteins, and casein corresponds to roughly 80% of the total protein in bovine milk . The initial values of casein in milk samples fluctuated from 2.6 to 3.0%, with Group B having the highest content and Group A having the lowest (Table 3). The observed values are consistent with those of Guo and Wang . As with protein, the content of casein was affected by feeding. The highest content was found in Group A at the end of the experiment, corresponding to an increase of 7.7%. In turn, no changes were observed in Group B after six months of the feeding trial. The results indicate that optimizing feed intake with ingredients rich in carbohydrates and organic acids such as molasses or Lba can ensure the necessary energy level to retain milk's nutritional value (casein in particular) during lactating. This observation is in line with those proposed by Emery as far back as four decades ago, in 1978 . The SCC is a direct marker of mastitis infection for individual cows and within herds and therefore was evaluated critically as an indirect indicator of cow udder health . To better reflect the state of animal health, the SCS values were calculated in this study and are depicted in Table 3. According to Shook and Schutz , having these numbers allows for achieving genetic improvement and better results in controlling mastitis resistance. As seen, the initial values of SCS varied from 3.2 and 2.3, with Group A at the initial stage of the experiment having the highest value and Group B having the lowest, respectively (Table 3). The estimation made available by Smith et al. was that cows with an SCS of 0-3 are generally considered to be uninfected. At the end of the experiment, the SCS values varied in the range from 3.0 to 3.5, with Group A showing the highest value while Group B showed the lowest. The most apparent increase in SCS values was found in Group B. Although, such an increase in SCS only marginally contributed to the reduction in milk yield, as proposed by Smith et al. . It has been proposed that the content of urea in milk can be utilized as a non-invasive input to a system to monitor the crude protein status in dairy cows on a regular basis . Therefore, urea content in milk samples was used in this study as a biomarker to estimate the availability of AAs in the diet of animals. The initial values of urea content varied from 23.3 to 23.5 mg dL-1. The observed values were consistent with those reported by Rzewuska and Strabel for the dairy cow in the first phases of lactation (Table 3). However, after six months of the experiment, following the developed dietary treatment schedule, the amount of urea in the milk of Group A and Group B decreased by 35.2% and 21.7%, respectively. Nevertheless, the observed values comply with data reported by Duinkerken et al., 2011 , indicating the range of urea in milk from 15.0 to 30.0 mg dL-1 as being optimal. Since the composition of molasses is mainly represented by carbohydrates while lacking essential AAs such as methionine, histidine, and lysine , a relatively higher value of urea content in Group B can be explained. Incorporating biotechnologically produced Lba into the diet of dairy cows resulted in ensuring the availability of readily digestible AAs essential for animals . Since the ECM is a generic productivity indicator that provides a clue on the value of milk based on the milk yield, fat, and protein content, this value is widely used to assess the overall quality of obtained milk as a function of dietary treatment . As seen, the ECM values at the initial stage of the experiment were significantly different (p <= 0.05) between the study groups (Table 3). The observed values were considerably higher than those reported by Guinguina et al., 2020, for dairy cows with a basic-feed diet while they were significantly lower than for dairy cows fed rumen-protected lysine as a supplement to the basic diet . The ECM values changed significantly (p <= 0.05) at the end of the study, corresponding to a percentage reduction of 11.4% and 23.2% for Group A and Group B, respectively. The main factor contributing to the decrease in ECM values was milk yield, which dropped the most in Group B fed with Lba. The report of Miller et al., 2021, indicates that molasses with 34% sucrose positively contributed to dairy cows' performances during the postpartum period, along with improved milk quality traits, by stimulating ruminal butyrate production and papillae development. Moreover, Ravelo et al. also concluded that by-products rich in sucrose or lactose in the diet of dairy cows promoted ruminal microbial fermentation, encouraging digestibility and increasing the pH of rumen fluids. Overall, the use of Lba in the diet of dairy cows during the lactation period favorably affected the performance and quality traits of milk; however, to achieve better results, the optimization of feed intake with ingredients rich in carbohydrates and proteins such as molasses and biotechnologically produced Lba, respectively, can deliver the necessary energy levels for increased milk production and its quality. 3.2. The Changes in Amino Acids and Their Quality Indices in Relation to Feeding Trial Met, Lys, and His have been identified as the most limiting AAs for lactating dairy cows , and their lack in the diet of animals leads to limited milk protein, fat production, and milk yield . Therefore, it has been proposed that supplementing the diet of lactating cows with rumen-protected AAs may be a prosperous approach for improving animals' performances and the quality traits of milk . The AA profile was analyzed via the HPLC-ESI-TQ-MRM-MS/MS approach, by-passing the derivatization step to elucidate the quality of proteins obtained in produced milk. The selective analysis confirmed the presence of 17 AAs in all milk samples except tryptophan due to its high oxidative degradation (Table 4). Furthermore, it was observed that Glu, Leu, Pro, and Lys were the prevalent representatives of AAs in milk protein. The observed values are consistent with those reported by Landi, Ragucci, and Di Maro . In the course of further study of the content of total AAs in milk protein, no significant difference between Group A and Group B was found at the beginning of the experiment. However, statistically significant differences (p <= 0.05) were established between Group A and Group B in the content of individual AAs such as Ile, Lys, and Val. After six months of the feeding trial, a significantly higher concentration of Ile and Val was detected in Group B, and the percentage increase corresponded to 5.9% and 3.3%, respectively. An increase in Tyr content by 6.5% and 4.3% was also observed in Group A and Group B, corresponding to the value of 4.9 g 100 g-1 in both groups. It is worth noting that the most apparent increase in the content of Ala was observed in Group B, indicating that cows responded favorably to Lba rather than to molasses. The study also noted a significant decrease (p <= 0.05) in Gly content by 10.5% and 5.3% in Group A and Group B, respectively. A similar observation was made by Li et al. 2019 , performing the metabolic profiling of yak mammary gland tissues and speculating that the decrease in Gly was related to the negative energy balance in yaks. The most pronounced decrease in Thr content was found in the milk of Group B, corresponding to 4.7%, while in Group A, a reduction amounted to 2.3%. A plausible explanation for having a reduction in Thr has been given by Tang et al., 2021 , indicating that the presence of this essential AA in high concentrations is vitally important to newborns since its primary function is to provide antimicrobial activity against pathogenic bacteria and to modulate the immune system response to viruses, while its gradual decrease takes place as the calf grows. Branched-chain AAs (BCAAs, valine, leucine, and isoleucine) belong to the group of exogenous AAs that must be supplied to the body through the diet . Multiple beneficial effects of BCAAs have repeatedly been proven , so the importance of these AAs in human nutrition is undebatable. The results of this study revealed a relatively similar sum of BCAAs in the milk sample at the beginning of the experiment with the dietary treatment. The content varied from 20.2 to 20.7 g 100 g-1, with Group A having the lowest value and Group B having the highest value. The observed values are consistent with those of Hulmi et al., 2010 . It is worth noting that the content of BCAAs in Group A after six months of the feeding trial decreased by 0.5%, while in Group B it increased by 2.4%. The predicted protein efficiency ratio (PER) is a valuable method providing crucial information on the quality of proteins in food systems. However, utilizing in vivo models to estimate PER is considered time-consuming and expensive . In this study, we attempted to predict the PER values based on mathematical equations using the information on amino acids from the milk samples. According to these models, the PER1 (Leu and Pro), PER2 (Leu and Tyr), and PER3 (Met, Leu, His, Tyr) values as functions of the AAs selected were estimated and they are depicted in Table 4. The results of the present study showed that PER1 values for protein isolates obtained from Group A and Group B before the feeding trial lay within the range between 3.1 and 3.2, respectively. Based on Friedman's classification, a PER < 1.5 is to be considered poor, from 1.5 to 2.0 is considered to be moderate, and > 2.0 is considered to be superior . Based on this proposal, the protein isolates can be classified as highly digestible, close to the values reported by Lee et al. for proteins of deboned chicken meat. In addition, Sarwar and Dupont and Tome support our observation, indicating that nearly 95% of milk protein is readily digestible within in vivo gastrointestinal tract models. It is worth noting that the observed values were far from those indicated for extruded, cooked, and baked yellow and green split pea flour . Since the PER1 values for Group A and Group B were close to each other after a feeding trial of six months, it is believed that animals received a balanced diet and even energy distribution within the entire experimental period. However, for calculation using Leu and Tyr, relatively higher values were determined for the PER2 index. The observed values after the feeding trial ranged from 3.2 to 3.3 for Group A and Group B, respectively. A relatively high value of Tyr can explain the difference between PER1 and PER2, reported to have roughly 99.0% true ileal digestibility . Group B tended to show a higher value after a feeding trial with Lba than Group A who were fed a high sucrose diet. The higher PER2 values compared to PER1 were mostly Leu-concentration-dependent. The predicted PER3 values were found to be different from those of PER1 and PER2 due to the inclusion of additional AAs, which were speculated to be more accurate. The assessed values after the feeding trial for both groups were statistically similar, corresponding to 2.7. However, after the feeding trial, the digestibility rate worsened despite the increase in Tyr and Ile, estimated by a percentage reduction of 11.1% and 3.7% for Group A and Group B, respectively. On the other hand, the increase in Phe and Ala concentrations and the rearrangement of AAs were the main factors that did affect the lowering of the digestibility rate of milk proteins. However, a decrease in PER3 values observed by Pastor-Cavada et al. seemed to be Met-content-dependent. According to PER3 values, the predicted digestibility of milk protein isolates above the standardized PER value for casein of 2.5 indicates its high bioavailability with a digestibility rate close to bean proteins, as Mecha et al. reported . Finally, the ratio of essential AAs to the total AAs at the beginning and end of the study was found in the range from 44.9 to 45.5% and from 45.6 to 45.8% for Group A and Group B, respectively. The observed ratio values of essential to total AA comply with the quality criteria outlined by the FAO/WHO . Given these numbers, it is attainable to state that milk obtained following lactating cows' dietary treatments should be considered to be an excellent source of amino acids that could provide the body with all essential AAs. 3.3. The Changes in Fatty Acids and Their Nutritional Indexes of Milk Lipids in Relation to Feeding Trial The composition of FAs in lipids recovered from milk cream is depicted in Table 5. In total, 29 FAs were identified and quantified, among which the dominance of palmitic acid (C16:0) from 31.0 to 34.7%, followed by oleic acid (C18:1n9c) from 18.4 to 20.9%, myristic acid (C14:0) from 11.8 to 12.9%, stearic acid (C18:0) from 8.5 to 10.1%, linolelaidic acid (C18:2n6t) from 1.5 to 2.3%, linoleic acid (C18:2n6c) from 1.6 to 1.8%, and behenic acid (C22:0) from 1.4 to 2.1% was found. The results are consistent with those of Mansson , indicating a similar descending order of FA content recovered from bovine milk. The results of the present study indicated that a high feed diet rich in sucrose (Group A) negatively affected the amount of individual FAs in the milk. The most apparent decline in the content of major FAs was observed for linoleic acid (C18:2n6t), corresponding to a 21.6% loss. In contrast, no changes for this polyunsaturated FA were found in Group B fed with biotechnologically obtained Lba. Similar changes were observed for behenic acid (C22:0), corresponding to a 21.6% loss and a 21.4% increase for Group A and Group B. A positive influence of biotechnologically obtained Lba was identified for FAs such as lauric (C12:0), tridecanoic (C13:0), myristoleic (C14:1), pentadecanoic (C15:0), and pentadecanoic (C15:1) acids. The negative effect of molasses supplementation on dairy cows' performances and milk composition by reducing milk yield, milk protein, lactose yield, and the composition of unsaturated FAs, in particular, was reported by Torres et al. . In some cases, there was a marked decrease in oleic acid after using molasses as an additive to feed lactating cows due to the interaction of molasses with buffers that negatively affected ruminal fermentation and consequently led to a loss in milk quality . However, after animals received a molasses diet, this experiment observed an increase rather than a decrease in oleic acid (C18:1n9c) concentration in milk. The content of CLA is feed-type-dependent since its concentration greatly varies from report to report . However, the CLA values found are in direct agreement with those reported by Brito et al. for cows fed chiefly grass. The CLA concentration in milk varied from 0.6 to 1.0%, with Group A fed with molasses having the highest value and Group B supplied with Lba having the lowest. After six months of the feeding trial, a significant reduction (p <= 0.05) in CLA was noted in Group A, corresponding to a 30.0% loss. On the other hand, the Lba positively affected CLA in Group B since as much as a 33.3% increase was observed. Relatively higher values of CLA in Group B can be explained presumably by the chemical composition of a liquid fraction rich in Lba, particularly the availability of linoleic acid that promoted the synthesis of CLA as reported by Gomez-Cortes et al. . Overall, the content of FAs in milk samples was affected by feeding. Without reference to the decrease in individual FAs, the higher values of MUFAs were achieved by the supplementation of lactating cows' diets with molasses (Group A). In contrast, the dietary inclusion of biotechnologically produced Lba in the diet promoted the increase in the content of SFAs and PUFAs in the milk. In the present study, health-related lipid indexes IA, IT, HH, and HPI were established to better reflect the health-promoting properties of milk lipids (Table 5). It was highlighted that IA and IT are great tools for assessing the potential contribution of FAs to human health . The lower IA and IT values, the less risk of developing cardiovascular diseases caused by blood vessel clogging. Performing mathematical analysis, the following IA indices were obtained from 2.8 to 3.6 and from 2.4 to 3.4 for Group A and Group B at the beginning and end of the study, respectively. The observed values are consistent with those of Lobos-Ortega et al. for fresh bovine milk estimated using near-infrared spectroscopy. IA values were statistically different (p <= 0.05) between Group A and Group B at the study's beginning and end. Up to 28.6% and 41.7% increases in IA values were observed for Group A and Group B, passing six months of the feeding trial, respectively. The lower IA value in Group B is explained by the statistically higher concentrations of individual MUFAs. Additionally, statistically higher CLA values in Group B reinforce this speculation as their superior anti-atherogenic, anti-platelet, and antioxidant properties have been reported multiple times by . However, the opposite results were obtained concerning the IT index, indicating statistically lower values for Group A than for Group B, corresponding to 3.0 and 3.5, respectively. To a greater extent, the observed IT values in both groups corresponded to those reported by Silva et al. and Sharifi et al. for milk from crossbred cows subjected to feed ad libitum and from high forage and nitrate-fed Holstein lactating cows, respectively. The enhancement of the rumen fermentation process and pH optimum achieved by supplementing the diet of Group A with readily digestible mono and disaccharides present in molasses perhaps promoted mammary de novo FAs rather than those with anti-atherogenic activity synthesis as proposed by Palmquist, Beaulieu, and Barbano . Further analysis revealed no statistically significant differences (p >= 0.05) between the HH values, indicating equal values for all samples investigated, corresponding to 0.5. For the first time, the HPI was proposed by Chen et al. as a quality marker of dietary fat, which is presently widely used in the analysis of dairy products. Furthermore, it is believed that products with higher HPI values are supposed to be more beneficial to human health . The HPI values were found in the range from 0.3 to 0.4, and no statistically significant differences (p >= 0.05) for both groups were revealed at the end of the feeding trial (Table 5). The observed values are in line with those reported by Kasapidou et al. for sheep fed in confinement with no access to grazing and by Chen and Liu for the cream of Holstein cows. 4. Conclusions The abundance of Lba in the whey fraction obtained by taking advantage of optimized fermentation conditions developed by the group of LBTU utilizing P. taetrolens DSM 21104 was confirmed chromatographically, corresponding to 11.3 +- 0.3 g L-1. A substantial yield of functional Lba made it attainable to enrich the diet of lactating cows (Group B) with the functional component, which has been used as an alternative to sugar beet molasses (Group A). The results of this study revealed an equally effective contribution of the Lba supplementation on dairy cow performance compared with the molasses. Milk quality indicators, e.g., protein and casein content, remained unaffected after six months of the feeding trial. Similar to molasses, the Lba contributed to the improvement in lipid synthesis as 7.1% and 14.7% higher lipid content was observed in the milk of Group B and Group A compared with the initial values, respectively. The content of essential AAs such as isoleucine and valine was significantly higher in the experimental group fed with Lba rather than molasses. A similar trend of increase was found for branched-chain AAs, indicating an increase of 2.4% compared with the initial value. It was found that Group B tended to show higher PER1, PER2, and PER3 values after feeding with Lba than Group A fed with a high sucrose diet. The content of FAs in milk samples was affected by feeding. The highest content of MUFAs was observed in Group A, which received molasses. In contrast, the dietary inclusion of Lba promoted the increase in SFA and PUFA content in the milk after six months of the feeding trial. To summarize, the use of Lba in the diet of dairy cows favorably affected the performance and quality traits of milk; however, to achieve better results, the optimization of feed intake with ingredients rich in carbohydrates and proteins such as molasses and biotechnologically produced Lba, respectively, can deliver the necessary energy levels for increased milk production and its quality. Author Contributions Conceptualization, D.R. (Diana Ruska), I.C. and J.Z.; data curation, J.Z.; formal analysis, V.R. and D.R. (Daina Rubene); funding acquisition, J.Z.; investigation, D.R. (Diana Ruska), V.R. and J.Z.; methodology, D.R. (Diana Ruska), V.R. and J.Z.; project administration, J.Z.; resources, J.Z.; software, V.R.; visualization, V.R.; writing--original draft, J.Z. and V.R.; writing--review and editing, V.R., K.J.-R. and I.C. All authors have read and agreed to the published version of the manuscript. Institutional Review Board Statement The Animal Welfare and Protection Ethics Board at Latvia University of Life Sciences and Technologies reviewed the application for conducting the study. As a result, the commission decided (decision no. 20/4) that there was no necessity for ethical approval because experiments were going to be completed on a commercial dairy farm following animal welfare guidelines, and the animals were not subject to pain, suffering, or lasting harm. Therefore, the study was conducted according to the Cabinet Regulation No. 743 of the Republic of Latvia guidelines focusing on ensuring animal welfare. Informed Consent Statement Not applicable. Data Availability Statement Data are available from authors upon reasonable request. Conflicts of Interest The authors declare no conflict of interest. Figure 1 Selected ion chromatogram in MRM mode represents the profiles of 18 amino acids and one metabolite of tryptophan in a standard solution at the concentration of 2.5 mM L-1. Note: 1--Cystine (Cys); 2--Aspartic acid (Asp); 3--Serine (Ser); 4--Glycine (Gly); 5--Threonine (Thr); 6--Glutamic acid (Glu); 7--Alanine (Ala); 8--Proline (Pro); 9--Histidine (His); 10--Lysine (Lys); 11--Arginine (Arg); 12--Valine (Val); 13--Methionine (Met); 14--Tyrosine (Tyr); 15--Isoleucine (Ile); 16--Leucine (Leu); 17--Phenylalanine (Phe); 18--Tryptophan (Trp); and 19--Tryptamine (Trm). animals-13-00815-t001_Table 1 Table 1 Physical-chemical characteristics of prepared lactobionic-acid-rich whey used in the experiment as a supplement to the basic feed of lactating cows. Quality Trait, % Lba Rich Whey, 1 kg Lba Rich Whey, 5 kg Molasse, 1 kg ** Carbohydrates 15.1 +- 0.7 * 75.6 +- 3.7 * 45.5-62.1 Crude protein 3.7 +- 0.1 18.7 +- 0.4 6.0-13.5 Fat 0.06 +- 0.01 0.2 +- 0.0 0.2 pH 5.6 +- 0.1 5.6 +- 0.1 7.1 LBA, g L-1 11.3 +- 0.3 56.5 +- 4.3 - Dry matter 22.5 +- 1.2 22.5 +- 1.2 75.1-84.0 Note: The superscript single asterisk * indicates the content of lactose solely. The composition of molasses was retrieved from Palmonari et al., 2020, and Saric et al. ** and expressed on a dry weight basis. animals-13-00815-t002_Table 2 Table 2 Physical-chemical characteristics of pre-concentrated commercially obtained mixed whey used as a carbon source for the production of lactobionic acid. Indices, g L-1 Raw Whey Total solids 20.7-25.1 pH 5.9-6.2 Lactose 15.3-18.0 Salts 2.2-2.5 animals-13-00815-t003_Table 3 Table 3 Average lactating cow milk productivity, milk quality traits, and energy-corrected milk values between the study group and phase. Quality Trait Study Group Group A Group B Study Phase Begin End Begin End Milk yield, kg d-1 35.7 +- 2.9 aA 28.8 +- 2.0 (<19.3) bA 37.4 +- 2.0 aA 28.8 +- 2.9 (<23.0) bA Fat content, % 3.4 +- 0.1 bB 3.9 +- 0.2 (>14.7) aB 4.2 +- 0.3 aA 4.5 +- 0.4 (>7.1) aA Protein, % 3.3 +- 0.1 bB 3.6 +- 0.1 (>9.1) aB 3.8 +- 0.2 aA 3.8 +- 0.2 (0)a A Casein, % 2.6 +- 0.1 bB 2.8 +- 0.2 (>7.7) aB 3.0 +- 0.1 aA 3.0 +- 0.1 (0) aA SCS 3.2 +- 0.8 bA 3.5 +- 0.8 aA 2.3 +- 0.4 aA 3.0 +- 0.4 aA Urea, mg dL-1 23.3 +- 0.7 bA 15.1 +- 0.9 (<35.2) aB 23.5 +- 2.0 aA 18.4 +- 0.9 (<21.7) bA ECM, kg d-1 32.5 +- 2.4 aB 28.8 +- 2.0 (<11.4) bA 39.7 +- 2.8 aA 30.5 +- 2.2 (>37.8) bB Note: Values are means +- SD of nine animals (n = 9). Different lowercase superscripts (a,b) in the same row indicate significant differences (Student's t-test; p <= 0.05) between the study phase. Different uppercase (A,B) superscripts in the same row indicate significant differences (Student's t-test; p <= 0.05) between study groups. Numbers in brackets indicate percentage increases (>) or decreases (<) in respective milk quality traits after the feeding trial. Group A--control group received molasses as a supplement to the feed; Group B--experimental group received biotechnologically obtained lactobionic acid as a supplement to the feed; SCS--somatic cell score; ECM--energy-corrected milk; and SD--standard deviation. animals-13-00815-t004_Table 4 Table 4 The profiles of amino acids in milk protein and their comparison between the group and study phase, g 100 g-1 DW. Amino Acid Study Group Group A Group B Study Phase Begin End Begin End Flavor Amino Acids Alanine (Ala) 3.1 +- 0.0 aA 3.2 +- 0.0 (>3.2) aA 3.1 +- 0.0 aA 3.3 +- 0.0 (>6.5) aA Arginine (Arg) 3.2 +- 0.0 aA 3.2 +- 0.0 (0) aA 3.3 +- 0.0 aA 3.3 +- 0.0 (0) aA Asparagine (Asp) 7.7 +- 0.1 aA 7.5 +- 0.2 (<2.6) bA 7.4 +- 0.1 aB 7.5 +- 0.0 (>1.4) aA Cysteine (Cys) 0.8 +- 0.0 aA 0.8 +- 0.0 (0) aA 0.8 +- 0.0 aA 0.7 +- 0.0 (<12.5) aA Glycine (Gly) 1.9 +- 0.0 aA 1.7 +- 0.0 (<10.5) bA 1.9 +- 0.0 aA 1.8 +- 0.0 (<5.3) aA Glutamine (Glu) 21.7 +- 0.2 aA 21.3 +- 0.1 (<1.8) aA 21.3 +- 0.1 aB 20.7 +- 0.2 (<2.8) bB Proline (Pro) 9.2 +- 0.1 aA 9.1 +- 0.0 (<1.1) aA 9.1 +- 0.1 aA 9.2 +- 0.1 (>1.1) aA Serine (Ser) 5.2 +- 0.0 aA 5.3 +- 0.0 (>1.9) aA 5.2 +- 0.0 aA 5.3 +- 0.0 (>1.9) aA SUM 52.8 +- 0.4 aA 52.1 +- 0.3 (<1.3) aA 52.1 +- 0.4 aB 51.8 +- 0.3 (<0.6) bB Essential Amino Acids Histidine (His) 2.9 +- 0.0 aA 3.0 +- 0.0 (>3.4) aA 2.9 +- 0.0 aA 2.8 +- 0.0 (<3.4) aA Isoleucine (Ile) 4.9 +- 0.0 bB 5.1 +- 0.0 (>4.1) aB 5.1 +- 0.0 bA 5.4 +- 0.0 (>5.9) aA Leucine (Leu) 9.3 +- 0.1 aB 9.1 +- 0.1 (<2.2) bB 9.5 +- 0.1 aA 9.5 +- 0.1 (0) aA Lysine (Lys) 8.2 +- 0.1 bB 8.6 +- 0.0 (>4.9) aA 8.4 +- 0.1 aA 8.1 +- 0.0 (<3.6) bB Tyrosine (Tyr) 4.6 +- 0.0 bA 4.9 +- 0.0 (>6.5) aA 4.7 +- 0.0 bA 4.9 +- 0.0 (>4.3) aA Threonine (Thr) 4.3 +- 0.0 aA 4.2 +- 0.0 (<2.3) aA 4.3 +- 0.0 aA 4.1 +- 0.0 (<4.7) bA Valine (Val) 6.0 +- 0.0 aA 5.9 +- 0.0 (<1.7) aB 6.1 +- 0.0 aA 6.3 +- 0.1 (>3.3) bA SUM 40.2 +- 0.2 aB 40.8 +- 0.1 (>1.5) aB 41.0 +- 0.2 aA 41.1+- 0.2 (>0.2) aA Flavor and Essential Amino Acids Methionine (Met) 2.3 +- 0.0 aA 2.4 +- 0.0 (>4.3) aA 2.3 +- 0.0 aA 2.3 +- 0.0 (0) aA Phenylalanine (Phe) 4.6 +- 0.0 aA 4.7 +- 0.0 (>2.2) aA 4.6 +- 0.0 aA 4.7 +- 0.0 (>2.2) aA SUM 6.9 +- 0.0 aA 7.0 +- 0.0 (>1.4) aA 6.9 +- 0.0 aA 7.0 +- 0.0 (>1.4) aA Branched-Chain Amino Acids 20.2 +- 0.2 aB 20.1 +- 0.1(<0.5) aA 20.7 +- 0.2 bA 21.2 +- 0.2 (>2.4) aA Amino Acids' Quality Indices PER1 3.1 aA 3.0 (<3.2) aB 3.2 aA 3.2 (0) aA PER2 3.3 aA 3.2 (<3.0) aA 3.3 aA 3.3 (0) aA PER3 2.7 aA 2.4 (<11.1) bB 2.7 aA 2.6 (<3.7) aA E/T, % 44.9 bB 45.5 (<1.3) aA 45.6 aA 45.8 (<0.4) aA Note: Values are means +- SD of triplicates (n = 3) of an average milk sample of nine animals (n = 9). Different lowercase superscripts (a,b) in the same row indicate significant differences (Student's t-test; p <= 0.05) between the study phase. Different uppercase (A,B) superscripts in the same row indicate significant differences (Student's t-test; p <= 0.05) between the study groups. Numbers in brackets indicate percentage increases (>) or decreases (<) in respective milk quality traits after the feeding trial. Group A--control group received molasses as a supplement to the feed; Group B--experimental group received biotechnologically obtained lactobionic acid as a supplement to the feed; PER--protein efficiency ratio; E/T, %--the ratio of essential amino acids (E) to the total amino acids (T); DW--dry weight; and SD--standard deviation. Branched-chain amino acids are the sum of essential amino acids, i.e., leucine, isoleucine, and valine. animals-13-00815-t005_Table 5 Table 5 Fatty acid profile, cholesterol content, and estimated nutritional indexes of milk lipids in relation to the feeding of lactating dairy cows, % DW. Fatty Acid Abbreviation Study Group Group A Group B Study Phase Begin End Begin End Undecanoic acid C11:0 n.d. n.d. n.d. n.d. Dodecanoic acid C12:0 6.4 aA 5.5 (<14.1) bA 6.0 aB 5.5 (<8.3) bA Tridecanoic acid C13:0 2.0 aA 1.7 (<15.0) aA 1.8 aA 1.8 (0) aA Tetradecanoic acid C14:0 11.8 bA 12.8 (>8.5) aB 11.8 bA 12.9 (>9.3) aA Tetradecenoic acid C14:1 2.5 aA 2.3 (<8.0) aB 2.4 aA 2.4 (0) aA Pentadecanoic acid C15:0 3.2 aA 2.8 (<12.5) aA 2.8 aB 2.6 (<7.3) aB Pentadecenoic acid C15:1 1.9 aA 1.7 (<10.5) aA 1.6 aB 1.6(0) aA Hexadecanoic acid C16:0 31.6 aB 31.0 (<1.9) aB 32.9 bA 34.7 (>5.5) aA Heptadecanoic acid C17:0 1.0 aA 1.0 (0) aA 0.9 aA 0.9 (0) aA Heptadecenoic acid C17:1 0.8 BLQ BLQ BLQ Octadecanoic acid C18:0 9.6 aA 10.1 (>5.2) aA 9.5 aA 8.5 (<10.5) bA Octadecenoic acid C18:1n9t BLQ 0.8 (>100) A 0.9 a 0.8 (<11.1) bA Octadecenoic acid C18:1n9c 18.4 bB 20.5 (>11.4) aA 20.9 aA 19.4 (<7.2) bB Octadecadienoic acid C18:2n6t 2.3 aA 1.7 (<21.6) bA 1.5 aB 1.5 (0) aA Octadecadienoic acid C18:2n6c 1.8 aA 1.7 (<5.6) aA 1.6 aA 1.6 (0) aA Octadecatrienoic acid C18:3n6c 0.7 a 0.7 (0) aA BLQ 0.7 (>100) A Octadecatrienoic acid C18:3n3c 0.8 aA 0.6 (<25.0) aA 0.7 aA 0.7 (0) aA Eicosanoic acid C20:0 0.4 a 0.4 (0) aA BLQ 0.2 (>100) B CLA, Octadecadienoic acid C18:2 0.7 aA 0.5 (<28.6) aA 0.6 aA 0.6 (0) aA CLA, Octadecadienoic acid C18:2 0.3 a 0.3 (0) aA BLQ 0.3 (>100) A Eicosenoic acid C20:1n9c 0.5 bB 0.1 (<80.0) aB 1.7 aA 0.2 (<88.2) bA Heneicosanoic acid C21:0 BLQ BLQ BLQ 0.4 (>100) Eicosadienoic acid C20:2n6c BLQ 0.5 (>100) BLQ BLQ Eicosatrienoic acid C20:3n6c BLQ 0.3 (>100) BLQ BLQ Eicosatetraenoic acid C20:4n6c BLQ 0.3 (>100) BLQ BLQ Eicosatrienoic acid C20:3n3c 0.3 BLQ (<100) BLQ 0.2 (>100) Docosanoic acid C22:0 2.1 aA 1.5 (<21.6) bA 1.4 bB 1.7 (>21.4) aA Eicosapentaenoic acid C20:5n3c BLQ 0.4 (>100) BLQ BLQ Docosadienoic acid C22:2n6c BLQ BLQ 0.1 BLQ Tetracosanoic acid C24:0 0.9 aA 0.4 (>55.6) bA 0.6 aB 0.3 (<50.0) bA Tetracosenoic acid C24:1n9c BLQ 0.6 (>100) 0.2 BLQ Docosahexaenoic acid C22:6n3c BLQ BLQ BLQ 0.9 (>100) SFAs 68.97 aA 67.20 (<2.6) bB 67.65 bB 69.20 (>2.3) aA MUFAs 24.11 bB 26.03 (>8.0) aA 27.80 aA 24.38 (<12.3) bB PUFAs 6.92 aA 6.77 (<2.2) aA 4.55 bB 6.42 (>41.1) aA CLA 1.0 aA 0.7 (<30.0) bB 0.6 bB 0.8 (>33.3) aA Cholesterol, mg 100g-1 DW 390.3 +- 27.1 bA 421.9 +- 47.7 (>8.1) aA 359.0 +- 12.1 bB 394.8 +- 1.8 (>10.0) aB PUFA/SFA 0.1 aA 0.1 (0) aA 0.1 aA 0.1 (0) aA IA 2.8 bA 3.6 (>28.6) aA 2.4 bB 3.4 (>41.7) aB IT 3.2 aA 3.0 (<6.3) aB 3.0 bB 3.5 (>16.7) aA HH 0.5 aA 0.5 (0) aA 0.5 aA 0.5 (0) aA HPI 0.4 aA 0.4 (0) aA 0.4 aA 0.3 (<25.0) aA Note: Values are means +- SD of duplicates (n = 2). Different lowercase superscripts (a,b) in the same row indicate significant differences (Student's t-test; p <= 0.05) between study phases. Different uppercase (A,B) superscripts in the same row indicate significant differences (Student's t-test; p <= 0.05) between study groups. SFA--saturated fatty acids; MUFA--monounsaturated fatty acids; PUFA--polyunsaturated fatty acids; CLA--conjugated linoleic acid; IA--index of atherogenicity; IT--index of thrombogenicity; HH--ratio of hypocholesterolemic to hypercholesterolemic levels; HPI--health-promoting index; BLQ--below limit of quantification; DW--dry weight; SD--standard deviation; and n.d.--not detected. Disclaimer/Publisher's Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

PMC10000127

The frequency, duration, and intensity of heat waves in Australia are increasing. To reduce the impact of heat waves on milk production, novel management strategies are required. Altering the forage type and amount offered affect the heat load on dairy cows and offer potential strategies to ameliorate the effects of hot weather. Thirty-two multiparous, lactating Holstein-Friesian cows were assigned one of four dietary treatments: chicory high amount, chicory low amount, pasture silage high amount, or pasture silage low amount. These cows were exposed to a heat wave in controlled-environment chambers. Cows that were offered fresh chicory had similar feed intake to cows that were offered pasture silage (15.3 kg DM/d). However, cows that were offered chicory produced greater energy-corrected milk (21.9 vs. 17.2 kg/d) and had a lower maximum body temperature (39.4 vs. 39.6 degC) than cows that were offered pasture silage overall. Cows that were offered the high amount of forage had greater feed intake (16.5 vs. 14.1 kg DM/d) and energy corrected milk yield (20.0 vs. 17.9 kg/d) than cows that were offered the low amount, as intended, but with no difference in maximum body temperature (39.5 degC). We conclude that feeding chicory instead of pasture silage to dairy cows shows promise as a dietary strategy to ameliorate the effect of heat exposure, and there was no advantage in restricting feed amount. heat stress cattle feed type feed amount Agriculture Victoria ResearchDairy AustraliaGardiner FoundationThis investigation was part of the Dairy Feedbase research programme funded by Agriculture Victoria Research; Dairy Australia; and Gardiner Foundation. pmc1. Introduction The frequency, duration, and intensity of heat waves in Australia are increasing . Dairy cows are particularly susceptible to heat stress, with both long-term effects on reproduction, health , and milk production . Overall, heat production as a result of ingesting feed is a combination of heat that is generated during fermentation in the rumen, energy expenditure during digestive processes and the metabolism of digestion end-products . Metabolic heat is influenced by the chemical nature of the end products of ruminal fermentation and digestion, which is in turn determined by the types and amounts of the different feeds consumed. For example, dietary fat does not ferment in the rumen and can therefore be expected to not produce fermentation heat. In contrast, starch and other readily fermentable carbohydrates produce heat in the rumen . Furthermore, fibrous feeds that ferment in the rumen to produce acetate result in greater metabolic heat production than feeds that ferment to produce propionate, as the metabolism of acetate generates more heat than propionate . The fiber within a feed comprises its structural carbohydrates, and the fiber concentration in a feed has been linked to differences in voluntary feed intake between forage types . There is evidence that feeding cows a low-fiber diet (<=30% neutral detergent fiber, NDF) during heat stress can have a positive effect on milk production compared with feeding cows a high-fiber diet (42% NDF) . It has also been observed that cows fed high-fiber (NDF) diets during hot weather consume less feed than cows offered low-fiber diets . Additionally, Miron et al. demonstrated that reducing the NDF concentration of the diet resulted in increased milk production and dry matter intake (DMI), and decreased symptoms of heat stress. Offering a highly digestible (low NDF) and palatable forage type to cows during periods of hot weather may improve voluntary feed intake and milk production. During the summer months, dryland dairy farming in southern Australia relies heavily on pasture silage. Chicory (Cichorium intybus L.) has a lower concentration of NDF in comparison to perennial ryegrass (Lolium perenne L.) . The high dry matter digestibility and fast rumen passage rate of chicory may provide cows with the opportunity to increase their voluntary feed intake . Cows grazing on chicory have been shown to produce more milk and milk solids than cows grazing on perennial ryegrass swards during summer and autumn . Minnee et al. found that the inclusion of chicory increased milk yields by 2.7 kg/day compared to when feeding on perennial ryegrass alone, and that when the metabolizable energy (ME) of perennial ryegrass was low (9.6 MJ/kg of DM) compared to chicory (12.3 MJ/kg of DM) as the proportion of chicory in the diet was increased to 40%, milk yield increased from 9.9 to 12.6 kg/cow per day. In addition, when the ME of the perennial ryegrass was greater (10.5 MJ/kg of DM) milk yield increased from 14.9 to 15.7 kg/cow per day. It is possible that feeding a large amount of chicory will result in a decline in milk fat concentration due to the low NDF concentration. However, there are studies in which chicory was fed at up to 15 kg of DM or 60% of the diet, with no negative effect on milk composition . In comparison to ryegrass silage, chicory is a suitable alternative forage for high-producing dairy cows during summer because it has lower NDF concentration and greater DM digestibility. The low structural carbohydrate concentration of chicory has the potential to ameliorate the increase in body temperature through reduced fermentation heat. This may improve milk yield responses during heat stress compared to when pasture silage is fed to dairy cows. The amount of feed offered during hot weather may be more critical to total heat production than the fiber concentration . For example, the heat production of beef heifers managed on a high feed intake was greater than those managed on a low feed intake . The relationship between intake of ME and heat production has been suggested to be curvilinear . While the feed intake of dairy cows has been reported to decline with increasing dietary NDF, the rate of decline is lower in hot weather . This suggests that there is potential to reduce heat stress responses by altering the forage type and amount offered to lactating dairy cows during a period of increased risk of heat stress. The aim of this experiment was to determine the impact of offering dairy cows a diet containing forages of different proportions of structural carbohydrates on the DMI, milk production and physiological responses in a controlled heat challenge. As it is common practice to feed pasture silage to dairy cows during summer as the main forage source, this was selected as the forage to compare with chicory, a forage with considerably lower structural carbohydrate concentration. We hypothesized that (1) cows offered fresh chicory would have greater feed intake and greater energy-corrected milk (ECM) but lower body temperature compared to cows offered pasture silage; (2) cows offered a high amount of forage would have greater feed intake, ECM and body temperature compared to cows offered a low amount of forage; and (3) during the heat challenge, the decrease in DMI and ECM of cows offered fresh chicory would be less than that of cows offered pasture silage. 2. Materials and Methods 2.1. Cows and Diets Thirty-two multiparous, lactating Holstein-Friesian cows producing 28.9 +- 3.36 kg milk/d (mean +- standard deviation) with 582 +- 46.8 kg body weight (BW), 147 +- 26.8 days in milk (DIM), 5.0 +- 1.32 years of age, 4.5 +- 0.17 body condition (8 point scale; 1 = skinny, 8 = fat; ) and 100 +- 3.2 heat tolerance breeding value (DataGene, Bundoora, Victoria, Australia; 100 = national breed mean) were assigned one of four treatment diets. The 4 treatment diets were (1) CH-H--5 kg DM grain mix plus 13 kg DM of fresh cut chicory, (2) CH-L--5 kg grain mix plus 10 kg DM of fresh cut chicory, (3) PS-H--5 kg DM grain mix plus 13 kg DM pasture silage (predominantly perennial ryegrass), or (4) PS-L--5 kg DM grain mix plus 10 kg DM pasture silage. The grain mix consisted of cracked wheat grain (Triticum aestivum L.) (815 g/kg DM), cracked lupins (Lupinus albus L.) (112 g/kg DM), minerals (51 g/kg DM), E Mag 523 (6 g/kg DM, Queensland Magnesia Pty Ltd., Toowong, Queensland, Australia), and limestone (16 g/kg DM). During the recovery period, the CH-H diet consisted of the grain mix plus ad libitum fresh cut chicory, and the PS-H diet consisted of the grain mix plus ad libitum pasture silage. The CH-L and PS-L diets remained unchanged. Water was available ad libitum at all times except during milking. Compositions of the main dietary ingredients are shown in Table 1. Cows were offered their grain mix during milking and were allowed up to 30 min to consume the grain mix. After this time, refusals were collected, cows were moved to the animal house (described later), and the forage was offered. Chicory was freshly cut at 05:00 and 13:30 at a height of 5 cm above ground using a front-mount mower (Pottinger NovaCat 306F; Alois Pottinger Maschinenfabrik GmbH, Grieskirchen, Austria) and loader wagon (Quantum 3500P; Claas GmbH & Co., Harsewinkel, Germany) mounted on a tractor (Arion 530; Claas GmbH & Co., Harsewinkel, Germany). Silage was prepared once per day in the early afternoon. Silage was collected from the fresh face of a bunk and placed into a mixer wagon (Jaylor 5100 Trailer; Jaylor, East Garafraxa, ON, Canada) for homogenization. Quantities for individual cow feeds were weighed into plastic boxes of 50 L. Boxes of the afternoon feed were set aside until required. Boxes of the morning feed were stored at 4 degC until required. 2.2. Experiment Design The experiment had two treatment factors, each with two levels--forage types (chicory vs. silage) and forage amounts (high vs. low), resulting in a 2 x 2 factorial structure. Although 32 cows were enrolled in the experiment, only 30 places were available in the controlled-environment chambers. The 4 dietary treatments (CH-H, CH-L, PS-H and PS-L) were assigned in a 5-row by 6-column design (Table 2) in which each row corresponded to a cohort, each column to a chamber, and the cells to individual cows. The treatment allocation was randomized according to the row-column design, by permutation of rows and permutation of columns. Provision for the possibility of problems due to animal health or behavior was made by starting cows in two batches 8 days apart. Cows in the same treatment from the next block were used to replace cows removed due to animal health and behavior. Cows that recovered were used in a subsequent block in the position of the same treatment. Block 5 was adjusted by choosing 6 cows from the remaining 8 to boost the number of animals in treatments where previously cows had failed to complete the heat challenge or had been removed for animal health or behavior issues. Each cohort of 6 cows contained all 4 dietary treatments plus two dietary treatments represented twice. The doubled-up treatments were selected in a way to give near-balance of treatments over the 5 cohorts. The 5 cohorts correspond to 5 calving-date blocks, cows were similar within cohort in their DIM, and treatment groups were balanced (in terms of mean and standard deviation) for cow body weight, 7 d milk yield, lactation number, balanced performance index (DataGene, Bundoora, VIC, Australia) and heat-tolerance breeding value. This was achieved using covariate design implemented in GenStat 19 (VSN International Ltd., Hemel Hempstead, UK). During the covariate period (days 1 to 4), cows were managed as a single group in a paddock at ambient conditions with measurements made of milk yield and composition and of BW. During this time, cows continued to be offered the same diet as the main research herd: 7 kg DM/cow/d of a grain mix (cracked barley grain 500 g/kg DM, solvent-extracted canola meal 260 g/kg DM, and cracked wheat grain 240 g/kg DM) during milking and about 9 kg DM/cow of ryegrass pasture and 5 kg DM/cow of pasture silage per day in a paddock. The adaptation to diet commenced on day 5. Cows were moved to the experiment facilities, where they were offered their forage in individual feed stalls for 2 periods of 3.5 h in a well-ventilated animal house and rested on a 560 m2, roofed, rubber-floored loafing pad adjacent to the feed stalls. All cows were transitioned to the high-amount version of their treatment diet over 3 days (days 5 to 7), and adapted to the high-amount version of their diet for 7 days (days 8 to 14); then, cows on the low-amount treatments had their feed-offer reduced to the low amount for the remainder of the experiment. Days 19 to 21 were taken as the base period. Cows were moved to controlled-climate chambers for the pre-challenge period on day 22, where measurements were made at thermoneutral conditions. The heat challenge was then conducted for 2 days (days 23 to 24). From 06:01 to 12:00, the set conditions were 30 degC and 50% RH (temperature humidity index, THI = 80.1); from 12:01 to 18:00, setpoints were 33 degC and 50%RH (THI = 84.2); and from 18:01 to 06:00, the setpoints were 26.0 degC and 60%RH (THI = 74.5). The recovery period was monitored under ambient conditions for 4 d (days 25 to 28). Cows on the high-amount diets were offered ad libitum forage during this period. To ensure animal welfare and health were not compromised during the experiment, the welfare status of individual cows was monitored using a novel heat-stress risk approach. The aim of this was to objectively identify the point where animals were at risk of not being able to recover from the heat challenge and to signal removal from the experiment to maintain animal health and welfare. Each cow was individually assessed for panting score, respiration rate, and rectal temperature multiple times per day during the heat challenge period. A panting score was assigned for each cow based on the scale of Gaughan et al. . To obtain respiration rate, cows were visually observed, and the number of breaths (flank movements) counted in a 20 s period were multiplied by three. Rectal temperature was measured at milking (06:00 h and 15:00 h) during the heat challenge period using a large animal thermometer (AG-Medix, LLC Animal Thermometer AG-102 R01, Wisconsin, USA). Value ranges of respiration rate, panting score and rectal temperature were then each assigned a heat-stress risk rating (Table 3) based on scientific thresholds reported in the literature and our previous observations during experiments conducted under similar conditions . The individual cows' risk ratings were then added together to give a heat-stress risk total (HSRT). Based on the cow's HSRT, additional monitoring actions were implemented to maintain animal welfare and health. These actions are detailed in Figure 1. If necessary, cows were cooled by opening the chamber doors and setting the conditions to thermoneutral (17 degC, 60% RH). Chamber doors were closed once the cow rectal temperature was below 41.0 degC or when conditions within the chamber were cooler than ambient, whichever occurred first, and cows were monitored using the heat-stress risk approach. Cooled cows stayed within their chamber at thermoneutral conditions for the remainder of their scheduled time in the chamber. Cows that were cooled had their recovery monitored from the day that chamber temperature was reduced. 2.3. Feed and Feed Analysis Feed was offered in two equal portions immediately following the morning and afternoon milkings. Feed refusals were collected and weighed after each feed. Dry matter concentration was determined on representative samples of each grain, and minerals were collected every Tuesday and Wednesday of the experiment, as well as on representative samples of chicory collected at every feeding, and samples of silage were collected every afternoon. These feed samples were dried in a forced draft oven at 105 degC for 24 h. Samples of the grains, chicory, and silage offered were collected daily; a 100 g sub-sample was taken and added to the weekly (Monday to Sunday) bulk sample for each feed type and stored at -18 degC. Bulked samples were subsequently oven-dried at 64 degC for 48 h, ground to pass through a 1.0 mm screen (MEP rotor mill; Retsch GmbH, Haan, Germany), and then analyzed for crude protein (CP), soluble protein, acid detergent fiber (ADF), neutral detergent fiber (NDF), lignin, non-fiber carbohydrate, starch, ash, total digestible nutrients (TDN), crude fat (ether extract, EE), sodium, potassium, calcium, magnesium, phosphorous, sulfur, and chloride by traditional chemical analytical methods according to the procedures of Dairy One . Metabolizable energy was calculated using Equation (1) . ME (MJ/kg) = 14.55 - 0.0155 x ADF (g/kg)(1) 2.4. Milk Production and Composition Cows were milked twice daily, at ~06:00 h and ~15:00 h. Throughout the experiment, milk yield was measured for each cow at each milking. When cows were not in the chambers, milk yield measurements were recorded using a DeLaval Alpro milk metering system (MM27BC; DeLaval International, Tumba, Sweden), and milk samples for composition analysis were collected during the covariate, pre-challenge, heat challenge, and recovery periods. When cows were in the controlled-environment chambers, milk yield measurements were made by collecting and weighing the milk from individual cows. Milk samples were analyzed for fat, protein, and lactose using a mid-infrared milk analyzer (Bentley FTS, Bentley Instruments, Chaska, MN, USA). Energy-corrected milk standardized to 4.0% fat and 3.3% protein was calculated using Equation (2) :ECM (kg/d) = (milk yield (kg) x (376 x fat% + 209 x protein% + 948))/3138,(2) Milk yields were assigned such that milk from a PM milking and the following AM milking were matched to feed intake and weather conditions from the day of the PM milking. 2.5. Physiology Body temperature was measured intravaginally (vaginal temperature) and recorded every 5 min during the covariate, pre-challenge, heat challenge, and recovery periods using temperature loggers (iButton DS1922L; Maxim Integrated, San Jose, CA, USA) as described by Garner et al. and set to high-resolution mode. Skin temperature was measured every day during the pre-challenge, heat challenge, and recovery periods at approximately 06:00 h and 15:00 h. The surface temperature of the cow was measured using a non-contact infra-red thermometer (Oricom HFS1000; Oricom International, South Windsor, NSW, Australia) in object temperature mode at each of the left paralumbar fossa (left flank), the left trapezius muscle (neck) outside of the left front leg at the mid-point of the radius (leg upper), and the outside of the left front leg at the mid-point of the metacarpus (leg lower). Using the same methods described in the HSRT, respiration rate and panting score were measured by visually observing each cow at approximately 05:45 h and 14:45 h every day during the pre-challenge, heat challenge, and recovery periods. An additional observation was made at 11:45 h on each day of the 2-day heat challenge. Rectal temperature was measured at 06:00 h and 15:00 h during the heat challenge period. 2.6. Blood Blood samples were collected from each cow at approximately 14:45 h via coccygeal venipuncture during the pre-challenge period (day 22), on day two of the heat challenge period (day 24) and once during the recovery period (day 26). On each occasion, three 10 mL blood samples were collected--one into a vacutainer containing potassium EDTA for plasma collection and two samples into a vacutainer containing clotting activators for serum collection (BD Vacutainer System, Plymouth, UK). Using a syringe, approximately 0.1 mL of blood from the EDTA tube was removed immediately after collection and inserted into the reader chip of an auto-calibrated, portable, blood gas analyzer (Epoc Host2 Zebra MC55A0, Epocal Inc. Ottawa, ON, Canada), as per manufacturer's instructions. Analytical variables determined were blood pH, partial pressure of CO2 (pCO2), partial pressure of O2 (pO2), Na+, K+, Ca++, Cl-, glucose, lactate, creatinine, and hematocrit (Hct); the calculated values of bicarbonate (cHCO3-), total CO2 (cTCO2), base excess of extracellular fluid, base excess of blood, hemoglobin (cHgb), and oxygen saturation (cSO2); and anion gap K+ (AGapK). The remaining sample was then placed on ice before centrifugation within 30 min of collection at 1500x g and 4 degC for 10 min. Plasma was decanted into storage vials and stored at -20 degC until analysis. The two samples for serum collection were kept at 25 degC for 1.5 h before centrifugation at 1300 x g and 25 degC for 10 min. Serum was decanted into storage vials and stored at -20 degC until analysis. A subset of serum samples was transported on ice to Regional Laboratory Services (Benalla, VIC, Australia) within 24 h of collection. Samples were analyzed for concentrations of beta-hydroxy butyrate (BHB), non-esterified fatty acids (NEFA), urea total protein, albumin, glucose, haptoglobin, and phosphorus using a Kone 20 XT clinical chemistry analyzer (Thermo Fisher Scientific, Waltham, MA, USA), with reagents supplied by Randox Laboratories (Crumlin, UK) for fatty acids, and BUN and Regional Laboratory Services (Benalla, VIC, Australia) for BHB, albumin, and total protein. Haptoglobin concentration was measured using a colorimetric rate assay . 2.7. Calculations and Statistical Analyses The THI was calculated using Equation (3) THI = Tdb + (0.36 x Tdp) + 41.2,(3) where: Tdb = hourly dry bulb temperature (degC); Tdp = dew point temperature (degC); RH = relative humidity (%); b = [log10(RH/100.0) + (17.27 x Tdb)/(237.3 + Tdb)]/17.27; Tdp = (237.3 x b)/(1.0 - b). Temperature and relative humidity data were collected every 1 min during the pre-challenge and recovery periods using Minnow 1.0 data loggers (Senonics LLC, Arvada, CO, USA) positioned 2 m above the ground on the fence line between the feed stalls and the loafing area. During the heat challenge, temperature and relative humidity data were recorded every 1 min by the control system of the controlled-climate chambers. A THI threshold of 68 was used to determine if weather conditions were imposing heat stress on the animals as this is the point that has previously been identified as being when weather conditions start affecting milk production . Duration of vaginal temperature greater than 38.8 degC was calculated as the number of minutes that vaginal temperature exceeded the 38.8 degC threshold on a given day (06:00 h to 05:59 h the next calendar day). The threshold of 38.8 degC was used because this was the mean vaginal temperature of cows during the thermoneutral period in the experiment of Garner et al. . Only data from cows that completed the heat challenge period were used in the analysis of treatment effects. Data from 27 cows (CH-H, n = 7; CH-L, n = 7; PS-H, n = 7; PS-L, n = 6) were used in the following statistical analyses. The effect of heat challenge (trend over time) on milk production variables, body (i.e., vaginal) temperature, and respiration rate were analyzed by examining the linear regression slopes for mean changes between pre-challenge and heat challenge periods, heat challenge and recovery periods, and pre-challenge and recovery periods. These response variables were averaged across the treatments for each cohort, and this data was used for the t-test to examine if the slope was significantly different from zero at a 5% level of significance. The cohort was used as a replicate since it provided independent replication with respect to time. Given our experiment design, examining the main effect of forage type involved a comparison between levels of forage type only, and examining the main effect of forage amount involved comparison between levels of forage amount only. The interaction examined how effect of forage type changes with the levels of forage amount. The effects of treatments (feed type, feed amount) and their interaction (feed type x feed amount) on response variables in each period and between any two periods were analyzed using Linear Mixed Models (LMM), with individual cows as the unit of analyses. The fixed effects included the main effect of feed type, the main effect of feed amount, and their interaction. The effect of covariate (same variable measured in covariate period if available) and effect of cohort were also fitted as fixed effects. The effect of cow was fitted as random effect and was used as a residual term. The response variables were constructed to address the hypotheses, with one datum per animal by averaging per animal in each period. Also, a weighted mean per animal over the 10 days of measurement (3-day base period, 1-day pre-challenge period, 2-day heat challenge period and 4-day recovery period) for milk production was constructed. The change variables were calculated for each individual cow by taking the difference between the mean value of a specific variable measured during the pre-challenge period and the value of the same variable measured during the heat-challenge period. Similar change variables were calculated between the first and second days of recovery (initial recovery). The Linear Mixed Models (LMM) used to analyze these variables can be written in the following form:y=m+byc+K+F+A+F.A+e where y is the response variable of interest, m is an overall constant (grand mean), yc is the covariate (same variable if available from the covariate period), b is the linear coefficient, K is the effect of cohort, F is the main effect of feed type, A is the main effect of feed amount, F.A is the interaction between feed type and feed amount, and e is the random error for an individual cow assumed to follow normal distribution with zero mean and constant variance. The LMM were fitted using a restricted maximum likelihood (REML) algorithm in GenStat. Histograms of residuals and plots of residuals versus fitted values were examined for normality of distribution with constant variance. Three cows that did not complete the heat challenge were excluded from the analyses. All statistical analyses were undertaken using GenStat (GenStat release 21, VSN International Ltd., Hemel Hempstead, UK). Any p-values of < 0.05 were considered significant, and those >=0.05 and <0.10 were considered a trend. The Duncan's letters indicating significant differences amongst the comparisons of treatment means were based on Fisher's unprotected Least Significant Difference (LSD) test. When Fisher's unprotected LSD is used, the comparisons are tested even when the p-value for the term generating these means is not statistically significant. 3. Results Weather conditions during the base period (ambient conditions) included an air temperature of 18.4 +- 3.97 degC (daily mean +- standard deviation), relative humidity of 79 +- 14.5%, and THI of 66 +- 5.1. During the pre-challenge (in chambers) period, the cows experienced an air temperature of 18.8 +- 0.44 degC, relative humidity of 68 +- 2.1%, and THI of 66 +- 0.6. During the heat challenge (in chambers) period, the cows experienced an air temperature of 26.5 +- 3.15 degC, relative humidity of 67 +- 5.0%, and THI of 76 +- 4.1. Weather during the recovery period (ambient conditions) included an air temperature of 17.0 +- 4.25 degC, relative humidity of 79 +- 15.0%, and THI of 64 +- 5.5. The daily pattern of THI experienced by the cows during the base period, pre-challenge, heat challenge and recovery periods is shown in Figure 2. The coefficient of variation in THI was 0.01 during the pre-challenge period, 0.05 during the heat challenge, and 0.09 during the recovery period. 3.1. Effect of Heat Challenge Daily changes in DMI, milk yield, ECM and maximum vaginal temperature during the pre-challenge, heat challenge and recovery periods are shown in Figure 3. The heat challenge induced heat stress symptoms in all cows (Table 4). Mean vaginal temperature and respiration rate were greater in all cows during the heat challenge compared to during other periods (p < 0.05). While there was no effect of the heat challenge on feed or on milk yield during the heat challenge, we note that minimum milk production across the 10 days of measurements occurred on the day immediately after the heat challenge. 3.2. Challenge Completion In response to adjusting cohort 5 to balance the cows completing the heat challenge, the two cows not subjected to the heat challenge included one on the CH-L diet and one on the PS-L diet. One cow (PS-H) scheduled for the heat challenge became ill prior to the heat challenge, and two cows (1 CH-H, 1 PS-L) had their heat challenge cut short because their heat-stress risk total exceeded the pre-determined threshold. 3.3. Dry Matter Intake Feed intake across the 10 days of measurements, inclusive of the pre-challenge, heat challenge, and recovery periods (Table 5), was not affected by feed type, but as intended, cows offered more feed ate more (p < 0.001). The metabolizable energy of the chicory was greater than the pasture silage. Thus, metabolizable energy intake (MEI) was greater in cows offered chicory than those offered pasture silage (p = 0.020). Mean amount of grain refused per cow per day across the entire experiment was 25 g DM, but this measure ranged from 0 to 2.5 kg DM/day for individual cows. During the pre-challenge period, DMI (Table 5) was not affected by feed type, but the cows offered the high amount of feed ate more than those cows offered the low amount (p < 0.001). Intake of ME (Table 5) was greater in cows offered chicory than those offered pasture silage (p = 0.019) and MEI was greater in cows offered the high amount of DM than in those offered the low amount of DM (p < 0.001). During the heat challenge, DMI was not affected by feed type, but cows offered the high amount of forage ate more than those offered the low amount (p = 0.008). Intake of ME was not affected by feed type (p = 0.489), but MEI was greater in cows offered the high amount of feed than those cows offered the low amount (p = 0.021). From the pre-challenge period to the heat challenge, DMI and MEI declined, and there was no effect of feed type or feed amount. During the initial recovery, DMI increased for cows offered chicory but continued to decline for cows offered pasture silage (p = 0.022). There was an interaction between feed type and amount for DMI during the initial recovery period such that DMI increased for cows offered the high amount of forage but not for those offered the low amount (p = 0.017). This reflects all cows on the high amount of forage being offered more during the recovery period, but only the cows offered the CH-H diet consumed this extra feed (Table 5). The intake of ME increased during the initial recovery period for cows offered chicory, but continued to decline for cows offered pasture silage (p = 0.031). There was an interaction between feed type and amount for DMI during the initial recovery period such that MEI increased for cows offered the high amount of feed but not for those offered the low amount (p = 0.013), reflecting the change in intake. Again, this reflects the cows offered the CH-H diet consuming more feed than the other cows. 3.4. Milk Yield Across the 10 days of measurements, cows offered chicory had greater yields of milk (p < 0.001), ECM (p < 0.001), fat (p = 0.017), protein (p < 0.001), and lactose (p < 0.001) than cows offered pasture silage (Table 6). The concentration of fat in their milk was lower (p < 0.001), and the concentration of protein was greater (p < 0.001) for cows offered chicory compared to cows offered pasture silage. Cows offered the high amount of feed produced more milk than cows offered the low amount (p < 0.004) but there was no effect on milk composition. There were interactions between type and amount of forage for milk yield (p = 0.003), ECM yield (p < 0.001), fat yield (p = 0.035), and protein yield (p = 0.021). During the pre-challenge period, milk yields (p = 0.002) and yields of ECM (p = 0.021) and milk protein (p < 0.001) were greater from cows offered chicory than from cows offered pasture silage, and there was no effect of feed type on milk fat yield. Compared to cows offered chicory, cows offered pasture silage had a greater concentration of milk fat (p = 0.003) but a lower concentration of milk protein (p = 0.017). There was no effect of feed amount on milk parameters. There was an interaction between forage amount and type on milk yield (p = 0.038). During the heat challenge, cows offered chicory had greater yields of milk (p < 0.001), ECM (p < 0.001), milk fat (p = 0.005), and milk protein (p < 0.001) than cows offered pasture silage. Cows offered chicory had a lower concentration of milk fat (p = 0.001) but a greater concentration of milk protein (p = 0.004) than cows offered pasture silage. Cows offered the high amount of feed had greater yields of milk (p = 0.006), ECM (p < 0.001), fat (p = 0.004), and protein (p = 0.001) than cows offered the low amount. There were interactions between type and amount of forage for milk yield (p = 0.014), ECM yield (p < 0.001), and fat yield (p = 0.003). From the pre-challenge period to the heat challenge, feed type had no effect on the change in milk parameters. Feed amount affected the change in yields of milk and milk components (p < 0.05). Cows offered the high amount of feed had a positive change in yields of milk, ECM, milk fat, and milk protein, whereas this change was negative for cows offered the low amount of feed (Table 6). There were no interactions. During the initial recovery, the only difference observed in milk parameters was that the increase in the yield of milk protein was greater for cows offered chicory than those offered pasture silage (p = 0.046). There were no interactions. 3.5. Body Temperature Across the 10 days of measurements, cows offered chicory had lower mean (p = 0.022) and maximum (p = 0.024) vaginal temperatures (Table 7) than cows offered pasture silage. Feed amount had no effect on vaginal temperature parameters. During the pre-challenge period, all vaginal temperature parameters were lower in cows offered chicory than in cows offered pasture silage (p < 0.006). There was no effect of feed amount on vaginal temperature parameters. During the heat challenge, cows offered chicory had a lower mean vaginal temperature (p = 0.048) and duration above 38.8 degC (p = 0.010) than cows offered pasture silage. They also tended to have a lower maximum vaginal temperature (p = 0.059) than cows offered pasture silage. Cows offered the high amount of feed had a greater maximum vaginal temperature (p = 0.011) and tended to have a greater mean vaginal temperature (p = 0.059) than cows offered the low amount. From the pre-challenge to the heat challenge, feed type had no effect on change in vaginal temperature parameters (Table 7). Cows offered the high amount of feed tended to have a greater increase in vaginal temperature (p = 0.069) than cows offered the low amount, and they did have a greater increase in maximum temperature (p = 0.006). During the initial recovery, cows offered chicory tended to have a lower change in mean vaginal temperature (p = 0.085) than cows offered pasture silage. There was no effect of feed amount on change in any vaginal temperature parameters. 3.6. Respiration Rate and Skin Temperature During the pre-challenge period, respiration rate (p = 0.063) tended to be greater, but left flank temperature (p = 0.092) and leg-upper temperature (p = 0.086) tended to be lower in cows offered chicory compared to those offered pasture silage (Table 8). Leg upper (p < 0.001) and lower (p = 0.014) temperatures were greater in cows fed the high amount of feed than in those fed the low amount. During the heat challenge, cows offered chicory tended to have a lower upper-leg temperature (p = 0.097) than cows offered pasture silage. Cows offered the high amount of feed had greater respiration rates (p = 0.035) and skin temperatures (p < 0.016) than cows offered the low amount. 3.7. Blood Analytes Select blood analytes are presented in Table 9, and a complete listing of blood analytes measured is available in Table S1 Blood parameters. During the pre-challenge period, glucose concentration tended to be lower (p = 0.062) in cows offered chicory compared to those offered pasture silage. Cows offered the high amount tended to have greater blood pH (p = 0.069) than those offered the low amount. During the heat-challenge, cows offered chicory had lower blood concentrations of BHB (p = 0.006) and glucose (p = 0.024) than cows offered pasture silage. Cows offered the high amount tended to have a lower concentration of haptoglobin (p = 0.090) than cows offered the low amount. 4. Discussion 4.1. Forage Type The cows that were offered fresh chicory had similar feed intake compared to the cows that were offered pasture silage during each period of the experiment, except for during the recovery period. However, the cows that were offered chicory produced more ECM and had a lower body temperature than the cows that were offered pasture silage. Thus, we can only accept the ECM and body temperature portions of our first hypothesis and reject the intake portion. During the recovery period, cows on the high feed amount were offered a greater quantity of feed, but only the cows that were offered chicory chose to consume this extra feed. This greater voluntary intake of feed may be explained by the lower body temperature experienced by the cows that were offered chicory or the higher dry matter digestibility and faster rumen passage rate of chicory compared to pasture silage. Also, we speculate that the lower dry matter concentration of the chicory may have resulted in greater palatability compared to the pasture silage. The energy corrected milk yield from the cows that were offered chicory was greater than that from the cows that were offered pasture silage during all periods of the experiment, including the heat challenge period. This response to feeding chicory is in agreement with previous reports wherein chicory was fed in place of fresh ryegrass . The ECM our cows that were fed chicory was 3.6 kg greater than our cows that were fed pasture silage, and this difference is mostly accounted for by the difference in intake of ME (13 MJ = 2.6 kg ECM, ). The greater ECM yield from the cows that were offered chicory compared to those that were offered pasture silage was driven by a greater milk yield and a greater concentration of milk protein, despite the concentration of milk fat being lower in milk from the cows that were fed chicory than those fed pasture silage. This lower milk fat concentration in the cows that were offered chicory is in contrast to previous reports wherein feeding chicory had no effect on the concentration of milk fat . In those studies, chicory was no more than 60% of the total diet, while the cows in our experiment were offered chicory at 73% of total DMI. The lower concentration of milk fat is thought to be due to the chicory diet having a lower concentration of NDF than the pasture silage diet, since the proportion of fiber in the diet has previously been correlated with the concentration of fat in milk . Vaginal temperature was lower over the 10 days of measurement in the cows that were offered chicory than in the cows offered pasture silage. This is consistent with the heat increment of the chicory being lower than that of pasture silage due to the lower concentration of fiber . While the increase in body temperature induced by the heat challenge was not affected by diet type, the cows that were offered chicory had a lower temperature during the pre-challenge period than the cows that were offered the pasture silage, thus they also had a lower body temperature during the heat challenge. This suggests that while feeding chicory may not affect the change in body temperature due to a heat challenge, the lower body temperatures during the heat challenge means that our cows that were fed chicory experienced less thermal stress than those that were offered pasture silage. This is supported by the fact that the chicory-fed cows had a lower body temperature but had a similar respiration rate and skin temperature to the cows fed pasture silage. It is plausible that the tendency for greater respiration rate of the cows that were offered chicory contributed to a lower body temperature compared to cows offered pasture silage. 4.2. Forage Amount The cows that were offered the high amount of forage had a greater DMI and ECM yield than the cows offered the low amount, as intended. However, there was no overall difference in body temperature for the 10 days of measurement. Thus, we partially accept our second hypothesis for DMI and ECM, but not for body temperature. Dry matter intake was expected to be greater in the cows that were offered the high amount of forage than in those offered the low amount because the cows offered the low amount had their intake deliberately restricted. However, the cows that were offered the high amount of feed were also expected to have a greater decline in DMI during the heat challenge than those offered the low amount , but this was not the case in our experiment. While we did see numerical treatment differences in the decline in the DMI of our cows, these differences were not significant, illustrating a large variation in response between individual cows. During the first few days of the recovery period, there was a tendency for the cows that were offered the high amount of forage to have a greater increase in DMI than the cows offered the low amount, but this result could be confounded by us allowing those cows on the high amount to have ad libitum intake of feed instead of the previously capped amount. There was also an interaction between feed amount and feed type, but we have not been able to untangle this. Energy-corrected milk was expected to be greater in the cows that were offered the high amount of feed than in those offered the low amount because of the greater nutrient intake. However, the difference in milk yield over the 10-day measurement period was less than the 4.4 kg predicted by the difference in ME intake (5 MJ/kg milk, ). This discrepancy could be due to a difference in energy partitioning during the heat challenge, which has been reported previously . Untangling the response in our experiment is complicated by the interaction observed between the effect of feed type and feed amount and is thought to be driven by the cows that were offered the PS-H diet not producing more milk than those cows offered the PS-L diet. The yield of ECM was observed to increase from the pre-challenge to the heat challenge in the cows that were fed the high amount of forage, but it decreased in the cows fed the low amount. This is despite the fact that the cows that were fed the high amount of forage had a greater maximum body temperature during the heat challenge than the cows fed the low amount. This greater temperature would be expected to have a greater depressive effect on feed intake in cows fed the high amount of forage compared to those offered the low amount. Our results suggest that reducing the amount of feed offered to cows during a heat challenge could result in favorable outcomes with respect to body temperature, but further work is necessary to elucidate the effect of high feed amount during heat challenge. Body temperature was expected to be affected by the amount of feed eaten, since heat production has been positively correlated with DMI , and a greater heat production could be expected to result in a greater body temperature. It is possible that the cows that were offered a high amount of feed could shed heat at a greater rate than the cows offered a low amount of feed, thereby maintaining their body temperature. However, we have no evidence to support this. During the heat challenge, those cows with a greater DMI did have a greater body temperature, greater respiration rates and greater skin temperatures, which does agree with heat production being positively correlated with MEI. This correlation was established in sheep in metabolism cages , so the difference in observations across periods of our experiment could be due to the correlation only being apparent when our cows were under thermal stress. Restricting the intake of our cows during a heat challenge did not affect the decline in intake from thermoneutral conditions, and there was a negative impact on milk production over all weather conditions. There was a beneficial effect of restricted intake on body temperature during the heat challenge, but we consider this single advantage to be insufficient to justify restricting feed intake as a strategy for managing hot weather events. 4.3. Heat Challenge In contrast to our third hypothesis, diet type had no effect on the decrease in DMI or ECM due to the heat challenge. The cows that were offered chicory had a greater observed numerical decline in DMI from the pre-challenge to the heat-challenge than the cows offered pasture silage, but the large variation in individual cow DMI meant that no statistical difference between the decline in DMI was identified. The low fiber concentration in chicory relative to pasture silage was expected to result in a greater cow DMI during the heat challenge period . However, there is a report showing the decline in intake by animals with high intakes of a low-fiber diet during hot weather being greater than for those animals offered a high-fiber diet . A possible explanation for our observations is gut fill. This is because the cows that were offered chicory consumed 106 kg of fresh material, while the cows offered silage only consumed 29 kg of fresh material due to the difference in dry matter of the forages (approximate DM: chicory 10%; pasture silage 35%). The cows that were offered chicory had a lower body temperature and tended to have lower skin temperatures than those offered pasture silage. This agrees with previous reports in which cows had lower rectal temperatures when fed low NDF forage compared to high NDF forage . The body temperature appears more closely related to the intake of structural carbohydrates than the proportions of individual structural carbohydrates per se, as more fibrous feeds produce greater amounts of acetate, generating significant heat in the rumen . It has been suggested that the amount of feed offered during hot weather may be more critical to total metabolic heat production than the fiber concentration of the feed . In this experiment, the loss in production during the heat challenge was more pronounced in the cows that were offered less feed. The change in ECM from the pre-challenge to the heat challenge period was positive for the cows that were offered the high amount of forage, being 2.39 kg ECM greater in chicory compared to pasture-silage-fed cows. In contrast, the change in ECM observed in the cows that were offered the low amount of feed was negative, with the reduction in ECM yield being 1.27 kg ECM less in the chicory-fed cows compared to those fed pasture silage. While the cows that were offered the low amount of pasture silage consumed less feed than those offered the high amount, they had similar milk yields throughout the experiment. The reason for this is unclear. A possible confounding effect is the change of environment on the day before the heat challenge, when cows were moved to the controlled-environment chambers. Any effect from the change in location was minimized by familiarizing the cows with the controlled-environment chambers before the start of the experiment. The reason for the observed increase in milk yield from the pre-challenge to the heat challenge period is not clear, but it may be due to our allocation of effects to days. We allocated milk records such that milk produced was matched to the feed consumed prior to that milking. The weather cycle, and hence the heat challenge, were synchronized with the feeding cycle. Despite this, we observed the minimum milk yield on the first day of the recovery, indicating that there was a lag effect of the heat challenge. A lag in the effect of hot weather has been recognized previously, with weather up to 4 days prior to a milking reported to affect the milk yield . Given the short duration of our heat challenge and the potentially long lag effect, we conclude that it is difficult to interpret the change from the pre-challenge to the heat challenge. We recommend that our 10-day average data be used to assess the merits of chicory against those of pasture silage. 4.4. Blood Biochemistry During the experiment, we measured only minor changes to the blood biochemical profiles of our cows due to each of feed type, feed amount, and exposure to the heat challenge. Before exposure to heat, blood pH tended to be greater in the cows that were fed the high amount compared to the low amount of feed, but it remained within the expected normal range (7.35 to 7.50; ). During the heat challenge, blood pH remained within normal limits but was greater than during the pre-challenge period. This suggests that there was a minimal disturbance of the acid-base balance of blood during the short period of thermal stress. During the pre-challenge period, blood glucose concentration tended to be lower in the cows that were fed chicory compared to pasture silage, and this trend became significant during the heat challenge. As previous research has shown that cows with greater milk yields have lower blood glucose concentrations , our results may indicate a greater uptake of glucose by the mammary gland of cows that are fed chicory to support the synthesis of greater milk volumes observed in this experiment. High concentrations of BHB in the blood have been correlated with oxidative stress and apoptosis in the liver . The cows in this experiment had BHB concentrations well below the threshold for ketosis of 1.0 to 1.4 mmol before and during the heat challenge. This indicates that the level of thermal stress induced by the heat challenge was mild. The lower concentration of BHB in the serum of the cows that were fed chicory compared to those fed pasture silage is likely explained by butyric acid in the silage being converted to blood BHB . 4.5. Feed Presentation The manner of offering feed to our cows leaves several questions. Feeding cool feed to hot cows may mean that our results are disconnected from what would have happened had the feed experienced the same environmental conditions as the cows. The chicory was harvested from ambient conditions, but conditions were cool. The silage was collected from the stack once daily in the afternoon and the PM feed was left to sit in ambient (cool) conditions, but the AM feed was stored at 4 degC until required. There is also the fact that our feeds were offered in individual bins where there was no competition as exists when cows are grazing. Added to this is the impact that hot weather, similar to that used to challenge the cows, has on the nutritional and palatability aspects of the forage . Given these deviations from conditions that could be expected on commercial farms, further research is required to validate these results under grazing scenarios where the forage is subjected to the same environmental conditions as the cows. 4.6. Heat Stress Risk Assessment under Experiment Conditions The use of the HSRT in this experiment, using multiple indicators of heat stress (respiration rate, body temperature, panting score) rather than a single method (body temperature only), resulted in more animals safely completing the heat challenge period and remaining under experimental conditions. The increased frequency of monitoring triggered by a combination of respiratory and internal measures of heat stress provided a good balance between the experiment requirements and the animals' welfare. This improved the proportion of animals that completed the heat challenge in this experiment (27 of 29 cows) compared with previous experiments (12 of 24 cows and 18 of 24 cows ) that used a body temperature threshold of 40.9 degC as the only criterion at which cows were to be cooled before the end of the heat challenge; this improved the statistical power and efficiency of the experimental design. We recommend the use of a heat-stress risk assessment that uses multiple indicators of heat stress such as the one described in this manuscript in future controlled heat-exposure experiments. 5. Conclusions Forage type affected the ECM yield and body temperature of the cows across the 10 days of measurement. The cows that were offered fresh chicory produced more milk and had a lower body temperature than those that were offered pasture silage, despite consuming similar amounts of feed. However, the type of forage offered had no impact on the change in DMI or ECM during the heat challenge. While feeding chicory may not affect the change in body temperature due to a heat challenge, the resulting lower body temperature during the heat challenge indicated that the cows that were offered chicory experienced less thermal stress than those that were offered pasture silage. The amount of forage that was offered had no significant effect on body temperature over the 10-day observation period. However, offering more forage resulted in a greater DMI and ECM yield than when a lower amount of forage was offered. Restricting the intake of our cows during the heat challenge did not affect the decline in intake from thermoneutral conditions, and there was a negative impact on milk production over all weather conditions. There was a beneficial effect of restricted intake on body temperature during the heat challenge, but we consider this single advantage to be insufficient to justify the restriction of feed intake as a strategy for managing hot weather events. It is important to note that our forage was not subjected to the same weather/environmental conditions as our cows and that our feeds were offered in individual bins where there was no competition--as there is when cows are grazing. There is a need to validate our current results, wherein the feed should be grazed as in a commercial farm setting and therefore subjected to the same environmental conditions as the cows. Acknowledgments This work would not have been possible without the support of the technical staff at Agriculture Victoria Research, Ellinbank Centre. We also thank Meaghan Douglas, Agriculture Victoria Research, for modelling the diet responses in CPM Dairy. Supplementary Materials The following supporting information can be downloaded at: Table S1: Blood parameters. Click here for additional data file. Author Contributions Conceptualization, S.R.O.W., J.B.G., P.J.M. and L.C.M.; methodology, S.R.O.W., J.B.G., P.J.M., M.C.H. and L.C.M.; formal analysis, M.C.H. and K.G.; investigation, S.R.O.W., J.B.G., P.J.M., M.C.H. and L.C.M.; resources, W.J.W.; data curation, S.R.O.W., M.C.H. and K.G.; writing--original draft preparation, S.R.O.W. and L.C.M.; writing--review and editing, S.R.O.W., J.B.G., W.J.W., P.J.M., K.G. and L.C.M.; visualization, S.R.O.W. and M.C.H.; supervision, W.J.W.; project administration, W.J.W. and L.C.M.; funding acquisition, W.J.W., and L.C.M. All authors have read and agreed to the published version of the manuscript. Institutional Review Board Statement Cows were cared for according to the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes (NHMRC, 2013). The animal study protocol was approved by the DJPR Agricultural Research & Extension Animal Ethics Committee (Protocol code: 2019-15, Approved: 15 October 2019). Data Availability Statement The Data are available from the corresponding author upon reasonable request. Conflicts of Interest The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results. Figure 1 Flowchart of actions taken depending on the heat-stress risk total (HSRT) of the cow. Figure 2 Mean environmental conditions experienced by the cows (blue line) and mean vaginal temperature of all cows (orange line) during the base period (B), pre-challenge (P), heat challenge (H), and recovery period (R). Shading bands show +- one standard deviation from the mean. The pre-challenge and heat challenge were generated in controlled-climate chambers. Cows were in ambient conditions at all other times. Figure 3 Mean daily dry matter intake (DMI), milk yield (Milk), energy-corrected milk yield (ECM) and maximum vaginal temperature (BTmax) of cows offered chicory (green line) or pasture silage (orange line) during the base period (B), pre-challenge (P), heat challenge (H), and recovery period (P). Each band shows +- one standard deviation from the forage-type mean. The pre-challenge and heat challenge were generated in controlled-climate chambers. Cows were in ambient conditions at other times. animals-13-00867-t001_Table 1 Table 1 Composition of main dietary ingredients (g/kg DM unless otherwise stated). Parameter Grain Mix 1 Chicory Pasture Silage Dry matter (g/kg as fed) 878 99 350 Crude protein 143 201 157 Soluble protein (% CP) 13.9 15.7 14.7 Acid detergent fiber 42 289 344 Neutral detergent fiber 93 338 479 Lignin 11 106 30 Non fiber carbohydrate 670 274 197 Starch 551 10 11 Ash 66 139 115 Total digestible nutrients 822 577 652 Calcium 15 16 6.3 Magnesium 7.4 5.4 2.0 Sodium 1.0 9.0 4.2 Potassium 4.7 33 35 Chloride 2.8 18 12 DCAD (meq./100 g DM) -3.8 44 55 Copper (mg/kg DM) 86 8.8 13 Sulfur 1.9 4.8 3.1 Crude fat 29 48 52 ME 2 (MJ/kg DM) 13.9 10.1 9.2 1 Grain mix consisted of cracked wheat grain (815 g/kg DM), cracked lupins (112 g/kg DM), minerals (51 g/kg DM), E Mag 523 (6 g/kg DM, Queensland Magnesia Pty Ltd., Toowong, QLD, Australia), and limestone (16 g/kg DM). 2 ME = metabolizable energy. animals-13-00867-t002_Table 2 Table 2 Blocking structure and treatment allocation to chambers for each of the 5 cohorts. Cohort Chamber 1 Chamber 2 Chamber 3 Chamber 4 Chamber 5 Chamber 6 1 CH-L PS-H CH-H PS-L CH-H PS-L 2 CH-L PS-L PS-H CH-H CH-H PS-H 3 PS-L CH-H PS-H PS-H PS-L CH-L 4 CH-H CH-L PS-L CH-H CH-L PS-H 5 PS-H PS-L CH-L CH-L PS-H CH-H animals-13-00867-t003_Table 3 Table 3 Calculating the heat-stress risk total of individual cows housed in controlled-climate chambers under hot conditions by rating risk of thermal stress measured by observations of panting score, respiration rate, and rectal temperature. Panting Score 1 Risk Rating Respiratory Rate 2 Risk Rating Rectal Temperature 3 Risk Rating 0 to 2.5 0 <=120 0 <=40.9 0 3 1.5 121 to 159 1 41.0 to 41.5 1 3.5 2 >=160 2 41.6 to 41.9 2 4 3 Variable 3 >=42 3 4.5 4 PS risk + RR risk + RT risk = HSRT 4 1 PS obtained by visual observation of animals according to . 2 RR--breaths per minute, obtained by visual observation. 3 RT, degC, obtained via large animal rectal thermometer. 4 HRST--Heat-stress risk total, calculated from the sum of risk from each of PS, RR and RT. animals-13-00867-t004_Table 4 Table 4 Comparisons of the effects of pre-challenge (P), heat challenge (H) and recovery (R) periods on means of total DMI (kg/day), milk yield (kg/day), ECM yield (kg/day), milk fat concentration (g/kg), milk protein concentration (g/kg), milk lactose concentration (g/kg), vaginal temperature (degC) and respiration rate (breaths/min). Item Period SED 1 Contrast p-Value 2 P H R P-H H-R P-R P-H H-R P-R n 27 27 27 Total DMI 15.2 14.5 15.6 0.702 0.389 0.602 0.296 0.035 0.566 Milk yield 20.0 20.1 18.7 0.412 0.335 0.466 0.598 0.013 0.064 ECM yield 3 19.4 19.4 18.0 0.690 0.336 0.663 0.956 0.015 0.115 Milk fat 0.77 0.78 0.71 0.041 0.016 0.039 0.864 0.013 0.194 Milk protein 0.62 0.60 0.58 0.014 0.009 0.016 0.277 0.105 0.078 Milk lactose 0.99 1.00 0.90 0.024 0.017 0.029 0.422 0.003 0.045 Vaginal temperature 38.7 39.1 38.8 0.026 0.050 0.025 <0.001 0.002 0.120 Respiration rate 32.3 78.5 44.9 3.188 4.190 2.189 <0.001 0.001 0.005 1 SED = standard error of difference between means for the following contrasts: pre-challenge to heat challenge (P-H); heat challenge to recovery (H-R) and pre-challenge to recovery (P-R). 2 The p-value is based on t-tests, as described in the method section. 3 ECM = energy corrected milk yield (kg/d). animals-13-00867-t005_Table 5 Table 5 Nutrient intake (kg/d unless specified) across the 10 days of measurement and the intake of dry matter (DMI, kg/d) and metabolizable energy (MEI, MJ/d) during selected periods of the experiment. Main Effect 1 Treatment Means 2 Feed Amount SEDm 3 p Value CH PS High Low Feed Amount Feed Amount Feed x Amount CH-H 4 CH-L PS-H PS-L SEDt 5 n 14 13 14 13 7 7 7 6 10-day mean 6 Total DMI 15.4 15.2 16.5 14.1 0.42 0.41 0.558 <0.001 0.246 16.9 b 14.0 a 16.1 b 14.2 a 0.58 CP 2.80 2.29 2.76 2.33 0.069 0.066 <0.001 <0.001 0.080 3.08 c 2.52 b 2.44 b 2.13 a 0.095 NDF 4.05 5.43 5.23 4.25 0.163 0.155 <0.001 <0.001 0.780 4.52 b 3.58 a 5.94 c 4.92 b 0.225 NFC 6.20 5.33 6.01 5.52 0.135 0.129 <0.001 0.001 0.074 6.58 c 5.83 b 5.45 ab 5.21 a 0.187 Starch 2.80 2.77 2.79 2.78 0.021 0.020 0.188 0.597 0.626 2.81 2.78 2.77 2.77 0.030 Fat 0.65 0.65 0.72 0.58 0.020 0.019 0.757 <0.001 0.660 0.72 b 0.58 a 0.72 b 0.59 a 0.028 MEI (MJ/d) 175 162 179 159 5.0 4.7 0.020 <0.001 0.149 190 b 161 a 169 a 156 a 6.9 Pre-challenge DMI 15.4 15.1 16.4 14.0 0.36 0.35 0.375 <0.001 0.510 16.8 b 14.1 a 16.1 b 14.0 a 0.51 MEI 176 163 179 159 4.9 4.7 0.019 <0.001 0.416 188 c 163 ab 171 b 154 a 6.8 Heat challenge DMI 14.2 14.7 15.4 13.5 0.69 0.67 0.420 0.008 0.420 15.4 b 12.9 a 15.4 b 14.0 ab 0.96 MEI 163 158 169 152 72 6.9 0.489 0.021 0.253 176 b 150 a 162 ab 154 a 10.0 Pre challenge to heat DDMI 7 -1.3 -0.3 -1.0 -0.6 0.65 0.64 0.186 0.518 0.632 -1.3 -1.2 -0.7 0.1 0.92 DMEI -12.4 -4.5 -10.3 -6.6 6.44 6.15 0.259 0.574 0.498 -12.0 -12.7 -8.5 -0.4 8.90 Initial recovery DDMI 8 1.0 -0.4 0.8 -0.2 0.62 0.60 0.022 0.086 0.017 2.4 b -0.3 a -0.7 a -0.1 a 0.87 DMEI 9.4 -3.6 8.2 -2.5 5.97 5.69 0.031 0.057 0.013 22.8 b -4.1 a -6.3 a -1.0 a 8.24 1 Main effects: CH = chicory, PS = pasture silage; 2 treatment means comparison was performed based on Fisher's unprotected LSD; 3 SEDm = standard error of the difference between main effects; 4 treatment diet means: CH-H = chicory high amount, CH-L = chicory low amount, PS-H = pasture silage high amount, PS-L = pasture silage low amount; 5 SEDt = standard error of the difference between treatments; 6 10-day period = (3-day base period, 1-day pre-challenge period, 2-day heat challenge period and 4-day recovery period); 7 D variable = (heat challenge variable--pre-challenge variable); 8 D variable = (recovery day 2 variable--recovery day 1 variable); a,b treatment means with different superscripts are different (p < 0.05). animals-13-00867-t006_Table 6 Table 6 Milk yield and yield of milk components (kg/d) and milk composition (g/kg) across the 10 days of measurement and during selected periods of the experiment. Main Effect 1 Treatment Means 2 Feed Amount SEDm 3 p Value CH PS High Low Feed Amount Feed Amount Feed x Amount CH-H 4 CH-L PS-H PS-L SEDt 5 n 14 13 14 13 7 7 7 6 10-day mean 6 Milk yield 21.9 17.2 20.5 18.7 0.55 0.53 <0.001 0.003 0.003 23.7 c 20 b 17.2 a 17.3 a 0.76 ECM yield 7 20.7 17.1 20.0 17.9 0.29 0.28 <0.001 <0.001 <0.001 22.6 c 18.9 b 17.3 a 16.9 a 0.41 Fat yield 0.78 0.72 0.79 0.71 0.024 0.023 0.017 0.002 0.035 0.85 b 0.72 a 0.73 a 0.71 a 0.033 Protein yield 0.71 0.50 0.65 0.56 0.022 0.022 <0.001 0.001 0.021 0.78 c 0.63 b 0.52 a 0.48 a 0.031 Fat concentration 36 43 39 39 1.5 1.6 <0.001 0.916 0.363 35 a 36 a 43 b 42 b 2.2 Protein concentration 32 29 32 30 0.7 0.7 0.001 0.055 0.353 33 c 32 bc 31 ab 28 a 1.0 Pre-challenge Milk yield 21.8 18.1 19.9 20.0 1.07 1.04 0.002 0.925 0.038 22.9 c 20.7 bc 16.8 a 19.3 ab 1.49 ECM yield 20.7 18.1 19.3 19.5 1.09 1.05 0.021 0.849 0.099 21.5 b 19.9 ab 17.0 a 19.2 ab 1.52 Fat yield 0.78 0.77 0.75 0.79 0.060 0.057 0.761 0.554 0.397 0.79 0.77 0.72 0.81 0.083 Protein yield 0.71 0.53 0.64 0.61 0.037 0.036 <0.001 0.356 0.101 0.76 b 0.66 b 0.52 a 0.55 a 0.051 Fat concentration 35 43 39 40 2.2 2.2 0.003 0.516 0.254 34 a 38 ab 44 b 43 b 3.1 Protein concentration 33 30 32 30 0.91 0.88 0.017 0.062 0.270 33 b 32 b 31 b 29 a 1.3 Heat challenge Milk yield 22.7 17.4 21.2 19.0 0.75 0.73 <0.001 0.006 0.014 24.9 c 20.6 b 17.5 a 17.3 a 1.05 ECM yield 21.6 17.1 20.7 18.0 0.58 0.56 <0.001 <0.001 <0.001 24.2 c 19 b 17.2 a 17.0 a 0.80 Fat yield 0.83 0.73 0.83 0.73 0.033 0.032 0.005 0.004 0.003 0.93 b 0.72 a 0.72 a 0.74 a 0.046 Protein yield 0.72 0.49 0.66 0.55 0.027 0.026 <0.001 0.001 0.057 0.8 c 0.63 b 0.51 a 0.46 a 0.038 Fat concentration 36 42 40 39 1.6 1.6 0.001 0.701 0.454 37 a 35 a 42 b 43 b 2.2 Protein concentration 31 28 31 29 0.9 0.8 0.004 0.025 0.220 32 b 31 b 30 b 27 a 1.2 Pre challenge to heat Milk yield 8 1.0 -0.7 1.3 -1.1 1.08 1.05 0.165 0.035 0.754 1.98 b -0.08 ab 0.68 ab -2.05 a 1.497 ECM yield 0.9 -1.0 1.4 -1.5 1.25 1.20 0.180 0.026 0.659 2.61 b -0.85 ab 0.22 ab -2.12 a 1.737 Fat yield 0.05 -0.04 0.07 -0.06 0.064 0.061 0.184 0.035 0.318 0.15 b -0.05 a -0.01 ab -0.07 a 0.088 Protein yield 0.00 -0.04 0.02 -0.06 0.034 0.033 0.214 0.032 0.794 0.04 b -0.03 ab 0.00 ab -0.09 a 0.047 Fat concentration 0.5 -0.8 0.9 -1.2 1.82 1.86 0.413 0.278 0.055 3.4 b -2.3 a -1.7 ab 0.0 ab 2.60 Protein concentration -1.2 -1.7 -1.3 -1.6 0.31 0.30 0.148 0.345 0.838 -1.1 -1.3 -1.5 -1.9 0.44 Initial recovery Milk yield 9 1.2 0.6 0.8 1.0 0.51 0.50 0.212 0.746 0.120 1.5 0.87 0.1 1.11 0.71 ECM yield 1.0 0.6 0.5 1.1 0.67 0.64 0.471 0.400 0.081 1.4 0.7 -0.28 1.55 0.931 Fat yield 0.01 0.01 -0.01 0.04 0.044 0.040 0.929 0.268 0.143 0.02 0.01 -0.05 0.08 0.061 Protein yield 0.07 0.04 0.05 0.06 0.014 0.014 0.046 0.671 0.100 0.08 b 0.06 ab 0.03 a 0.06 ab 0.020 Fat concentration -1.1 -0.4 -1.4 -0.0 2.39 2.43 0.842 0.566 0.393 -0.8 -1.4 -2.1 1.4 3.39 Protein concentration 1.7 1.7 1.6 1.8 0.35 0.33 0.910 0.522 0.948 1.6 1.8 1.6 1.8 0.48 1 Main effects: CH = chicory, PS = pasture silage; 2 treatment means comparison was performed based on Fisher's unprotected LSD; 3 SEDm = standard error of the difference between main effects; 4 treatment diet effects: CH-H = chicory high amount, CH-L = chicory low amount, PS-H = pasture silage high amount, PS-L = pasture silage low amount; 5 SEDt = standard error of the difference between treatments; 6 10-day period = (3-day base period, 1-day pre-challenge period, 2-day heat challenge period and 4-day recovery period); 7 ECM = energy-corrected milk yield (kg/day); 8 D variable = (heat challenge variable--pre-challenge variable); 9 D variable = (recovery day 2 variable--recovery day 1 variable); a,b treatment means with different superscripts are different (p < 0.05). animals-13-00867-t007_Table 7 Table 7 Daily mean and maximum vaginal temperature (VT; degC) and duration of vaginal temperature greater than 38.8 degC (min/day) across the 10 days of measurement and during selected periods of the experiment. Main Effect 1 Treatment Means 2 Feed Amount SEDm 3 p Value CH PS High Low Feed Amount Feed Amount Feed x Amount CH-H 4 CH-L PS-H PS-L SEDt 5 n 14 13 14 13 7 7 7 6 10-day mean 6 Mean VT 38.8 38.9 38.8 38.8 0.03 0.03 0.022 0.306 0.994 38.8 ab 38.8 a 38.9 b 38.8 ab 0.05 Maximum VT 39.4 39.6 39.5 39.5 0.05 0.05 0.024 0.718 0.626 39.4 a 39.5 ab 39.6 b 39.5 ab 0.07 Duration > 38.8 degC 569 687 646 609 67.1 61.9 0.095 0.556 0.832 580 557 712 661 90.8 Pre-challenge Mean 38.6 38.8 38.7 38.7 0.04 0.04 0.001 0.825 0.850 38.6 a 38.6 a 38.8 b 38.8 b 0.06 Maximum 39.2 39.3 39.2 39.3 0.05 0.05 0.005 0.317 0.045 39.1 a 39.2 b 39.4 b 39.3 b 0.07 Duration > 38.8 degC 312 634 468 478 99.7 91.9 0.005 0.915 0.974 306 a 319 a 631 b 637 b 134.8 Heat challenge Mean 39.1 39.3 39.3 39.1 0.10 0.09 0.048 0.059 0.705 39.1 ab 39 a 39.4 b 39.1 ab 0.14 Maximum 39.8 40.1 40.1 39.7 0.14 0.14 0.059 0.011 0.972 40.0 ab 39.6 a 40.3 b 39.9 ab 0.20 Duration > 38.8 degC 914 1233 1128 1020 110 101 0.010 0.303 0.954 965 a 863 a 1290 b 1176 ab 148.7 Pre challenge to heat DMean 7 0.42 0.46 0.53 0.35 0.099 0.094 0.608 0.069 0.763 0.49 0.34 0.57 0.36 0.138 DMaximum 0.63 0.73 0.90 0.46 0.148 0.141 0.448 0.006 0.428 0.91 b 0.35 a 0.89 b 0.58 ab 0.207 DDuration > 38.8 degC 602 599 660 542 115 106 0.984 0.282 0.978 659 544 660 539 155.1 Initial recovery DMean 8 0.09 0.16 0.15 0.10 0.041 0.039 0.085 0.183 0.961 0.12 ab 0.06 a 0.19 b 0.14 ab 0.056 DMaximum 0.14 0.21 0.22 0.13 0.075 0.071 0.382 0.170 0.046 0.27 b 0.01 a 0.18 ab 0.25 b 0.104 DDuration > 38.8 degC 126 194 150 170 114 105 0.566 0.853 0.895 123 129 176 211 154.1 1 Main effects: CH = chicory, PS = pasture silage; 2 treatment means comparison was performed based on Fisher's unprotected LSD; 3 SEDm = standard error of the difference between main effects; 4 treatment diet effects: CH-H = chicory high amount, CH-L = chicory low amount, PS-H = pasture silage high amount, PS-L = pasture silage low amount; 5 SEDt = standard error of the difference between treatments; 6 10-day period = (3-day base period, 1-day pre-challenge period, 2-day heat challenge period and 4-day recovery period); 7 D variable = (heat challenge variable--pre-challenge variable); 8 D variable = (recovery day 2 variable--recovery day 1 variable); a,b treatment means with different superscripts are different (p < 0.05). animals-13-00867-t008_Table 8 Table 8 Respiration rate (breaths per min) and skin temperatures (degC) at 14:45 h during selected periods of the experiment. Main Effect 1 Treatment Means 2 Feed Amount SEDm 3 p Value CH PS High Low Feed Amount Feed Amount Feed x Amount CH-H 4 CH-L PS-H PS-L SEDt 5 n 14 13 14 13 7 7 7 6 Pre-challenge Respiration rate 35 30 32 33 2.6 2.5 0.063 0.812 0.972 35 35 29 30 3.6 Left flank 30.7 31.3 31.1 30.9 0.37 0.35 0.092 0.605 0.936 30.8 30.6 31.4 31.2 0.50 Neck 30.0 30.3 30.4 29.9 0.33 0.31 0.134 0.106 0.055 29.9 a 30.0 a 31.0 b 29.7 a 0.44 Leg upper 28.1 28.3 28.7 27.6 0.24 0.23 0.086 <0.001 0.004 28.2 b 27.9 ab 29.2 c 27.3 a 0.32 Leg lower 26.8 27.4 27.8 26.4 0.52 0.49 0.178 0.014 0.740 27.6 b 26.1 a 28.0 b 26.8 ab 0.70 Heat challenge Respiration rate 85 72 87 70 7.4 7.0 0.117 0.035 0.776 92 b 78 ab 81 ab 63 a 10.2 Left flank 34.8 35.0 35.2 34.6 0.25 0.24 0.411 0.015 0.443 35.2 b 34.4 a 35.2 b 34.8 ab 0.35 Neck 34.9 34.8 35.2 34.5 0.27 0.26 0.633 0.008 0.607 35.4 b 34.5 a 35.1 ab 34.5 a 0.38 Leg upper 33.2 33.5 33.8 33.0 0.19 0.18 0.097 0.001 0.938 33.6 bc 32.9 a 33.9 c 33.2 ab 0.27 Leg lower 33.5 33.8 34.0 33.2 0.23 0.22 0.265 0.001 0.553 34 bc 33 a 34.1 c 33.4 ab 0.31 1 Main effects: CH = chicory, PS = pasture silage; 2 treatment means comparison was performed based on Fisher's unprotected LSD; 3 SEDm = standard error of the difference between main effects; 4 treatment diet effects: CH-H = chicory high amount, CH-L = chicory low amount, PS-H = pasture silage high amount, PS-L = pasture silage low amount; 5 SEDt = standard error of the difference between treatments; a,b,c treatment means with different superscripts are different (p < 0.05). animals-13-00867-t009_Table 9 Table 9 Selected blood parameters during selected periods of the experiment. Blood pH and serum concentrations of beta-hydroxy butyrate (BHB; mmol/L), non-esterified fatty acids (NEFA; mmol/L), glucose (mmol/L), haptoglobin (g/L), Na+ (mmol/L), and K+ (mmol/L). Main Effect 1 Treatment Means 2 Feed Amount SEDm 3 p Value CH PS High Low Feed Amount Feed Amount Feed x Amount CH-H 4 CH-L PS-H PS-L SEDt 5 n 14 13 14 13 7 7 7 6 Pre-challenge pH 7.41 7.44 7.45 7.40 0.023 0.022 0.173 0.069 0.519 7.43 ab 7.40 a 7.47 b 7.41 ab 0.032 BHB 0.59 0.65 0.61 0.63 0.061 0.060 0.292 0.684 0.456 0.55 0.62 0.66 0.64 0.086 NEFA 0.09 0.11 0.10 0.11 0.015 0.015 0.160 0.280 0.541 0.09 0.10 0.10 0.13 0.022 Glucose 3.0 3.3 3.2 3.1 0.11 0.10 0.062 0.328 0.721 3.1 ab 3.0 a 3.3 ab 3.2 b 0.15 Haptoglobin 0.21 0.22 0.22 0.22 0.008 0.008 0.301 0.968 0.435 0.21 0.22 0.23 0.22 0.012 Na 139 139 140 139 1.1 1.1 0.619 0.358 0.882 139 138 140 139 1.5 K 4.4 4.6 4.6 4.4 0.19 0.19 0.256 0.302 0.350 4.5 4.2 4.6 4.6 0.27 Heat challenge (H2) pH 7.45 7.46 7.46 7.46 0.020 0.019 0.531 0.758 0.053 7.43 7.48 7.48 7.45 0.03 BHB 0.47 0.59 0.53 0.53 0.041 0.040 0.006 0.975 0.271 0.45 a 0.49 ab 0.62 c 0.57 bc 0.057 NEFA 0.08 0.09 0.07 0.10 0.014 0.014 0.612 0.159 0.091 0.08 ab 0.08 ab 0.07 a 0.11 b 0.020 Glucose 3.2 3.4 3.3 3.3 0.105 0.102 0.024 0.571 0.406 3.2 ab 3.1 a 3.4 b 3.4 b 0.15 Haptoglobin 0.24 0.22 0.22 0.24 0.011 0.010 0.248 0.090 0.855 0.23 ab 0.24 b 0.21 a 0.23 ab 0.015 Na 140 141 141 140 0.593 0.566 0.296 0.119 0.413 141 140 142 140 0.82 K 4.8 4.8 4.1 4.8 0.179 0.171 0.760 0.953 0.777 4.9 4.8 4.8 4.8 0.25 1 Main effects: CH = chicory, PS = pasture silage; 2 treatment means comparison was performed based on Fisher's unprotected LSD; 3 SEDm = standard error of the difference between main effects; 4 treatment diet effects: CH-H = chicory high amount, CH-L = chicory low amount, PS-H = pasture silage high amount, PS-L = pasture silage low amount; 5 SEDt = standard error of the difference between treatments; a,b,c treatment means with different superscripts are different (p < 0.05). 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PMC10000128

Extranodal NK/T-cell lymphoma (ENKTL) is an aggressive extranodal non-Hodgkin lymphoma (NHL) with poor outcomes, particularly in advanced-stage and relapsed/refractory disease. Emerging research on molecular drivers of ENKTL lymphomagenesis by next-generation and whole genome sequencing has revealed diverse genomic mutations in multiple signaling pathways, with the identification of multiple putative targets for novel therapeutic agents. In this review, we summarize the biological underpinnings of newly-understood therapeutic targets in ENKTL with a focus on translational implications, including epigenetic and histone regulatory aberrations, activation of cell proliferation signaling pathways, suppression of apoptosis and tumor suppressor genes, changes in the tumor microenvironment, and EBV-mediated oncogenesis. In addition, we highlight prognostic and predictive biomarkers which may enable a personalized medicine approach toward ENKTL therapy. extranodal NK/T-cell lymphoma (ENKTL) biomarkers novel therapies This research received no external funding. pmc1. Background Extranodal NK/T-cell lymphoma (ENKTL) is a rare extranodal non-Hodgkin lymphoma (NHL) that primarily occurs in the upper aerodigestive tract and has an aggressive presentation, with locoregional invasion in the nasopharynx causing necrosis, hemorrhage, and impingement on anatomic structures including the orbits . The incidence of ENKTL is much higher in East Asia and Latin America than in Europe and North America, representing up to 15% of all NHL diagnoses, likely due to underlying geodemographic differences in human leukocyte antigen (HLA) genes and genetic susceptibility . Outcomes for ENKTL are generally poor, with 5-year overall survival (OS) rates of approximately 50% when treated with asparaginase-based multi-agent combination chemotherapy regimens such as SMILE but as low as 25-28% in high-risk patients as assessed by various prognostic indices of NK lymphoma . The majority of patients with ENKTL present with limited-stage, localized disease, in which outcomes are better, with 5-year and 10-year OS rates of 64-89% and 57%, respectively, utilizing combination chemotherapy and radiation . However, patients with relapsed or refractory disease have dismal outcomes, with median survival of less than 12 months . Despite advances in the frontline management of ENKTL with combination chemoradiotherapy approaches , there is a pressing need for novel and rational therapies, informed by the molecular biology of ENKTL, particularly for patients with relapsed/refractory disease, for patients who cannot tolerate intensive frontline induction therapy, and for patients with high-risk disease features and advanced-stage disease. 2. Evolving Understanding of the Biology of ENKTL Historically, the pathobiology of ENKTL and its molecular drivers have been poorly understood. Gene rearrangement studies suggest that the majority of ENKTL cases are of NK cell rather than T cell lineage . Although there are no apparent differences in clinical presentation or survival between NK or T cell lineages , there is emerging evidence that different signaling pathways are constitutively activated in each cell lineage, reflecting a complex and heterogeneous molecular driver landscape of ENKTL . Classical descriptions of ENKTL oncogenesis by canonical T and NK cell signaling have focused on the ubiquitous presence of EBV in ENKTL tumor cells, and it is posited that EBV infection induces the overexpression of the transmembrane oncoprotein LMP1 in infected NK and T cells, resulting in ligand-independent activation of the NF-kB and MAPK signaling pathways . These signaling pathways, with loss of tumor suppressor genes on chromosome 6q21, as is commonly observed in ENKTL, cause tumor cell proliferation and avoidance of apoptosis by regulation of c-MYC and survivin and also upregulate cell surface expression of PD-L1 to promote escape from immune surveillance . However, ENKTL cells do not experience immortalization in response to EBV infection, as is seen in B-cell lymphomas, but rather become sensitized to proliferative cytokines such as IL-2, suggesting that other molecular alterations must occur in a multistep process for EBV-infected NK or T cells to undergo malignant transformation . Recent advances in somatic next-generation and whole-genome sequencing have provided emerging evidence that ENKTL is characterized by genomic aberrations in multiple signaling pathways, including broad epigenetic and gene methylation changes . In this review, we summarize newly-understood putative driver mutations in ENKTL with a focus on translational implications and novel therapies with rational biologic underpinnings (Table 1), as well as prognostic and predictive biomarkers which may enable personalized therapeutic approaches (Table 2). 3. Epigenetic Aberrations in ENKTL It is increasingly observed that epigenetic modifiers are the largest group of mutated genes in ENKTL, with one meta-analysis of nine next-generation sequencing (NGS) studies finding that 25% of identified mutations in ENKTL affected epigenetic regulators . Epigenetic aberrations play a critical role in tumorigenesis by silencing tumor suppressor genes and altering the regulation of oncogenes. A cohesive understanding of how epigenetic changes drive the ENKTL clone is starting to emerge . 3.1. Promoter Hypermethylation In many types of cancer, promoter regions of tumor suppressor genes are frequently hypermethylated during carcinogenesis, resulting in transcriptional silencing via the recruitment of histone deacetylases (HDACs) and repressive chromatin formation . Comparison of ENKTL tumor cells and normal NK cells with methylation assays have demonstrated global promoter hypermethylation and gene silencing in ENKTL, with decreased mRNA transcription of 95 putative tumor suppressor genes, including BCL2L11 (BIM), DAPK1, PTPN6 (SHP1), TET2, SOCS6, and ASNS with known functions . Specifically, BIM sensitizes NK cell lines to chemotherapy-induced apoptosis, DAPK1 mediates p53-dependent apoptosis, SHP1, and SOCS6 inactivate the JAK/STAT signaling pathway, and TET2 silencing may contribute to early global hypermethylation in ENKTL, so it is easily understood how hypermethylation of multiple tumor suppressor promoters may create additive effects leading to tumor growth and malignant transformation . These findings have been confirmed in other studies, with the identification of additional tumor suppressor genes, including DLC1 involved in RAS signaling , PRDM1 involved in apoptosis and promotion of NK cell growth in cooperation with activating STAT3 mutations , PTPRK involved in JAK/STAT signaling and apoptosis , HACE1 involved in apoptosis , as well as several other genes (P73, hMLH1, CDKN2A, CDKN2B, RARb, PCDH10, DLEC1, CADM, DAL1) with currently unknown functions . A recent genome-wide DNA methylation and transcriptomic study by Mundy-Bosse et al. (2022) demonstrated that ENKTL cells likely represent a malignant transformation from NK cells normally present in mucosal tissues, with ubiquitous and profound DNA hypermethylation driven by EBV infection causing the arrest of normal NK cell differentiation . In this study, patient-derived xenograft (PDX) mouse models of ENKTL were treated with the DNA hypomethylating agent 5-azacytidine, which resulted in a significant decrease in tumor burden and an increase in OS compared to control mice. In addition, 5-azacytidine treatment resulted in a global reduction in DNA methylation and the emergence of mature NK cell markers, suggesting that treatment with hypomethylating agents may induce terminal differentiation of ENKTL cells, as is seen in patients with acute promyelocytic leukemia treated with all-trans retinoic acid . These findings confirm the results of other studies, which have found that exposure of ENKL cell lines to hypomethylating agents such as 5-azacytidine or decitabine restores the expression of these putative tumor suppressors . In a phase I study, one patient with ENKTL responded to a combination of 5-azacytidine and romidepsin with profound demethylation of their tumor after treatment . There is a strong pre-clinical rationale for the treatment of ENKTL with hypomethylating agents, although there may be discordant effects from other therapeutic agents. For example, treatment with 5-azacytidine upregulates genes associated with immunoregulatory functions, suggesting possible synergy with immunotherapy agents , while there is concern that hypomethylating agents may desensitize ENKTL cells to asparaginase by upregulating ASNS and also lead to lytic reactivation of EBV . 3.2. Epigenetic Regulatory Genes Previous studies of ENKTL have revealed mutations in genes that regulate epigenetic changes to the genome, including genes responsible for chromatin remodeling, histone acetyltransferases and methyltransferases, and DNA demethylases . One of the most frequently mutated genes in 17-32% of ENKTL cases is BCOR , a BCL-6 interacting corepressor which is involved in the epigenetic modification of histones via HDACs and which is suggested to be a tumor suppressor given frequent loss-of-function mutations seen in ENKTL and enhanced cell proliferation and IL-2 production when BCOR is silenced . MLL genes are also commonly mutated in up to 19% of ENKTL cases, specifically MLL2 (KMT2D) and MLL3 (KMT2C), which are involved in histone methylation and may be tumor suppressors in B-cell lymphomas, although the role of these epigenetic regulators specifically in the tumorigenesis of ENKTL is unknown . Other commonly mutated epigenetic regulatory genes in ENKTL include TET1/TET2, EP200, ASXL3, CREBBP, and ARID1A, with yet unknown functions in ENKTL biology . An epigenetic regulator gene with documented oncogenic function in ENKTL is EZH2, a histone methyltransferase that is not mutated in ENKTL but rather is aberrantly overexpressed and promotes NK tumor cell growth independently of its histone methyltransferase activity . This non-canonical activity of EZH2 is likely due to its direct phosphorylation by JAK3, which converts EZH2 from a gene repressor to a transcriptional activator of genes involved in cell proliferation . The JAK/STAT pathway is likely an upstream regulator of EZH2, as evidenced by studies demonstrating that treatment of ENKTL cells with the JAK3 inhibitor tofacitinib reduced EZH2 expression and decreased ENKTL cell growth . EZH2 is a known druggable target, with an FDA-approved EZH2 inhibitor (tazemetostat) for EZH2-wild type and EZH2-mutated follicular lymphoma . However, treatment of ENKTL PDX mice with tazemetostat alone as well as in combination with 5-azacytidine did not improve survival in one study and was ineffective in ENKTL cell lines , suggesting that inhibition of the EZH2 methyltransferase catalytic site does not affect its non-canonical and enzyme-independent oncogenic functions. EZH2 inhibitors with novel mechanisms of action, such as 3-deazaneplanocin-A (DZNep), which disrupts the metabolism of methyl donors required for histone methylation by EZH2 and also accelerates proteasomal degradation of EZH2, have demonstrated enhanced lymphoma cell apoptosis compared to tazemetostat . EZH2 inhibitors are a rational drug target in ENKTL, although there is pre-clinical evidence that combination with agents targeted at its upstream regulators, such as JAK/STAT inhibitors, may also cause ENKTL cell death in patients with high EZH2 tumor expression . Further, targeting downstream target genes of EZH2, such as cyclin D1, may also be efficacious, with one study demonstrating that combined inhibition of upstream JAK with ruxolitinib and CDK4/6 with ribociclib produced synergistic inhibition of ENKTL cell growth . 3.3. Epigenetic Biomarkers Large-scale somatic sequencing of ENKTL has enabled the description of new biomarkers which are both prognostic and predictive, although large-scale studies will be required for clinical validation. Mutations in MLL2 (KMT2D) and TET2 were associated with inferior prognosis in ENKTL , and overexpression of EZH2 was associated with higher tumor cell proliferation, advanced stage, and inferior survival . Further, the presence of KMT2D mutations in circulating tumor DNA (ctDNA) was prognostic and correlated with total metabolic tumor volume, suggesting that serial measurements of ctDNA could be used to monitor treatment response and the presence of residual disease . MicroRNAs (miRNAs) are short noncoding RNA sequences that inhibit the expression of target genes by suppressing translation or promoting mRNA degradation, and many have been found to be overexpressed in ENKTL as putative oncogenes as well as prognostic biomarkers . For example, elevated plasma levels of miR-221 are associated with inferior overall survival , and miR-155 is associated with disease response . Predictive biomarkers are particularly important for ENKTL to promote maximal response to therapy for patients with an aggressive and difficult-to-treat malignancy. In a study of 17 patients with ENKTL, patients with methylated PTPRK tumors who were treated with the SMILE protocol had significantly worse overall survival and a trend towards inferior disease-free survival . In addition, ASNS expression is strongly correlated with cell survival in response to asparaginase treatment, which may serve as a clinically useful biomarker to determine which patients will respond to asparaginase-containing chemotherapy or which patients with low ASNS expression may be treated with lower doses of asparaginase to avoid toxicity . miRNAs have been identified as predictive biomarkers with putative therapeutic targets; for example, a novel inhibitor of miR-155 induced apoptosis in ENKTL cell lines as well as xenografts by downregulation of STAT3 and VEGF signaling pathways and upregulation of the pro-apoptotic BRG1 . High expression of SNHG12, a long noncoding RNA (lncRNAs) sequence which is overexpressed in ENKTL, conferred cisplatin resistance in ENKTL cells and also may be responsible for multidrug resistance in ENKTL owing to its contribution to P-glycoprotein overexpression, which is a known mechanism for anthracycline resistance in ENTKL . Another lncRNA, brain cytoplasmic RNA 1 (BCYRN1), is also overexpressed in ENKTL and is associated with inferior PFS as well as resistance to asparaginase . 4. Cell Survival and Proliferation The second most frequently mutated genes in ENKTL NGS studies are those of the JAK/STAT signaling pathway, predominantly in ENKTL cells of NK cell lineage, while T cell lineage ENKTL more commonly exhibits mutations of the RAS/MAPK signaling pathway and aforementioned epigenetic regulators . Aberrant regulation of both of these signaling pathways results in ENKTL survival and proliferation , driven by mutations in JAK3, STAT3, STAT5B, and DDX3X . However, these mutations rarely occur together, suggesting that there are disparate molecular signaling pathways leading to ENKTL tumorigenesis and confirmed in contemporary studies identifying three distinct subtypes of ENKTL by gene expression signatures: the TSIM (tumor suppressor-immune modulator) subtype with JAK/STAT activation and PD-L1 overexpression; the MB (MGA-BRDT) subtype with MYC overexpression and MAPK activation; and the HEA (HDAC9-EP300-ARID1A) subtype with changes in histone acetylation and NF-kB activation . Each of these subtypes may be targeted by specific therapeutic agents based on activated signaling pathways, and putative targeted agents for each pathway are outlined as follows. 4.1. JAK/STAT Signaling Pathway Constitutive activation of the JAK/STAT pathway is responsible for ENKTL survival and proliferation, with activating mutations in STAT3 being the most common driver, followed by activating JAK3 mutations . As previously discussed, activation of the JAK/STAT pathway in ENKTL converts the histone methyltransferase EZH2 into a non-canonical transcriptional activator of genes involved in cell proliferation, with inhibition of JAK3 decreasing EZH2 expression and decreasing ENKTL tumor growth . There are multiple inhibitors of the JAK/STAT pathway which have been studied broadly in hematologic malignancies, including specifically in ENKTL . The JAK1/3 inhibitor tofacitinib has demonstrated inhibition of cell growth in JAK3-mutant and STAT-3 mutant ENKTL cells , as well as ENKTL cells with EZH2 overexpression . Similar pre-clinical results have been demonstrated with novel pan-JAK inhibitors and STAT inhibitors , although none of these have yet been studied in patients. Ruxolitinib, a JAK1/2 inhibitor that is already approved for use in hematologic malignancies, is a therapy of interest in ENKTL, given pre-clinical work that JAK1/2 inhibition can disrupt JAK/STAT signaling in STAT3-mutated or overexpressed ENKTL . A phase II biomarker-driven study of ruxolitinib in patients with relapsed/refractory T-cell lymphomas revealed high response rates of 45-53% in patients with activating JAK/STAT mutations or STAT3 overexpression, compared to low response rates of 13% without those biomarkers . Although no patients with ENKTL were included, this study provides a strong clinical rationale for the use of ruxolitinib and other JAK/STAT inhibitors in ENKTL cases with JAK/STAT aberrations and suggests that a biomarker-based therapeutic approach utilizing the aforementioned TSIM, MB, and HEA subtypes may be warranted. Further, in vitro studies suggest that ruxolitinib may be combined with novel TP53-MDM2 inhibitors in TP53-wild type disease, farnesyltransferase inhibitors in TP53-mutated disease, and with dexamethasone or novel MCL-1 inhibitors , or with the BCL2 inhibitor venetoclax or aurora kinase inhibitor alisertib in another study , to maximize activity and avoid long-term resistance. 4.2. DDX3X and RAS/MAPK Signaling Pathway DDX3X is an RNA helicase gene that is one of the most commonly mutated genes in ENKTL , with significant upregulation of the NF-kB and MAPK pathways found in DDX3X-mutated ENKTL tumors, suggesting that DDX3X is a possible tumor suppressor . The RAS/MAPK signaling pathway is broadly implicated in carcinogenesis by promotion of cell survival and proliferation and is upregulated in the MB subtype of ENKTL , likely related to overexpression of the EBV-associated LMP1 oncoprotein . Previous pre-clinical studies have found that inhibition of the MAPK pathway is not effective in vitro or in vivo in NHL cells, likely due to the regulatory complexity of the pathway . However, pre-clinical studies have found that statins inhibit cell growth and enhance chemotherapy-induced cytotoxicity in NK leukemia cell lines due to inhibition of the MAPK pathway, suggesting an opportunity for future combination studies . Of note, DDX3X mutations portend a poor prognosis in patients with ENKTL treated with CHOP-based chemotherapy , although not in patients treated with more contemporary asparaginase-based regimens . 4.3. NF-kB Signaling Pathway and Survivin There are multiple drivers of NF-kB signaling pathway upregulation in ENKTL, including DDX3X mutations as well as EBV-associated LMP1 oncoprotein expression . The NF-kB pathway is considered a major driver of tumorigenesis in lymphomas and specifically in ENKTL, likely related to overexpression of a downstream anti-apoptotic protein called survivin which is elevated in nearly all cases of ENKTL and is prognostic . There has been considerable study focused on the inhibition of the NF-kB pathway in ENKTL, particularly with the proteasome inhibitor bortezomib, which inhibits ENKTL in vitro . Clinical use of bortezomib has been restricted to small case series , including a phase II study of bortezomib, gemcitabine, ifosfamide, and oxaliplatin in 7 patients with newly-diagnosed ENKTL which demonstrated an overall response rate of 43% but a median progression-free survival of only 4 months . A phase II study of bortezomib in combination with CHOP chemotherapy included 10 patients with ENKTL, for which the complete response rate was much lower than other lymphoma subtypes at 30%, with ENKTL patients having rapid disease progression after the first two cycles, likely due to known anthracycline resistance in ENKTL . There is a suggestion that bortezomib may be effective if combined with asparaginase-based chemotherapy, as inhibition of the NF-kB pathway would downregulate CD25, which induces asparaginase resistance in ENKTL cell lines , and effective combinations of proteasome inhibitors with other chemotherapeutics or targeted agents merits further study. For example, pre-clinical data has identified that terameprocol and mithramycin, inhibitors of survivin and its related transcription factors, induce apoptosis of ENKTL cells, with mithramycin also suppressing ENKTL growth in a mouse xenograft model, both of which may be appropriate partners for multipronged inhibition of the NK-kB pathway . 4.4. C-MYC C-MYC is a well-known oncogene in lymphomas and is overexpressed in ENKTL, likely driven by LMP1 and EBV infection and causing upregulation of downstream targets EZH2 and RUNX3, the latter of which is a master transcriptional regulator and a putative oncogene in ENKTL . C-MYC inhibitors are of particular interest in multiple aggressive lymphomas, and treatment of ENKTL cell lines with a small-molecule novel MYC inhibitor caused downregulation of both MYC and RUNX3 and, subsequently, apoptosis . 4.5. PDGFRa Platelet-derived growth factor receptor alpha (PDGFRa) is a tyrosine kinase pathway that is overexpressed in ENKTL, possibly driven by the PI3K/Akt/mTOR and JAK/STAT pathways, and mediates cell survival . Previous pre-clinical work has demonstrated that imatinib, a tyrosine kinase inhibitor (TKI), inhibits ENKTL cell proliferation in vitro as well as in mouse models , supporting further study of TKIs in ENKTL. In addition, high PDGFRa expression is a prognostic biomarker of survival . 4.6. PI3K/Akt/mTOR Signaling Pathway The PI3K/Akt/mTOR signaling pathway, which is a master regulator of growth and survival in normal and malignant cells, is downstream of the JAK/STAT pathway and is upregulated in many types of lymphoma, including ENKTL, where it is driven by EBV infection and LMP1 . Inhibitors of multiple targets along this signaling cascade have been explored in lymphomas, particularly PI3K inhibitors, which have already been approved by the FDA for various relapsed/refractory lymphoma subtypes. Several small phase I studies of duvelisib and tenalisib in peripheral T-cell lymphomas (PTCLs) have demonstrated an ORR of ~50% , and a phase I/II trial of copanlisib and gemcitabine in PTCL included 3 patients with ENKTL with 2 partial responses and 1 complete response . Combination targeting of the PI3K/Akt/mTOR signaling pathway appears to be more effective due to the activation of alternative signaling pathways by negative feedback, with drug sensitivity profiling revealing that inhibition of both mTOR and JAK may be more effective than single agents in NK/T-cell lymphoma cell lines . As an example, a phase I/II trial of the mTOR inhibitor temsirolimus in combination with the Akt inhibitor and immunomodulatory agent lenalidomide in relapsed/refractory lymphomas demonstrated an ORR of 67% in 9 patients with T-cell lymphomas, a superior ORR compared to single-agent mTOR inhibition or lenalidomide alone . 5. Avoiding Autophagy Avoiding apoptosis, either via overexpression of anti-apoptotic proteins or suppression of tumor suppressor genes, has been identified as a significant contributor to tumorigenesis in ENKTL. The tumor suppressor TP53 is one of the most frequently mutated genes in ENKTL and is present in up to 63% of cases , with a worse prognosis in TP53-mutated ENKTL . Although genomic studies have identified numerous other putative tumor suppressor genes in ENKTL with functional suppression of cell growth in ENKTL cell lines, such as PRDM1 and FOXO3, there are not currently targeted agents to re-enable these tumor suppressors . However, histone deacetylase inhibitors have been explored extensively in lymphomas to reactivate intrinsic apoptotic pathways, with several HDAC inhibitor agents under exploration in ENKTL . Histone Acetylation and HDAC Inhibitors As previously discussed, HDACs are histone deacetylases that frequently lead to the inactivation of tumor suppressor genes when aberrantly activated during tumorigenesis . As such, HDAC inhibitors lead to the reactivation of downstream apoptotic pathways, including upregulation of TP53 and downregulation of the PI3K/Akt/mTOR pathway by the accumulation of autophagic factors . Given the known activity of the HDAC inhibitor romidepsin in T-cell lymphomas, a prospective pilot study of romidepsin, specifically patients with relapsed/refractory ENKTL, was conducted in 2013 . However, of the first 5 patients enrolled in the study, 3 patients developed treatment-emergent EBV reactivation manifesting as severe liver injury and 1 death, resulting in early termination of the trial. A correlative analysis confirmed that romidepsin caused a significantly greater increase in EBV reactivation in ENKTL cell lines compared to vorinostat . This early experience decreased interest in HDAC inhibitors for ENKTL, although there was a suggestion that not all HDAC inhibitors would cause the same degree of EBV reactivation given different HDAC isoform specificity of different inhibitors. Subsequent clinical studies have explored various HDAC inhibitors in ENKTL, including chidamide, which is of particular interest given it also inhibits the PI3K/Akt/mTOR and RAS/MAPK signaling pathways, with an ORR of 15-50% in small studies of 15-19 patients . There have also been responses in single patients to belinostat and vorinostat . Simultaneous targeting of the NF-kB signaling pathway with proteasome inhibitors was posited to reduce the risk of EBV reactivation in a phase II study of panobinostat and bortezomib, in which 1 of 2 patients with ENKTL had a partial response, and neither patient had EBV reactivation . Several other HDAC inhibitor combinations have been explored, including chidamide in combination with chemotherapy as well as chidamide in combination with immunotherapy, the latter to be discussed later. In the former study of 53 patients with ENKTL, the ORR was significantly higher when chidamide was combined with chemotherapy (40%) compared to chidamide alone (15%), confirming pre-clinical data that HDAC inhibitors sensitize lymphoma cells to chemotherapy-induced DNA damage . In a phase II randomized trial of 74 patients with newly-diagnosed, high-risk, early-stage ENKTL, patients were treated with radiation therapy followed by gemcitabine, dexamethasone, and cisplatin (GDP) either with or without chidamide . Response rates, PFS, and OS were similar in the control group of GDP alone as compared to the experimental arm of GDP combined with chidamide, suggesting that chidamide may need to be paired with a different chemotherapy backbone or studied in patients with specific biomarkers such as the HEA subtype of ENKTL which is enriched with histone acetylation mutations . In that vein, a phase II single-arm trial of 36 patients with ENKTL in first complete response were administered chidamide, cladribine, gemcitabine, and busulfan conditioning chemotherapy prior to autologous stem cell transplantation . The 4-year PFS of ENKTL patients was 73%, which was significantly higher than historical PFS rates of 33-40% after autologous transplantation and suggested that the optimum pairing of HDAC inhibitors with chemotherapy partners merits further study. Novel HDAC inhibitors with different mechanisms of action have also been developed, including nanatinostat in combination with valganciclovir , citarinostat in combination with the JAK/STAT inhibitor momelotinib in cell lines , and the histone acetyltransferase KAT5 inhibitor NU9056 in cell lines . 6. Tumor Microenvironment Mobilizing the tumor microenvironment (TME) is a major therapeutic advancement in cancer, particularly with checkpoint inhibitors which augment the PD-1/PD-L1 signaling pathway to induce a cytotoxic T cell immune response against tumor cells. In lymphomas, immunotherapy has been most successful in Hodgkin lymphoma, in which anti-PD1 treatment alters the TME by downregulating pro-survival factors rather than inducing an immune response . The role of the TME in ENKTL is under investigation, with previous studies identifying the upregulation of PD-L1 in ENKTL cells driven by EBV-mediated LMP1 and constitutive activation of the JAK/STAT pathway . However, there are additional elements of the TME in ENKTL that may be amenable to therapeutic targeting. 6.1. Immune Surveillance and the PD-1/PD-L1 Signaling Pathway PD-L1 is very commonly expressed in ENTKL in 56-80% of cases , with the interplay of PD-1 and PD-L1 in the TME of particular interest in the therapeutic targeting of ENKTL, with a study finding that PD-L1 is expressed on both tumor cells as well as tumor-infiltrating macrophages in the ENKTL TME, suggesting that ENKTL cells may additionally induce immunosuppression by upregulation of PD-L1 in macrophages . Commercial monoclonal antibodies against PD-1, such as pembrolizumab and nivolumab, have been studied in small cohorts of patients with relapsed/refractory ENKTL: ORR 100% in 7 patients using various radiographic and serologic response criteria , ORR 57% in 7 patients and in 2 out of 3 patients of whom 2 quickly died due to infections and poor performance status , as well as in case reports . However, the follow-up in these studies was very short at less than 6 months, with PFS and OS also measured in less than 6 months for most of the patients. Sintilimab, an anti-PD-1 antibody with a different binding site than pembrolizumab and nivolumab with greater affinity for PD-1, has been extensively studied in ENKTL. In an initial phase II study of sintilimab monotherapy in 28 patients with relapsed/refractory ENKTL, the ORR was 75% (CR 21%) with a 2-year OS of 79% with 30 months of median follow-up . This trial generated significant interest in sintilimab, given high survival rates in patients with an otherwise dismal prognosis, and also demonstrated that patients with pseudo-progression due to immunotherapy, which developed in 18% of patients in the study, did not have an inferior survival and benefitted from ongoing continuous treatment with sintilimab. Given the low complete response rates with sintilimab monotherapy, there have been several efforts to combine immunotherapy with chemotherapy and other agents to induce deeper and prolonged responses, including in the frontline setting. The initial experience combining various anti-PD-1 agents with peg-asparaginase, gemcitabine, and oxaliplatin (P-GemOx) in 9 patients with newly-diagnosed, advanced-stage ENKTL revealed an ORR of 89% (78% CR) with 1-year PFS and OS of 67% and 100%, respectively, with follow-up of 10.6 months . This led to a subsequent phase II study of sintilimab plus P-GemOx, with preliminary results of 6 patients revealing an ORR of 100% (33% CR) with only 7.8 months of follow-up . The short follow-up of these studies does not confirm if combination therapy improves the promising survival observed with sintilimab monotherapy, although a stage-adapted study of sintilimab plus chidamide induction followed by P-GemOx (plus radiation for early-stage disease) demonstrated an ORR of 100% for 19 patients with early-stage disease and 100% in 7 out of 9 patients with advanced-stage disease who were able to complete therapy . The 1-year PFS and OS rates were both 93% across the entire cohort, demonstrating that a non-chemotherapy pre-phase may be effective in patients with early-stage disease who present with poor performance status. However, given that the ORR was 100% after sintilimab and chidamide induction in the early-stage cohort and only 44% in the advanced-stage group, with 3 patients developing rapid progression during induction in the latter cohort, the appropriate choice and sequence of combination therapies need to be carefully selected based on stage as well as predictive biomarkers. The combination of sintilimab plus chidamide also has activity in the relapsed/refractory setting, with an ORR of 60% (CR 49%) and an 18-month OS of 76% in 38 patients . Other combinations have been explored, including anti-PD-1 tislelizumab with chidamide, lenalidomide, and etoposide in 8 patients (ORR 88%, CR 63%) , anti-PD-1 toripalimab with chidamide, etoposide, and thalidomide in 3 patients (ORR 100%, CR 67%) , case reports of sintilimab and atezolizumab with chidamide, and ongoing study of the hypomethylating agent azacitidine with tislelizumab and peg-asparaginase . A more recent 2022 abstract reported the results of 27 patients with relapsed and refractory ENKTL who were treated with various anti-PD-1 antibodies in conjunction with hypomethylating agents (either decitabine or azacytidine), finding an ORR of 63% (CR 59%), including high ORRs in patients who had previously received checkpoint inhibitors, with median PFS of 12.8 months . Sintilimab is not currently available in the United States, although there are other novel immunotherapy agents which are amenable to study, such as the anti-PD-L1 agent sugemalimab which received breakthrough therapy designation by the FDA for relapsed/refractory ENKTL in 2020 based on the GEMSTONE-201 trial, which preliminarily demonstrated an ORR of 46% (CR 37%) with 2-year OS of 55% in 80 patients with relapsed/refractory ENKTL . The anti-PD-L1 agent avelumab has activity in relapsed/refractory disease, with an ORR of 38% (CR 24%) among 21 patients, with expression of PD-L1 significantly associated with complete responses . A novel recombinant anti-PD-1 agent, geptanolimab, was studied in 102 patients with relapsed/refractory PTCL, with the highest ORR of 63% among the 19 patients with ENKTL. There are also novel agents under investigation, including the anti-PD-1 IgG4 antibody camrelizumab , as well as monoclonal antibodies for newly-described immune checkpoint receptors in NK cell lymphomas such as killer cell lectin-like receptor G1 (KLRG1) . The available data suggest that immunotherapy is a rational and efficacious treatment for both newly-diagnosed and relapsed/refractory ENKTL, although long-term follow-up is necessary to assess if responses as monotherapy or combination therapy are durable. There have been mixed results in ENKTL with regards to the prognostic value of PD-L1 in ENKTL, with one study finding that higher expression of PD-L1 expression was associated with inferior treatment response and survival in early-stage disease when defined as >=38% on histology , but improved survival in advanced-stage disease when defined as >=10% on histology . Given emerging data about different molecular subtypes of ENKTL, it is possible that a biomarker-driven approach may be warranted, with immunotherapy targeted towards patients with the TSIM subtype characterized by PD-L1 overexpression, or patients with mutated PD-L1 who have better responses to pembrolizumab . Recent whole-genome sequencing has further identified 4 TME subgroups of ENKTL: an 'immune tolerance' group with high numbers of Tregs more common in early-stage disease; two 'immune evasion' groups with frequent cytotoxic T cells and high PD-L1 expression; and an 'immune silenced' group with an exhausted immune response more common in advanced-stage disease . The 'immune silenced' group had the worst prognosis and also had the worst response to pembrolizumab, with the best responses seen in the 'immune tolerance' group. These data suggest that immunotherapy may be targeted towards patients with the aforementioned predictive biomarkers or potentially to patients with early-stage disease who are more likely to have immunotherapy-responsive phenotypes. 6.2. Angiogenesis Several important molecular drivers of angiogenesis are upregulated in ENKTL, including vascular endothelial growth factor (VEGF) and its genes (VEGFA, VEGFC, and VEGFR2), as well as the oncogene MET and its related hepatocyte growth factor (HGF), all of which are regulated by the JAK/STAT pathway . Anti-VEGF antibodies such as bevacizumab have been studied in lymphomas, including a study of bevacizumab plus CHOP in 39 patients with T and NK cell lymphomas with an ORR of 90% (CR 49%) which demonstrated significant treatment-limiting toxicities and poor durability . However, a phase II trial of bevacizumab, gemcitabine, peg-asparaginase, oxaliplatin, and dexamethasone in 43 patients with newly-diagnosed ENKTL demonstrated an ORR of 100% (CR 100% for early-stage and 88% for advanced-stage disease) with a 2-year PFS and OS of 83% and 79%, respectively . An oral tyrosine kinase inhibitor specific for the VEGF receptor VEGFR2, apatinib, is being studied in conjunction with the anti-PD-1 inhibitor camrelizumab in relapsed/refractory PTCL, with preliminary results demonstrating that 2 of the 3 patients with ENKTL achieved a partial response . Pre-clinical data also support the use of MET inhibitors, which induce enhanced helper T cell recognition of ENKTL cell lines and may be combined with immunotherapy , and oral MET inhibitors such as crizotinib have already been studied in other types of PTCL with an ORR of 90% in ALK-positive lymphomas . Serum VEGF levels are prognostic of PFS and OS in patients with ENKTL treated with non-anthracycline-based chemotherapy . 6.3. Cytokines and Chemokines The inflammatory milieu of the ENKTL TME is thought to contribute to lymphomagenesis, as EBV infection causes NK/T cells to become sensitized to growth-promoting cytokines such as IL-2 . Several serum cytokines are prognostic in ENKTL, including IL-18, which is associated with HLH, advanced-stage disease, and poor OS ; IL-10, which is associated with worse OS, low CR rate, and higher early relapse rate ; and IL-2Ra, IL-9, IL-15, and MIP-1a . In addition, interferon, IL-6, and IL-10 are all associated with serum EBV DNA load, a known prognostic biomarker in ENKTL, suggesting that EBV-induced cytokine storm may contribute to poor prognosis in ENKTL . Inhibition of proliferative cytokines has been proposed as a treatment strategy in ENKTL, although there is no clinical data. However, a recent study suggests that IL-10 contributes to gemcitabine resistance in ENKTL cell lines by regulation of drug resistance genes, suggesting that inhibition of IL-10 in select patients may be utilized to increase tumor sensitivity to chemotherapy . Chemokines are chemoattractant cytokines that also mediate the TME in EBV-driven malignancies and promote metastasis and tumor angiogenesis, with chemokine receptor 4 (CCR4), the receptor for chemokine ligand 17 and 22 (CCL17 and CCL22), prominently identified in ENKTL tumors . Mogamulizumab, a monoclonal antibody against CCR4, has demonstrated cytotoxicity in ENKTL cell lines and has clinical activity in other T cell malignancies, warranting further study . 6.4. S100A9 Recent proteomic analysis has identified a novel biomarker in ENKTL, S100A9, an immunosuppressive molecule that is overexpressed in serum and tumor samples in ENKTL and likely mediates tumorigenesis by upregulation of PD-L1 expression . High levels of S100A9 are associated with poor response to therapy, early relapse, and advanced stage, and it has been proposed as a possible therapeutic target. 7. Other Therapeutic Targets Given the abysmal prognosis of relapsed and refractory ENKTL, numerous targeted agents have been trialed in single patients or small cohorts with variable efficacy. However, there is pre-clinical rationale for the combination of these targeted agents with other active chemotherapeutics in ENKTL. 7.1. CD38 Cell surface protein CD38 is expressed in the vast majority of ENKTL cases, and high expression of CD38 is correlated with inferior responses and survival . The anti-CD38 monoclonal antibody daratumumab has been explored in relapsed/refractory ENKTL in a phase II study of 32 patients, which demonstrated an ORR of 25% (no CRs) with a very short response duration, with a median duration of response of 55 days and a 6-month OS of 43% . Although daratumumab has low single-agent activity, it is frequently used in combination with other therapies in multiple myeloma, and there is emerging pre-clinical evidence that daratumumab enhances the cytotoxicity of asparaginase in a PDX mouse model; in that same study, pretreatment of ENKTL cell lines with all-trans retinoic acid (ATRA) increased expression of CD38 and increased daratumumab-induced cytotoxicity, setting the stage for future combination studies . 7.2. CD30 The cell surface protein CD30 is also expressed in the majority of ENKTL cases , with an unclear role as a prognostic marker . Brentuximab vedotin (BV), a CD30-directed antibody-drug conjugate that is used extensively in lymphomas, has low single-agent activity in relapsed/refractory ENKTL, with a phase II study that included 7 patients with ENKTL demonstrating an ORR of 29% (1 patient with CR) . P-glycoprotein (MDR1) may mediate resistance to BV in ENKTL, and cyclosporine, an MDR1 inhibitor, may increase BV response rates . There are ongoing studies to combine BV with combination chemoimmunotherapy as is done in other lymphomas, as well as CD30-directed CAR T cell therapy which may provide therapeutic avenues . 7.3. EBV-Targeted Approaches Targeting EBV and its myriad viral proteins which drive ENKTL tumorigenesis, including LMP1 and LMP2, have been explored , particularly the use of antigen-specific cytotoxic T-lymphocytes (CTLs) as has been utilized in EBV-associated PTLD. In one study, LMP-targeted autologous CTLs were infused into patients with EBV-associated lymphomas, including 11 patients with ENKTL . The ORR was 67% (all CRs) in the 6 patients with active disease, with durable remissions of 4 or more years in 3 of the patients and no treatment-emergent toxicities. In the 5 patients who were already in remission and felt to be at high risk of relapse, all 5 patients had ongoing sustained remission after CTL infusion with no CTL-related toxicities, suggesting that consolidation with CTLs may also be a therapeutic approach. This has been demonstrated in a cohort of 26 patients who had undergone allogeneic transplantation for EBV-associated lymphomas and who adjuvantly received allogeneic donor-derived LMP-specific T-cells, which demonstrated a 2-year EFS of 57% . A similar study of adjuvant CTLs in 10 patients with ENKTL who were in a CR after induction therapy demonstrated a 4-year PFS and OS of 90% and 100%, respectively, suggesting that the post-remission timepoint may be a critical period to consolidate with CTL therapy . A more recent study explored the activity of a novel T cell product, baltaleucel-T, in which autologous peripheral T cells are stimulated with antigen-presenting cells containing peptide libraries of multiple EBV-associated proteins, including LMP1, LMP2, and multiple cytokines . Of the 54 patients with advanced-stage, relapsed ENKTL screened for the study, only 47 had adequate whole blood collection, and only 15 patients actually received baltaleucel-T treatment, with the other patients either having manufacturing failure and death or disease progression death before product administration. The ORR among 10 patients with measurable disease was 50% (CR 30%), with a median PFS of 12 months. Although there was activity of the product in relapsed ENKTL, manufacturing issues present a significant challenge in an aggressive and rapidly-progressive malignancy, and off-the-shelf allogeneic products are under investigation. Antiviral therapy against EBV requires the presence of lytic phase viral proteins, and HDAC inhibitors are able to activate the lytic cycle to sensitize EBV-infected cells to ganciclovir. A phase I/II study of nanatinostat with valganciclovir in 55 patients with relapsed/refractory EBV-positive lymphomas included 9 patients with ENKTL and demonstrated an ORR of 60% (27% CR) in all patients with T/NK cell lymphomas and an ORR of 63% in ENKTL patients . 8. Conclusions and Future Directions Next-generation sequencing and proteomic analyses over the past decade have significantly improved our understanding of the molecular landscape that induces ENKTL tumorigenesis and have revealed several novel therapeutic approaches. Given that EBV-driven DNA hypermethylation is ubiquitously observed in ENKTL, combinations of hypomethylating agents with other therapeutic agents, particularly immunotherapy, in which synergy has already been demonstrated, present promising opportunities for future clinical trials . These innovations have also revealed new predictive biomarkers in newly-diagnosed and relapsed disease as well as refined prognostic and predictive scoring systems, including composite single-nucleotide polymorphism signatures and identification of unique genetic clusters of ENKTL , which may enable a personalized medicine approach towards ENKTL therapy. Prospective trials which incorporate these biomarkers and novel therapeutics in rational combinations with existing standard-of-care treatment regimens will be vital to validate their clinical utility and new treatment paradigms for this aggressive malignancy. Author Contributions Conceptualization, A.M. and B.M.H.; writing--original draft preparation, A.M.; writing--review and editing, P.P. and B.M.H. All authors have read and agreed to the published version of the manuscript. Institutional Review Board Statement Not applicable. Informed Consent Statement Not applicable. Data Availability Statement All data can be found in the text. Conflicts of Interest A.M. declares no conflict of interest. P.P. declares research funding from Viracta Therapeutics and Teva; honoraria from Viracta Therapeutics, Innate Pharma, Daiichi, Kyowa, and Teva; scientific boards for Viracta Therapeutics, Innate Pharma, BeiGene, Kyowa, Morphosysi, ADCT, Loxo, and Ono; and consultancy for DrenBio. B.M.H. declares consultancy for Viracta Therapeutics. cancers-15-01366-t001_Table 1 Table 1 Putative driver mutations in ENKTL and potential therapeutic approaches. Identified Mutation and Prevalence (If Available) Biologic Significance in ENKTL Hypothetical Therapeutic Approach References Promoter hypermethylation BCL2L11 (BIM) sensitizes NK cell lines to chemotherapy-induced apoptosis hypomethylating agents (azacitidine, decitabine) DAPK1 mediates p53-dependent apoptosis PTPN6 (SHP1) inactivates the JAK/STAT signaling pathway TET2 9% contributes to early global hypermethylation SOCS6 inactivates the JAK/STAT signaling pathway ASNS downregulation is associated with increased sensitivity to L-asparaginase DLC1 RAS signaling PRDM1 6% apoptosis PTPRK 10% JAK/STAT signaling and apoptosis HACE1 apoptosis P73 , hMLH1, CDKN2A, CDKN2B, RARb, PCDH10, DLEC1, CADM, DAL1 currently unknown functions Epigenetic regulatory genes EZH2 61% aberrant overexpression results in tumor growth independent of its histone methyltransferase activity via non-canonical transcriptional activator 3-deazaneplanocin-A (DZNep) tazemetostat (minimal activity in ENKTL) CDK4/6 inhibitors (i.e., ribociclib) for downstream cyclin D1 inhibition BCOR 17-32% tumor suppressor: suppression leads to enhanced cell proliferation and IL-2 production via HDACs none MLL2 (KMT2D) and MLL3 (KMT2C) 13-19% tumor suppressor related to histone methylation none TET1/TET2, EP200, ASXL3, CREBBP and ARID1A 5-9% currently unknown functions none MicroRNAs (i.e., miR-155) miR-155 activates STAT3 and VEGF signaling to promote lymphomagenesis novel miRNA inhibitors (i.e., for mIR-155) Cell survival and proliferation JAK/STAT pathway (usually STAT3 or JAK3 mutations) 18% constitutive activation and downstream EZH2 upregulation tofacitinib ruxolitinib (combined with novel TP53-MDM2, farnesyltransferase, or MCL-1 inhibitors, or with BCL2 inhibitor venetoclax or aurora kinase inhibitor alisertib) novel pan-JAK and STAT inhibitors momelotinib (combined with HDAC inhibitor citarinostat) NF-kB signaling pathway and survivin 7% constitutive activation and downstream upregulation of anti-apoptotic protein survivin proteasome inhibitors (bortezomib, carfilzomib) survivin inhibitors (terameprocol, mithramycin) DDX3X and RAS/MAPK pathway 14% tumor suppressor: upregulation of downstream NF-kB and MAPK pathways, resulted in cell proliferation previous inhibitors of MAPK pathway not effective statins (i.e., fluvastatin, atorvastatin) C-MYC 45% oncogene causing downstream upregulation of EZH2 and RUNX3 novel small molecule MYC inhibitors (i.e., JQ1) PDGFRa pathway 89-91% upregulated by PI3K/Akt/mTOR and JAK/STAT pathways and mediates cell survival via tyrosine kinase activity tyrosine kinase inhibitors (i.e., imatinib) PI3K/Akt/mTOR pathway upregulated by JAK/STAT pathway and mediates cell survival PI3K inhibitors (i.e., duvelisib) Akt inhibitor lenalidomide mTOR inhibitors (i.e., temsirolimus) may be more effective in combination Avoiding autophagy HDACs inactivation of tumor suppressor genes HDAC inhibitors (i.e., romidepsin, vorinostat, chidamide, panobinostat, nantinostat, citarinostat) novel histone acetyltransferase inhibitors (KAT5 inhibitor NU9056) TP53 (63%), PRDM1, FOXO3 tumor suppressors none (putative inhibitors for TP53-mutated and TP53-wild type disease) Tumor microenvironment PD-1/PD-L1 pathway 56-80% PD-L1 overexpression on tumor cells and tumor-infiltrating macrophages enabling escape from immune surveillance PD-1 inhibitors (pembrolizumab, nivolumab, sintilimab, tislelizumab, toripalimab, geptanolimab, camrelizumab) PD-L1 inhibitors (atezolizumab, sugemalimab, avelumab) VEGF overexpression and tumor angiogenesis anti-VEGF monoclonal antibodies (i.e., bevacizumab) VEGF receptor inhibitor apatinib MET and HGF overexpression and tumor angiogenesis, invasion, and metastasis MET inhibitors (i.e., crizotinib) Cytokines (IL-2, IL-18, IL-10, IL-9, IL-15, MIP-1a, IL-6, interferon) growth promotion cytokine inhibitors (not currently being studied in ENKTL) Chemokines (CCR4) promote tumor angiogenesis and metastasis mogamulizumab S100A9 upregulates PD-L1 expression and escape from immune surveillance none Other therapeutic targets CD38 50% cell surface binding mediates cell adhesion and proliferation of lymphocytes daratumumab (ATRA may enhance daratumumab-induced cytotoxicity) CD30 57% cell surface binding stimulates the NF-kB and MAPK pathways with pro-survival and anti-apoptotic effects brentuximab vedotin EBV overexpression of the oncoprotein LMP1, leading to ligand-independent activation of the pro-survival and anti-apoptotic NF-kB and MAPK pathways autologous or allogeneic cytotoxic T-lymphocyte cellular therapies (i.e., baltaleucel-T) antiviral therapy (valganciclovir, in combination with HDAC inhibitors) cancers-15-01366-t002_Table 2 Table 2 Prognostic and predictive biomarkers in ENKTL. Identified Mutation Prognostic or Predictive Significance in ENKTL References MLL2 (KMT2D) prognostic mutation associated with inferior overall survival mutation in circulating ctDNA associated with total metabolic tumor volume TET2 prognostic mutation associated with inferior overall survival EZH2 prognostic overexpression associated with higher tumor cell proliferation, advanced stage, and inferior overall survival MicroRNAs (i.e., miR-221 and miR-155) prognostic high serum miR-221 associated with inferior overall survival high serum miR-155 associated with disease response PTPRK predictive tumors with methylated PTPRK promoter regions treated with SMILE protocol had an inferior overall survival ASNS predictive high expression associated with asparaginase resistance lnRNAs (i.e., SNHG12 and BCYRN1) prognostic predictive high SNHG12 expression associated with cisplatin resistance as well as multidrug resistance via P-glycoprotein high BCYRN1 expression associated with inferior PFS and resistance to asparaginase Gene expression signatures prognostic predictive three molecular subtypes of ENKTL: tumor-suppressor/immune-modulator (TSIM), MYC-related (MB) and histone epigenetic altered (HEA) superior survival in HEA subtype and inferior survival in MB subtype TSIM subtype predicts response to checkpoint inhibitors, MB subtype predicts response to MYC inhibitors, and HEA subtype predicts response to HDAC inhibitors JAK/STAT predictive activating JAK/STAT mutations or STAT3 overexpression predicts response to ruxolitinib TP53 prognostic predictive TP53 associated with inferior survival mutational status predicts response to agents in combination with ruxolitinib: novel TP53-MDM2 inhibitors in TP53-wild type disease and farnesyltransferase inhibitors in TP53-mutated disease DDX3X prognostic mutation associated with inferior prognosis and inferior response to CHOP-based chemotherapy but not asparaginase-based chemotherapy survivin prognostic overexpression associated with inferior survival C-MYC prognostic presence of C-MYC associated with inferior overall survival PDGFRa prognostic high expression associated with inferior PFS PD-L1 prognostic predictive mixed results: high expression associated with inferior treatment response and survival in early-stage disease but improved survival in advanced-stage disease mutated PD-L1 gene is associated with better responses to pembrolizumab Whole genome sequencing prognostic predictive four immune subgroups of ENKTL: an 'immune tolerance' group (high numbers of Tregs, early stage, best prognosis); two 'immune evasion' groups (high cytotoxic T-cells and high PD-L1); and an 'immune silenced' group (exhausted immune response, advanced stage, worst prognosis) 'immune silenced' group had the worse response to pembrolizumab; 'immune tolerance' group had the best response to pembrolizumab VEGF prognostic high serum VEGF levels associated with inferior PFS and OS Serum cytokines prognostic predictive high serum IL-18, IL-10, IL-6, IL-2Ra, IL-9, IL-15, MIP-1a all associated with inferior survival high serum IL-10 predicts resistance in gemcitabine S100A9 prognostic high serum and tumor levels associated with advanced stage, poor response to therapy, and early relapse CD38 prognostic predictive high expression associated with inferior survival predicts response to daratumumab CD30 prognostic predictive mixed results with unclear effect on prognosis predicts response to brentuximab vedotin single-nucleotide polymorphisms (SNPs) prognostic composite signature of 7 SNPs predicts PFS and OS genome-wide mutation and genomic copy number alterations (gCNAs) analysis prognostic seven distinct genetic clusters of ENKTL identified with differences in overall survival between clusters Disclaimer/Publisher's Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). 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PMC10000129

Internal organ weight is an essential indicator of growth status as it reflects the level of growth and development in pigs. However, the associated genetic architecture has not been well explored because phenotypes are difficult to obtain. Herein, we performed single-trait and multi-trait genome-wide association studies (GWASs) to map the genetic markers and genes associated with six internal organ weight traits (including heart weight, liver weight, spleen weight, lung weight, kidney weight, and stomach weight) in 1518 three-way crossbred commercial pigs. In summation, single-trait GWASs identified a total of 24 significant polymorphisms (SNPs) and 5 promising candidate genes, namely, TPK1, POU6F2, PBX3, UNC5C, and BMPR1B, as being associated with the six internal organ weight traits analyzed. Multi-trait GWAS identified four SNPs with polymorphisms localized on the APK1, ANO6, and UNC5C genes and improved the statistical efficacy of single-trait GWASs. Furthermore, our study was the first to use GWASs to identify SNPs associated with stomach weight in pigs. In conclusion, our exploration of the genetic architecture of internal organ weights helps us better understand growth traits, and the key SNPs identified could play a potential role in animal breeding programs. internal organ weight GWAS DLY pigs genetic architecture Key Technologies R&D Program of Guangdong Province project2022B0202090002 Project of Swine Innovation Team in Guangdong Modern Agricultural Research System2022KJ126 This research was supported by the Key Technologies R&D Program of Guangdong Province project (2022B0202090002) and the Project of Swine Innovation Team in Guangdong Modern Agricultural Research System (2022KJ126). pmc1. Introduction Body weight, which can reflect growth performance and thus affect economic efficiency, has attracted a lot of attention in animal breeding programs. The body weight of cattle is the sum of various elements, including fat weight, internal organ weight, muscle weight, and bone weight, among others. Of these components, internal organ weight constitutes 14% of the total body weight of cattle . The weight and size of an organ are salient features that serve as dependable predictors of its developmental progression, wherein an augmented organ mass typically alludes to a heightened degree of maturation. Accelerated organ development leads to a smoother coordination of internal organs during vital biological processes such as oxygen transport, blood circulation, lipid metabolism, and digestion. This refinement of these processes can positively impact growth and economic traits. Previous studies have shown that the internal organ weights of crossbred steer calves are strongly correlated with carcass growth rate . Moreover, in humans, internal organ weights have been shown to be positively correlated with body weight and height in normal Zambian adults . Thus, comprehending the genetic architecture of heart weight (Heart WT), liver weight (Liver WT), spleen weight (Spleen WT), lung weight (Lung WT), kidney weight (Kidney WT), and stomach weight (Stomach WT) will propel genetic progress and facilitate the successful implementation of breeding programs. Genome-wide association studies (GWASs) are widely used to identify quantitative trait loci (QTL) and candidate genes associated with complex traits in animals and plants. To date, the number of QTL associated with Heart WT, Liver WT, Spleen WT, Lung WT, and Kidney WT are 29, 31, 19, 5 and 8, respectively, and no QTL have been reported to be associated with Stomach WT in the pig QTL database (accessed on 15 November 2022). Previous studies reported 39 QTL to be associated with internal organ weight in four local pig populations and one commercial population . For instance, Zhang et al. showed that a 2 cM QTL on Sus scrofa chromosome 2 (SSC2) was significantly associated with Heart WT, and three QTL were associated with Liver WT, Lung WT, and Spleen WT. Although several studies have identified QTL to be associated with internal organ weight , the process of genetic improvement remains slow. The difficulty (and high cost) of obtaining phenotypes for internal organ weight studies has led to fewer studies on its genetic architecture. Moreover, previous studies conducted single-trait GWASs for internal organ weight to map the genetic markers and genes; however, internal organ development is mutually coordinated by each different organ, and the single-nucleotide polymorphisms (SNPs) in the genome may act on multiple organs at the same time. Therefore, it is difficult to identify SNPs and candidate genes that affect multiple internal organs simultaneously using single-trait GWASs. Therefore, herein, we performed multi-trait GWASs to identify polymorphic SNPs and improve statistical efficiency, which mainly depends on the genetic correlation between traits . In this manner, it was observed that the statistical efficiency was improved in the case of low trait correlations . Previous studies demonstrated the superiority of conducting multi-trait GWASs in terms of uncovering the genetic architecture of complex traits in animals. For instance, Zhou et al. performed multi-trait GWASs to identify 21 pleiotropic SNPs that were not detected via single-trait GWASs in three body size traits. In Simmental beef cattle, An et al. detected 29 pleiotropic SNPs that were functional in all three growth periods using multi-trait GWASs. To date, there are no studies that use multi-trait GWASs to analyze the genetic architecture of visceral weight. Herein, we performed multi-trait GWASs to compensate for the deficiencies associated with single-trait GWASs and to provide new insights into the genetic mechanisms of multi-organ co-development. The aim of this study was to map the genetic markers and candidate genes associated with internal organ weight in pigs. To this end, we conducted single-trait and multi-trait GWASs for six internal organ weight traits in 1518 crossbred commercial Duroc x (Landrace x Yorkshire) DLY pigs. The results from the current study advanced our understanding of the genetic basis for internal organ weight and further revealed the complexity of the genetic architecture of internal organ weight in pigs. Integrating SNP results from GWASs as a source of prior biological information in the improvement program enhances the selection process by assigning higher weight to key SNPs that are critical for improving internal organ weight traits. 2. Materials and Methods 2.1. Ethical Statement All animals used in this study were treated in accordance with the guidelines for the use of laboratory animals of the Ministry of Agriculture of China and with the approval of South China Agricultural University (Guangzhou, China), No. 2018F089. 2.2. Animal Samples and Phenotype Collection Experimental animals were selected from a DLY three-way crossbred commercial line with no overlapping blood relations, through random selection based on genealogy, in which 89 Duroc boars were mated with 397 Landrace x Yorkshire sows to produce a large number of offspring. All pigs were raised in four farms of the Guangdong Wens Food Group Co., Ltd. (Guangzhou, China). In brief, a total of 1518 individuals (757 boars and 764 sows) were reared with free access to water and feed and were fattened to 115 kg. They were euthanized in 13 batches with a 24 h interval between each batch and had an average slaughter age of about 7 months. After the pigs were euthanized, their phenotypes were recorded, and their internal organs were excised, emptied, flushed, blotted dry, and weighed immediately using an electronic scale with a range of 0.0 kg to 300 kg and accuracy of +-100 g. The scale was calibrated using the linear calibration method with 20% MAX or 60% MAX weight. The organ distribution is shown in Figure 1. R 4.2.1 software was used to test the normal distribution of the descriptive statistics of the internal organ traits. 2.3. Genotyping and Quality Control Ear samples were collected from all 1518 individuals, and genomic DNA was extracted from the ear tissue of each pig using a standard phenol-chloroform method and subsequently diluted to 50 ng/mL for the genotyping procedure, controlling the quality OD260/280 between 1.8 and 2.0. The 1518 DLY pigs were genotyped using the GeneSeek Porcine 50K SNP BeadChip (Neogen, Lincoln, NE, USA), which contained 50,703 SNPs. After genotyping, to ensure the accuracy and validity of the GWAS results, we performed a quality control (QC) procedure using the PLINK v1.07 software with the following parameters: individual call rate > 95%; SNP call rate > 99%; minor allele frequency > 1%; and p > 10-6 for the Hardy-Weinberg equilibrium test. Moreover, SNPs in sex chromosomes and unmapped regions were excluded. After QC, a final set of 31,941 eligible SNPs remained for subsequent single-trait and multi-trait GWASs. 2.4. Population Structure and Linkage Disequilibrium (LD) Estimation PCA was conducted using the GCTA software to assess the population structure, and PLINK v1.07 was used to calculate the LD decay distance, which was evaluated as the squared correlation of alleles (r2) with a window size of 1000. 2.5. Single-Trait and Multi-Trait Genome-Wide Association Studies The GEMMA software was used to implement the linear mixed model (LMM) for the single-trait GWAS of each internal organ weight trait, including heart weight, liver weight, spleen weight, lung weight, kidney weight, and stomach weight. GEMMA calculated the genomic relatedness matrix (GRM) between individuals to account for the population structure. The mixed linear model was as follows:y=Wa+Xb+u+e where y is a vector of phenotypic values for each internal organ weight; W is the correlation matrix of covariates (fixed effects), including the top five eigenvectors of PCA, farm, sex, and slaughter lot; a is a vector of corresponding coefficients including the intercept; X is the genotypic vector of the SNP markers; b denotes the effect size of the SNP markers; u is a random effects vector, u~MVNn (0, lt-1K); e is the residual vector, e~MVNn (0, t-1In); l is the ratio of the specified variance components; t-1 is the variance of the residuals; K denotes the kinship matrix; I is the unit matrix; n is the number of individuals in the DLY population; MVNn denotes the multi-dimensional normal distribution. Moreover, the GEMMA software was used to implement the multivariate linear mixed models (mvLMMs) for multi-trait GWASs to assess pleiotropic SNPs. The mvLMMs and LMMs were both implemented as described in previous studies . In the current study, the LMMs and mvLMMs in the single-trait GWAS and the multi-trait GWAS utilized the same covariates. The multivariate linear mixed models were as follows:Y=WA+xbT+U+E; G ~MNnxd(0, K,Vg), E ~MNnxd(0,Inxn,Ve) where Y is a matrix of six internal organs for 1518 individuals; W is a covariable matrix (fixed effects); A is a matrix of the corresponding coefficients; x is a vector that marks the genotypes; b is a vector of marker effect sizes for six internal organs' weights. U denotes the random effects; E is a matrix of errors; K denotes the kinship matrix; Vg denotes symmetric matrix of genetic variance component; I is an identity matrix; Ve denotes a symmetric matrix of the environmental variance component; MNnxd(0,V1,V2) denotes the nxd matrix normal distribution with mean 0; V1 denotes row covariance matrix; V2 denotes column covariance matrix. Furthermore, the Bonferroni correction can lead to an overcorrection and can be too conservative, this can result in a limited number of labeled association p-values that meet the standard across the genome. This can lead to a high false-negative rate. To address this issue, the false-discovery rate (FDR) was employed as a correction to the threshold . Thus, the threshold p-value was calculated as P=FDR*NM; the FDR was set to 0.01, N is the number of SNPs with p-value less than 0.01, and M refers to the total number of SNPs after quality control. Moreover, quantile-quantile (Q-Q) plots were constructed for the six internal organ weight traits to further assess the population structure. In addition, the PLINK v1.07 and Haploview v4.2 software were implemented to perform the haplotype block analysis in chromosomal regions with multiple significant SNPs. The default parameters of Haploview 4.2 (MAF > 0.05, Mendelian error < 2, and p-value < 10-3 for the HWE test) were used to define the linkage disequilibrium (LD) blocks of SNPs. 2.6. Estimation of Heritability and Phenotypic Variation In the present study, the restricted maximum likelihood (REML) method was used to assess the SNP-based heritability of each internal organ weight trait, and the percentage of phenotypic variation that could be explained by significant SNPs was calculated using GCTA software. SNP-based heritability and the percentage of phenotypic variation explained by significant SNPs were calculated as follows :y=Xb+g+e with var(y)=Agsg2+Ise2 where y is the phenotypic value of each internal organ weight trait; b is the vector of fixed effects, including the top five eigenvectors of PCA, farm, sex, and slaughter lot; X is an association matrix; g is the vector of total genetic effect of all the qualified SNPs for the 1518 DLY pigs; Ag is the genomic association matrix between different individuals; sg2 is the additive genetic variance captured by either the genome-wide SNPs or the selected SNPs; se2 refers to residual variance. 2.7. Candidate Gene Search and Function Analysis Our previous studies on this population showed that the average r2 of 0.2 is about 200 kb apart ; the range for searching for the functional gene closest to the position of the significant SNP is determined based on the LD decay distance (r2 = 0.2) of the populations . We used the "biomaRt" package in R, based on the Sus scrofa 11.1 genome version database accessed 20 September 2022). Genes nearest the significant SNPs are list in Tables. We conducted a search of both PubMed and the relevant literature to examine the correlation between the nearest peak SNPs of all the candidate genes and the internal organ weight traits being analyzed. 3. Results and Discussion 3.1. Phenotype Statistics and Heritability Estimation The descriptive phenotypic statistics and estimated heritabilities (h2) for analysis of the internal organ weights are listed in Table 1. The weight of internal organs is a crucial indicator of internal organ development and has a significant impact on organ function. In the current study, the average Heart WT, Liver WT, Spleen WT, Lung WT, Kidney WT, and Stomach WT in DLY pigs were 455.57 g, 1763.61 g, 212.54 g, 1020.54 g, 0.41 kg, and 727.68 g, respectively. The estimated heritabilities of Heart WT and Lung WT were the lowest at 0.21 +- 0.04 and 0.28 +- 0.04, respectively, and all other organ weights had had moderate to high estimated heritabilities, ranging from 0.36 +- 0.04 to 0.49 +- 0.04. Similar to the results of a previous study, the estimated heritabilities of Heart WT, Liver WT, Spleen WT, and Kidney WT were between 0.35 and 0.54, which were moderate to high estimations , indicating that the estimated heritabilities of the weight of an internal organ is generally high in pigs and there is considerable room for improving the genetic contribution through breeding. Furthermore, the coefficients of variation were the lowest for Lung WT and all other traits were relatively high, indicating individual heterogeneity, low trait selection intensity, and high breeding potential. Moreover, the genetic and phenotypic correlation coefficients among Heart WT, Liver WT, Spleen WT, Lung WT, Kidney WT, and Stomach WT are listed in Table 2. The results revealed moderate to low genetic correlations among the six internal organ weight traits. Heart WT had moderate genetic correlations with Liver WT, Lung WT, and Kidney WT, suggesting that these traits could be improved together in pig breeding programs. On the other hand, Stomach WT showed close to 0 genetic correlations with most of the other traits, indicating Stomach WT traits are less influenced by other traits when they are inherited. Therefore, reasonable breeding strategies need to be designed to improve internal organ weight traits. The phenotypic correlation results showed that the correlation coefficients between the phenotypes were at moderate to high levels, excluding the low phenotypic correlation coefficients between Lung WT and Liver WT, and Spleen WT and Kidney WT, especially the phenotypic correlation coefficients of Liver WT and Kidney WT were as high as 0.62. When selecting for a certain phenotype in pig breeding, it is advantageous to also consider other related traits. 3.2. Population Structure and LD decay Population stratification is known to lead to false-positive results in GWASs. To detect potential population stratification, we performed PCA and added the first five principal components to the covariates of the GWAS model to correct for the population structure. Moreover, our previous study showed that the LD decay coefficient of the analyzed DLY pig population with r2 decayed to 0.2 at a physical distance of 200 kb , indicating that the DLY population is diverse with a weak linkage between loci, which facilitates the detection of key SNPs for internal organ weight traits. In addition, Q-Q plots were generated for Heart WT, Liver WT, Spleen WT, Lung WT, Kidney WT, and Stomach WT to further assess population stratification . The expansion coefficients (lambda) of the Q-Q plots for all six internal organ weight traits were close to 1, and no overall systematic bias was observed, signifying a negligible effect of the DLY pig group structure on GWASs. 3.3. Single-Trait GWASs Single-trait GWASs were performed for the weight of the heart, liver, spleen, lung, kidney, and stomach. The results showed that 6, 4, 3, 4, 3, and 4 SNPs were significantly associated with the weight of each organ, respectively. The results of these single-trait GWASs are presented in Figure 2 and Table 3. Notably, it is the first time that significant SNPs associated with Stomach WT have been identified in pigs. Furthermore, on the basis of the LD decay map, a region of 200 kb before and after the key SNPs was defined as a region to screen for candidate genes . For heart weight, six significant SNPs were identified, located on SSC5, 6, 7, 12, and 14. These six SNPs surpassed the significance threshold of 1.01 x 10-4. Figure 2B shows an expansion coefficient lambda (l) of 1.006. Details of the significant SNPs are listed in Table 3. The most significant SNP, WU_10.2_12_6703865 on SSC12, explains 1.70% of the phenotypic variation and is about 44 kb downstream of the CD300LB gene. The CD300LB gene is a triggering receptor expressed on bone marrow cells that regulates the cytosolic process of bone marrow cells , and the CD300LB protein stimulated by T cells regulates DNMT3A mutation and alters immune cells in heart failure . For liver weight, four significant SNPs were detected on SSC4, SSC9, and SSC10 with a l of 0.999 . These four SNPs surpassed the significance threshold of 1.04 x 10-4. The top SNP, H3GA0028070, accounted for 2.10% of the phenotypic variance and is located within the TPK1 gene. A significant SNP, named ASGA0044340, 12 kb upstream of H3GA0028070, also located on TPK1, explained 0.82% of the phenotypic variation. According to reports, TPK1 is a cofactor of certain enzymes associated with the glycolysis and energy production pathways. It is involved in the metabolism of water-soluble vitamins and cofactors and the thiamine metabolic pathway, and mutations in TPK1 can cause thiamine metabolic dysfunction syndrome . In addition, knockdown of this gene can lead to glycogen storage dysfunction . However, no studies have shown TPK1 to be directly associated with liver development and weight in pigs. The GWAS results of Spleen WT identified three significant SNPs, located on SSC3, SSC9, and SSC18, with a l of 0.972 . All three SNPs surpassed the threshold of significance (p < 1.20 x 10-4). The top SNP, ALGA0098928, explained 2.22% of the phenotypic variation and is located within POU6F2. POU6F2 is a suppressor associated with nephroblastoma (WT) that regulates cell proliferation and specific differentiation . According to the RT-qPCR results, the expression of POU6F2 is associated with renal morphogenesis , suggesting that POU6F2 may be closely associated with spleen weight traits. For lung weight, four significant SNPs were detected on SSC1, SSC7, and SSC11 with a l of 1.008 . These four SNPs surpassed the significance threshold of 1.04 x 10-4. An SNP named ALGA0110225 explained 1.81% of the phenotypic variance and is located 97 kb downstream of PBX3, indicating that PBX3 and ALGA0110225 may both play a role in Lung WT. A literature review revealed that PBX3 is directly regulated by targeting NBPF10, miR-144, and miR-224, which are directly associated with lung cancer cell proliferation . In addition, overexpression of PBX3 promotes the proliferation of A549 cells (lung cancer histiocytes) . Therefore, we believe PBX3 to be a promising candidate gene for influencing Lung WT, and the regulatory mechanism needs further investigation. We performed GWASs with the Kidney WT trait in DLY pigs and detected three SNPs that were above the significance threshold (p < 1.03 x 10-4) . Figure 2J shows that the lambda is 0.987. WU_10.2_15_153747936 on SSC15 explains 1.97% of the phenotypic variation and is located 341 kb downstream of the HDAC4 gene. Because the LD decay distance is 200 kb, it follows that HDAC4 may not have a significant effect on Kidney WT traits. QTL and significant SNPs have not been previously reported in relation to Stomach WT. Thus, this study is the first GWAS on pig Stomach WT. Herein, four significant SNPs were identified for the DLY pigs that were above the significance threshold of p < 1.17 x 10-4 . Of the four significant SNPs, three SNPs were simultaneously located on SSC8 and both MARC0052872 and ALGA0106192 were located within the UNC5C gene. Furthermore, the distance between MARC0052872 and ALGA0106192 was only 21 kb, explaining 2.53% and 2.62% of the phenotypic variation, respectively. ASGA0101191 was 100 kb away from the aforementioned SNPs with a phenotypic variation value of 2.14% and was located within BMPR1B. A literature review revealed that UNC5C plays a dominant role in netrin-1/UNC5C-mediated axonal rejection and that its promoter region sequence binds to p53 and acts as a target of p53 to regulate apoptosis . As regards the BMPR1B gene, it has been shown that the BMP family is expressed in the early organ and tissue formation during mouse embryonic development . However, neither the BMPR1B nor the UNC5C gene is directly associated with internal organ weight traits. The above GWAS results show that none of the SNPs associated with internal organ weight overlapped with those previously reported QTL documented in the pig QTL database . This may have been due to the fact that most studies focused on the breeding of native Chinese pigs, and fewer studies were conducted on DLY three-way crossbred commercial populations with significant breed differences. Moreover, the significant SNPs did not overlap in the six traits, i.e., none of the SNPs were polymorphic, which may be related to the low genetic and phenotypic correlation between traits and the low density of genetic markers, which was further verified by multi-trait GWASs. 3.4. Haplotype Block Analysis Figure 3 shows the LD pattern of significant SNPs associated with Stomach WT. In this study, multiple SNPs associated with Stomach WT were in close proximity to each other, with two significant SNPs on SSC8, which is located in a 21 kb region within the UNC5C and BMPR1B genes (the gene function is described above). The insufficient density of 50K microarray markers resulted in a low number of SNPs with linkage disequilibrium, which limited the resolution of the genetic architecture of key SNPs for the trait to some extent. 3.5. Multi-Trait GWASs In order to improve the statistical effect, multi-trait GWASs were individually performed for each SNP by combining the joint analysis of six internal organ weight traits. This revealed the genetic factors with significant interactions among different traits in the same individual under the same environment. Manhattan plots of the multi-trait GWASs are shown in Figure 4. The multi-trait GWASs combining six internal organ weight traits identified four significant SNPs with polymorphisms affecting the phenotypes, ALGA0032998, H3GA0028070, MARC0052872, and ALGA0106192 . SNP ALGA0032998 explained 1.36% of the phenotypic variation and is located within the ANO6 gene. The overexpression of CCR7 was observed to enhance the migration of BxPC-3 cells under the induction of the ANO6 gene, which is a potential mediator of ANO6 expression through the ERK signaling pathway. This promotion of migration was also seen in pancreatic ductal adenocarcinoma cells . The single-trait GWAS described the effects of three SNPs (H3GA0028070, MARC0052872, and ALGA0106192) located on TPK1 and UNC5C genes on the weight of the liver and stomach. These SNPs were not only significant in the single-trait GWAS but were also found to be simultaneously associated with the weight of all six internal organs, suggesting that these four SNPs have pleiotropic effects. Furthermore, no additional SNPs, independent of the single-trait GWAS results, were found. Similar results were previously reported by Guo et al. , in which no additional SNPs, independent of the single-trait GWAS results, were detected in the multi-trait GWASs for backfat thickness, carcass weight, and body weight in the DLY and Duroc populations. The reasons for this situation are manifold. For example, the complexity of the genetic architecture of the internal organ weight trait and the low marker density result in a low number of SNPs reaching significant levels. This renders LD detection insufficient and increases the difficulty of screening for co-dominant SNP or QTL regions. Thus, a larger sample population and a higher marker density are required to screen for loci associated with internal organ weight. 4. Conclusions In this study, we conducted single-trait and multi-trait GWASs on the internal organ weights of 1518 DLY pigs. A total of 24 significant SNPs were detected in the single-trait GWAS results for six internal organ weight traits. The four significant pleiotropic SNPs identified via multi-trait GWASs were associated with six internal organ weight traits, confirming the results of the single-trait GWASs and improving our ability to reveal the genetic architecture of organ weight traits. TPK1, POU6F2, PBX3, UNC5C, and BMPR1B were highlighted as potential genes responsible for differences in Liver WT, Spleen WT, Lung WT, and Stomach WT among individuals according to their gene functions. In summary, the results of this study contribute to our understanding of the genetics of internal organ weight traits in DLY pigs by assigning higher weights to relevant SNPs and key genes in the genome. Author Contributions Conceptualization, E.Z. and G.C.; resources, E.Z., G.C. and Z.W.; investigation, Y.Y., S.Z., D.R., Y.Q., S.W. and J.W.; software and validation, X.L., Z.Z. and J.W.; formal analysis, X.L., J.W. and Z.Z.; data curation, Z.Z., S.W., J.W. and Y.Y.; writing--original draft preparation, X.L. and J.W.; writing--review and editing, E.Z., G.C., Z.W. and J.Y.; visualization, J.Y. and Y.Y. All authors have read and agreed to the published version of the manuscript. Institutional Review Board Statement All animals used in this study were treated in accordance with the guidelines for the use of laboratory animals of the Ministry of Agriculture of China and with the approval of South China Agricultural University (Guangzhou, China), No. 2018F089. Informed Consent Statement Not applicable. Data Availability Statement Data are contained within the article. Conflicts of Interest The authors declare no conflict of interest. Figure 1 Distribution of heart, liver, spleen, lung, kidney, and stomach in the pig. Figure 2 Manhattan and Q-Q plots of internal organ weight traits in the single-trait GWAS. (A) GWAS for Heart WT; (B) Q-Q plot for Heart WT; (C) GWAS for Liver WT; (D) Q-Q plot for Liver WT; (E) GWAS for Spleen WT; (F) Q-Q plot for Spleen WT; (G) GWAS for Lung WT; (H) Q-Q plot for Lung WT; (I) GWAS for Kidney WT; (J) Q-Q plot for Heart WT; (K) GWAS for Stomach WT; (L) Q-Q plot for Stomach WT. The x-axis represents the chromosome, and the y-axis represents the -log10 (p-value) value in the Manhattan plot of the GWAS. The Q-Q plot is plotted with the x-axis representing the actual measured value of -log10 (p-value) and the y-axis representing the observed value of -log10 (p-value) and labeled with the expansion factor lambda (l). Figure 3 Linkage disequilibrium blocks of important SNPs on SSC8 in DLY pigs. The value in the box is the degree of linkage disequilibrium between SNPs (r2). Figure 4 Manhattan plots of multi-trait GWAS results for six internal organ weight traits in the DLY population. The x-axis represents the chromosome, and the y-axis represents the -log10 (p-value) value in the Manhattan plot of the GWAS. The dashed line indicates the FDR-corrected threshold. Figure 5 Venn diagram showing the distribution of SNPs in the single-trait GWASs and multi-trait GWASs, highlighting the role of pleiotropic SNPs in multiple traits. animals-13-00808-t001_Table 1 Table 1 Phenotypic statistics and heritability estimates for Heart WT, Liver WT, Spleen WT, Lung WT, Kidney WT, and Stomach WT. Trait N Mean (+-SD) Min Max C.V.% a h2 (+-SE) Heart WT 1518 455.57 +- 78.06 217.1 868.8 17.13 0.21 +- 0.04 Liver WT 1518 1763.61 +- 271.83 993.9 2607.7 15.41 0.46 +- 0.04 Spleen WT 1517 212.54 +- 47.49 95.9 502.1 22.34 0.49 +- 0.04 Lung WT 1517 1020.54 +- 240.10 918.9 2090.4 2.18 0.28 +- 0.04 Kidney WT 1486 0.41 +- 0.08 0.17 0.84 19.51 0.36 +- 0.04 Stomach WT 1518 727.68 +- 129.97 495.7 1321.7 17.86 0.47 +- 0.04 a Coefficient of variation (C.V.). animals-13-00808-t002_Table 2 Table 2 Phenotypic correlations (above the diagonal) and genetic correlations (below the diagonal) among organ weight traits within the DLY population. Heart WT Liver WT Spleen WT Lung WT Kidney WT Stomach WT Heart WT 1 0.36 0.33 0.41 0.37 0.49 Liver WT 0.31 +- 0.11 1 0.34 -0.02 0.62 0.38 Spleen WT 0.23 +- 0.11 0.11 +- 0.09 1 0.15 0.33 0.41 Lung WT 0.33 +- 0.13 0.17 +- 0.11 0.04 +- 0.11 1 0.05 0.27 Kidney WT 0.30 +- 012 0.33 +- 0.09 0.07 +- 0.10 0.29 +- 0.12 1 0.43 Stomach WT 0.02 +- 0.12 0.03 +- 0.09 0.26 +- 0.09 0.02 +- 0.11 0.03 +- 0.10 1 animals-13-00808-t003_Table 3 Table 3 Significant SNPs and candidate genes for Heart WT, Liver WT, Spleen WT, Lung WT, Kidney WT, and Stomach WT in single-trait GWASs. Trait SSC SNP Position (bp) MAF p-Value PEV (%) a Candidate Gene Distance Heart WT 6 WU_10.2_6_126961053 137,008,535 0.257 1.88 x 10-5 2.09% ST6GALNAC3 Within 14 6_43731895 132,228,312 0.407 1.99 x 10-5 1.71% HTRA1 124,717 12 WU_10.2_12_6703865 6,682,110 0.389 6.40 x 10-5 1.70% CD300LB 44,143 5 ALGA0032998 76,317,972 0.264 8.30 x 10-5 1.36% ANO6 Within 7 WU_10.2_7_116585612 110,088,932 0.242 7.42 x 10-5 1.10% KCNK10 -29,135 12 12_5381300 5,426,010 0.319 6.50 x 10-5 0.53% CDK3 Within Liver WT 4 WU_10.2_4_20570494 19,550,555 0.249 2.15 x 10-5 2.11% CCN3 -35,758 9 H3GA0028070 113,152,352 0.09 7.54 x 10-5 2.10% TPK1 Within 9 ASGA0044340 113,140,343 0.118 3.51 x 10-5 0.82% TPK1 Within 10 WU_10.2_10_3469625 1,753,591 0.476 3.08 x 10-5 0.43% RGS21 Within Spleen WT 18 ALGA0098928 54,993,603 0.328 1.39 x 10-5 2.22% POU6F2 Within 3 ALGA0105765 20,525,651 0.216 4.74 x 10-5 0.78% HS3ST4 -19,582 9 WU_10.2_9_45824613 40,989,995 0.399 1.47 x 10-5 0.58% TTC12 Within Lung WT 11 ALGA0060656 8,759,687 0.231 6.24 x 10-5 2.76% FRY Within 1 ALGA0110225 266,708,292 0.278 1.46 x 10-5 1.81% PBX3 97,646 7 ASGA0035515 98,022,168 0.04 3.57 x 10-5 1.43% YLPM1 Within 1 MARC0089438 11,012,474 0.271 4.46 x 10-5 0.69% / / Kidney WT 15 WU_10.2_15_153747936 138,955,316 0.369 2.61 x 10-5 1.97% / / 4 WU_10.2_4_119054114 108,872,194 0.478 3.93 x 10-5 1.62% RAP1A 122,469 11 WU_10.2_11_20231427 19,917,312 0.221 8.86 x 10-5 0.88% SUCLA2 Within Stomach WT 8 ALGA0106192 124,443,641 0.064 1.54 x 10-5 2.62% UNC5C Within 8 MARC0052872 124,421,940 0.063 1.61 x 10-5 2.53% UNC5C Within 8 ASGA0101191 124,548,240 0.081 2.40 x 10-5 2.14% BMPR1B Within 2 MARC0018316 46,014,239 0.46 8.35 x 10-5 0.74% ARNTL Within a The percentage of phenotypic variance explained by each SNP. animals-13-00808-t004_Table 4 Table 4 Significant SNPs and candidate genes for Heart WT, Liver WT, Spleen WT, Lung WT, Kidney WT, and Stomach WT in multi-trait GWASs. SSC SNP Position (bp) MAF p-Value PEV (%) a Candidate Gene Distance 8 ALGA0106192 124,443,641 0.064 7.29 x 10-5 2.62% UNC5C Within 8 MARC0052872 124,421,940 0.063 5.61 x 10-5 2.53% UNC5C Within 9 H3GA0028070 113,152,352 0.088 5.51 x 10-5 2.10% TPK1 Within 5 ALGA0032998 76,317,972 0.266 3.88 x 10-5 1.36% ANO6 Within a The percentage of phenotypic variance explained by each SNP. Disclaimer/Publisher's Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

PMC10000130

Pectus excavatum is a deformity of the thorax characterized by ventrodorsal narrowing of the sternum bone and costal cartilages, which can lead to compression and cardiopulmonary alterations in dogs, presenting a high prevalence in brachycephalic breeds. The aim of this report was to describe two types of management for the noninvasive treatment of pectus excavatum in newborn puppies of the breeds French Bulldog and American Bully. The puppies presented dyspnea, cyanosis and substernal retraction during inspiration. The diagnosis was performed by physical examination and confirmed by chest X-ray. Two types of splints were performed (a circular splint with plastic pipe and a paper box splint on the chest), aiming at thoracic lateral compression and frontal chest remodeling. The management was effective for the conservative treatment of mild-grade pectus excavatum, resulting in the repositioning of the thorax and improvement of the respiratory pattern. congenital malformation dyspnea splint bandage thorax dog brachycephalic breeds This research received no external funding. pmc1. Introduction Pectus excavatum (excavated chest) is a congenital malformation of the development of the ventral chest wall, also known as the 'funnel chest' . It is characterized by abnormal growth of the sternum and costal cartilages, where there is a deviation and ventrodorsal narrowing of the thorax, which presents with depression . This deformity is described in humans, dogs, cats, lambs, calves, rabbits, and other species . In canine species, the incidence of pectus excavatum is 0.33% , and pectus excavatum has a high prevalence in brachycephalic breeds, such as French Bulldog, English Bulldog, Pekingese, Pug, Maltese, Shih-Tzu and American Bully, associated with the probable genetic component . In these breeds, pectus excavatum can be frequently diagnosed with other deformities, such as swimmer dog syndrome, and respiratory tract defects, such as nasal stenosis, tracheal hypoplasia, and soft palate hyperplasia (brachycephalic airway syndrome) . Its etiology is not well defined, and it is believed to be a genetic predisposition with a hereditary component . Some studies suggest shortening of the diaphragmatic tendon, congenital deficiency of the cranial muscles of the diaphragm and osteogenesis/abnormal chondrogenesis. However, the phenotype and a variety of conditions can also be associated, such as abnormalities in intrauterine pressure and a negative increase in intrathoracic pressure secondary to alterations in the upper airway (for example, airway obstruction from soft palate hyperplasia) . The clinical manifestations of pectus excavatum are variable and are associated with pulmonary alterations secondary to reduced thoracic space or pulmonary atelectasis by cardiac compression (cardiac compression puts pressure on the lung, causing the pulmonary tissue to collapse with loss of volume). In some cases, they may present with recurrent pneumonia, causing chronic respiratory disease . Changes in the cardiovascular system can be observed with deviation and compression of the heart, systolic or diastolic dysfunction, or related to other congenital cardiac alterations . The main clinical signs observed are dyspnea, tachypnea, hyperpnea, pallor or the cyanosis of mucous membranes, intolerance to manipulation or exercise, apathy, lack of appetite, vomiting or regurgitation, cough, heart murmur and arrhythmias ; however, some animals may be asymptomatic. Depending on the severity of cardiorespiratory signs, this condition can lead to high mortality in affected dogs . The diagnosis of pectus excavatum is made by physical examination, visualization and palpation of the depression formed in the sternum, which is confirmed by complementary exams, such as chest X-ray, computed tomography or magnetic resonance imaging. The evaluation of pectus excavatum can be performed by measuring the frontosagittal and vertebral indices on chest X-rays . Table 1 shows the normal thoracic indices in dogs of nonbrachycephalic breeds, brachycephalic breeds and cats. Based on these indices, pectus excavatum can be classified as mild, moderate or severe (Table 2). Figure 2 shows radiographs of neonatal puppies of brachycephalic breeds with normal thoraxes and with different degrees of pectus excavatum. Initial treatment consists of clinical management of respiratory distress and therapy for respiratory infections. Treatment of pectus excavatum can be conservative or surgical, taking into account the age of the animal and the severity of the deformity. The change to conservative treatment can be performed for mild to moderate cases, and the use of a splint to compress the thorax has been described as an option for newborn puppies , and surgery is required for cases that do not respond to the splint, severe cases, or older animals . In newborn pups, noninvasive treatment may be the first choice because constant bone growth allows the thorax to be reshaped quickly and effectively with corrective compression splints and bandages. The aim of this report was to describe the clinical evolution of two cases of pectus excavatum in newborn puppies with two types of conservative treatment. 2. Case Reports 2.1. Case 1--Splint and Circular Bandage on Chest A nine-day-old French Bulldog female from a kennel was treated at a veterinary clinic in Rio Verde, Brazil, with a 24 h history of respiratory distress. It was described by the tutor that from the beginning of dyspnea, the neonate presented with apathy, and he was reluctant to move, constantly vocalized and was in an orthopneic position. The newborn was fed exclusively breast milk and was kept with the other puppies on a mattress with blankets. Regarding maternal information, the bitch was primiparous, at 18 months of age, fed a super-premium commercial diet, vaccinated, dewormed, with no history of diseases during the gestational phase, and supplemented with folic acid (0.1 mg/kg, once a day, for 30 days after insemination). No drugs were administered during the gestational phase. The delivery was eutocic, with the birth of seven puppies. No alterations were observed in the other neonates. The kennel had a history of diagnosis of pectus excavatum in another litter of a bitch related to this mother. The kennel had 11 dogs. Two sister bitches (acquired for breeding with no history) that mated with different males had litters with pups showing pectus excavatum. In total, two litters and five puppies (one puppy from one litter and four from another) were diagnosed with this deformity. From this other litter, all four pups were also treated using the pipe splint; three survived, and one died. On physical examination, the patient presented with marked dyspnea, substernal retraction during inspiration (Video S1), hyperpnea, cyanosis, pulmonary crackling, heart rate (HR) 196 bpm, respiratory rate (RF) 72 mpm, body temperature 36.3 degC, blood glucose 132 mg/dL, weight 356 g, absent suction and rooting reflexes, normohydrate, and no changes in cardiac auscultation. Thoracic deformity was observed on physical examination of the chest, with depression in the caudal region of the sternum bone and narrowing ventrodorsal direction. In the outpatient treatment, oxygen therapy per mask with 40% oxygen was performed for two hours, and the bronchodilator aminofilin (24 mg/mL) was administered sublingually at a volume of 0.2 mL/100 g of weight. After stabilization of the patient, complementary tests were performed: hemogram and chest X-ray. Moreover, 0.5 mL of blood from the jugular vein was collected for the realization of the hemogram, which demonstrated leukocytosis (26,000 leukocytes/mL) with left deviation associated with neutrophilia and slight absolute lymphocytosis. On the chest X-ray , it was possible to observe the ventrodorsal deviation of the sternum in the medial and caudal region of the ventral thorax, diagnosing pectus excavatum. The frontosaginal and vertebral index corresponded to 1.8 cm (<=2.0 cm) and 10.2 cm (>9 cm), respectively, classifying the pectus as mild grade. In addition, cardiopulmonary alterations were found: pneumonia with alveolar pulmonary pattern, pulmonary hyperinflation, rounded cardiac silhouette (globose aspect) and cardiac deviation to the left. Figure 4 shows the chest X-ray of a neonate of the same litter, without thoracic alterations, to compare chest structures and aid in diagnosis. Clinical treatment was initiated with antibiotic therapy (ceftriaxone 50 mg/kg subcutaneously every 12 h for five days); inhalation with a bronchodilator (aminophylline 24 mg/mL, in the volume of 0.2 mL/100 g of weight, every 12 h, for three days); N-acetylcistein 3 mg/kg orally every 8 h for five days; anti-inflammatory (fluticasona aerosol propionate 250 mcg, an intranasal spray every 8 h for five days), and feeding with commercial breast milk substitute (Support Milk Dog(r)) by orogastric tube (using a urethral probe number 04), while the neonate presented absent sucking reflex, at a volume of 3 mL/100 g of weight and temperature of 37 degC, every three hours. A circular splint was made for the thorax in the form of a 'C' using a polyvinyl chloride (PVC) pipe to perform slight lateral compression of the thorax for the remodeling of the rib cage and sternum bone. The width and length of the PVC corresponded to the width and length of the patient's thorax. A ventral space (approximately 2 cm) was maintained to avoid ventral compression of the thorax on surfaces, allowing space for remodeling the thorax/sternum in the frontal direction. The splint was upholstered with cotton wool and medical tape on the sides to support and slightly compress the sides of the chest, as well as to avoid traumatic injuries to the patient, promoting comfort in a certain way. The splint was placed on the newborn's chest, with the open part of the PVC on the patient's back and fixed with medical tape bandage . The puppy remained hospitalized for eight hours. After placing the splint, the patient was released home, as he already presented improvement of the respiratory pattern (clinical evaluation). After 24 h, the newborn already had a sucking reflex and fed normally on the mother. During treatment, the use of the splint did not hinder breastfeeding . On the return, eight days after the beginning of management, chest remodeling was observed, with the thorax correctly aligned, with absence of depression in the sternum bone region and absence of respiratory distress. Video S2 demonstrates the respiratory pattern without alterations, with absence of substernal retraction during inspiration. A new X-ray was requested for a detailed evaluation of the rib cage for a follow-up of the pulmonary condition and cardiac evaluation. However, the tutor chose not to perform the requested exams. Periodic evaluations and the future castration of the patient were recommended, as well as of parents and littermates. Figure 9 demonstrates the healthy puppy at two months of age. Currently, all puppies in the litter remain healthy, and all have been spayed or neutered. 2.2. Case 2--Paper Box Splint on Chest A three-day-old male American bully dog was treated at the UNESP Veterinary Hospital, Botucatu, Brazil, with a history of dyspnea and cyanosis for two days. At birth, the pup was diagnosed with cleft palate (secondary palatoschisis, involving hard and soft palate, with shallow depth), did not ingest colostrum, and was fed exclusively with a commercial substitute of breast milk (Support Milk Dog(r)) by an orogastric tube (using urethral probe number 06), at a volume of 3 mL/100 g of weight and temperature of 37 degC every three hours. The newborn was separated from the mother and littermates on the day of birth and was kept in a plastic box with blankets and heated with a thermal bag. Regarding maternal information, it was a primiparous female, at 12 months of age, fed a super-premium commercial diet, vaccinated, and dewormed, without historic diseases during pregnancy. No drugs were administered during the gestational phase. It was reported that the pregnancy came from inbreed mating between siblings of the same litter. The dog underwent elective cesarean section at approximately 62 days of gestation, with the birth of 10 puppies. The estimate of gestational age was determined by measuring fetal diameters on maternal ultrasound: biparietal (cephalic) diameter and thoracic diameter. No alterations were observed in the other neonates. On physical examination, the patient presented dyspnea, substernal retraction during inspiration (Video S3), cyanosis during manipulation, HR 255 bpm, FR 30 mpm, body temperature 36.8 degC, glycemia 151 mg/dL, weak suction reflexes and breast search, strong vestibular straightening reflex, body weight 318 g, was normohydrate and without changes in cardiopulmonary auscultation. In the evaluation of the thorax, depression was observed in the caudal region of the sternum bone, and ventrodorsal narrowing was observed . Complementary tests, hemogram and chest X-ray, were performed. Blood (0.4 mL) from the jugular vein was collected for the hemogram, which was within the reference standards for age. The chest X-ray was performed in the right lateral and ventrodorsal positions, observing a ventrodorsal deviation of the sternum and diagnosing pectus excavatum. The frontosagittal and vertebral indices corresponded to 1.7 cm (<=2 cm) and 10 cm (>9 cm), respectively, classifying the pectus as mild grade. In addition, a rounded cardiac silhouette (globose aspect) and right cardiac deviation were observed. The treatment was performed with oxygen therapy per mask, with 100% oxygen for 10 min, and the sublingual administration of aminophylline 24 mg/mL in a volume of 0.2 mL/100 g of weight. As the neonate did not ingest colostrum, fresh frozen plasma was given subcutaneously from an adult dog, healthy and vaccinated, as a source of passive immunity, in a volume of 2 mL/100 g of weight . Feeding by orogastric tube was indicated until later surgery to correct the palatoschisis. Corrective management of pectus excavatum was performed by positioning the neonate in lateral decubitus inside a resistant paper box . In this case, for a neonatal puppy of 318 g, a medicine box was used (in a horizontal position), with dimensions of 6 cm high, 6 cm wide, and 11.5 cm long , aiming at the lateral compression of the thorax on surfaces so that the frontal region of the thorax would return to its normal positioning. The box was coated with an elastic bandage, promoting greater stiffness. A space was made in the box to insert and immobilize (with gauze and medical tape) the thoracic limbs of the newborn to keep it immobile in lateral decubitus, avoiding its movement and consequent ventral compression of the thorax. After finishing the splint and bandage, the patient was released home. The lateral decubitus change occurred every two hours. The neonate was fed by an orogastric tube during the use of the box and was positioned in ventral decubitus for feeding. On the return, six days after the beginning of management, the neonate presented a chest wall without alterations, observing the remodeling of the thorax, absence of depression in the sternum region , regular respiratory pattern, absence of substernal retraction during inspiration (Video S4) and pink mucous membranes. A new X-ray was requested for chest evaluation, but the tutor chose to perform the examination later. Periodic evaluations and future castration of the patient were recommended, as well as of parents and littermates. The puppy developed normally, and no chest deformations were observed. Figure 14 shows the healthy puppy at 30 days of age. 3. Discussion The occurrence of malformations in dogs is attributed to prenatal factors, related to genetic causes, which may be hereditary, or to maternal exposure to teratogenic agents during pregnancy, such as toxins, radiation, chemical agents, infectious diseases, mechanical influences or drugs . In the history and anamnesis of the cases described, there was no report of maternal exposure to teratogenic agents. However, in case 1, the kennel had a history of diagnosis of pectus excavatum in the litter of a female of the same family. Thus, the malformation of the French Bulldog puppy may be related to genetic factors of the kennel breed lineage. In humans and animals, approximately 40% of individuals with pectus excavatum have a first-degree member of the family affected by the deformity . Moreover, in dogs, it is known that the highest incidence of malformations, approximately 84.4%, is observed in purebred puppies and that brachycephalic breeds have a higher predisposition to manifest pectus excavatum, with a prevalence of 44% , indicating that this poor formation is related to genetic components in these breeds . In case 2, the neonate came from consanguineous mating between siblings of the same litter. The main effect of consanguinity (or inbreeding) is the loss of genetic variability and increased homozygosis, which can lead to the manifestation of deleterious genes. The consequence of the reproductive process with this degree of familial relationship is that both carry genes related to malformations, and when they mate, they increase the chance of transmission of these replicas to their young, with potential manifestation of these deformities in the litter . Therefore, as genetic defects can be inherited from one or both parents, being commonly described in purebred puppies , and pectus excavatum has a high prevalence in brachycephalic breeds, it is likely that the cause of malformation in the American Bully puppy is also related to genetic factors. In puppies, pectus excavatum is present at birth and is detectable immediately after delivery or in a few days . It is important to perform a thorough physical examination of newborn dogs for the presence of malformations and to guide the tutor or breeder in evaluating the presence of some abnormality in the inspection of litters. Early diagnosis is essential for rapid treatment and a greater chance of survival of patients, since this deformity may be progressive, with the worsening of clinical signs. Depression of the sternum bone in the pectus is identifiable and palpable by physical examination; however, it is important to perform imaging tests, as they help in the classification of the degree of impairment of the defect and in the diagnosis of cardiac and respiratory tract abnormalities associated with this malformation. For example, the globose aspect of the heart may be associated with cardiac compression caused by pectus excavatum, associated congenital heart diseases, or due to altered cardiac positioning, which may cause the heart to appear radiologically enlarged . Due to the cardiorespiratory alterations of the patients in this report, the continued evaluation by imaging tests, such as chest X-ray and echocardiogram, was recommended. An evident clinical sign in the pups of this report was substernal retraction during inspiration (retraction of the abdomen just below the sternum). Retractions can be observed in newborns and children, as they have a more flexible thorax than adults . Thoracic retractions indicate that the patient is having difficulty breathing (high intrathoracic pressure required during inspiration to expand the lungs), and the substernal type is associated with the degree of mild to moderate respiratory difficulty . It is important to include pectus excavatum as a differential diagnosis in neonatal puppies with dyspnea and substernal retractions, especially in brachycephalic dogs. In addition, because pectus excavatum is often underdiagnosed, pneumonia that does not respond adequately to treatment should be further investigated. Leukocytosis with left deviation associated with neutrophilia and slight absolute lymphocytosis was observed in the blood count of case 1. This alteration may be associated with the pneumonia presented by this puppy, associated with a possible secondary bacterial infection. The neonatal blood count should be interpreted based on the reference parameters for the age (in weeks) of the patients, as described by Bird, 2011 and Von Dehn, 2014 . Antibiotic therapy with ceftriaxone, a broad-spectrum beta-lactam drug considered safe for newborn dogs, was instituted. Cephalosporins and penicillins are frequently used classes of antibiotics recommended for the treatment of neonatal infections . The choice between conservative or surgical treatment can be dictated by the severity of the defect, age of the animal, stiffness of the costal arches and capacity of retraction of the sternal defect . The use of corrective splints as a noninvasive treatment was effective in the present cases (mild degrees) and is a feasible option to be performed in newborn dogs. This occurs due to the flexibility of the costal cartilages and sternum of the pups, which facilitates thoracic remodeling . Young animals with mild pectus excavatum may present a good response to the use of corrective thoracic bandages without surgical intervention and only with conservative management. However, the lateral-medial compression of the thorax should not be used in more severe cases, as it may exacerbate dorsal intrusion of the sternum into the thoracic cavity. In these cases, surgery is indicated ; an ostectomy of a section of the costal cartilages can be performed to allow the sternum to be realigned, and bone plaque can be used to keep the sternum in this position. Furthermore, splints with circular sutures on the sternum can be used. The sutures are tied to the splint to keep the thorax in a normal position and kept in place for three to four weeks . In a case report of pectus excavatum in an English Bulldog neonatal puppy, the use of the noninvasive PVC pipe splint was also effective, demonstrating the general clinical improvement of the patient, anatomical conformation of the thorax close to normal, and restoration of the appropriate respiratory pattern . However, the management of the box in the thorax, demonstrated in this report, is unprecedented and has not yet been described in the literature. Regarding the choice of which splint to use, although the two types are effective, in our routine neonatal care, we observed that puppies with intense respiratory distress benefit from the use of the box compared to the PVC pipe, since in the management of the pipe in the chest, some animals may become uncomfortable due to greater lateral compression. We suggest testing the management in which the puppy will become more comfortable, observing the absence of crying, restlessness or exacerbation of dyspnea. The length of stay with the splint is also variable; in our care routine, we already observe complete remodeling of the chest in two to eight days. For the removal of the splint, the thorax should be inspected daily, removing the splint to observe the absence of depth of the sternum region and improvement of the respiratory pattern. Because it is a hereditary malformation, it is not recommended that animals diagnosed with pectus excavatum be used for reproductive purposes, and spay/neuter is recommended, even if they do not show clinical signs . This information was emphasized to the case tutors, as well as oriented about avoiding the continuity of the parents in the reproductive process, recommending the spay or neuter of these and animals with a history of pectus excavatum in their litters. Congenital malformations have been diagnosed more frequently in newborn dogs . It is important that the veterinarian has knowledge about the clinical or surgical approach of malformations to indicate the best corrective conduct, as well as to explain to the tutor or breeder the possibility of managing/treating each defect. As preventive measures, care should be taken in the choice of parents, avoidance of inbreeding and animals with a history of genetic problems or defects, as well as maternal exposure to teratogenic factors during pregnancy. Prenatal care is essential to prevent malformations and reduce mortality rates in neonatal puppies . 4. Conclusions Management with corrective splints (PVC pipe and box) is effective for the conservative treatment of pectus excavatum in neonatal puppies, resulting in the remodeling of the thorax and improvement of the respiratory pattern. However, more severe cases may need corrective surgery. Each case should be evaluated individually. In the cases presented, pectus excavatum was probably related to genetic factors associated with racial predisposition. In dogs, this is a progressive condition with cardiorespiratory involvement and should be diagnosed early and treated as soon as possible to avoid the evolution of clinical signs and increase the chance of neonatal survival. Acknowledgments We thank Giovana Furquim and Julia Cristie da Silva for image concession. We thank the support of the Diagnostic Imaging sector of School of Veterinary Medicine and Animal Science, Sao Paulo State University (Unesp), Botucatu, Brazil. Supplementary Materials The following supporting information can be downloaded at: Video S1: Neonate with pectus excavatum presenting dyspnea and substernal retraction during inspiration; Video S2: Neonate presenting respiratory pattern without alterations, with absence of substernal retraction during inspiration after management of the pipe splint; Video S3: Neonate with pectus excavatum presenting dyspnea and substernal retraction during inspiration; Video S4: Neonate presenting regular respiratory pattern, with absence of substernal retraction during inspiration after management of the splint of the box. Click here for additional data file. Author Contributions Conceptualization, investigation, writing--original draft preparation, writing--review and editing, K.H.N.P.P., K.d.M.F., L.A.A.T. and M.L.G.L., methodology, software, validation, formal analysis, visualization, K.H.N.P.P., K.d.M.F., L.A.A.T., R.C.S., G.d.A.C., J.C.M., N.T.P., M.A.Z., E.O. and M.L.G.L.; supervision, resources, E.O. and M.L.G.L. All authors have read and agreed to the published version of the manuscript. Institutional Review Board Statement Ethical review and approval were waived for this study because the present case report is based on retrospective analysis of medical records of clients' dogs presented for routine clinical consultation assessments. Informed Consent Statement The owners of the present case report signed informed consent forms for the authorization of the use of personal data and privacy notices. Data Availability Statement Not applicable. Conflicts of Interest The authors declare no conflict of interest. Figure 1 Frontosagittal and vertebral thoracic indices. The frontosagittal index is the ratio between the width of the thorax at the height of the tenth thoracic vertebra (a) and the distance between the center of the ventral surface of the tenth thoracic vertebra and the closest point to the sternum (b). The vertebral index is the ratio between the distance from the center of the dorsal surface of the selected vertebra to the nearest point of the sternum (b) and the dorsoventral diameter of the center of the same vertebra (c) (Adamovich-Rippie figure; Culp, 2016 ). Figure 2 Chest X-rays of neonatal puppies of brachycephalic breeds. (A) Chest without alterations, indices--frontosagittal: 1.14 cm; vertebral: 13.4 cm. (B) Pectus excavatum of mild degree (arrow), indices--frontosagittal: 2 cm; vertebral: 11.25 cm. (C) Pectus excavatum of moderate degree (arrow), indices--frontosagittal: 3 cm; vertebral: 6 cm. (D) Severe pectus excavatum (arrow), indices--frontosagittal: 3.14 cm; vertebral: 5.8 cm. Figure 3 Chest X-ray. (A) Right side position and (B) left side position, demonstrating ventrodorsal deviation of the sternum bone (arrows); (C) Ventrodorsal position demonstrating narrowing of the thorax (arrows), pneumonia and left cardiac deviation. Figure 4 Normal thoracic radiographic examination. (A) Right side position; (B) Left side position; (C) Ventrodorsal position. Figure 5 (A) PVC pipe used for making the splint; (B) Ventral space (2 cm) necessary for remodeling the thorax (arrow) and use of cotton to upholstery the sides of the splint. Figure 6 (A) Ventral view of the splint in the patient; (B) Dorsal view of the splint in the patient. Figure 7 Patient nursing during splint use. Figure 8 (A) Patient before splint management, presenting chest depression in the sternum bone region (arrow). (B) Patient after splint removal presenting chest with the absence of ventrodorsal narrowing in the sternal region (arrow) after noninvasive treatment. Figure 9 Puppy at two months old. Figure 10 Ventrodorsal narrowing in the sternal region (arrow). Figure 11 Chest X-ray. (A) Right lateral position, demonstrating ventrodorsal deviation of the sternum bone (arrow). (B) Ventrodorsal position, demonstrating rounded cardiac silhouette and right cardiac deviation. Figure 12 Box management. (A) Resistant paper box with space to insert and immobilize the previous members. (B) Puppy immobilized in the lateral decubitus position. Figure 13 Patient after splint removal. Thorax presenting absence of ventrodorsal narrowing in the sternal region (arrow) after noninvasive treatment. Figure 14 Puppy at 30 days old. animals-13-00906-t001_Table 1 Table 1 Frontosagittal and vertebral thoracic indices in dogs of nonbrachycephalic breeds, brachycephalic breeds and cats . Indices Nonbrachycephalic Breeds Brachycephalic Breeds Cats Frontosagittal (cm) 0.8-1.4 1-1.5 0.7-1.3 Vertebral (cm) 11.8-19.6 12.5-16.5 12.6-18.8 animals-13-00906-t002_Table 2 Table 2 Classification of pectus excavatum in dogs and cats based on frontosagittal and vertebral thoracic indices . Pectus Excavatum Indices (cm) Frontosagittal Vertebral Mild <=2 >9 Moderate 2-3 6-8.99 Severe >3 <6 Disclaimer/Publisher's Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

PMC10000131

To investigate the association between skeletal muscle mass and adiposity measures with disease-free progression (DFS) and overall survival (OS) in patients with advanced lung cancer receiving immunotherapy, we retrospectively analysed 97 patients (age: 67.5 +- 10.2 years) with lung cancer who were treated with immunotherapy between March 2014 and June 2019. From computed tomography scans, we assessed the radiological measures of skeletal muscle mass, and intramuscular, subcutaneous and visceral adipose tissue at the third lumbar vertebra. Patients were divided into two groups based on specific or median values at baseline and changes throughout treatment. A total number of 96 patients (99.0%) had disease progression (median of 11.3 months) and died (median of 15.4 months) during follow-up. Increases of 10% in intramuscular adipose tissue were significantly associated with DFS (HR: 0.60, 95% CI: 0.38 to 0.95) and OS (HR: 0.60, 95% CI: 0.37 to 0.95), while increases of 10% in subcutaneous adipose tissue were associated with DFS (HR: 0.59, 95% CI: 0.36 to 0.95). These results indicate that, although muscle mass and visceral adipose tissue were not associated with DFS or OS, changes in intramuscular and subcutaneous adipose tissue can predict immunotherapy clinical outcomes in patients with advanced lung cancer. immunotherapy cancer muscle mass fat mass overall survival No financial support was received to conduct the present study, or for the preparation or publication of this manuscript. pmc1. Introduction In recent years, immune checkpoint inhibitors (ICIs) or immunotherapies, such as nivolumab, pembrolizumab and ipilimumab, have evolved rapidly in medical oncology. The utilisation of ICIs has become a key component for managing a variety of malignancies including lung cancer, resulting in an unprecedented survival advantage over standard therapies such as radiation therapy and chemotherapy. While chemotherapy acts directly on cancer cells inhibiting the cell cycle, ICIs are antibodies targeting the programmed death 1 (PD-1), programmed death-ligand (PD-L1) or cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), blocking key regulatory signals that dampen immune responses in the tumour microenvironment. As a result, ICIs counteract immune suppression allowing for tumour reactive T cells to mount an antitumour response utilising the patient's immune system to target the malignancy . These therapies have shown promising effects in the treatment of lung cancer, as well as a selection of other solid tumours and haematologic malignancies . Several studies have pointed out a significant relationship between immunotherapy and multiple variables on overall survival. Among potential factors, sarcopenia (i.e., progressive and generalised loss of skeletal muscle mass ) has emerged as an important prognostic factor in different groups of cancer patients . However, the relationship between sarcopenia and overall survival in patients treated with immunotherapy is still unclear . While studies present a significant association between sarcopenia and shorter overall survival , others have no significant relationship . For example, in a previous study with small-cell lung cancer patients receiving salvage anti-PD-1 immunotherapy (n = 105), patients presenting with low levels of muscle mass (i.e., sarcopenic patients) had a ~200% greater risk of all-cause mortality compared to those with higher levels of muscle mass . In contrast, there was no difference in overall survival between sarcopenic and non-sarcopenic patients with solid metastatic tumours treated with ICIs (n = 261) . Moreover, the prognostic value of obesity in various malignancies is unknown and remains controversial for the survival of various malignancies . Although previous studies indicated a potential association between body mass index (BMI) and overall survival in advanced cancer patients treated with immunotherapy , others have demonstrated no significant association between BMI and clinical endpoints . These conflicting results, potentially related to the obesity paradox (i.e., inconsistency concerning the role of obesity on survival), preclude us from understanding the role of fat mass components (i.e., visceral adipose tissue or subcutaneous adipose tissue) on survival in this population . For example, while visceral adipose tissue (VAT) secretes various cytokines and cytokine-like factors, which potentially enhance cancer progression , derived factors from the subcutaneous adipose tissue can increase insulin sensitivity and lipid metabolism potentially resulting in an improved survival . Therefore, although BMI is a much simpler and widely used tool in clinical practice, it does not reflect individual components of body weight such as fat distribution or muscle quantity and quality. As a result, this study aims to investigate the associations between measures of skeletal muscle mass, intramuscular adipose tissue, subcutaneous adipose tissue, visceral adipose tissue and visceral-to-subcutaneous adipose tissue index and changes throughout treatment with disease progression and overall survival in patients with advanced lung cancer receiving immunotherapy. 2. Materials and Methods 2.1. Study Population Retrospective analyses of computerised tomography (CT) imaging and electronic medical record data were performed for all patients treated with immunotherapy who presented to Fiona Stanley Hospital, Western Australia between March 2014 and June 2019. A total of 124 patients with lung cancer were identified on immunotherapy. Patients without CT imaging data were excluded from the final cohort, resulting in a total of 97 patients included for further analyses. Demographic, pathological and survival information were obtained via electronic medical record review. The duration of follow-up was 60 months from the first presentation to the date of death for deceased patients or the date of last documented encounter for surviving patients. Demographic and clinical data such as sex, age, BMI, smoking habits, Eastern Cooperative Oncology Group (ECOG) performance status (PS), distant metastases, cancer type, treatment regimens, progression-free survival (PFS) and overall survival (OS) were collected by self-report and medical records, respectively. Our study was approved by the Hospital Ethics Committee (RGS0000003289) and conducted in compliance with the Helsinki Declaration. 2.2. Assessment of Muscle Mass and Fat Mass Parameters CT scans were at a median of 20 [interquartile range (IQR): 8 to 31] days before commencing immunotherapy treatment. CT scans of the abdomen/pelvis were performed as part of recommended staging pathway and retrieved from the hospital imaging PACS/RIS system (version 6.7.0.6011; Agfa, Mortsel, Belgium). A single 3 mm axial slice through the middle of the L3 vertebral body was retrieved using the sagittal reformatted images with the morphologic L5/S1 junction as reference. These images were imported into SliceOmatic (version 5.0 Rev 12; TomoVision, Magog, QC, Canada) and analysed using the ABACS mode (version 6 Rev-7b; Voronoi Health Analytics, Coquitlam, BC, Canada). If there was an artifact at this level, the nearest artifact-free contiguous slice above or below this level was utilised. A visual colour-coded overlay was reviewed to assess for correct segmentation; any errors were manually corrected using Edit mode and following standard anatomic boundaries. Area measurements (cm2) were obtained by auto-segmentation using the default Hounsfield unit (HU) thresholds and skeletal muscle was determined in the range of -29 to 150 HU, including the skeletal muscle compartment of psoas, paraspinal and abdominal wall musculature. Intramuscular adipose tissue (IMAT) was determined in the range of -190 to -30 HU, visceral adipose tissue (VAT) in the range of -150 to -50 HU and subcutaneous adipose tissue (SAT) in the range of -190 to -30 HU. Visceral-to-subcutaneous adipose tissue ratio was defined as the ratio between VAT and SAT values. Values were normalised to height squared (m2) to derive skeletal muscle, IMAT, VAT, SAT and VAT/SAT indexes. For further analysis, the skeletal muscle index was analysed as a categorical variable with two levels corresponding to sarcopenia (skeletal muscle index < 43 cm2*m-2 and BMI < 25 kg*m-2, or skeletal muscle index < 53 cm2*m-2 and BMI >= 25 kg*m-2) and non-sarcopenia (skeletal muscle index >= 43 cm2*m-2 and BMI >= 25 kg*m-2, or skeletal muscle index >= 53 cm2*m-2 and BMI < 25 kg*m-2), as previously established . Considering the lack of cut-off values for adiposity measures, median values based on our sample were used to categorise patients with higher and lower levels of IMAT, VAT, SAT and VAT/SAT indexes. Relative changes (%) were calculated as indexfollow-upindexbaseline*100%, with a threshold of 10% utilised to categorise groups with the lowest and highest index changes throughout treatment. 2.3. Assessment of Outcomes The primary outcome was overall survival, defined as deaths as a result of any cause, while disease progression defined as an increase in the size of the tumour by 20% was secondary. Vital causes and causes of death were obtained via electronic medical record review. Follow-up time for overall mortality was calculated as the time from CT scans to death from any cause or the end of follow-up (i.e., 60 months following the time of the first scan). 2.4. Statistical Analyses Analyses were performed using SPSS v.27 (Armonk, IBM Corp., NY, USA) and R Core Team (2013). Differences in overall mortality between groups based on sarcopenia, IMAT, SAT, VAT and VAT/SAT variables were assessed using the Kaplan-Meier method and the log-rank test. Paired-sample t-test was used to compare values between the first and second CT scans during immunotherapy. The hazard ratios (HRs) for the associations of skeletal muscle index, IMAT, SAT, VAT and VAT/SAT ratio indexes with overall mortality and disease progression were estimated in separate models using Cox proportional hazards regression. Logistic regression was used to determine the impact of body composition components on the occurrence of adverse events >= grade 2. Odds ratios (ORs) and 95% CIs were reported. Models were adjusted for age, BMI, cancer type and stage. A p-value of <=0.05 was considered statistically significant and point estimates were presented with 95% confidence interval. 3. Results 3.1. Patient Characteristics Patient characteristics are presented in Table 1. Patients were 67.5 +- 10.2 years of age (mean +- standard deviation) with a BMI of 26.1 +- 4.9 kg*m-2. Most patients were overweight/obese (60.8%). The majority of patients had adenocarcinoma (62.9%), followed by squamous cell carcinoma (29.9%). Most patients were treated with second line immunotherapy (75.3%). A total of 81 patients were stage IV (84.4%) and had metastatic disease present in more than two sites (22.9%), bone (17.1%), lymph node (8.6%), liver (5.7%), adrenal (2.9%) and brain (2.9%). In this cohort, the most common immunotherapy agent was Nivolumab (58.8%), followed by Pembrolizumab (24.7%) and Atezolumab (16.5%). A total number of 96 patients had disease progression and died during follow-up (99.0%), with median disease progression of 11.3 (IQR: 4.9 to 20.4) months and 15.4 (IQR: 7.2 to 24.0) months, respectively. 3.2. Association of Body Composition Components with Disease Progression and Overall Survival The median IMAT, SAT, VAT and VAT/SAT ratio index values were 3.85, 55.43, 41.90 and 0.74 cm2*m-2, respectively. Multivariable models indicated no significant associations of sarcopenia, IMAT, SAT, VAT and VAT/SAT ratio indexes at baseline with 5-year disease progression (HR: 0.69-1.25, p = 0.199-0.877) and 5-year overall survival (HR: 0.69-1.34, p = 0.123-0.724) in patients with advanced lung cancer undergoing immunotherapy (Table 2). Kaplan-Meier analyses stratifying patients according to body composition components cut-off values on 5-year disease progression and overall survival are presented in Figure 1 and Figure 2, respectively (p = 0.061-0.606). A second CT scan was performed in 88 patients as presented in Table 3. Changes in skeletal muscle, IMAT, SAT, VAT and VAT/SAT ratio indexes were not statistically significant following a median time of 15.4 months after the first CT scan (IQR: 7.1 to 26.5 days). Although changes in sarcopenia, VAT and VAT/SAT ratio indexes were not associated with 5-year disease progression (HR: 0.63-1.24, p = 0.064-0.484), >10% increases in IMAT (HR: 0.60, 95% CI: 0.38 to 0.95) and SAT indexes (HR: 0.59, 95% CI: 0.36 to 0.95) were associated with improved 5-year disease progression (p = 0.028 and 0.029; Table 4). Patients with a >10% increase in IMAT index presented a median disease progression of 15.9 (IQR: 8.8 to 24.6) months vs. 11.7 (IQR: 5.5 to 19.0) months in patients with a <=10% decrease in IMAT index (Kaplan-Meier Log-Rank, kh2 = 4.2, p = 0.042). Likewise, patients who had a >10% increase in SAT index presented a median disease progression of 16.9 (IQR: 10.8 to 29.6) months vs. 10.2 (IQR: 4.7 to 18.8) months of patients who had a decrease in SAT index (Kaplan-Meier Log-Rank, kh2 = 5.3, p = 0.022). Kaplan-Meier analysis on 5-year disease progression is presented in Figure 3. Regarding overall survival, a >10% increase in IMAT was associated with improved 5-year overall survival (HR: 0.60, 95% CI: 0.37 to 0.95, p = 0.031; Table 5). Patients who had an increase of 10% in IMAT presented a median overall survival of 17.8 (IQR: 9.6 to 27.9) months vs. 15.5 (IQR: 8.5 to 23.2) months of patients who had a decrease in this outcome (Kaplan-Meier Log-Rank, kh2 = 3.4, p = 0.067). Kaplan-Meier analysis on 5-year overall survival is presented in Figure 4. 3.3. Association of Body Composition Components with Immune-Related Adverse Events Thirty-six adverse events (43.4%) were observed during immunotherapy. Of these, a total of 11 grade 2 (13.3%) and 5 grade 3 events (6.0%) were observed. No associations were observed between sarcopenia, IMAT, SAT, VAT and VAT/SAT ratio indexes with high-grade adverse events during immunotherapy (OR: 0.95-2.00, p = 0.279-0.947). 4. Discussion The present study reported the associations between radiological measures of muscle and adipose tissue with disease progression and overall survival in patients with advanced lung cancer receiving immunotherapy. The main findings were: (i) muscle mass index at the time of or during immunotherapy was not associated with disease progression or overall survival; and (ii) patients with lung cancer presenting with increases of 10% in intramuscular and subcutaneous adipose tissue following treatment were at a ~40% decreased risk of disease progression and overall survival compared to those presenting with lower levels, regardless of age, BMI, cancer type and stage. The significant association of sarcopenia with poor disease prognosis has been observed in several papers across different types of cancer . Interestingly, the majority of studies reporting such findings in the field of immunotherapy were undertaken in patients with lung cancer . As far as we know, this is one of the few studies undertaken in patients with lung cancer mainly with adenocarcinoma and squamous cell carcinoma (~93% of the sample). Our study indicates that sarcopenia is not significantly associated with disease progression or overall survival in this population with advanced cancer receiving immunotherapy. As observed in our results, the presence of sarcopenia at the start of immunotherapy or a reduction of 10% in skeletal muscle mass index were not associated with disease progression and mortality. However, this result disagrees with previous studies undertaken in patients mainly with non-squamous lung cancer , which indicate that tumour histology may affect the interaction between sarcopenia and immunotherapy in patients with advanced lung cancer. Nevertheless, lower levels of muscle mass may still affect other important components of immunotherapy such as inflammation, cachexia and physical disability. Consequently, more research is required to elucidate the importance of sarcopenia for other important clinical measures. The investigation of obesity in immunotherapy is challenging given the confounding factors associated with the obesity paradox and its role in cancer dynamics . We observed that intramuscular and subcutaneous adipose tissue could be a predictive marker for improved survival when increased throughout the treatment course. The subcutaneous adipose tissue derives a range of factors such as leptin that could act to improve insulin sensitivity and lipid metabolism . As a result, this could potentially increase overall survival in this group of patients and represent an important measure during the cancer survivorship. However, the result that an increase in intramuscular adipose tissue could improve survival was unexpected. While previous studies identified a significant association of intramuscular adipose tissue with shorter survival in women with non-metastatic breast cancer and men with hormone-sensitive prostate cancer , others did not observe a significant association in metastatic breast cancer or advanced non-small-cell lung cancer treated with immunotherapy . Moreover, previous studies have demonstrated that increased intramuscular fat is related to increased frailty and sarcopenia and impaired physical function . In addition, others indicate that increased intramuscular fat is associated with poor survival and increased risk of hospitalisation in older adults or critically ill patients . Therefore, the interaction between intramuscular fat and immunotherapy is yet to be determined in this setting. Interestingly, we also observed an unexpectedly longer 5-year disease progression compared to other large immunotherapy randomised controlled studies . While we observed a median disease progression time of 11.3 months, a range of 3.0 to 5.0 months was reported in these trials . The reasons are likely multifactorial and related to our smaller sample size and retrospective nature compared to these large randomised controlled trials . Additionally, we observed high PDL1% values in our sample (median of 60%) and this may also account to a long disease progression as PDL1% is associated with improved survival even when using monotherapy agents in advanced non-small-cell lung cancer. Other factors such as mixed cancer stages (~16% stage III) and treatment line (~25% first treatment line) are different than these previous immunotherapy trials and may affect disease progression. Our cohort also presented more favourable histology (i.e., adenocarcinoma) and tumour burden may be different as 40% did not present distant metastasis. These factors may play a role in disease progression. Some limitations are worthy of comment. The retrospective nature of the study and the heterogeneity of CT scans may limit our ability to extrapolate our findings to a large scale. Future studies should undertake prospective models to assess the influence of body composition changes on clinical endpoints, as well as reporting the time of body composition assessment. In addition, the lack of standardisation (i.e., cut-off values), and this is due to variability in underlying technique without clear standardisation, makes comparison difficult to assess radiological measures of muscle and adipose tissue and affects our ability to provide more meaningful recommendations based on our findings. Although the use of body composition is promising, critical and technical studies are required to understand the relationship of sarcopenia with clinical endpoints and to inform specific and tailored interventions in patients treated with immunotherapy. Finally, we could not estimate the impact of sarcopenic obesity in our sample. This is an emergent topic in oncology given the high risk of mortality and severe complications experienced by patients during systemic and surgical cancer treatments. Future studies are required to investigate the impact of sarcopenic obesity in lung cancer patients during immunotherapy and identify clinical management strategies for this population. 5. Conclusions In conclusion, our findings are that rather than muscle mass and visceral adipose tissue, changes in intramuscular and subcutaneous adipose tissue can predict immunotherapy clinical outcomes regardless of age, BMI, cancer type and stage. This result provides new insights into the assessment of body composition in patients with advanced lung cancer undergoing immunotherapy. Consequently, future research should seek to assess a larger sample size of patients undergoing immunotherapy to further elucidate the influence of body composition, specifically monitoring intramuscular and subcutaneous adipose tissues. Acknowledgments Pedro Lopez is supported by the National Health and Medical Research Council (NHMRC) Centre of Research Excellence (CRE) in Prostate Cancer Survivorship Scholarship. We also acknowledge the Fiona Stanley Hospital Clinic Trials Unit for supporting the imaging software Sliceomatic/ABACS. Author Contributions Conceptualization, A.K. (Azim Khan) and A.K. (Adnan Khattak); methodology, A.K. (Azim Khan), C.J.W., P.L., F.S. and A.K. (Adnan Khattak); formal analysis, A.K. (Adnan Khattak), C.J.W., P.L. and F.S.; data curation, A.K. (Azim Khan), C.J.W., A.A., S.O., A.R., S.A., P.L., F.S. and A.K. (Adnan Khattak); writing--original draft preparation, A.K. (Azim Khan), C.J.W., A.A., S.O., A.R., S.A., P.L., F.S. and A.K. (Adnan Khattak); writing--review and editing, A.K. (Azim Khan), C.J.W., A.A., S.O., A.R., S.A., P.L., F.S. and A.K. (Adnan Khattak). All authors have read and agreed to the published version of the manuscript. Institutional Review Board Statement The study was conducted in accordance with the Declaration of Helsinki, and approved by the Hospital Ethics Committee of Fiona Stanley Hospital (RGS0000003289). Informed Consent Statement Informed consent was obtained from all subjects involved in the study. Data Availability Statement The data used in the present study will be made available upon request to the corresponding author. Conflicts of Interest The authors declare no conflict of interest. Role of funding source Sponsors were not involved in the study design, analysis or interpretation of data, manuscript writing and decision to submit the manuscript for publication. Figure 1 Kaplan-Meier curves of 5-year disease progression according to (A) sarcopenia, (B) intramuscular adipose tissue, (C) subcutaneous adipose tissue, (D) visceral adipose tissue and (E) visceral-to-subcutaneous adipose tissue at baseline in patients with advanced lung cancer undergoing immunotherapy. Figure 2 Kaplan-Meier curves of 5-year overall survival according to (A) sarcopenia, (B) intramuscular adipose tissue, (C) subcutaneous adipose tissue, (D) visceral adipose tissue and (E) visceral-to-subcutaneous adipose tissue at baseline in patients with advanced lung cancer undergoing immunotherapy. Figure 3 Kaplan-Meier curves of 5-year disease progression according to changes in (A) sarcopenia, (B) intramuscular adipose tissue, (C) subcutaneous adipose tissue, (D) visceral adipose tissue and (E) visceral-to-subcutaneous adipose tissue in patients with advanced lung cancer undergoing immunotherapy. Figure 4 Kaplan-Meier curves of 5-year overall survival according to changes in (A) sarcopenia, (B) intramuscular adipose tissue, (C) subcutaneous adipose tissue, (D) visceral adipose tissue and (E) visceral-to-subcutaneous adipose tissue in patients with advanced lung cancer undergoing immunotherapy. cancers-15-01382-t001_Table 1 Table 1 Characteristics of patients with lung cancer. Characteristics Patients (n = 97) Age, mean +- SD, years 67.5 +- 10.2 Males, n (%) 55 (56.7%) Height, mean +- SD, cm 168.1 +- 8.7 Weight, mean +- SD, kg 73.8 +- 15.6 BMI, mean +- SD, kg*m-2 26.1 +- 4.9 BMI, categories, n (%) Underweight 4 (4.1%) Normal weight 34 (35.1%) Overweight 43 (44.3%) Obese 16 (16.5%) Current smoker, n (%) a 74 (83.1%) Tumour type, n (%) Adenocarcinoma 61 (62.9%) Adeno-squamous 1 (1.0%) Large cell carcinoma 2 (2.1%) Poorly differentiated 3 (3.1%) Pleomorphic 1 (1.0%) Squamous cell carcinoma 29 (29.9%) Cancer stage, n (%) a III 15 (15.6%) IV 81 (84.4%) Metastasis location, n (%) a No metastasis 14 (40%) Adrenal 1 (2.9%) Bone 6 (17.1%) Brain 1 (2.9%) Liver 2 (5.7%) Lymph node 3 (8.6%) >=2 sites 8 (22.9%) Treatment line, n (%) First line treatment 24 (24.7%) Second line treatment 73 (75.3%) Treatment, n (%) Atezolumab 16 (16.5%) Nivolumab 57 (58.8%) Pembrolizumab 24 (24.7%) ECOG, n (%) a 0 49 (51.0%) 1 34 (35.5%) 2 12 (12.5%) 3 1 (1.0%) Neutrophils, mean +- SD 6.6 +- 4.4 Lymphocytes, mean +- SD 1.5 +- 0.8 Haemoglobin, mean +- SD 125.2 +- 20.4 Platelets, mean +- SD 306.1 +- 122.0 Neutrophils-to-lymphocytes ratio, mean +- SD 6.9 +- 11.6 PD-L1 levels, median (IQR), % 60.0 (1.0 to 80.0) BMI, Body mass index; ECOG, Eastern Cooperative Oncology Group performance status; IQR, interquartile range; SD, standard deviation; a, missing data: current smoker, n = 8; cancer stage, n = 1; TNM, n = 16; metastasis location, n = 62; ECOG, n = 1. cancers-15-01382-t002_Table 2 Table 2 Hazard ratios and 95% confidence intervals for associations between measures of skeletal muscle, intramuscular adipose tissue, subcutaneous adipose tissue, visceral adipose tissue and visceral-to-subcutaneous adipose tissue indexes and 5-year risk of disease progression and overall survival. Variables N 5-Year Disease Progression 5-Year Overall Survival HR (95% CI) p-Value HR (95% CI) p-Value Sarcopenia Non-sarcopenic 44 Reference Reference Sarcopenic 53 1.22 (0.81 to 1.85) 0.338 1.29 (0.85 to 1.95) 0.227 Intramuscular adipose tissue index <=3.85 cm2*m-2 49 Reference Reference >3.85 cm2*m-2 48 0.83 (0.51 to 1.33) 0.430 0.88 (0.54 to 1.42) 0.594 Subcutaneous adipose tissue index <=55.43 cm2*m-2 49 Reference Reference >55.43 cm2*m-2 48 0.69 (0.43 to 1.10) 0.199 0.69 (0.43 to 1.10) 0.123 Visceral adipose tissue index <=41.90 cm2*m-2 49 Reference Reference >41.90 cm2*m-2 48 0.96 (0.58 to 1.60) 0.877 1.10 (0.66 to 1.83) 0.724 Visceral-to-Subcutaneous adipose tissue index <=0.74 cm2*m-2 48 Reference Reference >0.74 cm2*m-2 49 1.25 (0.82 to 1.90) 0.305 1.34 (0.88 to 2.04) 0.178 All models were adjusted for age, BMI, cancer type and cancer stage. 95% CI, 95% confidence intervals; HR, hazard ratio. cancers-15-01382-t003_Table 3 Table 3 Changes in skeletal muscle, intramuscular adipose tissue, subcutaneous adipose tissue, visceral adipose tissue and visceral-to-subcutaneous adipose tissue indexes during immunotherapy. Outcomes n Baseline Post-Assessment Mean Difference Mean +- SD Mean +- SD Mean +- SD (95% CI) Skeletal muscle index, cm2*m-2 88 43.8 +- 8.5 43.7 +- 9.4 -0.1 +- 6.6 (-1.5 to 1.3) Intramuscular adipose tissue index, cm2*m-2 88 4.5 +- 2.3 4.7 +- 2.2 0.2 +- 1.3 (-0.1 to 0.5) Subcutaneous adipose tissue index, cm2*m-2 88 64.2 +- 36.5 65.5 +- 38.6 1.3 +- 17.5 (-2.4 to 5.0) Visceral adipose tissue index, cm2*m-2 88 52.5 +- 34.7 54.6 +- 36.8 2.1 +- 18.9 (-2.0 to 6.2) Visceral-to-subcutaneous adipose tissue ratio, cm2*m-2 88 1.0 +- 0.7 1.0 +- 0.7 0.0 +- 0.3 (-0.1 to 0.1) cancers-15-01382-t004_Table 4 Table 4 Hazard ratios and 95% confidence intervals for associations between changes in measures of skeletal muscle, intramuscular adipose tissue, subcutaneous adipose tissue, visceral adipose tissue and visceral-to-subcutaneous adipose tissue indexes and 5-year risk of disease progression and overall survival. Variables N 5-Year Disease Progression 5-Year Overall Survival HR (95% CI) p-Value HR (95% CI) p-Value Sarcopenia >10% of change 74 Reference Reference <=10% of change 14 1.24 (0.68 to 2.23) 0.484 1.39 (0.77 to 2.52) 0.277 Intramuscular adipose tissue index <=10% of change 52 Reference Reference >10% of change 36 0.60 (0.38 to 0.95) 0.028 0.60 (0.37 to 0.95) 0.031 Subcutaneous adipose tissue index <=10% of change 61 Reference Reference >10% of change 27 0.59 (0.36 to 0.95) 0.029 0.64 (0.39 to 1.03) 0.066 Visceral adipose tissue index <=10% of change 48 Reference Reference >10% of change 36 0.66 (0.42 to 1.04) 0.075 0.67 (0.43 to 1.07) 0.093 Visceral-to-Subcutaneous adipose tissue index <=10% of change 50 Reference Reference >10% of change 34 0.63 (0.39 to 1.03) 0.064 0.61 (0.38 to 0.99) 0.045 All models were adjusted for age, BMI, cancer type and cancer stage. cancers-15-01382-t005_Table 5 Table 5 Odds ratio and 95% confidence intervals for associations between measures of skeletal muscle, intramuscular adipose tissue, subcutaneous adipose tissue, visceral adipose tissue and visceral-to-subcutaneous adipose tissue indexes and risk of treatment toxicity. Variables N Treatment Toxicity OR 95% CI p-Value Sarcopenia Non-sarcopenic 44 Reference Sarcopenic 53 2.00 0.59 to 7.66 0.279 Intramuscular adipose tissue index <=3.85 49 Reference >3.85 48 0.95 0.21 to 4.22 0.947 Subcutaneous adipose tissue index <=55.43 49 Reference >55.43 48 1.23 0.28 to 5.48 0.782 Visceral adipose tissue index <=41.90 49 Reference >41.90 48 1.35 0.30 to 6.50 0.696 Visceral-to-Subcutaneous adipose tissue index <=0.74 48 Reference >0.74 49 1.57 0.44 to 5.95 0.490 All models were adjusted for age, BMI, cancer type and cancer stage. Disclaimer/Publisher's Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

PMC10000132

We studied the activity budgets of seven Ailurus fulgens, at three zoos within Australasia, using video cameras, and in-person observations. Red panda in this study followed a crepuscular activity pattern, with another short peak of activity around midnight. Ambient temperature greatly affected panda activity patterns; red panda allocated more time to resting and sleeping when temperatures increased. This preliminary study suggests how environmental factors affect captive red panda, which will help better inform captive facilities, and how this might impact their wild conspecifics. activity budgets temperature effects ex situ management This research received no external funding and was internally funded through Lincoln University. pmc1. Introduction Red panda (Ailurus fulgens) are currently listed as an endangered species under the IUCN Red List and Appendix I in CITES . Numbers are estimated to be between 10,000 and 15,000, but due to a lack of ongoing monitoring and an elusive nature, these numbers could be as low as a few thousand . Red panda are found along the Himalayan Mountain range across Nepal, Bhutan, the northern rim of India, Myanmar, and three Chinese provinces: Sichuan, Yunnan, and Tibet . Red panda have proven to be an extremely popular exhibit animal. They are charismatic, easy to work with, and require relatively little floor space, and their primary food source grows almost anywhere . In 1977, the international studbook for red panda was created . Individuals that are part of the cooperative worldwide breeding program are accounted for, and their genetics are managed. In 2009, a species survival plan (SSP) was implemented by the AZA (Association of Zoos and Aquariums) small carnivore taxon advisory group (TAG). Captive populations appear strong in number, with ~800 genetically managed individuals worldwide , and ~500 unmanaged in China . However, fertility and mortality rates differ significantly across the globe. Mortality rates of cubs in Australasia are around 10%, while in North America they may reach up to 40% . Red panda were long considered to contain two sub-species, Ailurus fulgens and Ailurus styani/refulgens. However, recent genome sequencing by Hu et al. showed that they are actually two species, undergoing a bottleneck after the Penultimate Glaciation (194,000-135,000 years ago). Both species are high-altitude specialists (1400-4800 m) that find temperatures above 24 degC difficult and require protection when mean temperatures exceed 27 degC . Typically, the slightly larger Ailurus styani is found at lower altitudes, 1400-3400 m, in the southern Sichuan region of China , while the smaller A. fulgens is found between 1500 and 4800 m. The majority of A. fulgens habitat is found around 3000 m, with bamboo being a key factor for their distribution . Wild red panda activity budgets have mainly been studied in Chinese reserves and show a diurnal to crepuscular activity pattern, with less movement around midday . Yonzon and Hunter studied A. fulgens in Nepal and found consistent crepuscular patterns, which may have been affected by human and livestock use of the same areas and resources. Wei and Zhang argue that while it seems that captive red panda follow a more crepuscular lifestyle, this is due to keeper movements and feeding times. The aim of this study was to assess what factors may affect captive red panda behaviour and activity patterns. As two observational methods were used, we also aimed to see if there are any major differences in behaviours recorded between the two types. 2. Materials and Methods 2.1. Study Animals The research was conducted at three zoos in New Zealand and Australia (Table 1). In 2018, 23 zoos within Australasia were part of the Red Panda Species Protection Plan (SPP); 17 of them housed 51 red panda (all captive-born), with the other 5 planning to house red panda in the near future. This study follows 7 out of the 51 panda (13.7% of the population) currently (2018) housed in Australasian zoos. Two of the panda in this study, Chito and Pasang, were on an anti-inflammatory, once a day for their joints, due to their age. 2.2. Observation Methods During public opening hours, red panda were observed for 1.5 h, twice a day, by the same observer to avoid inter-observer biases. Observation times were varied throughout the day to construct a general picture of daily behaviour. We developed an ethogram (Table 2) and recorded every behavioural occurrence for all panda over 10 min periods. The behaviours were decided upon both before and during observations, which only one person conducted. We modified behaviour descriptions from other red panda studies . A definition of scent marking was created following Conover and Gittleman . There was a 5 min break between each 10 min observation period. This was an all occurrences, interval sampling technique, as behaviours could happen at any time during the observations, and the length of behaviours may be cut off by the end of the interval. Also included in the observations was the time of day that the behaviour occurred, current weather conditions, keeper presence, which panda performed the behaviour, and the total length of the behaviour in seconds (to the nearest 10 s). Video cameras were also used to supplement observations of red panda behaviour. A Blackeye camera (model: BE2-W) (Kinopta, Porirua, New Zealand) was set up outside each enclosure to record behaviour for the entire trial. At Auckland Zoo, this camera was on the enclosure's back wall, only accessible with a keeper escort, and showed the main latrine site, feeding area, and walkways. Because of the size of the enclosure at Hamilton Zoo, much of it was out of view of the camera, which necessitated a focal area to be chosen. This area encompassed a large tower, many aerial walkways, and a latrine site. The camera was set up on a "staff only" path. The camera could be checked every morning for battery life, and batteries swapped as needed. CWS (Currumbin Wildlife Sanctuary) was also placed on an easy-to-reach perimeter fence so that it could be checked daily. This camera focused on the front platform where the panda slept and where most of the aerial walkways were. Apart from CWS camera footage, which was able to view the entire enclosure and was always recording, all other observations are a proportion of the time spent viewing animals. If animals were off-screen or outside viewing times, these data points were excluded. This manuscript comes from a larger dataset , in which author K. Bugler carried out her master's thesis. After a period of observations, to gather baseline behaviours, camera traps were put inside enclosures. This is the only point of difference in this study. As camera traps may also affect behaviour, this manuscript only considered the data from before camera traps were placed. Therefore, our observations at CWS were condensed into three days and a week each at Auckland Zoo and Hamilton Zoo. Video cameras were set up to capture as much as possible. Areas included were often sleeping, resting, and main walking areas, 2.3. Data Analysis Time-series graphs (in Microsoft Excel, 2016) were created to show when active behaviours occurred throughout a typical captive red panda day. Chi-square tests for this dataset were carried out in R studio (version 1.3.959) . Models were created using R studio's "glm" function (from the packages MASS, version 7.3-51.6, and lme4, version 1.1-23). Length of time was the predictor variable, with behaviour as a factor (fixed effects). This formed a basic model, which was then fitted to three alternative generalised linear models to test the best fit: a normal distribution glm, a Poisson glm, and a negative binomial glm. Model fit was assessed using the AIC (Akaike information criterion) function to compare models, and the negative binomial distribution had the best fit. Using the drop1 function on the chosen model, we tested which variables were significant. This test is useful when multiple variables are included in the initial model. Variables are removed one by one from the model and then compared in the output, showing which variable had the least impact on the overall result. Other factors were temperature, date, time, weather, individual panda, and position in the enclosure. These factors were tested, by individual zoo, with the drop1 feature until the model had only significant terms. We then examined how behaviour and other factors affected the response (length of time) on means plots, using the "emmeans" function from the package emmeans (version 1.4.7) ctb (Producer, city, state abbr. if Canada or USA, country), as well as pairwise comparison tests to test for differences between levels of categorical factors. 3. Results 3.1. Active Behaviours Behaviours from all panda at all zoos were combined and showed a slightly crepuscular pattern of activity, mostly following keeper activity, and another shorter peak around midnight. Auckland Zoo direct observations showed a rise in activity in the afternoon until observations ceased. Compared to the camera footage, red panda were active all through the afternoon and into the early hours of the evening, significantly reducing activity around midnight. At Hamilton Zoo , camera footage and observations showed two peaks in activity around 0900-1100 and 1600-1800. Observational data points finished around 1600-1700 due to the zoo opening hours. At CWS , there was an increase in activity between 0800 and 0900, for both monitoring methods, coinciding with when keepers first arrived with food and clean water. However, the camera footage showed that the morning peak started much earlier, around 0500. There was another spike in activity in the afternoon, when keepers fed the panda again, and activity reduced further from 1830 onwards. 3.2. All Behaviours Sleeping was the most common behaviour at ~68% (observations) and ~44% (camera footage) of the time (Table 3). The next most common behaviours were locomotion (~13%/~22%), resting (~7%/~15%), eating (~6%/~12%), and grooming (~6%/~3%). There was a significant interaction between the observation method and behaviour type (2 = 923.56, DF = 17, p < 0.001). All three zoos, when analysed separately, showed a positive significant interaction between method of observation and behaviour (Auckland Zoo (2 = 110.41, DF = 7, p < 0.001), Hamilton Zoo (2 = 232.14, DF = 7, p < 0.001), Currumbin Wildlife Sanctuary (2 = 36.80, DF = 7, p < 0.001)). For sleeping, the most common behaviour, there was a significant difference between observational methods (T = 4.541, DF = 190.56, p = 9.912 x 10-6). There were overlaps in the duration of a behaviour between zoos , but no overlaps occurred when observational methods were compared . Pairwise comparisons showed differences in mean length (seconds) of behaviours between all zoos (Auckland/Currumbin: p < 0.0001) (Auckland/Hamilton: p < 0.0001) (Currumbin/Hamilton: p < 0.0001). The observational method showed differences in the mean length of behaviours at the different zoos. 3.3. Temperature Effects Daytime temperatures during observations (Table 4) ranged from 11 degC at Hamilton Zoo to 32 degC at CWS. When drop1 chi-square tests were carried out, the duration of behaviours was significantly affected by temperature (Auckland Zoo (2 = 977.66, DF = 1, p < 0.001), Hamilton Zoo (2 = 914.94, DF = 1, p < 0.001), and CWS (Temperature: 2 = 483.11, DF = 1, p < 0.01)). Short-duration behaviours, such as scent marking, locomotion, and eating, occurred less often in hotter conditions . Scent marking increased at Auckland Zoo between 21 degC and 24 degC, but was not observed at all over 29 degC at CWS . Locomotion length and frequencies changed with increasing temperatures . More time was given to resting and sleeping as the temperature increased. All behaviours, and durations of behaviour, were performed at all temperatures at Auckland Zoo and Hamilton Zoo . The most significant change in behaviours performed was seen at CWS, when temperatures increased above 29 degC. 4. Discussion We estimated captive red pa nda (A. fulgens) daily activity patterns and compared them between zoos. This was achieved by monitoring red panda behaviour through in-person observations and a continuously recording camera. When all observations were combined , there were three peaks of activity (camera footage). The first occurred around 0600, before the zoos opened to the public, and the second occurred at 1700-1800, when zoos closed. The third and shortest peak of activity occurred around midnight. When observational data were considered, we saw two small peaks at 0800 and 1600. A comparison of active behaviours between both monitoring methods revealed that observations alone clearly miss active behaviours, which was consistently the case when zoos were considered independently. Food presentation across zoos varied and likely affected when animals were active. At Auckland Zoo , no fruit was left out for panda; instead, keepers would enter the enclosure and wait in the feeding area, offering them "panda cake" (a mixture of specialty biscuits, soaked overnight, with baby food and pear juice). Entering the enclosure to offer grapes was also performed during public-focused encounters. In the afternoon, cut pears were put onto a hanging log with nails so that the panda had to manipulate them off the device to eat. This was the beginning of their peak activity period. Fresh-cut bamboo was always on offer. Hamilton Zoo had less prominent peaks in activity. There was an increase in activity around 0900 when keepers arrived with food and medication for Chito. Cleaning usually took place simultaneously. The fruit was placed in feeding stations that a panda would stand on top of a platform to open. As with all zoos, bamboo was always on offer. The activity was highest between 1500 and 1700, with another short peak between 2200 and 0000. The most obvious example of panda activity co-occurring with keeper activity was at CWS . Keepers would first enter the enclosure around 0800 with food and would wait until Pasang approached for his medicine. Cleaning would occur shortly afterwards. Keepers then arrived again around 1400 with another bowl of food that was removed around 1600. Cut bamboo was offered, but the observer never witnessed it being eaten. Pasang was not observed performing any active behaviours between 1130 and 1300, likely due to midday heat. Previous studies on wild A. fulgens show bimodal/crepuscular activity patterns . However, more recent studies show that a wild red panda's most active hours are around dawn and midday. Trail camera studies in the wild showed the highest peak of activity between 0400 and 0600, and another between 1000 and 1400 . The previous findings of a crepuscular pattern are consistent with camera footage at CWS. Pasang would sleep from 1900 to the early morning hours when he would scent mark before keepers arrived. Scent marking and other exploratory/territorial behaviours are considered positive indicators of red panda health in captivity . This bimodal pattern of activity during the day clearly followed keeper timings. The activity of captive red panda was influenced by keeper presence and management, only slightly deviating from wild patterns. Care should be taken around feeding times and other keeper activities. Our recommendations are to feed panda early in the day to stimulate natural foraging hours, but to also leave some food overnight in secure devices (so that rodents and birds cannot eat it all). Fresh bamboo should be on offer at all times. If it is drying out, it should be replaced more often and placed in water or cooler areas, as red panda ignore dried-out leaves. Recent wild studies of A. fulgens saw individuals allocating their time to avoid human/animal disturbances and areas with high disturbance . Krebs et al. found that red panda showed anticipatory behaviours before keepers arrived and increased movement once they left . Reid found that Chinese red panda were more active in daylight hours, with decreased movement around midday . Zhang et al. describe two activity peaks at 0700-1000 and 1700-1800 . Red panda were more active during daylight hours, with intermittent crepuscular movements. Johnson et al. found A. styani to be more crepuscular and nocturnal than other studies, although the panda studied may not have established a home range . If a majority of red panda time budget studies in captivity are from A. fulgens, this may account for the differences in wild studies with A. styani. When comparing the two observational methods, differences in the lengths of behaviours recorded occurred between the types rather than between zoos. Observations recorded longer behaviours . This is likely due to observations being carried out during the day, when red panda spent more time resting and sleeping, which are uninterrupted behaviours. Red panda in this study were less active (Table 3) than those in wild red panda studies. This is likely due to the increased foraging needs of wild red panda, but it could also be influenced by 2/7 panda in this study being on medication due to age-related illness. Studies of wild red panda observe active activity 45-60% of the time, dependent on season , whereas our study shows panda to be less active (19-36%). A more recent study found captive red panda at Rotterdam Zoo to be active 40-53% of the time, although the two pairs in the study were young breeding pairs, and perhaps best compared to the pair at Auckland Zoo (Khela and Ramesh). In captivity, panda are brought a selection of food, such as bamboo, fruits, specialised biscuits, and the occasional day-old chick or mouse. This increase in fruit in the diet may lead to more short rests, as the stomach fills more rapidly . This can be seen in wild red panda, which have a high frequency of short rests in spring when fruit and new, more nutritious bamboo shoots are available . Glatston (2011) summarises that because "captive diets do not occupy enough of the red panda's daily activity panda may compensate for this activity 'vacuum' by over-grooming and eating hair". We found that grooming occurred less than in other studies, and no stereotypy over-grooming was observed. Average temperatures in Nepali/Indian border red panda habitat do not exceed 14 degC . However, because temperatures were only reliably recorded during the daytime for this study (Table 4), we compare the maximum temperatures seen in the wild to this study. Ranges of temperature in Autumn in situ vary from 1.6 to 27.6 degC . In this study, Auckland Zoo and Hamilton Zoo observations took place in Autumn, and temperatures ranged from 11 to 25 degC during the day, putting them in a similar range to the Nepal/India border red panda habitat. Summer ranges at CWS differed from in situ ranges. In the first week of summer daytime temperatures reached 32 degC, at CWS. While the maximum temperature, in-situ, throughout all summer, was only 28.9 degC . Despite small sample sizes, results show interesting trends in red panda behaviour. Having n = 1 for CWS only hints at what might be happening for other red panda in this heat. This study indicates a need for further investigation into the impacts of temperature on red panda behaviour, both in captivity and the wild. The temperature had a significant effect on panda behaviour. The temperature range in New Zealand is more similar to their natural range than the zoos in Australia and much of the rest of the world. At CWS, there were fewer short-duration behaviours (e.g., locomotion) as temperatures (above 29 degC) increased . Loeffler states that "protection of red panda from temperatures above 27 degC is critical" . The AZA red panda care manual also states that heat stress in red panda is intensified by high humidity . Air-conditioned indoor areas or nest boxes are recommended in these conditions. Misters, which CWS turned on daily, are noted as being a way to provide cooled areas for red panda . Auckland Zoo and Hamilton Zoo may become less stable and genetically non-viable . 5. Conclusions Peaks in activity occurred early morning, early evening, and midnight. While activity correlated to keeper activity, it also coincided with zoo opening hours and the coolest part of the day. Red panda were more active outside of opening hours and avoided activity during the warmest parts of the day. Care should be taken around when food is presented and keeper interaction/cleaning is occurring. Research such as this provides husbandry managers with knowledge of the needs of endangered animals under their care. This study suggests that further investigation into which of these factors is the most important may help to improve captive conditions for red panda. These captive red panda were much less active than their wild counterparts, likely due to differences in diet. Some captive red panda are known to over-groom to fill this extra time; however, this was not seen in our study. An external thermometer that records temperatures at regular intervals should be added to complement continuous recording, as the temperature was an important factor for time spent moving. Further study on the impact of temperature on red panda activity may help to increase the knowledge for their husbandry manual . Acknowledgments The authors would like to thank Auckland Zoo, Hamilton Zoo, and Currumbin Wildlife Sanctuary for their enthusiastic involvement in this study. We would also like to acknowledge the masters thesis from the leader author, K. A. Bugler which can be found at: (accessed on 26 November 2022). Author Contributions Conceptualisation, K.A.B. and A.M.P.; methodology, K.A.B.; validation, K.A.B., A.M.P. and J.G.R.; formal analysis, K.A.B. and J.G.R.; investigation, K.A.B.; resources, A.M.P. and J.G.R. data curation, K.A.B. and J.G.R.; writing--original draft preparation, K.A.B.; writing--review and editing, K.A.B., A.M.P. and J.G.R.; supervision, J.G.R. and A.M.P. All authors have read and agreed to the published version of the manuscript. Institutional Review Board Statement Lincoln University ethical review and approval were waived for this study due to only observational data being collected. Informed Consent Statement Not applicable. Data Availability Statement Data for this manuscript can be found at: (URL accessed on 21 February 2023) Conflicts of Interest The authors declare no conflict of interest. Figure 1 Frequency of active behaviours for red panda at three zoos, in 10 min intervals. The red lines show when direct observations were carried out. R2 values are calculated from an nth order polynomial line. (A) All zoos combined; (B) Auckland Zoo; (C) Hamilton Zoo; (D) Currumbin Wildlife Sanctuary. Figure 2 The average duration (response) of behaviours carried out at individual zoos by method type, as determined by Emmeans plot output as a histogram (+95 CI) v 1.4.7. Figure 3 Showing how the duration of behaviours was influenced by temperature at different zoo sites. The five most common behavioural categories were picked for this graph as defecation, interactions, playing, and scent marking occurred infrequently. (A) Auckland Zoo, Adj R2 = 0.052105, Intercept = 954.88, Slope = -30.654, p = 1.7274 x 10-5; (B) Hamilton Zoo, Adj R2 = -0.0012626, Intercept = 264.26, Slope = -0.022011, p = 0.99485; (C) Currumbin Wildlife Sanctuary, Adj R2 = 0.024695, Intercept = -362.83, Slope = 18.718, p = 0.0011086. (black line: Estimated means, grey shadow: +95 CI) v 1.4.7. Figure 4 A linear model with a straight line fitted (R studio v 1.4.7) shows that the duration of locomotion events across all zoos was affected by temperature. Figure 5 A linear model with a straight line fitted (R studio v 1.4.7) shows that the duration of sleeping events across all zoos was affected by temperature. animals-13-00846-t001_Table 1 Table 1 Details of red panda studied and the enclosures they lived in. Zoo Panda Details Enclosure Details Enclosure Size Auckland Zoo 2.1.0 Ramesh, male, 9 years Khela, female, 6 years Tashi, male, 4 months Lots of large natural trees, most of which are not native to their natural habitat. Large pond (empty due to juvenile). One main covered platform where feeding occurred. Two nest boxes hidden from public view and an off-display den. 170 m2 Hamilton Zoo 1.2.0 Chito, male, 16 years Taylor, female, 10 years Jamuna, female, 4 years Multiple large exotic tree species, large (empty) pond, a series of off-ground walkways and ramps interconnecting four covered platforms, and a visible large den. One tall man-made tower. A large bamboo stand wrapped around the back half of the perimeter. 529 m2 Currumbin Wildlife Sanctuary 1.0.0 Pasang, male, 16 years Three main platforms at differing heights, interconnected via off-ground walkways. One platform had a roof and the other was fully enclosed, where food and water were given. Natural trees were small. Large off-display den with resting areas and water access. Several water misters. 117 m2 animals-13-00846-t002_Table 2 Table 2 Ethogram of red panda behaviours. Behaviour Explanation of Behaviour Locomotion Movement, on all four paws, that would shift an individual from one area to another, such as by running, walking, or climbing Resting Lying, sitting, or standing, eyes open, responsive to surroundings, staying in one area Sleeping Lying, curled in a ball or flat, eyes closed, unresponsive to surrounding noise or activity Eating Selection and chewing of food brought by keepers, usually fruit, bamboo, or pellets (grass or leaves already in the enclosure may also be consumed) Drinking Licking up water in an enclosure, either from an artificial bowl or stream Grooming/Scratching Repeated licking or chewing motions of fur or quick paw movements, with claw, across own body Scent marking Frequent rubbing of genitals and/or urination on objects around the enclosure Defecation Urination, or passing of bowel movement (usually at a latrine site) Playing Spontaneous actions that are voluntary and internally motivated. These actions are not associated with the direct need for survival, e.g., eating or predator avoidance. Can be one or more, but is always non-aggressive Interaction Between two panda, overall non-aggressive Keeper interaction Non-aggressive actions towards a keeper, such as taking food from a keeper or sniffing a keeper's boots In den Out of sight while in the den Aggressive behaviour Vocalisation, staring, or aggressive displays towards a conspecific or keeper. Aggressive displays include standing on hind paws with forepaws raised above the head, head bobbing, and slamming forepaws on ground (this behaviour was not seen during the study) animals-13-00846-t003_Table 3 Table 3 Time spent performing behaviours across zoos as determined by two observation methods. Percentages are calculated from the total time observed/observation type. Behaviour Observation Camera Zoo Defecation 0.24% 2.34% Auckland 0.14% 5.71% Hamilton 0% 0.17% 0% 1.65% CWS All zoos Eating 6.84% 28.50% Auckland 3.93% 21.66% Hamilton 7.86% 0.37% CWS 5.89% 12.02% All zoos Grooming 4.86% 0.63% Auckland 6.9% 1.71% Hamilton 4.49% 5.57% CWS 5.6% 3.69% All zoos Interaction 0% 0.01% Auckland 0.33% 0.95% Hamilton 0% 0% CWS 0.16% 0.16% All zoos Locomotion 13.99% 33.04% Auckland 11.57% 49.78% Hamilton 11.32% 6.63% CWS 12.83% 21.66% All zoos Playing 0.04% 3.05% Auckland 0% 0% Hamilton n/a n/a CWS 0% 0.9% All zoos Resting 6.8% 19.66% Auckland 2.85% 18.78% Hamilton 20.35% 10.75% CWS 7.3% 15% All zoos Scent marking 0.32% 0.08% Auckland 0.21% 0.92% Hamilton 0.41% 0.47% CWS 0.21% 0.45% All zoos Sleeping 66.92% 12.59% Auckland 74.27% 0.35% Hamilton 41.91% 76.19% CWS 67.66% 44.47% All zoos animals-13-00846-t004_Table 4 Table 4 Temperatures across facilities during the study period, from observational dataset. Showing only daytime temperatures (0930-1930 at Auckland Zoo, 1000-1600 at Hamilton Zoo, and 0715-1630 at CWS). Zoo Average Minimum Maximum Season Auckland Zoo 22.77 degC 18 degC 25 degC Early Autumn (March) Hamilton Zoo 15.5 degC 11 degC 18 degC Mid-Autumn (April/May) CWS 27.49 degC 23 degC 32 degC Early Summer (December) Disclaimer/Publisher's Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

PMC10000133

Premature regression of corpora lutea (PRCL) may adversely affect the outcome of hormonal ovarian superstimulation in small ruminants, and the total dose of exogenous gonadotropins used may be one of the causes of this condition. There were two major objectives of the present study: (1) to evaluate the effects of different superovulatory doses of porcine follicle-stimulating hormone (pFSH) on the biometry, blood perfusion (Doppler), and echotextural characteristics of luteal structures; and, (2) to determine the usefulness of biometric, vascular, and echotextural luteal variables, as well as measurements of circulating progesterone (P4) concentrations for early detection of PRCL in superovulated Santa Ines ewes. Twenty-seven Santa Ines ewes received an intravaginal P4-releasing device (CIDR) from Days 0 to 8 (Day 0 = random day of the anovulatory period). An IM injection of d-cloprostenol (37.5 mg) was given at the time of the CIDR insertion and withdrawal. On Day 6, all the ewes received 300 IU of eCG IM and were divided into three treatment groups (each n = 9): G100 (100 mg); G133 (133 mg); and G200 (200 mg of pFSH) administered IM every 12 h in eight injections. Transrectal ovarian ultrasonography and jugular blood sampling for serum P4 measurements were performed on Days 11 to 15. On the day of embryo recovery (Day 15), all the ewes underwent diagnostic videolaparoscopy and were classified, based on their luteal characteristics, into three response groups: nCL (ewes with normal CL only); rCL (ewes with regressing CL only); and ewes with both nCL and rCL following the superovulatory regimen. Our present results indicate that the total pFSH doses of 100 mg and 200 mg result in similar ovulatory responses and luteal function/biometrics, although the percentage of donor ewes with nCL was greater (p < 0.05) for G100 compared with the G200 animals. An application of 133 mg of pFSH was associated with diminished luteogenesis. Lastly, circulating P4 concentrations, ultrasonographic estimates of total luteal area, and CL pixel heterogeneity (standard deviation of numerical pixel values) are promising markers of luteal inadequacy in superovulated ewes. superovulation corpus luteum progesterone ovarian ultrasonography sheep Sao Paulo Research Foundation (FAPESP)17/04193-9 Brazilian Agricultural Research Corporation (EMBRAPA)#22.13.06.026.00.03 This research was funded by the Sao Paulo Research Foundation (FAPESP), grant number 17/04193-9 and by the Brazilian Agricultural Research Corporation (EMBRAPA), Project Superovi #22.13.06.026.00.03. pmc1. Introduction Multiple ovulation and embryo transfer (MOET) technology is an important reproductive biotechnology, widespread in large and small ruminant production systems; however, it still possesses numerous drawbacks, some of which may be due to the inconsistency of hormonal protocols used . The variability in responses to superovulatory protocols is one of its major disadvantages , and the total amount of exogenous gonadotropins used for superovulation appears to be one of the causative factors . Purified porcine follicle-stimulating hormone (pFSH) is a primary choice for MOET , as it effectively stimulates antral follicular growth and maturation , including upregulation of various angiogenic factors that play a role in the terminal development of ovulatory follicles and ensuing luteogenesis . Traditionally, total pFSH doses used for superovulatory treatments in small ruminants range from 200 mg to 256 mg , even though smaller doses have been shown to be just as effective . Recent studies have shown that reducing a total pFSH dose can be beneficial for superovulatory treatments in ewes . Loiola-Filho et al. observed a higher fertilization rate and percentage of transferable and freezable embryos recovered in Dorper ewes superovulated with 128 mg, rather than 200 mg, of pFSH. In addition, the number of viable embryos was less following the treatment with 133 mg compared with that after the application of 100 or 200 mg of pFSH in Santa Ines ewes . In Santa Ines ewes, the use of 100 mg of pFSH was associated with greater luteal development and ovarian blood perfusion than using 200 mg of pFSH . High pFSH doses, in addition to being costly , can alter the frequency of luteinizing hormone pulses , the endocrine function of ovaries , and the endometrial prostaglandin release , resulting in premature regression of corpora lutea (PRCL; ). They were also involved in the occurrence of late ovulations and slower luteal development, due to decreased ovarian blood perfusion compared with that after applying lower pFSH doses . Short-lived corpora lutea (CL) frequently occur in small ruminants raised in tropical or subtropical climates that undergo superovulatory treatments and may be associated with decreased embryo yields , because the proper secretory activity of CL is essential for sustaining normal embryo development throughout the pre-implantation period . To date, the premature regression of CL has mainly been detected with videolaparoscopy, although B-mode ultrasonographic biometrics and Doppler-detectable blood perfusion indices are also significant markers of luteal function . Changes in the progesterone (P4) secretory ability of CL can also be assessed with a computerized analysis of ovarian ultrasonograms . Considering all the observations above, we hypothesized that accurate and non-invasive detection of PRCL could be accomplished by determining the biometric, vascular, and echotextural characteristics of luteal structures, as well as the circulating concentrations of luteal P4, and that lowering a superovulatory dose of pFSH could rectify or reduce the incidence of PRCL in ewes. Our laboratories continue to focus on research projects initially undertaken by Rodriguez et al. and Maciel et al. to corroborate the mechanisms of luteal inadequacy in superovulated ewes. The objectives of the present study were to: (1) evaluate the effects of different superovulatory doses of porcine follicle-stimulating hormone (pFSH) on the biometry, blood perfusion (Doppler), and echotextural characteristics of luteal structures; and, (2) determine the usefulness of biometric, vascular, and echotextural luteal variables, as well as measurements of circulating progesterone (P4) concentrations, for early detection of PRCL in superovulated Santa Ines ewes. 2. Materials and Methods 2.1. Location and Animals The present experiment was carried out in November at the School of Agricultural and Veterinary Sciences, Sao Paulo State University, Jaboticabal, SP, Brazil (21deg15'18'' South and 48deg19'19'' West), after formal approval by the local ethics committee (protocol number 12062/14). Twenty-seven clinically healthy, multiparous, non-pregnant (last parturition over 6 months earlier), and non-lactating Santa Ines ewes, with a mean body weight of 45.2 +- 5.8 kg and aged 3.0 +- 1.0 years, were used in this study. The ewes were kept in paddocks under an intensive rearing system, with unrestricted access to mineral salt licks, drinking water, and corn silage, and received a balanced feed in the amount of 200 g/animal/day. 2.2. Experimental Design Estrus was synchronized in all the ewes with intravaginal devices containing 0.3 g of P4 (Eazi-Breed CIDR, Controlled Internal Drug Release, Pfizer, New Zealand) that were inserted on a random day of the estrous cycle or anovulatory period (Day 0) and left in place for eight days. On the days of the CIDR insertion and withdrawal (Day 0 and Day 8), all the animals received an IM injection of 125 mg of PGF2a analog (Cloprostenol Sodium; Sincrocio, Ourofino, Cravinhos, SP, Brazil). On Day 6 (48 h before the CIDR removal), the ewes were randomly allocated to the three superovulatory treatment groups: (1) G100 (n = 9): 100 mg of pFSH (Folltropin, Bioniche, Orangeville, ON, Canada); (2) G133 (n = 9): 133 mg of pFSH; and (3) G200 (n = 9): 200 mg of pFSH per donor ewe. The superovulatory pFSH doses were administered in eight consecutive IM injections (20, 20, 15, 15, 10, 10, 5, and 5% of the total pFSH dose) given at 12 h intervals. On Day 6 (first injection of pFSH), a single IM injection of 300 IU of equine chorionic gonadotropin (eCG; Novormon, Syntex, Luis Guillon, Province of Buenos Aires, Argentina) was also given. After the CIDR removal, the ewes were placed in paddocks with fertile rams fitted with crayon marking harnesses (1:5 ratio) for 3 days. Estrus detection was performed three times a day by observing female sexual receptivity to ensure that each ewe was mated two or three times during behavioral estrus. 2.3. Ultrasound and Videolaparoscopic Evaluations B-mode and color Doppler ultrasound assessments of CL formed after the superovulatory treatments were performed at 24 h intervals during the period corresponding to the early luteal phase, between Day 11 (1 or 2 days after ovulation) and Day 15 (day of embryo recovery). The transrectal ovarian ultrasonography utilized the MyLab Vet30 Gold ultrasound unit (Esaote, Genova, Italy), equipped with a multi-frequency linear-array transducer (6 to 8 MHz) stiffened with a hollow plastic extension tube . For the B-mode scanning, the main gain was set at 65% of the maximum value, and a single focal point was positioned in the region of interest; all the settings were kept constant throughout the study period. The color Doppler pulse repetition frequency (PRF) was 1.4 kHz; the color gain was set at 70% of the maximum value (or just below the background noise level recorded in a standing, motionless animal); the wall filter (WF) was 75 kHz; and the maximum scanning depth was 8 cm . A videolaparoscopy of the ovaries was performed on the day of embryo recovery, as previously described by Oliveira et al. . All the visualized CL were classified, based on morphological characteristics indicative of their functionality, as normal (reddish/pinkish luteal structures distinctly protruding above the surface of the ovary ) or regressing (<=5 mm in diameter, grossly pale, with little or no protrusion above the surface of the ovary ). 2.4. Computerized Analysis of Ultrasound CL Images Ultrasonographic images containing the largest cross-sectional area (B-mode) or color Doppler area were selected for each CL prior to biometric or vascular perfusion evaluations, respectively. Using the Adobe FireWorks software (Adobe, San Jose, CA, USA), the total luteal area and average area of all the luteal structures (measured in pixels and subsequently converted to mm2) were determined. The following luteal blood perfusion parameters or color Doppler indices were determined: total color Doppler or vascularization area in both ovaries of each ewe, mean color Doppler area per luteal structure, and color Doppler area percentage (color Doppler area/total cross-sectional area of all CL x 100%). The Image ProPlus analytical software (Media Cybernetics, Inc., San Diego, CA, USA) was used to compute the first-order echotextural characteristics of individual luteal structures, namely the numerical pixel values (NPVs) and heterogeneity (standard deviation of NPVs), as previously described by Viana et al. . 2.5. Blood Collection and Measurements of Serum P4 Concentrations Blood samples were collected before each ultrasonographic examination by jugular venipuncture using 10 mL vacuum tubes without anticoagulants (Becton Dickinson Diagnostics, Sao Paulo, SP, Brazil). All the samples were left for 6 to 12 h at room temperature before being centrifuged at 3000x g for 15 min; the harvested serum was transferred to polypropylene microtubes in two aliquots of the same volume and kept frozen at -20 degC. The serum P4 concentrations were measured using a commercial radioimmunoassay kit (MP Biomedicals, LLC, Diagnostics Division, Orangeburg, NY, USA). The sensitivity of the assay was 0.1 mg/mL, and the average intra-assay coefficient of variation was 18%. 2.6. Embryo Recovery The surgical embryo collection was performed 6 days after the onset of estrus, as previously described by Maciel et al. . After flushing both uterine horns, morphological evaluations of the recovered structures were conducted using a stereomicroscope at 20 to 50x image magnification. The detection of cleavage divisions was used as evidence for oocyte fertilization. All the embryos (compact morulae, early and expanded blastocysts) were classified using the International Embryo Transfer Society criteria for embryo viability as grades I to III (transferrable quality embryos) or grade IV (embryos with a delayed development and/or signs of morphological degeneration). The following additional end-points were determined for each ewe : total structure/ova/embryo recovery rates = total number of all recovered structures/ova/embryos (unfertilized ova, embryos of different developmental stages)/number of CL x 100%; viable embryo rate = number of viable embryos/total number of recovered ova/embryos x 100; and the proportion of unfertilized eggs/degenerated embryos = number of unfertilized ova or degenerated embryos/total number of ova/embryos recovered x 100%. 2.7. Statistical Analysis The statistical analyses utilized the Statistical Analysis System software (SAS Inst., Inc., Cary, NC, USA). The Cramer-von Mises test was used to verify the normality and homoscedasticity of the data. Whenever necessary, the raw data were transformed using the Box-Cox procedure; however, all the results are presented in the non-transformed format. All the single time-point variables were compared between the treatment groups (G100, G133, and G200) using a one-way analysis of variance (ANOVA) and Fisher's exact test, and the serial data were analyzed by a two-way repeated measures ANOVA to determine the main effects of the treatment (total pFSH dose used) and time (day of observation), and their interaction. When the main effects or the interaction term were significant, the differences among the individual mean values were assessed by the Tukey test. The Fisher exact test was used for the analysis of the proportions. An initial inspection of our results revealed that the ewes of the present study exhibited three distinctive patterns of luteal responses. Therefore, additional statistical analyses were conducted among the subsets of donor ewes that contained the normal CL (nCL) only, regressing CL (rCL) only, or both nCL and rCL to see if hormone measurements and ultrasonography could be used to detect the incidence of PRCL. Pearson's correlation test was used to estimate associations of ovarian biometric, echotextural, and Doppler variables with serum P4 concentration throughout the entire observation period. Statistical significance was considered as p value < 0.05. 3. Results The type of luteal structures present could not be determined in 4 out of 27 ewes studied, due to the limited mobility of the ovaries visualized with videolaparoscopy on Day 15 (2 ewes from the G133 and 2 animals from the G200 group). There were no differences (p > 0.05) in the number of luteal structures (total, nCL, and rCL) among the animals superovulated with 100, 133, or 200 mg of pFSH (Table 1). However, the proportion of ewes with nCL only was greater (p < 0.05) in the G100 compared with the G200 group (67% vs. 14%, respectively; Table 1). On Day 11, corpora lutea could be detected ultrasonographically in five out of nine ewes in the G100 and G200 groups and in six out of nine ewes in the G133 group, and, from Day 12 to Day 15, they could be detected in all the animals studied. There was a significant main effect of day for the serum P4 concentrations (p < 0.001), and a significant main effect of the treatment group for the total (p < 0.001) and mean luteal area . The circulating P4 concentrations increased (p < 0.05) from Day 14 to Day 15 in the G100 ewes and from Day 11 to Day 14 in the G200 group, and they were greater (p < 0.05) in the G100 than in the G133 animals on Day 15 . The total luteal area was significantly greater in the G100 and the G200 ewes compared with the G133 animals on Days 14 and 15 . Overall, the mean luteal area was greater (p < 0.05) in the G200 than in the G133, but the post-ANOVA (Tukey) test revealed no significant differences among the individual means over time or between the three treatment groups. There were no differences in the mean numerical pixel values or the pixel heterogeneity of the luteal structures in the ewes of the present study . There was a significant main effect of the group for the total Doppler area of luteal tissue ; it was significantly greater in the G200 compared with the G133 ewes on Days 14 and 15. No significant differences were observed for the mean luteal Doppler area and vascularization percentage of all the detected luteal structures . There were weak to moderate overall correlations among all the biometric, echotextural, and hemodynamic attributes of the ultrasonographically detected CL and circulating concentrations of P4 with the data pooled for all the observation days (Days 11 to 15; p < 0.05), except for the numerical pixel values and CL vascularization percentage (Table 2). The total number of CL did not vary (p > 0.05) among the three subsets of ewes with different CL types after superovulation (Table 3). The donor ewes with nCL only exceeded (p < 0.05) their nCL + rCL counterparts in the number of nCL (by 1.8-fold), and the ewes with rCL only had 6.7 times more prematurely regressing CL than the nCL + rCL group (p < 0.05). There was a significant main effect of day for the serum P4 concentrations (p = 0.004) and a significant CL type x day interaction (p = 0.04) for the total luteal area in ewes varying in their CL status . The circulating P4 concentrations increased (p < 0.05) from Day 11 to Day 14 in the nCL ewes and from Day 12 to Day 15 in the nCL + rCL group . Moreover, they were greater (p < 0.05) in the nCL compared with the rCL ewes on Day 15. The total luteal area increased (p < 0.05) from Day 12 to Day 14 in the nCL animals ; there were no significant fluctuations in the mean CL area . Lastly, there were no significant main effects of the CL type, time, or their interaction for the echotextural and hemodynamic (Doppler) CL characteristics in the ewes of the present study . The main effect of the CL type for the pixel heterogeneity approached significance , due mainly to the numerically highest CL pixel heterogeneity values in the rCL group. Uterine flushing was successful in 19 out of 23 donor ewes (82.6%; Table 4). Embryo collection was not attempted in four donor ewes with ovarian adhesions, precluding the assessment of ovaries and exteriorization of the reproductive tract. No structures were recovered from one G100 ewe, three G133 ewes, and one G200 group animal. There were no significant differences in the embryo quality or recovery rates among the three groups of ewes receiving the different superovulatory doses of pFSH (Table 4). 4. Discussion All the pFSH doses used for the superovulation of the Santa Ines ewes in this study induced similar ovulatory responses. Rodriguez et al. and Brasil et al. reported similar ovarian responses in Santa Ines ewes subjected to the same superovulatory regimens/total pFSH doses. The occurrence of PRCL was observed in all the treatment groups at the time of embryo recovery, with the proportion of donor ewes with nCL only being greater for the G100 compared with the G200 group. This is, at least partly, in agreement with an earlier study by Rodriguez et al. , who showed that the rate of PRCL formation was the greatest after the highest dose (200 mg) of superovulatory pFSH treatment. It was suggested that the use of higher pFSH doses might be associated with the presence of persistent estrogen-secreting anovulatory follicles responsible for the untimely prostaglandin F2a-release and luteal regression . The biometric characteristics of the luteal tissue, and particularly the total luteal area, were generally greater in the groups G100 and G200 compared with those in the G133 donor ewes, even though the last group received the intermediate dosage of pFSH (133 mg). This is an unexpected result, which indicates that the effect of pFSH dose on ovarian luteogenesis is not dose-dependent. Future studies to elucidate this issue are warranted. Based on this observation, the lowest and highest pFSH doses tested in the present experiment (100 and 200 mg) can effectively be used in the superovulatory/MOET programs to produce the same ovarian responses. However, the proportion of ewes with nCL only was significantly greater in the G100 ewes, so a lower dose (100 mg) may be a primary choice. The luteal biometry is indicative of the luteal P4 secretory function and, in the present experiment, all but one of the luteal biometric variables were positively correlated with serum P4 concentrations (numbers of regressing CL were negatively correlated with circulating P4 concentrations). The luteal function was clearly depressed in the G133 ewes, which was most likely due to the combined effects of the numerically lowest ovulation rate and diminished luteotropic support, resulting in smaller CL sizes. As with the luteal biometrics discussed above, there was a difference in the luteal blood perfusion (total Doppler signal) between the G100/G200 ewes and the animals allocated to the G133 group. Statistically, the difference between the G200 and G133 groups was more pronounced than that between the G100 and the G133. We speculate that the intermediate pFSH dose (133 mg) did not elicit the same effect on the expression of vascular endothelial growth factor (VEGF; ), responsible for the maturation and stabilization of blood vessels , as the lowest and highest pFSH doses used in this study. This manifested in significant inter-dose differences in the luteal Doppler area, especially one day before and on the day of embryo recovery. The presence of a vascular network in an organ or tissue is a prerequisite for its biological function. Its assessment by color Doppler ultrasonography can be used to determine the secretory activity of the endocrine glands . The vascular system is essential for providing oxygen, nutrients, hormones, and substrates necessary for ovarian steroidogenesis. In the ewes of the present study, luteal vascularization showed a positive association with serum P4 concentrations, which is in complete agreement with earlier studies in small ruminants . In the present experiment spanning the early luteal phase of hormonally superstimulated ewes, the serum P4 concentrations rose significantly prior to Days 14 to 15 in all the animals except for the G133, probably due to the differences in the rate of luteal tissue development. In addition, an increase in the circulating P4 concentrations was observed in all the ewes that had nCL, but it did not occur in the ewes with rCL only. Luteogenesis is associated with intense cell proliferation, as well as biochemical and vascular alterations in the CL, typically leading to increased P4 release. However, the increment in P4 secretion appears to precede an increase in the luteal tissue content of ovaries in superovulated ewes , even though the two parameters remain quantitatively correlated. The numerical pixel values (NPV), or pixel intensity, are proportional to the number of sound waves reflected by acoustic tissue interfaces , and NPVs are indicative of cell density within examined structures . Thus, with the progression of luteogenesis over the ultrasound evaluation days in this study, a corresponding increase in the NPVs of the luteal glands was expected. In a study by Davies et al. , the mean pixel intensity of CL showed an increase during the early luteal phase in non-prolific Western White Face ewes, and from the early to mid-luteal phase in prolific Finn sheep, and a decline at the time of luteolysis in both genotypes of sheep. In addition, both the total luteal area and mean pixel values were correlated with the pattern of serum concentrations of P4 from Days 3 to 15 after ovulation in the Western White Face ewes and from Days 3 to 14 in the Finn sheep. However, in the present experiment spanning the early diestrus stage in superovulated Santa Ines ewes, there were no significant changes in the CL pixel intensity among the different treatment groups, or among the CL with different fates after the present superovulatory treatments. As with the NPVs above, there were no differences in the mean pixel heterogeneity of the detected luteal structures in the ewes of the present study. Pixel heterogeneity (standard deviation of NPV) has been another echotextural variable used to assess luteal function in cattle , sheep , and goats . Siqueira et al. and Simoes et al. observed higher CL pixel heterogeneity at the beginning and the end of the luteal phase of the estrous cycle in cattle and goats, respectively. These findings are due mainly to cell differentiation that occurs in the CL during the luteal phase in these species . At the beginning of the luteal development, the number of cells is small, and, for that reason, the ultrasound image is heterogeneous . The same echotextural changes were observed at the end of the luteal phase due to structural luteolysis in cows and goats . However, the mean pixel heterogeneity of the CL did not differ throughout the luteal phase (Days 3 to 15 after ovulation) for both the Western White Face and Finn ewes . In cyclic ewes, the NPV, pixel heterogeneity, and percentage of the CL occupied by blood clots only declined from 12-24 h to 60-72 h after ovulation . Moreover, there were no significant correlations between the P4 concentrations and the pixel heterogeneity for either the Western White Face ewes or Finn sheep . The only variables analyzed in this study that may facilitate the detection of PRCL in superovulated ewes appear to be the serum P4 concentrations, total luteal tissue area, and, unlike in cyclic ewes , CL pixel heterogeneity. However, only the assessment of luteal pixel heterogeneity may provide sufficient information to identify the donor ewes with ovaries containing rCL only on Day 15 of the superovulatory protocol. The circulating P4 concentrations differed only between the nCL and rCL groups of ewes on Day 15, and a rise in the total luteal area and serum P4 concentrations occurred exclusively in the animals with nCL only by Day 14. Therefore, none of these metrics allows for the detection of ewes with rCL only by Day 14-15, and none of them permits the accurate identification of donor ewes with ovaries containing both nCL and rCL. The fact that nCL and rCL may co-exist in the same animal confirms that the mechanism behind PRCL may not only be related to the early release of uterine prostaglandin, but it may also be associated with individual variations among the preovulatory follicles and resultant CL. Such variations may include differences in estrogenicity and gonadotropic responsiveness among preovulatory antral follicles , or dissimilar sensitivity of CLs to luteolytic factors . Further studies on the etiology of luteal cell death during PRCL in ruminant species are warranted. In a previous study , using the same superovulatory regimen and doses, the proportion of unfertilized oocytes was greater in the G100 than in the G200 ewes, and the embryo variability rate was lower in the G133 compared with the G200 group. Only numerical (non-significant) differences were noted in the ewes of the present study that were a subset of the animals used by Maciel et al. ; this was probably due to considerable individual variation in superovulatory responses. To the best of the authors' knowledge, this is the first report of using minimally invasive techniques to diagnose prematurely regressing CL in superovulated ewes. Other studies aimed to establish the usefulness of the B-mode and color Doppler imaging modalities for detecting PRCL were not completely successful . Although the use of the color Doppler technique appeared to increase the accuracy of CL detection and enumeration in superovulated ewes, both ultrasound modalities failed to detect prematurely regressing CL, probably due to the fact that visual assessments rather than computer-assisted analyses of luteal ultrasonograms were performed . We suggest that the evaluation of the total area and pixel heterogeneity of individual CL may increase the accuracy of detecting PRCL, which will ultimately result in a practical, efficient, and non-invasive technique to prevent the occurrence and/or to minimize the adverse effects of PRCL in small ruminants undergoing hormonal ovarian superstimulation. 5. Conclusions The present results indicate that superovulatory treatment of ewes with the lowest total pFSH dose (100 mg) is associated with similar CL development to that achieved with the highest dose (200 mg), although the percentage of donor ewes with normal CL function only was greater after the treatment with a 100 mg dose. The luteal biometrics and Doppler indices of the forming luteal structures were correlated with their P4 secretory ability. The serum P4 concentrations, ultrasonographically determined luteal tissue area, and CL pixel heterogeneity appear to be the most valuable prospective markers of premature luteolysis in superovulated ewes. Author Contributions Conceptualization: J.R.B., M.G.K.R., G.S.M., G.B.V., J.F.d.F., P.M.B. and M.E.F.O. Methodology: J.F.d.F., P.M.B. and M.E.F.O. Software: J.R.B., G.B.V. and M.E.F.O. Formal analysis: J.R.B., M.G.K.R. and G.S.M. Investigation: J.R.B., M.G.K.R., G.S.M., G.B.V., J.F.d.F., P.M.B. and M.E.F.O. Data curation: J.R.B., G.B.V., P.M.B. and M.E.F.O. Writing--original draft preparation: J.R.B., P.M.B. and M.E.F.O. Supervision: P.M.B. and M.E.F.O. Project administration: J.F.d.F. and M.E.F.O. Funding acquisition: J.R.B. and M.E.F.O. All authors have read and agreed to the published version of the manuscript. Institutional Review Board Statement The animal study protocol was approved by the Ethics Committee of the School of Agricultural and Veterinarian Sciences, Sao Paulo State University, Jaboticabal, SP, Brazil (protocol no. 12062/14). J.F. Fonseca and M.E.F. Oliveira are CNPq fellows. M.E.F.O. is a FAPERJ fellow. Informed Consent Statement Not applicable. Data Availability Statement The results of this study were disseminated in the preliminary form at the 49th IETS Annual Conference in Lima, Peru (abstract 246; Reproduction Fertility and Development 35(2):252-253; DOI: 10.1071/RDv35n2Ab246). Conflicts of Interest The authors declare no conflict of interest. Figure 1 Mean (+-SEM) serum progesterone (P4) concentrations (A) as well as total (B) and mean luteal area (C) from Day 11 (1 or 2 days after ovulation or beginning of luteogenesis) to Day 15 (day of embryo recovery) of the superovulatory protocol in Santa Ines ewes superovulated with 100 (G100), 133 (G133), or 200 (G200) mg of pFSH given in eight decreasing doses. Different letters in the lower chart area indicate statistically significant differences: a-c--over time within the treatment groups; AB--between the groups. Numbers in parentheses (panel B) denote the numbers of ewes in which CL were detectable ultrasonographically on Day 11. Figure 2 Mean (+-SEM) numerical pixel values (A) and heterogeneity (B) of CL ultrasonographically detected from Day 11 (1 or 2 days after ovulation or beginning of luteogenesis) to Day 15 (day of embryo recovery) of the superovulatory protocol in Santa Ines ewes superovulated with 100 (G100), 133 (G133), or 200 (G200) mg of pFSH given in eight decreasing doses. Numbers in parentheses (panel A) denote the numbers of ewes in which CL were detectable ultrasonographically on Day 11. Figure 3 Total (A) and mean (B) luteal Doppler area as well as luteal vascularization percentage (total Doppler area/total luteal area x 100%) (C) from Day 11 (1 or 2 days after ovulation or beginning of luteogenesis) to Day 15 (day of embryo recovery) of the superovulatory protocol in Santa Ines ewes submitted to superovulatory treatment with 100 (G100), 133 (G133), or 200 (G200) mg of pFSH given in eight decreasing doses. Numbers in parentheses (panel A) denote the numbers of ewes in which CL were detectable ultrasonographically on Day 11. Figure 4 Mean (+-SEM) serum progesterone (P4) concentrations (A), as well as total luteal (B), and mean luteal area (C) from Day 11 (1 or 2 days after ovulation or beginning of luteogenesis) to Day 15 (day of embryo recovery) of the superovulatory protocol in Santa Ines ewes that had normal CL (nCL) only, regressing CL (rCL) only or both nCL and rCL following the superovulatory pFSH treatment. Different letters in the lower chart area indicate statistically significant differences: a-c over time within the treatment groups; AB--between the groups. Figure 5 Mean (+-SEM) numerical pixel values (A) and heterogeneity (B) of CL ultrasonographically detected from Day 11 (1 or 2 days after ovulation or beginning of luteogenesis) to Day 15 (day of embryo recovery) of the superovulatory protocol in Santa Ines ewes that had normal CL (nCL) only, regressing CL (rCL) only, or both nCL and rCL following the superovulatory pFSH treatment. Numbers in parentheses (panel A) denote the numbers of ewes in which CL were detectable ultrasonographically on Day 11. Figure 6 Total (A) and mean luteal Doppler area (B), as well as luteal vascularization percentage (total Doppler are/total luteal area x 100%) (C), from Day 11 (1 or 2 days after ovulation or beginning of luteogenesis) to Day 15 (day of embryo recovery) of the superovulatory protocol in Santa Ines ewes that had normal CL (nCL) only, regressing CL (rCL) only, or both nCL and rCL following the superovulatory pFSH treatment. Numbers in parentheses (panel A) denote the numbers of ewes in which CL were detectable ultrasonographically on Day 11. animals-13-00873-t001_Table 1 Table 1 Proportions (%) of animals with different types of corpora lutea [CL; normal (nCL) or regressing (rCL)] and mean (+-SEM) numbers of luteal structures determined laparoscopically on Day 15 of the superovulatory protocol in Santa Ines ewes superovulated with 100 (G100), 133 (G133), or 200 (G200) mg of pFSH administered in eight decreasing doses. Variable/Group G100 G133 G200 % of ewes with nCL only 67% (6/9) a 57% (4/7) 14% (1/7) b % of ewes with rCL only 11% (1/9) 0% (0/7) 29% (2/7) % of ewes with nCL and rCL 22% (2/9) 43% (3/7) 57% (4/7) Total no. of CL 13.8 +- 1.8 8.7 +- 1.1 14.2 +- 2.4 nCL 11.1 +- 2.9 8.6 +- 1.3 8.4 +- 3.2 rCL 2.7 +- 1.7 1.7 +- 1.0 6.8 +- 3.2 abp < 0.05. animals-13-00873-t002_Table 2 Table 2 Summary of overall correlations (Pearson product-moment) of CL biometric, echotextural, and Doppler parameters (input variables) with serum progesterone (P4) concentrations (output variable) in Santa Ines ewes superovulated with 100, 133, or 200 mg of pFSH administered in eight decreasing doses, during the period spanning Day 11 (1 or 2 days after ovulation or beginning of luteogenesis) to Day 15 (day of embryo recovery). Input Variable (x) Coefficient of Correlation p Value Total no. of CL 0.18 0.04 nCL 0.38 0.00005 rCL -0.19 0.04 Total luteal area 0.30 0.001 Mean luteal area 0.41 0.000003 Numerical pixel values 0.15 0.12 Pixel heterogeneity -0.29 0.002 Total Doppler area 0.31 0.0006 Mean Doppler area 0.33 0.0003 CL vascularization % 0.02 0.86 animals-13-00873-t003_Table 3 Table 3 Mean (+-SEM) number of CL [total, normal (nCL) or regressing (rCL)] determined laparoscopically on Day 15 (day of embryo recovery) of the superovulatory protocol in Santa Ines ewes with nCL only, rCL only, or both nCL and rCL following the superovulatory regimen. Variable/Group nCL only (n = 10) rCL only (n = 3) nCL + rCL (n = 10) p Value Total no. of CL 14.3 +- 2.2 16.7 +- 2.9 10.6 +- 1.4 0.21 nCL 14.3 +- 2.2 a - 8.1 +- 1.4 b 0.002 rCL * - 16.7 +- 2.9 a 2.5 +- 0.8 b <0.001 * One-way ANOVA on ranks; ab different letter superscripts within the same row indicate statistical differences (p < 0.05). animals-13-00873-t004_Table 4 Table 4 Embryo yields (per donor ewe) in Santa Ines ewes that underwent hormonal ovarian superstimulation with different total doses of pFSH (mean +- SEM). Variable/Groups G100 (n = 8) G133 (n = 8) G200 (n = 7) p Values No. of recovered structures 6.0 +- 1.3 4.1 +- 1.4 7.1 +- 1.4 0.32 No. of unfertilized eggs * 2.1 +- 1.0 1.0 +- 0.9 1.0 +- 0.7 0.58 No. of embryos 3.9 +- 1.5 3.1 +- 1.3 6.1 +- 1.3 0.31 No. of viable embryos (grades I-III) 2.6 +- 1.0 1.5 +- 0.9 4.4 +- 1.3 0.18 No. of degenerated embryos (grade IV) * 1.2 +- 0.6 1.6 +- 0.7 1.7 +- 0.5 0.86 Recovery rate (%) * 48.3 +- 9.6 48.1 +- 14.4 54.9 +- 12.6 0.91 Unfertilized eggs (%) * 39.3 +- 16.3 20.0 +- 17.0 12.7 +- 8.8 0.41 Viability rate (%) 42.3 +- 13.0 35.8 +- 14.9 61.9 +- 11.8 0.39 Degenerated embryos (%) 18.4 +- 7.6 44.2 +- 15.2 25.3 +- 5.6 0.19 * One-way ANOVA on ranks. Disclaimer/Publisher's Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

PMC10000134

Cancers (Basel) Cancers (Basel) cancers Cancers 2072-6694 MDPI 10.3390/cancers15051359 cancers-15-01359 Correction Correction: van de Velde et al. Vincristine-Induced Peripheral Neuropathy in Pediatric Oncology: A Randomized Controlled Trial Comparing Push Injections with One-Hour Infusions (The VINCA Trial). Cancers 2020, 12, 3745 van de Velde Mirjam Esther 1* Kaspers Gertjan J. L. 12 Abbink Floor C. H. 3 Twisk Jos W. R. 4 van der Sluis Inge M. 25 van den Bos Cor 23 van den Heuvel-Eibrink Marry M. 2 Segers Heidi 6 Chantrain Christophe 7 van der Werff ten Bosch Jutte 8 Willems Leen 9 van den Berg Marleen H. 1 1 Emma Children's Hospital, Amsterdam UMC, Vrije Universiteit Amsterdam, Pediatric Oncology, 1081 HV Amsterdam, The Netherlands 2 Princess Maxima Center for Pediatric Oncology, 3584 CS Utrecht, The Netherlands 3 Emma Children's Hospital, Amsterdam UMC, Amsterdam Medical Center, Pediatric Oncology, 1105 AZ Amsterdam, The Netherlands 4 Department of Epidemiology and Biostatistics, Amsterdam UMC, Vrije Universiteit Amsterdam, 1081 HV Amsterdam, The Netherlands 5 Department of Pediatric Oncology, Erasmus Medical Center Rotterdam/Sophia Children's Hospital, 3000 CB Rotterdam, The Netherlands 6 Department of Pediatric Hemato-Oncology, University Hospitals Leuven, 3000 Leuven, Belgium 7 Department of Pediatrics, Clinique du MontLegia, CHC, 4000 Liege, Belgium 8 Department of Pediatric Onco-Hematology, Universitair Ziekenhuis Brussel, 1090 Brussels, Belgium 9 Department of Paediatric Haematology-Oncology and Stem Cell Transplantation, Ghent University Hospital, 9000 Ghent, Belgium * Correspondence: mi.vandevelde@amsterdamumc.nl; Tel.: +31-(0)20-444-6206; Fax: +31-(0)20-444-5122 21 2 2023 3 2023 21 2 2023 15 5 135921 9 2022 29 11 2022 (c) 2023 by the authors. 2023 Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license ). pmcError in Table In the original publication, there was a mistake in Table 1 as published . The number of patients with death was swapped between the one-hour administration group and the push administration group. The corrected Table 1 appears below. The authors state that the scientific conclusions are unaffected. This correction was approved by the Academic Editor. The original publication has also been updated. cancers-15-01359-t001_Table 1 Table 1 Demographic and clinical characteristics of the participants at baseline. One-Hour Administration Group (n = 45) Push Administration Group (n = 45) Sex Male 26 (58) 24 (53) Female 19 (42) 21 (47) Age, years (mean(SD)) 9.06 (5.11) 9.29 (5.25) Age >= 5 years and included for ped-mTNS assessment 32 (71) 34 (76) Disease Acute lymphoblastic leukemia 29 (64) 29 (64) Hodgkin lymphoma 7 (16) 11 (24) Nephroblastoma 6 (13) 2 (4) Medulloblastoma 1 (2) 1 (2) Rhabdomyosarcoma 2 (4) 0 (0) Low-grade glioma 0 (0) 2 (4) Ancestry a Europe 36 (80) 37 (82) Eastern Asia 1 (2) 0 (0) Latin-America (including Caribbean) 1 (2) 2 (4) Middle-East (including Northern Africa) 3 (7) 3 (7) Sub-Saharan Africa 1 (2) 0 (0) Combination 2 (4) 3 (7) Missing 1 (2) 0 (0) No. (%) of patients needing VCR dose reductions or omissions 1 (3) 0 (0) No. (%) of patients using analgesics for neuropathic pain 9 (20) 11 (24) Relapse (No. (%)) 2 (4) 1 (2) Death (No. (%)) 3 (7) 1 (2) Values represent the number (%) of participants, unless indicated otherwise. SD: standard deviation; ped-mTNS: pediatric modified Total Neuropathy Score; VCR: vincristine, a: Ancestry was parent-reported; participants could only be included in one category. Disclaimer/Publisher's Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content. Reference 1. Van de Velde M.E. Kaspers G.J.L. Abbink F.C.H. Twisk J.W.R. van der Sluis I.M. van den Bos C. van den Heuvel-Eibrink M.M. Segers H. Chantrain C. van der Werff Ten Bosch J. Vincristine-Induced Peripheral Neuropathy in Pediatric Oncology: A Randomized Controlled Trial Comparing Push Injections with One-Hour Infusions (The VINCA Trial) Cancers 2020 12 3745 10.3390/cancers12123745 33322788

PMC10000135

Imbalances in the gut microbiota composition may lead to several reproductive disorders and diseases during pregnancy. This study investigates the fecal microbiome composition between primiparous and multiparous cows during non-pregnancy and pregnancy to analyze the host-microbial balance at different stages. The fecal samples obtained from six cows before their first pregnancy (BG), six cows during their first pregnancy (FT), six open cows with more than three lactations (DCNP), and six pregnant cows with more than three lactations (DCP) were subjected to 16S rRNA sequencing, and a differential analysis of the fecal microbiota composition was performed. The three most abundant phyla in fecal microbiota were Firmicutes (48.68%), Bacteroidetes (34.45%), and Euryarchaeota (15.42%). There are 11 genera with more than 1.0% abundance at the genus level. Both alpha diversity and beta diversity showed significant differences among the four groups (p < 0.05). Further, primiparous women were associated with a profound alteration of the fecal microbiota. The most representative taxa included Rikenellaceae_RC9_gut_group, Prevotellaceae_UCG_003, Christensenellaceae_R_7_group, Ruminococcaceae UCG-005, Ruminococcaceae UCG-013, Ruminococcaceae UCG-014, Methanobrevibacter, and [Eubacterium] coprostanoligenes group, which were associated with energy metabolism and inflammation. The findings indicate that host-microbial interactions promote adaptation to pregnancy and will benefit the development of probiotics or fecal transplantation for treating dysbiosis and preventing disease development during pregnancy. cow pregnancy primiparous and multiparous fecal microbiota 16S rRNA sequencing Science and Technology Department of Sichuan Province2021YFYZ0001 Innovative Research Team of Beef Cattle in Sichuan ProvinceSCCXTD-2022-13 This work was supported by the Science and Technology Department of Sichuan Province (2021YFYZ0001) and the Innovative Research Team of Beef Cattle in Sichuan Province (SCCXTD-2022-13). pmc1. Introduction Pregnancy is a wonderful and complex physiological process. In order to adapt to the growth and development of the fetus, drastic changes occur in maternal hormones, immunity, and metabolism before and after pregnancy. For mammals, progesterone (P4), estradioal (E2), follicle stimulating hormone (FSH), luteinizing hormone (LH), and Prolactin (PRL) are the main reproductive hormones to maintain and evaluate maternal pregnancy . Growth hormone, thyroid hormone, and sex hormones could also change with maternal pregnancy . The maternal immune system undergoes significant adaptations during pregnancy to avoid harmful immune responses against the fetus and to protect the mother and her future baby from pathogens . For example, the number of T cells during pregnancy is lower than before pregnancy . More nutrients are needed to be stored and consumed during pregnancy to meet the nutritional demands of the mother and fetus. Maternal metabolism changes to meet the nutritional requirements during pregnancy, the most obvious being the decrease in insulin sensitivity . Additionally, compared to multiparous women, primiparous women have more exaggerated physiological responses, resulting in higher weight gain and body fat gain than that of multiparous women during pregnancy . There are also many differences between primiparous and multiparous cows, including productivity, reproductive ability, energy balance, immune, metabolic, and hormonal responses . Gut microbiota can produce a variety of nutrients, such as amino acids, fatty acids, and vitamins, which play an important role in regulating host metabolism, energy balance, and immune response . With the changes of maternal hormones, immunity and metabolism during pregnancy, the composition and abundance of gut microbiota also shifted. The relative abundance of 21 genera of gut microbiota showed significant differences between non-pregnant and pregnant mice fed a standard diet. There were 4 abundant genera (present at greater than 1%) significantly increased and 5 rare taxa (present at lower than 0.5%) reduced during pregnancy compared to non-pregnant mice . For dairy cows, the fecal microbial communities change dramatically in bacterial abundance at different taxonomic levels among the 12 distinctly defined production stages in a modern dairy farm, especially between virgin cows and parous cows . Information on host-microbial interactions during pregnancy is emerging . Recent studies showed that gut microbiota can impact the synthesis and metabolism of a variety of substances during pregnancy, regulating body weight, blood pressure, blood sugar, blood lipids, and other physiological indexes, and even leading to some pregnancy complications . Parity has also been identified as one of the key determinants of the maternal microbiome during pregnancy. The difference in microbiome trajectories among different parities was significant in sows, with the greatest difference between zero parity and low parity animals. It was suggested that there are dramatic differences in the microbial trajectories of primiparous and multiparous animals . Compared to multiparous sows, primiparous sows had a lower gut microbiota richness and evenness during the periparturient period . Primiparous cows have different uterine and rumen microbiome compositions compared to multiparous cows . However, it is still unclear if parity impacts the maternal cow's gut microbiome during both non-pregnancy and pregnancy. In this study, the gut microbiome composition was investigated in fecal samples from primiparous and multiparous cows during non-pregnancy and pregnancy. It confirmed that there is an inherent shift in gut microbiota associated with pregnancy and differences in gut microbiota composition between primiparous and multiparous animals. The results will help develop strategies to improve the reproductive management of cows. 2. Materials and Methods 2.1. Ethics Statement The collection of biological samples and experimental procedures carried out in this study were approved by the Institutional Animal Care and Use Committee in the College of Animal Science and Technology, Sichuan Agricultural University, China (DKY20210306). 2.2. Sample Collection A total of 24 healthy Holstein cows were selected from one dairy herd under the same conditions in southwestern China, with the same feeding processes, similar body conditions, and similar body weight. According to their reproductive stages, the cows were divided into four groups: the cows before their first pregnancy (13 months, n = 6, BG); at their first pregnancy (the 4th month of pregnancy, 18 months, n = 6, FT); open cows with more than three lactations (30 days after parturition, 57 months, n = 6, DCNP); and pregnancy cows with more than three lactations (the 4th month of pregnancy, 60 months, n = 6, DCP). Animals were fed the total mixed ration (TMR) made according to NRC (2012) with the same feed raw material. None of the cows had received antibiotics in the last 3 months. All 24 fecal samples were obtained once from cow rectum content on the same day, transferred to separate sterilized 2 mL tubes, and stored immediately in liquid nitrogen. All samples were then transported to the laboratory and stored at -80 degC for further analysis. 2.3. DNA Extraction, PCR Amplification and Gene Sequencing Total genome DNA was extracted from fecal samples, the negative control (DNA free water), and the positive control (16S Universal E29), using a BIOMICS DNA Microprep Kit (Zymo Research, D4301, Irvine, CA, USA) according to the manufacturer's instructions. DNA concentration and purity were tested on 0.8% agarose gels. DNA yield was detected with a Tecan Infinite 200 PRO fluorescent reader (Tecan Systems Inc., San Jose, CA, USA). The 16S rRNA amplification covering the variable region V4-V5 was carried out using the primers 338F (5'-ACTCCTACGGGAGGCAGCAG-3') and 915R (5'-GTGCTCCCCCGCCAATTCCT-3') by a Thermal Cycler PCR system (Gene Amp 9700, ABI, Foster City, CA, USA). PCRs were performed in triplicate in a 25 mL mixture. The PCR products were diluted six times, quantified with electrophoresis on 2% agarose gel, and then purified by the Zymoclean Gel Recovery Kit (Zymo Research, D4008, Irvine, CA, USA). About 100 ng of DNA were used for library preparation. The library was prepared using the TruSeq(r) DNA PCR-Free Sample Preparation Kit (Illumina, San Diego, CA, USA), followed by quality evaluation on the Qubit@ 2.0 Fluorometer (Thermo Fisher Scientific, Waltham, MA, USA) and Agilent Bioanalyzer 2100 system (Agilent, Santa Clara, CA, USA). Library was finally paired-end sequenced (2 x 300) on an Illumina MiSeq PE300 platform (Illumina, San Diego, CA, USA). 2.4. Data Analysis The raw fastq files were merged using FLASH . The raw tags were analyzed using the QIIME (v1.9.0) pipeline . All tags were quality filtered. Sequences shorter than 200 nt with an average quality value less than 25, and those containing two or more ambiguous bases, were discarded. The clean tags were then mapped to the Gold database (accessed on 5 May 2021)) using UCHIME algorithm, followed by removal of the chimera sequences to identify the effective tags . The operational taxonomic units (OTUs) table was created at 97% similarity using the UPARSE pipeline . Representative sequences from each OTU were aligned to 16S reference sequences with PyNAST . The phylogenetic trees were drawn using FastTree . Annotation analysis was performed using the UCLUST taxonomy and the SILVA database . The abundance of OTUs was normalized using a standard sequence number corresponding to the sample with the least sequence. The comparison of OTU numbers used a one-way analysis of variance (one-way ANOVA), followed by the Bonferroni multiple comparisons test. The alpha diversity was calculated to analyze the complexity of species diversity in the sample, including observed species, Chao1, Shannon, Simpson, coverage, and Faith's PD. The beta diversity, weighted Unifrac and unweighted UniFrac, was calculated to evaluate the differences of samples in species complexity. Principal coordinate analysis (PCoA) was used to visualize differences in bacterial community composition among groups. The linear discriminant analysis coupled with effect size (LEfSe) was performed to identify the differentially abundant taxa between different groups. Pairwise comparisons were made using metagenomSeq. 3. Results 3.1. Sequencing Information In order to evaluate the effect of reproductive status on the cow fecal microbiota, the V4-V5 hypervariable regions of the 16S rRNA gene were sequenced in the microbial communities of 24 samples. A total of 705,988 raw PE reads were generated from these 24 samples (average: 29,416 +- 4914, range: 21,956-36,765). After quality control, 632,192 effective tags were obtained from 24 samples (average: 26,341 +- 4408, range: 19,472-32,926), with an average of 407.67 +- 0.92 bps per tag after the merging of overlapping paired-reads, quality filtering, and removing of chimeric sequences. By the 97% sequence similarity, 6842 OTUs were computationally constructed with 1727.38 +- 405.39 (range: 999-2788) as the mean number of OTUs per sample, and the mean number of OTU in DCNP group was significantly lower than that of BG and FT group (p < 0.01) . 3.2. Microbial Ecology of the Fecal Microbiome These 6842 OTUs taxonomically assigned to microbial 2 Kingdom, 17 phyla, 25 classes, 38 orders, 67 families, 168 genera, and 117 species. According to OTUs' number, the average abundance of each group at different category levels was evaluated . The fecal microbial communities were dominated by bacteria (84.58%), and archaea were only 15.42% abundant. The most abundant phyla across all 24 metagenomic libraries were Firmicutes (48.68%), followed by Bacteroidetes (34.45%), and Euryarchaeota (15.42%). Other less abundant phyla were Spirochaetes (0.85%), Tenericutes (0.42%), Proteobacteria (0.07%), Actinobacteria (0.06%), Fibrobacteres (0.02%), Cyanobacteria (0.02%), and Planctomycetes (0.01%) . At the genus level, there are 11 genera with more than 1.0% abundance, including Ruminococcaceae UCG-005 (21.91%), Methanobrevibacter (13.28%), Rikenellaceae RC9 gut group (10.13%), [Eubacterium] coprostanoligenes group (7.10%), Prevotellaceae UCG-004 (6.47%), Alistipes (5.52%), Ruminococcaceae UCG-013 (4.89%), Prevotellaceae UCG-003 (4.61%), Ruminococcaceae UCG-014 (1.78%), Methanocorpusculum (1.42%), Christensenellaceae R-7 group (1.12%) . 3.3. Microbial Diversity of the Fecal Microbiome The alpha diversity indexes, including observed species, Chao1, Shannon, Simpson, coverage, and Faith's PD, for four groups were calculated to estimate species richness and diversity . Compared to the BG and FT groups, the observable species, Chao1, and Faith's PD were significantly lower, and coverage was significantly higher in the DCNP group (p < 0.05, Kruskal-Wallis test), but without statistical significance in the DCP group (p > 0.05, Kruskal-Wallis test). Further, no statistically significant difference was shown among the four groups in Shannon and Simpson (p > 0.05, Kruskal-Wallis test). Based on the Jaccard and Bray-Curtis methods, principal coordinated analysis (PCoA) of beta diversity was further used to analyze compositional differences in fecal microbiota among four groups . The samples in the BG, FT, and DCP groups were clustered together according to their particular groups, while the samples in the DCNP group were spread out. The samples in the BG and FT groups tended to cluster together in accordance with PCoA results. Both Jaccard and Bray-Curtis distances showed significant differences among the four groups (ANOSIM, p < 0.01), except between groups DCP vs. DCNP (ANOSIM, p > 0.05). 3.4. Microbial Taxonomy and Function Analysis Linear discriminant analysis effect size (LEfSe) was used to discover the differential microbiota and estimate their effect size. Based on LEfSe, it restrictively analyzed the successfully annotated species and detected 60 taxa significantly different in abundance among four groups. There were 7 taxa significantly more abundant in the BG group, 17 in the FT group, 8 in the DCNP group, and 28 in the DCP group . The most representative taxa were Rikenellaceae and Rikenellaceae_RC9_gut_group in the DCP group, Prevotellaceae and Prevotellaceae_UCG_003 in the FT group, Christensenellaceae_R_7_group in the DCNP group, and Firmicutes, Clostridia, Clostridiales, and Ruminococcaceae in the BG group. The metagenomeSeq was further used to compare the abundance of OTUs between each group. The abundance of 4, 12, and 23 OTUs was significantly increased, while that of 1, 2, and 17 OTUs was significantly reduced in the FT, DCNP, and DCP groups compared with the BG group, respectively. In the three comparison groups, the abundance of six common genera (>1%), namely Prevotellaceae UCG-003, Ruminococcaceae UCG-013, [Eubacterium] coprostanoligenes group, Rikenellaceae RC9 gut group, Methanobrevibacter, and Ruminococcaceae UCG-005, was identified as a significant difference . There were 16 and 21 OTUs that were significantly increased, and 2 and 19 OTUs that were significantly reduced, in the DCNP and DCP groups compared with the FT group, respectively. A total of 8 common genera, such as the Christensenellaceae R-7 group, Ruminococcaceae UCG-014, Prevotellaceae UCG-003, Ruminococcaceae UCG-013, [Eubacterium] coprostanoligenes group, Rikenellaceae RC9 gut group, Methanobrevibacter, and Ruminococcaceae UCG-005, were observed to have significant differences . Furthermore, in the DCP group, the abundance of 4 OTUs decreased compared with the DCNP group. The relative abundance of 2 common genera, Methanobrevibacter and Prevotellaceae UCG-003, in the DCNP group was higher than that in the DCP group. 4. Discussion The reproductive efficiency and health of cows have always been priorities. The gut microbiota composition plays an important role in the reproductive performance throughout a female's lifetime. In humans, the gut microbiome has been considered to affect every stage and level of female reproduction, including follicle and oocyte maturation in the ovary, fertilization and embryo migration, implantation, the whole pregnancy, and parturition . The gut microbial communities can influence reproductive success from mate choice to healthy pregnancy and successfully producing offspring in animals . Recent studies reported that bovine vaginal and fecal microbiome associated with differential pregnancy outcomes . The fecal microbiome predicted pregnancy with a higher accuracy than that of the vaginal microbiome . In this study, the fecal microbiota were investigated in 4 different reproductive stages and revealed the dramatic changes in fecal microbiota diversity and composition among 4 groups using the sequencing of the 16S rRNA gene. In this study, Firmicutes, Bacteroidetes, and Euryarchaeota were the three most dominant phyla, and Ruminococcaceae UCG-005, Methanobrevibacter, and Rikenellaceae RC9 gut group were the three most dominant genera in the cow fecal samples. They were consistent with several earlier studies . In previous studies, Bacteroidetes (51.6~59.74%) and Firmicutes (27.6~38.74%) together comprised up to 81.6~93.20% of the cow fecal bacterial abundance . The phylum Euryarchaeota was predominant within the Archaea and accounted for around 0.25% of the cow fecal microbiota abundance . Ruminococcaceae UCG-005, Methanobrevibacter, and Rikenellaceae RC9 gut groups predominate in the Firmicutes, Euryarchaeota, and Bacteroidetes phyla, respectively. Ruminococcaceae UCG-005 and Rikenellaceae RC9 gut group usually had a relative abundance >8% of fecal microbiota in dairy cows. The genus Methanobrevibacter comprised more than 80% of the phylum Euryarchaeota in cow fecal Archaea . The age and pregnancy are two important factors contributing to the species richness and diversity of fecal microbiota. The alpha diversity index, observed species, Chao 1, coverage, and Faith's PD were significantly different among the BG, FT, and DCNP groups in this study. However, the cluster among four groups was significant, separating BG and FT groups from DCNP and DCP groups by PCoA based on Jaccard and Bray-Curtis distances. These also showed that the greatest differences in microbiome trajectories occurred between nulliparous and primiparous animals . Nulliparous animals had higher gut microbial diversity than that of primiparous animals, and pregnancy could increase gut microbial diversity . The effect of age is more related to calving. The increase in alpha diversity during pregnancy could be due to an increase in nutrient requirements during lactation. The first birth is the most important physiological change in a cow's life, and pregnancy increases metabolism. In order to further identify important taxa differed among groups, LEfse and metagenomeSeq analyses were conducted. LEfse analysis is helpful to discover the important differential taxa (biomarkers) and estimate their effect sizes. The LEfSe analysis revealed that the most differentially abundant taxa were in DCP, followed by FT, DCNP, and BG. The metagenomeSeq analyses showed that the comparisons with the most significant differences in microbial taxa are BG vs. DCP and FT vs. DCP, followed by FT vs. DCNP, BG vs. DCNP, BG vs. FT, and DCNP vs. DCP. These suggested that parturition experience is one of the most important factors to impact cattle gut microbiome trajectory. Previous study also reported that the most difference in microbiome trajectory occurred between nulliparous and low parity sows . There was significant difference between multiparous and primiparous cows on vaginal and uterine microbiotas . The most representative taxa were associated with energy metabolism and inflammation. Mice fed with high-fat diet increased the richness of gut microbial Rikenellaceae_RC9_gut_group. The high-fat diet also increased the risks of intestinal pathogen colonization and inflammation . Supplementation of probiotics increased the relative abundance of Prevotellaceae_UCG_003, which improved the energy status of the beef steers . Fibrolytic enzyme increased the relative abundance of Christensenellaceae_R_7_group, which improve the average daily gain and feed conversion ratio of lambs . The ruminococcaceae family is the predominant acetogen in the cattle rumen, which is related to cellulose and hemicellulose degradation . The carbohydrate resource and the fiber decomposition process in diet contribute to the different abundances of Ruminococcaceae UCG-005, Ruminococcaceae UCG-013, Ruminococcaceae UCG-014, and other Ruminococcaceae in cattle feces . Methanobrevibacter is another common inhabitant of the cattle rumen, which can reduce CO2 with H2 to form methane .The serum cholesterol concentration tended to be lower after feeding Eubacterium coprostanoligenes to germ-free mice . Thus, gut microbes are involved in changes in energy intake and immunity during cattle adaption to pregnancy. 5. Conclusions In conclusion, this study investigated the difference in fecal bacterial communities between primiparous and multiparous cows during non-pregnancy and pregnancy. The results revealed that pregnancy increased the relative abundance and diversity of fecal microbiota, while aging reduced those traits. In addition, primiparous were related to a profound alteration of the fecal microbiota. The most representative taxa included Rikenellaceae_RC9_gut_group, Prevotellaceae_UCG_003, Christensenellaceae_R_7_group, Ruminococcaceae UCG-005, Ruminococcaceae UCG-013, Ruminococcaceae UCG-014, Methanobrevibacter, and [Eubacterium] coprostanoligenes group, which were associated with energy metabolism and inflammation. In the future, further functional studies will be able to treat dysbiosis and prevent disease development during pregnancy by using probiotics or fecal transplantation. Author Contributions Conceptualization, S.L. and X.J.; methodology, Y.H.; software, S.C.; validation, Z.K. and J.W.; formal analysis, X.J.; investigation, X.J.; resources, S.L.; data curation, W.S.; writing--original draft preparation, X.J.; writing--review and editing, J.W. and W.S.; supervision, S.L.; project administration, X.J.; funding acquisition, S.L. All authors have read and agreed to the published version of the manuscript. Institutional Review Board Statement Not applicable. Informed Consent Statement Not applicable. Data Availability Statement The data presented in this study are available on request from the corresponding author. Conflicts of Interest The authors declare no conflict of interest. Figure 1 Histogram of the number of OTUs in the four groups. Different letters indicate a significant difference among groups (p < 0.05), while the same letter indicates no difference among groups (p > 0.05). Figure 2 The fecal microbiota composition of the four groups. Figure 3 Relative abundance of fecal microbiota at the phylum level in the four groups. Figure 4 Heatmap of high abundance at genus level in the four groups. Figure 5 Box-plot representation of alpha diversity among four groups. The number of observed OTUs (A), Chao1 (B), Shannon (C), Simpson (D), Coverage (E) and Faith's PD (F) among the four groups. Figure 6 Principal Coordinates Analysis (PCoA) using Jaccard distance (A) and Bray-Curtis distance (B) among the four groups. Figure 7 Histogram of the LDA scores for differentially abundant of fecal bacteria among the four groups. Figure 8 The differentially enriched OTUs among the four groups. Adjusted p < 0.05 (blue) or p < 0.01 (red) were considered significant. (A)-BG VS. FT, (B)-BG VS. DNCP, (C)-BG VS. DCP, (D)-FT VS. DNCP, (E)-FT VS. DCP, (F)-DNCP VS. DCP. Disclaimer/Publisher's Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

PMC10000136

Background: Liver transplantation is the only promising treatment for end-stage liver disease and patients with hepatocellular carcinoma. However, too many organs are rejected for transplantation. Methods: We analyzed the factors involved in organ allocation in our transplant center and reviewed all livers that were declined for transplantation. Reasons for declining organs for transplantation were categorized as major extended donor criteria (maEDC), size mismatch and vascular problems, medical reasons and risk of disease transmission, and other reasons. The fate of the declined organs was analyzed. Results: 1086 declined organs were offered 1200 times. A total of 31% of the livers were declined because of maEDC, 35.5% because of size mismatch and vascular problems, 15.8% because of medical reasons and risk of disease transmission, and 20.7% because of other reasons. A total of 40% of the declined organs were allocated and transplanted. A total of 50% of the organs were completely discarded, and significantly more of these grafts had maEDC than grafts that were eventually allocated (37.5% vs. 17.7%, p < 0.001). Conclusion: Most organs were declined because of poor organ quality. Donor-recipient matching at time of allocation and organ preservation must be improved by allocating maEDC grafts using individualized algorithms that avoid high-risk donor-recipient combinations and unnecessary organ declination. Eurotransplant extended donor criteria liver transplantation declined organs MELD extended right lobe liver transplantation hepatocellular carcinoma HCC The study received no external funding. pmc1. Introduction Liver transplantation is the only promising treatment for end-stage liver disease and patients with hepatocellular carcinoma (HCC). Although efforts have been made to expand the pool of available organs without compromising patient safety and ethical standards, too many organs are still declined for transplantation. Between 2016 and 2020, 10,113 liver donors were reported to Eurotransplant (ET), but 2428 (24%) of these organs were not used. During this time, 2116 (~30%) of all listed transplant candidates in ET died while waiting for an organ, and 462 candidates dropped out of the waiting list because they became too sick to receive a transplant . Similar trends have also been reported in the United States, where transplant candidates received a median of five liver offers before they died or dropped out of the waiting list, and 84% of the candidates who were removed from the waiting list because they became too sick to receive a transplant or died while waiting for an organ, received at least one offer before the terminal event . These patients received a median of two organ offers more than patients who were eventually transplanted, and the organs were refused, most commonly because of the donor quality or age . Similar trends have been reported in Germany . Efforts to improve access to transplantation have revealed differences in organ donation and organ acceptance practices . In Germany, donor and donor organ assessment is standardized by the German medical chamber (Bundesarztekammer (BAK)) and by the German Organ Procurement Organization (Deutsche Stiftung Organtransplantation (DSO)). For liver transplantation, ET allocation algorithms are also included in the BAK guidelines. Despite these detailed guidelines on organ allocation and the fact that all relevant information on organs procured in Germany is usually fully available to the transplant centers, the decisions are often subjective during allocation, and whether to accept or decline an organ offer is at the discretion of the local transplant team . Transplant surgeons typically prefer to avoid risk and decide whether to allocate a donor organ based on short-term results . Instead of accepting an organ that may be associated with a high risk of poor postoperative outcome, transplant surgeons tend to wait longer for a better graft, disregarding the potential benefits the organ may offer, and that there is no guarantee that a better organ will become available before the transplant candidate dies or is removed from the waiting list because the disease (e.g., HCC) progressed beyond the transplantation criteria . There are many reasons why an organ may be declined for transplantation. To better understand this complex situation, we analyzed the factors involved in organ allocation in our transplant center. We evaluated how organs were allocated for transplantation, why these organs were declined, and what happened to them after they were declined. 2. Materials and Methods All livers from brain-dead donors that were declined for transplantation by our center between 2016 and 2020 were reviewed. Data on donor and recipient characteristics, allocation procedures, reasons for declining, and the fate of declined organs were collected from a prospective database, a transplant registry, written and electronic medical records, ET allocation protocols, and DSO protocols. Organ offers were analyzed separately from organs that may have been offered more than once (one offer one case). The reasons why an organ was declined were extracted from handwritten and electronic allocation protocols and no organ offer was excluded from the analysis. The Ethics Committee of the University of Heidelberg approved the analysis (S-548/2012). 2.1. Organ Allocation Offered organs were characterized as primary, extended, or rescue allocations . 2.1.1. Primary Allocation Donor organs are reported to ET, which prioritizes organ allocation based on urgency and model for end-stage liver disease (MELD) scores . Liver transplant candidates are ranked on a match list and ET offers the organ to the first candidate on this list and contacts the corresponding center (primary allocation), where the local transplant surgeon decides whether to accept the organ or not. Two candidates receive the same organ offer, but the higher-ranked candidate is prioritized. This decision has to be made within 30 min and the reason for declining an organ is recorded . 2.1.2. Extended Allocation If the organ cannot be delivered to the transplant center in time or if the organ is refused by three different centers, ET offers the organ to regional transplant centers (patient-oriented extended allocation). The regional centers have to choose two patients from their waiting list and report them to ET within 30 min. The offer goes to the highest-ranked candidate . 2.1.3. Rescue Allocation If the extended allocation fails, the organ is offered to further centers on a first-come first-served basis (center-oriented rescue allocation). If no suitable recipient can be found within ET, the organ is offered to other countries. Together with the local coordinator, ET can decide to withdraw the organ . 2.1.4. Assessing Transplant Candidate Condition Transplant candidate condition was assessed using the laboratory model for end-stage liver disease (labMELD) score. A cut-off value of 20 differentiated between low-risk (labMELD < 20) and high-risk (labMELD >= 20) transplant candidates, as previously reported . Transplant candidates with standard exceptional MELD (eMELD) scores received MELD points that started at a fixed initial value. The eMELD score was upgraded as long as the defining condition persisted . The matchMELD score was calculated as the highest labMELD or eMELD score. 2.2. Reasons for Declining Organs for Transplantation A categorization system was developed to characterize the reasons for declining an organ. Multiple reasons for declining were possible and different offers may have been declined for the same reason. 2.2.1. Major Extended Donor Criteria Donor age > 65 years, biopsy proven macrovesicular steatosis >40%, estimated prolonged cold ischemia time (CIT) > 14 h, and extended right lobe (ERL) livers were considered major extended donor criteria (maEDC) . Organs declined because of maEDC were analyzed separately. 2.2.2. Size Mismatch and Vascular Problems Organs were declined because of size mismatch if the body mass index (BMI) of the donor and recipient were significantly different. Body surface area (BSA) was calculated using the formula BSA = weight (kg) 0.425 x height (cm) 0.725 x 0.007184. The body surface area index (BSAi) was calculated using the formula BSAi = donor BSA/recipient BSA, as previously reported . BMI and BSAi values were compared between organs declined because of size mismatch and organs declined for other reasons. Vascular problems considered valid reasons for declining an organ were short graft vessels, mismatch between vascular diameter of the donor and recipient, and vascular injury during the organ procurement phase. 2.2.3. Medical Reasons and Risk of Disease Transmission Organ offers from donors with severely abnormal liver function tests (transaminases more than three times the normal level [46-50 U/L] and bilirubin >3 mg/dL), severe hypernatremia (>165 mmol/L), livers from hemodynamically instable donors or from donors who have been reanimated over longer periods, and offers from donors with multiple comorbidities or from donors who drowned in salty water were considered high-risk and were declined. Organ offers from donors with recent malignancy and/or infectious diseases were declined because they were categorized as high or unacceptable risk for transplantation according to the Guide to the Quality and Safety of Organs for Transplantation or because the risk-benefit analysis did not justify acceptance for the recipient . 2.2.4. Other Reasons Rescue allocation organs were declined if the recipient's blood group was incompatible with that of the donor or if the recipient was not transplantable at the time of the allocation. 2.3. Fate of Declined Organs The fate of the declined organs was also analyzed and the reasons for declining an organ were compared between the group of further allocated and transplanted organs and the group of completely discarded organs. 2.4. Statistical Analysis IBM SPSS Statistics for Windows, Version 22.0 (IBM Corp., released 2013, Armonk, NY, USA) was used for statistical analyses. Continuous data are expressed as mean +- standard deviation (SD) or median and range, and categorical variables are shown as percentages. Independent t-test and Mann-Whitney U test for non-normally distributed data were used to compare continuous variables between groups. Categorical variables were analyzed using the Pearson chi-square test or Fisher exact test. A two-sided p < 0.05 was considered statistically significant. 3. Results 3.1. Allocation Type, Donor, and Recipient Characteristics Our transplant center was offered 1311 livers, and we declined 1086 organs that were offered 1200 times (462 primary, 180 extended, and 524 rescue allocation offers). Allocation type was missing for 34 organ offers. Of the 1086 offered organs, 863 were declined once, 99 twice, seven three times, and one four times. The median donor age was 59 years (range 0-96 years), and 56% of donors were male. The mean donor BMI was 27.21 +- 6.33 kg/m2, and the mean recipient BMI was 25.9 +- 5.82 kg/m2. The median transplant candidate age was 52 years, and 51% were male (Table 1). 3.2. Waiting List Dynamics During the study period, 203 liver transplantations were performed, and 255 of 600 transplant candidates on the waiting list either recovered or asked to be removed from the waiting list. Ninety-one transplant candidates died while waiting for transplantation, and 36 patients were removed from the waiting list because their disease had progressed beyond the transplantation criteria. Fifteen patients were referred to another center adjacent to their new place of residence. The mean labMELD score was 20.2 +- 9.79 and the mean eMELD score was 27.41 +- 6.15 (Table 1). In non-HCC patients listed for transplantation, the mean labMELD score was 21 +- 9.7, and 111 of 539 non-HCC patients were removed from the waiting list or died of their disease. Each non-HCC patient received at least one organ offer (median, 2; range, 1-26). A median of 9 days (range 1-437) passed between the last organ offer and death or removal from the waiting list in these candidates. In transplant candidates with HCC, the mean eMELD score was 27.41 +- 6.2, and 22 of 61 HCC patients were removed from the waiting list because they did not fulfill the criteria for transplantation anymore or died of their disease. Each transplant candidate with HCC received at least one organ offer (median, 2; range, 1-24). A median of 35 days (range 2-113) passed between the last organ offer and death or removal from the waiting list. 3.3. Reasons for Declining an Organ for Transplantation 3.3.1. Major Extended Donor Criteria A total of 336 (30.9%) of 1086 livers (368 of 1200 offers) with maEDC were declined. We declined 45 (4.1%) livers from older donors, 192 (17.7%) macroscopic fatty livers or livers with biopsy proven macrovesicular steatosis, 72 (6.6%) livers with a long estimated conservation time, and 48 (4.4%) split livers (Table 2). Interestingly, none of the organs from donors older than 65 years were split livers. The labMELD and eMELD scores were similar between transplant candidates who were offered livers from donors older than 65 years and transplant candidates who were offered livers from younger donors. Donors of steatotic livers were significantly older than donors of non-steatotic livers. Fewer steatotic livers with a longer estimated CIT were offered than non-steatotic livers with a longer CIT. None of the offered steatotic livers were split livers and most of these organs were primarily offered to transplant candidates with lower labMELD scores (Table 2). Donors of split livers were significantly younger than donors of full-size livers, and the estimated CIT was significantly longer in split livers. The labMELD scores between the groups were comparable, but split livers were predominantly allocated to female patients and patients with higher eMELD scores (Table 2). 3.3.2. Size Mismatch and Vascular Problems A total of 386 (35.5%) size mismatch livers of 1086 livers (427 of 1200 organ offers) were declined, and 6 livers (seven offers) with vascular problems were declined. The BMI and BSAi of both donor and transplant candidate were known in 669 organ allocation offers. As expected, BMI differences were greater between donors and candidates in the size mismatch group than in the control group (6.32 +- 5.96 vs. 4.10 +- 3.73, p < 0.001) (Table 3). BSAi extremes (<0.78 and >1.24) were significantly higher in the size mismatch group than in the control group (9.6% vs. 4.9%, p < 0.001, and 26.3% vs. 2.6%, p < 0.001, respectively) indicating an optimal estimation of the donor-recipient size mismatch. The BSAi of offered livers was in the normal range (0.78-1.24) for 539 organs. However, 180 organ offers were declined because of size mismatch despite the normal range of BSAi (0.78-1.24), indicating a suboptimal estimation of the donor-recipient size mismatch in 64.1% of the 281 organ offers that were declined based on BMI size mismatch estimation (Table 3). 3.3.3. Medical Reasons, Risk of Disease Transmission, and Other Reasons A total of 172 (15.8%) of 1086 livers (188 of 1200 organ offers) were declined because of medical reasons and risk of disease transmission, and 225 (20.7%) organs (253 offers) were declined because of other reasons. 3.4. Fate of Declined Organs 3.4.1. Further Allocated and Transplanted Organs A total of 430 (40%) of 1086 declined organs were allocated and transplanted in our center (n = 32; 7%) or in another transplant center (n = 398; 93%) . Of these 430 organs, 76 (18%) were declined because of maEDC, 190 (44%) because of size mismatch and vascular problems, 61 (14%) because of medical reasons and risk of disease transmission, and 103 (24%) because of other reasons. The fate of organs that were declined because of maEDC is summarized in Figure 2. 3.4.2. Discarded Organs A total of 547 (50%) of 1086 declined livers were completely discarded; 498 (91%) of these were not further allocated, and 49 (9%) livers were transferred and then discarded in another center. Of these 547 organs, 205 (37%) were declined because of maEDC, 148 (27%) because of size mismatch and vascular problems, 98 (18%) because of medical reasons and risk of disease transmission, and 96 (18%) because of other reasons . The fate of organs that were discarded because of maEDC is summarized in Figure 2. 3.4.3. Unknown Fate The fate of 109 (10%) of 1086 declined livers were unknown. Of these 109 organs offered from abroad, 16 (14%) were declined because of maEDC, 54 (50%) because of size mismatch and vascular problems, 13 (12%) because of medical reasons and risk of disease transmission, and 26 (24%) because of other reasons . The fate of organs that were declined because of maEDC is summarized in Figure 2. The reasons for organ declination and the fate of declined organs are summarized in Table 4. Significantly more grafts that were completely discarded had maEDC than grafts that were eventually allocated (37.5% vs. 17.7%, p < 0.001). Size mismatch and vascular problems were significantly more prevalent in the group of allocated organs than in the group of discarded organs (44.2% vs. 27.1%, p < 0.001 and 24% vs. 17.6%, p = 0.016, respectively). The fate of organs that were declined because of maEDC is summarized in Figure 2. 4. Discussion Since 1967, patient-oriented ET has facilitated international organ exchange for transplantation within Europe . A shortage of organs has driven transplant centers to accept maEDC livers for transplantation in recent years . These organs are acceptable for adult recipients with lower labMELD scores and recipients with HCC who are generally in a better condition because they do not affect patient survival . However, maEDC livers are associated with postoperative complications and affect graft survival, so careful donor-recipient matching is important . Still, far too many organs are declined . Every organ that is declined is a missed opportunity that increases mortality on the waiting lists. This study has shown that 50% of potentially suitable organs are declined. This indicates that decision-making is not standardized during allocation, and that whether to accept or decline an organ is at the discretion of the local transplant teams, who evaluate the organ risk based on the information available. Our results show that while an organ might be unsuitable for one recipient, it might be suitable for another. We also show that patient care can be improved and emphasize the need for optimized allocation protocols to avoid unnecessary declination of organs. This is particularly relevant to HCC patients and to maEDC grafts, which are becoming the new "standard" and need specific allocation policies. The median donor age has increased in ET, and >33% of the donors are now >=65 years old . The risk of graft loss increases linearly with donor age, but survival benefits have been reported in recipients of grafts from older donors, especially older recipients and recipients with HCC, who seem to be less affected by advanced donor age . The present study showed that the acceptance rate of livers from older donors is high, which may reflect the pressures of organ shortage. Macrovesicular steatosis >40% is the strongest predictor of graft loss, especially in combination with advanced donor age or longer CIT . However, HCC patients with labMELD scores <20 seem to be less affected by steatotic livers . In our study, steatotic livers were primarily offered to low-risk transplant candidates; however, most steatotic livers were completely rejected, even after being allocated to other centers. A prolonged CIT reduces graft survival, but this depends strongly on the underlying disease. A longer CIT reduces survival more in patients with hepatitis C-related cirrhosis; in contrast, patients with HCC and alcoholic cirrhosis with labMELD scores <20 can tolerate a longer CIT without significant impact on survival. This suggests that grafts with longer CITs should preferentially be allocated to these recipients . The present study showed that over two thirds of organs declined because of CIT were completely rejected. Most ERL grafts were declined because of donor-recipient size mismatch. However, ERL livers were also declined because they were offered to high-risk recipients. On the one hand, this decision is justified as ERL grafts have been associated with higher vasculobiliary complication rates, re-transplantation rates, and lower graft survival . On the other hand, patient survival has been shown to be unaffected by ERL livers, indicating that these grafts can be used safely . However, only optimal, high-quality organs are considered for split-liver transplantations, and a short CIT is essential for therapy success . The allocation process is very complex and the estimation of CIT before organ procurement is imprecise. Still, the conservation time is ~50% longer in split liver transplantation than in full size transplantation. With this in mind, ex situ split livers should be allocated to patients at the center performing the split procedure, whereas in situ split grafts can be allocated to different centers. In this way, conservation time can be reduced and allocation and prioritizing bias can be avoided . Split liver was considered a maEDC, but it was not a primary reason for declining an organ in our study. This may explain the high transplantation rate of 92%, but it may also reflect the pressures of organ shortage. We recently suggested an allocation algorithm that balances the number of maEDC with the recipient's condition . According to this algorithm, grafts with more than one maEDC should be allocated to low-risk transplant candidates and/or transplant candidates with HCC or alcoholic liver cirrhosis who are less vulnerable to the effects of maEDC . This leaves non-maEDC grafts free for recipients who need them, such as patients with hepatitis C-related cirrhosis or high-risk patients with labMELD scores of > 20. Most of the offered maEDC grafts were completely discarded, even after being allocated to other centers. This emphasizes the poor organ quality in ET and shows that maEDC organs are still considered unsuitable for transplantation and are discarded most of the time . However, this might also reflect a suboptimal allocation of maEDC grafts to suitable recipients, possibly because accurate maEDC data were missing at the time of allocation. Donor age and steatosis cannot be modified, but livers from older donors or fatty livers can be allocated more adequately. On the one hand, inspection-based liver assessment is a poor predictor of higher-grade steatosis . On the other hand, waiting for pathology results after organ procurement prolongs CIT unnecessarily. Each additional hour of cold storage increases the risk of 1-year graft loss by 3.4% after full-size liver transplantation and by 10% after extended right lobe liver transplantation (ERLT) . Therefore, to accurately assess the grade of steatosis, a biopsy should be taken and the steatosis grade of the donor liver (alone or in combination with other maEDC, especially CIT) should be considered even before allocation to avoid high-risk donor-recipient combinations and to ensure optimal recipients are selected for each organ. This would reduce the number of organs that are declined. Ideally, an organ should be transplanted into the intended recipient, but there should always be another candidate close to the transplant center with an uncomplicated anatomy to whom the liver can be reallocated without prolonging cold storage . Other centers were willing to accept some maEDC grafts that we had declined, such as organs from older donors and split livers, possibly because their candidates were more suitable for these grafts, or possibly because these centers used machine perfusion techniques that reduce ischemia reperfusion injury and increase organ viability . Although we cannot confirm that machine perfusion was used in these reallocations, our results suggest that optimizing allocation logistics and organ preservation can solve the problems of prolonged CIT, which is the only modifiable maEDC. Deeper understanding of HCC biology and artificial intelligence could ensure optimal organ allocation . Before declining organs because of size mismatch, more suitable parameters should be considered. Fukazawa et al. developed a model that estimated 3-year graft survival based on the BSAi in a collective of 24,509 patients . The authors suggested a BSAi range of 0.78-1.24 for avoiding adverse outcomes associated with size mismatch . In agreement with this, we also found that the BSAi may be a more sensitive indicator of size mismatch than BMI, as well as the fact that we may have been too cautious and declined too many organs because of size mismatch in the past. Most of the organs we have declined because of size mismatch were transplanted elsewhere, and we have now integrated the BSAi into our recipient assessment and organ allocation protocols. Only one third of the grafts that were declined for medical reasons were transplanted elsewhere, confirming that our decision to decline them was solid. However, it is not clear whether the eventual recipients of these organs were in fact suitable. This is a limitation of our study. Most of the competitive organ offers were accepted elsewhere because there was no matching recipient on our waiting list, or our recipients were non-transplantable. This identifies important communication gaps between transplant institutions that need to be overcome in the future to ensure optimal organ allocation. LabMELD scores among the listed non-HCC transplant candidates at the time of organ offer and the time that passed between their last organ offer and their death or removal from the waiting list suggest that these candidates were more ill than their labMELD score indicated at the time of organ offer. This shows that high-risk graft offers were declined and that transplant surgeons either waited for the clinical condition to improve or hoped for a better organ offer. This confirms the subjective nature of the decision-making process. 5. Conclusions The numbers of organs that were further allocated and transplanted shows that the ET allocation model provides equal opportunities for all transplant candidates. However, most organs were declined because of organ quality and for reasons that cannot be modified. High-quality donor organs are scarce, so transplant surgeons must make high-risk subjective decisions on whether to accept a potentially risky organ or wait for a better one that may not come before the patient dies or has to be removed from the waiting list because the disease, especially HCC, has progressed beyond the transplant criteria. To help with this challenge, donor-recipient matching at time of allocation and organ preservation must be improved. This would allow the avoiding of unnecessary organ transport and reduce costs and could be achieved by allocating maEDC grafts using individualized algorithms that avoid high-risk donor-recipient combinations and unnecessary organ declination. Importantly, the impact of CIT on post-transplant outcomes could be improved by managing CIT during organ procurement and by promoting communication between institutions during organ allocation. More transparency regarding decisions to accept or decline an organ may also help standardize organ allocation, and the donor pool could be expanded by accepting the policy that everybody is a donor unless they opt out. Author Contributions V.J.L. designed the study, collected and analyzed the data, and wrote the manuscript. S.A., E.K., O.G., E.A. and C.S. were responsible for data preparation and analysis. C.S., T.H., B.P.M.-S., U.M., S.P., F.L., D.-H.C., M.M., H.F. and M.G. participated in research and data analysis. A.M. co-designed the study, analyzed the data, and participated in the writing of the manuscript. All authors have read and agreed to the published version of the manuscript. Institutional Review Board Statement The study was conducted in accordance with the Declaration of Helsinki and approved by the Ethics Committee of the University of Heidelberg (S-548/2012) on 7 December 2012. Informed Consent Statement Informed consent was obtained from all subjects involved in the study. Data Availability Statement The data presented in this study are available on request from the corresponding author. Conflicts of Interest The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Abbreviations BAK, Bundesarztekammer; BMI: body mass index; BSA, body surface area; BSAi, body surface area index; CIT, cold ischemia time; DSO, German Organ Procurement Organization (Deutsche Stiftung Organtransplantation); EDC, extended donor criteria; eMELD, exceptional model of end-stage liver disease; ERLT, extended right lobe liver transplantation; ET, Eurotransplant; HCC, hepatocellular carcinoma; maEDC, major extended donor criteria; MELD, model of end-stage liver disease; SD, standard deviation. Figure 1 Flow diagram of declined organs (n = 1086). Fate of the declined organs (A-C) and reasons for declining an organ (*). maEDC, major extended donor criteria. Figure 2 Flow diagram and fate of organs declined because of major extended donor criteria (maEDC; n = 336; 31% of all declined organs). Multiple reasons for declining were possible, and different offers may have been declined for the same reason. CIT, cold ischemia time; maEDC, major extended donor criteria. cancers-15-01365-t001_Table 1 Table 1 Demographic characteristics and clinical donor and recipient parameters. Donor Characteristics Mean Median SD Range (min-max) Age (years) 57 59 20 0-96 BMI (kg/m2) 27.21 26.12 6.33 11-68 Weight (kg) 81 80 22.71 3-190 Height (cm) 171 174 17 52-200 Gender Female (n, %) Male (n, %) 475 (44) 611 (56) Recipient characteristics Mean Median SD Range (min-max) Age (years) 49 52 12 15-74 BMI (kg/m2) 25.94 25.03 5.82 17-53 Weight (kg) 77.5 75 22.34 42-185 Height (cm) 172 170 10 147-205 labMELD 20.2 18 9.79 6-40 eMELD 27.41 27.53 6.15 2-40 MatchMELD 23.86 23 9.11 7-40 Gender Female (n, %) Male (n, %) 330 (49) 346 (51) BMI, body mass index; labMELD, laboratory model for end-stage liver disease score; eMELD, standard exception model for end-stage liver disease score; matchMELD, either eMELD or labMELD score (the higher value was chosen for allocation purposes). cancers-15-01365-t002_Table 2 Table 2 Comparison of donor and recipient characteristics in cases of organs declined because of major extended donor criteria (maEDC) and organs declined for other reasons. Donor Age > 65 Years Yes (n = 45) No (n = 1041) p Female donor (n, %) 31 (68.9) 443 (42.6) <0.001 Donor age (mean +- SD) 81 +- 7.2 56 +- 19.2 <0.001 Donor BMI (mean +- SD) 26.2 +- 6 27.3 +- 6.3 0.25 Liver steatosis (n, %) 2 (4.4) 190 (18.3) 0.015 Split organs (n, %) 0 (0) 48 (4.6) 0.258 Estimated CIT > 14 h (n, %) 4 (8.9) 68 (6.5) 0.534 Female recipient (n, %) 21 (46.7) 273 (26.2) 0.005 Recipient age (mean +- SD) 46 +- 14 49 +- 12.5 0.174 Recipient BMI (mean +- SD) 26.4 +- 5.6 25.7 +- 5.6 0.53 labMELD (mean +- SD) 19.8 +- 8.7 20.8 +- 10 0.582 eMELD (mean +- SD) 28.2 +- 6 27.5 +- 5.9 0.774 Liver steatosis Yes (n = 192) No (n = 894) p Female donor (n, %) 85 (44.3) 389 (43.5) 0.873 Donor age (mean +- SD) 62 +- 15.8 55 +- 20.1 <0.001 Donor age > 65 years (n, %) 81 (42.2) 294 (32.9) 0.015 Donor BMI (mean +- SD) 29.5 +- 6.7 26.7 +- 6.1 <0.001 Split organs (n, %) 0 (0) 48 (5.4) <0.001 Estimated CIT > 14 h (n, %) 6 (3.1) 66 (7.4) 0.036 Female recipient (n, %) 25 (29.1) 269 (52.7) <0.001 Recipient age (mean +- SD) 52 +- 9 49 +- 13 0.012 Recipient BMI (mean +- SD) 27.7 +- 5.1 25.4 +- 5.6 <0.001 labMELD (mean +- SD) 18.3 +- 8.3 21.2 +- 10.1 0.007 eMELD (mean +- SD) 25.0 +- 3.2 27.8 +- 6.1 0.106 Long estimated CIT Yes (n = 72) No (n = 1014) p Female donor (n, %) 27 (37.5) 447 (44.1) 0.325 Donor age (mean +- SD) 55 +- 17.3 57 +- 19.7 0.361 Donor age > 65 years (n, %) 22 (30.6) 353 (34.8) 0.522 Donor BMI (mean +- SD) 26.4 +- 4.4 27.3 +- 6.4 0.141 Liver steatosis (n, %) 6 (8.3) 186 (18.3) 0.036 Split organs (n, %) 8 (11.1) 40 (3.9) 0.011 Female recipient (%) 12 (40) 282 (49.8) 0.35 Recipient age (mean +- SD) 47 +- 13.7 49 +- 12.5 0.309 Recipient BMI (mean +- SD) 25.6 +- 4.4 25.8 +- 5.6 0.902 labMELD (mean +- SD) 21.7 +- 12 20.7 +- 9.8 0.676 eMELD (mean +- SD) 32.4 +- 7.3 27.3 +- 5.8 0.090 Split livers Female donor (n, %) Yes (n = 48) No (n = 1038) p Female donor (n, %) 20 (41.7) 454 (43.7) 0.882 Donor age (mean +- SD) 36 +- 15.4 58 +- 19.2 <0.001 Donor age > 65 years (n, %) 0 (0) 375 (36.1) <0.001 Donor BMI (mean +- SD) 23.1 +- 3.2 27.4 +- 6.4 <0.001 Liver steatosis (n, %) 0 (0) 192 (18.5) <0.001 Estimated CIT > 14 h (n, %) 8 (16.7) 64 (6.2) 0.011 Female recipient (n, %) 25 (65.8) 269 (48.2) 0.044 Recipient age (mean +- SD) 44 +- 14 49 +- 12.4 0.010 Recipient BMI (mean +- SD) 24.8 +- 7.5 25.8 +- 5.4 0.289 labMELD (mean +- SD) 21 +- 11.7 20.7 +- 9.8 0.864 eMELD (mean +- SD) 31.3 +- 5.5 26.9 +- 5.8 0.006 BMI, body mass index; CIT, cold ischemia time; labMELD, laboratory model for end-stage liver disease score; eMELD, standard exception model for end-stage liver disease score; SD, standard deviation. cancers-15-01365-t003_Table 3 Table 3 BSAi analysis and comparison between organ offers declined because of size (BMI) mismatch and offers declined because of reasons other than size mismatch. Size Mismatch n = 281 Reasons to Decline Other than Size Mismatch n = 388 p BSAi BSAi < 0.78 (n, %) 27 (9.6) 19 (4.9) <0.001 BSAi = 0.78-1.24 (n, %) 180 (64.1) 359 (92.5) BSAi > 1.24 (n, %) 74 (26.3) 10 (2.6) BMI BMI (mean +- SD) 6.32 +- 5.96 4.1 +- 3.73 <0.001 BMI (median (range)) 5.12 (0.11-35.88) 3.27 (0.02-31.42) BSAi, body surface area index; BMI, body mass index. cancers-15-01365-t004_Table 4 Table 4 Reasons for declining an organ and comparison between organs that were reallocated and organs that were discarded. Reasons for Declining an Organ Further Allocated and Transplanted Organs n = 430 Discarded Organs n = 547 p maEDC (n, %) 76 (17.7%) 205 (37.5%) <0.001 Size mismatch and vascular problems (n, %) 190 (44.2%) 148 (27.1%) <0.001 Liver function and medical issues (n, %) 61 (14.2%) 98 (17.9%) 0.138 Other reasons (n, %) 103 (24%) 96 (17.6%) 0.016 maEDC, major extended donor criteria. 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PMC10000137

The growth hormone receptor (GHR) is a member of the cytokine/hematopoietic factor receptor superfamily, which plays an important role in the growth and development, immunity, and metabolism of animals. This study identified a 246 bp deletion variant in the intronic region of the GHR gene, and three genotypes, including type II, type ID, and type DD, were observed. Genotype analysis of structural variation (SV) was performed on 585 individuals from 14 yak breeds, and it was found that 246 bp deletion was present in each breed. The II genotype was dominant in all yak breeds except for SB yak. The association analysis of gene polymorphisms and growth traits in the ASD yak population showed that the 246 bp SV was significantly associated with body length at 6 months (p < 0.05). GHR messenger RNA (mRNA) was expressed in all the tested tissues, with significantly higher levels in the liver, muscle, and fat than in other organs. The results of transcription activity showed that the luciferase activity of the pGL4.10-DD vector was significantly higher than that of the pGL4.10-II vector (p < 0.05). Additionally, the transcription-factor binding prediction results showed that the SV in the runt-related transcription factor 1 (Runx1) transcription-factor binding site may affect the transcriptional activity of the GHR gene, regulating yak growth and development. This study showed that the novel SV of the GHR gene could be used as a candidate molecular marker for the selection of the early growth trait in ASD yak. Bos grunniens growth hormone receptor (GHR) gene growth traits SV 246 bp candidate molecular marker National Key R&D Program of China2021YFD1600200 Central Public-interest Scientific Institution Basal Research FundY2022LM10 Agricultural Science and Technology Innovation Program25-LZIHPS-01 China Agriculture Research System of MOF and MARACARS-37 Foundation for Innovation Groups of Basic Research in Gansu Province20JR5RA580 This study was supported by the National Key R&D Program of China (2021YFD1600200), Central Public-interest Scientific Institution Basal Research Fund (Y2022LM10), Agricultural Science and Technology Innovation Program (25-LZIHPS-01), China Agriculture Research System of MOF and MARA (CARS-37) and Foundation for Innovation Groups of Basic Research in Gansu Province (20JR5RA580). pmc1. Introduction Yak (Bos grunniens) mainly lives in the Qinghai-Tibet Plateau at an altitude of 3000-5500 m . In China, there are about 16 million yaks, accounting for about 95% of the world's population . It is an important and dominant animal species in plateau animal husbandry, which can provide meat, milk, fur, etc. . Among them, yak meat is one of the main resources of the plateau animal husbandry economy . Compared with cattle beef, yak meat is high in protein and energy, low in fat, and rich in amino acids, and the habitat of yak is least affected by humans. Therefore, yak meat is considered a natural green food, which meets people's demand for green and healthy meat quality . As a plateau variety, yak can protect the Qinghai-Tibet Plateau ecosystem and promote local economic development. Compared with cattle, the growth rate of yak is slow due to the lack of an efficient yak breeding program to improve growth traits, which affects the yield of yak meat. Therefore, it is of great significance to study the growth traits of yak. The growth hormone receptor (GHR) gene is an essential growth-related candidate gene that plays a critical role in the early stages of animal growth and development . The growth hormone (GH)-insulin-like growth factor (IGF) growth axis is crucial for the growth and differentiation of skeletal muscle . The GHR gene combines with GH to initiate the intracellular signal transduction mechanism, increase the expression of insulin-like growth factor 1 (IGF-1), promote cell proliferation, and affect the growth and development of animal skeletal muscle . In mice, GHR knockouts exhibit impaired skeletal muscle development characterized by a reduced number and area of muscle fibers and concomitant functional defects. GHR gene variants are associated with growth and development in livestock. The GHR gene is considered to be a molecular genetic marker of growth performance in fertile pigs . In sheep, the GHR gene variation is associated with growth traits . According to recent studies, the presence of genetic variations in the GHR gene is associated with economic traits, such as cattle growth and production . These studies suggest that genetic variation in the GHR gene plays an important role in regulating animal growth and development. Structural variation (SV) is an important and abundant source of genetic and phenotypic variation, including insertions/deletions (Indel), duplications, translocations, and copy-number variations (CNVs) . Compared with a single nucleotide variant (SNV), SVs could have a greater impact on the function and expression of genes . Up to now, many SVs are found to be associated with economic traits in domestic animals. For example, a deletion of 110 kb in the MER1 repeat containing the imprinted transcript 1 (MIMT1) gene is associated with abortion and stillbirth in cattle . The lysine acetyltransferase 6A (KAT6A) gene is associated with the development of different body size traits in sheep . In addition, a 225 bp deletion in the glutaminyl-peptide cyclotransferase-like (QPCTL) gene was associated with body weight and carcass traits in chickens . Upadhyay et al. detected an SV in the regulatory region of polypeptide N-acetylgalactosaminyltransferase-like 6 (GALNTL6) associated with feed efficiency and growth traits in cattle. However, there is no detailed study on SVs of the yak GHR gene. In this study, we found a 246 bp sequence deletion SV of the yak GHR gene. In order to identify the role of the GHR gene SV in yak populations in China, we analyzed the association between GHR 246 bp deletion variation and phenotypic traits to illustrate the genetic effect of GHR 246 bp deletion variation in yak breeds. This study provides useful information for the genetic protection and improvement of Chinese yak. 2. Materials and Methods 2.1. Ethics Statement This work has been approved by the Lanzhou Institute of Husbandry and Pharmaceutical Sciences; the grant number is No. LIHPS-CAAS-2017-115. In addition, we collected all blood samples and analyzed the data strictly following the guidelines for the Care and Use of Laboratory Animals. 2.2. Sample Collection Blood samples were collected from a total of 585 individuals of 14 yak breeds. The numbers of samples from each breed were as follows: Datong yak (DT, n = 22); Xueduo yak (XD, n = 21); Huanhu yak (HH, n = 21); Gannan yak (GN, n = 21); Tianzhu White yak (TZB, n =21); Niangya yak (NY, n = 18); Leiwuqi yak (LWQ, n = 21); Sibu yak (SB, n = 18); Muli yak (ML, n = 23); Jiulong yak (JL, n = 21); Maiwa yak (MW, n = 21); Zhongdian yak (ZD, n = 21); Bazhou yak (BZ, n = 21); and Ashidan yak (ASD, n = 315). We tracked the phenotypic data of these Ashidan yaks (i.e., body weight (BW, kg), withers height (WH, cm), body length (BL, cm), and chest girth (CG, cm)) at four age groups of growth (i.e., 6 months old, 12 months old, 18 months old, and 30 months old). The phenotype was measured using the standard method of measurement . Tissues, including the heart, muscle, liver, spleen, kidney, lung, and brain, were collected for expression profiling. 2.3. Genomic DNA (gDNA) Extraction and Polymerase Chain Reaction (PCR) Yak blood gDNA was extracted using the Tiangen whole-genome blood extraction kit (Beijing, China), and the DNA quality was detected using an ultra-microspectrophotometer (Thermo Fisher Scientific, USA). The gDNA was stored at -20 degC for further analysis. The primers for gene structure variation were designed by the National Center for Biotechnology Information (NCBI) online primer design software Primer-BLAST and synthesized by Qingke Zexi (Xi'an) Biotechnology company (Xi'an, Shaanxi, China) (Table 1). PCR was then performed. A total volume of 10 mL PCR mixture contained 1 mL of gDNA (50 ng/mL), 1 mL of each primer (10 mmol/L), 5 mL of Go Taq(r)Green Master Mix (Promega, Madison, WI, USA), and 2 mL of ddH2O. The PCR conditions were as follows: 95 degC for 3 min, 95 degC for 5 s, 58 degC for 30 s, 72 degC for 1 min for 30 cycles, and 72 degC for 5 min. The products were identified via 2% agarose gel electrophoresis. Six PCR products were selected and sequenced by Qingke Zexi (Xi'an) Biotechnology Company (Xi'an, Shaanxi, China). 2.4. RNA Extraction and Quantitative PCR Identification The total RNA of yak tissue was extracted using the Trizol method, and the integrity of RNA was identified with 1% agarose gel electrophoresis. cDNA was synthesized via reverse transcription using a translator first-strand cDNA synthesis kit (Roche, Shanghai, China) and stored at -20 degC for further analysis. Quantitative real-time polymerase chain reaction (RT-qPCR) was performed using Go Taq(r)qPCR and RT-qPCR Systems (Promega, Madison, WI, USA) to investigate the expression levels of the GHR messenger RNA (mRNA) in each tissue. Three replicates were selected for each sample. The primer information is provided in Supplementary Table S1. The RT-qPCR conditions were as follows: 95 degC for 3 min, 95 degC for 5 s, 64 degC for 20 s for a total of 40 cycles, and 72 degC for 30 s. The results were analyzed using the 2-DDCT method . 2.5. Cell Culture and Cell Transfection The 293T cell line was purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). High-sugar DMEM (Hyclone, Logan, UT, USA), fetal bovine serum (Gibco, Waltham, MA, USA), and 1% streptomycin/penicillin (Invitrogen, Waltham, MA, USA) were used. Plasmids were transfected into cells using ViaFectTMTransfection Reagent (Promega, Madison, WI, USA) according to the manufacturer's instructions. 2.6. Plasmid Construction and Prediction of Transcription-Factor Binding Sites (TFBSs) Based on the yak GHR 246 bp deletion locus, the luciferase reporter vector pGL4.10 was selected, and the pGL4.10-II (324 bp, containing the 246 bp deletion) and pGL4.10-DD (78 bp, lacking the 246 bp deletion) vectors were constructed to detect the transcriptional activity of GHR 246 bp deletion. In this study, the online software AnimalTFDB 3.0 ) (accessed on 10 February 2022) and JASPAR ) (accessed on 10 February 2022) were used for the prediction of TFBSs at the GHR 246 bp deletion locus. The runt-related transcription factor 1 (Runx1) binding sequence (TAAATGCAAA) was identified on the II genotype, and the pGL4.10-KO-Runx1 dual-luciferase reporter vector (324 bp) was constructed. The vector pGL4.75 (Promega, Madison, WI, USA) was used as an internal reference vector for determining the luciferase reporter system. 2.7. Statistical Analysis Gene frequency and genotype frequency were calculated using the following equations: Genotype frequency (FAiAj) = AiAj number of individuals/total number of samples; Allele frequency (FAi) = FAiAj + 1/2 FAiAj. The online website SHEsis was used to calculate Hardy-Weinberg equilibrium, heterozygosity, polymorphism information, and genetic variation content of GHR gene SV in the population. Meanwhile, we analyzed the association between the SV type of the GHR gene and the growth traits of Ashidan yak by using a one-way analysis of variance (ANOVA) and frequency-distribution histogram test. A mixed linear model used in the analysis is as follows: Yij = m + Gij + eij, where Yij is the observed growth trait; m is the population average of each trait; Gij is the fixed effect of SV genotype of the GHR gene; and eij is the random residual. The difference between the mean values was evaluated by using one-way ANOVA with a post-event least significant digit (LSD) multiple-comparison test. The software used for the statistical analyses was the IBM SPSS Statistics software (Version 23.0). Data are expressed as mean+- standard error (SE). 3. Results 3.1. Identification of SV of Yak GHR Gene A 246 bp deletion variation was identified in intron 6 of the GHR gene (NW_005393449.1: 3052434-3052680). Based on the designed primers (F1, R1), GHR-SV246 was amplified with PCR using yak gDNA as the template. The GHR-SV246 polymorphism was analyzed via PCR amplification and agarose gel electrophoresis of the product . The product length matched the expectation, and three genotypes were identified, namely II (778 bp), ID (778 bp and 532 bp), and DD (532 bp). The sequencing analysis of the amplified products showed that the sequencing results were consistent with the reference genome sequence. 3.2. Identification of SV of Yak GHR Gene The genotype frequencies and allele frequencies of GHR-SV246 in different yak breeds are shown in Table 2. The frequencies of different genotypes in Chinese yak populations were 0.723, 0.202, and 0.075, respectively. The II genotype was the dominant genotype in different populations except for SB yak; the allele frequencies were 0.824 and 0.176, respectively. Among them, the frequency of the I allele was the highest in the ML yak population (0.979) and the lowest in the SB yak population (0.500) (Table 2). The Ho ranged from 0.500 to 0.959, indicating high heterozygosity within different yak breeds, and the Ne ranged from 1.04 to 2.00. Classification based on the polymorphism information content (PIC value < 0.25, low polymorphism; 0.25 < PIC value < 0.5, medium polymorphism; PIC value > 0.5, high polymorphism) showed that the 246 bp deletion of the GHR gene exhibited relatively low polymorphism in ZD, ML, LWQ, NN, GN, and XD yak populations, while moderate polymorphism was observed in other yak populations (Table 3). 3.3. The Association of the 246 bp Deletion of GHR with Growth Traits of Yak In this study, the normal distribution of yak growth traits and the average value of the growth parameters were statistically analyzed (Supplementary Table S1). We also analyzed the association of GHR gene polymorphisms with the growth traits in yaks using a linear model. As shown in Table 4, the polymorphisms in the GHR gene were significantly associated with body length in 6-month-old ASD yaks (p < 0.05), while there was no significant relationship with other growth traits (p > 0.05). 3.4. Identification of Fluorescence Activity of GHR Gene Polymorphism in Yak To detect the transcriptional activity of different GHR-SV246 genotypes in cells, pGL4.10, pGL4.10-II, and pGL4.10-DD vectors were co-transfected into 293T cells with the internal reference plasmid pGL4.75. The fluorescence activity of the pGL4.10-II vector was significantly different from that of the blank control group pGL4.10 (p < 0.05) and was significantly lower than that of the pGL4.10-DD vector (p < 0.05) . 3.5. Effect of Transcription Factors on the Transcriptional Activity of Mutant Recombinant Plasmids In order to determine the effect of Runx1 transcription-factor binding with the 246 bp deletion on transcriptional activity in cells, the pGL4.10-KO-Runx1 luciferase reporter vector with mutant Runx1 binding site was constructed, and its fluorescence activity was identified. The luciferase activity of vector pGL4.10-KO-Runx1 was significantly lower than that of vector pGL4.10-II , indicating that the transcription factor Runx1 could be combined with the II genotype sequence, thus increasing its transcriptional activity in cells. 3.6. mRNA Expression Profile of the GHR Gene in Yak The expression levels of the GHR gene in different tissues were identified in yaks. There were significant differences in the expression of the GHR gene in various tissues of yaks, with the highest expression in fat, followed by the liver, muscle, and kidney tissues, and the lowest expression in the heart, spleen, and lung tissues . 4. Discussion SV is a major determinant of the phenotypic diversity of animals. Studies on chickens , sheep , and cattle have reported that SVs play a significant role in their genetic diversity. In poultry, the SVs of prolactin receptor (PRLR) and Lpin1 were significantly associated with chicken growth, carcass characteristics, and other economic traits. In cattle, the SVs of SIRT4 and NPM1 were significantly associated with bovine growth traits and meat quality . These studies show that SV is an important class of molecular genetic markers, which is of great value in revealing the genetic mechanism of the economic traits of livestock and poultry. The GHR gene is a member of the cytokine receptor superfamily . Some studies have pointed out that the GHR gene can regulate the growth of skeletal muscle by interacting with the growth hormone (GH) . In this study, we analyzed the expression of the GHR gene in different tissues of yak and found that the GHR gene was expressed in different tissues, with high expression in adipose tissue and moderate expression in the muscle. This is consistent with previous studies on GHR expression profiles . GHR mRNA expression levels in muscle and fat have a positive effect on post-weaning weight gain in cattle, while GHR mRNA expression in the liver has a negative effect on post-weaning weight gain in cattle . A study reported that there is a natural antisense transcript (GHR-AS) on the GHR gene of chickens, and its overexpression can promote the expression of myogenic differentiation 1 (MyoD) and myosin heavy chain (MyHC) and the differentiation of myoblasts, thus enhancing the differentiation of myoblasts . A previous study found associations between GHR gene polymorphisms and the growth traits in cattle at different ages , showing that GHR gene variation can affect the growth and production traits of cattle. However, few studies have reported an association between GHR gene variation and yak growth and production traits. In this study, we found a 246 bp structural variation site in intron 6 of the yak GHR gene. The genotyping results showed that the three genotypes of GHR genes existed in different yak breeds, and the analysis of genetic parameters suggested that the 246 bp deletion was in Hardy-Weinberg equilibrium in different yak breeds, which indicates that it is relatively stable in yak population. In ASD yak, this SV belongs to a moderate polymorphism, indicating that the degree of genetic variation is relatively large, and it has certain breeding potential. We found that individuals with the DD genotype showed higher body length at 6 months of age in ASD yak breeds. These data suggest that SVs in the GHR gene might influence the early development of yak. Introns are noncoding segments of genes that are removed via splicing during gene transcription and ultimately do not exist in mature RNA molecules . Introns play an important role in the regulation of gene expression networks. Ostrovsky et al. found that the intron 9 of the heparanase (HPSE) gene has a regulatory effect on its expression, and the existence of mutation sites in this intron has a significant impact on the regulatory effect of the intron. In this study, the transcriptional activity of the deletion sequence in the yak GHR gene was analyzed, and the luciferase activity of the pGL4.10-DD vector was significantly higher than that of the pGL4.10-II vector (p < 0.05). This finding indicates that there may be repressive TFBSs in the II genotype sequence, which interferes with the expression of the GHR gene by binding to transcription factors and affects the growth and development of yaks. Genomic variation in introns can affect gene expression through cis-element targets or by binding to other functional genes . Boriushkin et al. found a Kruppel-like factor 4 (KLF4) transcription-factor binding site located in the intron of the delta-like canonical Notch ligand 4 (DLL4) gene, which is a key regulator of angiogenesis. They also found that KLF4 can inhibit the expression of DLL4 via this binding site. The appearance of gene variant sites may introduce novel transcription-factor binding sites or destroy existing binding sites . In this study, in the II genotype sequence, we detected a Runx1 transcription-factor binding site, which was first discovered in acute myeloid leukemia . Runx1 has an important regulatory role in the growth and development of animal muscles. In mouse muscle cells, the overexpression of Runx1 inhibits myogenic differentiation but promotes myoblast proliferation . When skeletal muscle is injured, the expression of Runx1 is upregulated in skeletal muscle, thereby inhibiting the premature differentiation of primary myoblasts and promoting muscle regeneration . Runx1 inhibits Spi1 gene expression by interacting with Spil introns . In this study, we hypothesized that Runx1 has an effect on the transcriptional regulation of the GHR gene II genotype in yaks. This study found that changes in Runx1-TFBS reduced the fluorescence activity of the II genotype vector, indicating that the Runx1 could bind to the II genotype sequence and increase the transcriptional activity of the GHR gene. The luciferase activity of the pGL4.10-II vector was not significantly different from the pGL4.10 empty-vector fluorescence activity (p > 0.05). There may also be a binding site of an inhibitory transcription factor in the II genotype sequence. 5. Conclusions We detected and validated the SV of the GHR gene in yaks for the first time. The results indicated that SV of the GHR gene was significantly correlated with body length at 6 months of age in ASD yaks. Moreover, the transcriptional activity assay found significant differences among different genotypes, and transcription factor Runx1 promoted transcriptional activity in the II genotype. Our study provides primary evidence for the role of the GHR gene, which may be a molecular marker for early yak breeding in the future. Supplementary Materials The following supporting information can be downloaded at: Figure S1: Histogram of normal distribution of body weight frequency of yaks at different ages, Figure S2: Histogram of normal distribution of yak body length frequency at different months, Figure S3: Histogram of normal distribution of yak body height frequency at different months, Figure S4: Histogram of normal distribution of chest girth frequency of yaks at different ages, Table S1: The overall average of the growth parameters. Click here for additional data file. Author Contributions Conceptualization, F.W., X.W. and P.Y.; methodology, F.W. and X.W.; software, X.M., Q.Z. and Q.B.; validation, F.W., C.L. and P.Y.; formal analysis, F.W., X.W. and Q.B.; investigation, F.W. and Q.Z.; resources, C.L., M.C. and X.G.; data curation, F.W., X.W. and C.L.; writing--original draft preparation, F.W.; writing--review and editing, C.L., X.G. and X.W.; visualization, P.Y.; supervision, P.Y.; project administration, P.Y.; funding acquisition, P.Y. All authors have read and agreed to the published version of the manuscript. Institutional Review Board Statement This work has been approved by the Lanzhou Institute of Husbandry and Pharmaceutical Sciences, the grant number is No. LIHPS-CAAS-2017-115. Data Availability Statement This study did not report any data. Conflicts of Interest The authors declare no conflict of interest. Figure 1 The gel electrophoresis of different genotypes of GHR-SV246; M, GL DNA marker 2000; II, homozygous insertion type; ID, heterozygous type; DD, homozygous deletion type. Figure 2 The dual-luciferase activity of GHR-SV246 mutant recombinant plasmid in 293T cells; the same letter means no significant difference (p > 0.05); different letters indicate significant differences (p < 0.05). Figure 3 The dual-luciferase activity of GHR-SV246 recombinant plasmid in 293T cells. Figure 4 The expression level of GHR gene mRNA in different tissues of yak. animals-13-00851-t001_Table 1 Table 1 The information of primer sequence. Levels Gene Primer Sequence (5'-3') Product Length (bp) Tm (degC) DNA GHR F1 TCAGAGATGAGCAACAGTGCC 778 58.0 R1 TGCGTATCTACACCTGAGCAC F2 GGggtaccTGAGCAACAGTGCCCCATTT 324 64.9 R2 GAagatctTCACACACTCTAGACCTTAAAGCTG RNA F3 CAGCAGCCCAGTGTTATCCT 230 64.0 R3 AATGTCGCTTACCTGGGCAT b-actin F4 GCAGGTCATCACCATCGG 177 64.0 R4 CCGTGTTGGCGTAGAGGT F: forward primer; R: reverse primer. animals-13-00851-t002_Table 2 Table 2 Genotype and allele frequencies of GHR-SV246 in different Yaks. Breeds Sample Size/n Genotype Frequency Allele Frequency Description II ID DD I D Ashidan yak (ASD) 315 0.714 0.181 0.105 0.805 0.195 Domestic, Datong Qianghai, PRC Datong yak (DT) 22 0.636 0.364 0 0.818 0.182 Domestic, Datong Qianghai, PRC Xueduo yak (XD) 21 0.762 0.238 0 0.881 0.119 Domestic, Henan Qianghai, PRC Huanhu yak (HH) 21 0.524 0.286 0.190 0.667 0.333 Domestic, Haibei Qianghai, PRC Gannan yak (GN) 21 0.950 0.0500 0 0.977 0.0230 Domestic, Gannan Gansu, PRC Tianzhubai yak (TZB) 21 0.666 0.286 0.0480 0.810 0.190 Domestic, Tianzhu Gansu, PRC Niangya yak (NY) 18 0.889 0.111 0 0.944 0.0560 Domestic, Naqu Tibet, PRC Leiwuqi yak (LWQ) 21 0.714 0.286 0 0.857 0.143 Domestic, Changdu Tibet, PRC Sibu yak (SB) 18 0.500 0.500 0 0.500 0.500 Domestic, Sibu Tibet, PRC Muli yak (ML) 23 0.957 0.0430 0 0.979 0.0210 Domestic, Liangshan Sichuan, PRC Jiulong yak (JL) 21 0.667 0.0950 0.238 0.714 0.286 Domestic, Jiulong Sichuan, PRC Maiwa yak (MW) 21 0.500 0.0910 0.409 0.545 0.455 Domestic, Hongyuan Sichuan, PRC Bazhou (BZ) 21 0.667 0.238 0.0950 0.786 0.214 Domestic, Hejing Xinjiang, PRC Zhongdian yak (ZD) 21 0.571 0.333 0.0960 0.738 0.262 Domestic, Zhongdian Yunnan, PRC Total 586 0.723 0.202 0.0750 0.824 0.176 II, homozygous insertion type; ID, heterozygous; DD, homozygous deletion type. animals-13-00851-t003_Table 3 Table 3 The genetic parameters of Yak GHR-SV246. Breeds Sample Size/n HWE p-Value Genetic Parameters Description Ho He Ne PIC Ashidan yak (ASD) 315 0.493 0.686 0.313 1.45 0.265 Domestic, Datong Qianghai, PRC Datong yak (DT) 22 0.474 0.702 0.298 1.42 0.253 Domestic, Datong Qianghai, PRC Xueduo yak (XD) 21 0.365 0.790 0.210 1.27 0.187 Domestic, Henan Qianghai, PRC Huanhu yak (HH) 21 0.636 0.556 0.444 1.80 0.346 Domestic, Haibei Qianghai, PRC Gannan yak (GN) 21 0.109 0.955 0.0449 1.04 0.0439 Domestic, Gannan Gansu, PRC Tianzhubai yak (TZB) 21 0.486 0.692 0.308 1.44 0.260 Domestic, Tianzhu Gansu, PRC Niangya yak (NY) 18 0.216 0.894 0.106 1.12 0.100 Domestic, Naqu Tibet, PRC Leiwuqi yak (LWQ) 21 0.410 0.755 0.245 1.32 0.215 Domestic, Changdu Tibet, PRC Sibu yak (SB) 18 0.693 0.500 0.500 2.00 0.375 Domestic, Sibu Tibet, PRC Muli yak (ML) 23 0.102 0.959 0.0411 1.04 0.0403 Domestic, Liangshan Sichuan, PRC Jiulong yak (JL) 21 0.599 0.592 0.408 1.69 0.325 Domestic, Jiulong Sichuan, PRC Maiwa yak (MW) 21 0.689 0.504 0.496 1.98 0.373 Domestic, Hongyuan Sichuan, PRC Bazhou (BZ) 21 0.519 0.664 0.336 1.51 0.280 Domestic, Hejing Xinjiang, PRC Zhongdian yak (ZD) 21 0.575 0.613 0.387 1.63 0.312 Domestic, Zhongdian Yunnan, PRC HWE, Hardy-Weinberg equilibrium; Ho, homozygosity; He, heterozygosity; Ne, number of effective alleles; PIC, polymorphic information content. animals-13-00851-t004_Table 4 Table 4 The association analysis between different body size traits and different GHR genotypes of Yaks. Age Growth Trait SV Types p-Value II (0.714) ID (0.181) DD (0.105) 6 months (n = 315) Body weight 84.711 +- 0.692 83.754 +- 1.374 83.000 +- 1.806 0.601 Body length 91.667 +- 0.485 b 91.386 +- 0.963 b 95.242 +- 1.266 a <0.05 * Body height 94.356 +- 0.350 94.684 +- 0.695 94.758 +- 0.914 0.861 Chest girth 123.938 +- 0.513 124.175 +- 1.020 124.364 +- 1.341 0.944 12 months (n = 315) Body weight 82.698 +- 0.690 81.140 +- 1.389 83.970 +- 1.826 0.433 Body length 95.920 +- 0.332 95.807 +- 0.659 97.182 +- 0.866 0.370 Body height 90.502 +- 0.281 90.561 +- 0.558 91.121 +- 0.734 0.733 Chest girth 117.187 +- 0.332 117.281 +- 0.660 117.970 +- 0.867 0.701 18 months (n = 226) Body weight 122.329 +- 1.023 123.550 +- 2.014 120.548 +- 2.288 0.616 Body length 101.335 +- 0.455 102.600 +- 0.895 101.581 +- 1.017 0.454 Body height 102.903 +- 0.459 102.900 +- 0.903 102.258 +- 1.026 0.843 Chest girth 138.387 +- 0.827 142.350 +- 1.628 138.129 +- 1.849 0.084 30 months (n = 180) Body weight 154.944 +- 1.121 155.375 +- 2.621 159.273 +- 3.161 0.451 Body length 113.421 +- 0.509 112.906 +- 1.009 113.136 +- 1.217 0.893 Body height 99.286 +- 0.450 99.125 +- 0.892 100.955 +- 1.076 0.322 Chest girth 146.484 +- 0.730 147.125 +- 1.448 146.818 +- 1.746 0.919 a,b Values represent statistically significant differences at a level of p < 0.05; * values represent significant differences at a level of p < 0.05. 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PMC10000138

The microencapsulated mixture of organic acids and pure botanicals (OA/PB) has never been evaluated in goats. The aim of this study was to extend the analysis to mid-late lactating dairy goats, evaluating the effects of OA/PB supplementation on the metabolic status, milk bacteriological and composition characteristics, and milk yield. Eighty mid-late lactating Saanen goats were randomly assigned to two groups: one group was fed the basal total balanced ration (TMR) (CRT; n = 40) and the other was fed a diet that was TMR supplemented with 10 g/head of OA/PB (TRT; n = 40) for 54 days during the summer period. The temperature-humidity index (THI) was recorded hourly. On days T0, T27, and T54, the milk yield was recorded, and blood and milk samples were collected during the morning milking. A linear mixed model was used, considering the fixed effects: diet, time, and their interaction. The THI data (mean +- SD: 73.5 +- 3.83) show that the goats did not endure heat stress. The blood parameters fell within the normal range, confirming that their metabolic status was not negatively influenced by OA/PB supplementation. OA/PB increased the milk fat content (p = 0.04) and milk coagulation index (p = 0.03), which are effects that are looked on as favorable by the dairy industry in relation to cheese production. dairy goats nutrition sustainable farming supplementation blood profile milk performance This research received no external funding. pmc1. Introduction One aim of the livestock industry is to maximize productivity, while keeping antibiotic usage to a minimum to achieve more sustainable and higher quality production . Alternative feed additives, such as dietary acidifiers, essential oils, probiotics, and prebiotics, have been introduced as potential replacements for antibiotics and as nutritional strategies to prevent metabolic disorders in ruminants . Of the possible feed additives, organic acids (OA) such as citric acid and sorbic acid and pure botanicals (PB) such as thymol and vanillin have been widely used in the field of animal nutrition for their positive effects on production performances and the metabolic and immune statuses . The moderate-to-strong antimicrobial activity of OA occurs thanks to their ability to penetrate the bacterial cell wall and inhibit microbial enzymatic reactions and nutrient transport systems . The antioxidant and anti-inflammatory activities exerted by PB also occur through their capacity to impair the enzymatic systems involved in bacterial energy production and structural component synthesis . The PB vanillin is often used as a flavoring agent to improve the feed palatability . The specific combination of OA (citric and sorbic acids) and PB (thymol and vanillin) as a microencapsulated mixture (16.7% sorbic acid, 25% citric acid, 1.7% thymol, and 1% vanillin) within a slow-release lipid matrix (OA/PB) has already been shown to exert antimicrobial, anti-inflammatory, antioxidant, and immunomodulatory properties in, for example, supplementing the feed of weaned pigs with OA/PB improved growth performances and intestinal integrity by modulating inflammatory processes at both the local and systemic levels . A modulatory effect on the immune status was also shown in broiler chickens . The authors showed how dietary supplementation with this specific microencapsulated compound led to immunological benefits by increasing the in vitro activity of the peripheral blood leukocytes, as well as the heterophil and monocyte counts. Moreover, in a study carried out by Fontoura and colleagues , giving the same microencapsulated product to heat-stressed Holstein dairy cows was associated with improvements in the milk yield, the energy-corrected milk value, and milk nitrogen efficiency. Most of the available scientific literature explores the immunomodulatory and antioxidant properties of OA/PB, whereas, to our best knowledge, the information on OA/PB supplementation on metabolic status and milk performances in ruminants is still sparse. Therefore, the aim of the present study was to evaluate the effects of the dietary inclusion of this specific microencapsulated product containing OA/PB on the metabolic status (hematological and hematochemical parameters) and milk (milk yield, bacteriological characteristics, and milk composition) of mid-late lactation goats. We hypothesized that OA/PB supplementation may also have beneficial effects on the goats' metabolic status, as well as the milk yield and quality during the mid-late lactating phase. 2. Materials and Methods The experimental protocol was designed according to the guidelines of the current European Directive on the care and protection of animals (2010/63/EU), and it was approved by the Ethical Committee of the Department of Veterinary Sciences of the University of Turin (Italy, Prot. N. 0002184/2021). 2.1. Animals, Management, and Environmental Conditions The present study was conducted on a breeding farm that rears around 250 Saneen goats for milk production in north-west Italy. A total of 80 homogenous and clinically healthy Saanen goats in the mid-late lactation phase (110 days in milk), mean +- SD BCS of 3.4 +- 0.03 on a 5-point scale , were involved in the study. The feeding trial started at T0, corresponding to 105 days from the beginning of lactation, and lasted for 54 days (T54). The health status of the animals was monitored on a daily basis throughout the entire feeding trial (from T0 to T54) by a veterinarian expert in small ruminants. At T0, the dairy goats were randomly divided into two adjacent group pens, each covering an area of 80 m2. The two group pens were separated from each other by the central feeding lane and located inside a naturally ventilated shed that was open on two sides. The concrete floor was covered with a layer of wheat straw bedding. The animals had ad libitum access to tap water. Since the study was carried out in the summer period (July-August), a single data logger (ORIA, Parent-WA44) was installed in each group pen to monitor the environmental temperature and humidity, and the temperature-humidity index (THI) was recorded on an hourly basis. The THI was calculated according to the formula described in Salama et al. :THI = Td - (0.55 - 0.55 x RH) x (Td - 58) where Td is the dry bulb temperature (degF) and RH is the relative humidity (%). 2.2. Diets For 54 days (from T0 to T54), the control group (CRT; n = 40) of goats were fed the daily total mixed ration (TMR), whereas the treatment group (TRT; n = 40) received the diet that was TMR supplemented with 10 g/head/day of microencapsulated organic acids and pure botanicals (OA/PB; containing 16.7% sorbic acid, 25% citric acid, 1.7% thymol, and 1% vanillin in a matrix of hydrogenated fats; Aviplus R, Vetagro S.p.A., Reggio Emilia, Italy). The goats were administered the TMR once daily between 8.00 and 9.00 AM, after the morning milking. The feed was supplied by distributing it along the feeding lane next to each pen. The ration was formulated to cover the goats' nutritional requirements according to the Institute National de la Recherche Agronomic (INRA, 2018). The chemical analysis of the TMR was performed according to the methods described in the scientific literature . Briefly, TMR was ground to pass through a 1 mm sieve and stored in an airtight plastic container for subsequent dry matter (DM) (method number 943.01), ash (method number 924.05), CP (method number 954.01), aNDFom (method number 2002.04), and ADF (method number 973.18) determination . The chemical composition of TMR is shown in Table 1. 2.3. Blood and Milk Sampling Blood and milk samples were collected at T0, T27, and T54 during the morning mil-king, which took place between 6:00 and 8:00 AM. For the determination of the hematological parameters, blood samples were collected from the jugular vein into 10 mL vacutainer tubes (Vacutainers, Becton Dickinson) containing ethylenediaminetetraacetic (K3EDTA) acid as an anticoagulant. For the determination of the hematochemical parameters, a second set of blood samples was collected into 10 mL tubes containing a clot activator. The blood samples were stored at 4 degC and immediately transferred to the laboratory to proceed with the analyses as described in Section 2.4. Composite milk samples (pooled from the two hemi-udders) were manually collected for the bacteriological and milk composition assessments. Before milking, the teats were carefully cleaned with cotton wool impregnated with 70% ethanol according to the method described by Ariznabarreta et al. . After discarding the first streams of milk, two composite milk samples were collected into 50 mL Falcon sterile tubes (Falcon Conical Centrifuge Tubes, Tewsbury, MA, USA) from each lactating goat. All the samples were kept at 4 degC from the time of collection. One sample was used for the bacteriological analysis, which was carried out immediately upon arrival at the laboratory as described in Section 2.5. The second sample was used for determining milk composition as described in Section 2.5, which was always performed within 6 h of collection. At the end of each milking procedure, the milk yield (MY, kg) was recorded for each lactating goat, which was assessed using the electronic milk meter present in the milking room. 2.4. Analysis of Hematological and Hematochemical Parameters Whole blood samples from K3EDTA tubes were immediately processed for the blood counts using an automated blood analyzer (Melet-Schloesing-MS4, Osny, France) and according to the instructions provided by the manufacturer. The parameters analyzed were: erythrocyte count (red blood cell (RBC)); hemoglobin (Hb) concentration; mean corpuscular volume (MCV); the hematocrit (HCT); mean cell hemoglobin (MCH); mean corpuscular hemoglobin concentration (MCHC); platelets (PLT); mean platelet volume (MPV); leukocyte (white blood cell (WBC)), neutrophil (NEU), basophil (BAS), eosinophil (EOS), monocyte (MON), and lymphocyte (LYM) counts. Serum samples were centrifuged at 3500 rpm for 15 min at 20 degC and analyzed using an automated system photometer (I-Lab Aries Chemical Analyzer--Instrumentation Laboratory S.p.A, Werfen Company, Milano, Italy) for total protein (TP), aspartate aminotransferase (AST), alanine aminotransferase (ALT), creatinine (CREA), and blood urea nitrogen (BUN) determinations. The same operator always performed the analysis according to the internal quality control standards. 2.5. Analysis of Milk Bacteriological Characteristics and Milk Composition The bacteriological analysis was performed using 10 microliters (mL) of each milk sample spread on three different agar mediums, specifically: blood agar supplemented with 5% defibrinated sheep blood; Baird Parker agar supplemented with rabbit plasma fibrinogen on MacConkey agar; Tallium Kristalviolette Tossin agar. The plates were incubated at 37 degC for between 24 and 48 h. Suspected colonies were further tested in order to identify the microorganisms. These tests involved the microscopic examination of the colonies after Gram staining, the catalase test, and the API method (API system, BioMerieux, Italy). Isolated bacteria were subsequently tested for antimicrobial susceptibility using the disc diffusion method (Kirby-Bauer test) performed on Mueller-Hinton agar. Moreover, individual milk samples were analyzed using a MilkoScanTM 7 RM (Foss, Denmark) to determine their milk composition (casein, fat, lactose and urea content, milk coagulation index (MCI), somatic cell count, and fat-free dry matter). 2.6. Statistical Analysis Statistical analyses were carried out using the software JMPpro v16 (SAS Institute Inc., Cary, NC, USA). The Shapiro-Wilk test and Levene's test were performed to test the data for normality and homoscedasticity, respectively. Then, when necessary, somatic cell count data were logarithmically or Box-Cox transformed before further analysis . The THI data were subjected to a bivariate analysis, and the relationships between the variables were investigated using Pearson's correlations. A linear mixed effects model was constructed considering the following fixed effects: dietary treatment, sampling time, and their interaction. Each goat was then considered to be an experimental unit and used as the random variable for all the analyses, with T0 data used as the covariate. The normality of the data was checked once again for the resulting model residuals. Least squares means were separated using Student's t test adjusted p-values when at least one tendency F-test (p <= 0.10) was detected in the fixed effect interaction term. Finally, the X-test was implemented to study the incidence of bacteria in the milk samples. 3. Results 3.1. Animals and Environmental Conditions All the 80 Saneen goats enrolled in the present study remained healthy for the entire length of the experimental trial (from T0 to T54). The results from the bivariate analysis revealed no differences between the environmental data recorded in the two group pens (CTR and TRT), indicating that the enrolled animals were exposed to the same environmental conditions. The mean THI for the period spanning from T0 to T54 was 73.5 +- 3.83 (+-SD). 3.2. Hematological and Hematochemical Parameters None of the samples collected contained clots or had hemolyzed. Table 2 reports the hematological and hematochemical mean data for the two dietary group (CRT and TRT) at each sampling time (T0, T27, and T54) and the statistics from the linear mixed effects model considering the effect of the dietary treatment, sampling time, and their interaction. Considering the hematological parameters, the EOS and PLT levels were higher in CRT compared with those in TRT on the basis of the dietary treatment (p = 0.01 and p < 0.01, respectively). Moreover, the levels of EOS and PLT increased over time in both groups (p < 0.01 and p = 0.03, respectively). The NEU concentrations also differed between the two groups on the basis of the dietary treatment (p = 0.05), with lower values in CRT compared with those in TRT at T27 (45.3% vs. 47.0%, respectively), and the higher values in CRT compared with those in TRT at T54 resulted in a significant interaction between the dietary treatment and sampling time (54.9% vs. 45.7%, respectively; p < 0.01). The RBC count varied between the two groups on the basis of the dietary treatment (p < 0.01), with there being lower values in CRT compared with those in TRT at T27 (13.7 x 106/mL vs. 13.9 x 106/mL, respectively), and higher values in CRT compared with those in TRT at T54, causing a significant interaction between the dietary treatment and sampling time (13.6 x 106/mL vs. 13.1 x 106/mL, respectively; p < 0.01). An effect of the dietary treatment was also seen on the MCH, with the values being higher in CRT than they were in TRT (p < 0.01); however, while the mean MCH value increased over the sampling time in CRT, it decreased in TRT (p < 0.01), with a significant interaction with the between diet and sampling time at T54 (6.30 pg in CRT vs. 5.50 pg in TRT, p < 0.01). The MCHC was influenced by both the dietary treatment (p < 0.01) and the sampling time (p = 0.04). Specifically, the MCHC values were higher in CRT compared with those in TRT, with a significant interaction between the diet sampling times at T27 (33.0 g/dL vs. 31.2 g/dL, respectively; p < 0.01) and T54 (34.5 g/dL vs. 30.7 g/dL, respectively; p < 0.01). Differences between the two groups of animals on the basis of diet were also found in relation to HCT (p < 0.01), which was lower in CRT than it was in TRT at T27 (24.1% vs. 25.4%, respectively), whereas it was higher in CRT than it was in TRT at T54 (25.2% vs. 24.2%, respectively). The MON and LYM concentrations decreased in both CRT and TRT over the sampling time (p < 0.01 and p = 0.05, respectively). The Hb values differed between the two groups of animals on the basis of the dietary treatment, with them being higher in CRT than they were in TRT at both T27 (8.10 g/dL vs. 7.90 g/dL, respectively; p < 0.01) and T54 (8.25 g/dL vs. 7.35 g/dL, respectively; p < 0.01), and with a significant interaction between the dietary treatment and sampling time (p < 0.01). Regarding the hematochemical parameters, the CREA level was higher in CRT compared with that in TRT on the basis of the dietary treatment (p = 0.01), and the values increased in both groups according to the sampling time (p < 0.01). BUN also differed between the CRT and TRT, showing an interaction between the diet and sampling time at T54 (36.7 mg/dL and 34.3 mg/dL, respectively). The TP level was higher in CRT than it was in TRT due to the dietary treatment (p = 0.01). Moreover, the TP level increased in both groups according to the sampling time (p < 0.01). 3.3. Milk Bacteriological Characteristics and Milk Composition None of the milk samples collected at any of the sampling times (T0, T27, and T54) or from either animal group (CRT and TRT) were found to be contaminated. Table 3 reports the mean milk composition results according to dietary group (CRT and TRT) and sampling time (T0, T27, and T54). MY was influenced by the sampling time (p < 0.01): the level of it was lower in both groups with respect to T0 at T27 (2.82 kg in CRT and 2.77 kg in TRT), but then, it increased in both groups at T54 (3.09 kg in CRT and 3.14 kg in TRT). The milk fat content varied significantly according to the dietary treatment (p = 0.04), with it being higher in TRT compared with that in CRT at both T27 (2.56% and 2.12%, respectively) and T54 (2.24% and 2.04%, respectively). The MCI was also influenced by the dietary treatment (p = 0.03), it was lower in CRT compared with that in TRT at T54 (75.1% and 75.6%, respectively), resulting in a significant interaction between the dietary treatment and sampling time at T54 (p = 0.02). The % of lactose differed between the groups according to the sampling time (p < 0.01), it was higher in CRT compared with that in TRT at T27 (4.24% and 4.20%, respectively), but it was lower in CRT compared with that in TRT at T54 (4.09% and 4.11%, respectively). The concentration of milk urea also differed between the groups according to the sampling time (p < 0.01), with lower values in CRT compared with those in TRT at T27 (26.1 mg/dL and 27.0 mg/dL, respectively), but there were equal values in CRT and TRT at T54 (28.2 mg/dL and 28.2 mg/dL, respectively). The fat-free DM, the FCM, the FPMC, and the ECM were not influenced by the dietary treatment, but they did vary in both CRT and TRT on the basis of the sampling time (p < 0.01), showing reduced levels at T27 with respect to those at T0 and higher levels at T54. All the 80 Saneen goats enrolled in the present study remained healthy for the entire length of the experimental trial (from T0 to T54). The results from the bivariate analysis revealed no differences between the environmental data recorded in the two group pens (CTR and TRT), indicating that the enrolled animals were exposed to the same environmental conditions. The mean THI for the period spanning from T0 to T54 was 73.5 +- 3.83. 4. Discussion 4.1. Animals and Environmental Conditions To the best of our knowledge, the dietary supplementation of OA (citric and sorbic acids) and PB (thymol and vanillin) microencapsulated in a slow-release lipid matrix (OA/PB) has never previously been investigated in dairy goats. Some studies are available in the scientific literature on other livestock species, for which this specific microencapsulated compound was found to improve both productivity, in terms of growth performances and milk yield, and the physiological and immune statuses . It is well known that blood and milk parameters in healthy goats can vary in relation to several factors, such as breed, physiological status, and housing conditions . In particular, certain environmental conditions, namely an increase in the temperature-humidity index (THI), can negatively influence the health of ruminant species and their milk production by decreasing the dry matter intake and shifting glucose utilization away from milk synthesis . The scientific literature on the impact of heat stress in dairy goats is sparser than that which is available for dairy cows. In dairy cows, milk production already becomes negatively affected at THI values of 72-75 ; goats, on the other hand, are less sensitive to heat stress . The impact of heat stress on dairy goats depends on the breed and on the stage of lactation, with higher milk yield losses in early lactating goats compared with those in mid-late lactating goats . For example, a moderate-to-severe heat stress occurred in dairy Saanen goats following exposure to a THI of 81 or 89 for 4 consecutive days or a THI of 79 for 5 weeks . In the present study, Saanen dairy goats in the mid-late lactation phase received a dietary supplement of OA/PB for 54 days. As the study was carried out during the summer period (July-August), the THI was monitored on an hourly basis throughout the course of the feeding trial (from T0 to T54). The mean +- SD THI recorded was 73.5 +- 3.83, and the results of the bivariate analysis showed no differences between the environmental data recorded in the two group pens (CRT and TRT). These findings indicate that our goats were not exposed to a condition of heat stress at any time during the feeding trial, implying that any differences found in the blood and milk parameters evaluated were related to the dietary treatments and not the environmental factors. 4.2. Hematological and Hematochemical Parameters As previously stated, the effects of OA and PB have been mainly investigated in pigs and poultry, whereas the available information about their supplementation in ruminant feed is still limited. In our study, the challenge diet (TRT) was associated with decreases in the red blood cell (RBC) parameters, namely the RBC counts, HCT, Hb, MCH, MCHC, and PLT. Several stressors have been reported to cause a decrease in the mature RBC counts and the release of immature forms, called reticulocytes, characterized by a lower Hb content, higher volume, and, by consequence, a lower MCHC . The differences between TRT and CRT revealed in the present study are difficult to explain on the basis of the current scientific literature, and although the TRT group had lower RBC parameter values than the CRT group did, the results reflect a physiological rather than a pathological condition since they fell within the normal ranges for healthy goats . Moreover, although no differences in the WBC concentration were detected, we did observe changes in specific types of white blood cell, with fewer EOS and NEU in TRT compared with those in CRT due to the dietary supplementation. This finding may suggest that the OA/PB supplementation could also have an immunomodulatory effect on dairy goats, as previously confirmed in non-ruminant species . Concerning the effects of the OA/PB on the hematochemical parameters, the CREA and TP levels were higher in CRT compared with those in TRT as a result of the dietary treatment. TP and blood urea nitrogen (BUN) are indicators of protein metabolism in the organism. Abas et al. found that the supplementation of organic acids in the rations of yearling lambs resulted in higher TP and lower BUN values. No differences in the BUN or milk urea nitrogen concentration (MUN) were found between the two dietary groups in the present study, but the goats fed the TRT diet showed lower values of TP. Unfortunately, it was not possible to calculate the dry matter intake, but on the basis of the available scientific literature, we may speculate that OA/PB supplementation could lead to the more efficient utilization of dietary nitrogen in dairy goats, as previously seen in dairy cows . The increase in the CREA concentration in the blood over the sampling period in both the CRT and TRT groups was most likely derived from a lactation-induced increase in muscle metabolism and is probably related to the normal muscle turnover occurring in mid-late lactation dairy goats . Moreover, we found that the CREA concentration was lower in TRT compared with that in CRT, an effect of the dietary treatment. When a marked increase in the CREA concentration is observed, it is generally related to a decline in renal function. However, although the lower CREA levels observed in the goats is an encouraging result, the potential effect of OA/PB improving renal function should be explored in more detail. 4.3. Milk Parameters Although milk yield is expected to decrease over time as per the normal lactation curve for dairy goats , no defined trend was observed in the present study, probably because the period between the sampling times was too short to reveal any differences. Nonetheless, in the context of the dairy goat industry, the milk composition and its technological properties are traits of great interest since most of the milk produced is processed into cheese . Milk composition can be influenced by several factors such as breed, parity, nutrition, and environmental effects . The prime factor reported to influence milk composition is the presence of subclinical mastitis (followed by the animal being toward the end of the lactation phase) . For this reason, we evaluated the bacteriological characteristics of all the milk samples throughout the feeding trial, and the results confirmed the absence of any bacteriological contamination due to mastitis. Moreover, the milk fat content and milk coagulation index in the TRT group improved over time. Milk fat is an important factor for cheese yield and firmness, as well as in determining the color and flavor of dairy goat products . Lipid supplementation in goat diets is generally associated with a higher milk fat content ; however, our findings suggest that supplementation with OA/PB could be useful for increasing the milk fat content in the mid-late lactating phase of dairy goats. This could be due to an increase in fatty acid synthesis in the mammary gland , but it may also be due to the fact that the supplementation of the total mixed ration with OA positively influences microbial fermentation in the rumen , which has been shown to increase volatile fatty acid production and reduce methanogenesis in dairy cows . In fact, milk fat synthesis requires palmitate, which is synthetized from acetate . However, the microencapsulated OA/PB mixture should be rumen protected and, accordingly, only exert its effect in the small intestine. However, the mechanism through which the microencapsulated OA/PB exerts its effect on the gut mucosa of ruminants, improving gut health and the intestinal barrier integrity, is uncertain at present . Therefore, further studies are required to understand the mode of action of OA/PB in ruminants. Investigations should be directed at elucidating the effect of OA/PB supplementation on the gut microbiota, which is known to influence the gut environment and intestinal permeability . 5. Conclusions This is the first study to evaluate the effects of supplementing the dairy goat diet with OA/PB. The hematological and hematochemical parameters revealed no negative consequences of OA/PB supplementation. Moreover, the finding that OA/PB may support renal function is certainly of interest and worthy of further investigation. The results obtained demonstrate that OA/PB exerts positive effects on milk performance by increasing the fat content and the milk coagulation index. Since goat's milk is mainly used for cheesemaking, these findings encourage further research on OA/PB supplementation in the dairy goat diet. Author Contributions Conceptualization: A.G., G.M., R.P. and C.F.; methodology: S.B., F.C., F.R. and L.D. software: D.C.; data curation: A.G., F.R., G.B. and D.C.; writing--original draft preparation: A.G., F.R., V.S.d.l.M.-E. and D.C.; writing--review and editing: D.B., G.M., E.V., M.G. and V.B.; supervision: D.B., R.P. and C.F. All authors have read and agreed to the published version of the manuscript. Institutional Review Board Statement The study was approved by the Ethics Committee of the Department of Veterinary Sciences of the University of Turin (Italy, Prot. N. 0002184/2021). Informed Consent Statement Not applicable. Data Availability Statement The data that support the findings of this study are available from the corresponding author upon reasonable request. Conflicts of Interest Richard Paratte has a technical consultancy association with Vetagro S.p.A. The authors have not stated any other conflict of interest. animals-13-00797-t001_Table 1 Table 1 Chemical composition of the TMR. Values are expressed as means +- SD. TMR a Dry matter (DM), % 45.0 +- 1.15 Ash, % of DM 8.50 +- 0.25 Crude protein, % of DM 15.8 +- 0.54 Starch, % of DM 21.8 +- 1.71 aNDFom b, % of DM 33.8 +- 1.26 ADF c, % of DM 22.8 +- 0.81 ADL d, % of DM 3.62 +- 0.19 uNDF240 e, % of DM 9.85 +- 0.53 a TMR ingredients: 35.1 % meadow hay silage, 32.4% complementary pelleted feed (containing crude protein 18%, ether extract 5%, crude fiber 8%, crude ash 7.5%), 16.2% second-cut alfalfa hay, 10.8% second-cut meadow hay, and 5.40% wheat bran. The hay was evaluated for its quality and the absence of undesirable weeds . b sodium sulfite-treated NDF with ash correction. c Acid detergent fiber. d Acid detergent lignin. e Unavailable NDF estimated by 240 h in vitro fermentation. animals-13-00797-t002_Table 2 Table 2 Hematological and hematochemical parameters for CRT (control group, n = 40) and TRT (treatment group, n = 40) blood samples collected at the three sampling times (T0, T27, and T54). Data are expressed as means +- standard error of the mean (SEM). Item Diet Time p-Values T0 T27 T54 Diet Time DietxTime Covariance Mean SEM Mean SEM Mean SEM WBC, 103/mL CRT 13.8 3.35 12.4 4.10 12.6 5.27 0.71 0.37 0.08 <0.01 * TRT 12.2 3.17 11.2 3.29 11.3 3.81 EOS, % CRT 6.05 2.60 7.20 2.20 10.9 4.65 0.01 * <0.01 * 0.12 0.03 * TRT 2.60 1.83 6.10 2.88 9.05 2.40 NEU, % CRT 49.9 0.94 45.3 1.53 54.9 2.34 0.05 * <0.01 * <0.01 * <0.01 * <0.01 * TRT 49.2 0.93 47.0 1.10 45.7 0.88 BA, % CRT 0.45 0.50 0.50 0.60 0.40 0.50 0.54 0.29 0.03 * 0.02 * TRT 0.40 0.50 0.30 0.50 0.50 0.55 MON, % CRT 4.20 0.93 4.00 1.00 3.60 1.38 0.18 <0.01 * 0.13 <0.01 * TRT 4.00 0.78 3.40 0.85 3.45 0.80 LYM, % CRT 38.8 0.81 42.8 1.43 40.1 1.41 0.41 0.05 * 0.31 <0.01 * TRT 41.2 1.01 42.1 1.11 41.2 0.90 RBC, 106/mL CRT 13.5 2.04 13.7 2.15 13.6 6.21 <0.01 * 0.69 0.02 * <0.01 * <0.01 * TRT 14.2 1.74 13.9 1.33 13.1 1.79 MCH, pg CRT 6.15 0.40 5.90 0.50 6.30 0.75 <0.01 * <0.01 * <0.01 * <0.01 * <0.01 * TRT 6.10 0.78 5.70 0.60 5.50 0.88 MCHC, g/dL CRT 34.4 0.24 33.0 0.20 34.5 0.36 <0.01 * 0.04 * <0.01 * <0.01 * <0.01 * <0.01 * TRT 33.8 0.37 31.2 0.29 30.7 0.37 MCV, mm3 CRT 17.9 1.80 18.0 1.90 18.4 2.30 0.81 0.01 * 0.54 <0.01 * TRT 18.1 3.48 18.3 3.15 18.4 3.33 Hb. g/dL CRT 8.15 0.95 8.10 1.00 8.25 5.13 <0.01 * 0.63 0.01 * <0.01 * <0.01 * TRT 8.70 0.88 7.90 0.55 7.35 0.80 HCT, % CRT 23.5 3.10 24.1 2.50 25.2 13.0 <0.01 * 0.81 0.12 <0.01 * TRT 25.6 3.65 25.4 3.50 24.2 4.65 PLT, 103/mL CRT 1054 46.0 113 67.0 122 57.3 <0.01 * 0.03 * 0.13 <0.01 * TRT 114 82.0 94.5 73.5 99.0 98.0 CREA, mg/dL CRT 0.38 0.03 0.26 0.01 0.64 0.02 0.01 * <0.01 * 0.20 <0.01 * TRT 0.47 0.01 0.25 0.01 0.60 0.02 BUN, mg/dL CRT 41.3 1.16 33.2 1.21 36.7 1.10 0.85 0.03 * 0.01 * <0.01 * TRT 40.7 1.14 34.6 0.97 34.3 1.18 TP, g/L CRT 7.82 0.05 7.87 0.06 7.94 0.08 0.01 * <0.01 * <0.01 * <0.01 * <0.01 * TRT 7.65 0.05 7.19 0.17 7.86 0.06 ALT, units/L CRT 31.2 0.63 29.3 0.66 29.3 0.64 0.24 0.55 0.47 <0.01 * TRT 29.3 0.87 28.8 0.85 28.3 0.84 AST, units/L CRT 147 41.3 160 71.0 165 37.3 0.77 0.15 0.68 <0.01 * TRT 144.5 46.8 158 41.8 157 34 * Statistical significance: p <= 0.05. Abbreviations: WBC, white blood cell; EOS, eosinophils; NEU, neutrophil; BA, basophil; MON, monocyte; LYM, lymphocyte; RBC, red blood cell; MCH, mean corpuscular hemoglobin; MCHC, mean corpuscular hemoglobin content; MCV, mean corpuscular volume; Hb, hemoglobin; HCT, hematocrit; PLT, platelets; CREA, creatinine; BUN, blood urea nitrogen; TP, total protein; AST, aspartate transaminase; ALT, alanine aminotransferase. animals-13-00797-t003_Table 3 Table 3 Milk yield and milk composition for CRT (control group, n = 40) and TRT (treated group, n = 40) at the three sampling times (T0, T27, and T54). Data are normally distributed and are expressed as means +- standard error of the mean (SEM). Item Diet Time p-Values T0 T27 T54 Diet Time DietxTime Covariance Mean SEM Mean SEM Mean SEM MY, kg/milking CRT 2.98 0.10 2.82 0.10 3.09 0.10 0.75 <0.01 * 0.42 <0.01 * TRT 2.94 0.09 2.77 0.12 3.14 0.10 Total protein, % CRT 3.15 0.05 3.12 0.05 3.19 0.06 0.37 0.10 0.22 <0.01 * TRT 3.12 0.04 3.15 0.06 3.12 0.05 Casein, % CRT 2.38 0.05 2.35 0.05 2.41 0.05 0.25 0.13 0.51 <0.01 * TRT 2.34 0.03 2.37 0.05 2.35 0.04 Fat, % CRT 2.47 0.75 2.12 0.86 2.04 0.71 0.04 * <0.01 * 0.46 <0.01 * TRT 2.71 0.68 2.56 0.34 2.24 0.49 MCI, % CRT 75.4 0.37 75.2 0.31 75.1 0.29 0.03 * 0.29 0.03 * <0.01 * 0.02 * TRT 75.1 0.23 75.4 0.18 75.6 0.15 Lactose, % CRT 4.21 0.03 4.24 0.04 4.09 0.04 0.59 <0.01 * 0.10 <0.01 * TRT 4.18 0.03 4.20 0.03 4.11 0.03 SCC, 103/mL CRT 356 819 366 702 382 1181 0.94 0.64 0.58 <0.01 * TRT 488 769 574 770 697 1026 MUN, mg/dL CRT 37.6 1.50 26.1 1.32 28.2 1.14 0.08 <0.01 * 0.32 <0.01 * TRT 36.0 1.11 27.0 0.86 28.2 0.96 fat-free DM, g CRT 239 71.3 220 68.2 243 59.26 0.23 <0.01 * 0.87 <0.01 * TRT 232 60.1 223 61.0 244 60.8 FCM, kg CRT 2.61 0.73 2.49 0.70 2.62 0.59 0.14 <0.01 * 0.78 <0.01 * TRT 2.63 0.77 2.47 0.68 2.73 0.62 FPCM, kg CRT 2.39 0.65 2.22 0.75 2.50 0.60 0.09 <0.01 * 0.65 <0.01 * TRT 2.55 0.58 2.29 0.52 2.59 0.62 ECM, kg CRT 2.28 0.58 2.11 0.68 2.36 0.62 0.09 <0.01 * 0.68 <0.01 * TRT 2.42 0.57 2.18 0.51 2.45 0.58 * Statistical significance: p <= 0.05. Abbreviations: MY, milk yield; MCI, milk coagulation index; SCC, somatic cell counts; MUN, milk urea nitrogen; FCM, fat corrected milk; FPCM, fat and protein corrected milk; ECM, energy corrected milk. Disclaimer/Publisher's Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

PMC10000139

The rapid development of urbanization has changed landscape patterns and biological habitats severely and, therefore, affected biodiversity. In this study, we selected 75 townships in Lishui, a mountainous area in eastern China, to conduct bird surveys for two years. We analyzed the birds' composition characters in townships with different levels of development in order to identify the effects on bird diversity of the urban development level, land cover pattern, landscape pattern, and other factors. In total, 296 bird species from 18 orders and 67 families were recorded between December 2019 and January 2021. A total of 166 species of birds belonged to Passeriformes (56.08%). The seventy-five townships were divided into three grades by K-means cluster analysis. The average number of bird species, richness index, and diversity index were higher in G-H (highest urban development level) compared with the other grades. At the township level, landscape diversity and landscape fragmentation were the key factors that positively affected the bird species number, diversity index, and richness index. Landscape diversity had a greater effect than landscape fragmentation, particularly on the Shannon-Weiner diversity index. The diversity and heterogeneity of urban landscapes could be improved by constructing biological habitats in future urban development planning to maintain and increase biodiversity. The results obtained in this study provide a theoretical basis for urban planning in mountainous areas, and a reference for policymakers to formulate biodiversity conservation strategies, construct reasonable biodiversity patterns, and solve practical biodiversity conservation problems. bird diversity landscape features township urbanization Ministry of Ecology and Environment of China2110404 Project of Biodiversity Conservation in Lishui, Zhejiang ProvinceHXYJCP2021110648 This research was funded by the "Project supported by the biodiversity investigation, observation and assessment program (2019-2023) of Ministry of Ecology and Environment of China (2110404)" and the "Project of Biodiversity Conservation in Lishui, Zhejiang Province (HXYJCP2021110648)". pmc1. Introduction The rapid development of cities changes very much of our environment . Urbanization affects biological habitats, including the destruction and loss of habitats, thereby affecting the diversity of plants, insects, and vertebrates, especially birds . Reducing the impact of urban development on biodiversity and reaching a harmonious balance between man and nature is the goal of future urban development, and also a hot topic in biodiversity research . Birds are important components of biodiversity and the most common vertebrates in cities. Bird species are important indicators of the status of urban ecological environments and biodiversity . Given the importance of birds for urban ecological environments and the well-being of residents , it is particularly relevant to conduct ornithological research in the context of rapid urbanization. The present study analyzed the impacts of urban development on bird diversity . The research areas were mostly urban green spaces/parks , and locations with different levels of urban development were qualitatively evaluated . From the perspective of the whole urban area, expanding the sampling unit, such as considering a county or township as the smallest unit, can reduce the limitations caused by sampling and more comprehensively reflect the situation for a whole city. The environmental factors that affect bird diversity are considered to vary significantly at different spatial scales . On a large regional scale, climate, climate stability, and regional areas are among the main factors to affect bird diversity . At the local scale, the land-use pattern, landscape pattern, and socio-economic development may be more important for the distribution of biodiversity, especially bird diversity . At even smaller scales, such as urban green spaces and parks, the vegetation structure, green space areas, and habitat characteristics are major factors that influence bird diversity . Lishui is located in a mountainous area in a subtropical region of eastern China and was selected as the research site for this study because of its rich bird resources, with 370 species, and most of them are resident bird species (129 species), followed by winter migrants (112 species) . In this study, bird surveys were conducted for 2 years in 75 townships in Lishui. A quantitative method was used to classify the development grade of each township, and the key factors that could affect bird diversity distribution were identified. In particular, we aimed (1) to determine the characteristic bird diversity in townships with different development levels in a mountainous area of eastern China; (2) to identify the key factors that might affect bird diversity at the township level; and (3) to determine how to allocate land use and landscape patterns in a scientific and reasonable manner in order to maintain and increase urban biodiversity under inevitable urban expansion and development. The results obtained in this study may provide a theoretical basis for urban planning and biological habitat design in the future development of cities in mountainous areas. In addition, our findings could provide a reference to allow decision-makers to formulate biodiversity conservation strategies, construct reasonable biodiversity patterns, and solve practical problems in biodiversity conservation. 2. Materials and Methods 2.1. Study Area Lishui City (118deg41'-120deg26' E, 27deg25'-28deg57' N) in Zhejiang Province is a typical city in a mountainous region of eastern China . Lishui has a total area of 17,275 km2 and a population of 2707400 at the end of 2020. Lishui City is a subtropical monsoon climate zone. The average annual temperature is 17.8 degC. Lishui is located in the Wuyi mountain series in a subtropical region, and the forest coverage rate in the city is about 81.7%. In this mountain series, 3573 peaks are greater than 1000 m above sea level. The southwest mainly contains medium-sized mountains and the northeast is dominated by low mountains, with medium-sized mountains and the Bihu Plain, Songgu Plain, and Huzhen Plain in between. The main water systems are the Oujiang River, Qiantang River, and Feiyun River. Thus, Lishui was selected as a representative city for studying the urban bird diversity in a mountainous area . In the last 10 years, rapid urban construction development and urban land-scale expansion have occurred in Lishui. The urbanization rate was 61.8%. Between 2010 and 2020, the urban area increased by 280.2 km2, i.e., an increase of 67.5%, mainly due to losses of forest and farmland ecosystems. This rapid development has greatly impacted biodiversity, which may lead to the loss of habitat for some species and a decrease in bird diversity. In this study, bird diversity was investigated in 75 townships with different development levels, which were identified as research sites according to the township population, population density, township area, urbanization rate, and other indicators. 2.2. Bird Surveys Between December 2019 and January 2021, bird surveys were conducted in the 75 townships in Lishui during each quarter in the spring (April-May), summer (July-August), autumn (October-November), and winter (December-January). Two or three transects were selected in each township. In total, 188 transects were investigated. Surveys were conducted on sunny days during time periods with frequent bird activities (7:00-10:00 and 15:00 to sunset in the spring and autumn; 5:00-8:00 and 17:00 to sunset in the summer; 9:00-11:00 and 15:00 to sunset in the winter). The line transect method was used to conduct the bird surveys. The surveyor walked along the transects at a speed of 1-2 km h-1 and recorded the species and number of each species observed or heard within 50 m of the transect's center. Each transect had a length of 1-1.5 km. Bird species were identified and classified referring to "A Checklist of Classification and Distribution of the Birds of China" (Third Edition) . Conservation status (least concern, near-threatened, vulnerable, endangered, and critically endangered) was based on IUCN . 2.3. Selection of Environmental Factors and Data Acquisition Land-use data (2020) from Landsat TM/ETM images with a resolution of 30 m were used as the main data sources. Erdas 9.3 software was used to interpret and quantify the areas of various land-use types. The land-use factors included the following indicators: proportion of forest area (PFOR), proportion of shrub area (PSHR), proportion of grassland area (PGRA), proportion of wetland area (PWET), proportion of bare land area (PBAR), proportion of farmland area (PFAR), and proportion of urban area (PURN) . GIS technology and FRAGSTATS 4.1 software were used to analyze the landscape pattern structure in each township, where the numbers of patches and average patch areas were calculated. The landscape heterogeneity indexes included the landscape diversity index, landscape evenness index, and landscape fragmentation index . The urbanization index included the township populations, population densities, and township area size , where the data were derived from the Statistical Yearbook of Lishui in 2021. 2.4. Classification of Townships Quantitative classification of the township development grades allows us to more accurately explore the effects of the urban structure and landscape pattern on the biodiversity distribution . K-means clustering was conducted to divide the 75 townships into different development grades according to factors including the total population, population density, township area, and the proportion of the area comprising non-natural ecosystems (urban and farmland). The K-means clustering results were divided into four grades. The results of 5 clustering indexes of grades 1 and 4 were similar, so, we combined grades 1 and 4. Finally, the seventy-five townships in the study area were divided into three grades, where G-H represented townships with the highest degree of urban development, G-M represented townships with a moderate degree of urban development, and G-L represented townships with a low degree of urban development. The characteristics of the different grades are shown in Table 1. 2.5. Data Analysis 2.5.1. Bird Diversity The number of bird species, richness index, diversity index, and evenness index were used to measure the level of bird diversity in each township . (1) Bird species comprised the total number of bird species observed in four surveys. (2) The Shannon-Weiner diversity (HB) of bird species was calculated using the following formula:(1) HB=-i=1SPiln(Pi), where Pi is the ratio of the number of individuals from bird species i relative to the total number of individuals in the community and S is the number of bird species. (3) The richness index (RB) comprising the Pielou coefficient was calculated as:(2) RB =(SB - 1)/Ln(NB) where SB is the number of bird species and NB is the sum of the number of individuals of all bird species. (4) The evenness index (JB) was calculated as:JB = HB/HBmax,(3) where HBmax is the theoretical maximum diversity index and HBmax = Ln(SB). 2.5.2. Landscape Index According to previous research, the landscape heterogeneity indexes included the landscape diversity index, landscape evenness index, and landscape fragmentation index . (1) The Shannon-Weiner landscape diversity (H) was calculated as:(4) H=-i=1SPiln(Pi), where S is the total number of landscape types in a township and Pi is the proportion of the landscape area for type i relative to the total area. (2) The landscape evenness index (JB) was calculated as:J = H/Hmax,(5) where Hmax is the theoretical maximum diversity index and Hmax = Ln(S). (3) The landscape fragmentation index was calculated as:D = (N - 1)/NC,(6) where N is the total patch number and NC is the average patch area in all landscape types. 2.5.3. Statistical Analyses One-way analysis of variance was conducted to test for differences in the bird species, richness index, diversity index, and evenness index between townships with different grades . Species accumulation curves were used to assess whether the sample sizes were adequate and to estimate the community richness in towns with different grades by applying the "vegan" package in R 4.1.6. Variance inflation factors (VIFs) were calculated to assess the collinearity among environmental variables . After removing two variables with strong collinearity comprising the total population and landscape evenness index, the VIF values for all environmental variables were less than 3. Finally, the number of bird species, richness index, diversity index, and evenness index were used as response variables. A multiple stepwise regression analysis was conducted using 10 indicators comprising the township population density, township area, shrub area proportion, grassland area proportion, wetland area proportion, bare land area proportion, farmland area proportion, urban area proportion, landscape diversity index, and landscape fragmentation index, as predictor variables. All analyses were performed using the "vegan" package in R 4.1.6 and with IBM SPSS Statistics 25. 3. Results 3.1. Bird Diversity Between December 2019 and January 2021, 296 species belonging to 18 orders and 67 families were recorded in the study areas (Table 2). Most bird species belonged to Passeriformes (166 species, 56.08%), whereas Suliformes, Bucerotiformes and Trogoniformes had the lowest number of representatives (one species each). In particular, sixty-one of the bird species are under special protection in China, including seven species under first-class state protection and fifty-four species under second-class state protection. Among the birds under special protection, the top five orders were Accipitriformes (eighteen species, 29.5%), Passeriformes (ten species, 16.39%), Strigiformes (eight species, 13.11%), Falconiformes (five species, 8.20%), and Galliformes (five species, 8.20%). Among the IUCN Red List of Threatened Species, the least concerned species comprised the greatest proportion (84.12%, n = 249). Two critically endangered (CR) species were recorded comprising the Siberian crane (Grus leucogeranus) in Huzhen town and yellow-breasted (Emberiza aureola) bunting in Shitang town and Bihu town. Four endangered (EN) species were recorded comprising the scaly-sided merganser (Mergus squamatus), white-eared night heron (Gorsachius magnificus), Cabot's tragopan (Tragopan caboti), and black-faced spoonbill (Platalea minor). Six vulnerable (VU) species and thirty-five near-threatened (NT) species were also recorded. In terms of the different residence types, most were resident bird species (136 species, 45.95%), followed by winter passing migrants (58 species, 19.59%), visitors (55 species, 18.58%), and summer visitors (47 species, 15.88%). 3.2. Analysis of Bird Diversity in Townships with Different Grades The species accumulation curves obtained for the three grades were characterized as gradually flattening as the number of individuals increased . More individual birds made small contributions to the new species records, thereby suggesting the gradual saturation of bird species. The sampling integrity of the bird survey in each township was greater than (99%), which indicates that the overall data set was reasonable and suitable for further analysis. The bird species recorded in different township grades differed significantly (p < 0.001). The total number of species was highest in G-M (242 species), followed by G-L (229 species) and G-H (215 species). However, the average number of bird species was highest in each township in G-H (n = 7, mean +- standard deviation (SD) = 79.71 +- 18.33), and the average number of bird species decreased as the township development grade decreased (G-M, n = 23, mean +- SD = 59.87 +- 19.04; G-L, n = 45, mean +- SD = 50.13 +- 13.77). The bird diversity index (p = 0.003) and richness index (p < 0.001) differed significantly among township grades, but there was no significance. Figure 3 shows that the bird diversity index, evenness index, and richness index were highest in G-H. The bird richness index followed the order of G-H (n = 7, mean +- SD = 11.638 +- 2.179) > G-M (n = 23, mean +- SD = 9.165 +- 2.114) > G-L (n = 45, mean +- SD = 8.183 +- 1.821). The diversity index followed the order of G-H (n = 7, mean +- SD = 3.628 +- 0.255) > G-M (n = 23, mean +- SD = 3.427+- 0.325) > G-L (n = 45, mean +- SD = 3.258 +- 0.271). The evenness index followed the order of G-H (n = 7, mean +- SD = 0.831 +- 0.039) > G-M (n = 23, mean +- SD = 0.847 +- 0.052) > G-L (n = 45, mean +- SD = 0.840+- 0.052). In terms of the composition of birds, Passeriformes comprised the most abundant order in the three township grades (G-L > G-M > G-H). Figure 4 shows that the waterbird species represented by Anseriformes, Pelecaniformes, Gruiformes, and Charadriiformes were more abundant in G-H. Significantly more forest bird species represented by Accipitriformes and Strigiformes were found in G-L than in G-H and G-M. In Trogoniformes, the red-headed trogon was only recorded in G-M and G-L. 3.3. Environmental Variables Affect Bird Diversity As shown in Table 3, the multiple stepwise regression analysis results obtained two explanatory models for the three response variables comprising bird species, diversity index, and richness index. The model that included the landscape diversity index (H) and landscape fragmentation index (D) was best at explaining the number of bird species, diversity index, and richness index. The models are as follows:Ybird specie = 30.489+3.118 x H+14.327 x D(7) Ydiversity index = 2.948+0.045 x H+0.240 x D(8) Yrichness index = 6.168+0.354 x H+1.351 x D(9) Multiple stepwise regression analysis did not obtain a reasonable model for explaining the bird evenness index. The 10 predictor variables tested in this study had no significant effects on the bird evenness index. 4. Discussion 4.1. Bird Diversity in Different Township Grades Negative effects of urbanization on bird diversity have been confirmed in many previous studies . In this study, we determined bird diversity in townships with different grades of development; however, our results showed that the number of bird species, richness index, and diversity index were significantly higher in the townships with the highest level of development. This finding appears to be different from most previous studies. The study location is a mountainous area and the average proportion of the 75 townships covered by natural ecosystem areas is 78.67%. The townships in the study area with the highest level of urban development still had a high proportion of their areas covered by forest ecosystems (mean = 56.63%). Thus, appropriate urban development (mean = 16.12% of urban ecosystem area) is conducive to increasing the diversity of birds. This trend agrees with the moderate disturbance hypothesis, where biodiversity peaks under an intermediate level of disturbance , which can be achieved through urban wetland parks , contiguous farmland interspersed with forest land, and other patterns. Our results are consistent with the conclusions obtained by Marzluf et al., who studied areas with 20-90% forest coverage and found that bird diversity peaked under forest coverage of 50-60% . Palomino et al. also showed that the bird species richness was highest in areas with an urban building coverage of 15-28% . In conclusion, moderate urbanization increases the diversity of habitats, and thus the richness and diversity of bird communities. 4.2. Key Environmental Factors That Affected Bird Diversity The factors that affect urban bird diversity have been studied for many years. It is generally considered that habitat patterns such as landscape heterogeneity, landscape fragmentation, and habitat connectivity are the main factors that affect urban bird diversity at the urban scale . In the present study, the multiple stepwise regression models obtained based on the bird diversity index and various factors showed that landscape diversity and landscape fragmentation had significant positive effects on bird diversity, and they were the key variables that affected the number of bird species, diversity index, and richness index. In terms of the impact of landscape diversity, our findings are consistent with those obtained in most previous studies . It is considered that improving landscape diversity is conducive to increasing bird diversity. Townships with generally high landscape diversity have large areas covered by forest ecosystems, but other ecosystem types are also present, such as farmland, wetlands, and towns. Forests provide adequate food and habitat for most bird species , and the increment of other landscape types can enhance bird diversity by providing the habitats available for birds that are more dependent on wetlands, farmland, and towns, thereby providing more ecological niches and refuges . We also found that at the township level, bird diversity was strongly determined by landscape diversity and landscape fragmentation, and the effect of landscape diversity was stronger than landscape fragmentation. Similarly, De Camargo et al. suggested that the response of birds to habitat quantity is significantly stronger than that to landscape fragmentation at the landscape level . However, the positive or negative effect of landscape fragmentation has been debated. Numerous studies have shown that the relationship between landscape fragmentation and bird diversity varies in different threshold ranges . According to our findings, in the study location where the urban landscape area did not exceed 45%, increased landscape fragmentation improved bird diversity because the large and contiguous forests in this landscape pattern were divided by small farmland areas, towns, and other habitats, which increased the number of patches in the landscape. However, the increased landscape fragmentation also increased landscape diversity, thereby maintaining higher bird diversity and richness . Fahrig et al. also concluded that landscape fragmentation increases species richness by increasing landscape diversity. In addition, Deng et al. argued that bird densities are higher on small forested islands than on large islands . 4.3. Suggestions for Preserving Biodiversity during Urban Development Maintaining and even improving urban biodiversity must be considered during inevitable urban development planning. It has been shown that forest agricultural mosaic landscapes can enhance bird diversity in farmland landscapes, and wetland patches and mosaic patterns with surrounding forests can also promote the coexistence of organisms that depend on different habitats . Therefore, in urban planning and management, in addition to maintaining a high forest coverage rate, the heterogeneity and diversity of the landscape pattern should be increased in an appropriate manner to enhance local biodiversity, which can be achieved by designing urban biological habitats. The existing nature reserves, forest parks, wetland parks, and other protected natural areas in cities are all biological habitats with good foundations. In city planning, attention should be paid to the design of various types of habitats to improve the diversity of the landscape . Therefore, it is very important to increase the number of biological habitats comprising grassland, wetland, farmland, and other types in mountainous cities. In addition, special landscape types should be preserved and promoted. For example, the meadow landscape distributed in high-altitude areas in the current study area should be strongly protected to maintain the diversity of birds, such as the russet bush warbler (Locustella mandelli) and buff-throated warbler (Phylloscopus subaffinis), which are highly dependent on these landscape types. The two approaches that can be applied to establish biological habitats are ecosystem-centered and species-centered , where the former focuses on the overall design from the perspective of the landscape and the latter focuses on the design of biological habitats from the perspective of protecting a certain species or group. Birds have high activity levels and they are dependent on specific habitats. Most birds can select a fixed habitat that provides a suitable environment. The diversity of other groups can be indirectly protected by protecting and promoting certain bird habitats, thereby improving biodiversity. For example, in order to protect the scaly-sided merganser (assessed as EN level in the IUCN Red List), we can protect the river landscape where it lives, preserve the farmland and village landscapes around the river landscape as buffer zones to reduce excessive human disturbance, and treat the scaly-sided merganser as an umbrella species to improve the habitat quality for other birds in Anseriformes, which accounted for 5.41% of the birds in the study area . 5. Conclusions In this study, we investigated bird diversity and distribution patterns at the township level in Lishui, a mountainous area of eastern China. We obtained the following main results. (1) In the mountainous area, moderate urbanization increased the diversity of habitats, which may harbor an increased number of bird species and richness, and improve the diversity of bird communities. (2) At the township scale, bird diversity was determined by the combined effects of landscape diversity and landscape fragmentation, and the effect of landscape diversity was stronger than that of landscape fragmentation. (3) Under the premise of maintaining a high forest coverage rate, maintaining landscape diversity was conducive to increasing the number of bird species, richness, and diversity. Landscape fragmentation is improved by adding wetlands, grasslands, farmlands, urban parks, etc. In this case, the increase in landscape fragmentation might improve the number of bird species, richness, and diversity. In addition, due to data acquisition limitations, the predictor variables used to determine effects on bird diversity did not include socio-economic development level indexes, but the effects of these factors could be investigated in future research. Based on our conclusions, we suggest that in future urban planning and management, the heterogeneity and diversity of landscape patterns can be increased by constructing urban biological habitats to maintain and enhance local biodiversity. The results obtained in our study provide a theoretical basis for urban planning in similar mountain cities during future development. In addition, our results provide a reference for decision-makers to formulate better biodiversity conservation in city development plans and practical biodiversity conservation problems. Acknowledgments We would like to thank the Lishui Municipal Bureau of Ecological Environment for its support of this study and the people who participated in the bird field survey. Author Contributions Conceptualization, P.C., L.C. and W.Z.; methodology, W.Z. and H.Z. software, W.Z., Y.Z. and X.F.; validation, W.Z. and Y.W. formal analysis, W.Z.; investigation, W.Z. and S.Z.; resources, W.Z.; data curation, W.Z., Y.Z., X.F. and S.Z.; writing--original draft preparation, W.Z.; writing--review and editing, W.Z., P.C. and L.C.; visualization, W.Z., Y.Z. and X.F.; supervision, P.C. and L.C.; project administration, P.C.; funding acquisition, P.C. All authors have read and agreed to the published version of the manuscript. Institutional Review Board Statement Not applicable. Informed Consent Statement Not applicable. Data Availability Statement The data will be provided upon request to the corresponding author. Conflicts of Interest The authors declare no conflict of interest. Figure 1 Locations of 75 townships in Lishui. The black line represents the boundary of the Lishui administrative region and the green regions are the 75 townships (drawn by the author). Figure 2 Sample completeness curves for bird communities in three township grades. The filled circles represent the sample coverage. Red represents G-H, blue represents G-M, and green represents G-L. Figure 3 Comparison of average bird species (A), Shannon-Weiner index (B), Pielou index (C), and Margalef index (D) in three township grades. In the box plot, the black horizontal line represents the average values, the colored horizontal lines represent the median, dots represent the outliers. Figure 4 Comparison of bird species from different orders in the three township grades. Green represents G-L, red represents G-M, and blue represents G-H. The lengths of the different colored columns indicate the relative number of bird species from an order in different towns. animals-13-00882-t001_Table 1 Table 1 Average values (+- one standard error) for characteristics of each township grade. G-H Number of Towns: 7 G-M Number of Towns: 23 G-L Number of Towns: 45 Population Density 402 +- 186 122 +- 83 93 +- 61 Total Population 56923 +- 18080 18347 +- 5794 8314 +- 3694 Township Size 158.44 +- 61.57 181.98 104.98 PURN 16.12 +- 12.95 3.31 +- 0.94 2.65 +- 2.13 PFAR 27.25 +- 7.30 15.87 +- 7.25 16.35 +- 8.59 PFOR 49.29 +- 14.64 73.98 +- 9.31 75.31 +- 10.10 PSHR 1.89 +- 20.09 1.35 +- 0.90 1.08 +- 0.66 PGRA 2.91 +- 0.72 2.54 +- 2.27 2.85 +- 1.56 PWET 2.22 +- 0.41 2.57 +- 2.91 1.24 +- 1.40 PBAR 0.32 +- 0.15 0.38 +- 0.40 0.52 +- 0.35 Landscape Diversity Index 1.2037 +- 0.1057 0.9896 +- 0.5019 0.7741 +- 0.2066 Landscape Evenness Index 0.6186 +- 0.0543 0.5086 +- 0.2579 0.3978 +- 0.1062 Landscape Fragmentation Index 8.1571 +- 4.1850 4.3361 +- 0.3547 3.3444 +- 1.6081 animals-13-00882-t002_Table 2 Table 2 Compositions of bird communities observed in 75 townships. Orders Families Proportion of Total Families Species Proportion of Total Species PASSERIFORMES 38 56.72% 166 56.08% CHARADRIIFORMES 6 8.96% 22 7.43% ACCIPITRIFORMES 2 2.99% 18 6.08% ANSERIFORMES 1 1.49% 16 5.41% PELECANIFORMES 2 2.99% 14 4.73% GALLIFORMES 1 1.49% 8 2.70% STRIGIFORMES 1 1.49% 8 2.70% GRUIFORMES 2 2.99% 7 2.36% CUCULIFORMES 2 2.99% 7 2.36% PICIFORMES 1 1.49% 7 2.36% CORACIIFORMES 3 4.48% 6 2.03% FALCONIFORMES 1 1.49% 5 1.69% COLUMBIFORMES 1 1.49% 4 1.35% CAPRIMULGIFORMES 2 2.99% 3 1.01% PODICIPEDIPORMES 1 1.49% 2 0.68% SULIFORMES 1 1.49% 1 0.34% TROGONIFORMES 1 1.49% 1 0.34% BUCEROTIFORMES 1 1.49% 1 0.34% Total 67 100% 296 100% animals-13-00882-t003_Table 3 Table 3 Multiple linear regression models based on relationships between environmental variables (predictor variables) and the number of bird species, bird diversity index, and bird richness index (response variables). Response Variables Model Input Variables R2 F Significance Bird Species Model1 landscape fragmentation index 0.214 21.148 p < 0.001 Model 2 landscape diversity index 0.281 15.429 p = 0.007 landscape fragmentation index Bird Diversity Index Model 1 landscape fragmentation index 0.155 14.565 p < 0.001 Model 2 landscape diversity index 0.218 11.317 p = 0.011 landscape fragmentation index Bird Richness Index Model 1 landscape fragmentation index 0.184 17.722 p < 0.001 Model 2 landscape diversity index 0.221 11.474 p = 0.039 Disclaimer/Publisher's Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

PMC10000140

As one of the most abundant game species in Europe, European wild boar (Sus scrofa) populations prove highly adaptable to cultivated landscapes. The ongoing process of climate change and the high agricultural yields seem to further optimize the living conditions for this species. In long-term reproduction monitoring, we collected data on the body weight of wild boar females. Over an 18-year period, the body weight of wild boar females increased continuously, then stopped and decreased. It was possible to detect differences between the body weights of animals from forest and agricultural areas. For these areas, differences in body weight development also led to a significant distinction in the onset of puberty. We conclude that, even in a highly cultivated landscape, forested areas provide habitat characteristics that may strongly influence reproduction. Second, with dominant agricultural areas in Germany, wild boar reproduction has been favored in recent decades. Sus scrofa reproduction weight increase habitat climate factors Ministry of Food, Agriculture and Consumer Protection of Lower Saxony, Germany (Niedersachsisches Ministerium fur Ernahrung, Landwirtschaft und Verbraucherschutz)Verein der Forderer der Wildtierforschung an der Stiftung Tierarztliche Hochschule Hannover e. V.Deutsche Forschungsgemeinschaft (DFG, German Research Foundation)491094227 University of Veterinary Medicine Hannover, FoundationThis research was funded by the Ministry of Food, Agriculture and Consumer Protection of Lower Saxony, Germany (Niedersachsisches Ministerium fur Ernahrung, Landwirtschaft und Verbraucherschutz) and by "Verein der Forderer der Wildtierforschung an der Stiftung Tierarztliche Hochschule Hannover e. V." This Open Access publication was funded by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation)--491094227 "Open Access Publication Funding" and the University of Veterinary Medicine Hannover, Foundation. pmc1. Introduction The wild boar (Sus scrofa) is a popular and widely abundant game species , and populations have increased throughout Europe in recent decades . It is an opportunistic omnivore with a high adaptability to changing environmental conditions . The wild boar has by far the highest reproductive potential and fecundity of all ungulate species worldwide in relation to its body mass . Combined with a low natural adult mortality rate in temperate climates , it can achieve high population densities in a very short time period . Due to its increasing population, it has become a very important species from an economical point of view . It can transmit diseases (like the African swine fever) to livestock and cause damage to agriculture . Several aspects that contribute to wild boar population growth have been studied and discussed. Among them, the most important factors are a reproductive success or lower juvenile mortality due to optimal feeding and mild climatic conditions . Several parameters of wild boar reproductive biology, such litter size , sex ratio and parturition time (unpublished own data) are positively related to maternal body weight. A body weight increase in free-living wild boar populations was reported from different European habitats , arising the question if similar developments could be observed from study areas situated in parts of Germany that were dominated by a high extent of cultivated landscape. Therefore, the aim of this research was to investigate whether the body weight of female wild boars in some areas of northern Germany increased over an 18-year period and whether the increase in body weight was different among age classes and habitats. Here, the influence of external (e.g., climate and mast) and internal (e.g., age and maturity) factors on body weight was tested, and, in addition we also tested for a possible effect on the onset of puberty. We hypothesized that body weight increased in all age classes over the years and that comparisons between forest and agricultural habitats showed a greater weight increase in the latter. 2. Materials and Methods 2.1. Study Area The study area was situated in the East of the federal state of Lower Saxony (northern Germany) (52.36deg N, 10.35deg E) and comprised about 5500 km2 with altitudes ranging from 60 to 130 m asl . Based on data from the German Meteorological Service, the average annual temperature since 2000 was 9.7 degC, and the average annual precipitation was about 800 mm. The trend of mean temperature is increasing, and mean precipitation is decreasing . The study area comprised either forestal-agricultural habitats (fah) or represented a large contiguous forestal habitat . Within the total area, one large block of forest (430 km2) was interpreted as a separate territory, containing only 3% of agricultural areas, and thus, agricultural food was available for wild boars only to a limited amount. Forest composition was almost the same as in the total area, while the forested area additionally comprised 2% of heathland and 5% of settlement and infrastructure. This area could be interpreted as forestal habitat , while the agriculturally dominated part of the study area was about 5500 km2, with 61% auf agricultural areas and 22% of forest. The remaining 17% were buildings and infrastructure. The agricultural land was characterized by 80% of energy-enriched products (e.g., grain, sugar beets, oilseed, rape, maize, leguminoses and potatoes) in 2020. The remaining 20% consisted of heathland, fallow land and horticultural products . Since 2007, only slight changes within the different field crops were reported . The forest consisted of 18.9% oak (Quercus spp.), 11.2% beech (Fagus sylvatica), 7.6% other deciduous trees (e.g., Betula spp, Acer spp., Tilia spp., Sorbus aucuparia), 45.5% pine (Pinus spp.) and 16.6% other coniferous trees (Larix spp., Picea abies, Pseudotsuga menziesii). 2.2. Data Collection Data were collected from 3517 free-ranging hunted wild boar females in the study area during the hunting years from 2003 until 2020. Each hunting year (indicated as "year" in the following text) started in April and ended in March. Since the majority of samples derived from November-January, we focused our analyses on these months of the hunting year (n = 3.367, Table 1). The age of the wild boar females was identified by tooth determination . Following the age identification, female wild boars were grouped into three classes (n = 2024 piglets: <=12 months; n = 866 yearlings: >=13-24 months; n = 477 adults: >=25 months, Table 1). Their dressed body weight (without digestive system, heart, lungs, liver, reproductive tract, and blood, hereafter "bw") was recorded. Dissection and examination of the reproductive tract (uterus and ovaries) was performed according to our own protocols . Consequently, the onset of sexual maturity was assumed when ovaries had follicles >=0.4 cm in diameter. 2.3. Statistical Analysis We described with Tukey's Honest Significance Difference (HSD) test differences between mean bw in the three age classes both overall and within the two habitat types. Then, we used generalized linear models (GLM, ) with identity link to analyze the development of bw over the hunting years, including age class, habitat type, tree crop and weather variables (Table 2). Explanatory variables were checked for correlations among each other , but multicollinearity did not affect our analyses, as checked by correlations and variance inflation. Testing for both linear and unimodal relationships with the dependent variable (bw), we used the AIC (Akaike Information Criterion) from univariate models with each numerical weather variable to decide about their inclusion in the GLM. We stopped when adding a new variable did not result in a lower AIC value. We also included the hunting index as a measure of hunting intensity for each hunting year. We used a GLM with logit link and binomial error structure to find predictors of wild boar puberty. All analyses were performed using R software version 4.1.0 . 3. Results 3.1. Overall Body Weight Tukey's HSD test revealed highly significant differences between age classes in the whole sample. Mean bw for piglets, yearlings, and adults was 27.6 kg [sd 8.6, n = 2024], 54.0 kg [sd 10.5, n = 866] and 66.7 kg [sd 12.9, n = 477], respectively. In all age classes, we found an increase in bw that resulted in significant differences between some months, following a seasonal pattern during autumn and winter. A bw difference (diff) between November and January was significant in yearlings (Tukey's HSD test: p = 0.05, diff = -2.3 kg) and adults (Tukey's HSD test: p < 0.01, diff = -4.2 kg). In piglets, there was a significant bw difference between November and December (Tukey's HSD Test: p < 0.0001, diff = 3.1 kg) and, like in the other two age classes, between November and January (Tukey's HSD Test: p < 0.0001, diff = -3.9 kg). The weight increase in piglets from November to December was especially pronounced in the fh (diff = 5.0 kg) and, to a lesser extent, in the fah (diff = 2.3 kg). 3.2. Effect of Habitat on Body Weight Mean bw was higher in fah compared to fh habitats . The difference of 2.8 kg was significant (Tukey's HSD p > 0.01). Differences between habitat types were particularly pronounced in yearlings. In this age class, the mean bw of animals from the agricultural habitat was 8.0 kg higher than that from forest habitat (Tukey's HSD p > 0.01). In piglets and adults, the weight difference between habitats was 5.2 kg and 5.5 kg, respectively. 3.3. Temporal Trends in Body Weight and External Factors Bw differed per age class and habitat, but the overall trend was similar in both factor variables. In all models, the relationship of bw with hunting year was best described by a fourth-order polynomial that visually suggested a slight increase until about 2017, followed by a decrease until 2020 . Fluctuations in bw were more pronounced for wild boar in fh. Strong beech mast benefited especially adult animals in fh , while oak mast did result in differences in bw without any significance. Among all age classes, climate factors such as frost periods in February negatively affected bw, while higher precipitation in May and July had a positive impact . The inclusion of the hunting index further improved the final model, with the bw being negatively correlated with this predictor of hunting pressure , which was again true for all age classes. 3.4. Predictors of Wild Boar Puberty In our data, piglets reached puberty approximately at a bw of 30 kg, and in fh, this was achieved at an age of 11 months, whereby piglets of fah seemed to achieve the crucial body weight at a lower age, i.e., about 8-9 months . As a result, in fah, 80 percent of the 8-month-old piglets had already reached puberty, while in forest areas, only 64 percent did. In years with beech masts, the probability of puberty was slightly higher than in years without mast . 4. Discussion During the last years, a permanent weight increase in female wild boars of all age classes in Lower Saxony culminated in 2017 and decreased from thereon for unknown reasons, therefore, partly supporting our hypothesis on a permanent weight increase of the species in the wild. The unexpected halt and decrease in body weight might be in line with extremely arid summers occurring since 2018 in the region . By highlighting a significant distinction of body weight between animals from forestal (fh) vs. agricultural (fah) habitats, our second hypothesis proved right. In Poland, a similar increase of wild boar body weight has been reported, and the distance to forestal areas also turned out to be a significant factor . Interestingly, the difference in body weight has an additional impact on puberty achievement, and here, mast seeding events significantly improve the condition of animals situated in forestal habitats. This is supporting our results from earlier studies, indicating that the occurrence of splendid masts of oak or beech result in a higher reproductive outcome, especially in forestal areas . Additionally, the precocious puberty of female and male wild boars in our study area supports the theory of a high share of piglets in reproduction. Anyway, the proven distinction of habitats longs for discussion. Various studies reflect that wild boars' spatial behavior is highly adaptive and diverse , especially among different European countries . In our study, we focused on adjacent areas of agricultural habitats and woodland, where the woodland was characterized by its consistency. We assume that wild boar populations inhabiting agricultural areas near the edge of the forest habitat do enter it and vice versa but that the majority of the animals inhabiting the pure forest area is concentrating on the forest, which is also providing enough forage, especially under climate change conditions . This is supported by the fact that puberty is reached by piglets inhabiting agricultural dominated areas at lower ages, while mast occurrence influences the onset of puberty, especially among forest-inhabiting animals. While the hybridization of domestic pigs and wild boars might lead to an adjustment in reproductive traits in the latter, there is no indication that hybridization might be responsible for the habitat-dependent differences we found in this study. In Europe, hybridization mainly occurred during the outside feeding of domestic pigs in the Roman Empire and Middle Ages , and in some countries, it was also observed after World War II or even recently . A wide variation in the degree of ongoing hybridization in European countries is stated, referring to the type of pig farming as one explaining factor . In this context, Germany is one of the countries where industrial pig farming is most common and, therefore, ongoing hybridization is less likely. Nevertheless, the differences in the reproductive performance of wild boars between Northern and Southwestern Germany that were found in former studies were not tested for differences in hybridization with domestic pigs so far , and studies that precisely perform German wild boar genome analyses would certainly be helpful to interpret reproduction in this species more accurately. While both sexes of wild boars seem to reach puberty on highly weight-dependent conditions and have gained weight continuously over the last years, it was surprising to observe that mast seeding events in forestal habitats appear to additionally influence the onset of puberty. The meaning of mast seeding events for wild boar reproduction is controversially discussed, and although, e.g., wild boars in France also seem to respond to mast seeding events positively through a higher breeding proportion, the effect seems to be caused mainly by their weight increase . This could be due to the difference in habitats, with the French Mediterranean forest seemingly serving as a main feeding source for wild boars , while our study area has been dominated by agricultural land use from the beginning of the monitoring. The high adaptability of wild boars might lead to aligned feeding strategies, in which the occurrence of mast seeding events might not provoke an imminent increase of body weight but resolve in a short-time adjustment of body fat and fatty acids, discussed as "flush feeding" in pigs , that also seems to have different outcomes depending on the timing and reproductive status of the animals . Few large contiguous forestal habitats are situated in Northern Germany, and with climate change, the persistence of comparable forests is threatened . Whether additional reproductive parameters, such as the time of farrowing and litter size of wild boars, will also prove to be habitat-dependent in our study areas will, therefore, be part of our future research. An additional impact could be shown for various external factors, such as precipitation in June and frost periods in February, leading to diminished body weights, whereas higher temperatures in March and April, as well as precipitation in May, were improving body weight. It seems reasonable that cold periods in February, with possibly farrowing females or piglets among the rut, lead to a reduced body weight of a high percentage of animals. Potentially, animals will search for shelter and be less active in foraging . Likewise, it can be assumed that strong precipitation in June may lead to crop failure or at least poor harvests as a consequence and thus also results in a decrease in body weight. The meaning of seasonality in wild living species is an important topic in wildlife research , and in the wild boar, questions about reproductive seasonality still remain unsolved . Additionally, little is known for weight development of wild boars in other European habitats, and further research can be helpful to evaluate the importance of these factors in Europe's future climate. 5. Conclusions Within a long-term study on the reproduction of female wild boars, we were able to gain outstanding results on basic features such as body weight development and received valuable insights on the impacts of external and internal factors on puberty. We, therefore, explicitly propose to establish long-term monitoring of wildlife species to a far greater extent. Acknowledgments We would like to thank The Forestry Seeds Advisory Board (FSB) at Oerrel, Lower Saxony, the Forestry Commission Offices in Lower Saxony, the state office for statistics (NLS), and the German Meteorological Service (DWD) for providing their data. We want to further thank all the hunters for their cooperation and help in collecting data, especially the Forestry Commission Offices Oerrel, Unterluss and Wolfenbuttel as well as the "Verwaltung Gunther Graf v. d. Schulenburg". Special thanks belong to Angelika Niebuhr for her support and care of the database and to all the volunteers, students and ITAW staff who helped to collect samples throughout all the years. Author Contributions Conceptualization, F.G. and O.K.; methodology, F.G.; software, T.L.; validation, T.L., O.K. and F.G.; formal analysis, F.G.; investigation, F.G. and C.M.; resources, U.S.; data curation, F.G.; writing--original draft preparation, F.G.; writing--review and editing, C.M. and O.K.; visualization, T.L.; supervision, U.S.; project administration, F.G. and O.K.; funding acquisition, F.G. and O.K. All authors have read and agreed to the published version of the manuscript. Institutional Review Board Statement Ethical review and approval were waived for this study due to animals' origin from regularly performed hunting. Informed Consent Statement Not applicable. Data Availability Statement Data for land use and climate are available online via and (accessed on 1 February 2023). Conflicts of Interest The authors declare no conflict of interest. Figure 1 Map of Germany (left), indicating the site of the study area in Lower Saxony, Germany (right part of the figure), showing forestal-agricultural habitat (fah, outlined in orange) around the large contiguous forestal habitat (fh, outlined in green) where wild boar females of this study were sampled. Figure 2 Bw (weight) of wild boar females (all ages) increased with hunting year until about 2017, followed by a decline that was less pronounced in agricultural dominated habitat fah (n = 2837). Figure 3 Strong beech mast (numbers represent levels according to categorization from forestry commission office Oerrel, Lower Saxony; 1 = no, 2 = part, 3 = half, 4 = full mast) resulted in (significantly) positive effects on bw only for adult wild boar females in fh (n = 680, p = 2.77 x 10-7 for beech = 4, age = adult, habitat = fh). Figure 4 Bw (weight) of wild boar females of all ages was negatively influenced by days of frost in February (FrostFeb), lower precipitation in May (Nmai, precipitation in mm) and July (Njuli, precipitation in mm) and strong hunting pressure (HI, ratio of hunting bag/hunting area). Figure 5 Different probabilities of puberty in the two landscape types (forest = fh, agriculture = fah), as described by age of piglets. Figure 6 Probability of puberty slightly decreased with increasing overall bw over years (left) and slightly increased with beech mast (right; numbers represent levels according to categorization from forestry commission office Oerrel, Lower Saxony; 1 = no, 2 = part, 3 = half, 4 = full mast). animals-13-00898-t001_Table 1 Table 1 Distribution of wild boar females according to age and habitat. Habitat Piglets Yearlings Adults Habitat Total fh 306 184 103 593 fah 1718 682 374 2.774 total 2024 866 477 3.367 animals-13-00898-t002_Table 2 Table 2 Predictors of wild boar body weight used in this study. Variable Measure/Explanation Year numerical (2003-2020) Age class three nominal levels: piglets, yearlings, adults Habitat two nominal levels: forested (fh), agricultural (fah) Oak crop four ordinal levels: (1-4) Beech crop four ordinal levels: (1-4) Precipitation monthly sum in mm Temperature monthly average (degC) Hunting Index wild boar annually shot per km2 Disclaimer/Publisher's Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

PMC10000141

Competitions involving sled dogs are rapidly growing and body temperature assessment could represent a prompt and non-invasive method of screening for potential pathological conditions during or after activity. The aim of this clinical study was to evaluate if thermography is able to monitor the post-competition ocular and superficial body temperature variations during a sled dog competition. It subsequently compared the data relating to the ocular temperatures in different race types: mid-distance (30 km) and sprint (<=16 km). Results showed a statistically significant increase in post-competition ocular temperature of both eyes, regardless of the length of the race. The relative increase in the temperatures of the other body surfaces was lower than the expected values, probably due to the influence of environmental and subjective factors such as the type of coat of the Siberian Husky or subcutaneous fat. Infrared thermography has therefore proved to be useful method in sled dog competition conditions for screening superficial temperature variations, as the investigation is normally conducted in an external environment and often in demanding work conditions. sled-dog thermography Siberian Husky sled dog This research received no external funding. pmc1. Introduction Research on the history of the sled dog suggests that the use of the dog as a draft animal appears to date back to at least 8000 to 9000 years ago and it reached a particularly high level among the nomadic tribes of Siberia (Chukchi and Samoyed) . Today, the sled dog is limited to tourist, recreational or competitive activities; in particular, there are three main sports disciplines: sleddog, pulka and skijoring. Races vary enormously from long-distance racing (from 250 to 1600 km in length), such as the well-known Iditarod in Alaska or the Finnmarkslopet and the Femundlopet in Norway, to mid-distance racing (30-250 kms) or sprint racing (generally 4-20 km). Recently, the athlete role of canines has seen great improvement in terms of media and scientific involvement, with an increase in knowledge of the physiological requirements of sled dogs, promoting and encouraging physical well-being and veterinary clinical health surveillance during competitive events . However, although a standard official protocol to monitor sport dog fitness has not yet been approved, it is usual practice for veterinarians working in dog sled competitions to perform microchip recognition and a pre-start clinical evaluation. Environmental parameters in sledding competition contexts limit the use of many diagnostic tools, either portable or non-invasive instruments, which could allow immediate and reliable results and help diagnostic screening during clinical examinations in the field. The assessment of body temperature in the sled dog represents a non-invasive monitoring method of canine thermoregulation during activity, consequently acquiring a value of non-invasive clinical screening of potential pathological conditions. During intense physical activity, the energy produced by muscular effort is generally converted into metabolic heat: in the dog, heat is then dissipated through the respiratory system, evaporation processes , salivation and painting , resulting in water loss that could lead to signs of dehydration and increased body temperature . In competitive and environmental contexts such as those reported for sled dogs, the rectal temperature measurement, generally performed in clinical trials, necessarily requires canine containment and can be extremely uncomfortable for the dog. The thermographic technique was used for the first time in equine medicine by Smith and it is currently widely used especially for the diagnosis and follow-up of pathologies involving the locomotor system . Additionally, in the sporting context, the use of a thermal imaging camera is a valid aid during equine training for monitoring muscle warm-up and verifying the presence of asymmetries in thermal distribution . Thermography has also recently been applied in canine sports training and could represent a non-invasive screening tool for pathological conditions, such as potential injuries to the musculoskeletal system or stress diseases that will then require further diagnostic investigations . However, to the best of the authors' knowledge, no specific publications have been produced on thermography application in sled dogs. The aims of this study were to: 1. evaluate thermography as a screening tool to monitor the potential variation of the pre-and post-race superficial body and ocular temperatures in sled dogs; 2. compare the ocular temperatures obtained in different competitions (mid-distance race and sprint race, of 30 km and <=16 km of length, respectively, according to Italian national regulations). 2. Materials and Methods Ethical approval for this study was obtained by the Ethical Committee of the University of Bologna (protocol number: ID 914/2018). Thirty-three Siberian Huskies (10 intact females, 2 spayed females and 21 intact males), mean age 63.9 +- 27.08 months (median 65 months), mean weight 21.27 +- 3.59 kg (median 20 kg) and Body Condition Score between 3.5 and 5.5 (following guidelines for Body Condition Score of International Sled Dog Veterinary Medical Association), attending the First Cortina International trial sporting event (Italy) on 26-27 February 2021, were included in this study. Fourteen dogs attended the mid-distance competition and nineteen dogs the sprint races on tracks equal to or less than 16 km. Soundness status was routinely evaluated before the competition by a licensed doctor in veterinary medicine. Thermographic images were taken using a portable thermo camera, FLIR T540 24deg (T540, FLIR Systems Inc., Danderyd, Sweden), with infrared (IR) resolution 464 x 348 pixels. The field of view was 53deg x 41deg. Images were recorded and analysed with FLIR Tools software (FLIR Systems Inc., Danderyd, Sweden). The investigation was carried out in the stakeout of each team. Pre-competition thermographic scans (T0) were acquired up to two hours before the start, compatibly with the arrival of the mushers in the Cortina. The post-competition images (T1) were taken no later than 20 min after the end of the race from 10:30 to 12:00. The thermal images were taken out of direct sunlight and wind after removing the harness immediately after the race. The camera was handheld at approximately 1 metre away from the dog head (ocular measurements) and 1.5 m up from other regions in order to include all the regions of interest (ROIs). Images were taken on dogs tied into the stakeout or kept on a leash by the musher in standing position. Generally, the musher was allowed to support the dog's head to measure eye temperatures. A total of six thermographic images were acquired for each dog: a. face region; b. forelimb cranial view; c. right lateral body; d. left lateral body; e. hindlimb caudal view; f. lumbo-sacral region dorsal view. For each thermal image, one or more ROIs have been identified, for a total of 15 ROIs (Table 1). The ocular temperature was evaluated as the average value resulting from the three different values obtained: the first one, by the hotspot centred on the medial canthus of the eye and the second and third ones by the mean value of two perpendicular lines, drawn from the dorsal to the ventral margin of the eye and from the medial ocular canthus to the lateral one . All data were submitted to descriptive (mean +- standard deviation-SD and median) and analytic statistical analysis. A paired T-test was applied to investigate differences between the post-race temperature in the different ROIs. Dogs were also divided into two groups according to the distance of the race (mid-distance (30 km) and sprint (<=16 km) and the Mann-Whitney U test was used to compare the difference in ocular temperatures at the end of the race (T1). The significance level was set for a p value <= 0.05. All data were analysed using Stata version 16 software (StataCorp, College Station, TX, USA-2019). 3. Results In total, 66 ocular thermographic images and 429 superficial body images were analysed. The environmental temperature ranged from -5 degC to +3 degC and from 8 degC to 10 degC when data at T0 and T1 were collected, respectively. Considering the entire group of dogs, in the pre-competition phase (T0) the mean temperature of the right eye was 32.98 +- 1.68 degC, while in the left eye the mean value was 32.83 +- 1.57 degC; in the post-race (T1) the mean temperature was 35.08 +- 0.95 degC and 34.95 +- 0.88 degC for the right and left eye, respectively . When comparing the mid-distance (30 km) and sprint (<=16 km) groups of dogs, the ocular temperature mean value at T0 in the mid-distance group was 32.49 +- 1.34 degC in the right eye and 32.17 +- 1.37 degC in the left eye; the ocular temperature mean value at T0 in the sprint group was 33.34 +- 1.84 degC in the right eye and 33.32 +- 1.56 degC in the left eye. The ocular temperature mean value at T1 in the mid-distance group was 35.24 +- 0.96 degC in the right eye and 35.24 +- 0.56 degC in the left eye; the ocular temperature mean value at T1 in the sprint group was 34.97 +- 0.95 degC in the right eye and 34.74 +- 1.01 degC in the left eye (Table 3). The statistical analysis showed a significant increase in ocular temperature in all dogs examined (N = 33) between T0 and T1 for both the right and left eyes (p < 0.0001). No significant difference in the post-race ocular temperature between the mid-distance (30 km) and the sprint category group (<=16 km) were observed for either eye, right (p = 0.49) or left (p = 0.12). The mean +- SD and median values of body surface temperature in the different ROIs are reported in Table 4. A statistically significant increase in the temperature of the body surfaces of all ROIs between T0 and T1 was recorded (p < 0.05) . 4. Discussion The aims of this study were to investigate if thermography could be used as a screening technique to (1) monitor the variation post-competition in superficial body temperature and ocular temperatures and (2) evaluate if ocular temperatures changed in different distance competitions. The results showed that thermography was able to monitor the increase in body temperatures secondary to physical activity: a statistically significant increase in superficial and ocular temperatures was reported, but no significant differences were detected for ocular temperatures when mid-distance and sprint competitions were compared. Body temperature variations during exercise have been widely documented in domestic mammals, as an effect of the increase in muscle metabolism with changes in respiratory and heart rates , and thermal loss during exercise may alter normal homeostasis. Changes in body temperature, as an index of the physiological response to exercise, has to therefore be correctly monitored; however, it should be performed without generating stress in the dog, especially in a competitive context. For this reason, thermography could represent a suitable non-invasive tool for screening. The use of thermography in the context of sled dogs is not yet widespread, even if this technique has generally been used during clinical examinations of dogs and for research aims in La Grande Odysseee . In our experience, during sled dog competitions, the infrared camera for temperature measurement was easy to manage and particularly helpful, as it is a non-invasive portable screening tool that allows quick screening of superficial temperature changes. Image acquisition often occurs in challenging working conditions for climatic and organisational reasons; consequently, the T0 acquisition was often determined by the availability of the mushers for the dog's examination, while at T1 the main difficulty was related to the simultaneous arrival of different teams participating in our study. This standard protocol resulted in a limited increase (5-10 min) in the whole clinical examination, routinely scheduled before the competition. However, the thermographic evaluation was carried out before the physical examination in order to avoid any clinical manipulation, especially regarding the musculoskeletal system, which could generate artefacts. The distance between camera and patient was set at 1 metre for ocular images and 1.5 m for other ROIs. Although most publications on thermography for clinical purposes describe distances less than or equal to 1 metre from the target region, in accordance with the guidelines prepared by the American Academy of Thermology, which report an ideal distance from the subject of 3-8 feet (0.9-2.4 m), we have standardised a distance to 1 and 1.5 m to allow us to correctly include in the frame (wide-angle photographic lens) all ROIs that would then be used within each specific projection. However, other studies on dogs have evaluated body surface temperatures with a dog-camera distance varying between 0.3 and 0.67 m . The operator who acquired the images was a well-trained and experienced practitioner. Vainionpaa and collaborators (2012) reported that, although the thermographic technique does not depend on the operator who performs the examination or interprets it, it is fundamental that the operator is adequately trained to reduce the differences related to the methods of acquisition and interpretation of thermal images . The medial canthus of the eye has been reported as an anatomical point with the highest temperature on the eye, also compared to the lateral canthus . Other studies have measured the ocular temperature within a rectangular area that included the ocular globe and about 1 cm of the eyelids or only at the level of the lacrimal caruncle . We decided to apply a measurement technique similar to that described by Elias, which, in our opinion, was easier to perform in the field . The applied technique allowed the examination of several points of the eye surface in order to perform a wide mapping of the ocular temperature. Thermal images to measure body surface temperature were acquired on standing dogs obtaining five projections: front limbs in cranial view, right lateral body projection, left lateral body projection, hind limbs in caudal view and dorsal view of the lumbosacral region. These five projections made it possible to analyse 13 ROIs. Other studies have identified in the thermal images the areas centred on the main anatomical regions, standardised according to the width of the main muscle groups (neck, shoulder, arm, back, rib, rump and thigh) or by using specific sub-regions centred around specific muscle groups . We decided to identify the ROIs as generic anatomical areas, as our aim was to confirm by thermography the systemic superficial heating of the dogs (body and eyes) after races and not a specific involvement of individual muscle groups during activity. Our data confirmed a statistically significant increase in ocular and body temperature after physical activity, regardless of the distance of the race; an evident uniformity of ocular temperatures resulted between the post-race values, while larger standard deviations of the means of the superficial body temperatures were observed, as they probably were more affected by environmental factors. The values of the eye temperatures collected in the post-race outdoor environment were found to be similar to those measured in indoor conditions with controlled temperature and humidity on dogs that exercised on a treadmill . Rizzo and collaborators reported that the mean value for both eyes was 33.29 +- 1.6 degC at rest and 35.49 +- 0.52 degC after 10 min of treadmill exercise . In our study, the comparison between the two different typologies of races revealed no difference at T1, leading us to conclude that ocular temperature increases regardless of the distance travelled during the run. These data were in line with results of other previous thermographic studies, which had shown that ocular temperature increases uniformly during exercise , as well as results described by Phillips et al. (1981) relative to rectal temperature in running sled dogs . Moreover, ocular temperature between the post-race increased by 2.1 degC on average, in line with that reported by Rizzo and collaborators . In contrast, Zanghi (2016) observed differences in post-exercise eye temperature increase depending on breeds . For practical reasons due to competition rules, the ocular temperature was not compared with the rectal temperature, which would have allowed an effective monitoring of changes in the internal body temperature ; however, measurement of rectal temperature is not part of the routine pre-race examination. With particular attention to superficial body temperature, our results showed a significant increase post-competition in all examined ROIs. However, pre-race superficial body temperatures were lower in the CDDX ROIs (mean and standard deviation 5.11 +- 2.78 degC), CDSX (5.29 +- 2.82 degC) and LGS (4.99 +- 3.34 degC). It has been speculated that the body surface temperature of mammals is about 5 deg C lower than rectal temperature , but our results significantly deviate from the expected values. The thickness and type of coat of the Siberian Husky may have influenced the measurement of the body surface temperature, limiting a real surface measurement, worsened by the presence of snow or sleet on the coat. The coat of the Siberian Husky is composed of a dense undercoat slightly topped by guard hairs. These characteristics allow the interposition of an insulating layer of air between the skin and the hair, limiting the propagation of thermal radiation . The preparation of the dogs, according to the protocol for thermographic examination performed in a closed and controlled environment by Soroko and collaborators (2021), involves combing the hair one hour before the exercise session : this practice would make it possible to flatten the hair and limit the isolation layer of air. However, this manipulation was not feasible in our context. Relative to the higher temperature in the ROIs of the right anatomical regions (ODX, LDX, CDX and GDX) observed at T1, we hypothesise that it could be due to the sun position that for most of the race radiated the right side of the dogs. Environmental parameters may affect thermal detection during physical activity: significant association was observed between the environmental and body temperatures in sled dogs competing between -9 and 25 degC . Philips and collaborators showed that sled dogs are mostly affected by mid-high ambient temperatures (>=15 degC), with an average body temperature increase of up to 4.6 degC, in contrast to the 2 degC mean increase when ambient temperatures were <15 degC . Sled dogs tolerate environmental temperatures below 0 degC well, as these dogs spend a lot of time and compete outdoors . Nevertheless, in our study it was not possible to exclude that the range of environmental temperature variation between 0 and 10 degC during the race did not partially influence the final evaluations. Relative to ocular temperature, Zanghi B.M. (2016) observed a return to the physiological value within 30 min indoors in Beagle dogs, unlike Labrador Retrievers. Differently, rectal temperature returned to physiological values in 15 min in Beagles and in 30 min in Labrador Retrievers . Another study conducted by Diverio and collaborators (2016) monitored rectal temperature in avalanche dogs: these authors reported activity-induced rectal temperature changes that returned to the normal range two hours after the end of the research activity . Similar results on working dogs were also observed by Spinella and collaborators (2021) . The main limitation of this study concerned the acquisition of thermal images outdoors, as it was not possible to recreate standard environmental conditions to limit the influence of external factors such as ambient temperature, snow-covered ground and sunlight, which could cause artefacts. However, our aim was to obtain information in real race conditions. Only Siberian Husky dogs were included in this study, because this breed is consolidated from an ethnographic and health point of view and, therefore, guarantees a more homogeneous sample . The Husky's undercoat provides excellent thermal insulation and may give a biased result, but on the other hand all the breeds used in this specific activity have similar haircoat features. Moreover, as previously reported, the anticipation of activity could be associated with an increase in rectal temperature in pre-race timing , but this does not appear to have affected the thermography's ability to determine the temperature changes before and after the race. Furthermore, the dogs examined at the stakeouts were sometimes initially lying in the snow: in the literature, it is reported that ice on the fur surface could affect thermal images, particularly when it melts, as the humidity could increase surface evaporation . Finally, the number of included dogs (33 dogs) represented only 7.5% of the total sample that took part in the competition; although a good number of dogs were examined, these were not equally distributed between the races. 5. Conclusions Thermography is a useful method during the initial screening and in the follow-up during clinical visits in sled dog sports competitions, especially in the pre-competition, where it is advisable to minimize stress. Ocular temperature was found to be more reliable than the temperature of the other superficial regions. Our data confirmed that thermography can detect the increase in post-race ocular temperature, which was not correlated to the distance travelled by the dogs. Thermographic evaluations of other superficial areas also showed an increase in post-competition temperatures secondary to physical exertion. However, this increase was less than the expected values, probably highlighting the limit represented by individual and environmental factors. Author Contributions Conceptualization, G.S., G.C. and S.V.; methodology, G.S, A.G., G.C., S.M. and S.V.; software, V.M.; validation, G.S., S.M. and S.V.; investigation, A.G., G.C., V.M. and S.M.; data curation, G.S., G.C. and S.V.; writing--original draft preparation, G.S., G.C. and S.V.; writing--review and editing, G.S., A.G., S.M. and S.V. All authors have read and agreed to the published version of the manuscript. Institutional Review Board Statement Ethical approval for this study was obtained by the Ethical Committee of the University of Bologna (protocol number: ID 914/2018). Informed Consent Statement We received oral consent by the owner, who voluntarily accepted to include their dogs in the study. Data Availability Statement All data are contained within this article. Interested qualified researchers may request further information by contacting the corresponding author. Conflicts of Interest The authors declare no conflict of interest. Figure 1 Five-and-a-half-year-old, female, Siberian Husky dog. Example of ocular measurement as the average value resulting from the three different values obtained on the hotspot centered on the medial canthus of the eye and on two perpendicular lines, drawn from the dorsal to the ventral margin of the eye and from the ocular medial canthus to the lateral one. The brighter colors (red, orange, and yellow) indicate warmer temperatures (more heat and infrared radiation emitted), while the purples and dark blue/black indicate cooler temperatures (less heat and infrared radiation emitted). Moreover, all commas inserted in the measurements within the figure are indicated according to the Italian style, therefore they must be understood as points to indicate decimals following the British style (e.g., "33,9" in Italian style has to be intended as "33.9" following the British style). Sp = Spot. Li = Line. Regarding the other ROIs, elliptical areas corresponding to the main body regions were identified within each image, as well as lines (major axis of the ellipse) and points within the areas: the final surface temperature was obtained from the average of the measurements obtained in areas, lines and points . Figure 2 Five-and-a-half-year-old, female, Siberian Husky dog. Pattern of measurement of left homobrachial region (OSX), thoraco-lumbar region and left dorsal portion of the ventro-lateral region of the abdomen (LSX) and left thigh region (CSX). For each ROI, the body surface temperature was measured using three techniques: by points of interest (A), by lines (B) and by areas (C). The brighter colors (red, orange, and yellow) indicate warmer temperatures (more heat and infrared radiation emitted), while the purples and dark blue/black indicate cooler temperatures (less heat and infrared radiation emitted). Moreover, all commas inserted in the measurements within the figure are indicated according to the Italian style, therefore they must be understood as points to indicate decimals following the British style. Sp = Spot. Li = Line. El = elliptical area. Figure 3 Five-year-old, female, Siberian Husky dog. Comparison of macroscopic thermographic aspect of ocular image before (T0) and after competition (T1). The brighter colors (red, orange, and yellow) indicate warmer temperatures (more heat and infrared radiation emitted), while the purples and dark blue/black indicate cooler temperatures (less heat and infrared radiation emitted). Figure 4 Five-year-old, female, Siberian Husky dog. Comparison of macroscopic thermographic aspect of left lateral view of the body surface before (T0) and after competition (T1). The brighter colors (red, orange, and yellow) indicate warmer temperatures (more heat and infrared radiation emitted), while the purples and dark blue/black indicate cooler temperatures (less heat and infrared radiation emitted). -2.0 = -2.0. animals-13-00854-t001_Table 1 Table 1 Description of the fifteen ROI taken into consideration in the examination and relative anatomical regions as reported by Shaller in Illustrated Veterinary Anatomical Nomenclature in 1992 . Regions of Interest (ROIs) Anatomical Regions 1 Right eye 2 Left eye 3 Right pectoral subregion of the sternal region (PDX) Presternal region 4 Left pectoral subregion of the sternal region (PSX) Presternal region 5 Right homobrachial region (ODX) Scapular region, region of scapular cartilage, supraspinatus region, infraspinatus region, shoulder joint region 6 Left homobrachial region (OSX) Scapular region, region of scapular cartilage, supraspinatus region, infraspinatus region, shoulder joint region 7 Right lateral view of the thoraco-lumbar region and right dorsal portion of the ventro-lateral region of the abdomen (LDX) Region of the thoracic vertebrae, lumbar region, paralumar fossa 8 Left lateral view of the thoraco-lumbar region and left dorsal portion of the ventro-lateral region of the abdomen (LSX) Region of the thoracic vertebrae, lumbar region, paralumar fossa 9 Lateral view of the right thigh region (TDX) Region of thigh 10 Lateral view of the left thigh region (TSX) Region of thigh 11 Lateral-caudal view of the right leg region (LeDX) Crural region, region of common calcaneal tendon, popliteal region 12 Lateral-caudal view of the left leg region (LeSX) Crural region, region of common calcaneal tendon, popliteal region 13 Caudal view of the thigh and right leg region (TLDX) Region of thigh, crural region, region of common calcaneal tendon, popliteal region 14 Caudal view of the region of the thigh and left leg (TLSX) Region of thigh, crural region, region of common calcaneal tendon, popliteal region 15 Caudal view of the lumbar region, sacral region and dorsal portion of the hip or gluteal region (LSG) Lumbar region, sacral region, region of the root of the tail, gluteal region, regio clunis animals-13-00854-t002_Table 2 Table 2 Eye temperature: mean value +- SD and median obtained in T0 and T1 in the entire group. All values are expressed in degC. Data in T1 were significantly increased (*). (ROI) T0 T1 Mean Value +- SD Median Mean Value +- SD Median RIGHT EYE 32.98 +- 1.68 33.20 35.08 +- 0.95 * 34.90 LEFT EYE 32.83 +- 1.57 33.00 34.95 +- 0.87 * 35.00 animals-13-00854-t003_Table 3 Table 3 The table summarizes the mean +- SD and median temperature values of the right and left eye in T0 and T1 in dogs that participated in the mid-distance and sprint competition. All values are expressed in degC. No significant differences were observed between T1 values comparing mid-distance and sprint races. Right Eye Left Eye T0 Mean Value +- SD Median Mean Value +- SD Median Mid-distance 32.49 +- 1.34 31.90 32.17 +- 1.37 31.85 Sprint 33.34 +- 1.84 34.10 33.32 +- 1.56 33.80 T1 Mid-distance 35.24 +- 0.96 34.95 35.24 +- 0.56 35.30 Sprint 34.97 +- 0.95 34.90 34.74 +- 1.01 34.70 animals-13-00854-t004_Table 4 Table 4 Mean +- SD and median temperature values of the ROIs in T0 and T1. T0 T1 ROI Mean +- DS Median Mean +- DS Median PDX 6.02 +- 3.33 5.90 14.41 +- 4.64 * 14.40 PSX 6.47 +- 3.14 6.90 13.20 +- 2.46 * 13.20 ODX 8.45 +- 2.38 8.80 24.51 +- 9.46 * 21.10 OSX 8.25 +- 3.22 8.60 18.97 +- 5.54 * 19.70 LDX 7.21 +- 2.69 7.70 27.81 +- 11.12 * 25.00 LSX 7.30 +- 3.41 7.30 21.55 +- 6.91 * 21.00 TDX 7.91 +- 2.60 8.00 24.73 +- 10.79 * 22.40 TSX 7.92 +- 3.14 7.70 18.78 +- 4.96 * 19.00 LeDX 9.79 +- 3.52 9.50 20.39 +- 7.23 * 17.95 LeSX 10.45 +- 3.72 10.10 17.68 +- 4.87 * 17.30 TLDX 5.11 +- 2.78 4.80 14.97 +- 6.44 * 14.40 TLSX 5.29 +- 2.82 5.40 12.79 +- 5.07 * 11.60 LSG 4.99 +- 3.34 5.40 25.98 +- 8.83 * 24.30 All values are expressed in degC. (PDX, right pectoral subregion of the sternal region; PSX, left pectoral sub-region of the sternal region; ODX, right homobrachial region; OSX, left homobrachial region; LDX, right lateral view of the thoraco-lumbar region and right dorsal portion of the ventro-lateral region of the abdomen; LSX, left lateral view of the thoraco-lumbar region and left dorsal portion of the ventro-lateral region of the abdomen; TDX, lateral view of the right thigh region; TSX, lateral view of the left thigh region; LeDX, lateral-caudal view of the right leg region; LeSX, lateral-caudal view of the left leg region; TLDX, caudal view of the thigh and right leg region; TLSX, caudal view of the region of the thigh and left leg; LSG, caudal view of the lumbar region, sacral region and dorsal portion of the hip or gluteal region). All values in T1 resulted statistically significant increased compared to T0 (*). 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PMC10000142

Rumen degradation kinetics of 25 feedstuffs (six protein feeds, nine energy feeds and ten roughages) were first determined using the nylon bag technique in situ and the differences of degradation characteristics fitted with five or seven time points measuring data were evaluated with the goodness of fit (R2) of degradation curves. Protein and energy feeds were incubated for 2, 4, 8, 16, 24, 36, 48 h, roughages were incubated for 4, 8, 16, 24, 36, 48, 72 h, where three and six data sets of five time points were screened out, respectively. Only the degradation parameters a (rapidly degraded proportion), b (slowly degraded proportion) and c (degradation rate of slowly degraded proportion) of several feeds at five time points were significantly different from those at seven time points (p < 0.05), and the others were not significant (p > 0.05). The R2 of the degradation curves obtained at five time points was closer to 1, indicating that the fitting obtained at five time points was more accurate in predicting the real-time rumen degradation rate of feed. These results indicate that it is feasible to determine the rumen degradation characteristics of feedstuffs by only setting five measuring time points. nylon bag technology rumen degradation sheep simplified method time points selection China Agriculture Research SystemCARS-39 This study was supported by grants from the China Agriculture Research System (CARS-39). pmc1. Introduction Though the nylon bag technique has been used extensively for evaluating the rumen degradation profile of feedstuffs, discrepancies are commonly found between the studies deriving from intralaboratory on the aspect of measurement procedures. The difference in any factor of bag size, feed particle and amount, sampling rule, times of sampling, washing method, and mathematical calculation would likely affect the results. To enhance the comparability of the results from different studies and assure the reliability of the measurement, standard procedures were recommended and improved more than once . However, the incubation times recommended procedures which might remarkably influence the accuracy and efficiency of the evaluation. Orskov suggested that concentrates should be cultured in rumen for 2, 6, 12, 24 and 36 h; Lindberg recommended concentrates for 2, 4, 8, 16 and 24 h, and roughages for 2, 4, 8, 16, 24, 36, 48 h; AFRC recommended that concentrates should be cultured for 2, 6, 8, 24, 48 h, and roughages for 2, 6, 8, 24, 48, 72 h. Michalet-Doreau and Vanzant suggested that the required number of time points should be able to describe the curve. The recent studies that used the nylon bag method to determine rumen degradation characteristics of feedstuff on sheep are briefly summarized in Table 1. It is evident that various numbers and hours of sampling were used in different studies, with the number of sampling time point being mostly in the range of 5-9 and the longest incubation duration being 72 h in most research, such as A.R. Seradj , L. Tao , B. Ghorbani , Xiaogao Diao , and so on . Systematic studies are in need to make clear the influence of sampling hours and sampling times on the results of the rumen nylon bag technique. In this study, rumen degradation characteristics of 25 feeds (six protein feeds, nine energy feeds, and ten roughages) were determined on fistulated sheep by the nylon bag technique with the setting of seven sampling time points. The degradation parameters a, b, c obtained at five time points were compared with those obtained at seven time points. Furthermore, the R2 of degradation curve estimated with five or seven time points were compared, to investigate the feasibility of reducing the number of sampling time point in the measurement of rumen degradation parameters of feedstuffs. The outcome of this study would enrich the database of nutritive value of feedstuffs in sheep, and provide a reference for the application of the nylon bag technique. 2. Materials and Methods All animal management and experimental procedures followed the animal care protocols approved by the Animal Care and Use Ethics Committee of China Agricultural University. 2.1. Animals and Diets Eight ruminally fistulated Wether sheep aged 2 to 3 years old with an average live weight of 57.4 +- 2.4 kg were selected and divided into two groups. They were then placed into a group of 4 sheep (i.e., four replicates) to determine rumen degradation parameters of different feeds. The animals were fed twice daily at 8:00 and 17:30, with free access to clean water. Sheep were fed a ration (DM basis) consisting of 45.00% soybean stem, 25.00% wheat straw, 18.56% corn, 4.95% soybean meal, 4.95% wheat bran, 0.62% CaHPO4, 0.31% NaCl, 0.31% sodium bicarbonate, and 0.3% premix. 2.2. Samples Preparation and Nutrient Analysis A total of 25 feedstuffs collected around the country were used in the present study and the nutritional compositions are presented in Table 2. To prepare feed samples, raw materials were dried at 65 degC for 48 h in a forced-air oven and then milled through a 1 mm sieve for chemical analysis and 2.5 mm sieve for in situ degradation. The concentrations of dry matter (DM), organic matter (OM), and fat were analyzed according to the methods of AOAC . Nitrogen (N) content was measured by the Kjeldahl method using a FOSS semi-automatic nitrogen analyzer, and crude protein (CP) content was calculated as N x 6.25. The contents of neutral detergent fiber (NDF) and acid detergent fiber (ADF) were analyzed using an automatic fiber analyzer (A2000i, Ankom Technology, Macedon, NY, USA) following the methods described by Van Soest et al. . 2.3. In Situ Nylon Bag Experiment The in situ degradabilities of DM, CP, and OM in the 25 feeds were determined according to the procedure described by Mehrez and Orskov . A given amount of feed sample, i.e., 3 g for protein feeds, 3 g for energy feeds, or 5 g for roughages, was weighed into the nylon bag (48 mm pore size, 6 cm x 10 cm bag size) in duplicate, 1 feed was cultured in rumen of each sheep and a total of 14 nylon bags. The tied bags were placed into the rumen before the morning feeding at 0800 and removed at the given time points. Differently, the incubation time points for protein feeds or energy feeds were set as 2, 4, 8, 16, 24, 36, 48 h, while the roughages were incubated for 4, 8, 16, 24, 36, 48, 72 h. After the removal from the rumen, the bags were immediately washed under running water till the flow-out water was clear, in order to stop microbial fermentation . Then, the bags with clean residue were dried to a constant weight at 65 degC for 48 h and weighed. The residues were further ground through a 1 mm sieve for nutrient analysis. 2.4. Calculations of Degradation Kinetics Parameters The kinetic parameters of in situ degradation were calculated based on the measured degradabilities at all 7 time points or 5 selective time points. The data of instant degradability were fitted using the following exponential equation:Y = a + b (1 - e-ct)(1) where Y is the nutrient disappearance at time point t, a is the rapidly degradable fraction, b is the potentially degradable fraction, c is the degradation rate of fraction b (%/h), and t is the time (h) of incubation. ED = a + bc/(c + k)(2) where a, b, and c are the same as those in Equation (1), and k is the rumen outflow rate. In this study, the rumen outflow rate was set by referring to previous studies, i.e., roughages 3.14%/h , DDGS 3.99%/h, silage feeds 2.53%/h, and oil-seed-meals 5%/h . To compare the difference of the degradation kinetic parameters deriving from the calculation with 5 time points or 7 time points data, the potential combinations of 5 time points were screened out as follows: 1 2, 16, 24, 36, 48 h, 2 2, 8, 16, 24, 48 h, 3 2, 8, 16, 36, 48 h for protein feeds and energy feeds, and 1 4, 16, 36, 48, 72 h, 2 4, 16, 24, 48, 72 h, 3 4, 16, 24, 36, 72 h, 4 4, 8, 16, 24, 72 h, 5 4, 8, 16, 36, 72 h, 6 4, 8, 16, 48, 72 h for roughages. The selection was as follows: by referring to the relevant literature and observing the rumen degradation rate curve, it was found that the longest incubation time of concentrates and roughages in the rumen were 48 h and 72 h, respectively. At this time, the degradation curve tended to be flat. The rumen degradation rate of 16 h could be used to calculate the small intestine digestibility of feeds. Considering the properties of the feeds, the degradation rate of protein and energy feeds in the rumen was faster, while that of roughages was slower. In addition, according to the research basis of our laboratory, 2 h and 4 h were selected as the shortest culture time of protein/energy feeds and roughages, respectively. Therefore, the shortest and longest time points, and 16 h of feed culture in the rumen, were kept. In addition, in order to reduce the stress caused by excessive density of time points, the protein/energy feeds culture for 4 h were removed. The degradation parameters "a", "b", and "c" were calculated at 5 time points in the same way as Formula (1). 2.5. Reasonable Criteria for Selection The criteria for selecting the optimal combination were as follows, 1 when the degradation parameters (a, b, c) obtained at 5 time points were closer to the values obtained at 7 time points (the difference was not significant), it indicated that the selected time point combination was the best; 2 when the R2 of fitting curve of DM, CP, and OM obtained from 5 time points was closer to 1, it indicated that the combination was the best. 2.6. Statistical Analyses The data concerning nutrients disappearance and kinetic parameters a, b, and c were analyzed using the general linear model (GLM) procedure of SAS. The differences of degradation parameters were calculated with SPSS. Difference was considered significant when p < 0.05. 3. Results 3.1. The Parameters a, b, c and ED of DM, CP and OM of the Feedstuff The parameters a, b, c and ED of 25 feeds are summarized in Table 3, Table 4 and Table 5. In general, the a, b, c and ED of DM, OM, and CP of each feedstuff obtained at five time points were different from those obtained at seven time points (p < 0.05). For the protein feeds, the "a" of CP and OM of CSM, the "b" of CP, and the "a" of OM of CGM, the "a" of DM of DDGS, and the "b" of CP of SOM obtained at five time points of 1, 2, 3, 4 were significantly different from those obtained at seven time points (p < 0.05). The ED of CP of CGM and ES obtained at five time points of 1, 2, 3, 4 were significantly different from those obtained at seven time points (p < 0.05). There were no significant differences in other degradation parameters (p > 0.05). For energy feeds, the "a" and "b" of DM, CP, and OM of BY and WT, the "b" of DM of HS, the "a" and "b" of DM of HY9 and GWC, the "a" of DM of YC, and the "b" of DM of YWB obtained at five time points of 1, 2, 3, 4 were significantly different from those obtained at five time points (p < 0.05). The ED of DM, CP, and OM of CBS, the ED of DM of YWB and GWC obtained at five time points of 1, 2, 3, 4 were significantly different from those obtained at seven time points (p < 0.05). There were no significant differences in other degradation parameters (p > 0.05). For roughages, the "a" and "b" of CP of RG, the "a" and "b" of DM of RSW, the "a", "b", and "c" of OM of RSW and TP, the "b" of DM of OG and CS, the "c" of OM of CS, the "a" of DM of TP, and the "a", "b", and "c" of CP of TP obtained at five time points of 1, 2, 3, 4, 5, 6 were significantly different from those obtained at seven time points (p < 0.05). The ED of DM and OM of RSW and the ED of DM, CP, and OM of TP obtained at five time points of 1, 2, 3, 4, 5, 6 were significantly different from those obtained at seven time points (p < 0.05). There were no significant differences in other degradation parameters (p > 0.05). 3.2. The R2 of Fitted Curves of DM, CP, and OM Obtained at Different Time Points The R2 of rumen degradation fitted curves of DM, CP, and OM in the protein feeds, energy feeds, and roughages of 5 or 7 time points are illustrated in Figure 1, Figure 2 and Figure 3. As seen in the figures, the R2 of degradation curves fitted with 5 or 7 time points of each feed was greater than 0.9. Additionally, the R2 of 1 2, 16, 24, 36, 48 h was closer to 1 for protein and energy feeds, the R2 of 6 4, 8, 16, 48, 72 h was closer to 1. The closer the value of R2 was to 1, the better the fitted curve fit the observed value (rumen degradation rate of feeds). 4. Discussion The degradation parameters of various types of feeds were different in the rumen, and the effect of different time points combination (1,2 and 3 of protein feeds and energy feeds, 1, 2, 3, 4, 5, and 6 of roughages) on the "a", "b", "c" and ED of feeds was also different. The rate and extent of DM fermentation in the rumen were important determinants of the degree of ruminal digestion , and they could be affected by the factors of processing, feed property, animal physiological status, etc. For protein feeds, compared with the degradation parameters "a", "b", and "c" of DM and OM, the degradation parameters of CP at five time points had a greater effect, which may have been related to the structure of protein in feeds. For energy feeds, compared with the parameters obtained at seven time points, the effects of different combinations of five time points on degradation parameters were different. Roughages generally have higher NDF and ADF contents. The main components of cell wall NDF and ADF exerted a dramatical limiting effect on the digestibility of forage. For TP, the "a" of DM, the "a" and "c" of CP and OM at five time points (1, 2, 3, 4, 5 and 6) were significantly lower than that at seven time points; the ED of DM, CP, and OM were significantly higher than that at seven time points. Five time points may not have been suitable to evaluate TP, and the specific reasons need to be further studied. In conclusion, using five time points to evaluate rumen degradation characteristics will lead to changes in degradation parameters ("a", "b", and "c"), but has little effect on ED. Feed nutrients mainly include rapidly degraded proportion (a), slowly degraded proportion (b), and unstable proportion. The changes of a, b, and c would affect the ED. Although the ED was affected by outflow rate, for this study, the base diet of the sheep was consistent; thus, the effect of fewer samples in nylon bags on outflow rate was negligible. Additionally, ED was one of the most important data points for evaluating the nylon bag method and should be retained. By comparing the degradation parameters obtained at five or seven time points, it was found that the selection of different time points would lead to significant differences in a, b, and c, but it did not show in ED, which may have been because the influence of different time points on the a and b of the feed was cancelled out in the calculation of ED. The nylon bag technique is a common method to evaluate the nutritional value of ruminant feeds, but the standardization of its measurement procedure needs further improvement. Generally, the more time points measured, the more accurate degradation parameters and ED obtained, and the higher fitting degree of degradation curve obtained. The dynamic degradation curve would evidently present the fermentation characteristics of the feeds in the rumen. Michalet-Doreau and Vanzant suggested that the number of time points could be used to describe the curve. The British AFRC recommended that the nylon bag method be used to determine the rumen degradation characteristics of concentrate and roughage at five time points. In the present study, the rumen degradation curve of nutrients could be obtained at five time points and little gap between the curves fitted with five or seven time points data were found, suggesting a strong feasibility of the simplification of time points in the nylon bag technique. In addition, given that too many time points will much increase workload and cause serious stress to the experimental animals, animal welfare in experimental protocols would be improved and the cost and labor would be reduced by reducing measurement time points. On the other hand, setting too many time points with short time intervals would likely affect the normal function of the rumen and, consequently, affect the test results, in that frequent extraction and placement would lead to the long-time exposure of rumen microbes to the external environment. In this study, it was feasible to use five time points to calculate the degradation parameters "a", "b", and "c" of feeds, and according to the reasonable selection criteria (the difference was not significant, the degradation parameters were closer to seven time points, and the R2 of fitted curve was closer to 1), for protein and energy feeds, the degradation parameters obtained by using 1 2, 16, 24, 36, 48 h were closer to those obtained by using seven time points, and the R2 of the fitting curve obtained was better than the seven time points, for roughages, the degradation parameters obtained by using 6 4, 8, 16, 48, 72 h were closer to those obtained by using seven time points, and the R2 of the fitting curve obtained was better than the seven time points. 5. Conclusions The results of this study showed that rumen degradation parameters ("a", "b", and "c") varied with different time points, but had little effect on ED, and the R2 of fitted curves obtained five time points was closer to 1. It is feasible to determine the rumen degradation characteristics of feedstuff at five time points. Moreover, the optimal combinations of five rumen incubation time points were found to be 2, 16, 24, 36, 48 h for protein and energy feeds, and 4, 8, 16, 48, 72 h for roughages. Author Contributions Conceptualization, validation, and resources, W.Z.; writing--original draft preparation, S.L.; software and validation, L.H.; technical support and revision of the manuscript, F.M. All authors have read and agreed to the published version of the manuscript. Institutional Review Board Statement The study was conducted in accordance with guidelines of the Animal Care and Use Committee of China Agricultural University (Beijing, China, protocol code AW12212202-1-1). Informed Consent Statement Not applicable. Data Availability Statement Publicly available data sets were analyzed in this study, and these have been referenced in the manuscript. Conflicts of Interest The authors declare no financial or commercial conflict of interest. Abbreviations RSM (rape seed meal); CSM (cotton seed meal); CGM (corn gluten meal); ES (expanded soybean); DDGS (distillers' dried grains with soluble); SOM (soybean meal); BR (brown rice); BY (barley); WT (wheat); HS (Heilongjiang sorghum); HY9 (Heilongjiang corn Heyu 9); GWC (Guizhou white corn); YC (Yunnan corn); YWB (Yunnan wheat bran); CBS (spouting corn bran); WS (wheat straw); RG (rye grass); RSW (radish straw); BS (beckmannia syzigachne); SS (soybean stem); CWR (Chinese wild rye); RS (rye straw); OG (orchard grass); CS (corn silage); TP (turnip root). Figure 1 The R2 of fitted curve of DM, CP, and OM of protein feeds of 5 or 7 time points. (A-C), respectively, represent the R2 of fitted curve of DM, CP, and OM in the rumen of protein feeds. The number 0 = 7 incubation time points, 1, respectively, the R2 of fitted curve of 2, 16, 24, 36, 48 h; 2, respectively, the R2 of fitted curve of 2, 8, 16, 24, 48 h; 3, respectively, the R2 of fitted curve of 2, 8, 16, 36, 48 h. Figure 2 The R2 of fitted curve of DM, CP, and OM of energy feeds of 5 or 7 time points. (A-C), respectively, represent the R2 of fitted curve of DM, CP, and OM in the rumen of energy feeds. The number 0 = 7 incubation time points, 1, respectively, the R2 of fitted curve of 2, 16, 24, 36, 48 h; 2, respectively, the R2 of fitted curve of 2, 8, 16, 24, 48 h; 3, respectively, the R2 of fitted curve of 2, 8, 16, 36, 48 h. Figure 3 The R2 of fitted curve of DM, CP and OM of roughages of 5 or 7 time points. (A-C), respectively, represent the R2 of fitted curve of DM, CP, and OM in the rumen of roughages. The number 0 = 7 incubation time points, 1, respectively, the R2 of fitted curve of 4, 16, 36, 48, 72 h; 2, respectively, the R2 of fitted curve of 4, 16, 24, 48, 72 h; 3, respectively, the R2 of fitted curve of 4, 16, 24, 36, 72 h; 4, respectively, the R2 of fitted curve of 4, 8, 16, 24, 72 h; 5, respectively, the R2 of fitted curve of 4, 8, 16, 36, 72 h; 6, respectively, the R2 of fitted curve of 4, 8, 16, 48, 72 h. animals-13-00947-t001_Table 1 Table 1 Comparison of recent studies on the application of nylon bag technique for rumen degradability in sheep. Items Bag Repliacation Incubation Conditions Diet Feeding Level Material Pore Size Sample Grind Size Animal Number of Animals Number of Bags Number of Times Times A.R.Seradj -- ad libitum Dacron bags 45 mm -- Rasa Aragonesa ewes 4 1 5 4, 8, 12, 24, 48 h (Moringa oleifera) L.Tao -- -- -- -- -- Hancrossbred ewes 6 -- 5 6, 12, 24, 48, 72 h (roughages) B.Ghorbani -- maintenance level polyamide 40 +- 10 mm -- Zel ewes 3 4 -- 1, 3, 6, 12, 24, 36, 48, 72, 96 h (sesame meal, soybean meal) Xiaogao Diao 70:30 maintenance nylon cloth 48 mm 2.5 mm (roughages) Yunnan semi-fine wool sheep 4 2 7 2, 4, 8, 16, 24, 36, 48 h (72 for roughages) S.Rjiba-Ktita -- ad libitum -- 50 mm 3 mm Barbarine rams 4 2 6 3, 6, 24, 48, 72, 96 (seagrass, macroalgae, barley grain and barley grass) M.A.Harahap -- maintenance level -- 46 mm -- local Ettawah cross bred goats 2 3 7 0.75, 1.5, 3, 6, 12, 24, 48 V.Palangi 60:40 -- polyester mesh bag 47 mm 2 mm Ghezel sheep 3 2 7 0, 4, 8, 16, 24, 36, 48 h (barley) Mohammad Farhad Vahidi 70:30 ad libitum heat-sealed nylon bag 50 mm 2 mm Shal sheep 3 2 5 0, 24, 48, 72, 96 h (lignocellulosic forages) Biwei Jiang -- ad libitum -- -- 40 mesh (roughages) tan sheep 3 2 7 3, 6, 12, 24, 36, 48, 72 h (roughages) Rodrigo A.C. Passetti -- -- R1020 50 mm 4 mm (roughages) ewe lambs 3 -- 9 1, 3, 6, 12, 24, 36, 48, 72, 120 h (roughages) -- means no date information was involved. animals-13-00947-t002_Table 2 Table 2 Routine nutrients in common feeds for sheep (DM basis). Items OM (%) CP (%) Fat (%) ADF (%) NDF (%) RSM 92.44 38.01 11.56 44.85 58.88 CSM 93.04 50.01 0.70 12.08 22.32 protein feeds CGM 98.88 65.05 0.59 10.33 40.21 ES 94.66 39.62 23.51 11.88 24.63 DDGS 95.02 28.70 10.85 18.00 41.83 SOM 93.18 49.77 1.60 10.38 18.68 BR 99.12 12.42 1.12 2.19 4.69 BY 98.62 13.84 0.71 3.70 27.55 WT 98.02 13.45 1.52 3.65 12.73 HS 98.39 10.92 2.91 6.40 14.56 energy feeds HY9 98.78 8.17 3.05 4.65 12.14 GWC 98.24 10.44 4.93 1.22 7.72 YC 98.60 9.31 3.16 3.41 9.69 YWB 94.03 17.95 3.71 13.92 44.61 CBS 96.43 18.68 1.98 17.32 59.80 WS 91.75 9.31 2.13 40.07 70.26 RG 88.12 9.90 1.73 33.48 56.80 RSW 86.62 12.44 1.43 33.47 45.84 BS 91.24 14.33 2.36 32.61 52.69 roughages SS 89.88 12.87 1.44 35.21 48.35 CWR 93.08 10.15 0.70 44.66 72.33 RS 94.91 15.99 2.57 38.99 66.39 OG 88.93 16.92 1.85 34.82 63.96 CS 95.46 10.33 3.42 27.35 47.13 TP 92.32 17.23 0.91 19.08 26.93 animals-13-00947-t003_Table 3 Table 3 The degradation parameters of dry matter, crude protein, and organic matter in rumen of six protein feeds (%). DM CP OM a b c ED a b c ED a b c ED 0 24.31 29.34 0.055 39.39 9.75 40.12 ab 0.089 a 35.11 22.44 31.48 0.054 b 39.93 RSM 1 22.70 31.51 0.050 38.52 11.83 41.31 a 0.057 b 33.89 20.44 30.24 0.089 a 39.81 2 23.24 30.38 0.056 39.22 12.13 37.48 b 0.087 a 35.92 20.68 31.59 0.080 a 40.07 3 23.48 29.62 0.055 39.00 10.59 38.65 ab 0.100 a 36.31 20.10 30.83 0.090 a 39.91 0 15.44 54.74 0.064 46.12 10.94 c 66.86 0.056 a 46.02 13.21 b 54.03 0.072 45.10 CSM 1 16.60 54.33 0.060 46.23 16.32 a 66.82 0.042 b 46.75 16.95 a 53.30 0.058 45.62 2 16.50 53.73 0.060 45.81 16.59 a 64.53 0.042 b 45.93 16.94 a 52.53 0.059 45.31 3 15.94 55.10 0.061 46.29 14.47 b 66.28 0.049 ab 47.34 16.37 a 53.79 0.060 45.78 0 16.72 a 39.79 0.076 42.67 8.58 a 67.30 a 0.023 42.67 a 16.82 a 40.98 0.072 42.98 CGM 1 14.07 ab 41.59 0.084 42.23 8.84 ab 63.78 b 0.024 32.79 b 14.35 b 42.49 0.078 42.51 2 13.99 ab 41.12 0.091 42.60 7.91 b 66.22 ab 0.024 32.72 b 14.42 b 42.08 0.084 42.98 3 13.67 b 41.53 0.093 42.76 10.15 a 52.16 c 0.033 33.64 b 14.19 b 42.00 0.087 42.95 0 25.71 68.69 0.043 60.62 16.81 67.72 a 0.034 60.62 a 24.23 67.95 a 0.047 57.46 ES 1 23.81 68.53 0.046 56.68 16.37 68.29 a 0.032 43.17 b 23.79 67.12 ab 0.049 56.94 2 24.13 66.27 0.047 56.17 17.72 63.71 b 0.033 42.86 b 23.80 65.63 b 0.049 56.22 3 23.58 68.48 0.047 56.76 16.35 64.64 b 0.037 43.84 b 23.49 67.98 a 0.048 56.68 0 26.44 b 50.96 a 0.039 50.90 12.35 38.83 b 0.036 30.79 24.74 bc 57.60 a 0.037 50.73 ab DDGS 1 29.45 a 50.19 ab 0.031 51.55 13.07 36.07 c 0.040 31.17 29.45 a 50.19 bc 0.031 51.55 a 2 29.56 a 49.70 ab 0.030 50.77 12.34 37.30 bc 0.037 30.34 26.46 b 49.03 c 0.035 49.44 b 3 27.80 ab 47.88 c 0.040 51.83 12.93 41.88 a 0.028 30.31 23.72 c 51.28 b 0.047 51.54 a 0 30.45 63.41 a 0.037 57.23 14.23 ab 76.13 b 0.032 43.63 29.48 64.83 0.033 55.28 SOM 1 30.60 62.65 ab 0.037 57.33 16.08 a 70.67 c 0.034 44.68 28.97 65.33 0.029 55.09 2 30.79 61.38 b 0.037 56.81 13.88 b 77.35 b 0.032 43.95 29.29 64.19 0.033 55.00 3 29.85 62.08 ab 0.040 57.44 15.30 ab 82.39 a 0.026 43.20 28.70 63.55 0.036 55.35 a-c indicate that there are significant differences of ED of the same feed in the same column (p < 0.05). The same applies in the following tables. 0 respectively 7 incubation time points; 1 respectively 2, 16, 24, 36, 48 h; 2 respectively 2, 8, 16, 24, 48 h, 3 respectively 2, 8, 16, 36, 48 h. animals-13-00947-t004_Table 4 Table 4 The degradation parameters of dry matter, crude protein, and organic matter in rumen of nine energy feeds (%). DM CP OM a b c ED a b c ED a b c ED 0 13.13 a 78.86 ab 0.046 50.97 9.86 a 81.21 b 0.045 48.46 13.34 a 79.31 0.045 50.69 BR 1 11.77 ab 78.02 b 0.050 50.94 8.07 ab 81.98 ab 0.048 48.01 11.65 ab 79.18 0.048 50.43 2 11.00 b 80.29 a 0.048 50.37 7.93 b 81.97 ab 0.047 47.73 11.22 b 79.77 0.047 49.79 3 10.17 b 80.00 a 0.051 50.72 8.78 ab 83.35 a 0.043 47.41 10.62 b 80.59 0.048 50.22 0 33.95 a 62.97 c 0.086 73.62 a 18.2 a 74.88 c 0.061 58.84 33.86 a 63.12 c 0.088 73.83 a BY 1 27.90 c 67.06 b 0.109 73.91 a 14.87 b 78.89 a 0.058 57.30 27.68 b 67.34 b 0.111 74.13 a 2 28.76 c 68.66 ab 0.085 72.05 ab 15.47 b 76.71 b 0.062 57.90 28.67 b 68.81 ab 0.086 72.24 ab 3 28.10 c 69.45a 0.088 72.35 a 14.8 b 77.38 ab 0.064 58.27 28.00 b 69.64 a 0.089 72.55 a 0 37.43 a 55.96 c 0.089 73.23 20.81 a 73.66 d 0.078 ab 65.48 a 40.14 a 51.76 d 0.102 a 74.75 a WT 1 34.18 b 60.60 a 0.079 71.36 14.90 b 82.51 a 0.068 b 62.30 b 34.32 b 60.42 a 0.079 c 71.27 c 2 34.36 b 60.10 a 0.087 72.46 15.61 b 79.91 b 0.077 ab 64.03 ab 34.18 b 58.86 bc 0.095 ab 72.70 bc 3 35.08 b 58.22 b 0.086 71.82 15.69 b 79.05b c 0.078 ab 63.79 ab 34.43 b 58.20 c 0.094 ab 72.48 bc 0 9.73 b 83.32 b 0.040 46.48 b 11.67 a 78.98 b 0.041 46.43 ab 14.98 b 80.31 a 0.037 49.12 HS 1 10.22 a 77.75 c 0.049 48.50 a 8.00 c 83.41 a 0.039 44.71 b 17.92 a 73.07 b 0.041 50.84 2 7.48 b 86.21 a 0.043 47.14 ab 9.97 ab 77.21 b 0.043 45.53 ab 15.28 b 80.87 a 0.037 49.40 3 8.75 ab 86.00 a 0.039 46.33 b 9.84 ab 78.62 b 0.042 45.82 ab 15.53 b 80.38 a 0.036 49.23 0 16.39 a 79.85 a 0.036 ab 49.77 a 23.28 a 72.2 ab 0.026 47.76 12.80 b 71.24 ab 0.055 ab 50.14 bc HY9 1 14.36 bc 72.30 c 0.043 ab 47.71 bc 21.11 b 73.8 a 0.026 46.48 15.47 a 63.45 d 0.072 a 52.96 a 2 13.55 cd 74.14 b 0.041 ab 47.04 bc 21.74 ab 71.59 b 0.027 47.02 12.96 b 72.66 a 0.054 ab 50.69 b 3 15.61 ab 80.55 a 0.030 b 46.07 c 21.33 b 67.9 c 0.031 47.11 13.35 b 70.72 c 0.054 ab 50.20 bc 0 36.72 a 57.9 a 0.038 61.80 a 31.42 51.57 c 0.040 54.15 27.28 ab 65.48 b 0.044 a 57.54 ab GWC 1 29.67 b 53.37 b 0.051 56.67 b 29.78 55.06 a 0.037 53.23 26.13 b 66.57 b 0.043 a 57.06 ab 2 30.29 b 50.97 c 0.055 56.97 b 30.07 54.48 ab 0.038 53.67 26.41 b 66.03 b 0.044 a 57.35 ab 3 29.73 b 51.88 bc 0.057 57.37 b 29.71 52.87 bc 0.042 53.72 28.46 a 69.73 a 0.033 b 56.39 b 0 29.07 a 57.05 b 0.033 51.62 27.70 30.84 0.057 a 44.03 26.51 71.58 a 0.033 54.57 YC 1 25.79 b 60.54 a 0.034 50.21 27.52 31.07 0.056 a 43.91 24.77 71.77 a 0.034 53.92 2 27.12 b 56.58 b 0.036 50.96 27.59 31.08 0.056 a 44.05 25.00 71.22 ab 0.035 54.23 3 26.91 b 56.34 b 0.038 51.09 28.57 30.90 0.047 b 43.54 24.71 69.61 b 0.037 54.27 0 32.86 43.82 b 0.053 ab 77.20 a 29.42 a 58.34 b 0.151 75.70 25.85 44.2 ab 0.077 ab 54.54 1 32.30 46.91 a 0.038 b 55.03 b 24.8 b 62.34 a 0.185 76.09 26.64 45.27 a 0.059 b 53.59 YWB 2 34.45 39.63 c 0.047 ab 55.86 b 29.44 a 59.52 b 0.138 75.62 27.27 41.83 c 0.073 ab 54.36 3 32.47 40.33 c 0.060 a 56.76 b 28.98 a 58.42 b 0.147 74.90 25.98 43.51 abc 0.080 a 55.06 0 18.54 a 49.17 a 0.038 ab 39.40 b 31.89 50.31 a 0.026 48.56 b 18.07 a 50.02 a 0.036 ab 38.82 b CBS 1 18.13 a 47.81 ab 0.040 ab 42.06 a 29.91 49.68 a 0.031 51.51 a 17.60 ab 49.02 ab 0.038 ab 41.48 a 2 17.90 ab 48.31 ab 0.039 ab 41.78 a 30.82 46.97 b 0.033 51.98 a 17.49 ab 49.23 ab 0.037 ab 41.28 a 3 16.10 b 47.16 b 0.051 a 42.56 a 30.91 47.06 b 0.032 51.93 a 15.80 b 47.63 b 0.049 a 41.96 a a-d indicate that there are significant differences of ED of the same feed in the same column (p < 0.05). The same applies in the following tables. 0 respectively 7 incubation time points; 1 respectively 2, 16, 24, 36, 48 h; 2 respectively 2, 8, 16, 24, 48 h, 3 respectively 2, 8, 16, 36, 48 h. animals-13-00947-t005_Table 5 Table 5 The degradation parameters of dry matter, crude protein, and organic matter in rumen of 10 roughages (%). DM CP OM a b c ED a b c ED a b c ED WS 0 7.30 a 39.02 0.035 27.28 11.35 ab 42.33 0.020 27.77 6.19 a 40.02 a 0.034 26.55 1 6.13 ab 37.37 0.042 27.43 9.56 b 42.19 0.023 27.26 5.42 ab 37.86 b 0.041 26.86 2 6.71 a 38.12 0.037 27.23 9.63 ab 42.89 0.022 27.21 6.13 a 38.71 ab 0.035 26.62 3 6.64 a 39.24 0.036 27.46 9.88 ab 42.72 0.021 27.10 5.98 a 39.78 ab 0.035 26.83 4 6.13 ab 37.37 0.042 27.43 9.56 b 42.19 0.023 27.26 5.42 ab 37.86 b 0.041 26.86 5 5.60 ab 38.71 0.043 27.93 11.55 a 41.15 0.020 27.76 4.54 ab 39.61 ab 0.042 27.21 6 4.60 b 38.09 0.049 27.85 11.03 ab 40.02 0.023 27.95 3.75 b 38.87 ab 0.047 27.11 RG 0 18.48 56.82 0.040 50.18 26.11 a 40.04 b 0.044 49.34 14.88 ab 59.57 a 0.039 47.63 1 19.50 55.45 0.040 50.39 20.35 b 44.50 a 0.054 48.53 16.38 a 58.15 ab 0.037 47.84 2 18.82 55.78 0.042 50.64 19.89 b 45.29 a 0.055 48.72 15.1 ab 58.46 ab 0.041 48.31 3 19.48 55.03 0.040 50.34 20.59 b 45.19 a 0.052 48.66 16.24 a 57.22 b 0.039 47.79 4 18.00 56.66 0.042 50.36 20.02 b 46.25 a 0.054 49.30 14.01 b 59.59 a 0.041 47.90 5 18.95 55.97 0.039 49.99 20.47 b 44.94 a 0.054 48.96 15.79 ab 58.61 ab 0.036 47.22 6 17.90 56.73 0.042 50.39 18.82 b 45.88 a 0.061 49.16 14.05 b 59.72 a 0.041 47.91 RSW 0 18.69 e 80.71 a 0.072 a 66.14 a 22.54 b 55.64 a 0.056 57.88 25.77 a 54.86 d 0.090 a 66.79 a 1 22.16 d 59.13 b 0.056 ab 59.97 b 22.95 ab 55.66 a 0.051 57.48 21.51 de 61.29 a 0.052 b 59.72 b 2 25.13 bc 56.54 c 0.046 b 58.61 b 22.90 b 55.14 abc 0.053 57.48 22.16 cde 59.72 ab 0.052 b 59.40 b 3 23.47 cd 57.98 bc 0.051 b 59.25 b 22.42 b 55.41 ab 0.055 57.58 20.90 e 61.20 a 0.055 b 59.91 b 4 27.70 a 52.19 e 0.046 b 58.63 b 23.7 ab 53.46 c 0.053 57.36 23.83 bc 56.68 c 0.053 b 59.42 b 5 23.32 cd 57.36 bc 0.057 ab 60.35 b 23.71 ab 54.22 abc 0.052 57.52 22.89 cd 59.12 b 0.053 b 60.07 b 6 25.72 b 55.03 d 0.050 b 59.60 b 24.83 a 53.66 bc 0.048 57.30 25.5 ab 57.07 c 0.046 b 59.27 b BS 0 13.88 c 31.52 ab 0.084 ab 36.81 11.20 b 29.66 0.044 28.47 12.42 35.83 ab 0.070 37.11 1 16.62 a 28.40 e 0.079 ab 36.96 12.02 ab 30.73 0.034 27.88 13.93 34.16 b 0.068 37.27 2 14.51 bc 30.62 bc 0.095 a 37.50 11.10 b 29.74 0.045 28.42 13.05 35.04 ab 0.073 37.54 3 14.93 abc 30.53 bc 0.090 a 37.57 10.99 b 30.09 0.044 28.50 13.28 34.87 ab 0.072 37.51 4 13.90 c 32.50 a 0.080 ab 37.24 11.11 b 29.96 0.044 28.56 12.32 36.41 a 0.069 37.35 5 16.10 ab 29.74 bc 0.069 b 36.55 12.53 ab 30.08 0.033 27.99 13.44 35.05 ab 0.064 36.98 6 16.30 ab 29.05 cd 0.070 b 36.32 13.32 a 30.77 0.028 27.74 13.37 35.00 ab 0.065 36.96 SS 0 15.75 ab 47.92 ab 0.087 bc 50.90 20.56 55.38 ab 0.075 59.54 12.87 a 48.93 bcd 0.085 b 48.51 1 13.83 b 48.81 ab 0.116 a 52.26 22.45 53.01 c 0.077 60.03 10.02 b 50.75 ab 0.114 a 49.82 2 15.83 a 47.32 ab 0.105 ab 52.26 21.66 53.91 bc 0.079 60.29 12.77 a 48.62 cd 0.100 ab 49.73 3 15.60 ab 46.89 b 0.108 a 51.95 21.93 53.74 bc 0.078 60.27 12.87 a 47.93 d 0.101 ab 49.42 4 15.69 ab 48.97 a 0.084 c 51.38 20.49 56.11 a 0.074 59.84 12.63 a 50.38 abc 0.082 b 49.11 5 15.95 a 47.85 ab 0.086 c 50.97 21.78 54.47 abc 0.070 59.43 12.27 a 49.77 abcd 0.086 b 48.75 6 15.26 ab 48.82 ab 0.088 bc 51.19 21.74 54.27 abc 0.071 59.34 11.31 ab 51.02 a 0.089 b 49.00 CWR 0 14.63 43.28 cd 0.024 33.24 31.16 30.34 a 0.034 46.09 11.98 37.26 bc 0.014 23.18 1 14.99 44.75 bc 0.021 33.13 30.77 26.66 b 0.045 46.47 12.11 32.68 d 0.017 23.41 2 15.46 44.56 bc 0.021 33.11 31.50 27.09 b 0.037 46.19 12.53 43.01 a 0.010 23.15 3 15.06 42.71 d 0.023 33.21 31.62 26.79 b 0.037 46.09 12.32 37.71 b 0.013 23.18 4 15.91 45.60 b 0.018 32.70 31.38 27.05 b 0.037 45.99 12.48 43.12 a 0.010 22.90 5 15.22 45.16 bc 0.020 32.85 30.40 27.12 b 0.045 46.30 11.83 32.83 d 0.017 23.27 6 15.93 52.10 a 0.015 32.62 29.76 27.62 b 0.048 46.51 12.10 35.70 c 0.014 23.22 RS 0 34.49 34.98 abc 0.021 48.12 32.70 a 34.97 ab 0.043 52.78 32.39 35.15 abc 0.022 46.38 1 34.24 33.30 cd 0.022 48.03 30.39 b 36.06 a 0.051 52.76 32.31 33.87 c 0.022 46.35 2 34.67 35.12 ab 0.019 47.87 31.48 ab 36.00 a 0.043 52.35 32.73 35.84 ab 0.019 46.20 3 34.36 33.51 bcd 0.022 47.98 31.65 ab 35.07 ab 0.044 52.10 32.40 34.38 bc 0.021 46.33 4 35.39 36.43 a 0.016 47.79 33.27 a 33.23 b 0.043 52.42 33.12 36.61 a 0.017 46.16 5 34.58 33.17 d 0.022 48.06 31.39 ab 34.25 ab 0.053 52.94 32.37 33.85 c 0.022 46.39 6 35.04 36.07 a 0.017 47.90 30.42 b 35.74 a 0.057 53.39 32.86 36.20 ab 0.018 46.24 OG 0 11.86 b 67.42 a 0.057 55.28 12.77 bc 74.14 ab 0.058 60.90 14.86 ab 62.18 a 0.054 54.16 1 13.19 ab 65.35 bc 0.060 56.19 12.07 cd 73.46 b 0.068 62.39 13.71 b 62.80 a 0.058 54.43 2 14.34 a 64.30 c 0.057 55.75 15.94 a 69.97 d 0.057 61.08 15.61 ab 61.19 ab 0.052 53.71 3 13.89 a 64.68 c 0.058 55.88 14.87 a 72.31 bc 0.059 62.15 14.76 ab 62.19 a 0.054 54.03 4 12.57 ab 65.93 abc 0.056 54.87 14.34 ab 71.15 cd 0.057 60.13 16.11 a 60.08 b 0.052 53.62 5 11.61 b 67.38 a 0.058 55.30 10.51 d 75.86 a 0.064 61.51 13.73 b 62.86 a 0.058 54.49 6 12.43 ab 66.58 ab 0.056 55.06 11.78 cd 74.07 ab 0.062 61.00 14.86 ab 61.52 ab 0.055 54.10 CS 0 35.33 b 36.29 a 0.053 59.63 ab 36.53 37.58 0.080 64.86 34.35 a 37.24 b 0.049 c 59.56 ab 1 37.52 a 33.61 b 0.051 60.04 a 36.23 37.93 0.079 65.00 35.87 a 35.25 c 0.054 ab 59.92 a 2 37.61 a 33.25 b 0.052 59.96 a 37.25 36.87 0.074 64.71 26.09 b 43.95 a 0.071 a 58.48 abc 3 37.18 ab 34.35 b 0.052 60.35 a 37.02 36.99 0.075 64.71 35.75 a 35.79 bc 0.055 ab 60.11 a 4 35.56 b 36.13 a 0.051 57.87 bc 37.38 36.19 0.077 63.06 34.87 a 36.54 bc 0.052 ab 57.59 c 5 35.70 ab 36.39 a 0.049 57.88 bc 36.23 37.71 0.081 63.42 34.49 a 37.43 b 0.052 ab 57.83 bc 6 36.97 ab 34.90 b 0.044 57.32 c 36.37 37.69 0.080 63.46 35.95 a 35.68 bc 0.046 c 57.21 c TP 0 11.83 b 82.86 abc 0.043 50.11 d 18.61 a 78.66 d 0.064 a 62.55 b 21.71 a 76.52 d 0.074 a 67.14 b 1 15.55 a 84.38 a 0.046 69.82 a 15.71 b 83.35 bc 0.036 b 64.60 a 10.47 d 89.13 a 0.049 b 69.37 a 2 17.06 a 82.27 bc 0.045 69.81 a 14.41 b 85.34 a 0.039 b 66.38 a 16.34 c 83.32 b 0.046 b 69.93 a 3 15.96 a 84.01 ab 0.046 69.99 a 15.08 b 82.10 c 0.040 b 65.47 a 18.73 b 80.20 c 0.045 b 70.23 a 4 16.14 a 81.86 c 0.041 62.40 b 15.42 b 81.75 c 0.038 b 60.39 c 18.37 b 80.30 c 0.044 b 64.96 c 5 15.44 a 82.35 bc 0.041 61.83 bc 15.64 b 83.17 bc 0.037 b 60.35 c 17.82 bc 81.94 bc 0.043 b 65.27 c 6 15.23 a 84.57 a 0.036 60.17 c 14.97 b 84.42 ab 0.036 b 60.06 c 18.52 b 81.24 c 0.041 b 64.28 c a-e indicate that there are significant differences of ED of the same feed in the same column (p < 0.05). 0 respectively 7 incubation time points; 1 respectively 4, 16, 36, 48, 72 h; 2 respectively 4, 16, 24, 48, 72 h; 3 respectively 4, 16, 24, 36, 72 h; 4 respectively 4, 8, 16, 24, 72 h; 5 respectively 4, 8, 16, 36, 72 h; 6 respectively 4, 8, 16, 48, 72 h. 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PMC10000143

This review aims to consolidate the relevant published data exploring the amino acid (AA) requirements of Nile tilapia, Oreochromis niloticus, and to reach a new set of recommendations based on those data. There are still inconsistencies in lysine, sulfur-containing AA, threonine, tryptophan, branched-chain AA, and total aromatic AA recommendations in data that have appeared since 1988. This review finds that strain, size, basal diet composition, and assessment method may have contributed to the inconsistencies in AA recommendations. Currently, the expansion of precision AA nutrition diets for Nile tilapia is receiving more attention because of the demand for flexibility in widespread ingredient substitutions which will allow compliance with environmentally sustainable principles. Such approaches involve changes in diet ingredient composition with possible inclusions of non-bound essential and non-essential AAs. Increasing the inclusion of non-bound AAs into Nile tilapia diets may modify protein dynamics and influence AA requirements. Emerging evidence indicates that not only essential but also some non-essential amino acids regulate growth performance, fillet yield, and flesh quality, as well as reproductive performance, gut morphology, intestinal microbiota, and immune responses. Thus, this review considers current AA recommendations for Nile tilapia and proposes refinements that may better serve the needs of the tilapia industry. amino acid requirement ideal amino acid ratio growth performance health status Nile tilapia This research received no external funding. pmc1. Introduction Global tilapia production is projected to continue growing until 2031 through the sustainable management and utilization of natural resource principles based on improved nutrition practices . In this way, the growth performance, reproduction, and health of tilapias have been improved by genetic selection breeding programs coupled with precision nutritional strategies to meet this increasing demand. However, this need has generated challenges for the tilapia industry, including concerns of food security, food safety, feed ingredient shortages, diseases, and environmental issues. Emerging evidence suggests a variety of economically and environmentally sustainable feedstuffs for use in aquafeeds . One of these groups is the plant-protein feedstuffs; however, vegetable feedstuffs may contain antinutrients and limiting amounts of certain amino acids (AAs) that may impair protein synthesis . Consistently, the deficiency of a single essential AA may impair several physiological functions and, subsequently, growth performance . Emerging evidence suggests that AAs also act as signaling molecules regulating protein synthesis and energy metabolism in the Nile tilapia, Oreochromis niloticus . Therefore, the provision of tilapia feeds closely matching optimum AA requirements is a crucial strategy for overcoming various challenges, including optimizing sustainable raw materials, lowering feeding costs, and attenuating nitrogen loss into the environment. Fish, like other animals, synthesize body proteins from AAs that are provided in the diet as well as some AAs that can be synthesized in the body from precursors. Those which must be provided in the diet due to lack of endogenous synthesis capabilities have traditionally been referred to as dietary indispensable or essential amino acids (EAAs). Classical fish nutrition textbooks consider arginine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, and valine as nutritionally essential AAs to maintain normal physiological functions of cells and tissues . These EAAs are most critical to provide in the diet because a deficiency of any one can limit protein synthesis, which is often manifested as reduced weight gain as well as other specific deficiency signs. Another group of AAs commonly referred to as dispensable or non-essential amino acids (NEAAs) includes alanine, asparagine, aspartate, cysteine, glutamate, glutamine, glycine, proline, serine, and tyrosine. Those NEAAs traditionally have been classified as such because they can be synthesized in the body from precursor biochemicals. They also may be found in dietary protein and used for synthesizing tissue proteins as well as participating in various metabolic pathways. Emerging evidence has indicated that dietary supplementation of NEAAs also may have beneficial effects on the growth performance , reproduction , health , and flesh quality of Nile tilapia that are fed plant-based diets. Some AAs may be classified as conditionally essential amino acids (CEAAs) because their rates of use may exceed their rates of synthesis under certain physiological conditions. AAs in this category include glutamate, glutamine, glycine, proline, and hydroxyproline, as well as the sulfonic acid taurine. The CEAA term also has been applied where the reduction or elimination of certain protein feedstuffs in the diet which are rich in certain AAs requires the supplementation of such AAs to prevent growth reduction or other metabolic impairment. The participation of AAs in various metabolic processes beyond protein synthesis such as cell signaling, gene expression, and metabolic regulation has led to the term "functional" AAs, which can include EAAs, CEAAs, and NEAAs . The relevance of the concept of functional AAs to aquatic animal nutrition has been established and is beginning to receive heightened attention. Notably, many relevant outcomes on AA requirements of tilapia have been published since the last National Research Council (NRC) edition in 2011 . However, the summary of AA requirements of tilapia has not been updated for more than a decade. Variables such as fish strain and size, feed composition and processing technology, as well as feed management and statistical methods, may influence experimental estimates of AA requirements of Nile tilapia and may explain some of the inconsistencies in AA recommendations throughout the literature. Importantly, some of these new data provide insights for optimal production efficiency, welfare, health, and flesh quality responses. Thus, this review comprehensively consolidates the relevant data on dietary AA requirements, identifies the shortfalls, and recommends directions for future research in tilapia nutrition. 2. Methodology The studies selected in this review were completed since 1988, and the impacts of single AA additions on the growth performance of Nile tilapia were determined in most of them, although statistical models using the deletion method were applied to estimate AA requirements in the remaining studies. Where data permit, the impacts of dietary AA recommendations based on growth performance, reproduction, and health responses are tabulated. Also, all data are expressed on a dry-matter basis. Generally, growth rate, feed conversion ratio, and protein retention efficiency were identified as the most appropriate parameters for deriving AA recommendations, and their simple mean (+-standard deviation) recommendations were used to summarize the AA recommendations reported during the past 34 years. While it is well-established that feedstuffs contain more total than digestible amino acids, few studies have reported dietary amino acid requirements that consider amino acid digestibility. Therefore, in this review, we focused on data based on diets' total amino acid content. Furthermore, relevant Nile tilapia production stage ranges for the recommendations are included as footnotes. Standard deviations of the mean AA values are recorded to illustrate the variations caused by units in expressing requirements (g/kg diet or % crude protein). These mean values are somewhat problematic because AA requirements are expressed on a total basis, and variations in respective fish size ranges, feed ingredients and diet composition, response variable, and statistical methods for estimating AA requirements are the main influential factors on these outcomes. Recent studies have used various estimation methods to arrive at AA recommendations for Nile tilapia, including linear, quadratic, and polynomial broken-line regressions. An alternative method proposed to determine the optimal AA ratio for pigs and applied for other species such as Atlantic salmon, Salmo salar , and Nile tilapia is the deletion method. This method involves monitoring the nitrogen balance as AAs are reduced from the diet and assuming that reducing a non-limiting AA does not affect nitrogen retention . This approach differs from the conventional procedure by allowing for the determination of the requirements for all essential AAs in one set of experiments and is well-accepted as an efficient and rapid tool to estimate the ideal AA profile in Nile tilapia . Additionally, there are some studies in which more than one assessment method was applied to compare the impacts of different methods on estimated AA requirements. Broken-line models generated 27% of tabulated recommendations, while both linear and broken-line assessments generated 9%. Additionally, polynomial regressions generated 50%, while the deletion method generated 14% of the recommendations. Finally, we established recommendations for the dietary essential AA profile (plus cystine and tyrosine) for each Nile tilapia production stage relative to dietary protein, as these values have not yet been established for all AAs and production stage ranges. For this, we considered previously established dietary protein requirement values in the literature for each production stage. 3. Results 3.1. Amino Acid Composition of Tilapia Tissues The AA composition of eggs and whole bodies of Nile tilapia at different production stages are shown in Table 1. Lysine was the dominant essential AA, while glutamic acid was the major non-essential AA. However, there is little information in the literature about the impact of dietary AA supplementation on egg and tissue AA profiles. Previous research identified that the AA composition of eggs varies with dietary protein . Conversely, early studies have reported that dietary AA supplementation does not alter the whole-body AA composition of nursery, pre-growout, and growout Nile tilapia . 3.2. Dietary Amino Acid Recommendations for Nile Tilapia The AA requirements recommended for some Nile tilapia production stages are presented in Table 2. Of note, there are tangible differences across the literature, and the genesis of these inconsistencies could include genetic differences between strains, diet composition, fish management, rearing conditions, and the statistical method by which AA requirements were assessed. The AA requirements of Nile tilapia were published by the NRC (2011) and are considered one of the main references for AA recommendations. However, genetic improvements and higher performance objectives set by the modern tilapia industry have motivated researchers to review the NRC (2011) recommendations over the past decade. Notably, a number of recent studies have been published, although different experimental methodologies have been used. Several experiments were conducted in which the requirement of one single or multiple AAs was determined, as shown in Table 2. Based on data displayed in Table 1, the dietary recommendation of each essential AA (plus cystine and tyrosine) was computed relative to the dietary crude protein content and shown in Table 3. For this, we considered the dietary protein content for each fish production stage based on previous values established for Nile tilapia. Previous evidence shows that the culture system influences the dietary protein requirement of Nile tilapia broodstock. Thus, based on the percentage of each AA relative to dietary protein, the dietary AA recommendations for Nile tilapia broodstock are displayed in Table 4. The concept of ideal protein for domestic animals was first proposed by Mitchell over 60 years ago and remains relevant in poultry and pig nutrition . The ideal protein concept refers to dietary protein with an AA profile that exactly meets an animal's requirements . According to this concept, dietary protein should have an AA profile that exactly meets the animal's requirements, as shown in Table 5. Lysine is used as a reference AA to express the requirements for other AAs, which simplifies diet formulation, as solid requirement data for most AAs are not readily available . Additionally, the concept of ideal AA ratios was introduced for Nile tilapia in 1994 to aid in the development of cost-effective feed formulations . Of note, the ideal protein concept also has been applied to reduce dietary protein and optimize fishmeal-free diets for Nile tilapia. Importantly, further studies are required to continuously update ideal AA profiles considering the ideal protein concept in Nile tilapia diets. 3.3. Importance of Amino Acids in Nile Tilapia 3.3.1. Lysine Lysine is the first limiting essential AA in typical corn, wheat, and cereal coproduct-based diets for Nile, and supplementation of feed-grade lysine has been largely adopted in practical and experimental diets for Nile tilapia. Lysine's primary metabolic role in protein synthesis also is the basis for it to be the reference AA in computing ideal AA ratios. Interestingly, an early study demonstrated that the effectiveness of using intact lysine from high-lysine corn protein concentrate was not significantly different from that of crystalline lysine in Nile tilapia . It is noteworthy that accurate estimations of dietary lysine requirements are critical because recommendations for the balance of AAs based on the ideal protein concept are expressed as ratios to lysine (Table 5). In this sense, dietary lysine is generally considered the first limiting essential AA. As shown in Table 3, there is a relatively low variation in dietary lysine recommendations for body weight gain of fish. On the basis of body weight gain, the simple means of total and digestible lysine intakes are 14 and 12 mg/g body weight gain, respectively. Previous work indicated that lysine utilization efficiency remained relatively high (+63%) in nursery and pre-growout Nile tilapia and decreased (48%) in growout Nile tilapia . In this sense, the dietary lysine requirements for the maintenance of nursery, pre-growout, and growout Nile tilapia have been established to be 2.7, 45.1, and 56.3 mg lysine/kg0.8 body, respectively . Previous work determined that growout Nile tilapia required an intake of 23 mg of lysine to deposit 1 g of body weight gain . Moreover, lysine has been shown to improve body weight, feed efficiency, and fillet yield in Nile tilapia . Additionally, high quantitative lysine requirements have been described for Nile tilapia reared in saline water , with Nile tilapia reared in brackish water (8%0) showing higher lysine requirements than those raised in 0%0 water (23 vs. 21 g/kg diet, respectively) . 3.3.2. Sulfur-Containing Amino Acids Methionine and cysteine (cystine forms from two cysteine residues) are the total sulfur-containing AAs considered in tilapia feeds. Of note, methionine is considered the first limiting AA in Nile tilapia fed cereal-based diets, particularly soybean-meal-rich diets . Methionine plays an essential role in cellular metabolism as a methyl donor and acts as the precursor to cysteine . Notably, methionine is one of the most supplemented feed-grade AAs in fish feeds, including those for Nile tilapia . In addition, the total sulfur-containing AA requirement of Nile tilapia is often met by supplementing methionine and considering the replacement values of cysteine and methionine in tandem. Previous research identified that cystine could spare up to 49% of the methionine requirement on a molar sulfur basis in diets for Nile tilapia . Moreover, reports on quantitative methionine requirements for body weight gain are very similar when expressed as individual methionine contents (CV of 13%) compared to methionine-plus-cystine contents (CV of 12%). These suggest that total sulfur-containing AAs may be used to express the dietary requirement, along with considering the minimum level of dietary methionine. The optimal methionine-plus-cystine requirement is well established in the literature, averaging 9.7 g/kg diet (3.5% of crude protein), as displayed in Table 2. A previous study established methionine utilization efficiencies of 0.76 and 0.55 and determined the methionine maintenance requirements of 3.12 and 16.5 mg methionine/kg0.7 body for pre-growout and growout Nile tilapia, respectively . Importantly, a previous work determined that growout Nile tilapia required an intake of ~15.2 mg of methionine to deposit 1 g of body weight gain . 3.3.3. Threonine Threonine is a potential limiting AA in conventional corn-, wheat-, soybean-, and coproducts-based diets fed to Nile tilapia . Feed-grade threonine is commercially available and may be included in diets for Nile tilapia. Threonine is an important AA because it is prominent in intestinal mucin secretion and in the production of antibodies , as well as influencing digestive and absorptive capacities and antioxidant status in the intestine . However, the underlying mechanisms of action of threonine on mucin secretion and intestine health status in Nile tilapia are not fully understood, although many studies have reported the positive impacts of adequate and excess provision of dietary threonine on Nile tilapia performance. Noteworthily, the optimal threonine requirement is well established in the literature and averages 12 g/kg diet (4.3% crude protein), as shown in Table 2. An early study identified the dietary threonine requirement as lower for body weight gain (10.5 g/kg diet; 3.6% crude protein) compared to fillet yield (11.5 g/kg diet; 4% crude protein) in growout Nile tilapia . Additionally, these authors also established that growout Nile tilapia require an intake of 15.9 mg of threonine to deposit 1 g of body weight. 3.3.4. Tryptophan Tryptophan is considered a potential limiting AA in conventional plant-based diets, particularly in Nile tilapia fed corn and its coproducts . Feed-grade tryptophan is also commercially available for inclusion in Nile tilapia aquafeeds. In addition to protein synthesis, tryptophan plays an important role in producing several metabolites, mainly the neurotransmitter/neuromodulator serotonin and the hormone melatonin in teleost fish . Thus, as a precursor of serotonin, dietary tryptophan has been linked to various behavioral patterns . Consistently, adequate tryptophan supplementation leads to reduced aggressive behavior and stress in Nile tilapia , resulting in positive effects on growth, feed efficiency , and survival . In this review, the tryptophan requirement averages 3.1 g/kg diet (1% crude protein), as shown in Table 2. Previous work established that pre-growout Nile tilapia require an intake of ~3 mg of tryptophan to deposit 1 g of body weight . 3.3.5. Branched-Chain Amino Acids Isoleucine, leucine, and valine are branched-chain AAs that attract less attention, as their requirements are usually met by typical protein feedstuffs in conventional Nile tilapia diets. In addition, it is important to elaborate diets with a balanced profile of branched-chain AAs because interactions between leucine and valine exist, and imbalances have been reported to depress performance . In this sense, it is possible that branched-chain AA interactions may have influenced the tabulated requirement values. Variations in branched-chain AA recommendations are summarized in Table 2. Growing evidence has suggested the optimal branched-chain AAs ratio (Ile:Leu:Val) to be 1:1.3:0.9 in diets for pre-growout Nile tilapia . However, very few studies have been designed to investigate the interactive effects of branched-chain AAs in Nile tilapia. Feed-grade isoleucine, leucine, and isoleucine have yet to become economically feasible. In addition to protein synthesis, an adequate supply of leucine and valine is important for maintaining immune responses . Recently, a study reported that leucine and valine improved intestinal function, enhanced digestive and absorptive capacities, and positively regulated glucose and fatty acid metabolism in the liver, thereby improving the growth performance of Nile tilapia . Dietary isoleucine, leucine and valine requirements average 10.5 g/kg diet (3.8% crude protein), 11.8 g/kg diet (4.2% crude protein), and 10.1 g/kg diet (3.6% crude protein), respectively, as described in Table 2. Previous studies reported that Nile tilapia require intakes of ~15 , ~18 , and 19 mg of isoleucine, leucine, and valine, respectively, to deposit 1 g of body weight. 3.3.6. Arginine Arginine has received little consideration in Nile tilapia because its requirement is usually met by typical protein feedstuffs in conventional Nile tilapia aquafeeds. It is noteworthy that the optimal arginine requirement is well established in the literature and averages 14.8 g/kg diet (5.1% crude protein), as shown in Table 2. Early works showed that Nile tilapia required an intake of 20-27 mg of arginine to deposit 1 g of body weight . Additionally, a previous study found that arginine at 16.7 g/kg diet stimulated the mRNA expression of myogenic regulatory factors (MRFs), growth hormone (GH), insulin-like growth factors (IGFs) , and consequently hypertrophic muscle processes, supporting enhanced growth of Nile tilapia . Also of note, a previous study found that Nile tilapia fed 23.9 g arginine/kg diet exhibited higher immune responses and survival when challenged by Streptococcus agalactiae . Furthermore, emerging evidence suggests that arginine positively changes intestinal microbiota, activates intestinal fatty acid oxidation, and alleviates triglyceride accumulation in intestinal tissue and intracellular cells of Nile tilapia . Additionally, the beneficial effect of 29 g arginine/kg diet on liver health was reported in Nile tilapia reared under high-density (500 fish/m3; 63 +- 20 g body weight) conditions in floating net cages . On the contrary, the above authors also reported that a higher level of arginine (41 g/kg diet) promoted the incidence of liver necrosis, further supporting the observation that excess arginine may lead to increased plasma ammonia concentration, decreasing the excretion efficiency of this metabolite, as described in Jian carp, Cyprinus carpio var. Jian . However, more extensive research is necessary to investigate the effects of excess arginine on the nitrogen excretion of Nile tilapia. 3.3.7. Histidine Histidine is considered a marginally limiting AA in typical plant-based protein feedstuffs used in Nile tilapia diets . Feed-grade histidine is generally not commercially available for use in Nile tilapia aquafeeds. As shown in Table 2, dietary histidine requirements average 7.3 g/kg diet (3.4% crude protein), and previous studies reported that Nile tilapia require an intake of ~9 mg of histidine to deposit 1 g of body weight. An early work reported the positive effects of adequate histidine supplementation on muscle growth by hypertrophy and hyperplasia in pre-growout Nile tilapia . Another study established that histidine also increased mRNA levels of muscle growth-related genes, myoblast determination protein (MyoD), and myogenin, as well as protein synthesis of growout Nile tilapia . Furthermore, emerging evidence has identified the antioxidant capacity of histidine to improve flesh quality attributes in grass carp , while another study reported influences on fillet quality of growout Nile tilapia . However, few studies have been conducted to evaluate the health status of Nile tilapia in response to dietary histidine supplementation. This approach is of major importance in applying the precision nutrition concept in Nile tilapia operations. 3.3.8. Total Aromatic Amino Acids The academic community has not extensively explored the dietary total aromatic AA (phenylalanine plus tyrosine) requirements of Nile tilapia, possibly because both AAs are not considered marginally limiting in feed ingredients typically used for tilapia. Therefore, feed-grade phenylalanine and tyrosine are not commercially available for diet supplementation. However, it is possible that in low-protein diets, phenylalanine may be a limiting AA for Nile tilapia. Importantly, the total aromatic AA requirements of Nile tilapia can be met by supplementing phenylalanine and also considering phenylalanine and tyrosine in tandem because tyrosine can spare some of the dietary phenylalanine otherwise required for tyrosine synthesis . In this regard, the total aromatic AA requirement is influenced by dietary tyrosine levels. Recent work determined the tyrosine replacement value for phenylalanine on a molar basis to be 37% in Nile tilapia . Another recent study confirmed that adequate phenylalanine supplementation influenced growth rate in nursery Nile tilapia . As displayed in Table 2, the optimal phenylalanine-plus-tyrosine requirement averaged 18.3 g/kg diet (5.9% crude protein). A recent study reported that Nile tilapia require an intake of ~35 mg of phenylalanine plus tyrosine to deposit 1 g of body weight . 3.3.9. Non-Essential Amino Acids Growing evidence suggests that non-essential AAs are closely related to the growth performance, health, and flesh quality of Nile tilapia. Non-essential AAs assume more important roles in fish fed plant-rich diets because of their more limited presence . Additionally, anti-nutritional factors in vegetable ingredients may have adverse effects on AA digestion and absorption and also impair fish health . Emerging evidence has identified that glutamine plays crucial roles in growth and intestinal function and enhances leucocyte function in Nile tilapia . In addition, early studies identified that supplementation of glycine could enhance the antioxidant ability of Nile tilapia and has beneficial effects on growth . Previous research reported a positive association between dietary taurine intake and lipid digestion/absorption with carbohydrate and AA metabolism that promoted enhanced growth performance of Nile tilapia . Along with these beneficial effects, taurine plays an antioxidant role which may have positive effects on flesh quality attributes as well as on reproductive performance . Of note, it has been well established that carnitine plays a central role in regulating the lipid b-oxidation of long-chain fatty acids to produce energy in fish species such as zebrafish, Danio rerio , and this may explain the decreased mesenteric and fillet fat accumulation in growout hybrid tilapia . In addition, carnitine supplementation was found to improve antioxidant functionality in flesh quality attributes and ameliorate or prevent induced liver, intestine, and gill histopathological lesions in Nile tilapia . 4. Conclusions and Implications The environmental impact of fish farming is becoming a major challenge that could warrant restrictions on tilapia production. Higher nitrogen excretion levels into the environment are an increasing issue, and well-balanced aquafeeds have been identified as a potential solution. Of note, most studies have estimated the requirement of one individual AA at a time without considering the interactive effects of other dietary AAs. Therefore, it is important to design AA requirement assays for Nile tilapia that take AA interactions into consideration. A lack of attention to protein synthesis dynamics in non-protein-bound AA diets is important because they are supplemented at relatively higher levels in plant-based diets. Therefore, it is important to investigate the digestive dynamics of proteins as well as protein synthesis while maintaining good health status and flesh quality. Moreover, it is evident that AA nutrition of broodstock needs more attention in terms of the impact of AAs on reproductive performance responses. Interestingly, some new studies have reported that non-essential AAs improve growth and reproductive performance as well as the health of fish. Worthy of note is the fact that emerging evidence shows that genomic approaches constitute an important tool for better understanding the underlying mechanisms of fish growth, behavior, and health in order to estimate the AA requirements of Nile tilapia. However, it is clear that the AA requirements of Nile tilapia might vary as a consequence of numerous experimental conditions, including fish strain, size, culture system, and the basal diet used. Notably, genetic selection in Nile tilapia has improved growth performance, fillet yield, and feed efficiency. Therefore, dietary formulations should consider increased amino acid requirements. Interestingly, the current survey highlighted low variations in lysine recommendations relative to protein content, considering that lysine is used as the reference AA in applying the ideal protein concept. This review also identified that emerging studies considered this concept to determine multiple AA requirements using the deletion method. More comprehensive data in the literature are needed for AA requirements through growout in tilapia production in addition to confirming previously established values for lysine, methionine, and threonine. This review observed variations in AA recommendations for different commercial production parameters such as weight gain, feed efficiency, and fillet yield. Therefore, it is important to choose the most appropriate parameter or combination of parameters that represents the business model of the tilapia industry. Finally, these considerations indicated that well-balanced AA diets might be useful for improving the economic and ecological sustainability of tilapia farming into the future. Acknowledgments The authors are grateful for the support of their corresponding organizations. Author Contributions W.M.F. and T.P.d.C., conceptualization, methodology, software, validation, formal analysis, investigation, resources, and data curation; W.M.F., writing--original draft preparation, writing--review and editing, visualization, supervision, and project administration; D.M.G.III, writing--review and editing. All authors have read and agreed to the published version of the manuscript. Institutional Review Board Statement Not applicable. Informed Consent Statement Not applicable. Data Availability Statement The data presented in this study are available on request from the corresponding author. Conflicts of Interest The authors declare no financial and personal circumstances of interest with other people or organizations that may be perceived as inappropriately influencing the representation or interpretation of data and in the writing of the article. The funder had no role in the design of the study, in the collection, analysis, or interpretation of data, in the writing of the manuscript, or in the decision to publish the results. animals-13-00900-t001_Table 1 Table 1 Amino acid profile (g/100 crude protein) of eggs and whole bodies of Nile tilapia. AA Egg 1 Fry 2 Nursery 3 Pre-growout 4 Growout 5 Mean +- SD EAA Arg 4.7 3.7 4.2 5.4 6.3 4.9 +- 1.0 His 1.9 5.2 2.3 2.1 1.5 2.6 +- 1.5 Ile 2.8 3.2 3.9 3.9 4.3 3.6 +- 0.6 Leu 5.9 6.8 6.0 6.5 7.1 6.5 +- 0.5 Lys 5.8 6.0 7.2 6.7 7.4 6.6 +- 0.7 Met 2.9 5.3 2.0 2.3 2.5 3.0 +- 1.3 Phe 2.2 3.5 3.2 3.7 3.5 3.2 +- 0.6 Thr 4.5 3.7 3.3 3.9 4.1 3.9 +- 0.4 Trp n.d. n.d. 0.9 0.9 0.8 0.9 +- 0.1 Val 4.4 4.4 4.4 4.6 4.7 4.5 +- 0.1 NEAA Ala 8.4 7.1 5.6 6.1 7.0 6.8+- 1.1 Asp 8.1 7.0. 8.2 8.2 8.1 8.2 +- 0.1 Cys n.d. n.d. 0.9 0.7 0.7 0.8 +- 0.1 Glu 9.4 11.8 12.1 12.0 14.5 12.0 +- 1.8 Gly 4.3 n.d. 6.8 7.3 9.5 7.0 +- 2.1 Pro 7.2 7.2 n.d n.d n.d 7.2 +-0.0 Ser 8.8 5.2 3.2 3.6 3.6 4.9 +- 2.3 Tyr 2.7 3.4 2.9 3.0 2.8 3.0 +- 0.3 Abbreviations: EAA, essential amino acid; NEAA, non-essential amino acid; n.d., non-determined; 1 Egg composition of Nile tilapia females fed a 350 g/kg crude protein diet ; 2 Yolk-sac resorbed Nile tilapia fry of 12 mg body weight ; 3 Body weight of ~1 g ; 4 Body weight of ~44 g ; 5 Body weight of ~829 g . animals-13-00900-t002_Table 2 Table 2 Amino acid recommendations for Nile tilapia. Amino Acid Fish Stage Dietary Requirement Response P:E Reference g/kg Diet (DM) % Protein Arg 1 11.8 4.2 WG 26.8 1 18.2 6.2 WG n.p. 1 13.6 4.9 WG 21.4 2 16.7 5.2 WG 18.5 2 13.7 4.9 NR 16.1 Mean +- SD 14.8 +- 2.6 5.1 +- 0.7 18.7 +- 2.7 His 1 4.8 1.7 WG 26.8 2 4.8 1.8 NR 16.1 2 8.2 3.1 WG 15.4 3 8.8 2.8 WG 19.4 Mean +- SD 6.7 +- 2.2 2.4 +- 0.7 19.4 +- 5.2 Ile 1 8.7 3.1 WG 26.8 2 9.1 3.3 NR 16.1 1 13.7 5.0 WG 19.6 Mean +- SD 10.5 +- 2.8 3.8 +- 1.0 20.8 +- 5.5 Leu 1 9.5 0.34 WG 26.8 2 13.5 0.48 NR 16.1 1 12.5 0.43 WG n.p. Mean +- SD 11.8 +- 2.1 4.2 +- 007 21.5 +- 7.6 Lys 1 14.3 5.1 WG 26.8 2 16.5 5.9 NR 16.1 3 15.1 6.0 WG 19.2 2 18.0 5.6 WG 18.5 Mean +- SD 16.0 +- 1.6 5.7 +- 0.4 20.2 +- 4.6 Met 1 7.5 2.7 WG 26.8 3 6.8 2.3 WG 20.6 1 9.1 3.2 WG n.p. 1 8.1 2.9 WG 19.1 Mean +- SD 7.9 +- 1.0 2.8 +- 0.4 22.2 +- 4.1 Met +Cys 1 9.0 3.2 WG 26.8 3 11.2 3.8 WG, FY 20.6 1 10.0 3.5 WG n.p. 1 8.5 3.0 WG 19.1 Mean +- SD 9.7 +- 1.2 3.4 +- 0.4 22.2 +- 4.1 Phe 1 10.5 3.8 WG 26.8 1 1 8.8 3.0 WG 21.4 1 12.1 3.5 WG 20.9 Mean +- SD 10.5 +- 1.2 3.4 +- 0.4 23.0 +- 3.3 Phe + Tyr 1 15.5 5.6 WG 26.8 1 1 18.6 6.4 WG 1 20.6 5.9 WG Mean +- SD 18.2 +- 2.6 6.0 +- 0.4 23.0 +- 3.3 Thr 1 10.5 3.8 WG 26.8 1 13.3 4.7 WG 2 13.5 4.8 NR 16.1 3 11.5 4.0 WG, FY 22.7 Mean +- SD 12.2 +- 1.4 4.3 +- 0.5 21.9 +- 5.4 Trp 1 2.8 1.0 WG 26.8 2 2.4 0.9 NR 16.1 1 3.4 1.1 WG 19.3 1 3.8 1.2 WG 18.5 1 3.1 1.0 WG n.p. Mean +- SD 3.1 +- 0.5 1.0 +- 0.1 20.2 +- 4.6 Val 1 7.8 2.8 WG 26.8 2 9.7 3.5 NR 16.1 1 12.7 4.5 WG 17.4 Mean +- SD 10.1 +- 2.5 3.6 +- 0.9 20.1 +- 5.8 Abbreviations: SD, standard deviation; DM, dry matter; WG, weight gain; NR, nitrogen retention; FY, fillet yield; P:E, protein-to-energy ratio (g/MJ gross or digestible energy); n.p., non-presented; 1 Hybrid tilapia, Oreochromis niloticus x Oreochromis aureus. animals-13-00900-t003_Table 3 Table 3 Dietary amino acid recommendation (g/kg diet) for Nile tilapia 1. Amino Acid Production Stage 2 Fry Nursery/Pre-Growout Growout Crude Protein (g/kg Diet) 3 460 350 320 Arg 23.5 17.9 16.3 His 11.0 8.4 7.7 Ile 17.5 13.3 12.2 Leu 19.3 14.7 13.4 Lys 26.2 20.0 18.2 Met 12.9 9.8 9.0 Met + Cys 15.6 11.9 10.9 Phe 15.6 11.9 10.9 Phe + Tyr 27.6 21.0 19.2 Thr 19.8 15.1 13.8 Trp 0.5 0.4 0.3 Val 16.1 12.3 11.2 1 Each amino acid is estimated relative to crude protein (mean value) displayed in Table 2; 2 Previously established for Nile tilapia as nursery (1.6 to 30 g of body weight), pre-growout (31 to <=220 g of body weight), and growout (>220 g of body weight) ; 3 Mean values previously established for Nile tilapia fry , nursery and pre-growout , and growout Nile tilapia . animals-13-00900-t004_Table 4 Table 4 Dietary amino acid recommendations (g/kg diet) for broodstock Nile tilapia 1. Amino Acid Crude Protein (g/kg Diet; Dry Matter) 350 2 380 3 400 4 Arg 17.9 19.4 20.4 His 8.4 9.1 9.6 Ile 13.3 14.4 15.2 Leu 14.7 16.0 16.8 Lys 20.0 21.7 22.8 Met 9.8 10.6 11.2 Met + Cys 11.9 12.9 13.6 Phe 11.9 12.9 13.6 Phe + Tyr 21.0 22.8 24.0 Thr 15.1 16.3 17.2 Trp 0.4 0.4 0.4 Val 12.3 13.3 14.0 1 Each amino acid is estimated relative to crude protein (mean value) displayed in Table 2; 2 Broodstock raised in earthen pond ; 3 Broodstock raised in recycling system ; 4 Broodstock raised in water salinity up to 14%0 . animals-13-00900-t005_Table 5 Table 5 Dietary amino acid profile (% of lysine) based on the ideal protein concept for Nile tilapia. Amino Acid Fish Production Stage 1 Mean +- SD Nursery 2 Pre-Growout 2 Growout 2 Lysine 100 100 100 10 0 +- 0 Arginine 86 125 81 97 +- 24 Histidine 30 34 34 33 +- 2 Isoleucine 56 57 51 55 +- 3 Leucine 84 96 66 82 +- 15 Methionine 41 64 3 41 3 49 +- 13 Phenylalanine 64 101 4 70 4 78 +- 20 Threonine 103 93 89 95 +- 7 Tryptophan 16 24 23 21 +- 4 Valine 60 76 73 70 +- 9 Abbreviation: SD, standard deviation; 1 Previously established for Nile tilapia as nursery (1.6 to 30 g of body weight), pre-growout (31 to <=220 g of body weight), and growout (>220 g of body weight) ; 2 Data established for nursery , pre-growout , and growout Nile tilapia by deletion method; 3 Methionine plus cystine; 4 Phenylanaline plus tyrosine. Disclaimer/Publisher's Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

PMC10000144

Passerine nestlings frequently suffer from sub-optimal food conditions due to climate change-induced trophic mismatch between the nestlings and their optimal food resources. The ability of nestlings to buffer this challenge is less well understood. We hypothesized that poor food conditions might induce a higher immune response and lower growth rate of nestlings, and such physiological plasticity is conducive to nestling survival. To test this, we examined how food (grasshopper nymphs) abundance affects the expression of interferon-g (IFN-g), tumor necrosis factor-a (TNF-a), interleukin-1 b (IL-1b) genes, plasma IGF-1 levels, body mass, and fledging rates in wild Asian short-toed lark (Alaudala cheleensis) nestlings. Linear mixed models revealed that nymph biomass significantly influenced the expression of IFN-g, TNF-a, and IL-1b genes, and the level of plasma IGF-1. The expressions of IFN-g, TNF-a, and IL-1b genes were negatively correlated with nymph biomass and plasma IGF-1 level. Plasma IGF-1 level, nestling body mass growth rate, was positively correlated with nymph biomass. Despite a positive correlation between the nestling fledge rate and nymph biomass, more than 60% of nestlings fledged when nymph biomass was at the lowest level. These results suggest that immunity and growth plasticity of nestlings may be an adaptation for birds to buffer the negative effects of trophic mismatch. nestling immunity growth trophic mismatch physiological plasticity National Natural Science Foundation of China31872246 32071515 This research was funded by the National Natural Science Foundation of China, grant numbers 31872246 and 32071515. pmc1. Introduction In the middle and high latitudes of the Northern Hemisphere, the nestling growth period of passerines must be synchronized with the peak abundance of insect larva prey to maximize nestling growth and survival . Climate change-induced spring temperature increases can advance the timing of insect development , whereas the timing of breeding in birds is regulated more by photoperiod than temperature , resulting in a trophic mismatch between nestlings and insect larvae . Nestlings hatching outside the peak period of insect larvae abundance may suffer malnutrition, posing a significant challenge to nestling survival . Although adults increase their feeding efforts to satisfy nestling nutritional requirements , little is known about how nestlings cope with the stress caused by trophic mismatch. Whether nestlings that hatch during periods when food is less abundant buffer such nutritional challenges using their own resources remains unknown. Understanding how nestlings cope with trophic mismatch may provide insight into the ability of birds to adapt to climate change. Phenotypic plasticity is the ability of organisms to change gene expression in response to short-term environmental challenges . Several food-controlled experiments have demonstrated physiological plasticity in nestlings in response to changes in food conditions . For example, when food is insufficient, House sparrow (Paaser domesticus) nestlings can prioritize bone growth, whereas Zebra finch (Taeniopygia guttata) nestlings can adjust their growth rate during different developmental stages . Therefore, the physiological plasticity in nestlings could be an important mechanism for coping with sub-optimal food conditions. When food is scarce, the limited resources of nestlings must be allocated between growth and survival . The immune system is critical for the survival of animals because it serves as a defense mechanism against pathogenic organisms . Pathogen infection experiments indicate that maintaining immune function consumes a significant amount of nutrients and energy, which can inhibit growth . Other studies suggest that short periods of poor nutrition enhance nestling immunity . These findings suggest that temporary food shortages may activate immune response while reducing growth to ensure the short-term survival of nestlings. Immune cytokines and insulin-like growth factor-1 (IGF-1) are important immune and growth factors in birds. Immune cytokines are small soluble protein molecules produced by immune cells such as T lymphocytes and macrophages in response to immunogen or other stimuli . Studies in humans have shown that inadequate nutrition can induce macrophages to produce immune cytokines . Interleukin (IL), interferon (IFN), and the tumor necrosis factor (TNF) superfamily are the primary cytokines. For example, IL-1 and TNF-a can activate T and B lymphocytes, stimulating the production and release of antibodies , whereas IFN can stimulate peripheral cells to inhibit virus replication . Insulin-like growth factor 1 (IGF-1) is an important indicator reflecting bird growth . For example, plasma IGF-1 level was positively correlated with the nestling growth rate of wild Great tits (Parus major) . IGF-1 activates protein synthesis by changing gene transcription levels and increases muscle mass by promoting cell growth and differentiation . An increase of immune cytokines, especially IL-1, can activate the hypothalamic-pituitary-adrenal axis, leading to the secretion of corticosterone, which has been known to inhibit the activity of IGF-1 . Therefore, immune cytokines inhibit the activity of IGF-1. This physiological pathway could explain why maintaining immunity may slow down growth. Individual variation in hatching dates frequently occurs in passerine bird populations . Variation in hatching dates is considered an ecological benefit because it is a way of partitioning niches along the time axis to avoid competition for breeding resources within a population . While most adults tend to synchronize the nestling hatch date with the insect larva peak, some nestlings hatch outside the peak period of insect abundance . This natural variation among individuals provides a model for evaluating the ability of nestlings to cope with food challenges. The Asian short-toed lark (Alaudala cheleensis) is the dominant passerine of the Hulun-Beir grassland in Inner Mongolia, China. Asian short-toed larks exhibit significant within-population variation in the timing of breeding, as is typical of passerines. Most individuals begin egg-laying in mid-May, while others start later in late April or May . Asian short-toed lark nestlings are predominantly fed grasshopper nymphs (Orthoptera) . The proportion of nymphs in the diet of nestlings hatching outside the period of peak grasshopper nymph abundance was significantly lower than that of nestlings hatching within the period of peak grasshopper nymph abundance. The proportion of beetles and grass seeds in nestlings' diets increased with decreasing nymph abundance. Moreover, the nutrients and energy that nestlings obtained from alternative diets were significantly lower than that from grasshopper nymphs. Plasma glucides, amino acids, tricarboxylic acid (TCA) cycle metabolites, and some fatty acids of the nestlings hatched under medium or low nymph abundance conditions were significantly lower than those of nestlings hatched under high nymph abundance conditions . We found that Asian short-toed larks in Hulun-Beir, Inner Mongolia, are affected by climate change-induced trophic mismatch, where abnormally high or low spring temperatures in some years caused significant asynchrony between peak hatching and peak grasshopper nymph abundance . The peaks of nymph abundance in 2014 and 2016 occurred 12 days earlier and later than in 2015, respectively, which resulted in a complete mismatch between the hatching peak and the nymph peak period in these two years . As a result of this "trophic mismatch", most nestlings hatched outside the peak period of grasshopper nymph abundance and suffered poor food conditions. Therefore, Asian short-toed larks are a suitable case species for investigating the capacity of nestlings to respond to food challenges. In this article, using the Asian short-toed lark population as a model, we hypothesize that poor food conditions may induce a higher immune response and lower growth rate of nestlings, and such physiological plasticity is conducive to nestling survival. To test this hypothesis, we examined IFN-g, TNF-a, and IL-1b expression, plasma IGF-1, body mass, and fledge rate per nest in a field population of Asian short-toed lark nestlings that hatched during different periods of grasshopper nymph abundance and analyzed the relationships between these physiological and ecological indicators. 2. Materials and Methods 2.1. Study Site The study area is located in the Hulun Lake National Nature Reserve (47deg45'50'' N-49deg20'20'' N, 116deg50'10'' E-118deg10'10'' E) in the northeastern part of the Inner Mongolian Autonomous Region, China. This is a semiarid, steppe region that has long, severe winters and short summers. The mean annual temperature, precipitation, and potential evaporation are -0.6 degC, 283, and 1754 mm, respectively. The dominant plant species are Stipa krylovii, Leymus chinesis, and Cleistogenes squarrosa. 2.2. Data Collection We monitored Asian short-toed lark nests in the study area daily from 15 April to 15 June 2019, recording nestling hatching dates, body mass, age, brood size, and the number of nestlings that survived to fledgling. Fledge rate of nestlings is the percentage of a fledgling number relative to the total nestling number hatched in one day, excluding the preyed nestlings. The relative abundance of grasshopper nymphs in the study area was quantified by catching these in an insect net on 10 parallel, 2 m x 100 m sampling transects, spaced 10 m apart, daily. Captured nymphs were dried in a drying oven at 70 degC for 24 h and weighed to determine their biomass. The mean daily nymph biomass was the average daily biomass obtained from all 10 transects. We used "nymph biomass proportion" (NBP), the proportion of the daily nymph biomass relative to the total biomass measured on all survey days, as a measure of daily grasshopper nymph abundance. Totally 135 nestlings were recorded. Asian short-toed lark nestlings can fly 8 days after hatching. We use the NBP on the fourth day after sample nestlings hatched as an indicator of the quality and quantity of food available to that nestling. Daily NBP and the number of newborn nestlings are shown in Figure 1. The daily mean ambient temperature during the experiment varied from 7.54 to 17.39 degC. Because variation in the mean ambient temperature during different periods of the study may also have affected the immune status of nestlings , we also measured the nest temperature daily at 6:00 am with a FLIR C2 infrared thermometer when nestlings were four days old. 2.3. Nestling Blood Samples A 100 uL peripheral blood sample was collected from the first hatched four-day-old nestling in each nest at 6:00 am. The brachial wing vein of each nestling was punctured with a disinfected 23 G needle within 1-3 min of capture, and blood exuding from the puncture site was collected into heparinized microcapillary tubes. The skin around the puncture site was disinfected with medical alcohol before and after puncturing. Pressure was applied to the puncture site for 1 min with an alcohol-soaked cotton wool swab to staunch bleeding. Blood samples were centrifuged at 4000 r/min for 20 min to separate the plasma and blood cells. The resultant plasma and blood cells were snap-frozen in liquid nitrogen and stored at -80 degC. 2.4. Cytokine Gene Expression Analysis The total RNA of blood cell samples was isolated from 1 mL TRIzol reagent in 200 mL chloroform, centrifuged at 12,000 r/min for 5 min at 4 degC, after which about 200 mL of the supernatant was transferred to a clean RNase-free centrifuge tube. 200 mL chloroform was then added to the tube, which was centrifuged at 12,000 r/min for 15 min at 4 degC. 150 mL of the supernatant was then transferred to a clean RNase-free centrifuge tube to which 150 mL isopropanol was added and the tube centrifuged at 12,000 r/min for 15 min at 4 degC. The resultant RNA pellet was washed with 400 mL 75% ethanol (dissolved in DEPC water) two times, briefly air dried for 5 min, and dissolved in 30 mL DEPC water. Total RNA quality and quantity were evaluated using agarose gel electrophoresis (AGE) and a NanoDrop 2000 spectrophotometer. After validation, 2 mg of the total RNA sample was reversely transcribed to cDNA using M-MLV Reverse Transcriptase with oligo dT primers. The cDNA was used as a template for RT-qPCR. Primers were designed based on IFN-g, TNF-a, and IL-1b mRNA sequences in NCBI using Primer 5 software (Table 1). The RT-qPCR protocol was as follows; pre-degeneration at 95 degC for 1 min, degeneration at 95 degC for 15 s, annealing at 59 degC for 15 s and extension at 72 degC for 40 s, for a total of 40 cycles. After the reaction, a melting curve analysis from 55 to 95 degC was applied to ensure the consistency and specificity of the amplified product. The GAPDH expression was confirmed stable under all treatments, and this gene was consequently used as the reference gene to normalize mRNA levels among samples. RT-qPCR was performed twice in triplicate. The values of the average cycle threshold (Ct) were determined, and Ct scores for gene transcripts in each sample were normalized using the DCt scores for GAPDH and expressed as the fold change in gene expression using the equation 2-DDCT. 2.5. Plasma IGF-1 Analysis IGF-1 concentration was measured using enzyme immunoassay kits from Enzo Life Sciences (New York, NY, USA). Bound IGF-1 was separated from the binding protein with 12.5% 2 mol/L HCl and 87.5% ethanol solution according to the methods in Sparkman et al. . The kits have been validated for the Asian short-toed lark by serial plasma dilutions. All serum samples were 1:5 diluted (10 mL of the sample and 40 mL of the sample dilution) and added to sample wells in triplicate. The respective intra-plate coefficients of variation were 5.2% and 7.7%. 2.6. Statistical Analysis The effects of nymph biomass, nest temperature, and their interaction on the plasma IGF-1 levels, IFN-g, TNF-a, and IL-1b gene expression levels in the blood cells of nestlings were analyzed with linear mixed models (LMMs). Regression analysis is used to analyze the relationship between nymph biomass and plasma IGF-1, the expression of cytokine genes, body mass, and survival rate per nest. p-values < 0.05 were considered significant. The LMM analyses were performed in SPSS version 24 (SPSS Inc., Chicago, IL, USA), and the regression analyses were performed in R (version 3.6.2). 3. Results 3.1. Blood Cell Cytokine Gene Expression of Nestlings The LMMs of the effects of NBP and nest temperature on IFN-g, TNF-a, and IL-1b gene expression indicate that only NBP significantly influenced the expression of all three genes (Table 2). Quadratic polynomial regression indicates a significant, negative correlation between NBP and IFN-g, TNF-a and IL-1b gene expression . 3.2. Plasma IGF-1 Levels and Body Mass of Nestlings The LMMs of the effects of NBP and nest temperature on nestling plasma IGF-1 concentration indicate that NBP significantly influenced the level of IGF-1 while temperature did not (Table 2). Quadratic polynomial regression indicates a significant positive correlation between NBP and plasma IGF-1 concentration . The body mass of one to seven-day-old nestlings increased with NBP increase and approximated a cubic polynomial regression curve . We categorized NBP as high (NBP>5%), medium (2% < NBP <= 5%), and low (NBP <= 2%) based on Figure 1 and assigned nestlings to these groups. The body mass-age relationships of the three groups of nestlings approximated logistic regression curves . The daily mean slope of the curve (growth rate) was 1.49, 1.72, and 2.11 in the NBP > 5%, 2% < NBP <= 5%, and NBP <= 2% groups, respectively . 3.3. Correlation between Blood Cell Cytokine Gene Expression and Plasma IGF-1 of Nestlings Quadratic polynomial regression indicates that there was a significant, negative correlation between plasma IGF-1 concentration and blood cell IFN-g, TNF-a and IL-1b gene expression . 3.4. Fledge Rate of Nestlings Quadratic polynomial regression shows that the fledge rates of nestlings increased with NBP increase (R2 = 0.98, p < 0.001), and the lowest fledge rate was more than 60% . 4. Discussion The expression of IFN-g, TNF-a, and IL-1 b genes was negatively correlated with nymph biomass, indicating that the immune systems of nestlings were activated by stimulating immune cells to release cytokines when food conditions were limited. At the same time, there was a significant positive correlation between plasma IGF-1 level and NBP, indicating that the secretion of IGF-1 of nestlings was inhibited when food conditions were limited. The negative correlations between the three kinds of cytokines and IGF-1 indicate that, as found in available studies , a higher immune response may inhibit the secretion of IGF-1 in nestlings. These results confirm that poor food conditions can induce a higher immune response, which may negatively influence the growth of nestlings. Some available studies in humans have shown that malnutrition can stimulate macrophages to produce cytokines . When the energy intake is low, glycolysis, the first stage of the glucose decomposition process, is enhanced to meet energy demands. Studies have shown that increasing glycolytic enzymes, such as pyruvate kinase 2 and glyceraldehyde-3-phosphate dehydrogenase, can induce macrophages to increase the release of IL-1 and TNF-a . Therefore, poor nutrition may increase the expression of immune cytokine genes in nestlings by increasing glycolysis. In addition, the secretion of IGF-1 is also closely related to the energy level of the cells; low energy levels can inhibit IGF-1 secretion by activating the AMPK pathway . In agreement with our results, some experimental studies have also shown that nestlings can maintain immunity and reduce tissue growth during short-term food restriction. The authors interpreted these observations as showing that the energy required for maintaining normal immunity is minor compared with the energy required for growth . Unlike previous studies, our study tested the gene expression of immune cytokines, signaling molecules that activate the immune system . The gene expression of IFN-g, TNF-a, and IL-1 b increased in nestlings under poor food conditions, indicating that more nestling energy was used in the immune response rather than the immune system needing less energy. Therefore, the results of immune cytokines and IGF-1 levels showed that the energy required for the growth of nestlings was insufficient in poor food conditions, and the energy required for maintaining immune function might be given priority. The relationship between the body mass of nestlings and NBP and the growth rate difference among different NBP groups implies that the lower IGF-1 level retards the growth of nestlings. In contrast, the results show that nestling survival rates positively correlated with NBP, indicating that poor food conditions cause higher nestling mortality. Nonetheless, the fact that up to 60% of nestlings could fledge even when NBP was at its lowest indicates that most of the nestlings in poor food conditions can survive despite a relatively light body mass. These results, together with the cytokine results, suggest that poor food conditions induced higher immune response might ensure most of the nestlings survive in poor food conditions despite a lighter body mass, thus partially buffering the effect of trophic mismatch. However, such physiological plasticity may only work in the short term because prolonged food shortage caused by severe trophic mismatch will lead to a decline in immune function due to a lack of protein, energy, and other essential nutrients . In other words, the physiological plasticity of nestlings appears insufficient to buffer the severe food shortage induced by trophic mismatch. Under the climate change scenario, extreme inclement weather occurs frequently and is seasonally heterogeneous . For example, a long-term study found that a cold spring could seriously reduce the abundance of insect larvae, reducing the survival rate of a migrating bird species by more than 50% . According to our previous study, the Asian short-toed larks in our study area have experienced severe trophic mismatch induced by warm and cold springs in 2014 and 2016, in which the peak of nymph abundance was 6 days earlier and later than the peak hatchings in 2014 and 2016, respectively . Although the mismatch days are the same between the two years, the nestling survival rate in 2016 is lower than in 2014. These facts suggest that extreme weather may lead to a more severe food shortage for the nestlings, which may not be mitigated by the physiological plasticity of nestlings. Inter-sibling competition is a possible factor affecting the survival of nestlings in poor food conditions. Limited food causes increased inter-sibling competition manifesting in increased begging behavior, which means that individuals in the same nest may receive different amounts of food and consequently differ in nutritional status . The nestlings with weak competitiveness may be unable to obtain essential energy and nutrients to survive. Therefore, although the plasticity of immunity and growth may maintain the short-term survival of some nestlings, it cannot guarantee the survival of severely malnourished individuals. Some available studies on other bird species might support this deduction. The studies on Barn swallows (Hirundo rustica) and Marsh harriers (Circus aeruginosus) showed the inter-sibling immune response difference . Another study on the House sparrow (Passer domesticus) showed that nestlings that received less food from parents showed a weaker immune response . More studies on the physiological and begging behavior differences among siblings of Asian short-toed larks need to be done in the future. Similar to our body mass result, some studies have found a reduction in body size due to trophic mismatch-induced malnutrition . The ecological consequence of this morphological variation is species-specific. A study on the Blue-footed booby (Sula nebouxii) showed that the body mass of nestlings decreased when they suffered trophic mismatch, but there was no significant decrease in longevity and reproductive performance in smaller fledglings . On the contrary, a study on a long-distance migrant species, Red knot (Calidris canutus), found that the shorter bills of smaller offspring have consequences at their tropical wintering grounds where shorter-billed individuals have reduced survival rates because their bills couldn't reach deeply buried Loripes . Therefore, the plasticity of immunity and growth may only be a short-term response of nestlings, which could benefit the short-term survival of nestlings suffering trophic mismatch. Due to the different environmental conditions and life history of different species, the long-term fitness consequence of smaller body size needs species-specific research. 5. Conclusions Our results show that a poor food condition stimulates the release of cytokines and inhibits the secretion of IGF-1, reducing the body mass of nestlings. These results indicate that nestlings have immunity and growth plasticity when food is limited. The fact that more than 60% of nestlings could survive even when NBP was at its lowest indicates that the plasticity of immunity and growth may make most of the nestlings survive in poor food conditions, thus partially buffering the effect of climate change-induced trophic mismatch. Such plasticity could only benefit the short-term survival of nestlings suffering trophic mismatch. At the same time, long-term fitness consequence of smaller body size needs species-specific research at different adult life history stages. Acknowledgments We are grateful to Muren Wu, Songtao Liu, and Huashan Dou in the Hulun Lake National Nature Reserve for their help in the field study. Author Contributions Conceptualization, S.Z.; methodology, G.L. and X.L.; investigation, X.Z. and G.L.; writing--original draft preparation, X.Z. and G.L.; writing--review and editing, G.L.; supervision, S.Z.; project administration, S.Z.; funding acquisition, S.Z. All authors have read and agreed to the published version of the manuscript. Institutional Review Board Statement The animal study protocol was approved by the Hulun Lake National Reserve Administration and the biological and Medical Committee of the Minzu University of China (Permission code: 2020021AA). Informed Consent Statement Not applicable. Data Availability Statement The data used in the present study are available from the corresponding author upon reasonable request. Conflicts of Interest The authors declare no conflict of interest. Figure 1 Daily nymph biomass proportion (NBP) and the number of newborn Asian short-toed lark (Alaudala cheleensis) nestlings. NBP is the proportion of the daily grasshopper nymph biomass relative to the total biomass measured on all survey days. Figure 2 The relationship between grasshopper nymph biomass proportion (NBP) and the relative expression of the interferon-g (IFN-g) (a), tumor necrosis factor-a (TNF-a) (b), interleukin-1b (IL-1b) (c) genes of Asian short-toed lark (Alaudala cheleensis) nestlings (n = 45). NBP is the proportion of the daily grasshopper nymph biomass relative to the total biomass measured on all survey days. The blue area is the confidence interval. Figure 3 The relationship between nymph biomass (NBP) and plasma insulin-like growth factor-1 (IGF-1) concentrations of Asian short-toed lark (Alaudala cheleensis) nestlings (n = 45). NBP is the proportion of the daily grasshopper nymph biomass relative to the total biomass measured on all survey days. The blue area is the confidence interval. Figure 4 The relationship between nymph biomass proportion (NBP) and body mass of Asian short-toed lark (Alaudala cheleensis) nestlings (n = 45). 1d, 2d, 3d, 4d, 5d, 6d, and 7d indicates the age of nestlings in days. NBP is the proportion of the daily grasshopper nymph biomass relative to the total biomass measured on all survey days. Figure 5 The relationship between body mass and age (days after birth) of Asian short-toed lark (Alaudala cheleensis) nestlings (n = 45). NBP is the proportion of the daily grasshopper nymph biomass relative to the total biomass measured on all survey days. The shaded area is the confidence interval. Figure 6 The relationship between plasma IGF-1 concentration and relative expression of the interferon-g (IFN-g) (a), tumor necrosis factor-a (TNF-a), (b) interleukin-1b (IL-1b), (c) genes of Asian short-toed lark (Alaudala cheleensis) nestlings (n = 45). The blue area is the confidence interval. Figure 7 The relationship between nymph biomass proportion (NBP) and fledge rate of Asian short-toed lark (Alaudala cheleensis) nestlings (n = 45). NBP is the proportion of the daily grasshopper nymph biomass relative to the total biomass measured on all survey days. The blue area is the confidence interval. animals-13-00860-t001_Table 1 Table 1 Primer sequences and anticipated size of amplified products. Gene Forward Primer Size (bp) Accession No. TNF-a F:5-CCGCCCAGTTCAGATGAGTT-3 R:5-GCAACAACCAGCTATGCACC-3 130 MF000729.1 IFN-g F:5-TGAGCCAGATTGTTTCGATG-3 R: 5-CTTGGCCAGGTCCATGATA-3 248 NM_205149.1 IL-1b F:5-ACTGGGCATCAAGGGCTACA-3 R:5-GCTGTCCAGGCGGTAGAAGA-3 142 NM_204524.1 GAPDH F:5-CACTGTCAAGGCTGAGAACG-3 R:5-GATAACACGCTTAGCACCA-3 187 NM_204305.1 animals-13-00860-t002_Table 2 Table 2 Results of linear mixed models of the effects of nymph biomass proportion (NBP) and nest temperature on the expression of the interferon g (IFN-g), tumor necrosis factor-a (TNF-a), interleukin-1b (IL-1b) genes and the plasma insulin-like growth factor-1 (IGF-1) of Asian short-toed lark (Alaudala cheleensis) nestlings. NBP is the proportion of the daily grasshopper nymph biomass relative to the total biomass measured on all survey days. Response Variable Explanatory Variable F p IFN-g NBP 64.015 <0.001 Nest temperature 1.004 0.323 NBP x Nest temperature 0.262 0.611 TNF-a NBP 59.585 <0.001 Nest temperature 3.343 0.075 NBP x Nest temperature 0.049 0.826 IL-1b NBP 53.372 <0.001 Nest temperature 3.584 0.066 NBP x Nest temperature 2.578 0.117 IGF-1 NBP 20.675 <0.001 Nest temperature 2.342 0.134 NBP x Nest temperature 0.001 0.970 Disclaimer/Publisher's Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

PMC10000145

Preference and performance trials were conducted to investigate the effects of extruded corn with different degrees of gelatinization on the feed preference, growth performance, nutrient digestibility, and fecal microbiota of weaning piglets. In the preference trial, 144 piglets who were 35 days old were weighed and allotted to six treatments with four replications per treatment. Piglets in each treatment group were allowed to choose two of the following four corn-supplemented diets: conventional corn (NC) or extruded corn with low (LEC; 41.82% gelatinization), medium (MEC; 62.60% gelatinization), or high (HEC; 89.93% gelatinization) degrees of gelatinization for 18 days. The results showed that the piglets preferred diets supplemented with a low degree of gelatinization of extruded corn. In the performance trial, 144 piglets who were 35 days old were weighed and allotted into four treatments with six replications per treatment. Piglets in each treatment were fed one of the four diets for 28 days. The results showed that LEC and MEC decreased the feed:gain ratio at 14-28 days and 0-28 days, respectively, and increased the apparent total tract digestibility (ATTD) of crude protein compared with NC. Meanwhile, LEC increased the total protein and globulin content in the plasma on day 14, and MEC increased the ATTD of ether extract (EE) compared with NC. Extruded corn with low and medium degrees of gelatinization increased the abundance of Bacteroidetes at the phylum level and Lactobacillus, Alloprevotella, Prevotellaceae_UCG-03, and Prevotella_2 at the genus level. The results showed that extruded corn can improve feed preference, increase growth performance and nutrient digestibility, and modify gut microbiota, and the ideal degree of gelatinization is approximately 41.82-62.60%. extrusion corn gelatinization choice feeding palatability growth rate fecal microbiota piglets Key Research Project of Zhejiang province2021C02007 2022C02043 This study was supported by grants from the Key Research Project of Zhejiang province (Grant No. 2021C02007 and 2022C02043). pmc1. Introduction Weaning stress is normally associated with decreased feed intake and growth performance due to abrupt changes in the piglet's environment and diet . It takes up to three weeks for piglets to re-establish their pre-weaning levels of energy intake . Thus, feed with high digestibility and good palatability is crucial to ensure a fast initiation of feeding after weaning and maintain a continuous supply of energy. The nutritional improvement of corn is of interest to producers because corn plays an important role in the piglet diet because it is rich in starch and a major source of energy. Extrusion is a thermal and mechanical process that results in several starch chemical changes, including changes to the crystalline structure and gelatinization degree that could increase enzyme susceptibility and flavor . Therefore, corn extrusion may be an effective way to improve nutrition utilization and palatability in the piglet diet. Many experiments have focused on the effect of gelatinized vs. non-gelatinized corn or its supplementary ratio, yet few results have been published regarding the appropriate gelatinization degree. Different gelatinization degrees of extruded corn may result in different fermentable substrates in the hindgut which change the gut microbiota. Further, starch with different gelatinization degrees would change pellet properties, such as hardness and water absorption indexes, which may affect piglets' chew work and thus influence palatability . Our hypothesis is that the composition of gut microbiota and feed palatability may vary with changes in gelatinization degrees. We noticed that findings in previous studies were inconsistent. Kotara et al., found that the average daily gain (ADG), average daily feed intake (ADFI), and nutrient digestibility increased linearly when the gelatinization degree increased from 23.8% to 81.9% . In contrast, Hongtrakul et al., showed a quadratic decrease in ADG and ADFI and a quadratic increase in the digestibility of dry matter (DM), nitrogen, and gross energy (GE) when the gelatinization degree increased from 14.5% to 89.3% . Therefore, in the present study, we aimed to quantify the palatability of extruded corn with different degrees of gelatinization using a two-way choice-preference trial. The impact of extruded corn with different gelatinization degrees on the growth performance, nutrient digestibility, and fecal microbiota of piglets was also studied in a performance trial. 2. Material and Methods 2.1. Extruded Corn All experimental materials of corn from northeast China were selected from a single patch. Corn was grounded using a hammer mill with a 2.0 mm screen and then extruded using a single-screw extruder (EXT-200S, Beijing Modern Yanggong Machinery S&T Development Co., Ltd., Beijing, China). To obtain low, medium, and high gelatinization degrees of the extruded corn, the samples were steam-cooked at 105 degC for 60 s, 80 s, and 100 s with 17%, 17.5%, and 17.5% moisture content and then extruded at 100 degC, 110 degC, and 120 degC for 5 s, respectively. After this, the corn samples were cooled using a counter-flow cooling procedure and ground though a 1 mm screen to generate mashed corn. 2.2. Experiment 1: Preference Trial 2.2.1. Experimental Animals, Design, Diet, and Feeding Management A total of 144 piglets (35 days old; 72 barrows and 72 gilts; Duroc x Large White x Landrace) with 9.45 +- 0.76 kg body weight (BW) were used to perform an 18-day two-way choice test. The piglets were allotted to six dietary treatments with four replicates per treatment and 6 piglets per replicate (including three barrows and three gilts). The four diets used in the experiment included a basal diet containing 52.5% conventional corn (NC, 12.65% gelatinization) and three extruded corn diets containing 12.5% conventional corn and 40% extruded corn with low (LEC; 41.82% gelatinization), medium (MEC; 62.60% gelatinization), and high (HEC; 89.93% gelatinization) degrees of gelatinization. All four diets were tested in pairs to form six treatments: (i) NC vs. LEC, (ii) NC vs. MEC, (iii) NC vs. HEC, (iv) LEC vs. MEC, (v) LEC vs. HEC, and (vi) MEC vs. HEC. The pen used in the experiment had an area of 2.7 x 1.8 m and was equipped with two feeders on each side and an equidistant independent water supply at the opposite wall. The feeders in each pen were switched to the opposite side every three days to avoid any differences associated with the location. Water and feed were provided ad libitum throughout the experimental period. The diets were offered in pellet form, and the composition of the basal diet is presented in Table 1. 2.2.2. Growth Performance and Feed Preference Measurement Body weight and diet consumption were measured at the end of the experiment to calculate ADFI, ADG, and the feed:gain ratio (F:G). The relative preference for each individual test diet was the ratio of the amount of feed intake of that diet to the total sum of the amount of feed intake of the two diets offered simultaneously in the pair combination. 2.3. Experiment 2: Performance Trial 2.3.1. Experimental Animal, Design, Diet, and Feeding Management A total of 144 piglets (34 days old; 72 barrows and 72 gilts; Duroc x Large White x Landrace) with 9.24 +- 0.55 kg BW were used in a 28-day performance trial. Piglets were weighed and allotted to four dietary treatments and fed with one of the four diets also used in Experiment 1. Each treatment consisted of six replicates, and each replication had 6 piglets (including three barrows and three gilts). The piglets were housed with separate feeders and drinkers. Water and feed were provided ad libitum throughout the experimental period. The diets were offered in pellet form, and the composition of the diets was the same as in Experiment 1, as presented in Table 1. Chromic oxide, which is an indigestible marker, was added to the diets at an inclusion rate of 4 g/kg feed during the last four days of the experimental period. 2.3.2. Growth Performance Measurement Piglets were weighed on days 0, 14, and 28, and feed consumption was recorded at the same time on a pen basis to calculate ADFI, ADG, and F:G. 2.3.3. Nutrient Digestibility Measurement Feces and feed were collected from each pen every day during the final four days. The whole feces obtained from each pen were pooled, and H2SO4 (10%) at 10% (v/w) was added to part of the feces to determine the crude protein (CP). All feces samples were then dried at 105 degC for 24 h and homogenized before sampling and analysis. The concentrations of nutrients in the feed and feces were analyzed according to the method of AOAC (2007) , including ether extract (EE; method 920.39; AOAC Int., 2007), CP (method 920.39; AOAC Int., 2007), DM (method 930.15; AOAC Int., 2007), ash (method 942.05; AOAC Int., 2007), and chromic oxide (method 930.15; AOAC Int., 2007). The organic matter (OM) was calculated as DM minus Ash. The apparent total tract digestibility (ATTD) of CP, EE, DM, and OM was calculated using the following equation:ATTD% = [(Nutrient/Cr2O3)diet - (Nutrient/Cr2O3)feces]/(Nutrient/Cr2O3)diet x 100%(1) 2.3.4. Serum Physical Indexes and Feeding Hormones Measurement One barrow and one gilt from each pen were selected for the collection of blood samples from the anterior vena cava on day 14 and at the end of the experiment. The serum samples were centrifuged at 3000x g for 10 min at 4 degC and stored at -80 degC until analysis. The concentrations of total protein (TP), albumin (ALB), and total cholesterol (TC) were detected using biuret colorimetry, the bromocresol green method, and the cholesterol oxidase method, respectively. All of the above kits (TP, code no. A045-1-1; ALB, code no. A028-2-1; TC, code no. A111-1-1) were purchased from the Nanjing Jiancheng Bio-Engineering Institute (Nanjing, China). The concentrations of globulin (GLB) were calculated using TP minus ALB. Glucagon-like peptide-1 (GLP-1) and leptin were determined using porcine-specific ELISA kits (GLP-1, code no. H294-1, Nanjing Jiancheng Bio-Engineering Institute, Nanjing, China; leptin, code no. H174, Nanjing Jiancheng Bio-Engineering Institute, Nanjing, China). 2.3.5. 16S rDNA Gene Sequencing Measurement On day 28, fecal samples were collected via rectal massage from two pigs (one gilt and one ballow) per pen and pooled into one sample for high-throughput sequencing. The total DNA from each fecal sample was extracted using a MicroElute Genomic DNA kit (D3096-01, Omega Inc., Norwalk, CT, USA), according to the manufacturer's instructions. The V3-V4 fragment of 16S rRNA was amplified using total DNA as a template, and the conserved primers used were 319F (5'-ACTCCTACGGGAGGCAGCAG-3') and 806R (5'-GGACTACHVGG GTWTCTAAT-3'). Raw data were processed using Greengenes, and sequential analysis, comparison, and annotation were performed using the Ribosomal Database Project (Version 11.3). Illumina MiSeq 2 x 300 bp paired-end was used for sequencing. Operational taxonomic units (OTUs) with a similarity threshold of 97% were determined using R version 3.1.0. Moreover, b-diversity analyses based on the QIIME version 1.7.0-dev were performed to elucidate fecal microflora. 2.4. Statistical Analysis Analyses of BW, ADG, ADFI, and F:G in experiment 1 were performed using one-way ANOVA with a line segment detector (LSD) using SPSS software (version 22.0; SPSS Inc., Chicago, IL, USA). Feed consumption and relative preference in experiment 1 between two diets of a paired combination were analyzed using a t-test (SPSS 22.0). Feed consumption, relative preference associated with each individual diet in experiment 1 and growth performance, serum parameters, and nutrient digestibility in experiment 2 were analyzed using linear and quadratic regressions, and then the differences among the treatments were also analyzed using one-way ANOVA with LSD (SPSS 22.0). All statistics and data visualizations for the microbiota were analyzed using R statistical software and related R software or Qiime 1.7.0. Comparisons of alpha diversity and relative abundance of phyla and genera were performed using the Kruskal-Wallis and Wilcoxon rank sum tests. Principal coordinate analysis (PCoA) was performed using weighted UniFrac metrics. Linear discriminant analysis (LDA; >3.0) was performed using the linear discriminant analysis effect size (LEfSe). Data were presented as means and pooled SEM. For BW, ADG, ADFI, F:G, feed preference, ATTD of the nutrients, and fecal microflora, a pen was used as the experiment unit. For plasma parameters, the individual piglet was used as the experiment unit. Differences were considered significant at p < 0.05, and a tendency was revealed at 0.05 < p < 0.1. 3. Results 3.1. Growth Performance in Experiment 1 In the two-way choice test, piglets who were given a choice of the degree of gelatinization of the extruded corn showed limited effects on growth performance, including BW, ADG, ADFI, and F:G (Table 2). 3.2. Relative Preference and Feed Consumption in Experiment 1 As shown in Table 3, the LEC piglets showed a higher relative preference and feed consumption when they were offered a combination of NC/LEC and LEC/MEC (p < 0.05). LEC showed a higher trend in relative preference and feed consumption when piglets were offered a combination of LEC/HEC (0.05 < p < 0.01). In the whole feed preference trial, diets supplemented with LEC were the most preferred corn (67.02%) with the highest feed consumption (408.95 g/d). Multiple comparisons showed that LEC markedly increased relative preference and feed consumption compared with that of NC, MEC, and HEC (p < 0.05). As the gelatinization degree increased, the relative feed preference and feed consumption quadratically increased (p < 0.05). 3.3. Growth Performance in Experiment 2 As shown in Table 4, the gelatinization degree markedly affected F:G during the periods of 14-28 days and 0-28 days. Diets supplemented with extruded corn with low or medium gelatinization degrees markedly decreased F:G in the periods of 14-28 days and 0-28 days compared with conventional corn or extruded corn with a high degree of gelatinization (p < 0.05). As the gelatinization degree increased, F:G quadratically decreased in the period of 14-28 days (p < 0.05). No significant differences were observed in BW, ADG, or ADFI among the four treatments (p > 0.05). 3.4. Nutrient Digestibility The nutrient digestibility results are summarized in Table 5. The piglets in the LEC and MEC groups showed a higher ATTD for CP than those in the NC and HEC groups (p < 0.05). The ATTD of EE also markedly increased in the MEC group compared with that of the NC and HEC groups (p < 0.05). As the gelatinization degree increased, the ATTD of CP and EE quadratically increased (p < 0.05). 3.5. Serum Physical Indexes and Feeding Hormones As shown in Table 6, the LEC group showed the highest level of TP and GLB content in the plasma and markedly increased compared with that of the NC group on day 14 (p < 0.05). On day 28, no significant difference was observed in the plasma contents of TP, ALB, GLB, and total cholesterol among the four groups (p < 0.05). The plasma levels of ORX, GLP-1, and LEP showed no differences on days 14 and 28 among all groups (p < 0.05). As the gelatinization degree increased, no linear or quadratic changes were observed in the serum indexes on days 14 and 28. 3.6. Fecal Microflora The effects of the extruded corn on the microbial communities are shown in Figure 1. The MEC group tended to have an increased number of observed species (p = 0.090) and Chao-1 index (p = 0.106) . Piglets in the four treatment groups shared 1795 OTUs of fecal microflora in the Venn diagram . NC, LEC, MEC, and HEC contained 237, 45, 72, and 55 OTUs, respectively. The beta-diversity analysis by principal coordinate analysis showed that the microbiota in the NC group were separated from the LEC, MEC, and HEC groups . The microbiota in the piglets who were fed the extruded corn were highly clustered, regardless of the degree of gelatinization . The microbial community structure is presented in Figure 2. At the phylum level, the most abundant phyla were Bacteroidetes and Firmicutes, with an average relative abundance of over 95% of the total bacteria in the piglets. No significant difference was observed in the relative abundance of Firmicutes; however, the relative abundance of Bacteroidetes increased from 46.05% (NC) to 50.37% (LEC), 50.78% (MEC), and 51.92% (HEC) (p < 0.05) . There was no significant difference in the relative abundance ratio of Firmicutes to Bacteroidetes between the groups . At the genus level, Prevotella_9 (7.79%), Lactobacillus (7.70%), and Prevotellaceae_NK3B31_group (7.19%) were the top three genera . Significant differences in the top 15 genera are shown in Figure 2d. The LEC group had an increased in the relative abundance of Lactobacillus, Prevotellaceae_NK3B31_group, Prevotellaceae_UCG-003, Allopreotella, Prevotella_1, and Prevotella_2 compared with the NC group (p < 0.05). The MEC group showed a marked increase in Lactobacillus, Allopreotella, Prevotellaceae_UCG-003, and Prevotella_2 abundance compared with that of the NC group (p < 0.05). The HEC group showed an increase in Prevotellaceae_NK3B31_group, Allopreotella, and Prevotella_1 abundance compared with that of the NC group (p < 0.05). LDA effect size was performed to detect specific bacterial taxa with significantly different abundances among the NC, LEC, MEC, and HEC groups . Twelve distinct bacterial taxa were identified using a log LDA score threshold of 3.0. Prevotella 6 and Ignatzschineria were found to be enriched in the microbiota of the NC group. Lactobacillus, Prevotellaceae_UCG_003, Prevotellaceae_UCG_001, Campylobacter, and Succinivibrio spp. were enriched in the MEC group. Alloprevotella, Prevotella1, Prevotella_2, Coprococcus3, and Lachnospira were enriched in the LEC group. Prevotellaceae_NK3B31_group, Ruminococcaceae_UCG_008, Ruminococcus_2, Oscillibacter, Fournierella, and Ruminiclostridium_9 were enriched in the HEC group. Significant differences in the top 15 genera are shown in Figure 3. The LEC and MEC groups markedly increased their relative abundance of Lactobacillus, Alloprevotella, Prevotellaceae_UCG_003, and Prevotella_2 compared with the NC group (p < 0.05). Meanwhile, the LEC and HEC groups also showed a higher abundance of Prevotellaceae_NK3B31_group and Prevotella_1 than the NC group (p < 0.05). 4. Discussion In the two-way choice preference tests, piglets preferred feed containing extruded corn, especially the feed with a 41.82% gelatinization degree. This result was similar to that of Sola-Oriol et al., who noted that extrusion increased pigs' preferences for several cereals, including corn and naked oats, when the inclusion rate was 30% or 60% . As expected, when the degree of gelatinization of the extruded corn reached 89.93%, the preference decreased to 41%, which was similar to that of conventional corn. This could be a result of the increased feed hardness. Hardness can indicate the maximum force that piglets must compress during chewing. The extrusion process gelatinizes starch and improves its adhesive property, which acts as a binder during pelleting and thus increases the pellet hardness . As the degree of gelatinization increased, pellet hardness increased, which decreased feed preference . Starch with a high degree of gelatinization is normally associated with a high water absorption index, which may stick to the mouth, and a greater amount of saliva may be needed to swallow the feed, which affects piglets' chewing capability . The addition of extruded starch to diets has beneficial effects on nutrient digestibility. Yong et al., reported that supplementary extruded corn increased the in vitro and in vivo apparent digestibility of CP, GE, DM, and starch, both in vitro and in piglets . Liu et al., suggested that the extrusion process increases the ATTD of DM and GE in corn and broken rice in weaning piglets . Several factors can be attributed to increased nutrient digestibility, such as increased enzyme susceptibility, reduced starch resistant content, and decreased starch granules . In the present study, an increased ATTD of both CP and EE was observed in diets containing extruded corn. We also noted that diets with a medium degree of starch gelatinization (62.60%) resulted in the best digestibility, whereas a high degree of starch gelatinization contributed to low digestibility. These results aligned with Adb et al.'s study, which reported that CP, EE, and DM digestibility in pigeons increased when the starch gelatinization degree decreased from 73.6% to 53.1% . Hongtragul et al., also noted significant quadratic variation between the degree of starch gelatinization and nutritional digestibility . The decreased nutritional digestibility of highly gelatinized corn may be related to intestinal viscosity. Normally, extruded corn with a high degree of gelatinization has high water solubility which decreases the chyme viscosity . The decreased viscosity reflects a short mean transit time, resulting in a reduction in nutrient digestion and subsequent absorption. Over-processing during extrusion may also promote a Maillard reaction between amino acids and glucose which enhances the encapsulation of starch granules via the protein and resistant starch content and thus reduces its digestibility . The effects of extruded cereal on the growth performance of piglets varies. Lawlor reported no beneficial effects of extrusion on the growth performance of piglets with different initial body weights . Similar results were reported in Mateos's study, which used rice and maize diets . Hongtragul's study showed the positive effects of extruded corn as the piglets' ADG and ADFI increased . Kotara also found that ADG, ADFI, and G:F increased linearly when the degree of starch gelatinization increased from 23.8% to 81.9% in barley, wheat, and maize diets . One possible explanation for these differences could be the variation in extruded corn quality caused by extrusion conditions, such as steam flow, temperature, and pressure. In the present study, although ADG and ADFI showed no differences among all groups, F:G markedly decreased when the degree of gelatinization of the extruded corn was 41.82% and 62.60%. The nutrient digestibility results were consistent with those of F:G and might be the main reason for the improved feed utilization in the LEC and MEC groups. Meanwhile, we also noted that feed preference had limited effects on ADFI when the piglets were fed a single feed. This difference may be because feed intake is influenced by multiple factors, such as nutritional composition, palatability, and nutritional availability . In the present experiment conditions and diet compositions, nutritional requirements had a stronger effect on feed intake than palatability when piglets were fed the single feed. The TP is composed of ALB and GLB. ALB is synthesized by the liver and significantly correlated with nutritional status . GLB is synthesized by immune organs and plays an important role in immune responses . In the present study, TP and GLB showed significant differences on day 14, with the highest levels in the LEC group, followed by the MEC group. These results were consistent with the results of F:G and nutrient digestibility, which indicated an increased nutritional status and an increased immunity. GLP-1 and leptin are peripheral appetite regulatory peptides that play important roles in the regulation of feeding behavior. Zhuo et al., noted that extrusion ingredients, including corn and broken rice, elevated the plasma levels of GLP-1 and peptide YY . However, the GLP-1 and leptin levels showed no differences among the four treatments in our present study. These differences may exist because the blood in the experiment was collected after fasting, and the hormone level had dropped to a low point, which made the difference not significant. The extrusion process elevates nutrient digestibility in the small intestine and alters fermentable substrates in the large intestine, thereby changing the gut microbial community structure. In weaning piglets, the extrusion process of corn or broken rice tends to increase the microbial diversity, as indicated by the increasing Chao-1 and ACE indices . Our present study identified a similar result: extruded corn with a medium degree of gelatinization (62.60%) tended to increase the observed species and Chao-1 index. Generally, higher microbial diversity is associated with higher resilience to environmental challenges and better disease resistance . A study conducted by Moen showed a converse result as cereal extrusion decreased the microbiota diversity in growing pigs . These differences might be due to the growth period or extrusion condition and require further study. Bacteroidetes are Gram-negative bacteria that perform a main role in the maintenance of a healthy state and sophisticated homeostasis safeguarded by microbiota . In the present study, extrusion increased the relative abundance of Bacteroidetes, regardless of the degree of gelatinization. Although Bacteroidetes are normally related to inflammation and obesity-driven dysbiosis , a study by Vadder et al., indicated that Bacteroidetes strongly correlate with increased levels of short chain fatty acid (SCFA) . The increased abundance of Bacteroidetes can also provide anti-inflammatory effects with certain probiotics . To further identify the effects of increased Bacteroidetes on piglets, we compared the microbial abundance at the genera level among the four groups. One important finding was that Prevotella, which is an important genus that belongs to Bacteroidetes, increased in the piglets who were fed extruded corn. A study by Mach et al., reported that the presence of Prevotella was associated with increased luminal secretory immunoglobulin A, indicating a positive influence on mucosal immunity . Prevotella is also reported to be capable of metabolizing fibers into SCFAs, such as acetate, propionate, and butyrate . Therefore, piglets with a high abundance of Prevotella may be better adapted to plant-derived feeds and may confer a performance advantage , which in turn may explain the improved feed conversion ratio of extruded corn in the present study. We also noted that extruded corn increased the fecal Lactobacillus abundance in piglets, and this effect was positively correlated with the degree of gelatinization. Lactobacillus is regarded as a beneficial bacterium that inhibits the growth of some pathogens, indicating healthier gut flora in extruded corn-treated piglets . Wang also found a positive relationship between gelatinized starch and Lactobacillus in the sorghum-based diets of growing pigs . 5. Conclusions The piglets in this study preferred diets containing extruded corn with a gelatinization degree of 41.82% when they were given a choice. Corn extrusion increased the feed conversion ratio, improved the ATTD of CP and EE, and increased the abundance of Bacteroidetes, Lactobacillus, and Prevotella in the piglets. Under the experimental conditions, the ideal gelatinization degree of extruded corn for the piglets was approximately 41.82-62.60%. Acknowledgments The authors want to thank Hangzhou Zhengxing Animal Husbandry Co., LTD and Deqiu Lu from Harbin PuFan Feed Co., Ltd. for their assistance during the feeding and sampling process. Author Contributions B.D. and Z.X. designed the protocol of the study; B.D., X.L., J.W. and K.Q. performed the project administration; J.W. and Q.M. formulated the diets; J.W., X.L. and X.T. performed the data analysis; B.D., J.W., X.T. and X.D. wrote and modified the manuscript. All authors have read and agreed to the published version of the manuscript. Institutional Review Board Statement Animal studies were performed in accordance with the guidelines of the Zhejiang Farm Animal Welfare Council of China and approved by the Ethics Committee of the Zhejiang Academy of Agricultural Sciences (No.2020112). Informed Consent Statement Not applicable. Data Availability Statement The data are available on request from the corresponding author. Conflicts of Interest The authors declare no conflict of this study. Figure 1 Summary of the microbial community from the fecal samples of piglets in experiment 2 (n = 6). (A) is the Shannon index reflecting species diversity; (B) is the Chao-1 index reflecting species diversity; (C) is a Venn diagram summarizing the number of common and unique observed taxonomic units (OTUs) in the microflora; and (D) is the PCoA plot based on weighted UniFrac distances. Figure 2 Microbial community structure from the fecal samples of piglets in experiment 2 (n = 6). (a) represents the relative abundances of the top 15 bacterial strains at the genus level (b) is the ratio of Firmicutes/Bacteroidetes. (c) is the top 10 relative abundances of bacterial strains at the phylum level. (d) represents the LEfSe analysis showing the LDA scores (>3.0). Different superscripts indicate significant differences (p < 0.05). Figure 3 Genus with significant differences in the top 15 from the fecal samples of piglets in experiment 2 (n = 6). Different superscripts indicate significant differences (p < 0.05). a,b values differ significantly (p < 0.05). animals-13-00922-t001_Table 1 Table 1 Composition and nutrition levels of the basal diets (as-fed basis). Ingredient Content Nutrient Levels (2) Content Maize 52.5 Crude protein 18.35 (18.13) Dehulled soybean meal 16.5 DE (MJ/kg) 14.51 Extrusion full-fat soybean 10 Calcium 0.76 (0.78) Broken rice 15 Phosphorous 0.62 (0.65) Fish meal 2 Lysine 1.31 (1.35) Calcium carbonate 1.1 Methionine + Cystine 0.72 (0.70) calcium hydrophosphate 0.8 Threonine 0.87 (0.81) Sodium chloride 0.25 Coline chloride 0.1 Lsine hydrochloride 99% 0.42 DL-methionine 98 0.12 Threonin 98% 0.18 Thyptophan 99% 0.03 Premix (1) 1.00 Total 100.00 (1) Premix provided the following per kilogram of basal diet: Cu 125 mg, Zn 70 mg, Fe 100 mg, Mn 40 mg, Se 0.3 mg, I 0.4 mg, vitamin A 7500 IU, vitamin D3 750 IU, vitamin E 25 IU, vitamin K3 2.0 mg, vitamin B1 1.88 mg, vitamin B2 3.75 mg, vitamin B6 2.19 mg, vitamin B12 0.025 mg, nicotinic acid 25 mg, D-pantothenic acid 15.6 mg, folic acid 2.0 mg, and biotin 0.19 mg. (2) Nutrient levels outside the brackets were calculated values, and the numbers in the brackets were analyzed the levels. animals-13-00922-t002_Table 2 Table 2 Growth performance of piglets offered different gelatinization degrees of extruded corn in a two-way choice test in experiment 1 1. Treatment 1 NC/ LEC NC/ MEC NC/ HEC LEC/ MEC LEC/ HEC MEC/ HEC SEM p-Value Initial BW, kg 9.43 9.46 9.44 9.40 9.46 9.43 0.15 0.403 Final BW, kg 16.04 15.30 15.60 14.77 15.60 15.37 0.29 0.196 ADG, g 366.83 324.33 341.92 298.08 341.33 339.08 9.7 0.221 ADFI, g 633.33 557.87 600.93 537.96 620.14 555.09 19.67 0.439 F:G 1.74 1.73 1.75 1.81 1.81 1.64 0.04 0.848 1 NC, basal diet; LEC, basal diet with 40% extruded corn with a low (41.82%) degree of gelatinization; MEC, basal diet with 40% extruded corn with a medium (62.60%) degree of gelatinization; HEC, basal diet with 40% extruded corn with a high (89.93%) degree of gelatinization; ADG, average daily gain; ADFI, average daily feed intake; and F:G, feed gain ratio. animals-13-00922-t003_Table 3 Table 3 Feed consumption and relative preferences of piglets offered different gelatinization degrees of extruded corn in experiment 1. Treatment 1 Free Choice a/b Relative Preference, % Feed Consumption, g/d a b SEM p-Value a b SEM p-Value 1 NC/ LEC 35.22 64.78 6.38 0.004 223.15 410.19 44.11 0.017 2 NC/ MEC 37.44 62.56 7.75 0.106 208.56 349.31 44.25 0.115 3 NC/ HEC 51.14 48.86 8.38 0.904 312.73 288.19 51.31 0.831 5 LEC/ MEC 66.98 33.02 7.00 0.001 360.42 177.55 38.71 0.003 4 LEC/ HEC 69.31 30.69 11.34 0.085 456.25 163.89 85.46 0.083 6 MEC/ HEC 55.01 44.99 7.35 0.537 299.77 255.32 39.33 0.612 Individual diets Relative preference, % Feed consumption, g/d NC 41.26 +- 8.62 b 248.15 +- 56.40 b LEC 67.02 +- 2.26 a 408.95 +- 47.93 a MEC 50.02 +- 15.35 b 275.54 +- 88.41 b HEC 41.51 +- 9.57 b 235.80 +- 64.41 b 1 NC, basal diet; LEC, basal diet with 40% extruded corn with a low (41.82%) degree of gelatinization; MEC basal diet with 40% extruded corn with a medium (62.60%) degree of gelatinization; and HEC, basal diet with 40% extruded corn with a high (89.93%) degree of gelatinization. a,b values within rows with different superscript differ significantly (p < 0.05). animals-13-00922-t004_Table 4 Table 4 Effect of different gelatinization degree of extruded corns on growth performance of piglets in experiment 2. Treatment 1 NC LEC MEC HEC SEM p-Value Treatment Linear Quadratic BW, kg Day 0 9.24 9.23 9.26 9.24 0.12 0.371 0.990 1.000 Day 14 12.05 12.15 12.18 12.20 0.12 0.911 0.674 0.909 Day 28 17.60 18.65 18.09 18.16 0.19 0.313 0.475 0.366 ADG 0-14 d 200.68 208.45 208.40 211.55 28.54 0.918 0.573 0.826 14-28 d 396.31 464.54 422.76 425.53 54.48 0.291 0.585 0.298 0-28 d 298.49 336.50 315.58 318.54 31.89 0.338 0.450 0.344 ADFI 0-14 d 434.33 436.35 415.48 426.98 36.74 0.558 0.527 0.758 14-28 d 724.40 771.63 703.57 781.75 88.21 0.434 0.470 0.713 0-28 d 579.37 603.99 559.52 604.37 56.45 0.465 0.726 0.859 F:G 0-14 d 2.17 2.13 2.03 2.04 0.27 0.397 0.242 0.489 14-28 d 1.84 a 1.67 b 1.66 b 1.85 a 0.18 0.028 0.918 0.039 0-28 d 1.95 a 1.80 b 1.77 b 1.91 a 0.17 0.015 0.653 0.101 1 NC, basal diet; LEC, basal diet with 40% extruded corn with a low (41.82%) degree of gelatinization; MEC basal diet with 40% extruded corn with a medium (62.60%) degree of gelatinization; HEC, basal diet with 40% extruded corn with a high (89.93%) degree of gelatinization; ADG, average daily gain; ADFI, average daily feed intake; and F:G, feed gain ratio. a,b values within rows with different superscript differ significantly (p < 0.05). animals-13-00922-t005_Table 5 Table 5 The effects of different gelatinization degrees of extruded corn on the ATTD of CP and EE in piglets in experiment 2. Treatment 1 NC LEC MEC HEC SEM p-Value Treatment Linear Quadratic CP, % 81.44 b 83.47 a 83.45 a 82.39 b 0.23 <0.001 0.157 <0.001 EE, % 83.76 b 84.57 ab 85.64 a 83.46 b 0.26 0.008 0.975 0.013 DM, % 76.90 a 74.04 c 77.50 a 75.80 b 0.31 <0.001 0.831 0.622 OM, % 78.01 b 76.96 b 79.17 a 77.39 b 0.25 0.004 0.980 0.813 1 NC, basal diet; LEC, basal diet with 40% extruded corn with a low (41.82%) degree of gelatinization; MEC basal diet with 40% extruded corn with a medium (62.60%) degree of gelatinization; HEC, basal diet with 40% extruded corn with a high (89.93%) degree of gelatinization; CP, crude protein; EE, ether extract; DM, dry matter; and OM, organic matter. a,b Values within rows with different superscript are differ significantly (p < 0.05). animals-13-00922-t006_Table 6 Table 6 The effects of different gelatinization degrees of extruded corn on the plasma parameters of piglets in experiment 2. Treatment 1 NC LEC MEC HEC SEM p-Value Treatment Linear Quadratic TP (g/L) 14d 57.27 b 61.10 a 59.74 ab 58.73 ab 0.51 0.047 0.339 0.558 28d 56.19 55.99 57.23 57.55 0.38 0.414 0.919 0.752 ALB (g/L) 14d 22.99 24.05 23.85 23.89 0.19 0.201 0.751 0.675 28d 22.34 22.58 22.65 23.06 0.15 0.495 0.753 0.396 GLB (g/L) 14d 34.28 b 37.05 a 35.89 ab 34.84 b 0.37 0.034 0.200 0.430 28d 33.85 33.41 34.57 34.49 0.27 0.365 0.991 0.941 TC (mmol/L) 14d 0.95 0.96 0.71 0.80 0.05 0.245 0.353 0.572 28d 1.07 0.83 0.79 0.98 0.05 0.177 0.930 0.467 GLP-1 (pmol/L) 14d 28.61 29.24 27.65 30.17 0.81 0.773 0.453 0.764 28d 33.79 34.06 33.22 36.76 1.46 0.582 0.786 0.892 Leptin (ng/mL) 14d 6.99 7.07 6.78 7.38 0.20 0.805 0.155 0.159 28d 7.97 8.22 8.09 8.47 0.32 0.543 0.180 0.388 1 NC, basal diet; LEC, basal diet with 40% extruded corn with a low (41.82%) degree of gelatinization; MEC basal diet with 40% extruded corn with a medium (62.60%) degree of gelatinization; HEC, basal diet with 40% extruded corn with a high (89.93%) degree of gelatinization; TP, total protein; ALB, albumin; GLB, globulin; TC, total cholesterol; and GLP-1, glucagon like peptide-1.a,b values within rows with different superscript differ significantly (p < 0.05). 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PMC10000146

Bamboo part preference plays a critical role in influencing the nutrient utilization and gastrointestinal microbiota composition of captive giant pandas. However, the effects of bamboo part consumption on the nutrient digestibility and gut microbiome of geriatric giant pandas remain unknown. A total of 11 adult and 11 aged captive giant pandas were provided with bamboo shoots or bamboo leaves in the respective single-bamboo-part consumption period, and the nutrient digestibility and fecal microbiota of both adult and aged giant pandas in each period were evaluated. Bamboo shoot ingestion increased the crude protein digestibility and decreased the crude fiber digestibility of both age groups. The fecal microbiome of the bamboo shoot-fed giant pandas exhibited greater alpha diversity indices and significantly different beta diversity index than the bamboo leaf-fed counterparts regardless of age. Bamboo shoot feeding significantly changed the relative abundance of predominant taxa at both phylum and genus levels in adult and geriatric giant pandas. Bamboo shoot-enriched genera were positively correlated with crude protein digestibility and negatively correlated with crude fiber digestibility. Taken together, these results suggest that bamboo part consumption dominates over age in affecting the nutrient digestibility and gut microbiota composition of giant pandas. giant panda bamboo part aging microbiome nutrient digestibility Chengdu Giant Panda Breeding Research FoundationCPF2014-03 Chengdu Research Base of Giant Panda Breeding2021CPB-B07 Sichuan Science and Technology Program2020YFH0145 2021YFH0157 National Key Research and Development Program of China2022YFD1301300 This work was supported by the project of Chengdu Giant Panda Breeding Research Foundation (CPF2014-03), Chengdu Research Base of Giant Panda Breeding (2021CPB-B07), Sichuan Science and Technology Program (2020YFH0145, 2021YFH0157), and National Key Research and Development Program of China (2022YFD1301300). pmc1. Introduction The giant panda (Ailuropoda melanoleuca) is a highly specialized herbivorous species of ursid that consumes bamboo as the primary and almost exclusive diet. Unlike most herbivores, the giant panda has no apparent internal gastrointestinal adaptions to its bamboo-dominated diet, and exhibits a short digestive tract with a rapid passage of digesta, which is similar to the gastrointestinal tract morphology of most carnivores . The extremely high amount of bamboo consumption each day and low energy expenditure can partly explain how giant pandas persist solely on bamboo, a high fibrous plant with low nutritional value and digestibility . However, the giant panda has been shown to lack homologs of the enzymes needed for the degradation of structural carbohydrates, the key component of bamboo . It has thus been believed that the utilization and extraction of nutrients from the bamboo diet largely depends on the gut microbiome of the giant panda, as the giant panda gut microbiome has been found to exhibit a high abundance of putative genes involved in carbohydrate degradation, suggesting high utilization potential of structural polysaccharides . Both wild and captive pandas exhibit seasonal changes in bamboo part preference, with shoots consumed in spring and summer, leaves in autumn and winter, and culms in the transition period, namely later winter and early spring . Dietary changes are an important factor influencing the composition and function of the gut microbiome . Evidences have been accumulated to show the giant panda's gut microbiota are shaped by the seasonally-driven shifts in bamboo part preference, as the nutrient content in different parts of bamboo varies significantly, with higher cellulose, hemicellulose, and starch, as well as lower proteins, in the leaves and culms than in shoots . Gut microbiota has been shown to significantly affect the nutrient utilization capacity and health status of the host . In captive giant pandas, the apparent digestibility of bamboo parts differed significantly, resulting in different degrees of nutrient retention used by gut microbes in the hindgut . Therefore, the changes in gut microbiome elicited by different bamboo part consumption would significantly affect the nutrient digestibility of the giant pandas. Aging is an inevitable biological process in an organism that leads to an increased risk of many diseases . In terms of longevity, captive giant pandas generally have a lifespan of almost 30 years, and individuals older than 20 are considered to be "geriatric" because the reproduction process of the giant panda generally ends after this age . Aging has been proven to significantly shape the structure of gut microbiota and affect the immune and metabolic functions of giant pandas . Likewise, impaired digestive function and higher risk of gastrointestinal disorders have been recognized in aged giant pandas . The seasonal variation in bamboo part consumption has been shown to significantly affect the nutrient digestibility of captive giant pandas . However, little is known about the effects of bamboo part preference on aged giant pandas, especially the changes of gut microbiome and nutrients digestibility. To address this issue, the nutrients digestibility and gut microbiota composition were compared between adult and older captive giant pandas when fed exclusively with a diet comprising of either shoots or leaves. 2. Materials and Methods 2.1. Ethics Statement All protocols for the present study that involved animal care and treatment were approved by the Institutional Animal Care and Use Committee of Chengdu Research Base of Giant Panda Breeding (No. 2020010). 2.2. Study Subjects and Animal Husbandry A total of 11 adult (aged 9-17 years, average age was 13) and 11 geriatric (aged 20-37 years, average age was 25) captive giant pandas were the subjects of the present study. All subjects were singly housed at the Chengdu Research Base of Giant Panda Breeding (CRBGPB, Chengdu, Sichuan, China), and all were considered healthy and were not under any medical treatment during the study period. The ambient temperature was maintained at 15 degC-22 degC, and the air humidity was 65-75%. All giant pandas were fed according to the normal husbandry practices of the CRBGPB as described in Wang et al. . Bamboo was provided to giant pandas three times each day (08:00, 14:00, and 20:00). In the present study, giant pandas were given free access to bamboo and water, and the specific bamboo part was offered according to the seasonal shifts. In CRBGPB, bamboo shoots of Phyllostachys nidularia Munro were consumed by pandas in autumn and bamboo leaves of Bashania fargesii were provided to pandas in winter. In addition to the supply of bamboo parts, dietary supplements were provided daily and of the same mass to all subjects. In this study, both adult and geriatric pandas were provided with bamboo shoots for 3 months and bamboo leaves for 3 months: bamboo shoot-fed adult (AS), bamboo leaf-fed adult (AL), bamboo shoot-fed old (OS), and bamboo leaf-fed old (OL) giant pandas. 2.3. Sample Collection At the last day of each period during which pandas were offered the corresponding bamboo part, fecal samples were collected from each giant panda. For each panda, the spontaneous excreted fecal samples were collected within 10 min of defecation after the feeding in the morning. To avoid contamination, samples were collected only after the floor was cleaned and disinfected. Furthermore, the outer layer of feces that contacted the floor was discarded and only fecal parts that did not touch the floor were kept and stored at -80 degC pending further analysis. 2.4. Apparent Nutrient Digestibility Measurement During the last three days of each single-bamboo-part consumption period, the apparent nutrient digestibility of the corresponding bamboo part was determined in both adult and older giant pandas. The amount of ingested food and excreted feces of each individual giant panda was weighed. The bamboo samples that pandas consumed and fecal samples were collected twice a day, weighed, and immediately stored at 4 degC. During the next day, corresponding proportions of fecal samples were kept and mixed according to the amount of daily excreted feces. Finally, about 1 kg of bamboo leaves and 1.5 kg of the corresponding fecal samples, as well as 5 kg of bamboo shoots and the corresponding fecal samples, were kept at -80 degC for long-term storage. The bamboo and fecal samples were dried, ground, and sieved through a 0.45 mm sieve, then mixed, sampled, and stored at -20 degC. The chemical components of the bamboo and fecal samples were determined according to the AOAC analysis method . An oven drying method was adopted to measure the dry matter (DM) content, the Kjeldahl method was used to determine the crude protein (CP) content, the Soxhlet extraction method was applied to evaluate the ether extract (EE) content, the continuous extraction of samples by dilute acids and bases was used to measure crude fiber (CF), and lastly, the oxygen bomb calorimeter calorimetric method was used to analyze the gross energy (GE) concentration of bamboo and fecal samples. The calculation equation of apparent nutrient digestibility was as follows:Apparent digestibility= Daily intake x Nutrient substance (Bamboo)- Daily feces x Nutrient substance (Feces)Daily intake x Nutrient substance (Bamboo) 2.5. Genomic DNA Extraction from Feces and Sequencing The genomic DNA of each fecal sample was isolated with the QIAamp Fast DNA Stool Mini Kits (Qiagen, Beijing, China) following the manufacturer's instructions. The integrity and concentration of obtained DNA samples were assessed visually by agarose gel electrophoresis or measured using a NanoDrop ND-1000 device. Sterilized water was used as a negative control sample, and was included in the DNA isolation process, which showed no detectable PCR product. The common primers 515F and 806R were used to amplify the V4 region of the bacterial 16S rRNA gene, and the resulting PCR products were pooled and purified by using the Agencourt AMPureXP beads (Beckman Coulter, Brea, CA, USA) along with the MinElute PCR Purification Kit (Qiagen, Beijing, China). After pooling and purification, these amplicons were then used to construct Illumina libraries with the Ovation Rapid DR Multiplex System 1-96 (NuGEN, San Carlos, CA, USA). All of the sample libraries were sequenced on the Illumina MiSeq platform with a PE250 sequencing strategy (Novogene, Beijing, China). The raw data were deposited in the NCBI BioProject database with the accession number PRJNA916390. 2.6. Fecal Microbiota Analysis The raw Illumina data were processed by Mothur software v1.3.6 (MI, USA) . The high-quality paired-end sequences, which were obtained by removing the primer and barcode sequence, and also the low-quality reads, were assembled into tags with overlapping relationships. The library size of each sample was randomly subsampled into the minimum sequencing depth to minimize the biases caused by sequencing depth between samples. The USEARCH v7.0.1001 was applied to cluster tags into OTUs based on 97% cut-off. The representative sequence of each OTU cluster was used for taxonomic classification against the Ribosomal Database Project database with RDP v2.6 . The OTU abundance table and the OTU taxonomic assignment table laid out from the Mothur software were processed with R studio v3.4.1 to calculate alpha diversity indexes of communities, as well as the beta diversity index and the Bray-Curtis distance . The structural dissimilarity of the microbiota communities across the samples were visualized by non-metric multidimensional scaling (NMDS) analysis based on the Bray-Curtis distance matrix. 2.7. Statistical Analysis For nutrient digestibility parameters, the statistical analysis was performed using SAS version 9.4 (SAS Institute Inc., Cary, NC, USA). Giant panda was considered the experimental unit for all analyses (n = 11 per treatment), and the results were expressed as means and SEM. The main effects of bamboo part and age, and the interaction between bamboo part and age were determined via two-way ANOVA. After transforming non-normal distributed data to approximately conform to normality by SAS software, the alpha indexes including Observed species, Chao 1, Shannon and Simpson index as well as the relative abundance of top 10 phyla and top 30 genera were tested for significance with the one-way ANOVA, followed by Tukey's test to evaluate the differences between treatments. Data were presented as mean +- SE. The intragroup statistic differences in beta diversity based on the Bray-Curtis distance were assessed using the one-way ANOSIM test with 10,000 permutations. Spearman's correlation between the gut microbiota composition and nutrient digestibility parameters were calculated by the ggcor package within R software version 3.6.1 . Only correlations with Spearman's coefficient r > 0.5 and p < 0.05 were used to generate the network graph, which was visualized and manipulated by Gephi version 9.2 . The differences were considered statistically significant when the p values were less than 0.05. 3. Results 3.1. Bamboo Part and Age Affect Apparent Nutrient Digestibility of Giant Pandas A significant effect of age (F = 4.86, df = 1, p = 0.04) on the dietary gross energy utilization efficiency was observed showing that aged giant pandas had weaker energy extraction capacity from their diet compared to their younger counterparts (Table 1). There was a significant effect of bamboo part ((F = 203.23, df = 1, p < 0.001) for crude protein digestibility, indicating that bamboo shoot ingestion increased the crude protein digestibility of both adult and aged giant pandas (Table 1). There was a significant effect of bamboo part (F = 13.65, df = 1, p = 0.001) and age (F = 11.44, df = 1, p = 0.002) as well as a significant bamboo part x age interaction (p < 0.05) for ether extract digestibility (Table 1). This demonstrates that bamboo shoot feeding increased ether extract digestibility of aged rather than adult giant pandas when compared to bamboo leaf ingestion. Results indicated that bamboo shoot-fed giant pandas had lower crude fiber digestibility than bamboo leaf-fed counterparts (F = 16.06, df = 1, p < 0.001, Table 1). 3.2. Bamboo Part and Age Affect Fecal Microbial Profiles of Giant Pandas After the pre-processing of raw reads, high-quality tags were generated from all samples ranging from 57,136 to 91,531, which were subsampled to 57,136 to avoid the bias induced by the sequencing depth between samples. A total of 3,728 OTUs were obtained by clustering these tags at a 97% similarity cutoff. The fecal microbiome of the bamboo shoot-fed giant pandas exhibited greater observed species (F = 4.65, df = 3, p = 0.01), Chao1 (F = 56.08, df = 3, p < 0.001), Shannon (F = 62.11, df = 3, p < 0.001), and Simpson index (F = 5.01, df = 3, p = 0.005) values than the bamboo leaf-fed counterparts regardless of age . The inter-group Bray-Curtis distance was significantly higher than the intra-group when giant pandas were fed with different bamboo parts independent of age (F = 25.49, df = 5, p < 0.001), otherwise there was no difference in the inter-group and intra-group Bray-Curtis distances . The NMDS-based map also showed that the fecal microbiome of giant pandas could be sorted into two clusters by bamboo part consumption rather than age , indicating the dominant role of bamboo part consumption in shaping the fecal microbiome of both adult and old giant pandas. The predominant phyla in feces of AS, AL, OS, and OL pandas were Firmicutes and Proteobacteria . Bamboo shoot feeding was found to decrease the relative abundance of Firmicutes and increase the relative abundance of Proteobacteria in adult giant pandas rather than old giant pandas compared to bamboo leaf consumption . Additionally, bamboo shoot feeding increased the relative abundance of Acidobacteriota, Actinobacteria, and Chloroflexi as well as decreased the relative abundance of Bacteroidetes in both adult and old giant pandas compared to bamboo leaf feeding . At the genus level, Escherichia-Shigella and Clostridium_sensu_stricto_1 were the two most abundant bacteria in feces of all four groups . The relative abundance of Cellulosilyticum, Citrobacter, Enterococcus, Lactococcus, Pantoea, Ralstonia, Raoultella, Acinetobacter, Bradyrhizobium, Leuconostoc, Massilia, and Providenicia were higher in feces of bamboo shoot-feeding giant pandas than the bamboo leaf-feeding group regardless of age . Bamboo shoot intake was found to decrease the relative abundance of Streptococcus, Lachnospiraceae_NK4A136_group, and Terrisporobacter in feces of both adult and old giant pandas compared to bamboo leaf consumption . Bamboo shoot feeding increased the relative abundance of Helicobacter and decreased the relative abundance of Clostridium_sensu_stricto_1 in feces of adult giant pandas rather than the old group . Compared to bamboo leaf consumption, the decreased abundance of Escherichia-Shigella and increased abundance of Turicibacter, Hafnia-Obesumbacterium, and Weissella were observed in bamboo shoot-fed old giant pandas rather than the adult group . 3.3. The Correlation between Fecal Microbiota and Nutrient Digestibility in Giant Pandas The genus Streptococcus and Lachnospiraceae_NK4A136_group were significantly positively correlated with crude fiber digestibility, whereas the genus Lactococcus, Turicibacter, Raoultella, Citrobacter, Enterococcus, Pantoea, Cellulosilyticum, Weissella, Providencia, and Hafnia-Obesumbacterium were significantly negatively correlated with crude fiber digestibility . The genus Streptococcus, Terrisporobacter, and Lachnospiraceae_NK4A136_group were significantly negatively correlated with crude protein digestibility, whereas the genus Lactococcus, Turicibacter, Raoultella, Citrobacter, Enterococcus, Ralstonia, Pantoea, Cellulosilyticum, Weissella, Providencia, Helicobacter, Hafnia-Obesumbacterium, Massilia, Bradyrhizobium, Leuconostoc, and Acinetobacter were all significantly positively correlated with crude protein digestibility . The genus Providencia was significantly positively correlated with ether extract digestibility . 4. Discussion Despite exhibiting a carnivore's characteristic simple gastrointestinal tract, giant pandas acquire the majority of the required nutrients from bamboo. Because of the limited digestibility of plant cellulose by the giant panda genome, it was suggested that the gut microbiome may play a vital role in the digestion of this highly fibrous bamboo diet . Seasonal dietary shifts in bamboo part selection have been observed in both wild and captive giant pandas, and have been shown to extensively shape the host microbiome . The bamboo part preference during different seasons has been shown to significantly influence the nutrient digestibility of adult captive giant pandas, which is associated with changes in the gut microbiota composition . Owing to the improvements in husbandry and veterinary care, the number of geriatric pandas in zoological institutions has increased in recent years. The aging process in giant pandas elicits a significant change in the gut microbiome, indicating that geriatric pandas exhibit a different gut microbiota composition than younger pandas . While studies in humans and other animals have shown that there may exist an interaction between diet and aging in regulating host phenotype and shaping gut microbiota composition , such information in different bamboo part-fed geriatric and adult pandas remains unknown. Unlike studies in other animals showing similar nutrient digestibility between adult and senior individuals , lower energy digestibility was found in aged giant pandas compared to the adults in the present study, indicating the declined energy extraction capacity from food in aging giant pandas. Giant pandas feed almost exclusively on bamboo, of which the different plant parts exhibit significantly different nutrient compositions . Wang et al. showed that the bamboo part exerted a significant effect on nutrient digestibility in giant pandas. Bamboo shoots consumption has been shown to increase the crude protein digestibility and decrease the crude fiber digestibility of giant pandas . Consistently, higher crude protein digestibility and lower crude fiber digestibility were observed in bamboo shoot-fed adult and geriatric giant pandas compared to those fed with bamboo leaves in the present study, which might be attributed to the inhibition of crude protein utilization induced by the higher level of fiber in bamboo leaves . In rodent models, the aging process was found to decrease lipid absorption through reducing the pancreatic lipase activity . In this study, bamboo shoot consumption increased the ether extract digestibility in aged giant pandas rather than in adults compared to bamboo leaf feeding. This finding might be related to the lower lipase activity in the small intestine of senior giant pandas and the higher ether extract content in bamboo leaves. Compared with adults, the ether extract in bamboo leaves was too high for aged giant pandas to fully digest, resulting in the lower digestibility of ether extract in senior pandas fed with bamboo leaves than those fed with bamboo shoots . Accumulated evidences have demonstrated the possible role of the gut microbiota in the regulation of nutrient harvest in humans and monogastric animals . More typically, as the giant panda lacks enzymes for the digestion of bamboo, it has thus been suggested that the giant panda appears to have no alternative but to rely on symbiotic gut microbes to extract nutrients from its highly fibrous bamboo diet . A previous study contended that dietary shifts induced changes in nutrient digestibility in captive giant pandas and were associated with the alteration of the microbiota composition . Both bamboo plant part and age have been shown to play a critical role in shaping the gut microbiota profile in captive giant pandas , however the interaction between bamboo plant part and age on intestinal microbiota composition, as well as the relationship between the interaction-induced gut microbiota shifts and nutrient digestibility of the captive giant pandas, remains unknown. Consistent with the previous study showing a more diverse gut microbiome in bamboo shoot-fed giant pandas than their counterparts , we found that bamboo shoot feeding increased the observed species, Chao1, Shannon, and Simpson indexes in both adult and old giant pandas. This indicates that there is a more abundant and diverse microbiome in bamboo shoot-fed giant pandas. Research showed that the elderly pandas exhibited lower bacterial species richness and diversity than the younger individuals . However, in this study, the main effect of age on the alpha diversity indices of microbiome in giant pandas was not observed, which is inconsistent with findings in rodents in which the microbial composition was generally affected by age rather than diet . This indicates the predominant role of dietary shifts rather than age in shaping the gut microbiota of giant pandas. The dissimilarity distance analysis in the present study also confirmed that the fecal microbiota of giant pandas could be sorted into two clusters by bamboo part independent of age. It has been demonstrated that phyla Firmicutes and Proteobacteria were the most predominant bacteria in the fecal microbiome of giant pandas . In the present study, bamboo shoot feeding decreased the abundance of Firmicutes and increased the abundance of Proteobacteria in the adult group rather than the geriatric group compared to bamboo leaf feeding. This is contradictory with the previous finding that the relative abundance of Proteobacteria was the highest in the bamboo-leaf fed giant pandas . However, in vivo studies in rodents revealed that bamboo shoot-derived components promoted the colonization of bacteria belonging to Proteobacteria and decreased the abundance of Firmicutes bacteria in the gut . The contradictory results might stem from the different study subjects or use of different bamboo species. Previous studies in monogastric animals showed that the relative abundance of Acidobacteriota was positively correlated with the intake amount of dietary protein and the relative abundance of Bacteroidetes was negatively correlated with dietary protein level . In the present study, the higher abundance of Acidobacteriota and lower abundance of Bacteroidetes were observed in bamboo shoot-fed giant pandas regardless of age, which might be attributed to the higher amount of protein in bamboo shoots than bamboo leaves . Consistent with the previous findings , the genera Escherichia-Shigella and Clostridium_sensu_stricto_1 were predominantly present in the fecal microbiome of giant pandas in this study. Bamboo shoot consumption has been shown to decrease the abundance of Escherichia-Shigella and increase the abundance of Weissella in the feces of giant pandas . Our study further revealed that the bamboo shoot feeding-induced changes in Escherichia-Shigella and Weissella abundances were only observed in aged giant pandas. In addition, the decreased abundance of Clostridium_sensu_stricto_1 was observed in bamboo shoot-fed adults rather than geriatric giant pandas compared to the bamboo leaf group. This finding was consistent with the previous study showing the higher abundance of Clostridium_sensu_stricto_1 in the bamboo leaf consumption stage versus bamboo shoot consumption stage . The inconsistent findings demonstrate that the genus Clostridium_sensu_stricto was not significantly enriched in the bamboo leaf stage and showed low sensitivity to the host's seasonal dietary changes . These contradictory results regarding the effects of bamboo part consumption on predominant genera abundance in giant pandas further suggest that the distribution of bacteria at the genus level in giant pandas might be dependent on the interaction effect of dietary shifts and age of the host. Seasonal variations in bamboo part selection has been shown to shape the bacteria distribution at the genus level of giant pandas . The abundances of genera Cellulosilyticum, Lactococcus, and Streptococcus were significantly affected by the consumption of different bamboo parts . Consistently, in this study, bamboo shoot feeding significantly increased the abundance of Cellulosilyticum, Lactococcus and other genera as well as decreased the abundance of Streptococcus in feces of both adult and aged giant pandas compared with bamboo leaf ingestion. In monogastric animals, the shifts in gut microbiota composition were found to closely correlate with nutrient digestibility . The genus Streptococcus was positively related to crude fiber digestibility in pigs . In this study, the genera Streptococcus and Lachnospiraceae_NK4A136_group were positively correlated with crude fiber digestibility in giant pandas, indicating the critical role of these two genera in the utilization of crude fiber of bamboo. High protein diets and ingredient consumptions have been shown to increase the abundance of the genera Turicibacter and Lactococcus in rodents . In the present study, the genera Turicibacter, Lactococcus, and other genera were positively correlated with the crude protein digestibility of giant pandas, which indicates that these bacteria may be important for the protein utilization of the bamboo parts. Taken together, the gut microbiota composition of giant pandas was mainly shaped by bamboo part consumption rather than age. 5. Conclusions In conclusion, bamboo shoot feeding increased the crude protein digestibility and decreased the crude fiber digestibility of giant pandas regardless of age. Bamboo part consumption dominated over age in shaping the gut microbiota composition of giant pandas. The shifts in taxa distribution at genus level might be responsible for the bamboo part-induced nutrient extraction alterations. Supplementary Materials The following supporting information can be downloaded at Table S1: the dominant phyla (average abundance > 1%) in different groups; Table S2: the dominant genera (average abundance > 1%) in different groups. AS, adult giant panda fed with bamboo shoots; AL, adult giant panda fed with bamboo leaves; OS, old giant panda fed with bamboo shoots; OL, old giant panda fed with bamboo leaves. Click here for additional data file. Author Contributions Experimental design and data interpretation, Y.Y., M.L. and H.Y.; sample collection and pre-processing, W.Z., G.X., R.L. and Y.D.; writing--original draft preparation, Y.Y. and W.Z.; writing--review and editing, M.L. and H.Y. All authors have read and agreed to the published version of the manuscript. Institutional Review Board Statement The animal study protocol was approved by the Institutional Animal Care and Use Committee of Chengdu Research Base of Giant Panda Breeding (No. 2020010, 2020-4-17). Informed Consent Statement Not applicable. Data Availability Statement The research data in this study has been deposited in the NCBI BioProject database with the accession number PRJNA916390. Conflicts of Interest The authors declare no conflict of interest. Figure 1 Effects of bamboo part consumption and age on alpha diversity indices of fecal microbiome in captive giant pandas. (A) Observed species. (B) Chao1 index. (C) Shannon index. (D) Simpson index. AS (n = 11), adult giant pandas fed with bamboo shoots; AL (n = 11), adult giant pandas fed with bamboo leaves; OS (n = 11), old giant pandas fed with bamboo shoots; OL (n = 11), old giant pandas fed with bamboo leaves. In each panel, values are presented as mean +- standard error; bars with different letters denote a significant difference (p < 0.05). Figure 2 The beta diversity index and structure analysis of fecal microbiome in giant pandas. (A) The inter-group Bray-Curtis distance. (B) Non-metric multidimensional scaling (NMDS) ordination plot derived from the Bray-Curtis distances in feces of giant pandas. AS (n = 11), adult giant pandas fed with bamboo shoots; AL (n = 11), adult giant pandas fed with bamboo leaves; OS (n = 11), old giant pandas fed with bamboo shoots; OL (n = 11), old giant pandas fed with bamboo leaves. In panel (A), values are presented as mean +- standard error; bars with different letters denote a significant difference (p < 0.05). Figure 3 Effects of bamboo part consumption and age on the taxa distribution at the phylum level in giant pandas. (A) Heat map of relative abundance of phyla. (B,C) Significantly different phyla in feces among four groups. AS (n = 11), adult giant pandas fed with bamboo shoots; AL (n = 11), adult giant pandas fed with bamboo leaves; OS (n = 11), old giant pandas fed with bamboo shoots; OL (n = 11), old giant pandas fed with bamboo leaves. In panel (B,C), values are presented as mean +- standard error; bars with different letters mean significant difference (p < 0.05). Figure 4 Effects of bamboo part consumption and age on the taxa distribution at the genus level in giant pandas. (A) Heat map of relative abundance of top 30 genera. (B,C) Significantly different genera in feces among four groups. AS (n = 11), adult giant pandas fed with bamboo shoots; AL (n = 11), adult giant pandas fed with bamboo leaves; OS (n = 11), old giant pandas fed with bamboo shoots; OL (n = 11), old giant pandas fed with bamboo leaves. In panel (B,C), values are presented as mean +- standard error; bars with different letters mean significant difference (p < 0.05). Figure 5 Network analysis between the top 30 genera and nutrient digestibility in giant pandas. The nodes were colored according to nutrient digestibility and genus. Only correlations with Spearman's coefficient r > 0.5 and p < 0.05 are shown. animals-13-00844-t001_Table 1 Table 1 Effects of bamboo part and age on apparent nutrient digestibility of captive giant pandas. Parameters Adult Giant Panda Old Giant Panda SEM p-Value Shoots Leaves Shoots Leaves Bamboo Part Age Bamboo Part * Age Dry matter (%) 26.56 32.67 32.67 26.44 3.66 0.99 0.99 0.10 Gross energy (%) 41.11 ab 43.00 a 35.89 ab 28.11 b 4.56 0.52 0.04 0.30 Crude protein (%) 81.78 a 58.67 b 83.11 a 53.56 b 1.95 <0.01 0.31 0.10 Ether extract (%) 52.56 a 47.22 a 49.00 a 12.22 b 0.77 <0.01 < 0.01 <0.01 Crude fiber (%) 14.67 bc 27.67 a 7.78c 22.01 ab 4.26 <0.01 0.07 0.89 Note: Within a row, values with different letter superscripts indicate significant difference (p < 0.05). * means the interaction between bamboo part and individual age. 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PMC10000147

The Calabrian Alpine newt (Ichthyosaura alpestris inexpectata) is a glacial relict with small and extremely localised populations in the Catena Costiera (Calabria, Southern Italy) and is considered to be "Endangered" by the Italian IUCN assessment. Climate-induced habitat loss and recent fish introductions in three lakes of the Special Area of Conservation (SAC) Laghi di Fagnano threaten the subspecies' survival in the core of its restricted range. Considering these challenges, understanding the distribution and abundance of this newt is crucial. We surveyed the spatially clustered wetlands in the SAC and neighbouring areas. First, we provide the updated distribution of this subspecies, highlighting fish-invaded and fishless sites historically known to host Calabrian Alpine newt populations and two new breeding sites that have been recently colonised. Then, we provide a rough estimate of the abundance, body size and body condition of breeding adults and habitat characteristics in fish-invaded and fishless ponds. We did not detect Calabrian Alpine newts at two historically known sites now invaded by fish. Our results indicate a reduction in occupied sites and small-size populations. These observations highlight the need for future strategies, such as fish removal, the creation of alternative breeding habitats and captive breeding, to preserve this endemic taxon. amphibian Urodela conservation Ichthyosaura alpestris inexpectata Calabria Italy endemism invasive fish morphometrics Regione Calabria103/2021 This study was supported by research grant number 103/2021, funded by Regione Calabria, FESR-POR Calabria 2014-20120-Azione 6.5.A.1-Sub Azione 1. pmc1. Introduction The biodiversity crisis is particularly dramatic in freshwater ecosystems . This biodiversity crisis requires actions and conservation measures at multiple geographical scales, global, regional, and local, considering both conservation priorities and limited resources . The most often used criteria to establish conservation priorities are taxon rarity and endemism (e.g., ). IUCN criteria consider the limited number of localities of a given taxon as a key factor for assigning high-risk categories , while other conservation projects (e.g., the EDGE of Existence programme) involve other evaluation frameworks, such as including endangered species that embody a substantial portion of distinct evolutionary heritage. The insular nature of lentic freshwater habitats has produced a high rate of endemism with small geographic ranges (and reference therein). Amphibians are nowadays subject to a plethora of possible threats and pressures, ranging from alien species' introduction to pollution, emerging infectious diseases and habitat loss, which sometimes act in synergy and result in detrimental effects at both individual and community levels (e.g., ). Alien species in freshwater habitats are among the greatest threats to native biodiversity . Indeed, for amphibians in general and newts in particular, fish introduction is considered a determining factor of pond-breeding populations' decline and extinction . Naturally, the impact of alien species is particularly alarming for native taxa with a restricted range. Ichthyosaura alpestris inexpectata (Dubois & Breuil, 1983), known as the Calabrian Alpine newt, is an endemic taxon of Southern Italy considered to be a post-glacial relict with small and fragmented populations . It represents a unicum of high biogeographical and conservation value, with populations exhibiting genetic variation likely originating from the Middle Pleistocene and possessing limited genetic diversity, which supports a long-standing isolation hypothesis . Since the early 1980s, the newt has been known in five localities in the north of the Catena Costiera (a coastal chain representing the first mountain range of the Calabrian Apennines), in ponds and lakes of varying depths localised in clearings within beech forests at an altitude ranging from 850 to 1135 m a.s.l. . All these sites currently fall within the Natura 2000 Network established under the EU Habitats Directive (92/43/EEC) (Table 1). The Calabrian Alpine newt is assessed as "Endangered (EN)" in the Red List of Italian Vertebrates because of the low number of occurrence localities, the continuous degradation and loss of the few sites due to the impact of human activity, and the recent introduction of allochthonous ichthyofauna in its core range. Since 2017, in fact, three fish species, Cyprinus carpio, Gambusia holbrooki and Carassius sp., have been observed in three newt breeding sites in the Special Area of Conservation (SAC) "IT9310060 Laghi di Fagnano", originally devoid of fish fauna . Moreover, other natural pressures, such as the transformation of habitats due to silting phenomena and drought, represent worrying critical issues , as was also found for another peninsular Italian amphibian species (and references therein). The aims of this study are threefold. First, we aim to provide an updated framework of the presence/distribution, status and threats of the Calabrian Alpine newt. Second, we aim to supply baseline ecological data for each inhabited site derived from recent systematic monitoring in the SAC Laghi di Fagnano. Third, we aim to provide the first demographic and population parameters. Specifically, we surveyed all spatially clustered wetlands in the protected area where the focal species was previously observed and the two recently discovered sites, thus giving the most updated information about I. a. inexpectata breeding habitats (e.g., habitat features and aquatic macroinvertebrate assemblages). Moreover, we collected data regarding abundance, body size and body condition in four sites (two new and two historical), expanding the information on morphometrics and sex ratio to obtain the first perceptions of demography. 2. Materials and Methods 2.1. Study Taxon The Alpine newt Ichthyosaura alpestris (Laurenti, 1768) is widely distributed in most of western and central Europe and the Balkans . In Italy, this urodele has a discontinuous distribution with three subspecies, two of which have a large distribution area and one of which is extremely localised. Ichthyosaura a. alpestris (Laurenti, 1768) is widespread in the Alpine area, I. a. apuana (Bonaparte, 1839) occurs in the Langhe hill system, Turin hills, Oltrepo' Pavese (Northern Italy), the Tuscan-Emilian Apennines, the Maritime and Apuan Alps and a restricted and disjointed area within the Laga Mountains (Central Italy); conversely, I. a. inexpectata has a very limited range in Southern Italy . Adults of this medium-sized newt present an aquatic lifestyle in spring, enabling them to breed in ponds with aquatic and riparian vegetation, transparent waters and short periods of winter frost. Reproduction occurs mainly in May and June, and no autumn breeding events are known to date . Paedomorphic phenotypes were observed in Lago Due Uomini and Laghicello . Available data on ecological requirements, phenology and life history traits, and demographic information for I. a. inexpectata are scarce or outdated. Knowledge of feeding habits only refers to the population of Laghicello . In addition, for this site, the rough estimation of a few hundred individuals of I. a. inexpectata (210-300) is the only one reported in the literature . The same authors assumed that the total number of mature individuals in all populations (from Laghicello and Fagnano lakes) could be estimated between 840 and 1200 . Much less is known about populations in Laghi di Fagnano, which have never been assessed. 2.2. Study Area and Site Description We surveyed seven lentic habitats, ranging from temporary ponds to permanent lakes . Five sites fall within the Natura 2000 site "Laghi di Fagnano", which protects a natural system of lakes and ponds with an elevation range from 1000 to 1100 m a.s.l. There are nine other amphibian species within this network of wetlands, in addition to I. a. inexpectata (Table 2). Due to this species diversity, the site was recognised as an Area of National Herpetological Relevance by the Societas Herpetologica Italica in 1998 (AREN--ITA022CAL002) . The Special Area of Conservation covers a surface area of around 19 ha, and it extends mainly along the slopes of Monte Caloria (Fagnano Castello) along a sub-plateau belt in which high-grade metamorphic rocks outcrop. The local climate is strongly influenced by the warm and humid air masses from the Tyrrhenian Sea, which give rise to thick fog formations and frequent and abundant rainfall that help mitigate the long, dry summer . The site falls within the lower supratemperate bioclimatic belt with upper subhumid ombrotype . The ponds and lakes network are within beech forest formations (Fagus sylvatica), included in habitat 9210*, with a rich shrub and herb layer. The climatic conditions and soil characteristics allowed the formation of peat bogs consisting of a layer of sphagnum and a mosaic of different plants of humid and marshy environments linked with dynamic successions due to variations in water level . A detailed description of the habitats and vegetation in the SAC is available at (accessed on 12 January 2023). Briefly, a higher and constant water depth of up to 2 m characterises "Lago Due Uomini" (hereinafter referred to as DU), which hosts submerged vegetation of Potamogeton natans and a massive presence of Phragmites australis. "Lago Trifoglietti" (TR) is a silting peat bog consisting of a thick moss layer of Sphagnum auriculatum with various species of Carex rostrata, C. paniculata, Juncus effusus, Lysimachia vulgaris, Eupatorium cannabinum and, in the most flooded areas, P. natans and Eleocharis palustris. Moreover, the lake holds a unique palynological archive that has yielded important information on the dynamics of Holocene vegetation and climate in southern Italy . A spring flows into the lake from the north, whereas an outflow runs southward. To prevent dry-up during summer and complete infilling, the municipality of Fagnano Castello built a small earthen dam in 2000 that led to the formation of a small underlying pond called "Trifoglietti inferiore" (TRIF). "Lago Fonnente" (FO) refers to an area including the main lake and an adjacent flooded meadow, which dries up in the dry season, and a pond, historically called "Fosso Armando" (FA) which is adjacent but not connected with the other wetlands. FO shows well-preserved marsh vegetation with sedges and rushes and is characterised by Typha latifolia and P. natans. During the study period, the contiguous flooded meadow appeared almost completely dry in June, when numerous dead larvae of all three species of newts and tadpoles of Hyla intermedia were found. Therefore, the site was not visited further in subsequent samplings. The new breeding sites of the Calabrian Alpine newt are constituted by two woodland ponds found in the localities of Piano di Zanche (PZ) and Pantano Lungo (PL) (municipality of Fagnano Castello) during a herpetological survey in 2015 and, after discovering, visited sporadically. The description of these habitats is given in Section 3. 2.3. Sampling Procedures We sampled the five sites in the SAC Laghi di Fagnano and the two new ponds from April to October 2022, during and after the newts' breeding season (May-July), once a month. PZ and PL were visited six times (except in May) due to a weather change (sudden thick fog) preventing sampling. All visits were conducted during daylight hours. Newt sampling techniques differed across sites due to the intrinsic features of each. Sites TR and DU are the most extensive, and due to the morphology and presence of dense aquatic vegetation, these cannot be easily and completely surveyed. Therefore, the visual encounter survey method (VES) was used for the lakes TR and DU by walking across the entire wadable surface of the wetlands for at least 30 min, so as to cover, in that time, the entire pond perimeter (~14 m/min). While searching for newts, we also reported the detection of fish. For FO, FA, TRIF, PZ and PL sites, survey protocol required at least two observers to enter the wetland and capture animals using dip netting. At least three removal passes lasting 20 min were made during a single visit, removing the individuals captured at each pass to avoid multiple counts and applying the removal sampling scheme to estimate population abundance (see below). All the captured newts were handled with latex gloves and temporarily kept in plastic containers filled with water from the pond and measured for body mass (to the nearest 0.01 g using digital scales), snout-vent length (SVL) and total length (with a ruler to the nearest 0.1 cm). A randomly selected subset of captured adults (n = 25) for PL in June was measured, so as to make the captured individuals' number homogeneous. The sex was determined by typical secondary sexual characters. Recognition of newt species during larval stages was based on the comparative staging tables of Bernabo and Brunelli . Paedomorphic newts were distinguished by gill slits and three pairs of external gills, and evident secondary sexual characteristics . Once all measurements and data had been collected, newts were released into the capture pond. At each sampling session, other amphibian species occurring and breeding in the investigated aquatic sites were also recorded. 2.4. Habitats' Characteristics and Macroinvertebrates Assemblages We described the features of the seven sampled aquatic habitats with different hydrological balances and subjected to different pressures (fish-containing and fishless ponds) by recording the size, the water permanence, the distances between each pond and the water's physical-chemical characteristics. To evaluate the biodiversity levels and trophic availabilities of the four breeding water bodies where adult newts currently occur, we investigated the structure of macroinvertebrate assemblages. Therefore, macroinvertebrate sampling was conducted in June and October, following a time-space-standardised quantitative procedure. In each monitoring site, we sampled three main mesohabitats in fordable zones (emergent and submerged aquatic vegetation, littoral and central sediments) using a Surber-type sampler (square opening: 25 cm x 25 cm) (Scubla s.r.l., Remanzacco UD, Italy) equipped with a 60 cm net of 250 mmesh and a plastic gathering glass. All samples were sieved through a 0.45 mm sieve (Endecotts Ltd., London, UK) and immediately stored in refrigerated containers with 80% ethanol. Once in the laboratory, macroinvertebrates were separated from sediments through a stereomicroscope. All organisms were counted and identified at the family level to estimate the following parameters and indices: abundance (N), taxonomic richness (TR), Shannon-Wiener Index (H') and Margalef Index (D) . Water physical-chemical parameters were assayed twice during the study, in the early summer and autumn, with three measurements along a transect at about 10-20 cm below the surface water. Specifically, temperature, conductivity, pH and total dissolved solids were determined with a multiparameter probe (Hanna Instruments, model HI991300). Dissolved O2 and relative saturation percentage were measured with a portable oxygen meter (Hanna Instruments, model HI98193). 2.5. Data Analysis We analysed the dataset, collecting information on adults of the Calabrian Alpine newts captured in TRIF, FA, PZ and PL (Table 2). The sample size from the four populations in several months was too small since we captured only larvae and <3 individuals; therefore, for each population, we pooled the total number of captured males and females during the sampling period. Operational sex ratio in each sampling was expressed as the proportion of males over the total adult number (males/(males + females)); deviations from equality were assessed via a two-tailed binomial test when more than ten newts were caught. To assess the body condition (a proxy for physical condition, overall health and fitness ) of the four populations of Calabrian Alpine newts, we calculated the scaled mass index (SMI) . This index provides a comparable measure across individuals of different sizes by rescaling body mass measurements to a standard length (here calculated for SVL) accounting for allometric growth . We calculated SMI separately for males and females within each site, thus avoiding the scaling issue that results when comparing BCIs across groups known to differ in size . SMI is computed as follows (with Mi and Li being the body mass and the linear body length, respectively, bSMA the scaling exponent estimated via the SMA regression of M on L, and L0 the mean length of the study population):M^i=Mi[L0Li ]bSMA We quantified the scaling exponent bSMA in R by using 'smatr' package from ln-transformed data. The normality of morphometric data (body mass, SVL, total length and SMI) was analysed via the Shapiro-Wilk test and the homogeneity of variances was analysed via the Brown-Forsythe test. Data fit normal distributions and resulted homoscedastic; therefore, we used a one-way analysis of variance (ANOVA) followed by Tukey's multiple comparisons test to determine whether there was a difference in body size and body condition between sex within populations and comparing females and males, respectively, among populations. We also tested the effect of fish presence, macroinvertebrate diversity (in terms of Shannon index), site and month on the SMI using a linear model. We used OriginPro, Version 2022b (OriginLab Corporation, Northampton, MA, USA) to produce plots. We estimated newt abundance through the most recent field capture data to obtain updated information about TRIF, FA and PL (sites where sufficient numbers of adult newts were found). Considering that the sampling method we used can be ascribed to the constant probability of capture (each pass does not lower or increase the probability of capture of the subsequent pass, and the chance of sampling each individual is constant, both for their detectability and/or population stability) and the removal of captured individuals was performed at each pass, we used the whole capture dataset, setting the removal method = 'Zippin' . The population size was estimated through the 'FSA' package in R. We considered the highest population estimate in the month of greater detectability of adult individuals (present in the water during the breeding season). 3. Results 3.1. Current Distribution Framework and Habitats' Characteristics Based on our multi-year observations (1984-1992, 2005, 2015-2018) and during a sampling visit in June 2022, as well as from information gathered from locals and landowners, the occurrence of the Calabrian Alpine newt population reported in the SAC "Pantano della Giumenta" is seriously questionable, since the last observation refers to the early 1980s . In Laghicello, the presence of Alpine newts was verified during a single visit in April 2022; no fish were introduced, but the site appeared subjected to advanced natural silting phenomena (I. Bernabo pers. obs.). During surveys conducted in 2022 in three study sites in the SAC Laghi di Fagnano (FO, TRIF and FA), Calabrian Alpine newts were observed at least once for each life stage, whereas in DU and TR we did not observe any . A true absence was not proven, and we could not exclude its presence in the deep central areas of the lakes, which cannot be sampled except with methods other than those used. Individuals of the common carp (Cyprinus carpio) and of the Eastern mosquitofish (Gambusia holbrooki) were highly abundant in the lakes DU and TR, respectively; the presence of cyprinid fish (Carassius sp.) and G. holbrooki was also recorded in FO and TRIF, respectively, but at a low density (Table 2). The two new records were confirmed to be breeding sites : PZ is a temporary pond, and the water comes almost exclusively from rainfall; conversely, PL is a large woodland puddle that presents a mostly stable hydroperiod thanks to a water flow from a spring. Both sites have significant tree cover and low O2 levels due to a high plant debris load; woods of Fagus sylvatica dominate the vegetation around the two wetlands, and no water vegetation (macrophyte or algae) is present. Water bodies' environmental features (measured during the last field campaigns) are summarised in Table 2 and in Table S1. Considering the linear distances among clustered wetlands in the SAC of Laghi di Fagnano and the new sites, we found that the nearest was PL (835 m away from FA), whereas PZ occurs at a greater distance (1321 m from TRIF) . About the amphibian community, among the ten species occurring in the SAC, only Bombina pachypus was not found. Rana dalmatina was the most common species (in 100% of the sites), followed by Lissotriton italicus and Triturus carnifex, and then Pelophylax sinkl esculentus (71%). Salamandra salamandra, Rana italica, Hyla intermedia and Bufo bufo were detected in 43% of the aquatic sites (Table 2). Syntopy of I. a. inexpectata with L. italicus occurred in five sites (FO, TRIF, FA, PL and PZ), and it also lived with T. carnifex in four ponds (FO, TRIF, FA and PZ). Regarding aquatic macroinvertebrate assemblages, a total of 43 families, 15 orders, and 7 classes of invertebrates, cumulatively, were identified in the four study sites (TRIF, FA, PZ and PL). The taxonomic list of all organisms collected at each monitoring site is reported in Table S2. All ponds in both sampling campaigns showed high diversity indices, indicating stable habitats for aquatic invertebrates. In particular, PL showed the highest values of diversity indices (TR: 25, H': 3.056, D: 3.050), followed by FA (TR: 15, H': 2.727, D: 2.261) and TRIF (TR: 17, H': 2.555, D: 2.119), whereas PZ had the lowest ones (TR: 12, H': 2.439, D: 1.591). Moreover, the macroinvertebrate community biodiversity increased in terms of number of taxa from June to October, due to the significant input of trophic resources associated with woody/leaf litter, with an overall autumnal increase of 15.7%. 3.2. Population parameters In TRIF, we captured 53 adult Calabrian Alpine newts from May to September, with a maximum of 34 individuals captured in June (20 males and 14 females; estimated abundance 37 +- 3, 95% C.I. = 30-44) , whereas we observed numerous larvae from late summer to autumn, with many of these showing evidence of predation (i.e., tails and legs partially nipped). TRIF was the only site where we detected paedomorphic individuals (four males and three females); paedomorphic males and females had an average SVL (+- SE) of 38.8 +- 0.4 mm and 41.8 +- 0.5 mm, respectively. In FA, 30 adult newts were captured from April to June, with a maximum of 15 captured adults in April (10 males and 5 females; estimated abundance 20 +- 8, 95% C.I. = 5-35) when the pond, characterised by a highly variable water level, presented its maximum water level . The pond was almost completely dry (level of about 20 cm) from the end of July until October, and only abundant larvae were observed. In PZ, we collected eight adults (five males and three females) of Calabrian Alpine newts in June, whereas in September, we found one female with a few larvae . The complete drying of the pond was detected in July and August. In PL, we detected adult Calabrian Alpine newts every month except May (see Materials and Methods); larvae were observed from the end of July to October, including overwintering larvae. Altogether, we captured 129 adults; the highest number of individuals was observed in June (33 males and 35 females) . The estimated maximum abundance of newts was 88 +- 14 (estimate +- SE; 95% C.I. = 62-115). In individual samples with N >= 10, our results showed a male-biased sex ratio of 0.59 (p = 0.256) in TRIF. In FA, the proportion significantly differed from 0.5 both in April (0.67) and June (0.73) (all p < 0.05), whereas, in PL, a significant deviation from 50:50 toward males (0.65) occurred only in April (p < 0.05). In all populations except for PZ (due to its small size), females were confirmed to have larger body sizes than males (F7,116 = 10.50, Tukey's multiple comparisons test, p < 0.001) . Overall, the average (+-SD) SVL was 47 +- 3.5 mm in males and 53 +- 4.2 mm in females, whereas the average total length was 81 +- 5.4 mm and 95 +- 5.5 mm, respectively. Sex differed statistically in body mass within sites (F7,116 = 17.86, Tukey's multiple comparisons test, p < 0.001). The mean body mass was 2.6 +- 0.5 g in males and 4.4 +- 1.1 g in females, showing no intra-sexual divergence between populations (F7,116 = 0.55, Tukey's multiple comparisons test, p > 0.05). There were no differences among populations in females' or males' body mass, SVL or total length . SMI differed statistically between sexes in each site, and males exhibited the lowest body condition index (Tukey's multiple comparisons test, all p < 0.001); neither male nor female body conditions varied significantly among the four populations . Moreover, we found no significant effect of fish presence, macroinvertebrate diversity, site or month on the SMI (R2 adj. = 0.073, p > 0.05 for all of the factors considered). 4. Discussion 4.1. Updated Distribution, Species Occurrence and Habitat Characteristics In recent years, no effort has been directed towards surveying and monitoring the Calabrian Alpine newt. Our results update the information on the newt distribution, providing records of local disappearance from three historical sites and the discovery of two new breeding ponds. The Calabrian Alpine newt was traditionally considered to occur in five localities (see Table 1): three in the SAC Laghi di Fagnano (FO, DU, TR), one in the locality Laghicello and one at the SAC "Pantano della Giumenta". Their occurrence in this protected area, where a goldfish population (Carassius auratus) has been stable for almost twenty years, has not been confirmed since 1984, and the population has to be considered extinct. In Laghicello, the endemic newt still occurs. In contrast, in two of the three historically known breeding sites in the SAC Laghi di Fagnano, DU and TR, the newts were not recorded after fish introduction in 2017 and 2019, respectively. However, past surveys (until 2018) applying VES, when the two lakes were fishless, allowed the detection of all newt species. Thus, VES may not be the most efficient way to detect newts in these two studied lakes and could lead to false absences. Furthermore, to avoid predation risk from fish, amphibians can increase shelter use and reduce activities , resulting in a lower probability of being detected and, likewise, reduced foraging activities and reproduction, two essential fitness components . On the other hand, it is also well known that newts avoid fish-invaded habitats . Another Alpine newt subspecies, the Bosnian Alpine newt I. a. reiseri (Werner, 1902) from the Prokosko Lake, was dramatically reduced or, more probably, disappeared from the lake following fish introductions . In our case, if the event is not a local extinction, it is a demographic reduction since the detection probability is also a function of the abundance . The progressive modification or loss (early desiccation and silting phenomena) of the few suitable habitats in which I. a. inexpectata breeds and the substantial threat posed by the introduction of invasive fish may lead to reproductive failure. This could result in the extinction of the endemic subspecies and those of other newts' populations of community and conservation concern. Fish eradication would also bring benefits to the two other newt species that are syntopic with the Calabrian Alpine newt, i.e., Triturus carnifex (Annex II and IV) and Lissotriton italicus (Annex IV), which are protected under the Habitats Directive and categorised as NT and LC by the IUCN, respectively. Knowledge of taxon distribution in a given area underpins most conservation efforts and planning . In the context of the severe threat to the survival of the Calabrian Alpine newt, the discovery of two new occurrence localities (PZ and PL) is crucial for the newts' persistence and highlights the importance of constant field monitoring to assess the species distribution at a local scale , which is a dynamic trait, determined by local extinction and new colonisation phenomena. These new breeding sites are small woodland ponds formed on natural depressions in well-preserved beech forests, away from the main roads and excessive tourism. Local landscape features include forest continuity and a network of ponds that allow the permeability of matrix habitats for dispersion. The linear distance and the narrow differences in altitude (about 200 m) separating the new ponds from the known ones of Laghi di Fagnano suggest that these sites may have been colonised through the dispersal of newts from the nearby locations. Indeed, the closest connection pathway may have been about 0.8 km from FO and FA toward the northeast for PL and about 1.3 km from TR and TRIF toward the southeast for PZ. Although fidelity to the breeding site is reported in the Alpine newt , movement capabilities over long distances, exceeding 1 km and up to 4 km, have also been reported . Future studies should map all wetlands in the vicinity of the SAC and consider inter-pond movements, migrations and dispersal to disentangle newt population dynamics and better understand how to plan adequate conservation measures, such as pond creation. In addition, conducting investigations with new approaches, such as eDNA, could be helpful in detecting newts' occurrence in suitable habitats. At high altitudes (i.e., in the Alps), I. alpestris usually lives in oligotrophic sites where the water is very clear. In the Apennines, on the other hand, it inhabits mid-altitude sites where water is frequently turbid, the maximum temperature is high and the environment is generally unpredictable . We provided the first information on water parameters where the subspecies I. a. inexpectata breeds. The new ponds can be considered oligotrophic environments based on physical-chemical measurements and total hardness (the total amount of Ca and Mg ranged between 3.29 and 11.4 mg/L) . Dissolved oxygen values are very low, suggesting a strong insaturation, most likely due to the scarce primary productivity and the high oxygen consumption triggered by a large amount of autochthonous and allochthonous plant detritus in the ponds. Here, we describe the first data on the potential feeding resources available for the Calabrian Alpine newt. Overall, the four studied ponds showed high diversity and a well-structured macroinvertebrate assemblage. Our biodiversity indices values are comparable to those calculated for other natural lakes and ponds and higher than those measured in artificial, garden and urban ponds . In October, the macroinvertebrate assemblages showed, in each pond, an increase in shredding invertebrates feeding on organic detritus derived from the riparian vegetation . With regard to hydrological variation, there was a drop in the water level in October at all sites, particularly in FA and PZ. However, this lower volume of water did not negatively affect the macroinvertebrate assemblages and seemed adequate to sustain the ecological niches occupied by these organisms. Most invertebrates found in breeding ponds are involved in the diet, during the aquatic phase, of adults, paedomorphs and metamorphs of any subspecies of I. alpestris . Typically, the Alpine newt shows generalist dietary habits and exhibits seasonal plasticity; it exploits temporary resources and differing prey taxa in relation to their cycles of availability and local abundance . However, an individual shift towards a more specialist feeding strategy in artificial sites in response to increasing prey diversity has been reported . In general, the abundance of invertebrate prey in the aquatic breeding sites allows newts not to leave these habitats to forage, a relevant factor for their conservation status . Our preliminary analysis reported no significant correlation between the body condition index, prey species availability and fish presence. The contribution of the newts to the feeding ecology may be elucidated only with dietary studies that may also clarify the trophic strategy of the Calabrian Alpine newt. Newts act as predators in naturally fishless ponds ; therefore, it is also essential to investigate if the unavoidable trophic competition with introduced fish may become a decisive limiting factor. Indeed, knowledge of the trophic ecology of threatened and protected newts is crucial to evaluating possible threats , especially when dealing with the effects of fish introduction, and thus to adopting appropriate conservation policies . 4.2. Population Parameters The ecology and demographic parameters of the Calabrian Alpine newt are scanty or outdated. Although long-term monitoring programmes are required for more robust estimations of trends in their distribution and abundance, our survey confirmed that the overall population of the Calabrian Alpine newt in its core range might be much smaller than hitherto assumed. Indeed, the only available study by Dubois and Ohler in 2009 on the population of Laghicello roughly estimated counts of between 210 and 300 individuals. The authors supposed that the total number of mature individuals at the four historically known sites (Table 1) is probably much less than four times the estimated population size of Laghicello. Altogether, 90 adult newts were caught in the ponds within the SAC Laghi di Fagnano, whereas 138 adults were sampled at the two new sites. The highest number was found in permanent ponds. A few adults and larvae were found in PZ; this pond may completely dry out in the warm season and is replenished by late summer rainfall. The detection of larvae of the three newts at the end of September confirmed a late summer reproduction in this water body. However, its interannual hydroperiod is not well-studied, and it remains unclear what reproduction and metamorphosis success occurs at this pond. PL was confirmed to be a suitable breeding habitat of high conservation value for the Calabrian Alpine newt and other amphibians; here, the most abundant population is present. Population monitoring and demographic estimates in the breeding sites of Laghi di Fagnano are pivotal for the persistence of the Calabrian Alpine newt after fish introduction. To ensure accurate population size estimates, we plan to adopt a monitoring scheme using aquatic funnel traps in lieu of dip-netting and a capture-mark-recapture approach. Throughout the breeding season, we found statistically significant sex bias in caught newts towards males in FA (every sampling) and PL (in April). In each sampling event, males outnumbered females. The observation of distorted sex ratios in amphibians using counts or captures may reflect an actual ecological trait of the studied populations but may also be an artefact due to different capture probabilities between sexes , and references therein]. In other populations of Alpine newts, sex ratios have been reported to be either female-biased . According to Lanza and coauthors , both sexes remain in the water throughout the breeding season. However, males were more numerous than females at the breeding sites early in the year, and females outnumbering males in late spring and early summer represents a weaker trend in many newt species, including the Alpine newt . Given our data, we can speculate that the sex ratio in the studied ponds during the breeding season is quite dynamic and is determined by locally specific factors. Further studies are needed on such dynamics, which may vary between different types of water bodies (e.g., temporary vs. permanent, temperature, food availability and habitat quality), influencing mating behaviour and the duration of the breeding season. Body condition is a measure of an animal's foraging success and physiological state . In our estimations of body condition, remarkable intersexual differences were found within each population. Males' and females' body conditions of Calabrian Alpine newts did not vary significantly among the four study ponds, which were both with and without fish and had differences in their hydrological regime. Further long-term monitoring studies are required to determine the seasonal and inter-annual variation in SMI values for each population and the effects of fish presence on newts' body condition. Our results on body size are comparable with those available in the literature on inexpectata and other subspecies. Sexual size dimorphism was detected in our study populations, with females being longer and heavier than males, as previously reported in the literature . 4.3. Threats, Conservation Measures and Active Management We suppose that within a few years, fish may cause the extirpation of the Calabrian Alpine newt and other native amphibian species from otherwise viable habitats. Meanwhile, the temporary ponds, although protected from the introduction of fish due to their hydroperiod, could likely disappear or become unsuitable habitats for amphibians in a changing climate. Omnivorous fish, such as carp, can cause an increase in water turbidity by damaging plant communities and accelerating eutrophication processes, thus compromising the habitat choice and reproduction of several amphibian species . Predatory fish cause numerous adverse effects, such as direct predation on eggs and larvae, injuries due to predation attempts, resource competition, and changes in activity patterns and micro-habitat use . Furthermore, both fish categories may damage native amphibian populations by pathogen transference . We found paedomorphic Calabrian Alpine newts only in TRIF, and to date, paedomorphs have been reported to occur in two localities: Lago Due Uomini and Laghicello . Facultative paedomorphosis is frequent in several Italian populations of I. alpestris and is favoured in permanent aquatic habitats and where prey are abundant . Evaluating the occurrence and incidence of alternative paedomorphic phenotypes should be of interest, considering the impacts of competition and predation. Indeed, it has been documented that introduced fish have determined the decline of metamorphs and the disappearance of paedomorphs from many newt sites in Europe . A recent study showed several detrimental effects of the invasive G. holbrooki on paedomorphs of Lissotriton graecus, which exhibited avoidance behaviour and higher metamorphosis rates in the presence of fish . The eradication or control of non-native invasive fish species in the lakes of Fagnano are undoubtedly unique and powerful conservation tools to improve the status of endemic and protected newts as well as the whole amphibian community. Given the different ecological and biological conditions of each water body, a targeted strategy combining several fish management techniques is necessary. Eradication programmes should include physical removal and chemical approaches after a risk analysis to decide which strategy should be chosen and what the likelihood of success is. Considering the relatively high financial costs of eradication, the prioritisation of fish removal from ponds of higher suitability for newts could buffer the impact of fish presence . Frog and salamander populations are expected to recover within a few years after fish removal from lakes . The proximity of stable newt-breeding populations that can act as both sources and sinks may strongly favour the long-term persistence and newt recolonisation of lakes that have been returned to a fishless condition . Therefore, in this emergency context, it is mandatory to create a network of small satellite artificial and semi-natural ponds, spatially connected to the historical sites, where to maintain newt populations until programmes to remove fish from lakes can be carried out. The identification of sites where to create suitable habitats should be realised through field surveys and the application of the most recent ecological modelling techniques to indicate the areas with major potential for retaining water, and also predicting changes in temperature and precipitation. Regarding the newly discovered localities, both sites deserve regular interventions to guarantee water permanence and reduce silting phenomena by enlarging and deepening the ponds and removing excessive biomass accumulation. Furthermore, continuous surveillance is desirable to quickly notice any introduction of fish. Finally, to deal with the 'emergency' situation, priority conservation action should be taken to preserve the Calabrian Alpine newt by implementing an ex situ conservation programme with research and conservation facilities in scientific institutions or zoos in Italy and Europe. The necessary ex situ initiatives will complement and support the in situ conservation of this highly endangered taxon, also considering that its disjunct distribution and its narrow range make this Alpine newt, by far, the most relevant biogeographic and conservation-related taxon compared to all of the other five Ichthyosaura alpestris subspecies . Thus, the conservation of the Calabrian Alpine newt is not only important in terms of protecting taxonomic diversity but, being an evolutionarily significant unit, it assumes relevance for the purpose of better conserving the whole species' genetic diversity . 5. Conclusions Updated distribution information and ecological data are mandatory to start a specific management plan for an endemic taxon on the brink of extinction, which is deserving of urgent conservation actions. Our observations facilitate conservation and management activities in the SAC Laghi di Fagnano and the surrounding area. The radical ecological upheaval following fish introductions undermines the survival of the Calabrian Alpine newt and the other two syntopic pond-breeding newts of community and conservation interest. An effective long-term management strategy to restore disturbed habitats by removing fish cannot be further postponed, including pond creation to provide all the amphibian species with alternative habitats. Local biodiversity and ecological functions greatly benefit from the eradication or control of non-native fish. Intensive monitoring and a systematic collection of data to ascertain more in-depth key ecological requirements and population status over a long period are also essential. Acknowledgments We thank Eleonora Bozzo, Giuseppe De Bonis, Maria Prigoliti and Carlo Terranova for their help in conducting the fieldwork and Antonio Lucadamo for statistical advice. We are grateful to Giovanni Aramini (Environment and Territory Department of the Calabria Region) for his support to the problem of the conservation of the Calabrian Alpine newt. We are also grateful to the three reviewers, whose comments greatly improved the quality of our research. Supplementary Materials The following supporting information can be downloaded at: Figure S1: Representative images of the new breeding sites of Ichthyosaura alpestris inexpectata; Table S1: Water physical-chemical parameters measured in the study ponds; Table S2: Macroinvertebrates taxonomic list and relative abundance; Table S3: Morphometric parameters of Ichthyosaura alpestris inexpectata from the study sites. Click here for additional data file. Author Contributions Conceptualisation, I.B.; methodology and formal analysis, I.B., V.C., A.R., A.C. and M.I.; investigation, I.B., V.C., A.C., M.G.S. and S.T.; data curation, I.B. and V.C.; writing--original draft preparation, I.B., A.R., V.C. and M.I.; writing--review and editing, I.B., V.C., M.I., F.A., A.R., A.C., M.B. and S.T.; supervision, M.B. and S.T. All authors have read and agreed to the published version of the manuscript. Institutional Review Board Statement Permits for field work and sampling procedures were obtained by the Italian Ministry of the Environment (prot. number: 0008263--25 January 2022). Data Availability Statement Not applicable. Conflicts of Interest The authors declare no conflict of interest. Figure 1 Italian ranges (minimum convex polygons based on ) of the two Italian endemic subspecies of the Alpine newt Ichthyosaura alpestris, namely, I. a. apuana (light blue) and I. a. inexpectata (green); in dark grey, the Calabria region, where our target subspecies (I. a. inexpectata) occurs. Figure 2 (a) Distribution and putative population sizes in the known localities of Ichthyosaura alpestris inexpectata based on literature data and historical observations; see Table 1 for details; (b) Results from recent surveys. Figure 3 (a) Water bodies' network within the SAC "Laghi di Fagnano" (Laghicello and Pantano della Giumenta sites are excluded from the view) and (b) the distances between each site (including all); the two new sites and the correspondingly shorter distances from which the Calabrian Alpine newt possibly colonised them are highlighted through boxes (full and dashed lines, respectively). Figure 4 Captures of adults of Ichthyosaura alpestris inexpectata at four of the study sites during the sampling period. The months with no water are highlighted in grey. Asterisks mark a significant deviation from evenness, * p < 0.05, ** p < 0.01 (Fisher's exact probability test); arrows indicate when larvae were found. Figure 5 Boxplots of: SVL (a), total length (b), body mass (c), and SMI (d) for I. a. inexpectata males and females in the four sites. The horizontal white bars and circles represent median and mean values, respectively. The box is determined by the 25th and 75th percentiles, and the whiskers refer to the minimum and maximum values observed within data for each population. Outliers are shown as grey circles. One-way ANOVA with post hoc Tukey tests: * p < 0.05, ** p < 0.01; *** p < 0.001. animals-13-00871-t001_Table 1 Table 1 Summary of the data related to known sites. The last year of observation of Ichthyosaura alpestris inexpectata for each site is reported. Maximum number of adults captured or observed is based on information retrieved from the available literature or collected ante 2022 in the DiBEST database. Trifoglietti: TR = large pond Trifoglietti; TRIF = small pond Trifoglietti Inferiore. Fonnente: FO = Lake Fonnente; FA = Fosso Armando. Site Code N2000 Municipality Locality Year of Last Observation Maximum Number Captured/Observed (1983-2018) References IT9310058 Pantano della Giumenta Malvito Stagno C/Pantano dorato 1983 4 IT9310060 Laghi di Fagnano Fagnano Castello Trifoglietti (pond TR and pond TRIF) 2018 (TR) and 2022 (TRIF) 23 Due Uomini 2018 21 Fonnente (lake FO and pond FA) 2022 25 IT9310061 Laghicello San Benedetto Ullano Laghicello 2022 69 animals-13-00871-t002_Table 2 Table 2 Geographical locations, morphometric parameters and environmental characteristics measured in the study ponds. Site name: Fonnente (FO), Due Uomini (DU), Trifoglietti (TR), Trifoglietti inferiore (TRIF), Fosso Armando (FA), Piano di Zanche (PZ), Pantano Lungo (PL). The approximate area was calculated using Google Earth Pro satellite images (May 2022). Pond water depth (considering the maximum seasonal depths measured in April-May 2022), pond regime (P: permanent; T: temporary pond subject to drying out at least during a single survey), shading (i.e., the extent of shading of the site during the surveys) and presence or absence of fish are reported. Life stage: adults = A, larvae = L; overwintering larvae = OL; paedomorphs = P. Species abbreviations: Tla: Typha latifolia; Pna: Potamogeton natans; Pau: Phragmites australis; Cve: Carex vesicaria; Sau: Sphagnum auriculatum; Cro: Carex rostrata; Cpa: C. paniculata; Jef: Juncus effusus; Lvu: Lysimachia vulgaris; Eca: Eupatorium cannabinum; Epa: Eleocharis palustris; Lit: Lissotriton italicus; Tca: Triturus carnifex; Ssa: Salamandra salamandra; Hin: Hyla intermedia; Pes: Pelophylax sinkl esculentus; Rda: Rana dalmatina; Rit: R. italica; Bbu: Bufo bufo. Site Name FO DU TR TRIF FA PZ PL Lat. N Long. E 39deg33'24'' 16deg1'13'' 39deg33'9'' 16deg1'22'' 39deg32'54'' 16deg1'2'' 39deg32'53'' 16deg1'27'' 39deg33'23'' 16deg1'18'' 39deg32'11'' 16deg1'34'' 39deg33'48'' 16deg1'30'' Altitude (m a.s.l.) 1050 1077 1048 1045 1055 880 1010 Area (m2) 3561 20,679 10,800 520 827 742 1 235 1 Depth (m) 1 - 1.5 2 0.9 1.2 0.7 1.1 Permanence P/T 3 P P P T T P Shading (%) 20 20 30 80 20 90 90 Fish presence Carassius sp. Cyprinus carpio G. holbrooki G. holbrooki - - - Aquatic vegetation Tla, Pna Pna, Pau, Cve Sau, Cro, Cpa, Jef, Lvu, Eca, Epa, Pna Cve, Jef Jef absent absent I. a. inexpectata L - - A, L, P A, L A, L A, L, OL Other breeding species Tca, Lit, Pes, Rda, Hin Tca, Bbu, Pes, Rda Hin, Pes, Rda Lit, Tca, Ssa, Pes, Rda, Rit, Bbu Lit, Tca, Ssa, Hin, Pes, Rda, Rit Lit, Tca, Bbu, Rda Lit, Ssa, Rda, Rit 1 Area calculated by measuring the maximum length and width and assuming an elliptical shape. 2 From . 3 Temporary as referred to the adjacent flooded meadow. 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PMC10000148

Developing sound breeding programs for aquaculture species may be challenging when matings cannot be controlled due to communal spawning. We developed a genotyping-by-sequencing marker panel of 300 SNPs for parentage testing and sex determination by using data from an in-house reference genome as well as a 90 K SNP genotyping array based on different populations of yellowtail kingfish (Seriola lalandi). The minimum and maximum distance between adjacent marker pairs were 0.7 Mb and 13 Mb, respectively, with an average marker spacing of 2 Mb. Weak evidence of the linkage disequilibrium between adjacent marker pairs was found. The results showed high panel performance for parental assignment, with probability exclusion values equaling 1. The rate of false positives when using cross-population data was null. A skewed distribution of genetic contributions by dominant females was observed, thus increasing the risk of higher rates of inbreeding in subsequent captive generations when no parentage data are used. All these results are discussed in the context of breeding program design, using this marker panel to increase the sustainability of this aquaculture resource. seriola inbreeding genotyping-by-sequencing ANID, Ministry of Science, Chile1191189 University of Chile1074/2022 Strategic Science Investment Fund of the NZ Ministry of Business, Innovation and EmploymentThis research was funded by FONDECYT grant Ndeg 1191189, from ANID, Ministry of Science, Chile, and by the ENLACE Ndeg 1074/2022 grant from the University of Chile and by the Strategic Science Investment Fund of the NZ Ministry of Business, Innovation and Employment. pmc1. Introduction The yellowtail kingfish (Seriola lalandi) is a pelagic carnivorous fish that inhabits tropical and temperate waters of the Southern Hemisphere and the Northern Pacific, with known populations in Australia, New Zealand, Japan , the Southeast China Sea , the Mediterranean Sea , and the Pacific coast of South America . As the demand for yellowtail kingfish continuously grows and fishery quotas have reached maximum levels , commercial aquaculture production of this species has been successfully established in Australia , the Netherlands, and Denmark, while the establishment of new farms has been planned in New Zealand as well as North and South America . The complete production cycle of S. lalandi has been successfully set up along the northern coast of Chile . However, relatively little attention has been paid to a proper understanding of the genetic structure and diversity of both natural and captive populations of this species. In countries such as Germany, the Netherlands, and the USA, commercial aquaculture has been established using juveniles from single farms (particularly from Chile, Juan Lacamara, Pers. Comm.), where broodstock were initially sourced from natural fisheries, often with little or no attention to genetic diversity . Nevertheless, such practices are not compatible with the long-term viability of the industry . Traditional pedigree-based breeding programs for S. lalandi can also prove challenging. Similar to many other species, S. lalandi is a communal broadcast spawner that readily breeds in captivity without requiring hormonal inductions or gamete stripping for in-vitro fertilization (IVF) . While this trait is highly beneficial for practical husbandry and production purposes, the absence of controlled matings prevents the holding of progeny by family, which allows for simple (non-molecular method) pedigree tracking. Combined with their high fecundity rate and propensity for skewed parental contribution , there is a high risk that high inbreeding rates arise in breeding programs, leading to a rapid reduction in effective population size (Ne) . Furthermore, the difficulty in securing an even representation of all the broodstock in single production batches adds complexity for accurately assessing genetic parameters. For example, heritability and genetic correlations for harvest traits estimated in a commercial population were in most cases not statistically significant, which is likely due to the low number of parents (8 sires and 6 dams) used for estimation of such parameters . For this reason, it is necessary to develop accurate marker panels to assess parentage while using pedigrees as a tool to control the rates of inbreeding and maintain genetic diversity in sustainable breeding programs. Genetic studies for paternity testing have so far been carried out using microsatellite markers developed from other species of the same genus . Using heterologous microsatellites can introduce biases when assessing population variability; for instance, null alleles and homoplasy may falsify the genetic structure and parentage testing. The aim of this study is to develop a novel genotyping-by-sequencing (GBS) marker panel for parentage testing using single nucleotide polymorphism (SNP) data obtained from a genotyping array and a whole S. lalandi genome assembly, as presented elsewhere . We focused our attention on the performance of this panel under real experimental conditions, considering the information of progeny data from two source populations, one native to Chile and another to New Zealand (NZ). This information was used to calculate predicted inbreeding rates using the genetic contribution theory and variation in family size. The results are discussed from the perspective of developing breeding programs for "these new" aquaculture species that are effective in increasing the genetic gain while constraining the rates of inbreeding. 2. Materials and Methods 2.1. Production System and Data Sampling Fish from Chile were sampled from a captive S. lalandi broodstock population held in Acuinor S.A. Company's facilities in Caldera City, located in the Atacama Region. These fish were captured from a wild population at Punta Frodden as well as from different locations near Caldera (27.0deg S; 70.8deg W) and kept to conform to the base population of the national S. lalandi breeding programs (56 dams and 51 sires). The individuals were arranged in four independent breeding units (R1, R2, R3, and R4 with about 20-30 fish per tank). Hence, they were exposed to different photoperiods and temperature increases to ensure the availability of larvae throughout the entire year. The progeny was reared under standard farm conditions, and the fish were kept until harvest at about 3 Kg in different Recirculating Aquaculture Systems (RAS) units. We sampled fin clips from all Chilean broodstock and 161 randomly sampled progeny from the different breeding units (R1, R2, and R3) produced by mass spawnings. All fish were anaesthetized before sampling using MS-222 as part of the management plan to measure production variables. Fish from New Zealand (n = 31) were sampled from a captive broodstock originated from the east coast of the Northland province (35deg S, 174deg E) North Island and was kept in captivity for 2 to 8 years. The animals had been held at the NIWA Northland Aquaculture Centre, located in Ruakaka. Fin clips were kept frozen or in >70% ethanol before DNA extraction. Genomic DNA was extracted using the NucleoSpin(r) Plant II kit (Macherey-Nagel(r), Duren, Germany) according to the manufacturer's instructions. The samples were quantified using a Qubit fluorometer (Thermo Fisher Scientific, Waltham, MA, USA) with the Qubit dsDNA BR Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA). DNA was normalized to a final concentration of 2 ng/mL. 2.2. Genotyping-By-Sequencing (GBS) Panel Construction The marker panel was developed using the genomic resources within the national breeding program of S. lalandi. To develop the genetic resources needed for its implementation, we first produced an in-house genome assembly for the species. This reference was developed by sequencing a single male with a coverage of 70x using a mixture of single and mate Illumina pair-ends reads. The draft genome was assembled using MaSuRCA with the default parameters. An additional 34 individuals (17 males and 17 females) were sequenced at 10x coverage to discover SNPs using the scaffolds obtained by MASURCA and the procedures explained below. These fish were also used to discover SNPs associated with the sex determination gene, that appeared to be causal . We used 90 K SNPS selected (out of more than 5 million SNPs discovered) to develop the 90 K-SNP genotyping array (constructed by ThermofisherTM, Waltham, MA, USA). Scaffolds were anchored to linkage groups with CHROMONOMER using the linkage distance between 90 K SNPs markers as obtained from the genotyping array using LEP-MAP (we used information on 200 progeny from 10 full sibs families to generate the linkage map ). The assembled genome comprised 24 linkage groups with a total genome size of about 657 Mb encompassing 95% of the total expected genome. More detailed information will be provided in a separate study (in preparation ). Selected markers used for the construction of the GBS panel were obtained from genotype data of the base (founder) population of the national Chilean breeding programme of S. lalandi. In this case, a total of 300 SNPs were selected using genotype data obtained from the 90 K-SNP-genotyping array of the S. lalandi broodstock population as obtained from data from the broodstock population genotyped with the 90 K-SNP-genotyping array. The SNPs (280 markers) used for parentage assignment in the panel were evenly placed across all 24 linkage groups. The markers were selected based on informativeness using a minimum allele frequency (MAF) of 0.34. The average marker spacing is 2 Mb . All the markers selected for the panel followed Hardy-Weinberg equilibrium in the broodstock population and showed no linkage disequilibrium within linkage groups. We included 20 markers in the vicinity of the diagnostic SNP used for sex prediction (these markers were obtained from a genome-wide association analysis as explained above and will be published in a separate study ). For obtaining the actual genotypes of the progeny, a targeted GBS protocol was used. A total of 300 primer pairs were developed based on proprietary software from Thermo Fisher (accessed on 20 January 2021); Thermo Fisher Scientific, Waltham, MA, USA). A total of 192 libraries were prepared using AgriSeqTM HTS Library Kit (Thermo Fisher Scientific). Libraries were genotyped using the Ion 540 Chef kit along with the Ion 540 Chip (~80 million reads) kit (Thermo Fisher Scientific). Sequences were mapped to the S. lalandi genome assembly using BWA . SAM files were sorted with SAMtools , and PCR duplicates were removed using SAMBAMBA . SNPs were identified using FreeBayes accessed on 30 March 2021) with default settings. The initial set of SNPs identified was filtered using vcftools based on the following criteria (derived from the FreeBayes output): (1) a Phred-scaled SNP quality score with significance greater than 30; (2) minimum allele frequency of 0.49; (3) maximum percentage of missing values of 0.20; and (4) the maximum number of alleles as 2. 2.3. Genotype Detection and Parentage Analysis Using the GBS Panel SNP variation statistics were obtained separately for the different datasets (New Zealand (n = 31) and Chile (n = 161)) when using the GBS panel. We calculated minimum allele frequency (MAF), Hardy-Weinberg kh2 statistics, and observed heterozygosity (He) using PLINK . The cumulative probability of exclusion (CPE) for single-parent (CPE-1) and both-parent inference (CPE-2) were calculated using formulae of Jamieson and Taylor , as well as the polymorphic information content (PIC) with equations obtained from . Parentage assignment was carried out using data from the Chilean broodstock (which was genotyped by the 90 K genotyping array) and progeny (161, as explained above) with AlphaAssign . This procedure relies on a maximum likelihood approach to infer parents (sires or dams) using default parameters. In practice, sex is predicted at the progeny level with the GBS marker panel, making it possible to independently assign sires or dams in the next generation when selecting potential broodstock from the progeny available. This is expected to increase the accuracy of the procedure by reducing the number of possible single-parent (sire or dam) and progeny pairs when calculating the likelihood . Since we did not use the GBS panel for genotyping the parents, we obtained genotypes of the parental generation by extracting the markers selected for the GBS panel from data obtained from genotypes of broodstock using the 90 K-SNP-genotyping array. The predicted rates of inbreeding were calculated using two different methods. The first one uses genetic contribution theory, assuming random selection and discrete generations . In this case, an estimate can be obtained by using the following approximation (Equation (1)):(1) DF=14[ ri2] where ri is the mean parental genetic contribution for each parent calculated, from generation 0 to generation 1, using the predicted pedigree. The mean genetic contributions were obtained from the additive relationship values between parents and progeny and then averaged over the total number of progenies. The values of ri summed over each sex (dam or sires) equal 0.5. We also used the method derived by for estimating inbreeding effective size that incorporates the offspring contribution (Equation (6), without selfing from ), as:(2) Ne=2S-2 (ki2)2S-1 where S is the total number of progenies, and ki is the family size for each of the parents (males and females). This method is subject to relatively large standard errors when the number of parents is large and the number of offspring is small (giving an upward bias to Ne, as obtained from simulations ). The estimates of the rates of inbreeding and effective size were obtained as follows: (3) DF=12Ne The effective numbers of founders (ENF), was calculated as :(4) ENF=1/[ ri] All these calculations were carried out using PEDIG and Excel spreadsheets, using the pedigree predicted with AlphaAssign. Estimates of Ne and predicted rates of inbreeding were obtained using information from all the breeding units together as well as separately. 3. Results 3.1. Genotyping-By-Sequencing (GBS) Marker Panel Assessment We assessed the informativeness of the GBS marker panel across the different populations analyzed (New Zealand and Chile). After sequencing, a total of 188 out of 192 samples passed with a minimum sample call rate of 97% (with an average of 98%). The average coverage was 1152x per marker. Seven SNP markers did not pass the quality control since they had a low call rate and were excluded. One additional marker showed a MAF equal to 0 in the progeny. Therefore, a total of 272 markers were mapped to the 24 chromosomes using BWA . These markers were used in the final parentage analysis based on the GBS panel, and 20 markers were used for sex prediction. The list of SNPs and their detailed information is given in Supplementary Table S1. When examining the 272 markers used for paternity testing, MAF values ranged from 0.11 to 0.50, with an average of 0.36 for the entire dataset . The percentage of SNPs with a MAF between 0.40 and 0.50 was 36% and 40%, respectively, for the Chilean and New Zealand populations. In addition, the average polymorphic information content (PIC) was 0.50 and 0.53 for the Chilean and New Zealand populations, respectively. The realized average linkage disequilibrium (r2) between adjacent marker pairs within chromosomes was 0.05 and 0.04 for the Chilean and New Zealand populations, respectively. In addition, the CPE for single-parent (CPE-1) and both-parent (CPE-2) inference were in all the populations higher than 0.999. 3.2. Parentage Testing and Distribution of Genetic Contributions The average likelihood difference between unrelated parents (sires or dams), which is calculated to assign parentage, was substantially higher than in the case of chosen candidate parents using all marker data . The parents that were not assigned to offspring had values for the likelihood difference near zero. We tested individuals from New Zealand (as progeny) with putative parents from Chile. In this analysis, no putative sires or dams were assigned to the individuals coming from the New Zealand sample population, so the rate of false positives was negligible (data not shown). We tested different scenarios to assess the effect of the number of markers and the performance of the maximum likelihood approach for assessing parentage . This was achieved by randomly deleting markers used to infer parentage with PLINK using the thin option. We found a linear trend between the number of markers and the difference between the likelihood for a specific set of markers and the full set of markers (the ratio of the average difference between the likelihood for a reduced set of markers and the full set of markers was 20 to 100%). The linear regression coefficient was about 0.04 units per marker . The probability of non-assignment (using the number of parents not assigned due to a reduced likelihood difference from the total number of parents assigned) is highly dependent on the number of markers. The threshold for accurately assigning parental data was about 150 markers. . The parental assignment revealed an extreme asymmetry in the parental contributions, particularly concerning the females . In the more extreme case, a dam was indicated as the single parent of almost all the progeny in one of the production batches . The mean number of offspring per dam (17 out of 55 dams were assigned) was 12, with a very high variance (293). The males contributed more evenly to the gene pool. The average offspring count of males (36 out of 51 males were assigned) was 6, but with a much lower variance (24). The predicted rates of inbreeding varied significantly between methods, especially when considering individual breeding units. Overall, it was lower when using the method using genetic contributions (Table 1). Nonetheless, the predicted rates of inbreeding were in general very high when considering specific production groups, which is not surprising given the very low numbers of parents contributing within each batch, ranging between F1 4% and 7% and F2 ranging between 6% and 12% (Table 1). When considering all the groups together, the predicted rate of inbreeding was about 2.1% and 3.3% for DF1 and DF2, respectively. This gave the values of the effective population sizes of about 23.8 and 15.1 for Ne1 and Ne2 (see Table 1). The effective number of founders was about one-third the number of breeders contributing progeny in the next generation (Table 1). 4. Discussion Molecular parentage testing in aquaculture has been traditionally carried out using microsatellites . However, the general lack of species-specific standardized panels prevents efforts to automate the process, resulting in microsatellites not being routinely used for parentage analyses as a tool to control inbreeding. Furthermore, many microsatellites developed on congeners (e.g., S. quinqueradiata) are not usable due to their low informativeness and the presence of null alleles. Indeed, our research team showed that 10 out of 25 non-focal microsatellites are useless for parentage analysis . Additionally, microsatellite panels are not useful for sex determination, requiring a separate step of high-resolution melt PCR . For all these reasons, it is important to develop marker panels using SNPs, which are faster to obtain, more accurate, and cheaper when compared with microsatellites. In this study, the realized value for the cumulative probability of exclusion for a single or both parents was 1, suggesting that the GBS marker panel of 272 markers can be used very efficiently for parentage testing in captive S. lalandi populations. In fact, when using only 150 SNPs as markers, there is enough power to perform parentage analysis , with a rate of non-assignment lower than 5%. We also found that false positives are rare, since when performing parentage testing on an unrelated population, no related parents were obtained when more than 150 markers were used. 4.1. Predicted Rates of Inbreeding in S. lalandi The predicted rates of inbreeding calculated in this study should be interpreted as long-term estimates and not as forecasts of average inbreeding in the next generation. Furthermore, these rates should be thought of as rough estimates as they only rely on information from a relatively low number of single spawning events over limited time spans. Therefore, the genetic contributions are not at their asymptotic values, leading to an underestimation of the rates of inbreeding in the long term. This can be seen in Table 1, where values based on genetic contributions are smaller than estimates from another study . Some unintentional selection is likely ongoing in the population since, in large production batches, individuals are graded to optimize feeding practices while decreasing the rates of cannibalism . This preselection phase may explain, to a certain degree, the high variance of some of the parents assigned. Nevertheless, specific females seem to dominate single spawning events since they appear in half-sib maternal families with several males, which is in accordance with what had previously been observed in this species . Therefore, inbreeding is of concern in this species since, without intervention, it will be extremely high within a short time, and strategies to control its rates are needed. Overall, the predicted inbreeding rates in our breeding units were generally very high. In all cases, values of the effective population size were much smaller than the ones required to reduce the risk of extinction in species with high reproductive output . In practice, a cumulative number of spawnings should be used in order to secure a sustainable breeding program (see below). This means that the generation interval will be higher when compared with other species (i.e., salmonids), since multiple egg batches should be kept over longer time spans. There is some scope to constrain the rates of inbreeding by avoiding mating between related individuals, at least in the short term. In our case, since the founders of the population are the actual broodstock used for parentage testing, it is straightforward to select replacements based on the minimum inbreeding (using the progeny candidates) using a rotational mating scheme. This system has been devised in the classical paper by Kimura & Crow and applied to Coho salmon (Oncorhynchus kisutch) to replace broodstock . Nevertheless, this methodology would only be useful for the short term, and a high rate of inbreeding has been observed in the long term . In this mating system, broodstock was kept in a few separate groups, and males were transferred sequentially and circularly between neighboring groups, which were kept isolated . In the simplest system to be considered, n males and n females were arranged alternately so that each potential sire or dam mated with individuals of a neighboring reproduction unit. In this classical system, the second generation is the product of a half-sib or full-sib mating (when dams are also replaced). We have applied this replacement method by sequentially assigning sires and dams to unrelated reproduction units (R1, R2, R3, and R4). This procedure gave null inbreeding in the second generation, as expected since the co-ancestry between breeders within tanks was still 0 (only unrelated founders are available in generation 0). Further investigation is needed to understand the consequences of this type of mating in the long term. 4.2. Low-Density Marker Panels, Genomic Prediction, and Two-Stage Selection Programmes We have shown that specific progeny batches represent a reduced number of founders from the previous generation, even though the number of progenies can be large (since potentially millions of eggs are produced in each spawning event). This situation will lead to a series of practical problems when implementing genomic breeding programs in practice. First, if a reduced number of batches are produced in each generation, it will be difficult to predict accurate breeding values using SNPs since the associations between markers and quantitative trait loci (QTL) would not represent associations at the population level (most of the information will come from a small number of full-sib families). Secondly, progeny from single spawning events are related to half-sib maternal families, and therefore (when mated), they will produce inbred offspring. In the case of S. lalandi, several batches are produced within a year, but the progeny of each batch mostly originates from a single dominant female . For these reasons, it is important to include selection candidates from as large a number of batches as practicable in order to represent a significant proportion of the genomic variation at the population level. Secondly, one major issue in developing breeding programs for S. lalandi using genomic information is the genotyping cost of using, in particular, SNP-genotyping arrays. The price of the GBS marker panel is about 10% of the cost of the full genotyping array, giving some scope for implementing profitable selection programs, as we detailed as follows. We have previously found that in salmonids, a two-stage selection program gave the most profitable breeding results at the expense of maximum genetic gains and constrained rates of inbreeding . Therefore, an essential part of the design should follow a procedure for maintaining high levels of genetic variability by keeping a larger number of production batches in the first stage of selection while selecting for the traits of interest in different stages of the life cycle. This can be completed in the early stages, when the fish are still comparatively small in size and, hence, easier to manage (when fish can be handled in large batches from different spawnings at lower body weights, for example, at 10 months). At this stage, a set of diverse production batches can be sampled based on the trait of interest and then subjected to paternity testing for culling related individuals, based on a procedure minimizing coancestry levels between chosen candidates. The parentage testing using the GBS marker panel in this stage is essential to reconstruct the pedigree of the population and secure enough individuals of each sex differing by some degree of sexual dimorphism for body weight (females appear to be heavier than males during maturation) . A second stage should be performed by selecting individuals based on the genomic breeding values of the selected population while applying optimal contribution selection . In this case, breeding values should be predicted for harvest weight (usually 3-5 kg.) using information from a reference population and a denser genotyping array or a modification of a GBS genotyping incorporating SNP associated with traits of interest or using imputation. The exact proportions of individuals to be used for either stage require further investigation, as does the assessment of the optimum selection intensity to maximize profit . Another possibility is to use a smaller number of markers in genotyping panels for selecting individuals in a single stage (usually 3-5 kg) using imputation. This will decrease the genotyping costs while possibly maintaining the rates of gain, as has been suggested before . Nonetheless, it is difficult to constrain the inbreeding rate in these scenarios while using a reduced number of production batches for selecting individuals as potential selected broodstock candidates. Further research on this subject is required to disentangle the factors enabling sustainable rates of genetic gain. 5. Conclusions In this study, we developed a marker panel to perform parentage testing as well as sex determination ("Martinez manuscript in prep.") in S. lalandi. This resource is essential when implementing breeding programs, since controlling inbreeding rates requires not only parentage testing but also predicting candidate sex. We used markers spanning all the linkage groups devised using recombination data from a reference genome. This enabled us to select markers with high variability and minimal linkage disequilibrium. The validation in two unrelated populations (New Zealand and Chile) suggests that only half of the markers are required for parentage testing with sufficient power. No false positives were detected when considering cross-parentage testing between populations. This panel is key to developing sound breeding strategies that constrain the rates of inbreeding in the short and long term. This is particularly important considering the difficulties in carrying out breeding programs in species subjected to communal rearing and uncontrolled reproduction. These achievements will increase the viability of this new aquaculture resource and, as such, the success of the associated business activities. Supplementary Materials The following supporting information can be downloaded at: Table S1: Position and Minimum allele frequency for each SNP used in paternity testing. The position was obtained by using . Click here for additional data file. Author Contributions Conceptualization, V.M.; methodology, V.M.; validation, V.M. and N.G.; formal analysis, V.M.; investigation, V.M., N.G. and A.S.; resources, A.S.; data curation, V.M.; writing--original draft preparation, V.M.; writing--review and editing, V.M., N.G. and A.S; project administration, V.M.; funding acquisition, V.M. All authors have read and agreed to the published version of the manuscript. Institutional Review Board Statement All procedures involving the handling and treatment of animals were approved by the Bioethics Committee of the Veterinary Science Faculty, University of Chile, Chile (Permit Number. 10-2015). Informed Consent Statement Not applicable. Data Availability Statement The marker data is available upon request to the corresponding author. All the primers used for sequencing will be available for sequencing using Ion 540 chef upon request. The reference genome will be available upon request. Conflicts of Interest The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results. Figure 1 (a) Number of markers per chromosome (Y-axis) versus chromosome (linkage group: LG) size in Mb (assume 1cM equals Mb: X-axis). The linkage group 14 (LG14) included the markers for sex determination. (b) Markers used for the GBS panel and MAF. The outer layer shows the genome, the second layer (rectangle) shows the position of the markers, while the third, fourth, and fifth layers the observed MAF for the New Zealand and Chilean populations. Figure 2 Histogram of the likelihood difference between the chosen candidate (sires or dams) and unrelated sires or dams for each progeny evaluated using all markers included in the panel (272 SNPs). Figure 3 (a) Ratio of the average difference between the likelihood (PL) of the chosen parent and an unrelated individual for a different number of markers (expressed as a proportion when using all markers); (b) Proportion of non-assignment (PNA) given a different number of markers used for parentage testing. Figure 4 Bubble graphs showing the mean genetic contributions of single dams (a) and sires (b) across different breeding units. The Y-axis denotes the proportion of offspring per dam and sire. animals-13-00913-t001_Table 1 Table 1 Predicted rates of inbreeding and effective population size (Ne) for different production batches. DF1 (Predicted rate of inbreeding: Wooliams and Thompson, 1991); Ne1 (Ne: Wooliams and Thompson, 1991); DF2, (Predicted rate of inbreeding: Waples and Waples, 2005); Ne2 (Waples and Waples, 2005); ONF (Observed number of females); ONM (Observed number of males); NF (Number of contributing females); NM (Number of contributing males); ENF, (Effective number of founders); Ekf (Average family size of females); Ekm (Average family size of males); Vkf (Variance of family size of females); Vkm (Variance of family size of males). R3' corresponds to a different production batch from the breeding unit R3. Breeding Unit DF1 Ne 1 DF2 Ne 2 ONF ONM NCF NCM ENF Ekf Ekm Vkf Vkm R1 0.06 7.9 0.12 4.1 18 13 5 9 4 7 4 192 17 R2 0.06 8.6 0.09 5.8 15 9 5 10 4.9 12 3 292 6 R3 0.07 7.4 0.11 4.5 13 15 3 6 3.7 12 4 96 1 R3' 0.04 12.7 0.06 9.2 13 15 7 14 7.1 9 3 115 21 Overall 0.02 23.8 0.03 15.1 46 35 17 41 13.3 12 6 293 25 Disclaimer/Publisher's Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

PMC10000149

Osteosarcoma is considered the most common bone tumor affecting children and young adults. The standard of care is chemotherapy; however, the onset of drug resistance still jeopardizes osteosarcoma patients, thus making it necessary to conduct a thorough investigation of the possible mechanisms behind this phenomenon. In the last decades, metabolic rewiring of cancer cells has been proposed as a cause of chemotherapy resistance. Our aim was to compare the mitochondrial phenotype of sensitive osteosarcoma cells (HOS and MG-63) versus their clones when continuously exposed to doxorubicin (resistant cells) and identify alterations exploitable for pharmacological approaches to overcome chemotherapy resistance. Compared with sensitive cells, doxorubicin-resistant clones showed sustained viability with less oxygen-dependent metabolisms, and significantly reduced mitochondrial membrane potential, mitochondrial mass, and ROS production. In addition, we found reduced expression of TFAM gene generally associated with mitochondrial biogenesis. Finally, combined treatment of resistant osteosarcoma cells with doxorubicin and quercetin, a known inducer of mitochondrial biogenesis, re-sensitizes the doxorubicin effect in resistant cells. Despite further investigations being needed, these results pave the way for the use of mitochondrial inducers as a promising strategy to re-sensitize doxorubicin cytotoxicity in patients who do not respond to therapy or reduce doxorubicin side effects. osteosarcoma cancer chemotherapy doxorubicin drug resistance metabolism mitochondria targeting mitochondrial alterations University of PadovaRAGA_BIRD2020_01 AIRCIG 2018-ID. 21403 This research was funded by the University of Padova, grant number RAGA_BIRD2020_01 and AIRC, grant number IG 2018-ID. 21403 project P.I. Nicola Baldini. pmc1. Introduction Osteosarcoma is a severe bone tumor mostly affecting children and adolescents . Its worldwide incidence rate reaches four cases per million per year . Current therapeutic management includes surgery, radiotherapy, and chemotherapy . The standard of care for chemotherapy is represented by methotrexate, cisplatin, and doxorubicin, or a combination of these three agents . Doxorubicin (DXR) is a chemotherapeutic agent intercalating DNA and belongs to the anthracycline family. The mechanism of action consists of the inhibition of DNA topoisomerase II, which blocks DNA replication hampering the recombination of DNA double-strand. Recently, another mode of action for DXR has been proposed: this mechanism involves increased reactive species of oxygen (ROS) levels which causes DNA damage and leads to cell death . The successful use of doxorubicin in clinical therapy has been jeopardized by severe toxicity, such as cardiotoxicity , and the onset of drug resistance . The main known mechanisms of DXR resistance involve increased drug efflux due to P-glycoprotein expression, which is also associated with a worse outcome , decreased drug influx as well as drug compartmentalization into lysosomes , and enhanced DNA repair or detoxification systems . In the last decades, even the deregulation of energy metabolism has been described as a hallmark of cancer and drug resistance. This phenomenon is also called metabolic reprogramming and it involves several alterations in metabolic pathways, such as increased glycolysis, a shift toward the pentose phosphate pathway, as well as enhanced lipid synthesis, higher exploitation of glutamine pathway, and mitochondrial alterations . Several studies highlighted the central role that mitochondria play in cancer development and progression , and in the onset of drug resistance, suggesting that targeting the mitochondrial pathway could represent a promising strategy to overcome drug resistance . Previous studies in our laboratory already identified that mitochondrial alterations are strictly linked with chemotherapy resistance. In particular, we demonstrated that ovarian and osteosarcoma cisplatin-resistant cancer cells presented defects in mitochondrial morphology, in particular reduced mitochondrial mass, suggesting an enhanced activation of mitophagy and a central role played by mitochondria in cisplatin resistance . Similarly, in DXR-resistant cells with a multidrug-resistant (MDR) phenotype, we also demonstrated a lower mitochondrial activity . In light of these considerations, our hypothesis is that DXR resistance in osteosarcoma cell models could be driven also by mitochondrial dysfunctions. The purpose of this paper is to characterize the mitochondrial phenotype of two osteosarcoma cell lines, MG63 and HOS, analyzing both sensitive and DXR-resistant clones with the aim of identifying alterations in resistant clones that could be exploited for cancer therapy. 2. Materials and Methods 2.1. Cell Lines The HOS and MG63 cell lines were kindly provided by IRCCS Istituto Ortopedico Rizzoli (IOR, Bologna, Italy), and derived from human osteosarcoma. The cell lines included in our research are commercially available; for this reason, it is not necessary to obtain ethical approval. The DXR-resistant variants HOS DXR 10 ng/mL, HOS DXR 30 ng/mL, HOS DXR 100 ng/mL, as well as MG63 DXR 30 ng/mL and MG63 DXR 100 ng/mL, were obtained by continuous exposure to increasing DXR (Teva Pharmaceutical Industries, Tel Aviv, Israel) concentrations, as reported by Avnet and coworkers . Approximately 2-3 weeks (4-5 passages) were required to establish adequate growth at each DXR concentration. The cells were grown in Iscove's Modified Dulbecco's Medium (IMDM) medium (Corning, New York, NY, USA) supplemented with 10% fetal bovine serum (FBS) (Thermo Fisher Scientific, Waltham, MA, USA), 4 mM glutamine (Corning, New York, NY, USA), 100 U/mL penicillin, and 100 mg/mL streptomycin (Corning, New York, NY, USA), in humidified condition at 5% CO2 and 37 degC. Cells were collected every 2 days with the minimum amount of 0.25% trypsin-0.2% EDTA (Euroclone, Milan, Italy). 2.2. Mitochondrial Network by Confocal Microscopy Cells were grown on glass coverslips for 24 h in a complete medium. Then, cells were fixed with paraformaldehyde 4% in phosphate buffer saline solution (PBS), stained with 20 nM Mitotracker Orange (ex: 554 nm/em: 576 nm) (Thermo Fisher Scientific, Waltham, MA, USA) for 30 min and Hoechst 33342 (Thermo Fisher Scientific, Waltham, MA, USA) for 20 min. Images were acquired by confocal microscopy LSM 800 and software ZEN 2.1 60x objective and analyzed by ImageJ version 1.53t (U.S. National Institutes of Health, Bethesda, MA, USA). 2.3. Mitochondrial Membrane Potential by Flow Cytometry Cells were seeded in 12-well plates and grown in complete medium, washed with PBS, detached with 0.25% trypsin-0.2% EDTA and centrifuged for 5 min at 1200 rpm. Cells were stained with rhodamine-123 10 mM (Sigma-Aldrich, St. Louis, MO, USA) and incubated for 15 min. Fluorescence intensity was analyzed using an Epics XL flow cytometer. Mean fluorescence intensity (MFI) values were obtained using the EXPO 32 software. 2.4. Mitochondrial Mass by Flow Cytometry Cells were seeded in 12-well plates and grown in complete medium, washed with PBS, detached with 0.25% trypsin-0.2% EDTA and centrifuged for 5 min at 1200 rpm. Cells were resuspended with nonyl acridine orange (NAO) 1.25 mM (Sigma-Aldrich, St. Louis, MO, USA) and incubated for 15 min. Fluorescence intensity was analyzed using an Epics XL flow cytometer version 3.0. Mean fluorescence intensity (MFI) values were obtained using the EXPO32 software (Applied Cytometry Systems, Dinnington, Sheffield, UK). 2.5. Quantitative Real-Time PCR Total mRNA was isolated with TRIzol (Themo Fisher, Waltham, MA, USA) as previously described by Chomczynski and Sacchi and quantified with Nanodrop. The relative expression of each gene was determined by quantitative real-time PCR (EcoTM Illumina, Real-Time PCR system, San Diego, CA, USA) using One Step SYBR PrimeScript RT-PCR Kit (Takara Bio, Otsu, Shiga, Japan) and the primers designed as follow: PGC-1a: F 5'-ACACAGTCGCAGTCACAACAC-3' R 5'-GGAGTGGTGGGTGGAGTTAGG-3'; NRF1: F 5'-GCTTGCGTCGTCTGGATGG-3' R 5'-GTAACCCTGATGGCACTGTCTC-3'; NRF2: F 5'-TTCCTTCAGCAGCATCCTCTCC-3' R 5'-AATCTGTGTTGACTGTGGCATCTG-3'; TFAM: F 5'-AACAACGAAAATATGGTGCTGAGG-3' R 5'-CAAGTATTATGCTGGCAGAAGTCC-3'; BNIP3: F 5'-GAATTTCTGAAAGTTTCCTTCCA-3' R 5'-TTGTCAGACGCCTTCCAATA-3'. Linearity and efficiency of PCR amplifications were assessed using standard curves generated by serial dilution of complementary DNA; melt-curve analysis was used to confirm the specificity of amplification and the absence of primer dimers. All genes were normalized to CALNEXIN or GAPDH designed as follows: CALNEXIN: F 5'-GAAGGGAAGTGGTTGCTGTG-3'; R 5'-GATGAAGGAGGAGCAGTGGT-3'; GAPDH: F 5'-AATCCCATCACCATCTTCCA-3' R 5'-TGGACTCCACGACGTACTCA-3'. Expression levels of the indicated genes were calculated by the DDCt method using, respectively, the dedicated StepOne software version 2.1 (Applied Biosystems, Waltham, MA, USA) or EcoTM Software v4.0.7.0 (Illumina Inc., San Diego, CA, USA). 2.6. Western Blotting The Lowry method was used to evaluate the protein content after the cells had been seeded in 6-well plates, cultured in complete media, and lysed using ice-cold lysis buffer supplemented with protease inhibitor cocktails (Roche Molecular Biochemicals, Mannheim, Germany) (Biorad DC Protein Assay, MA, USA). In the running buffer, 25 mg of protein from each sample was placed onto a polyacrylamide gel and electrophoretically separated. The proteins were blotted onto a Hybond-P PVDF membrane following electrophoresis (Amersham Biosciences, Buckinghamshire, UK). The membrane was exposed to anti-TOM20 (mouse, 1:1000; AbCam, Cambridge, UK), anti-VDAC1 (mouse, 1:1000; AbCam, Cambridge, UK), and anti-BNIP3 (rabbit, 1:1000; AbCam, Cambridge, UK) after blocking with a 10% skim milk solution. Following washing, the membrane was incubated with HRP-conjugated anti-rabbit secondary antibody (1:3500; PerkinElmer, MA, SUA) or anti-mouse secondary antibody (1:10000; PerkinElmer, MA, USA). According to the manufacturer's instructions, the signal was seen using an improved chemiluminescent kit from Amersham Biosciences, and then it was examined using a Molecular Imager VersaDoc MP 4000 (Biorad, Hercules, CA, USA). Proteins were normalized to b-ACTIN (mouse, 1:7000, AbCam, Cambridge, UK) and GAPDH (rabbit, 1:2000, Cell Signaling, Danvers, MA, USA). 2.7. Mitochondrial ROS Levels by Flow Cytometry A constant number of cells were seeded, and after 48 h, 5 mM Mitosox (Invitrogen, Waltham, MA, USA) was added and incubated for 30 min at 37 degC. Cells were then washed in PBS solution, detached using 0.05% trypsin and 0.02% EDTA, centrifuged for 10 min at 2000 rpm, and resuspended in a solution of PBS, 2% FBS, and 0.02% sodium azide (Sigma-Aldrich, St. Louis, MO, USA). Cells were then examined directly using a 488 Argon laser-equipped Epics XL Coulter Systems (Beckman Coulter, Brea, CA, USA). Dead cells were electronically gated out using forward versus side scatter profiles, and a minimum of 104 cells of interest were then examined further. Analysis using the EXPO 32 software produced the percentages of the fluorescent cells' values (Beckman Coulter, Brea, CA, USA). 2.8. Apoptosis Detection by Flow Cytometry Cells were seeded in 6-well plates and grown in complete medium, washed with PBS, detached with 0.25% trypsin-0.2% EDTA and centrifuged for 5 min at 1200 rpm. Cells were stained with FITC Annexin V (InVitrogen, Waltham, MA, USA) and Propidium Iodide according to manufacturer's instructions, and then diluted with Annexin V-binding buffer (InVitrogen, Waltham, MA, USA). Fluorescence intensity was analyzed using BD FACS Aria instrument. 2.9. Cell Viability Assays 2.9.1. Crystal Violet Assay Crystal Violet Assay exploits the ability of this dye to bind the DNA and proteins of adherent cells. For the assay, cells were seeded in 96-well plates, and, following overnight incubation, cells were treated with DXR (1-10-50-100-250-500 ng/mL) and quercetin (0.1-1-5-10-25-50-100-200 mM). After 24, 48, and 72 h of treatment, cells were fixed with paraformaldehyde 4% in PBS. Then, cells were stained with Crystal Violet (Sigma-Aldrich, St. Louis, MO, USA) solution 0.1 % and resuspended in acetic acid 10% in water. The absorbance (Abs) was measured at 590 nm using a Victor3X multilabel plate counter (Wallac Instruments, Turku, Finland). 2.9.2. Trypan Blue Assay Cells were plated on 12-well plates and, following overnight incubation, were exposed to different rotenone treatments (0.01-0.1-1 mM) according to the experimental protocol. After treatments, cells were washed, detached with 0.25% trypsin-0.2% EDTA, and suspended in trypan blue (Sigma-Aldrich, St. Louis, MO, USA) at a 1:1 ratio in a medium solution. Cells were counted using a chamber Burker hemocytometer. 2.9.3. Hoechst Cell Count in Hypoxic Conditions Cells were plated on 96-well plates and, following overnight incubation, were exposed to 100 mM cobalt chloride (Sigma-Aldrich, St. Louis, MO, USA). 24 and 48 h from incubation with cobalt chloride, cells were stained with Hoechst 33258 2.25 mg/mL (Thermo Fisher Scientific, Waltham, MA, USA) for 20 min. To induce hypoxia from oxygen deprivation, cells were plated on 96-well plates and, following overnight incubation, were moved to the Environmental Chamber of ImageXpress Pico Automated Cell Imaging System (Molecular Devices, San Jose, CA, USA) at 2% O2 concentration. For both the chemically induced hypoxia and hypoxia from oxygen deprivation, cells were stained with Hoechst 33258 2.25 mg/mL for 20 min at different time points. Images were acquired by fluorescent microscopy ImageXpress Pico Automated Cell Imaging System with a 10x objective. Analysis software detected the total number of cells based on Hoechst positive nuclei. 2.10. Statistical Analysis The statistical analysis of experimental results was performed using GraphPad Prism version 7 (GraphPad, San Diego, CA, USA.). An appropriate post-hoc test (Bonferroni, Tukey--Kramer, or Sidak multiple comparison tests) was used after the one-way or two-way analysis of variance (ANOVA) of the data. For the purpose of assessing statistical significance, a p-value of less than 0.05 was taken into account. 3. Results 3.1. DXR-Resistant Clones Demonstrate Changes in Mitochondrial Morphology and Loss in Mitochondrial Membrane Potential Mitochondrial morphology can be modified in response to environmental and cellular stresses, such as DXR treatment. This process is regulated by fusion and fission dynamics and plays a pivotal role in cellular activity . The evaluation of mitochondrial network of sensitive and resistant osteosarcoma cell lines by confocal microscopy was performed through the orange-fluorescent probe Mitotracker Orange, which stains mitochondria with a membrane potential-dependent accumulation. Figure 1a shows the mitochondrial network of sensitive MG63 and resistant clones, DXR 30 ng/mL, and DXR 100 ng/mL, while Figure 1b refers to HOS and relative DXR-resistant clones, DXR 10 ng/mL, DXR 30 ng/mL, and DXR 100 ng/mL. Images suggested no difference in the mitochondrial network, which is similar and filamentous in both sensitive and resistant cancer cells. However, the membrane potential is lower in resistant clones compared to sensitive cells. To confirm the data obtained by confocal microscopy, the mitochondrial membrane potential of sensitive and resistant osteosarcoma cells was evaluated in both MG63 and HOS cell lines through flow cytometry analysis. Cells were stained with Rhodamine 123, a fluorescent probe sequestered by active mitochondria in a membrane potential fashion. Figure 1c,d shows that all resistant clones significantly reduced membrane potential compared to sensitive clones, thus confirming results obtained with confocal microscopy images. These preliminary results suggest an alteration in the mitochondrial status of DXR-resistant osteosarcoma cells, indicating that further investigations are needed to understand the molecular mechanisms responsible for these changes. 3.2. The Mitochondrial Mass Was Lower in DXR-Resistant Clones than in Sensitive Cells: Is Mitochondrial Biogenesis or Mitophagy the Involved Pathway? Mitochondria are dynamic organelles and changes in mitochondrial mass reflect unbalance between mitochondrial biogenesis and mitophagy . Increased or decreased mitochondrial mass has been correlated with cancer . Nonyl acridine orange (NAO), a fluorescent probe binding cardiolipin, was used to assess the mitochondrial mass of sensitive and DXR osteosarcoma cells by flow cytometry. Results showed a significant decrease of NAO fluorescence percentage in all resistant clones compared to sensitive cancer cells . To confirm these data, we even assessed the protein expression of VDAC1 and TOM20, which are already considered specific markers of mitochondrial mass. As it is possible to observe in Figure 2c,d, the expression of both proteins is significantly decreased in resistant cells with respect to their sensitive counterparts. The uncropped Western Blot images can be found in Figures S1 and S2. Considering that the equilibrium between mitochondrial biogenesis and mitophagy regulates mitochondrial mass , we analyzed some genes involved in both these pathways to dissect which is the process involved in mitochondrial alteration. For mitochondrial biogenesis, we checked the mRNA levels of PGC1-a, TFAM, and NRF-2. As Figure 3a,b shows, the expression of both PGC1-a and TFAM decreases in MG63 DXR-resistant cells, and TFAM levels are lower in HOS DXR-resistant clones. These results suggest that reduced biogenesis could be responsible for the observed reduction of mitochondrial mass in resistant clones. As further confirmation, the protein expression on BNIP3, a marker of mitophagy , downregulated in the MG63-DRX resistant clones, thus excluding the involvement of mitophagy as the process responsible for reduced mitochondrial mass . The uncropped Western Blot images can be found in Figure S2. Furthermore, we evaluated the mRNA levels of BNIP3 in HOS cell lines, as reported in Figure 3d. The results showed that only HOS DXR 30 ng/mL cancer cells slightly increase the expression, but not in a significant way. 3.3. DXR Osteosarcoma Cells Show Decreased mtROS Levels and Different Response to Hypoxia Cellular homeostasis is also regulated by the oxidative status, and alterations such as an increase in ROS production represent a factor involved in cell death. In the context of chemotherapy, it is known that the mode of action of several chemotherapeutic agents, such as cisplatin and DXR, is related to oxidative stress induction, which leads to cell death. On the other side, one of the mechanisms of drug resistance is the increased antioxidant system, which counteracts the enhanced chemotherapy-induced oxidative stress , and that might be obtained by reducing the biogenesis ROS-producing mitochondria. Therefore, we analyzed the ROS content. As confirmation, results in Figure 4a,b showed ROS decrease in DXR-resistant osteosarcoma cells compared to sensitive counterparts. The reduced mitochondrial mass and biogenesis and the reduced ROS production in resistant cells suggest that these cells have reduced mitochondrial oxidative phosphorylation. To validate this hypothesis, we measured the cellular response to low-oxygen (2%) or chemically induced hypoxia (with CoCl2). CoCl2 stabilizes hypoxia-inducible factors 1a and 2a under normoxic conditions and simulates a hypoxic microenvironment . As confirmation of the hypoxia-like metabolic state in CoCl2-treated cells, mRNA expression levels of the indirect hypoxia marker CAIX were induced in all cells tested . We then counted the number of cells after incubation with both low oxygen tension and CoCl2. Before 144 h, cell growth was mostly unaffected by both types of hypoxia, with the exception of sensitive cells treated with CoCl2 . In contrast, after longer exposure (at 144 h under low O2 tension), all cell types were significantly affected compared with the normoxic condition . Overall, it can be concluded that resistant cells appear to be much less dependent on oxygen metabolism than sensitive cells, as attested by both CAIX expression levels and cell numbers at 48 h when treated with CoCl2 . 3.4. DXR-Resistant Osteosarcoma Cells Show Different Behavior under Metabolic Stresses DXR-resistant osteosarcoma cells revealed a different mitochondrial profile with respect to their sensitive counterpart. Accumulating evidence showed that mitochondrial dysfunctions can be highlighted when oxidative metabolism is increased, such as glucose deprivation and galactose addition in the medium. The next step of this work was to evaluate the response of sensitive and DXR-resistant cancer cells under two metabolic stresses, glucose, and glutamine deprivation. MG63 DXR-resistant clones significantly reduced cell viability after 24 h in both glucose-free/galactose 5 mM, and glutamine-free media . On the other side, among the resistant HOS clones, only HOS DXR 100 ng/mL cells resulted to be affected by these metabolic stresses . These results, taken together, suggest mitochondrial dysfunctions in DXR-resistant cancer cells, which are more dependent on glucose and glutamine pathways with respect to sensitive counterparts. 3.5. Targeting Mitochondrial Biogenesis to Restore DXR Sensitivity Our results highlighted a different mitochondrial profile of DXR-resistant osteosarcoma cells compared to sensitive cells, in particular demonstrating reduced mitochondrial membrane mass, potential, and activity. Further investigations assessed that the process responsible for decreased mitochondrial mass in resistant cells is likely the reduction of mitochondrial biogenesis. In line with these observations, we reasoned to verify whether the induction of mitochondrial biogenesis was able to re-sensitize DXR effect in osteosarcoma-resistant clones. The previous literature reported that the natural compound quercetin is an inducer of mitochondrial biogenesis . We evaluated cell viability of both sensitive and DXR-resistant cancer cells following 24, 48, and 72 h of the combined treatment with quercetin 0.1-200 mM and DXR, at the respective concentration of each resistant cancer cell lines (DXR 10-30-100 ng/mL). Results showed that the association with quercetin re-sensitized the effect of DXR in MG63 and HOS DXR-resistant cancer cells , by enhancing the percentage of late apoptotic cells . In addition, we also demonstrated that quercetin 10 mM enhanced TFAM expression following 72 h of treatment . These preliminary data suggested that the stimulation of mitochondria biogenesis could improve DXR efficacy in resistant cells; therefore, the combined treatment between mitochondrial biogenesis inducers and the chemotherapeutic drug represents a promising pharmacological approach to overcoming drug resistance. However, further studies will be necessary to identify a selective inducer of this pathway. 4. Discussion Mitochondria play a pivotal role in cell life, coordinating several signaling processes such as the tricarboxylic acid (TCA) cycle, fatty acid oxidation (FAO), and oxidative phosphorylation (OXPHOS) , and regulating energy homeostasis and cell death via apoptosis. Recent studies correlated mitochondria and cancer, suggesting that mitochondrial dysfunctions are responsible for tumor development and metastasis progression and also for alterations in chemotherapy's response. Evidence reported that several chemotherapeutic agents accumulate into mitochondria, thus causing mtDNA damage . These organelles are considered one of the main targets of DXR, through abnormalities in mitochondrial shape and functionality and mitochondrial death mediated by apoptosis. Considering the pivotal role of mitochondria in tumor development and in DXR pharmacological activity, this work aimed to characterize the mitochondrial phenotype of sensitive and DXR-resistant osteosarcoma cells. Once alterations were identified, the goal was to target them to restore drug sensitivity in resistant cells. Several studies reported that alterations in the mitochondrial network, membrane potential, and mass are correlated with chemoresistance or cancer aggressiveness in different types of tumors such as colon, breast, and gastric cancers . These aspects reflect the functional and healthy state of these organelles and could become indicators of an unhealthy and dysfunctional status. Our results showed that DXR-resistant cancer cells decreased both the mitochondrial number and the membrane potential, although their morphology remains unchanged. The mitochondrial membrane potential (DPsm) is a thorough indicator of mitochondrial function, and its dysregulation causes an increase in reactive oxygen species production, with consequent oxidative damage to DNA and other cellular structures, and ultimately results in cell death . It is interesting to note that several cell models have shown a decrease in mitochondrial membrane potential after exposure to DXR in vitro , and our data are in line with these findings. In fact, all DXR-resistant osteosarcoma cells showed a reduced mitochondrial membrane potential with respect to the sensitive counterpart. It is known that DXR is a redox-active substance that can interact with a number of enzymes, such as mitochondrial complex I, to produce large quantities of ROS and the semiquinone radical . Our results indicate that mitochondria of resistant cells present lower levels of mitochondrial ROS production. Furthermore, DXR-resistant osteosarcoma cells also showed a reduction of mitochondrial mass, confirmed both with NAO fluorescence and the decreased expression of two mitochondrial markers, VDAC1 and TOM20. It is known that mitochondrial mass is regulated by the equilibrium between mitochondrial biogenesis and mitophagy. Alterations of these processes have been correlated with chemoresistance. Peroxisome proliferator-activated receptor-coactivator 1 (PGC-1) and mitochondrial transcription factor A (TFAM), two mitochondrial transcription factors, mediate an increase in mitochondrial number in response to cellular injury . According to several studies, the mitochondrial biogenesis pathway's genes and proteins are upregulated, and these changes cause cancer chemoresistance. Increased expression of TFAM and PGC-1a was assessed in tumors unresponsive to cisplatin and sorafenib; it was also demonstrated that the depletion of these proteins restored the sensitivity to the drugs . On the other side, a recent study in our laboratory demonstrated that the increased expression of BNIP3, which is a protein involved in the mitophagy process, is related to cisplatin resistance. Both genetic and pharmacological approaches through inhibition showed a re-sensitization of resistant cancer cells to the chemotherapeutic agent, supporting the idea that targeting mitochondrial dynamics could be a valuable strategy in chemosensitization. Interestingly, our data demonstrated that DXR-resistant osteosarcoma cells decreased mRNA levels of TFAM gene compared to their sensitive counterpart, thus suggesting that the downregulation of mitochondrial biogenesis could be the process involved in the DXR resistance onset. In line with this, the evaluation of the mitophagic marker BNIP3 shows that resistant cells didn't present significant alterations, prompting us to exclude an increased mitophagy as the mechanism responsible for a reduced mitochondrial mass. This result is not very surprising because in previous studies, we have already observed that osteosarcoma cells do not tend to activate autophagy as an adaptation mechanism to stressful conditions . Having identified the downregulation of mitochondrial biogenesis genes as a possible mechanism of resistance, we sought to combine DXR treatment with a drug that may induce the activation of this process and thus re-sensitize DXR-resistant cells. Quercetin is a natural flavonoid already known to be an inducer of mitochondrial biogenesis . In our DXR-resistant cell model, combining DXR with quercetin re-sensitized the resistant cells to DXR. However, quercetin is also a monocarboxylate transporter (MCT) inhibitor . Since MCTs play a significant role in maintaining intracellular pH by transporting excess protons outside cancer cells, it is also possible that quercetin treatment acidifies the cytosol in resistant cells. The subsequent increase of the pH gradient at the plasma membrane may facilitate the cellular intake of DXR, which is a weakly basic molecule, and thus increase its intracellular accumulation and toxicity . However, in this study, we also showed that quercetin treatment increases TFAM levels in MG63-resistant cells, confirming that, in these cells, quercetin treatment has the potential to restore DXR sensitivity by inducing mitochondrial biogenesis. In conclusion, our preliminary results pave the way for TFAM inducers or other mitochondrial inducers as possible DXR sensitizers. The combination of compounds acting on different targets represents a promising strategy in the context of chemoresistance therapy because it allows for a reduction in the dosage of single drugs, thus alleviating severe toxicities and at the same time enhancing the therapeutic effectiveness. 5. Conclusions Taken together, these data highlighted a different mitochondrial profile between sensitive and DXR-resistant osteosarcoma cells. In particular, resistant clones showed a decreased mitochondrial mass and reduced expression of mitochondrial biogenesis genes. The phenotyping of the mitochondrial profile allows the identification of altered pathways, which can be exploited by novel pharmacological approaches that selectively target mitochondrial dysfunctions in resistant cancer cells. In this work, the treatment of osteosarcoma cells with quercetin, a natural compound able to induce mitochondrial biogenesis, caused a re-sensitization of DXR-resistant cells to the chemotherapeutic agent. These preliminary results open the window to the development of promising pharmacological strategies to overcome DXR resistance in osteosarcoma. Supplementary Materials The following supporting information can be downloaded at: Figure S1: Western blot HOS; Figure S2: Western blot MG63; Table S1: Cytotoxic effect of doxorubicin in MG63 cell lines; Table S2: Cytotoxic effect of doxorubicin in HOS cell lines. Click here for additional data file. Author Contributions Conceptualization, I.G., M.C., S.A., E.R., M.M.; methodology, all authors; software, I.G., M.C., M.T.; data curation, I.G., M.C., M.T.; writing-original draft preparation, I.G., M.C., S.A.; writing-review and editing, V.C., E.R., M.M., S.A., N.B.; visualization, all authors; supervision: S.A., M.M., E.R.; project administration, S.A., M.M.; funding acquisition: S.A., M.M., E.R., N.B. All authors have read and agreed to the published version of the manuscript. Institutional Review Board Statement Not applicable. Data Availability Statement The data presented in this study is available within the article and the Supplementary Materials file. Conflicts of Interest The authors declare no conflict of interest. Appendix A Figure A1 (a) Relative mRNA expression levels of CAIX gene in MG63, MG63 DXR 30 ng/mL, and MG63 DXR 100 ng/mL cells following treatment with CoCl2. mRNA levels of CAIX are normalized to b-ACTIN. The horizontal dotted line refers to normalized untreated cells. Data represent mean +- SD of four different experiments. ## p < 0.01, treated vs. untreated sensitive cells. ** p < 0.01, *** p < 0.001, **** p < 0.0001 treated vs. untreated resistant cells. (b) Relative mRNA expression levels of CAIX gene in HOS, HOS DXR 10 ng/mL, and HOS3 DXR 30 ng/mL cells following treatment with CoCl2. mRNA levels of CAIX are normalized to b-ACTIN. The horizontal dotted line refers to normalized untreated cells. Data represent mean +- SD of four different experiments. #### p < 0.0001, treated vs. untreated sensitive cells. *** p < 0.001, **** p < 0.0001 treated vs. untreated resistant cells. Figure A2 Analysis of cell growth of sensitive and resistant clones under hypoxia for various exposure times. We counted the total number of HOS, HOS DXR 10 ng/mL, and HOS DXR 30 ng/mL cells after CoCl2 (a,b) treatments by Hoechst staining with an automated cell count system and at 24 h. Data represent mean +- SD of four different experiments. * p < 0.05, ** p < 0.01, resistant vs. sensitive clones after CoCl2 treatment. Figure A3 (a) Representative cytograms of apoptotic cells analyzed with Annexin V-PI staining. MG63, MG63 DXR 30 ng/mL and MG63 DXR 100 ng/mL cells were treated 72 h with quercetin 10 mM. The graphs represent the quantification of early (b) and late apoptosis (c) by showing the ratio between treated and untreated. (d) Representative cytograms of apoptotic cells analyzed with Annexin V-PI staining. HOS, HOS DXR 10 ng/mL, HOS DXR 30 ng/mL, and HOS DXR 100 ng/mL cells were treated 72 h with quercetin 10 mM. The graphs represent the quantification of early (e) and late apoptosis (f) by showing the ratio between treated and untreated. Data are shown as mean +- SD of three different experiments. * p < 0.05, treated vs. untreated cells. Figure A4 Relative mRNA expression levels of TFAM gene in MG63, MG63 DXR 30 ng/mL, and MG63 DXR 100 ng/mL cells following 72 h of treatment with quercetin 10 mM. mRNA levels of TFAM are normalized to GAPDH. The horizontal dotted line refers to normalized untreated cells. Data represent mean +- SD of three different experiments. * p < 0.05, treated vs. untreated cells. Figure 1 (a) Images representing mitochondrial network morphology of sensitive and resistant clones: MG63, MG63 DXR 30 ng/mL, and MG63 DXR 100 ng/mL. (b) Images representing mitochondrial network morphology of sensitive and resistant clones: HOS, HOS DXR 10 ng/mL, HOS DXR 30 ng/mL, HOS DXR 100 ng/mL. Mitotracker Orange (ex:554 nm/em:576 nm) was used to stain mitochondria, while Hoechst 33342 was used to stain nuclei. Images were acquired by confocal microscopy LSM 800 and software ZEN 2.1 60x objective. Scale bar = 20 mm. (c) Evaluation of mitochondrial membrane potential by flow cytometry. Representative cytograms and graphical quantification showing the percentage of fluorescence intensity of Rhodamine 123 (10 mM) in MG63, MG63 DXR 30 ng/mL, and MG63 DXR 100 ng/mL. The horizontal dotted line refers to normalized untreated cells. (d) Evaluation of mitochondrial membrane potential by flow cytometry. Representative cytograms and graphical quantification showing the percentage of fluorescence intensity of Rhodamine 123 (10 mM) in HOS, HOS DXR 10 ng/mL, HOS DXR 30 ng/mL, HOS DXR 100 ng/mL cells. The horizontal dotted line refers to normalized untreated cells. Data represent mean +- SD of three different experiments. **** p < 0.0001, resistant vs. sensitive cells. Figure 2 Evaluation of mitochondrial mass by flow cytometry and western blot. (a) Representative cytograms and graphical representation showing the percentage of fluorescence intensity of NAO 1.25 mM in MG63, MG63 DXR 30 ng/mL, and MG63 DXR 100 ng/mL cells. The horizontal dotted line refers to normalized untreated cells. (b) Representative cytograms and graphical representation showing the percentage of fluorescence intensity of NAO 1.25 mM in HOS, HOS DXR 10 ng/mL, HOS DXR 30 ng/mL, HOS DXR 100 ng/mL cells. The horizontal dotted line refers to normalized untreated cells. Data represent mean +- SD of three different experiments. **** p < 0.0001, resistant vs. sensitive cells. (c) Representative images and graphical quantification of protein expression of VDAC1 and TOM20, normalized to GAPDH in MG63, MG63 DXR 30 ng/mL, and MG63 DXR 100 ng/mL cells. The horizontal dotted line refers to normalized untreated cells. (d) Representative images and graphical quantification of protein expression of VDAC1 and TOM20, normalized to b-actin in HOS, HOS DXR 10 ng/mL, HOS DXR 30 ng/mL, HOS DXR 100 ng/mL cells. The horizontal dotted line refers to normalized untreated cells. The uncropped Western Blot images can be found in Figure S2. Data represent mean +- SD of four different experiments. * p < 0.05, ** p < 0.01, **** p < 0.0001, resistant vs. sensitive cells. Figure 3 (a) Relative mRNA expression levels of genes related to mitochondrial biogenesis in MG63, MG63 DXR 30 ng/mL, and MG63 DXR 100 ng/mL cells. mRNA levels of all genes are normalized to GAPDH. The horizontal dotted line refers to normalized untreated cells. (b) Relative mRNA expression levels of genes related to mitochondrial biogenesis in HOS, HOS DXR 10 ng/mL, HOS DXR 30 ng/mL, HOS DXR 100 ng/mL cells. mRNA levels of all genes are normalized to CALNEXIN. The horizontal dotted line refers to normalized untreated cells. (c) Representative images and graphical quantification of protein expression of BNIP3, normalized to GAPDH in MG63, MG63 DXR 30 ng/mL, and MG63 DXR 100 ng/mL cells. The horizontal dotted line refers to normalized untreated cells. The uncropped Western Blot images can be found in Figure S3. (d) Relative mRNA expression levels of BNIP3 in HOS, HOS DXR 10 ng/mL, HOS DXR 30 ng/mL, HOS DXR 100 ng/mL cells. mRNA levels of all genes are normalized to CALNEXIN. The horizontal dotted line refers to normalized untreated cells. Data represent media +- SD of three different experiments. * p < 0.05, ** p < 0.01, *** p < 0.001, resistant vs. sensitive cells. Figure 4 (a) Evaluation of ROS levels by flow cytometry. Representative cytograms and relative quantification showing the fluorescence intensity of Mitosox 5 mM in MG63, MG63 DXR 30 ng/mL, and MG63 DXR 100 ng/mL cells. The horizontal dotted line refers to normalized untreated cells. (b) Evaluation of ROS levels by flow cytometry. Representative cytograms and relative quantification showing the fluorescence intensity of Mitosox 5 mM in HOS, HOS DXR 10 ng/mL, HOS DXR 30 ng/mL, HOS DXR 100 ng/mL cells. The horizontal dotted line refers to normalized untreated cells. Data represent media +- SD of three different experiments. * p < 0.05, ** p < 0.01, *** p < 0.001, resistant vs. sensitive clones. Figure 5 Analysis of cell growth of sensitive and resistant clones under hypoxia for various exposure times. We counted the total number of MG63, MG63 DXR 30 ng/mL, and MG63 DXR 100 ng/mL cells (a,c-f) and HOS, HOS DXR 10 ng/mL, and HOS DXR 30 ng/mL cells (b,g-j) after CoCl2 (a,b) low O2 tension (2%) (c-j) treatments by Hoechst 33258 staining with automated cell count system and at different time points, 48 (a-e), 72 (b-f), 96 (c-g), and 144 h (d-h). Data represent mean +- SD of four-eight different experiments. * p < 0.05, ** p < 0.01, *** p < 0.001, hypoxia vs. normoxia conditions; # p < 0.05, CoCl2 vs. untreated conditions, *** p < 0.001, **** p < 0.0001, resistant vs. sensitive clones after CoCl2 treatment. Figure 6 (a,b) Evaluation of cell viability by Trypan blue assay in MG63, MG63 DXR 30 ng/mL and MG63 DXR 100 ng/mL cells after 24 h in glucose-free/5 mM galactose medium (a) and glutamine deprivation (b). The horizontal dotted line refers to normalized untreated cells. (c,d) Evaluation of cell viability by Trypan blue assay in HOS, HOS DXR 10 ng/mL, HOS DXR 30 ng/mL, and HOS DXR 100 ng/mL cells after 24 h in glucose-free/5 mM galactose medium (c) and glutamine deprivation (d). The horizontal dotted line refers to normalized untreated cells. Data represent mean +- SD of four different experiments. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 resistant vs. sensitive cells. Figure 7 Evaluation of cell viability in MG63, MG63 DXR 30 ng/mL, and MG63 DXR 100 ng/mL following treatment with DXR (a-c) and quercetin combined with DXR 30 and 100 ng/mL, respectively (d-f) for 24 (a-d), 48 (b-e), and 72 h (c-f). Data represent mean +- SD of three different experiments. ** p < 0.01, **** p < 0.0001, resistant vs. sensitive cells; # p < 0.05, #### p < 0.0001, DXR100 vs. DXR 30 clones. Figure 8 Evaluation of cell viability in HOS, HOS DXR 10 ng/mL, HOS DXR 30 ng/mL, and HOS DXR 100 ng/mL following treatment with DXR (a-c) and quercetin combined with DXR 10, 30 and 100 ng/mL, respectively, (d-f) for 24 (a-d), 48 (b-e), and 72 h (c-f). Data represent mean +- SD of three different experiments. ** p < 0.01, **** p < 0.0001, resistant vs. sensitive cells; ## p < 0.01, ### p < 0.001, #### p < 0.0001, DXR 100 vs. DXR 30 clones, or DXR 30 vs. DXR 10, or DXR 100 vs. DXR 10 clones. cancers-15-01370-t001_Table 1 Table 1 Cytotoxic effect of quercetin (calculated as IC50) after 24, 48, and 72 h of treatment in MG63, MG63 DXR30 ng/mL, and MG63 DXR100 ng/mL cell lines. Cell Line IC50 Quercetin (mM)a 24 h 48 h 72 h MG63 124.87 +- 9.03 68.42 +- 4.53 64.71 +- 3.87 MG63 DXR30 ng/mL 110.87 +- 2.46 47.03 +- 8.13 * 35.70 +- 2.34 ** MG63 DXR100 ng/mL 100.62 +- 7.50 deg 84.41 +- 7.54 56.84 +- 8.60 a Values were determined by regression analysis and are expressed as mean +- SD of three different experiments. * p < 0.05, ** p < 0.01, MG63 DXR30 ng/mL vs. MG63 cells; deg p < 0.05, MG63 DXR100 ng/mL vs. MG63 cells. cancers-15-01370-t002_Table 2 Table 2 Cytotoxic effect of quercetin (calculated as IC50) after 24, 48, and 72 h of treatment in HOS, HOS DXR 10 ng/mL, HOS DXR 30 ng/mL, and HOS DXR 100 ng/mL cell lines. Cell Line IC50 Quercetin (mM) a 24 h 48 h 72 h HOS 339.23 +- 6.70 226.80 +- 10.07 224.24 +- 4.72 HOS DXR10 ng/mL 536.25 +- 11.46 **** 265.30 +- 0.95 *** 286.04 +- 1.00 **** HOS DXR30 ng/mL 458.51 +- 3.02 degdegdegdeg 270.48 +- 3.57 degdegdegdeg 262.12 +- 3.39 degdegdegdeg HOS DXR100 ng/mL 792.65 +- 7.36 #### 236.77 +- 4.16 248.01 +- 2.88 #### a Values were determined by regression analysis and are expressed as mean +- SD of three different experiments. *** p < 0.001, **** p < 0.0001, HOS DXR 10 ng/mL vs. HOS cells; degdegdegdeg p < 0.0001, HOS DXR 30 ng/mL vs. HOS cells; #### p < 0.0001, HOS DXR 100 ng/mL vs. HOS cells. Disclaimer/Publisher's Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

PMC10000150

In order to study the smart management of dairy farms, this study combined Internet of Things (IoT) technology and dairy farm daily management to form an intelligent dairy farm sensor network and set up a smart dairy farm system (SDFS), which could provide timely guidance for dairy production. To illustrate the concept and benefits of the SDFS, two application scenarios were sampled: (1) Nutritional grouping (NG): grouping cows according to the nutritional requirements by considering parities, days in lactation, dry matter intake (DMI), metabolic protein (MP), net energy of lactation (NEL), etc. By supplying feed corresponding to nutritional needs, milk production, methane and carbon dioxide emissions were compared with those of the original farm grouping (OG), which was grouped according to lactation stage. (2) Mastitis risk prediction: using the dairy herd improvement (DHI) data of the previous 4 lactation months of the dairy cows, logistic regression analysis was applied to predict dairy cows at risk of mastitis in successive months in order to make suitable measurements in advance. The results showed that compared with OG, NG significantly increased milk production and reduced methane and carbon dioxide emissions of dairy cows (p < 0.05). The predictive value of the mastitis risk assessment model was 0.773, with an accuracy of 89.91%, a specificity of 70.2%, and a sensitivity of 76.3%. By applying the intelligent dairy farm sensor network and establishing an SDFS, through intelligent analysis, full use of dairy farm data would be made to achieve higher milk production of dairy cows, lower greenhouse gas emissions, and predict in advance the occurrence of mastitis of dairy cows. smart dairy farm system nutritional grouping greenhouse gas reduction mastitis prediction dairy cows Beijing innovation team of Livestock Industry SystemBAIC05-2023 This work was conducted with financial support from the Beijing innovation team of Livestock Industry System (BAIC05-2023). pmc1. Introduction With the rapid development of modern science and technology and the continuous improvement of communication technology, the development and application of the Internet of Things (IoT) have gradually been penetrating every aspect of life . It is estimated that there would be about 2.5 billion bytes of data every day, which is beyond the capacity of human computing. Artificial intelligence (AI), big data, and machine learning (ML) emerged as the times require . Traditional agriculture has gradually entered the era of intelligent agriculture after the era of mechanization and automation . Smart animal husbandry combines farm management with technologies such as IoT, 5G, ML, and AI to improve the production level of animal husbandry and promote profound changes in animal husbandry . Smart dairy farming is a specific application of animal husbandry intelligence, and it is also the mainstream trend in the modernization of dairy farms. Nowadays, ever more dairy farms are covered with large areas of 5G networks and sensors . Precise feeding is performed regularly and quantitatively using automated technology , and dairy farms are managed automatically and intelligently using technologies such as automatic ventilation and drainage and air quality detection . Dairy farms generate more and more data while the dairy farm scale expands . For example, radio frequency identification technology is used to recognize individual dairy cows and obtain information such as the location and body temperature . Different models (Wood model, Ali Schaeffer model, etc.) are used to predict 305d milk yield of cows and then evaluate the effect of genetic evaluation of dairy cows . The movement of dairy cows can be precisely calculated by a collar monitoring system to reveal whether animals are in heat . Different data streams represent different information, and most have their own collection and analysis systems. Most dairy farms are not able to effectively utilize data streams from different sources to extract their full value. Determining how to effectively integrate and utilize these data streams for dairy farm management has become a challenge . Studies have shown that the management of the entire farm can be improved by integrating these data streams in real time, which in turn enables data-driven decision making on the farm . In China, almost every large dairy farm has 2-3 management systems. The levels of dairy farm management systems are uneven and there is no well-recognized system that can efficiently integrate different sources of data flow and propose corresponding improvement measures for dairy farms. In order to solve this problem, we developed a system for data collection, processing, and analysis, namely a smart dairy farm system (SDFS), and illustrated the concept of the SDFS through two application scenarios: (1) nutritional grouping of dairy cows through cluster analysis for precise nutritional management, improving milk production and reducing greenhouse gas (GHG) emissions; (2) risk prediction of dairy cow mastitis through logistic regression to identify the dairy cows with mastitis risk in order to take measurements in advance. Through the overall analysis of dairy farm data, the SDFS could make better use of dairy farm data and improve the management level of dairy farms. However, farm data are a huge treasure, and the data used in this paper are only a part of the huge data of farms, so we need to constantly excavate, analyze, and interpret the data behind the farms and constantly promote the application of intelligent systems in farm production management. 2. Materials and Methods 2.1. SDFS Establishment and Data Collection 2.1.1. Setup of SDFS A dairy farm in the Beijing area with a herd of 2500 cows, including 1256 lactating cows, was selected. The dairy farm is designed with an intelligent sensor network structure, and dairy farm managers can achieve remote and precise monitoring and control via computer and mobile phone. The dairy farm was equipped with a large number of diversified intelligent devices for cow monitoring and management, such as radio frequency identification (RFID) electronic ear tags . A weighing system is combined with RFID technology to obtain the weight information of individual cows, which is sent to a management computer, providing management staff with basic information for the management of cattle farms. The farm also has a total mixed ration (TMR) precision feeding system and unmanned aerial vehicle (UAV) photography system . The installation of sensors for monitoring temperature, humidity, wind speed, and GHGs (such as methane (CH4) and carbon dioxide (CO2)) enables the monitoring of environmental parameters in the dairy farm . The dairy farm data are transmitted to the computer terminal of the SDFS (as a database) through sensors, and the relevant data can be extracted from the database for advanced analysis and visual presentation. The system enables the timely collection and analysis of data collected by the dairy farm sensor network. The SDFS includes automatic monthly reports, annual reports, growth performance, DHI data, reproductive performance, individual cow value display and analysis, nitrogen and phosphorus emissions, and GHG emissions. Two application examples (nutrient grouping and mastitis prediction) ae used below to illustrate the application of the SDFS. 2.1.2. Data Collection in Dairy Farm The data collected mainly included the following: (1) individual information: cattle pedigree information, body weight, appearance score, body condition score, etc.; (2) farm information: farm location, stalls, cattle barns, environment, etc.; (3) cattle management: recording routine events in herds, such as heat, insemination, calving, and disease prevention and control events; (4) feed: including diet composition, average delivery and feed residues per day, and dry matter intake (DMI); (5) dairy herd improvement (DHI) parameters: milk yield, milk fat percentage, milk protein percentage, fat/protein ratio, milk fat content, milk protein content, and somatic cell count (SCC) were recorded on monthly test days . 2.2. Application Scene of Smart Dairy Farm System 2.2.1. Nutrition Grouping Based on the individual cow information, DMI, and DHI data collected by the SDFS, Nutrient Dynamic System Professional Software (NDS, developed by Rum&n srl), which adopted the Cornell Net Carbohydrate and Protein System (CNCPS6.55) , was used to calculate metabolic protein (MP) and metabolic energy (ME) as two of references for nutritional grouping. Two hundred seventy lactating cows were divided into 9 pens by cluster analysis as the nutritional group (NG). The control groups were assigned to 9 pens with 30 cows in each pen according to the original grouping (OG; divided according to milk production of cows) method in the farm, in which cows were grouped according to lactation stages. The corresponding diets calculated by NDS were provided for each pen (Table 1 and Table 2). Since the monitoring of methane and carbon dioxide is greatly influenced by the environment, space, and air flow, the stability of the data detected by this sensor needs to be further improved. Therefore, the methane and carbon dioxide data in this study mainly used the predicted value of CNCPS system, and the stability was guaranteed to a certain extent . N intake was calculated and CH4 and CO2 emissions were also estimated by NDS . 2.2.2. Mastitis Prediction The mastitis prediction was carried out using the original DHI records of the dairy farm from 1 January 2019 to 31 December 2021. In general, SCC greater than 200,000/mL was used as the criterion for subclinical mastitis in dairy cows . From the original DHI records, records without SCC were deleted. Due to the skewed distribution of the SCC values and the heterogeneous variance, SCC was first transformed into somatic cell score (SCS), which was close to a normal distribution, before the subsequent analysis . The conversion formula was as follows:SCS = log2(SCC/100000) + 3. To improve the accuracy of model predictions, the values of SCS ranging from 0 to 10 were used . According to the SCC values, cows were divided into a healthy group and a mastitis risk group (Table 3). According to the SCC in DHI data, when SCC <= 200,000/mL, it indicates a healthy condition; when 200,000/mL < SCC <= 500,000/mL, it indicates subclinical mastitis, when SCC > 500,000/mL, it indicates clinical mastitis . In this paper, both subclinical and clinical mastitis were named as the mastitis risk group. Altogether, 2555 DHI records were used for regression analysis. The following independent variables were chosen: parity, days in milk (DIM), and milk indicators of the previous four lactation months (milk yield, milk protein percentage, milk fat percentage, lactose percentage, fat/protein ratio). A training set (70%) and a validation set (30%) were created from the collected data. The training set was used to filter the independent variables (equation 1) by bidirectional elimination stepwise regression. The parameters with statistically significant effects on the prediction of mastitis in dairy cows were obtained (Table 4), and corresponding coefficients were substituted into the logistic regression equation for further analysis using the validation set:(1) logit(P)=log(P1-P)=b0+b1X1+b2X2+b3X3+b4X4+...b26X26 where P represents the probability of positive results; 1 - P represents the probability of non-positive results. P1-P is the strength of the disease as a statistical indicator, called the odds ratio (OR), used to estimate the effect of the independent variable on disease. X1 is parity, X2-X5 represent the amount of milk produced during the first 1-4 lactation months, X6-X9 represent milk fat percentage in the first 1-4 lactation months, X10-X13 represent the protein rate of the first 1-4 lactation months, X14-X17 represent lactose rate in the first 1-4 lactation months, X18-X21 represent the fat-to-egg ratio of the first 1-4 lactation months, X22-X26 represent the natural months of the first 1-5 lactation months; b0 is a constant, and b1-b26 are the regression coefficients of each variable (X1-X26). The receiver operating characteristic (ROC) curve was plotted to reflect the prediction accuracy of predictive variables in the dairy cow mastitis risk assessment model. The X-axis is specificity (percentage of healthy cows that tested negative) and the Y-axis is sensitivity (the percentage of risk cows that correctly tested positive), The area under the curve (AUC) was calculated. An AUC of the prediction model between 0.50 and 0.70 indicates that the effect is average, an AUC between 0.70 and 0.90 is considered to indicate a good model, and an AUC higher than 0.90 is considered to indicate an excellent model . 2.3. Statistical Analysis First of all, the data were preliminarily sorted using Excel and SPSS 21.0. Cluster analysis was performed using the hclust function in the stats package of Rx64 (version 4.0.5) for nutritional parameters (milk production, parity, DIM, MP, ME, etc.). For mastitis prediction, the DHI data were analyzed using ANOVA in SPSS 21.0. Logistic regression analysis was performed using the general linear model (GLM) function, and the ROC curve was drawn using the PROC package of Rx64 software. 3. Results 3.1. Application of SDFS 3.1.1. Nutrient Grouping As shown in Table 5, compared with OG, the milk production of NG increased significantly (p < 0.05), except that the milk production in the mid-lactation group of the second parity was not significantly different. In general, the use of NG can significantly improve the milk yield of dairy cows at the same stage. As shown in Table 6, the N intake of dairy cows in NG was higher than that in OG; N production and N efficiency in NG were highly significantly increased than that in OG (p < 0.01). Compared to OG, overall N efficiency in NG increased by 1.98%. Generally speaking, the N intake of cows increased after NG treatment. The GHG emissions are shown in Table 7. CH4 and CO2 emissions of dairy cows in NG were lower than those in OG. In general, the use of NG resulted in a decrease in dairy cow methane and carbon dioxide emissions. 3.1.2. Mastitis Prediction The risk factors related to mastitis in the experimental dairy farm are shown in Table 8. OR values indicated the fitting degree of the model was good (Hosmer-Lemeshow (p > 0.05)). The results showed that milk yield in the second lactation month (p < 0.05), fat percentage in the first and third lactation months (p < 0.05), and natural month in the fifth lactation month (p < 0.05) had significant effects on mastitis risk in dairy cows. The predictive value of the mastitis risk assessment model was 0.773. The accuracy was 89.9%, the specificity was 70.2%, and the sensitivity was 76.3% . 4. Discussion Recently, the utilization of 5G and IoT technology has become a major trend in the development of animal husbandry . This study formed a large sensing network by applying the concept of precision animal husbandry and installing various types of equipment on dairy farms. Data are collected through various sensors, and IoT facilitates the transmission of data from the network to the SDFS for data analysis, forming the basis for the transformation of intelligent dairy farms into smart management. 4.1. Nutrition Grouping Currently, most dairy farms usually only consider the lactation stage when grouping cows to determine feed ration, resulting in less accurate feed nutrition on dairy farms . Studies have shown that grouping dairy cows according to their actual nutritional needs can increase the utilization rate of N in the diet , and milk production, so as to reduce GHG emissions . In this study, for dairy cows in NG, milk production significantly increased (p < 0.05). The dairy cows with different needs were fed different TMR formulations, which greatly improved the N efficiency and increased milk production. This is in agreement with the results of Cabrera et al. . Nutritional grouping of dairy cows could improve the nutritional accuracy of diet, prevent nutrient loss, reduce dietary costs, and increase milk production. Nutritional grouping had better theoretical nutritional accuracy, thereby reducing nutritional loss due to dietary and environmental influences . GHG emissions (CH4, N2O, CO2) are currently a research hotspot all over the world . In this study, dairy cows in NG had CH4 and CO2 emissions lower than those in OG, and the N efficiency increased by 1.98% on average. Kalalantari et al. . reported an increase in N efficiency by 2.7% when cows were divided into multiple nutritional groups and fed with an average MP lower than requirements. The methane emission also decreased. The reason for this discrepancy could be that the authors fully considered the cow's body weight (BW), BCS (the range of 2.0-4.5 to ensure the accuracy of the model), and NEL in their study, while in this study these factors were not in consideration. Therefore, the nutritional grouping of lactating dairy cows can fully consider the physical condition and nutritional needs of dairy cows and provide different diets for different groups to better meet the nutritional needs of dairy cows and lower GHG emissions . 4.2. Smart Prediction of Mastitis As dairy cows produce ever more milk, cow mastitis has been becoming one of the most important diseases that restrict the development of the global dairy industry . It is estimated that economic losses due to mastitis in dairy cows account for 38% of the total direct cost of common production diseases on dairy farms . Risk assessment and timely prevention of mastitis in cows are essential to ensure the health of cows and the consistent improvement of raw milk quality . Recent studies on mastitis prediction have focused on factors such as year, month, and farm . Individual information about cows has not been taken into account, making the prediction less practical. This study screened the factors that could predict the mastitis risk in the fifth lactation month based on the previous four lactation months using DHI data of individual cows. The milk yield in the second lactation month and fat percentage in the first and third lactation months were influential risk factors for mastitis and could accurately predict the risk of mastitis in the fifth lactation month. In this way, the incidence of mastitis in the dairy farm could be predicted two months in advance. This is expected to have a certain guiding significance for dairy farms. The ROC curve is a comprehensive representation of a model's accuracy. Sensitivity and specificity are indispensable indicators that reflect authenticity . The area under the curve (AUC) is the diagnostic value of the model. The larger the area is, the better the diagnostic performance of the model is . The predictive specificity of this study was similar to Cavero's predictive specificity (74.9%), which was calculated for mastitis prediction using data from 478 cows from neural networks and automated milking systems . Sun et al. reported a result of 87% true positives using artificial neural networks, which was similar to this study. The predictive value of this study was lower than the predictive value (AUC) of 0.93 reported by Jadhav et al. based on a 214-cow dataset. This may be due to the authors using more variables such as mammary and bedding hygiene and milking methods in their study. In practice, bedding hygiene status, teat shape, and udder hygiene also affect the occurrence of mastitis in dairy cows, interfering with the accuracy of individual dairy cow mastitis risk assessment . However, the prediction process could be always developing. With continuous accumulation and aggregation of more data and real-time data streams, the accuracy of model prediction could be improved over time. According to logistic regression and ROC curve description, we can find the risk indicators of dairy mastitis. Significantly, this equation does not apply to all cases; our team is still studying further, hoping to find a general equation that can be updated automatically by continuously merging the past data to detect the incidence of cow disease in time. 5. Conclusions This study combined IoT technology with dairy farm management to set up an SDFS. All kinds of data in the dairy farm will be intelligently captured by various sensors and transmitted to the SDFS in time for corresponding integration analysis. The applications of the SDFS were demonstrated in two aspects. NG according to the nutritional needs of dairy cows could improve nutritional accuracy, thus leading to increased milk production and N efficiency and reduced CH4 and CO2 emissions, so as to mitigate the environmental impacts. A mastitis prediction model was established using DHI data to identify potential cows at risk of mastitis in advance, thus reducing economic losses. By fully interpreting the hidden value of dairy farm data, the SDFS could help in the better management of dairy farms and promote the application of intelligent systems in dairy farm production. At present, our data mining of dairy farms is not comprehensive enough; we still need to continue to work hard. Author Contributions Conceptualization, K.G.; data curation, T.H. and J.Z.; formal analysis, J.Z. and X.Z.; funding acquisition, K.G.; investigation, T.H. and X.Z.; methodology, T.H. and J.Z.; project administration, K.G.; supervision, K.G.; validation, Y.C., R.Z. and K.G.; writing--original draft, T.H.; writing--review and editing, T.H., J.Z., X.Z., Y.C., R.Z. and K.G. All authors have read and agreed to the published version of the manuscript. Institutional Review Board Statement The study was approved by the Animal Care and Ethics Committee of Beijing University of Agriculture (protocol code BUA 2101030, date of approval: 30 January 2021). Informed Consent Statement Not applicable. Data Availability Statement The data presented in this study are available on request from the corresponding author. The data are not publicly available. Conflicts of Interest The authors declare no conflict of interest. Figure 1 Structure of smart dairy farm sensing network. Figure 2 ROC curve of mastitis prediction. animals-13-00804-t001_Table 1 Table 1 Diet composition and routine nutrient composition--original farm grouping. Ingredient TMR Diet Formulation (kg) Pens (OG) 1 2 and 3 4 and 7 5 and 8 6 and 9 Corn silage 23.00 23.00 23.31 23.32 23.21 Alfalfa hay 2.34 2.20 2.31 2.40 2.31 Oat hay 2.38 2.18 2.39 2.50 2.39 Corn grain fine 3.10 2.70 3.35 2.92 3.21 Soybean meal 2.93 2.66 3.32 3.00 3.12 Soft wheat bran 1.294 1.263 1.45 1.38 1.32 Soybean steam flaked 1.280 1.291 1.44 1.38 1.32 Beet pulp pellet 1.287 1.312 1.46 1.37 1.36 Calcium salt of fatty acids 0.10 0.00 0.40 0.10 0.15 Sugarcane molasses 0.51 0.35 0.60 0.30 0.56 Cottonseed meal 0.50 0.40 0.62 0.30 0.60 Premix 0.40 0.55 0.45 0.55 0.40 Nutritional composition ME (Mcal/day) 56.29 52.87 61.51 56.21 58.28 MP (g/day) 2518.80 2344.80 2732.50 2484.10 2604.02 CP (%) 16.76 16.46 17.10 16.63 17.02 Crude fat (%) 4.00 3.69 4.97 4.04 4.14 NFC (%) 37.85 37.32 37.34 37.02 37.80 NDF (%) 33.98 34.49 32.97 34.30 33.65 peNDF (%) 24.77 25.23 24.03 25.01 24.23 Starch (%) 24.26 24.15 24.24 23.92 24.14 Predicted DMI (kg/cow per day) 21.66 20.60 23.30 21.81 22.60 1-3 stand for diets for early, middle, and late stages of first lactation, respectively; 4-6 stand for early, middle, and late stages of second lactation, respectively; 7-9 stand for early, middle, and late stages of third lactation and over, respectively. animals-13-00804-t002_Table 2 Table 2 Diet composition and routine nutrient composition--nutrient grouping. Ingredient TMR Diet Formulation (kg) Pen (NG) 1 2 3 4 5 6 7 8 9 Corn silage 22.00 22.15 22.00 22.20 22.20 22.30 22.60 22.43 22.41 Alfalfa hay 2.20 2.20 2.00 2.21 2.21 2.31 2.40 2.28 2.28 Oat hay 2.00 2.00 2.00 2.34 2.34 2.32 2.52 2.22 2.28 Corn grain fine 3.32 3.14 2.84 3.54 3.43 3.22 3.66 3.57 3.36 Soybean meal 3.34 3.20 2.87 3.54 3.44 3.24 3.69 3.58 3.34 Soft wheat bran 1.17 1.20 1.27 1.57 1.37 1.16 1.44 1.38 1.36 Soybean steam flaked 1.13 1.11 1.27 1.43 1.23 1.13 1.54 1.37 1.27 Beet pulp pellet 1.30 1.30 1.21 1.40 1.20 1.11 1.46 1.36 1.26 Calcium salt of fatty acids 0.40 0.25 0.10 0.44 0.33 0.25 0.50 0.43 0.25 Sugarcane molasses 0.62 0.60 0.40 0.62 0.60 0.60 0.60 0.56 0.56 Cottonseed meal 0.58 0.50 0.40 0.60 0.54 0.58 0.60 0.60 0.60 Premix 0.41 0.45 0.40 0.43 0.43 0.35 0.40 0.38 0.38 Nutritional composition of diet ME (Mcal/day) 57.85 55.90 52.82 61.55 58.99 57.01 63.75 61.76 58.62 MP (g/day) 2572.00 2491.80 2336.40 2764.50 2630.60 2526.70 2863.90 2766.10 2622.80 CP (%) 17.25 17.07 16.98 17.46 17.28 17.18 17.56 17.57 17.37 Crude fat (%) 4.91 4.39 4.09 5.11 4.71 4.43 5.28 5.06 4.46 NFC (%) 38.01 38.04 37.83 37.52 37.90 38.05 37.33 37.74 37.90 NDF (%) 32.19 32.77 33.63 32.35 32.52 33.00 32.36 32.26 32.91 peNDF (%) 22.93 23.52 24.28 22.44 23.29 24.26 22.67 22.65 23.51 Starch (%) 24.47 24.37 24.59 23.96 24.28 24.23 23.70 24.25 24.29 Predicted DMI (kg/cow per day) 22.14 21.64 20.73 23.64 22.88 22.30 24.14 23.47 22.92 1-3 stand for diets for early, middle, and late stages of first lactation, respectively; 4-6 stand for early, middle, and late stages of second lactation, respectively; 7-9 stand for early, middle, and late stages of third lactation and over, respectively. animals-13-00804-t003_Table 3 Table 3 Disease grouping of Holstein cows. Group SCC 1, 104/mL SCS 2 Health Group SCC <= 20 SCS <= 4 Risk Group SCC > 20 SCS > 4 1 SCC: somatic cell count. 2 SCS: somatic cell score. animals-13-00804-t004_Table 4 Table 4 Risk factors of mastitis in Chinese Holstein cattle in the Beijing area. Variable Abbreviation Name State of illness Health 0 Risk 1 Milk yield 2 cnl2 X3 Fat percentage 1 rzl1 X6 Fat percentage 2 rzl2 X7 Fat percentage 3 rzl3 X8 Protein percentage 1 dbl1 X10 Lactose percentage 4 rtl4 X17 Fat/protein ratio1 zdb1 X18 Fat/protein ratio 3 zdb3 X20 Month 5 yuefen5 X26 animals-13-00804-t005_Table 5 Table 5 Effects of farm group and nutrient group strategies on milk production of dairy cows. Parity Pen Stage Milk yield p Value OG 1 NG 2 1 1 Early 37.45 + 3.45 a 39.28 + 2.90 b 0.031 2 Mid 35.78 + 4.22 a 37.87 + 3.19 b 0.035 3 Late 33.10 +-2.90 a 34.84 +-2.33 b 0.013 2 4 Early 40.17 +-4.07 a 42.29 +- 2.98 b 0.025 5 Mid 38.34 +- 4.22 39.74 +- 3.47 0.165 6 Late 36.48 +- 3.70 a 38.20 +- 2.75 b 0.046 >=3 7 Early 41.22 +- 4.36 a 43.58 +- 3.83 b 0.030 8 Mid 39.20 +- 3.81 a 41.64 +- 4.55 b 0.029 9 Late 37.55 +- 3.38 a 39.31 +- 3.14 b 0.041 Different lowercase letters in the same row differ significantly (p < 0.05). 1 OG: original grouping. 2 NG: nutritional grouping. animals-13-00804-t006_Table 6 Table 6 Effects of different grouping strategies on dietary N utilization. Parity Pen Stage N Intake (g) N Production (g) p Value N Efficiency (%) p Value Changes in N Efficiency 3 (%) OG 1 NG 2 OG NG OG NG 1 1 Early 598.88 617.10 18.22 201.94 +- 0.90 B 216.19 +- 0.76 A <0.001 33.72 +- 0.15 B 35.03 +- 0.12 A <0.001 3.88 2 Mid 579.68 597.89 18.21 197.86 +- 1.06 B 210.80 +- 1.06 A <0.001 34.13 +- 0.18 B 35.26 +- 0.18 A <0.001 3.31 3 Late 546.03 565.11 20.50 184.87 +- 0.90 B 194.38 +- 1.16 A <0.001 33.86 +- 0.17 B 34.40 +- 0.20 A <0.001 1.59 2 4 Early 654.1 666.13 12.03 224.40 +- 0.70 B 233.28 +- 0.83 A <0.001 34.31 +- 0.11 B 35.02 +- 0.12 A <0.001 2.07 5 Mid 624.9 634.84 9.94 215.92 +- 1.07 B 219.03 +- 1.07 A <0.001 34.55 +- 0.17 34.50 +- 0.17 0.248 -0.14 6 Late 598.03 610.95 12.92 203.08 +- 1.01 B 212.16 +- 0.95 A <0.001 33.96 +- 0.17 B 34.73 +- 0.15 A <0.001 2.27 >=3 7 Early 677.66 691.39 13.73 231.61 +- 1.09 B 237.45 +- 1.39 A <0.001 34.18 +- 0.16 B 34.37 +- 0.15 A <0.001 0.56 8 Mid 645.23 663.15 17.92 218.02 +- 0.96 B 228.40 +- 1.99 A <0.001 33.79 +- 0.15 B 34.49 +- 0.15 A <0.001 2.07 9 Late 623.76 635.36 11.6 210.00 +- 0.96 B 217.73 +- 1.53 A <0.001 33.67 +- 0.15 B 34.40 +- 0.14 A <0.001 2.17 Different uppercase letters in the same row differ highly significantly (p < 0.01). 1 OG: original grouping. 2 NG: nutritional grouping. 3 Changes in N efficiency = (NG-OG)/OG. animals-13-00804-t007_Table 7 Table 7 Methane and carbon dioxide emissions from cattle for group and nutrient group strategies. Parity Pen Stage CH4, g/d/Head Difference (%) CO2, kg/d/Head Difference (%) OG 1 NG 2 OG NG 1 1 Early 451.46 450.78 -0.15 13.16 13.14 -0.15 2 Mid 445.10 443.55 -0.35 12.97 12.95 -0.15 3 Late 432.10 428.40 -0.86 12.51 12.46 -0.40 2 4 Early 474.76 472.24 -0.53 13.90 13.82 -0.58 5 Mid 461.90 459.06 -0.61 13.50 13.46 -0.30 6 Late 453.69 452.21 -0.33 13.23 13.10 -0.98 >=3 7 Early 486.24 485.58 -0.14 14.26 14.18 -0.56 8 Mid 475.23 469.33 -1.24 13.86 13.72 -1.01 9 Late 463.48 458.97 -0.97 13.56 13.39 -1.25 1 OG: original grouping. 2 NG: nutritional grouping. animals-13-00804-t008_Table 8 Table 8 Risk factors of mastitis. Variable Abbreviation B 1 OR 2 95%CI 3 p Value Milk yield 2 cnl2 0.14 1.15 1.01 1.31 0.031 Fat percentage 1 rzl1 1.90 6.72 0.93 48.79 0.036 Fat percentage 2 rzl2 0.95 2.60 0.77 8.71 0.127 Fat percentage 3 rzl3 1.20 3.32 1.41 7.81 0.003 Protein percentage 1 dbl1 -2.73 0.07 0.00 1.46 0.054 Lactose percentage 4 rtl4 -4.02 0.02 0.00 0.15 0.000 Fat/protein ratio 1 zdb1 -4.53 0.01 0.00 4.16 0.102 Fat/protein ratio 3 zdb3 -2.55 0.08 0.01 1.19 0.043 Month 5 yuefen5 0.14 1.15 1.02 1.30 0.021 1 b = regression coefficient. 2 OR = odds ratio. 3 CI = confidence interval. Disclaimer/Publisher's Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

PMC10000151

Background: The majority of locally advanced cervical cancers (LaCC) are causally related to HPV. We sought to investigate the utility of an ultra-sensitive HPV-DNA next generation sequencing (NGS) assay--panHPV-detect--in LaCC treated with chemoradiotherapy, as a marker of treatment response and persistent disease. Method: Serial blood samples were collected from 22 patients with LaCC before, during and after chemoradiation. The presence of circulating HPV-DNA was correlated with clinical and radiological outcomes. Results: The panHPV-detect test demonstrated a sensitivity and specificity of 88% (95% CI-70-99%) and 100% (95% CI-30-100%), respectively, and correctly identified the HPV-subtype (16, 18, 45, 58). After a median follow up of 16 months, and three relapses all had detectable cHPV-DNA at 3 months post-CRT despite complete response on imaging. Another four patients with radiological partial or equivocal response and undetectable cHPV-DNA at the 3-month time point did not go on to develop relapse. All patients with radiological CR and undetectable cHPV-DNA at 3-months remained disease free. Conclusions: These results demonstrate that the panHPV-detect test shows high sensitivity and specificity for detecting cHPV-DNA in plasma. The test has potential applications in assessment of the response to CRT and in monitoring for relapse, and these initial findings warrant validation in a larger cohort. cervical cancer plasma HPV DNA response prediction circulating DNA next generation sequencing The Royal Marsden NHS Foundation Trust and The Institute of Cancer ResearchRMH Biobank was supported by the National Institute for Health Research Royal Marsden and Institute of Cancer Research Biomedical Research Centre and the Clinical Research FacilityA67 B014 Cancer Research UK ProgrammeC46/A10588 C7224/A13407 C7224/A23275 C7224/A28724 Rosetrees Trust, Oracle Cancer Trust and NIHR Senior Investigator FellowshipThis work was undertaken in The Royal Marsden NHS Foundation Trust and The Institute of Cancer Research. The Royal Marsden NHS Foundation Trust received a proportion of its funding from the NHS Executive; the views expressed in this publication are those of the authors and not necessarily those of the NHS Executive. This work (BRC grant no. A67 and B014) and sample processing which was done by the RMH Biobank was supported by the National Institute for Health Research Royal Marsden and Institute of Cancer Research Biomedical Research Centre and the Clinical Research Facility BRC grant no. A67. The authors acknowledge the support of Cancer Research UK Programme Grants C46/A10588, C7224/A13407, C7224/A23275 and C7224/A28724 (CRUK ICR/RM RadNet Centre of Excellence). KJH acknowledges support from Rosetrees Trust, Oracle Cancer Trust and NIHR Senior Investigator Fellowship. pmc1. Background Locally advanced cervical cancer (LaCC) FIGO (2019) stage IB3-IVA is treated with external beam radiotherapy (EBRT) and concomitant cisplatin, followed by image guided adaptive brachytherapy (IGABT). In contemporary series this leads to excellent 5-year local control rates of 89%, and 5-year overall survival of 65% across all stages . When relapse occurs, it is most often metastatic but can include cases of salvageable para-aortic lymph node relapse or oligometastatic disease. A smaller proportion of patients will also experience persistent or recurrent disease within the cervix or uterus, amenable to salvage surgery . The identification of persistent disease and early relapse is challenging and largely relies on multiparametric functional imaging for which differentiating between disease and treatment related changes is difficult . Often, recurrent or persistent disease may not be detected radiologically until wider metastasis has developed. On the other hand, sometimes, unnecessary salvage surgery is performed, which can lead to considerable morbidity. Therefore, there is a need to establish an alternative marker of active disease, for use as a monitoring tool to identify patients with either persistent or emerging disease, to complement and stratify existing clinical and radiological tools. The majority of LaCC are caused by the human papilloma virus (HPV). During carcinogenesis, the viral DNA that is present in the tumour cell (integrated into the tumour DNA or in episomal form) is released into the blood following tumour lysis and can be measured. Studies have been published demonstrating the utility of circulating tumour DNA as a method of investigating and monitoring tumour biology and clinical status ; circulating HPV-DNA (cHPV-DNA) can potentially be used as a detection marker for HPV-related LaCC. Published studies have used polymerase chain reaction (PCR) based techniques to detect cHPV-DNA in patients with LaCC at diagnosis; with variable sensitivity of 24-83% . The potential of cHPV-DNA as a marker of disease response following radical chemo-radiotherapy in LaCC has not been extensively evaluated. We developed an ultra-sensitive HPV DNA next generation sequencing (NGS) assay, panHPV-detect, with the ability to comprehensively detect circulating DNA of high-risk HPV genomes (16, 18, 31, 33, 35, 45, 52 and 58) and assess its relationship with disease status and response. We have previously validated this assay in patients with locally advanced anal cancer undergoing CRT. Here, we validate the assay in prospectively collected plasma DNA at serial time points in patients with LaCC treated with primary CRT. 2. Materials and Methods Informed consent was obtained from patients with LaCC planned to receive chemoradiation. The Institutional board (Ref. no. CCR 4157) and ethics committee (Ref. no. 14/NE/1055) approved the study. Treatment consisted of external beam radiotherapy to the pelvis (and para-aortic lymph nodes if common iliac lymph nodes involved) to a dose of 45 Gy in 25 fractions, concomitant cisplatin 40 mg/m2 weekly, and image guided adaptive brachytherapy to a combined target dose >=85 Gy EQD2. Involved lymph nodes were treated to 55-57.5 Gy in 25 fractions using a concomitant boost technique. Pelvic examination, multiparametric MRI and FDG PET/CT were performed at baseline, end of external beam chemoradiotherapy (week 5) and at 3 months post completion of brachytherapy. In the case of complete local response, further imaging with MRI and CT thorax was deferred until 12 months from completion of treatment, although patients continued to be followed with pelvic examination every 3 months. Standard practice for patients with negative PET/CT but equivocal MRI was to perform repeat MRI 3 months later, i.e., at 6 months. To identify locally persistent or recurrent cervical disease any patients with persistent FDG avidity at any time and corresponding MRI changes underwent examination under anaesthetic and cervical biopsy. Patients with regional or distant metastatic relapse were identified on serial imaging at the timepoints described. Serial plasma samples (20 mL) were collected at baseline (before CRT), at week 6-7 (during brachytherapy) and 3 months following completion of treatment in Streck(r) tubes. Samples of pooled plasma from a set of five healthy pre-screened individuals with no cancer or history of cancer purchased from CTLS (Clinical Trial Laboratory Services), London and 19 patients with breast cancer enrolled in the prospective sample collection study (PlasmaDNA, CCR3297, REC Ref No: 10/H0805/50) were used as negative controls. None of the negative controls were known to have pre-cancerous lesions. Written informed consent was obtained from all control participants. 2.1. Blood Samples: Plasma Processing and DNA Extraction 20 mL of blood was centrifuged at 1600x g for 10 min within 48 h of collection. The resulting plasma was then centrifuged again at 1600x g for 10 min, aliquoted and frozen at -80 degC. DNA was extracted from 5 mL of plasma using the QIAamp Circulating Nucleic Acid Kit (Qiagen, Manchester, UK) according to manufacturer's instructions. DNA was eluted in 50 mL of AVE buffer and stored at -20 degC. Plasma DNA was quantified using a Bio-Rad QX200 ddPCR system, using ribonuclease P (RNase P) as a reference gene as previously described . 2.2. Tumour Biopsies: HPV DETECTION in Tumour Formalin fixed paraffin embedded tumour blocks of the diagnostic biopsy samples were obtained. Eight 10 mm nuclear fast red-stained slides and two haematoxylin and eosin (H&E) stained slides were obtained from representative FFPE blocks. Tumour content, cellularity and suitable areas of tumour were marked for macro-dissection. DNA was extracted using the AllPrep DNA FFPE Kit (Qiagen, Manchester, UK) followed by cDNA synthesis using the Omniscript Reverse Transcription kit (Qiagen, Manchester, UK). 2.3. cHPV-DNA Sequencing in Plasma and Tissue (panHPV-Detect Assay Design) PanHPV-detect was designed as follows. Representative sub-lineages from each of the eight high risk HPV genotypes associated with 98% of cervical cancers (16, 18, 31, 33, 35, 45, 52 and 58) were aligned using CLUSTAL O (1.2.1) multiple sequence alignment programme accessed on 30 November 2020). Diagnostic single nucleotide polymorphisms (SNP) were identified for each sub-lineage and used as a guide for primer design using the Ampliseq Designer (ThermoFisher Scientific, Manchester, UK). Eight primer sets targeting the human genes ACTINB, GAPDH, HPRT were also included in the panel to act as positive controls for library preparation and sequencing efficiency. A total of 2043 SNP targets were submitted; 235 targets were missed thus providing 88.5% coverage. The amplicon length of 140 bp was specified with the primary aim of detection of circulating free DNA fragments in plasma. The panel consisted of 548 primers divided into two pools (Table 1). Ion torrent libraries were prepared using an Ion Ampliseq library preparation kit 2.0 (ThermoFisher Scientific) according to manufacturer's instructions using 5 ng of tissue DNA or 3 ng of plasma DNA per primer pool. Reads were aligned to an amalgamated reference containing scaffolds for each of the HPV genotypes as well as the reference human targeting genes using TMAP on the Ion Torrent machine. Bedtools v2.23.0 was used to extract on-target reads from the aligned files with a minimum overlap of 90% with amplicons in the panel. Additionally reads with a mapping quality of <30 were removed using samtools v1.2 . Reads were split into those covering human and HPV amplicons and coverage of each portion and each genotype was calculated individually. We performed an initial validation of panHPV-detect by testing its ability to correctly identify the HPV sub-type in cervical cancer tissue samples. We obtained tumour DNA extracted from cervical cancer tissue previously typed for the HPV sub-type using polymerase chain reaction (RT-PCR) for E6 and E7 mRNA using validated assays. Five samples for each of the eight high-risk HPV sub-types were obtained from the Scottish HPV archive. Samples from five HPV negative patients, with head and neck cancer recruited in the head and neck cancer cohort of this study and 19 patients with breast cancer enrolled in the prospective sample collection study (PlasmaDNA, CCR3297, REC Ref No: 10/H0805/50) were used as negative controls. HPV status was confirmed as negative with E7 RT-PCR performed on RNA extracted from the samples. None of the negative controls were known to have pre-cancerous lesions. Written informed consent was obtained from all participants. Following the initial validation, we investigated whether panHPV-detect was able to detect cHPV-DNA in pre-treatment plasma of patients recruited in the study. We then tested the concordance of HPV subtype identified by panHPV-detect in plasma with the HPV subtype identified in the tumour biopsies collected from the patients at diagnosis. 2.4. Sample Size Calculation The primary endpoint for this pilot study was the feasibility of detecting cHPV-DNA at baseline in patients undergoing CRT with radical intent. Our pilot study in HPV+ head and neck cancer demonstrated 100% sensitivity and 93% specificity in detecting cHPV-DNA in patients' plasma at baseline (pre-treatment) using the original NGS assay (HPV-detect) . To prove 85% sensitivity assuming that the true sensitivity is 99% and to have 80% power given two-sided type I error of 0.05, would require at least 19 HPV+ patients, all of them expected to be labelled as positive by NGS. Since ~85% of LaCC patients are HPV+, therefore, the study required 22 patients. The secondary endpoint was to assess the potential of cHPV-DNA to predict response to treatment. 2.5. Data Analyses Sensitivity and Specificity were calculated using contingency tables. These were calculated with the tissue HPV status and sub-type obtained from the patient's tumour biopsy as the gold standard test. All statistical analyses were performed using GraphPad Prism version 7 software. To classify HPV+ and using panHPV-detect in tissue, we set a threshold whereby a sample was classified positive if there were ten reads present from more than 30% of the different HPV amplicons for each sub-type. To assess the threshold for the number of amplicons needed for positive panHPV-detect readout in plasma--a ROC analysis was used. In the first step, HPV status and subtype was assigned in tissue using E7 mRNA to separate the two groups. The number of amplicons with greater than ten reads at baseline was input for each patient to find a suitable threshold for this parameter. Sorting the values in both HPV+ and negative groups and averaging adjacent values in the sorted list generated a list of thresholds. Based on the ROC analysis, a threshold that gave the greatest sensitivity and specificity for each HPV subtype was selected as the threshold for classification of plasma as HPV DNA positive. Based on the ROC analysis, a threshold of 6.5 amplicons with more than ten reads and 7.5 amplicons in more than ten reads was set for HPV16 and HPV18, respectively. These were the thresholds that gave the greatest sensitivity and specificity and were selected as thresholds for classification of plasma as HPV DNA positive. There were insufficient plasma samples for the other rarer HPV subtypes to provide a statistically robust threshold and therefore, an empirical and pragmatic (between 6.5 and 7.5 amplicons) threshold of seven amplicons with greater than ten reads were set for the other subtypes. 3. Results Tumour DNA from 39/40 pre-typed tumour samples (five of each of the HPV sub-types 16, 18, 31, 33, 35, 45, 52 and 58) obtained for validation was of satisfactory quality for the initial tissue validation. PanHPV-detect demonstrated 100% sensitivity and specificity in correctly identifying the HPV sub-type in tumour DNA and negative controls (Table 2). Twenty-two patients were recruited into the study. The median age was 48 (range 33-88). Patient characteristics are described in Table 3. Tumour tissue and baseline blood samples were available for 20 patients. In two patients' plasma samples were of poor quality and unsuitable for analysis. Tumour tissue in 18/20 patients was positive for HPV. cHPV-DNA was detected by panHPV-detect in 16/18 plasma samples. The tissue sample from one patient was positive for HPV59, which was not included in panHPV-detect. cHPV-DNA was not detected in plasma of HPV negative patients. Therefore, panHPV-detect demonstrated a sensitivity and specificity of 88% (95% CI-70-99%) and 100% (95% CI-30-100%), respectively. In 16 samples with detectable cHPV-DNA, the following sub-types were correctly identified as follows--HPV16--11 samples, HPV18--5 samples, HPV45--2 samples and HPV58--3 samples. Some samples had multiple HPV-subtypes. See Table 4. The median follow-up for all patients was 16 months (range 11-29). A total of 16 of the 22 patients recruited in the study had a complete response (CR) and six patients had equivocal disease according to MRI and PET-CT assessment at 3 months following completion of CRT. Of the 16 patients with detectable cHPV-DNA at baseline, cHPV-DNA was detected in plasma at 3 months in the two patients with residual disease and one patient with clinical CR. Of the two patients with detectable cHPV-DNA and radiological residual disease, one had disease in the para-aortic nodes and the other had residual disease in the cervix. The para-aortic disease was treated with radiotherapy. The other patient was referred for salvage surgery; however, this was not undertaken due to significant comorbidities. The patient with detectable cHPV-DNA and clinical CR at the 3-month time-point had clinical relapse at 14 months post-treatment. This was treated with salvage surgery. Four patients with radiological partial or equivocal response and undetectable cHPV-DNA at the 3-month time point and eight patients with CR and undetectable cHPV-DNA at the 3-month time-point were disease free at the last follow-up appointment. One patient with CR and undetectable cHPV-DNA at the 3-month time-point relapsed at 14 months post-CRT. This was successfully treated with salvage surgery. Of the 16 patients with detectable cHPV-DNA at baseline, panHPV-detect accurately predicted presence/absence of residual disease (macroscopic/microscopic) in 15 out of the 16 patients. 4. Discussion We have designed "panHPV-detect" a novel NGS based method for detection and tracking of cHPV-DNA from eight high-risk HPV genotypes. The assay was initially tested in banked HPV-typed cervical tissue samples. Following this, we tested this assay in a prospective observational biological sample collection study in patients undergoing radical CRT and we have demonstrated high sensitivity and specificity. Tracking cHPV-DNA in sequential samples through and after CRT accurately predicted response and residual disease suggesting the potential of panHPV-detect to enhance clinical decision-making. Compared to PCR-based assays, amplicon-based NGS of multiple regions of the viral genome is able to detect cHPV-DNA at baseline, with higher sensitivity and specificity. To our knowledge this is the first study to use NGS to detect cHPV-DNA in patients undergoing CRT for LaCC. Previously published studies used PCR for cHPV-DNA detection, with reported sensitivity of detecting cHPV-DNA at baseline of between 24-83% (Table 5). The majority of these studies used primers specific to HPV16 and 18. Although, 16 and 18 are the oncogenic HPV subtypes in approximately 80% of the HPV associated LaCC, excluding the other high-risk sub-types reduces the sensitivity of the assay. Despite using an identical PCR primer set for the seven commonest high-risk sub-types, two studies demonstrate sensitivity of 12% and 65% . Our NGS based assay which is able to detect multiple HPV genomic regions across the eight most common high-risk sub-types, demonstrates a superior sensitivity to the PCR based technique. We envisage several uses for cHPV-DNA in LaCC. Firstly, as an adjunct to standard-of-care MRI and/or PET-CT scans, cHPV-DNA could potentially confirm complete response at 3 months, precluding the requirement for repeated scans and biopsies. This would result in a significant health economic impact and avoid unnecessary patient morbidity and anxiety. This is supported in our study by the observation that 14 out of 16 patients with detectable baseline cHPV-DNA in our study had undetectable cHPV-DNA at 3 months post-CRT. Out of the 14, four had partial or equivocal radiological response; however, no evidence of disease relapse was observed on subsequent imaging and these patients remain relapse free at the last follow-up. Secondly, in other cases of equivocal imaging and where biopsy is either not possible or negative, cHPV-DNA could be used to confirm both local and metastatic disease. Three patients in our study had detectable cHPV-DNA at 3 months and subsequently had confirmed relapse. To our knowledge only three other studies correlated cHPV-DNA levels to disease response. In the study by Campitelli et al. high cHPV-DNA levels in 2 patients (out of their 16 patients with post-CRT blood samples) correlated with disease relapse and in the study by Yang et al. 5 patients (out of their 21 patients with post-CRT blood samples) had detectable cHPV-DNA all of whom had disease relapse. Elevated cHPV-DNA levels at 3-months following CRT in our study and the ones by Campitelli et al. and Yang et al. accurately predict residual disease and/or disease relapse (10/53 patients with detectable cHPV-DNA at 3 months, the three studies combined). More recently Leung et al. evaluated an NGS technique with 100% sensitivity and 67% specificity for detection of recurrence. Patients considered to have residual or recurrent cervical disease may be evaluated for salvage surgery. Although repeat cervical biopsy is undertaken prior to surgery, in the post CRT setting this is not targeted and difficult to interpret. Some patients will therefore proceed to surgery without positive biopsy and ultimately will not have pathological evidence of disease on hysterectomy specimen. Surgical complications of wound infection and healing are increased after CRT. Another role for cHPV-DNA is in the monitoring or follow up phase. Patients with persistent cHPV-DNA at 3 months and negative imaging could be triaged to more intensive imaging follow up, hopefully to capture early emergence of relapse sites potentially amenable to targeted treatment. Furthermore, using more sensitive assays such as plasma cHPV-DNA as a marker for detection of true high-risk patients could aid the design of adjuvant therapy studies. Using panHPV-detect we were able to accurately identify the specific sub-type of the cHPV-DNA, which possibly indicates the causative oncogenic sub-type. Studies have suggested that the causative HPV sub-type can affect prognosis with the alpha-7 (HPV18, 39, 45) sub-types having inferior outcomes compared to alpha-9 (HPV16, 31, 33, 52, 58) sub-types following CRT and worse prognosis following primary surgery in cervical cancers related to HPV 18 . Therefore, identification of the correct sub-type driving the oncogenic process may provide prognostic information. This was a pilot study to examine the potential of a novel NGS assay to detect cHPV-DNA before radical therapy and identify the causative HPV sub-type. This study did not have adequate power to so. However, it confirms the findings from studies by Campitelli et al. and Yang et al. highlighting the potential of cHPV-DNA in predicting disease response following CRT. 5. Conclusions cHPV-DNA has significant potential as a biomarker of response following radical intent CRT and of recurrence during surveillance for patients with LaCC. Furthermore, panHPV-detect can identify cHPV-DNA with high sensitivity and specificity enabling the use of cHPV-DNA as a biomarker to become a reality, albeit further validation in larger multi-centre studies is required. Such validation studies of panHPV-detect in patients undergoing curative intent CRT in LaCC are currently in development. Acknowledgments The authors also acknowledge The Scottish Human Papillomavirus Investigators network (SHINe) and The Scottish HPV archive for providing tissue samples for validation. Author Contributions S.L.: conceptualisation, project administration, data curation, resources, formal analysis, visualisation, manuscript editing; J.L.: investigation, manuscript review and editing; R.J.C.: investigation, manuscript review and editing; I.G.M.: investigation, manuscript review and editing; N.M.: investigation--sequencing; K.H.: supervision, resources, manuscript review and editing; K.V.: investigation, resources, manuscript review; E.M.: resources, manuscript review; N.T.: resources, manuscript review; S.A.B.: conceptualisation, project administration, formal analysis, manuscript editing. All authors have read and agreed to the published version of the manuscript. Institutional Review Board Statement This study was reviewed and approved by the Committee for Clinical Research (Review board) at the Royal Marsden Hospital NHS trust (Ref: CCR 4157). The study was also reviewed and approved by Health Research Authority (HRA) North-east--York Research ethics committee (REC)--Ref-14/NE/1055, approval letter dated 19 May 2017. Informed Consent Statement All participants provided written informed consent for participation in this study. Data Availability Statement Data are contained within the article. Conflicts of Interest J.L., R.J.C., I.G.M., N.M., K.H., K.V., E.M. have no declaration. S.L. declares honaria from MSD, GSK and Elekta. cancers-15-01387-t001_Table 1 Table 1 Details of panHPV-detect panel primer pools. HPV Primers in Pool 1 Primers in Pool 2 Total HPV16 40 39 79 HPV18 41 41 82 HPV31 33 32 65 HPV33 35 35 70 HPV35 15 14 29 HPV45 34 34 68 HPV52 38 38 76 HVP58 36 35 71 cancers-15-01387-t002_Table 2 Table 2 Sensitivity and specificity of panHPV-detect assay following initial validation of panHPV-detect in cervical cancer tissue samples obtained from another biobank that were confirmed HPV positive. Refer to text for details on negative controls. Tissue HPV Status--PCR panHPV-detect Positive Negative Total Present 39 0 Absent 0 24 Total 39 (Sensitivity 100%) 24 (Specificity 100%) 63 cancers-15-01387-t003_Table 3 Table 3 Patient characteristics for the cohort. Variable Number of Cases Number of patients 22 FIGO Stage I A 0 B 2 II A 2 B 15 III A 0 B 1 IV C 0 A 1 B 0 Pack-years (Includes ex Smokers) NS (Never Smoked) 15 <=20 4 >20 3 cancers-15-01387-t004_Table 4 Table 4 Details the HPV status in tissue at diagnostic pathology and in tissue and plasma using panHPV-detect. Patient Trial ID HPV-Status Tissue Diagnostic Pathology HPV-Status panHPV-Detect Tissue HPV-Status panHPV-Detect Plasma CCR 4157 (C01) 18 18 18 CCR 4157 (C02) 16 16 16 CCR 4157 (C03) 16 16 16 CCR 4157 (C04) 16 16 16 CCR 4157 (C05) neg neg neg CCR 4157 (C06) neg neg neg CCR 4157 (C07) 16 16 16 CCR 4157 (C08) 16 tissue block not available 16 CCR 4157 (C09) 18 18 not detected CCR 4157 (C010) 45 45 45 CCR 4157 (C011) unknown 16,58 16, 58 CCR 4157 (C012) not detected 18 18 CCR 4157 (C013) 59 not in panel NA CCR 4157 (C014) unknown 16,58 16, 58 CCR 4157 (C015) 16 D 16 no BL blood CCR 4157 (C016) 18 16,18 16, 18 CCR 4157 (C017) not done 16,18 16, 18 CCR 4157 (C018) 18 18 no BL blood CCR 4157 (C019) 18 no block 18 CCR 4157 (C020) unknown 16,58 16, 58 CCR 4157 (C021) 18 16,18 16, 18 CCR 4157 (C022) 45 45 45 cancers-15-01387-t005_Table 5 Table 5 Studies reporting PCR techniques for HPV DNA detection. Author Technique Stage Sample Size Sensitivity Specificity HPV Sub-Types Serial Samples Cheung PCR Non-metastatic 138 55.8% NR 16, 18 No Hsu PCR Non-metastatic 112 24.1% 100% 16, 18 No Satish PCR Non-metastatic 58 11.8% 100% 16, 18, 31, 33, 35, 45, 58 No Yang PCR Non-metastatic 50 50% 85% 16, 18 Yes Wei PCR Non-metastatic 23 65% 100% 16, 18, 31, 33, 35, 45, 58 No Jaberipour PCR Metastatic and Non-metastatic 69 23.5% 90% 16, 18, 33, 52 No Campitelli PCR Metastatic and Non-metastatic 16 81.25% 100% 16, 18 Yes Jeannot PCR Metastatic and Non-metastatic 47 83% 100% 16, 18 No Current study NGS Non-metastatic 22 88% 100% 16, 18, 31, 33, 35, 45, 52 and 58 Yes Disclaimer/Publisher's Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

PMC10000152

Approximately 15% of breast cancers are classified as HER2-positive, with an amplification of the ERBB2 gene and/or an overexpression of the HER2 protein. Up to 30% of HER2-positive breast cancers shows heterogeneity in HER2 expression and different patterns of spatial distribution, i.e., the variability in the distribution and expression of the HER2 protein within a single tumour. Spatial heterogeneity may potentially affect treatment, response, assessment of HER2 status and consequently, may impact on the best treatment strategy. Understanding this feature can help clinicians to predict response to HER2-targeted therapies and patient outcomes, and to fine tune treatment decisions. This review summarizes the available evidence on HER2 heterogeneity and spatial distribution and how this may affect current available treatment choices, exploring possible opportunities for overcoming this issue, such as novel pharmacological agents, belonging to the group of antibody-drug conjugates. HER2 breast cancer spatial distribution heterogeneity resistance This research received no external funding. pmc1. Introduction Approximately 15% of breast cancers (BCs) harbour an amplification of the ERBB2 gene, which encodes human epidermal growth factor receptor 2 (HER2) . Four main biological entities of BC could be identified, based on the gene expression profile: luminal A, luminal B, HER2-enriched and basal-like . In clinical practice, a surrogate classification is adopted, based on the evaluation of three key predictive biomarkers: two hormone receptors (HR), namely estrogen receptor (ER) and progesterone receptor (PgR), and HER2 overexpression and/or amplification . Thus, experts commonly define three different BC subtypes: luminal-like (ER-positive and/or PgR-positive and HER2-negative), HER2-positive (HER2 overexpression/amplification, with any ER and PgR expression), and triple negative (ER-negative, PgR-negative and HER2-negative) tumours. According to the latest ASCO/CAP guidelines (2018), the evaluation of HER2 status is defined by an immunohistochemistry (IHC) score as follows: 0 or 1+ for HER2-negative, 2+ for HER2-equivocal and 3+ for HER-positive . HER2-equivocals are further examined by in situ hybridization (ISH) assay to categorize tumours as either HER2-negative or HER2-positive (Table 1). ASCO/CAP guidelines state the predictive role of HER2, helping clinicians to identify patients with HER2-positive tumours who would benefit from anti-HER2 targeted treatment, compared to patients affected by HER2-negative BC . Indeed, targeted therapies capable of binding HER2 and blocking the downstream signalling, such as trastuzumab, pertuzumab, lapatinib and trastuzumab emtansine (T-DM1), have significantly changed the prognosis of HER2-positive BC . The mechanisms of resistance and sensitivity to anti-HER2 therapies in HER2-positive BC have been thoroughly investigated and reported , however, in this context little is known about the role of HER2 heterogeneity defined by ISH . Indeed, HER2 expression could be heterogeneous, showing different patterns of spatial and temporal distribution. HER2 temporal heterogeneity is beyond the scope of this review . HER2 spatial heterogeneity refers to the variability in the distribution and expression of the HER2 protein within a single tumour. This means that different areas within the same tumour may have different levels of HER2 expression, thus making the targeting of the protein potentially less effective. This is one of the reasons that may lead to treatment resistance. Spatial heterogeneity may also make it more difficult to accurately assess the HER2 status of a tumour and, accordingly, to determine the best treatment approach. Understanding the spatial heterogeneity of HER2 expression within a tumour can be important to predict response to HER2-targeted therapies, to fine tune treatment decisions and to predict patient outcomes . Moreover, the different patterns of HER2 spatial distribution could play a role in the efficacy of anti-HER2 drugs. The activity of some new pharmacological agents, belonging to the group of antibody-drug conjugates (ADC), may represent an opportunity for overcoming this issue. This review summarizes the available evidence on HER2 heterogeneity and spatial distribution and how they may affect current available treatment choices. 2. Overview of the Current Management of HER2-Positive Breast Cancer The current management of HER2-positive BC in early and in metastatic settings relies on anti-HER2 targeted therapies. In the early setting, the choice of (neo)adjuvant treatment is based upon the expected tumour sensitivity to pre-specified agents, individual risk of relapse and long-term toxicities. For tumours with a diameter less than 1 cm and without clinical lymph nodes involvement, surgery can be proposed. If a pathological stage I is confirmed, a treatment de-escalation could be planned (weekly paclitaxel for 12 administrations plus trastuzumab for 1 year), according to the results of the APT trial (3-yr invasive disease free survival (iDFS) 98.7%, confidence interval (CI) = 97.6-99.8) . In case of an (unexpected) pathological stage II or III, the preferred choice is adjuvant trastuzumab (plus pertuzumab) for 1 year with docetaxel/carboplatin (TC) or anthracyclines/cyclophosphamide/paclitaxel (ACT) . For tumours of at least 1-2 cm in greatest diameter, a neoadjuvant approach is the standard of care with chemotherapy plus trastuzumab +/- pertuzumab, with the possibility of fine tuning the treatment choice based on the individual risk of relapse . If pathological complete response (pCR) is not achieved after neoadjuvant treatment, T-DM1 has to be offered based on the results of the KATHERINE trial (3-yr iDFS 88.3% vs. 77.0%, hazard ratio 0.50 CI 0.39-0.64, p < 0.001) . In case a pCR is obtained, trastuzumab (plus pertuzumab) should be administered for a total of 12 months. For triple positive tumours (i.e., HR-positive and HER2-positive tumours), endocrine therapy should be added as per international guidelines . In the advanced setting, the CLEOPATRA clinical trial sets a new standard of care, represented by pertuzumab, trastuzumab and docetaxel, followed by maintenance pertuzumab/trastuzumab (median overall survival (mOS) 56.5 months vs. 40.8 months, hazard ratio 0.68 CI 0.56-0.84, p > 0.001) . Upon disease progression, patients could receive a new treatment option with trastuzumab deruxtecan (T-DXd) or tucatinib/capecitabine/trastuzumab (preferred choice in case of active brain metastases) . Indeed, data from the DESTINY-Breast03 trial established T-DXd as the second-line preferred choice in patients previously treated with a taxane and trastuzumab, given the benefit in both progression-free survival [PFS] and OS: for PFS, 28.8 months vs. 6.8 months compared to T-DM1 (hazard ratio 0.33, 95% CI 0.26-0.43, p < 0.0001); OS not reached (CI 40.5 months-not estimable) vs. OS not reached (34.0 months--not estimable), hazard ratio 0.64, (CI 0.47-0.87, p = 0.0037) . Even if the randomized phase II HER2CLIMB enrolled patients who had previously received trastuzumab and T-DM1, the PFS and OS benefits in patients with active or stable brain metastasis suggest its possible use for selected patients in this setting . At the time being, prospective data on the best treatment sequence for patients in third line and beyond are lacking. Certainly, there is the possibility to use tucatinib or T-DXd if not previously used . The use of T-DM1 is possible as well . Later lines may include the tyrosine kinase inhibitor (TKI) lapatinib, preferably in combination with capecitabine, and of the pan-HER TKI neratinib plus chemotherapy (Food and Drug Administration [FDA] approved, not European Medical Agency (EMA) approved), margetuximab might be used as well (FDA approved, not EMA approved) . Another, although old-fashion possibility, is to keep using trastuzumab beyond disease progression, but in combination with a different chemotherapeutic agent (e.g., vinorelbine, capecitabine, eribulin) . Endocrine therapy could be added to anti-HER2 treatments if BC also expresses HR . 3. The Resistance to Anti-HER2 Treatments HER2 is a transmembrane protein receptor belonging to the HER family . All members of this family are receptor tyrosine kinases (RTK) with an analogous structure, characterized by an extracellular ligand-binding domain, a transmembrane domain, and an intracellular tyrosine kinase domain. The binding between HER receptors and their ligands induces both homodimeric and heterodimeric interactions between family members, with consequent activation of downstream pathways involving phosphatidylinositol 3-kinase (PI3K) and RAS. When PI3K is stimulated, it leads to activation of protein kinase B (AKT) and mammalian target of rapamycin (mTOR), promoting cell proliferation, growth, and survival . RAS activates downstream proteins, namely the RAF, MEK and ERK. The oncogenic role of HER2 is mainly associated with the ERBB2 gene overexpression (located on chromosome 17q21), increasing the number of HER2 heterodimers on the cell's surface with the hyperactivation of oncogenic pathways, ultimately driving cell cycle progression, angiogenesis, invasiveness and metabolic reprogramming . In this section, the main mechanisms of resistance to anti-HER2 targeted therapy are summarized . 3.1. Impaired Binding to HER2 To function properly, each drug should be capable of accessing its binding site located on the HER2 protein. Low levels of HER2 and HER2 mutations determine a well-known lack of efficacy for the traditional anti-HER2 drugs . Indeed, variants in the structure of HER2 proteins may hinder the correct function of these agents. One example is the expression of specific HER2 splicing variants, which compromise the successful binding of monoclonal antibodies such as trastuzumab. Specifically, when the extracellular domain of HER2 is cleaved by the metalloproteinase ADAM10, the result is the 95HER2 isoform, which is unable to bind antibodies, but is sensitive to TKIs such as lapatinib . Given the absence of standardized methods to detect HER2 variants, their presence is not routinely assessed . Another proposed mechanism of impaired binding for monoclonal antibodies is the masking of the HER2 binding site. For example, membrane-associated mucin 4 (MUC4), which could be expressed by tumour cells or other cells in the tumour microenvironment (TME), hides the trastuzumab binding site on the extracellular domain IV of HER2 . 3.2. HER2 Mutations Activating mutations in the ERBB2 gene are detected in 2-3% of primary BCs (regardless of HER2 amplification) and in 3-5% of mBC; they are more frequent in lobular cancer and are associated with worse prognosis . The majority of HER2 mutations are located in the tyrosine kinase and in extracellular domains . In clinical practice, these variants cannot be detected by standard assays (IHC and FISH) and may only be detected through genome sequencing. Pre-clinical and clinical data support the hypothesis that these mutations have a role in the resistance to anti-HER2 treatment and to endocrine therapy . In particular, regarding HER2-positive BC, variants of the intracellular domain confer specific resistance to anti-HER2 TKIs, determining their inability to block HER2 function. L755S is the most common mutation and represents a mechanism of resistance to lapatinib , but it could be overcame by neratinib . Mutations L755S, V777L, D769Y and K753E are responsible for resistance to trastuzumab . In HR-positive HER2-negative BC, somatic HER2 mutations may lead to resistance to endocrine therapy , potentially conferring sensitivity to anti-HER2 TKIs . The SUMMIT trial (NCT01953926) showed that the combination of neratinib plus fulvestrant is clinically active in heavily pre-treated HER2-mutant HR-positive mBC patients (including those pre-treated with fulvestrant and CDK4/6 inhibitors): ORR was 30%, clinical benefit rate was 47% and mPFS was 5.4 months . Since genomic analyses suggested that resistance to neratinib may occur via mutant allele amplification or secondary HER2 mutations, the trial was amended to explore dual HER2 targeting with the combination of neratinib, trastuzumab plus in this subgroup. This strategy showed a clinical benefit rate of 47% and a mPFS of 8.2 months . 3.3. Altered Intracellular Signalling It is reasonable that resistance to anti-HER2 treatment derives from the constitutive activation of downstream signalling pathways of HER2. In this way, HER2-amplified cells become HER2-indipendent for their proliferation and survival, and the inhibition of the signalling cascade given by anti-HER2 treatment is bypassed. One example is represented by activating mutations in the alfa catalytic subunit of PI3K (PI3KCA), found in up to 20% of HER2-positive BCs . This gene is known to be involved in PI3K/AKT/mTOR cascade, activated by HER2 overexpression, and its upregulation may lead to increased cell proliferation and tumour progression . Recent evidence demonstrates that enrichment of MAP kinase pathway mutations can promote a switch in pathway dependence from PI3K/AKT to MEK/ERK, leading to resistance to anti-HER2 therapies . In addition, parallel pathways may be boosted; for instance, the hyperactivity of ER enhances the PI3K/AKT/mTOR pathway and determines acquired resistance to anti-HER2 targeted treatment . Aberrant activation of tyrosine kinase SRC and enhancement of the Cyclin D1-CDK 4/6 (cyclin dependent kinase) axis are other mechanisms of resistance described for both trastuzumab and TKIs . Moreover, different RTKs can heterodimerize with HER2, especially EGFR/HER1 and HER3; the overexpression of these partners promotes the formation of HER2/EGFR and HER2/HER3 heterodimers, escaping the inhibition of HER2 homodimerization given by monoclonal antibodies. The formation of heterodimers is stimulated by neuroregulin-1 (NRG1), which has been associated with resistance to T-DM1 and to TKIs . A crosstalk between the ER and the HER2 pathway has been described; indeed, approximately 50% of HER2-positive BCs overexpress ER and the crosstalk affect response to therapy and outcome. Finally, the overexpression of other proteins involved in the balance between cell death and survival could compensate the loss of HER2 function . 3.4. Other Mechanisms of Resistance Anti-HER2 therapy is subject to multidrug resistance mechanisms, specifically caused by P-glycoprotein, ABCG2 (more commonly known as BCRP (Breast Cancer Resistance Protein)), and multidrug resistance protein (MRP). The presence of drug efflux pumps on the tumour cell wall also reduces the intracellular cytotoxic action of the warhead of ADCs (for example, DXd and DM). Moreover, the heterogeneity in the expression of HER2 could impair the effectiveness of anti-HER2 drugs . Among trastuzumab's mechanisms of action, one of the most important is its ability to trigger antibody-dependent cytotoxicity (ADCC), which occurs when trastuzumab is bound to breast cancer cells and its Fcg region is recognized by immune cells expressing FcgRs. Several factors, such as polymorphisms in the Fcy receptor, may impair this binding and the immune activity of trastuzumab. The quantity of immune cells that are present in TME could also modulate the ADCC activity ; thus, combinations of anti-HER2 treatment and immunotherapy are being evaluated in several trials, so far with little success . In this regard, the composition of the TME, which includes the surrounding non-cancerous cells and the extracellular matrix, is crucial for the development of the resistance to anti-HER therapies . In order to maximize ADCC activity, a new agent was developed and recently approved by the FDA: margetuximab, a human/mouse chimeric IgG1 anit-HER2 monoclonal antibody. This drug has a trastuzumab backbone, with an engineered Fc-domain, in which the substitution of five amino acids boosts binding to FcyRIIIA (CD16A, a low affinity stimulatory receptor of macrophages and natural killer cells), and reduces the binding to the inhibitory Fc receptor FcYRIIB (CD32B) . In order to overcome some of the resistances described above, a combination of anti-HER2 targeted and non-targeted therapies are currently used in clinical practice (for example, pertuzumab, trastuzumab and chemotherapy). Albeit the reasons for the resistance to anti-HER2 antibody combinations are currently unknown, some hypotheses indicate that a constitutive activation of downstream signalling pathways may play a major role . 4. HER Heterogeneity, HER2 Spatial Distribution and Anti-HER2 Therapy Resistance As already mentioned, one of the possible mechanisms of resistance to anti-HER2 therapies is the intratumour heterogeneity in HER2 spatial distribution, that has been deeply investigated in BC . In this paragraph, main definitions of HER2 heterogeneity and HER2 spatial distribution are outlined; a brief summary of the 2018 ASCO/CAP guidelines for HER2 evaluation is provided for the reader in Table 1. HER2 genetic heterogeneity is defined as subclonal diversity within the tumour with an overall reported incidence from 5% to 30% and in 1-34% of HER2-positive BCs . Diagnostic guidelines have proposed different definitions for this concept . It is defined by ASCO/CAP (2013) guidelines as the presence of >=10% to <50% tumour cells with a ratio >= 2.0 when using dual probes or >=6 HER2 signals/cell when using single probes, selecting 2-4 representative invasive tumour areas . Vance et al. define a tumour as heterogeneous if there are more than 5% but less than 50% of infiltrating tumour cells with an ISH ratio higher than 2.2 . Regarding the HER2 spatial distribution, three distinct patterns of distribution of cells with heterogeneous HER2 expression have been described: "clustered type", "mosaic type" and "scattered type" . While in the first type two different tumour clones (one with HER2 amplification and the other with normal HER2 status) could be identified, the second type displays a diffuse intermingling of cells with different HER2 expression. The latter type is characterized by isolated HER-positive cells in a HER2-negative field . To overcome this issue, and to categorise these tumours in the two main groups used in clinical practice (positive vs. negative), current guidelines consider BC samples as HER2-positive by IHC testing if there is an aggregate population of amplified cells composing > 10% of the total tumour cell population . The recently introduced concept of HER2-low expression level has not been yet defined by ASCO/CAP guidelines, although these patients have been shown to benefit from ADCs . Probably, a possible low intensity of HER2-expression should be taken into account while handling IHC assay results to ascertain the heterogeneity. Discordance of HER2 expression between primary and residual tumour has been reported after the neoadjuvant therapy and has been associated with a lack of pathologic complete response (pCR) . In BC, therapy administration may lead to partial loss of selected cell populations, in particular HER2-targeted treatment may result in death of HER2-positive cell populations and survival of HER2-negative cells, resulting in HER2-heterogeneity . Interestingly, the study of Filho et al. assessed HER2 heterogeneity in different areas of pre-treated HER2-positive early BC biopsies followed by T-DM1 administration, where 10% of samples were classified as those with heterogenous HER2 expression, none of which achieved a pCR compared to non-heterogenous BCs, where pCR was achieved in 55% of the cases . The so-called "Subtype switch" after neoadjuvant treatment has been frequently observed and reported in literature with the highest frequency of HER2-enriched-to-luminal-A, and this effect has been found to be reversible after anti-HER2 therapy discontinuation . Finally, when performing the IHC HER2-testing assay, one should be aware of possible errors, that may result in staining heterogeneity, due to various pre-analytical issues, such as fixation time, antibody clones selection and antigen retrieval systems . To overcome the above-mentioned issues, a rigorous control and laboratory standardization is mandatory . 5. HER2 Heterogeneity and Response to Different Anti-HER2 Treatments The pathological feature of HER2 heterogeneity has deep clinical implications, although its definition is still under debate . Indeed, tumours with HER2 heterogeneity have shown to be less sensitive to anti-HER2 targeted treatments, both in metastatic and in early settings. For example, a shorter time to progression and lower OS has been reported during treatment with trastuzumab . Novel drugs, such as ADCs, have raised intriguing hints . Table 2 recapitulates these drugs and their mechanism of action. In the MARIANNE clinical trial, T-DM1 showed non-inferior, but not superior, efficacy and better tolerability compared to taxane plus trastuzumab for first-line treatment of HER2-positive advanced BC . A post-hoc exploratory analysis suggested that homogeneous HER2 IHC staining was associated with numerically longer median PFS than focal/heterogeneous staining in the HER2-positive population (IHC 2+ or 3+) . In early-stage BC, HER2 heterogeneity is an independent factor predicting incomplete response to anti-HER2 neoadjuvant chemotherapy . In the KRISTINE trial, neoadjuvant T-DM1 plus pertuzumab led to a lower pCR rate (44.4% vs. 55.7%; p = 0.016), compared to docetaxel, carboplatin, trastuzumab plus pertuzumab in HER2-positive stage II-III BC. Authors highlighted that BC samples from the 15 patients who experienced locoregional progression had lower HER2 expression and higher HER2 heterogeneity as compared to those from other patients in the T-DM1 arm . The already mentioned study by Filho et al. was a phase II clinical trial, specifically designed to prospectively assess the impact of HER2 heterogeneity on response to targeted therapy. Authors defined HER2 heterogeneity as an area of HER2 amplification in more than 5% but less than 50% of tumour cells, or a HER2 negative area by ISH. One hundred sixty-four patients with centrally confirmed HER2-positive early-stage BC were treated with neoadjuvant T-DM1 plus pertuzumab. HER2 heterogeneity was detected in 10% of the cases; pCR was 55% in the non-heterogeneous subgroup and 0% in the heterogeneous group (p < 0.0001). Single cell ERBB2 FISH identified the fraction of HER2 non amplified cells as the driver of resistance . In another study, 37 HER2+ BCs were analysed first with combined immunofluorescence and ISH followed by a validated computational approach. Samples were analysed before and after neoadjuvant HER2-based treatment, in order to study tumour evolution as well. This study confirmed that heterogeneous tumours were associated with significantly shorter DFS and fewer long term survivors . These data suggest that in the absence of systemic chemotherapy, the bystander killing effect (i.e., the unintentional payload diffusion from antigen-positive tumour cells to adjacent antigen-negative tumour cells) of T-DM1 may not be sufficient to eradicate heterogeneous HER2 BC. Novel ADCs, such as T-DXd, have been specifically designed to enhance the possibility to have a bystander activity: preclinical evidence highlights the cruciality of a cleavable linker that could release the payload from the antibody moiety and of a hydrophobic payload that could diffuse through the cell membrane towards neighbouring cells. In this way, the cytotoxic compound could be potentially delivered also to antigen-negative cells, providing higher chances of efficacy on heterogeneous tumours (Table 3). However, another layer of complexity should be added. Indeed, in all the cases reported above, BC could be heterogeneous or not heterogeneous, but remains still HER2-positive per definition (IHC 3+ and/or FISH amplified). Recently, the evidence that a bystander killing effect could play a role also in HER2-low and HER2-null (IHC 0) tumours set the biological rationale for ambitious translational and clinical studies. One example is the phase 3 DESTINY-Breast04 trial, in which T-DXd efficacy was evaluated in 557 heavily pre-treated patients with HER2-low expressing advanced BC (494 ER-positive, 63 ER-negative) . In the ER-positive cohort, the mPFS was 10.1 vs. 5.4 months (hazard ratio = 0.51, p <= 0.001), and the OS was 23.9 vs. 17.5 months, favouring T-DXd compared with treatment of physician choice (hazard ratio 0.64, p = 0.003). In the intention-to-treat (ITT) population, median PFS was 9.9 vs. 5.1 months, (hazard ratio = 0.50, p <= 0.001), and OS was 23.4 vs. 16.8 months (hazard ratio 0.64, p = 0.001). According to these results, T-DXd has been approved by the FDA and the EMA for the treatment of HER2-low BC patients. Furthermore, in the phase 2 DAISY clinical trial (NCT04132960) 179 patients with heavily pre-treated advanced BC were treated with T-DXd in order to assess the activity of the drug, its mechanism of action, and to identify biomarkers associated with drug response or drug efficacy. The study design provided three arms: Cohort 1, HER2 IHC 3+ (n = 68); Cohort 2, HER2-low (n = 73); Cohort 3, HER-null IHC 0 (n = 38) . Biopsy of metastatic sites was performed at baseline, during treatment and at tumour progression. Primary endpoint was the best overall response in each cohort. At a median follow-up of 15 months, the overall response in the HER2-positive cohort was 70%, 37.5% and 29.7% in HER2-positive, HER2-low and HER2-null cohort, respectively. mPFS was 11.1, 6.7 months (6.9 in HR-positive and 3.5 in HR-negative) and 4.2 months (4.5 in HR-positive and 2.1 in HR-negative), for each cohort. During the European Society of Medical Oncology (ESMO) Breast Cancer Congress 2022, a translational analysis of the DAISY trial was disclosed, investigating T-DXd efficacy according to HER2 expression status . These molecular analyses showed that cancer cells expressing low levels of HER2, but not HER2 IHC 0 cells, can internalize T-DXd (p < 0.053). By applying artificial intelligence to digital pathology, the predictive value of HER2 spatial distribution was investigated using weakly supervised and clustering algorithms on HER2-positive whole slide imaging (WSI) at baseline (n = 61). Clustering algorithms in the HER2-positive cohort identified a cluster associated with a lower best overall response (BOR) (p < 0.007), demonstrating that a high percentage of HER2 IHC 0 cells and their spatial distribution were associated with no response to treatment (p = 0.0008). At progression, a decreased HER2 expression was observed in 13/25 (52%) patients (p < 0.07) . Although these data are intriguing, results from the full publication are awaited. 6. Further Directions The study of the heterogeneity and spatial distribution of HER2 has brought some important results but has also raised some intriguing clinical hints for the treatment of HER2-positive and HER2-low BC. In particular, the advent of ADCs has disclosed the possibility to overcome HER2 intratumoural heterogeneity. Further studies on the definition of HER2 heterogeneity are mandatory to improve patients' outcomes. Three main issues could be identified in order to address further research: (1) improving the assessment of HER2 heterogeneity; (2) dissecting the complexity of the interaction between HER2-positive cells and other cell types in the TME; (3) identifying new drugs capable of overcoming resistance. Firstly, nowadays HER2 assessment is based on a qualitative IHC evaluation. Therefore, the study of quantitative assays to evaluate HER2 is currently under investigation. For instance, one study provides the use of The HERmarkTM Breast Cancer Assay (HERmark, Monogram Biosciences, South San Francisco, CA, USA), that is a quantitative method with a dual-antibody, proximity-based approach. This evaluation provides accurate quantification of the HER2 protein in tissue samples using a dual-antibody, proximity-based immunoassay approach, the VeraTagTM technology (Monogram Biosciences), to make precise and quantitative measurement of total HER2 protein expression with greater sensitivity and specificity than IHC . Moreover, other possibilities to assess HER2 are looming on the horizon. For instance, intriguing clinical trials (NCT02095210) are evaluating the uptake distribution of the HER2-binding radiolabelled agent [68Ga] ABY-025 by PET imaging in patients harbouring a biopsy-identified HER2 expression. Secondly, some intriguing analysis using spatial transcriptomic technologies have already been performed to study the spatial-gene expression profiles of HER2-positive tumours . Moreover, spatial proteomic characterization of HER2-positive BCs through neoadjuvant chemotherapy was demonstrated to be predictive of response in 57 HER2-positive BCs from the neoadjuvant TRIO-US B07 clinical trial. In this trial, samples were collected pre-treatment, after 14-21 days of HER2-therapy and at surgery. The proteomic changes after one cycle of therapy could predict pCR, outperforming transcriptomic changes . Single cell analysis has also been performed to identify HER2-low patients by capturing the spatial distribution of HER2 expression across the entire tumour from a subset of samples from ISPY1 and 2 trials. In this way, researchers could identify 84 patients affected by triple negative breast cancer (TNBC) expressing HER2-low who could potentially benefit from anti-HER2 drugs . Among them, 17 patients expressed HER2 at low levels and a previously published HER2-responsive gene expression signature . Finally, new pharmacological approaches have been proposed. In particular, mathematical models could be used in order to hypothesize which treatment combinations and schedules could be more effective in this patient population . Possible approaches exploit the combination of HER2-targeting and non-targeting agents and the use of pharmacological compounds capable of reducing intratumoural heterogeneity and phenotypic plasticity, such as histone deacetylase (HDAC), histone demethylase, and bromodomain inhibitors . Innovative platforms, such as bispecific antibodies, may combine proprieties of different drugs in order to overcome resistance. An example is zanidatamab, a humanised, bispecific monoclonal antibody directed against two non-overlapping domains of HER2 (i.e., the trastuzumab binding domain and the pertuzumab binding domain) that is currently under investigation for early stage and metastatic HER2-positive BC (NCT05035836, NCT04224272, NCT02892123). Zanidatamab zovodotin (zanidatamab-based antibody conjugated with zovodotin, an auristatin cytotoxic agent) is also being tested in a phase I trial (NCT03821233) and may represent a possible way to overcome the resistance determined by the heterogeneous expression of HER2. Another attempt supports adaptive therapy, in which the competition of drug-resistant and drug-sensitive cells is limited by alternating therapy and no treatment . 7. Conclusions The discovery of the HER2 protein and anti-HER2 targeted treatments has completely changed the prognosis of a subgroup of patients affected by BC. Since the introduction of trastuzumab in clinical practice, the treatment armamentarium has been expanded with the development of new pharmacological compounds such as the novel ADCs, resulting in progressive and important improvements in clinical outcomes for patients. The efforts of the scientific community should be addressed to a sharper dissection of this entity and to fully understand the implications of the heterogeneity in HER2 expression and its spatial distribution. In this context, new horizons will be defined by novel methods to dissect BC heterogeneity and innovative drugs targeted to overcome these mechanisms of resistance, such as modulators of histone proteins or bispecific antibody-drug conjugates. In this way, the best treatment choice could possibly be achieved, potentially de-escalating therapies in those patients with HER2-homogeneous expressing tumours and escalating treatment in HER2 heterogeneous ones. Acknowledgments M.I. was supported by Fondazione Umberto Veronesi. Author Contributions Conceptualization, F.G. and E.M.; methodology, F.G., E.M., A.C.S. and C.C. (Chiara Corti); software, F.G., E.M. and A.C.S.; validation, C.C. (Carmen Criscitiello), G.C., N.F. and E.M.; formal analysis, F.G. and A.C.S.; investigation, F.G., A.C.S. and C.C. (Chiara Corti); resources, F.G., A.C.S. and C.C. (Chiara Corti); data curation, F.G., A.C.S. and C.C. (Chiara Corti); writing--original draft preparation, F.G., A.C.S., C.C. (Chiara Corti), M.I., N.F. and E.M.; writing--review and editing, N.F., N.B., S.D., G.C. and E.M.; visualization, E.M.; supervision, E.M., N.F. and G.C.; project administration, E.M.; funding acquisition, not applicable. All authors have read and agreed to the published version of the manuscript. Conflicts of Interest GC: Honoraria for speaker's engagement: Roche, Seattle Genetics, Novartis, Lilly, Pfizer, Foundation Medicine, NanoString, Samsung, Celltrion, BMS, MSD; Honoraria for providing consultancy: Roche, Seattle Genetics, NanoString; Honoraria for participating in Advisory Board: Roche, Lilly, Pfizer, Foundation Medicine, Samsung, Celltrion, Mylan; Honoraria for writing engagement: Novartis, BMS; Honoraria for participation in Ellipsis Scientific Affairs Group; Institutional research funding for conducting phase I and II clinical trials: Pfizer, Roche, Novartis, Sanofi, Celgene, Servier, Orion, AstraZeneca, Seattle Genetics, AbbVie, Tesaro, BMS, Merck Serono, Merck Sharp Dome, Janssen-Cilag, Philogen, Bayer, Medivation, Medimmune. CCr served in advisory/consultancy/speaker roles for Roche, Pfizer, Eli Lilly, Novartis, AstraZeneca, Daiichi Sankyo, Gilead, Seagen and MSD. EM has acted as a consultant and/or has received funding for travel expenses and/or accommodation to Pierre Fabre, Genomic Health, Celgene, Roche, Eisai, Mylan, Exact Sciences, Merck Sharp & Dohme (MSD) Oncology, AstraZeneca, Daiichi Sankyo, Lilly, Novartis, Seagen, and Pfizer. SD has acted as a consultant for and/or has received funding for travel expenses and/or accommodation from AstraZeneca, Daiichi Sankyo, Lilly, Novartis, and Pfizer. NB has received honoraria for consulting and/or has received funding for travel expenses and or accommodation from Lilly, Roche and Novartis. NF has received honoraria for consulting, advisory role, speaker bureau, travel, and/or research grants from Merck Sharp & Dohme (MSD), Boehringer Ingelheim, Novartis, AstraZeneca, Daiichi Sankyo, GlaxoSmithKline (GSK), Gilead, Diaceutics, Adicet Bio, and Sermonix. These companies had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, and/or in the decision to publish the results. All other authors declare no potential conflicts of interest. Figure 1 Mechanism of resistance to anti-HER2 targeted therapy. (1) Resistance to monoclonal antibodies (right, red box). Numerous mechanisms of resistance have been described. First, low levels of HER2 expression determine few receptors to bind for Ab, thus lowering the effectiveness of Ab. Changes of the extracellular binding domain could generate resistance as well: the most studied variant is p95HER2, an isoform deriving from the cleavage of the extracellular domain by ADAM10 that causes the impossibility of HER2 binding for trastuzumab. Another mechanism of resistance is the constitutive activation of the PI3K pathway (not shown in the box), depending on the alteration of the alpha catalytic subunit of PI3K. Different RTKs can heterodimerize with HER2, especially EGFR and HER3; their overexpression promotes the formation of HER2-EGFR and HER2-HER3 heterodimers, activating the pathway. The increase in the ER expression, as well as the alterations of CD1 or CDK4-6, stimulates cell growth and survival, reducing the inhibitory effect of monoclonal antibodies. (2) Resistance to ADCs (centre, green box): besides the mechanisms of resistance shared with other classes of drugs, some escapes are peculiar for ADCs. For example, the presence of drug efflux pumps on the cell wall reduces the intracellular cytotoxic action of the warhead of ADCs (for example, Dxd and DM). NRG-1 overexpression stimulates the dimerization of EGFR-HER3 and the activation of PI3K pathways in tumours treated with T-DM1 (not shown in the figure). (3) Resistance to TKIs (left, blue box): low HER-2 expression, activation of CD1 and CDK4-6, increased ER, constitutive activation of PI3K pathway and the overexpression of NRG1 are mechanisms of resistance shared with other drug classes. Mutations of the binding site in the intracellular domain confer specific resistance, determining the inability of TKIs to block HER2 function. The most frequent mutations are L755S and T798I, that cause resistance to lapatinib. Abbreviations: ADCs: antibody-drug conjugates; EGFR: epidermal growth factor receptor; HER2: human epidermal growth factor receptor 2; ER: estrogen receptor; NRG1: neuro regulin 1; CDK: cyclin dependent kinase. Created with BioRender.com (www.biorender.com, accessed on 23 December 2022). Figure 2 Patterns of distribution of cells with heterogeneous HER2 status. Clustered type (left panel): two different tumour clones, one with HER2 amplification and the other with normal HER2 status, could be identified. Mosaic type (central panel): diffuse intermingling of cells with different HER2 expression. Scattered type (right panel): isolated HER-positive cells in a HER2-negative field. Abbreviations: HER2: human epidermal growth factor receptor 2. Created with BioRender.com (www.biorender.com, accessed on 4 February 2023). cancers-15-01385-t001_Table 1 Table 1 Algorithm for evaluation of HER2 expression according to the 2018 ASCO/CAP guidelines. HER2 Status Score IHC (Definition) ISH (Definition) deg^ HER2-positive 3+ (Circumferential membrane staining, complete, intense, in > 10% of TCs) Amplified (Average HER2 copy number >= 6.0 signals/cell) 2+ * (Weak/moderate complete membrane staining in > 10% of TCs) HER2-negative 2+ # (Weak/moderate complete membrane staining in > 10% of TCs) Not Amplified (Average HER2 copy number < 4.0 signals/cell) 1+ (Incomplete membrane staining, faint/barely perceptible in > 10% of TCs) 0 (No staining or incomplete membrane staining, faint/barely perceptible in <=10% of TCs) Abbreviations: TC: tumour cells; HER2: human epidermal growth factor receptor 2; *: if ISH positive; #: if ISH negative; deg: Note: in clinical practice, the evaluation of the status of HER2 by ISH is performed only when IHC score is 2+; ^: if ISH result is between 4 and 6, breast cancer is classified as HER2-negative. cancers-15-01385-t002_Table 2 Table 2 Antibody-drug conjugates targeting HER2 currently available for the treatment of breast cancer. Drug Antibody Linker Payload Bystander Effect Indication Trastuzumab emtansine Trastuzumab Non-cleavable DM1 (microtubule inhibitor) No HER2-positive BC Trastuzumab deruxtecan Trastuzumab Cleavable DXd (topoisomerase-1 inhibitor) Yes HER2-positive BC HER2-low BC Abbreviations: DXd: deruxtecan; DM1: emtansine; HER2: human epidermal growth factor receptor 2. cancers-15-01385-t003_Table 3 Table 3 Summary of trials investigating agents in heterogeneous HER2 expression in breast cancer. HER2 Expression Treatment Trial Phase Setting Primary Endpoint Ref. HER2-negative (HER2 IHC: 0) T-DXd DAISY (NCT04132960) 2 Advanced BOR 30.6% (95%CI: 22.7-45.4) HER2-Low (HER2 IHC 1+ OR HER2 IHC 2+ ISH non ampl) T-DXd DAISY (NCT04132960) 2 Advanced BOR 33.3% (95%CI: 56.7-79.8) T-DXd vs. TPC DESTINY-Breast06 (NCT04494425) 3 Advanced Ongoing // T-DXd vs. TPC (NCT03734029) 3 Advanced (>1-2 line) mPFS * 9.9mo vs. 4.9mo HR 0.50 (95% CI: 0.40-0.63; p < 0.0001) HER2-positive (HER2 IHC 2+ and ISH ampl OR HER2 IHC: 3+) T-DM1 vs. lapatinib + capecitabine EMILIA (NCT00829166) 3 Advanced (2nd line) mPFS 9.6 mo vs. 6.4 mo; HR: 0.65 (95% CI, 0.55-0.77, p < 0.001) T-DM1 vs. TTZ + taxane MARIANNE (NCT01120184) 3 Advanced (1st line) mPFS 14.1 mo vs. 13.7 mo (non-inferiority) HR: 0.91 (97.5% CI, 0.73-1.13) T-DM1 vs. TTZ KATHERINE (NCT01772472) 3 Post-neoadjuvant 13y-iDFS 88.3% vs. 77%; HR: 0.50 (95% CI, 0.39-0.64; p < 0.001) T-Dxd DAISY (NCT04132960) 2 advanced BOR 69.1% (95% CI: 39.1-54.2) Combinations of T-DXd and other agents DESTINY-Breast07 (NCT04538742) 1b-2 Advanced (1st line) ongoing // T-DXd vs. T-DM1 DESTINY-Breast03 3 Advanced (2nd line) mPFS 28.8 vs. 6.8 mo; HR: 0.33 (95% CI, p = <0.0001) T-DXd DESTINY-Breast01 (NCT03248492) 2 Advanced (>=3 line) ORR 60.9% (95% CI, 53.4-68) Trastuzumab Duocarmazine TULIP (NCT03262935) 3 Advanced (>=2 line) mPFS 7 mo (95% CI, 5.4-7.2) vs. 4.9 mo TPC (4-5.5); HR: 0.64 (95% CI, 0.49-0.84, p = 0.002) ARX788 ACE-Breast01 and ACE-Pantumour01 (NCT03255070) 1 Advanced ORR 74% (14/19) ACE-Breast-01 67% (2/3) ACE-Pan tumour-01 T-DM1 + pertuzumab NA 2 neoadjuvant pCR 55% in the non heterogeneous subgroup and 0% in the heterogeneous group (p < 0.0001) # Abbreviations: T-DXd: trastuzumab deruxtecan; T-DM1: trastuzumab emtansine; HER2: human epidermal growth factor receptor 2; ISH: in situ hybridization; TPC: treatment of physician choice; mo: months, TTZ: trastuzumab; iDFS invasive disease-free survival; ORR: objective response rate, IHC: immunohistochemistry, ampl: amplifed, HR: hazard ratio, BOR: Best Overall Response, PFS: Progression Free Survival; OS: overall survival; HR: hazard ratio; * among all patients; #: adjusted per hormone receptor status; Ref: reference. 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PMC10000153

Endolimax is a genus of intestinal amoebae which stands among the least known human protists. Previous studies on amoebic systemic granulomatosis of a marine fish (Solea senegalensis) resulted in the unexpected characterization of a new organism which was related to Endolimax and named E. piscium. The existence of multiple reports of systemic granulomatosis caused presumptively by unidentified amoebae in goldfish lead us to investigate the organism involved in goldfish disease. Analysed goldfish presented small whitish nodules in the kidney, which correspond to chronic granulomatous inflammatory reactions with a ring-layer of amoebae in the periphery. Amoebae were amitochondriate and were located in a parasitophorous vacuole within macrophages, as previous studies on this condition in goldfish and other freshwater fish pointed out. SSU rDNA characterization confirmed a new Endolimax lineage which appears closely related to E. piscium, but the molecular evidence, distinct pathological features and lack of ecological overlapping between the hosts support their assignment to a new species, E. carassius. The results support the existence of a considerable unexplored diversity of Endolimax spp. among fish, and their proper characterization can contribute to an understanding of Archamoebae evolution and pathogenic potential. parasites fish archamoebae systemic granulomatosis Carassius auratus Iodamoeba This research received no external funding. pmc1. Introduction Endolimax nana is a long-known endo-commensal or parasitic archamoebae of human and non-human primates. Reports have also described Endolimax spp. in birds, amphibians, reptiles, and even in insects (reviewed in ). However, these descriptions were based on superficial morphological observations and under a general assumption of host specificity for the different animal lineages. Until recently, a single complete SSUrDNA sequence of a monkey isolate (Cercocebus albigena), obtained in pioneering molecular phylogenetic studies of Entamoebidae , remained the only E. nana reference genotype available in public repositories. However, a recent PCR survey of human faecal samples using a panel of intestinal amoeba primers detected several new SSU rDNA segments from Endolimax nana genotypes grouping in two phylogenetically distinct human and non-human primate lineages . Further studies into Endolimax sp. genetic diversity have recently been published, including the release of quality Sanger reads of cloned rDNA genes from human, wastewater and swine E. nana SSU rDNA genotypes . A high degree of intrageneric variation at the SSU rDNA locus has been found for these new Endolimax nana sequences, and also for the closely related genus Iodamoeba . Both genera remain as the least well-known human parasitic protists and are largely unexplored in terms of morphology, taxonomy, genetic diversity, host specificity, and epidemiology . Previous research on the characterization of a cryptic granulomatous systemic disease (GSD) in cultured Senegalese sole (Solea senegalensis) revealed a new amoeba species clustering with Endolimax genotypes in phylogenetic analyses, which was named E. piscium . Specific studies on the ultrastructure, pathology, diagnostics, and transmission of E. piscium have been presented elsewhere . Besides the morphological and genetic differences with terrestrial Endolimax sp., the most noteworthy feature of E. piscium is its ability to breach the intestinal barrier causing systemic disease, which manifests in sole as conspicuous muscle lumps and nodules resembling abscesses in different organs . However, covert infections in the intestinal epithelium of asymptomatic fish have also been revealed by specific studies with in situ hybridization techniques (ISH) . Amoebae can cause severe diseases in fish, mainly affecting the gills but also systemic infections in different species, as reviewed in . Among these, several case reports of GSD in goldfish (Carassius auratus) have existed since 1977, including observations of amoebae-like organisms . A retrospective review of these reports after the full characterization of E. piscium suggested that the causative agent could be related to E. piscium and the aim of this work was to search for and characterize Endolimax-like parasites in goldfish. A new lineage of Endolimax was found and characterized. Phylogenetic, morphological and pathological data demonstrate a close relationship between E. piscium and the goldfish lineage, but support them being independent species. 2. Materials and Methods Fish and biological samples. Goldfish were obtained from commercial pet-store suppliers for the Universitat Autonoma de Barcelona (UAB) School of Veterinary Medicine practical training programs in fish diseases. They were maintained in glass tanks with individual closed recirculation filters and were subject to periodical monitoring and correction of their water physical-chemical quality parameters through partial water changes. Fish showing signs of distress or used during students practical skills training programs were sacrificed with an overdose of anaesthetic with 2-Phenoxyethanol (Panreac Applichem, Barcelona, Spain), which was progressively introduced into the water for 2 min up to a concentration of 4 mL.L-1. They were necropsied and examined following standard anatomopathological routines. Samples of the different organs were fixed in 10% Neutral Buffered formalin and embedded in paraffin, and sections were stained with haematoxylin and eosin for histological examination. Parallel samples were taken in 80% ethanol for retrospective studies with molecular techniques. Additional samples of kidney from three specimens were processed for transmission electron microscopy (TEM) studies. The samples were fixed in EM grade 2.5% glutaraldehyde (Merck, Darmstadt, Germany) in 0.1 M cacodylate buffer (pH 7.4, Sigma-Aldrich, Madrid, Spain), and processed as previously described . Ultra-thin sections were examined using a Jeol 1400 120 KV transmission electron microscope. Measurements were taken from structures of amoeba cells observed via TEM (n = 7). SSU rDNA sequencing and phylogenetic inference. Ethanol-fixed samples from goldfish kidneys with presumptive amoebiasis (as determined by the macroscopical and histopathological examination) were used for DNA extraction using a spin microcolumn and silica-based commercial kit (Roche Diagnostics). Primary amplification of amoebae SSU rDNA was carried out as described previously . Briefly, subterminal primers MM18Sf and MM18Sr were used in 50 uL PCR reactions containing 1x Taq buffer with 2.5 mM MgCl2, 0.2 mM deoxyribonucleotide triphosphate (dNTP) and 1 U High-Fidelity Taq DNA polymerase, and 25 pmol of each primer. Cycling conditions consisted of an initial denaturation (3 min 94 degC) and 35 amplification cycles (94 degC per 1 min, 55 degC per 1 min, 72 degC per 1 min) followed by a final, 8 min incubation at 72 degC. Amplification products were analysed on TAE agarose gels, and the amplicons were ligated into a plasmid vector (PCR4-TOPO, Invitrogen, Waltham, MA, USA), which was used to transform competent E. coli. Transformants were selected on LB-agar plates, and plasmids were purified from overnight cultures in liquid media. The presence of the expected insert's size was confirmed by restriction digestion analysis with EcoRI enzyme. Both strands of cloned products were Sanger sequenced using M13F and M13R primers, and additional walking primers s1, sx, r1 and r2 were designed from the partial reads . The sequences were assembled and curated by eye inspection of the electropherograms using the software Geneious Prime v2022.2.2. The final consensus sequence was inserted in an alignment with selected Amoebozoa sequences, which was refined by eye under ARB software according to secondary structure criteria. Aligned positions were sampled using different manually built masks varying in the stringency of the alignment homology criteria. The final dataset consisted of 1547 positions. The datasets were analysed for phylogenetic inference using maximum likelihood (ML) and Bayesian estimation methods. ML analyses were carried out using different algorithms as implemented in PhyML and RaxML programs and Bayesian inference was conducted in MrBayes v3.2.6 . Variations of alignment sampling masks, taxa sampling, substitution models and parameters were explored to test robustness and the stability of the reconstructions but a general time-reversible model (GTR) with a gamma-distributed variation rate (6 categories) + invariable sites (G+I) was chosen in final analyses. qPCR diagnosis and ISH. Upon detailed inspection of the new genotype, the repertory of probes designed for E. piscium and routinely used for qPCR and ISH diagnostics of this species in Senegalese sole were evaluated in silico and potentially cross-reacting probes were tested in vitro. SYBR-based qPCR using primers Ep459fQL18 and Ep634rQL20 was tested with a total of 29 goldfish samples procured from the UAB training programs, with selected Senegalese sole samples from the Institute of Aquaculture "Torre de la Sal" (IATS) fish parasitology diagnostics service collection and with both cloned targets. The goldfish samples processed for qPCR consisted of a portion of the posterior part of the visceral package including mostly intestinal tissue, but did not include kidney. In addition, histological sections with confirmed granulomatous lesions (mostly kidney) were selected for in situ hybridization studies. Both procedures were carried out as previously described . 3. Results 3.1. Morphological and Histopathological Observations Small whitish nodules, ranging between 50 and 450 mm in diameter, were observed in the kidney of infected goldfish . Histologically, the nodules consisted of chronic granulomatous inflammatory areas, with a large core of homogeneous necrotic tissue surrounded by fibroblasts and macrophages . Calcified material was observed among necrotic material in the core of some granulomas . Macrophage centres were usually observed at the periphery of the granulomas . Amoebae were detected in some granulomas within macrophages, among the external inflammatory cells and fibroblasts, but generally in a very small number . In situ hybridization using E. piscium probes clearly labelled these amoebae with a dark purple precipitate . The histological sections stained using ISH clearly showed that the parasites were located as a ring layer surrounding the granuloma core and they also corroborated the small number of parasite cells in most of the lesions . Apparently, extracellular amoeba were also detected at the periphery of one granuloma . At the ultrastructural level, amoebae were located inside macrophages, and surrounded by a parasitophorous vacuole. Parasite stages were mostly round in shape with an average diameter of 3.478 +- 0.379 mm. They contained one vesicular nucleus (1.265 +- 0.110 mm in diameter) displaying a large central nucleolus (diameter 0.476 +- 0.076 mm) . The nucleus was demarcated by a double nuclear membrane with prominent pores and small areas of associated peripheral chromatin . The parasites' cytoplasm contained small glycogen granules, often forming aggregates , and membranes resembling rough endoplasmic reticulum . Other structures characteristic of amoebae such as digestive vacuoles containing particulate material or products of lysosomal action were commonly observed . Expelled trophic materials filling the peripheral space of the parasitophorous vacuole were often present . Neither mitochondria nor mitosomes were observed. 3.2. SSU rDNA Characterization and Phylogenetic Inference Primers MM18sf and MM18sr amplified a ~3000 bps product from infected goldfish kidney. The product obtained from a single fish was cloned and Sanger sequenced and the consensus from several reads, with 2934 bps, was deposited in Genbank with accession number ON646298. The most significant matches in Blast searches against NCBI databases (highest Bit-Score) were Endolimax piscium clones, with which the new genotype shared an 87.5% pairwise identity with 100% coverage. These were followed by hits with segments of Iodamoeba sp., Endolimax sp. And other available archamoebae genotypes, reaching high pairwise homology (up to 93.7%) along the matching segment located between nts. 2016-2302 of the new sequence. The Endolimax piscium ISH probe 1999L24 Amsolea was 100% homologous with the goldfish SSU rDNA genotype, whereas the other probe included in the same procedure (1575L24 Amsolea) presented seven mismatches. Of the qPCR primers, the forward Ep459fQL18 was 100% homologous and the reverse primer Ep634rQL20 presented a single mismatch, which suggested a high likelihood of cross-species amplification using this test. This lead us to explore the melting curves of the amplicons in both genotypes, and to predict sufficient differences in the curves to discriminate both genotypes after amplification . This prediction was further verified with qPCR using clinical samples and cloned products from both genotypes. Out of 29 goldfish samples tested, 24 were PCR-positive with the E. piscium test (Ct cut-off value < 38) and the resulting amplicon melting curves matched the in silico predictions with minor Tm peak differences . Phylogenetic analyses resolved the new goldfish genotype sister to Endolimax piscium, and the fish genotypes clustered with other available genotypes from mammals in a well-supported Endolimax clade . In general, two distinct terrestrial Endolimax lineages were well supported although the inclusion of short sequence segments, particularly those from NGS studies from wastewater obtained in , resulted in the poor resolution of these OTUs. Using the longest representative quality sequences, the first lineage included swine isolates generated in a single study and Endolimax sp. (H80028) with a human faecal origin. The other major lineage included the reference E. nana AF149916 and three isolates from human stool. Iodamoeba sp. genotypes branched off as sister to the Endolimax spp. clade in our analyses. This arrangement was consistently resolved by phylogenetic inference using Bayesian methods and different implementations of maximum likelihood methods and substitution models. However, the support values for the main nodes were sensitive to taxon sampling and, particularly, to the stringency of the alignment trimming criteria. 3.3. Description of the Species: Endolimax carassius n. sp. Type host: Carassius auratus Locality: The parasite was detected in Carassius auratus from commercial ornamental fish suppliers in Barcelona, Spain. Previous reports in North America, Central Europe and Israel. Presumed to be distributed worldwide Location in the host: Systemic: mostly found within the inflammatory layer at the periphery of granulomatous lesions in the kidney. Detected via qPCR in samples from asymptomatic fish with a high prevalence (83%); Material deposited: A partial SSU rDNA (2934 bps) comprising the coding region between helixes 1-48 was deposited in GenBank with Accession no. ON646298; Taxonomic remarks: High morphological and pathogenic resemblance with Endolimax piscium but with distinct SSU rDNA genotype (87.5% pairwise identity). Phylogenetic clustering with terrestrial Endolimax sp. genotypes and with genotypes of the genus Iodamoeba as the closest sister group. 4. Discussion It is almost 50 years ago since cases of GSD attributed to amoebae were first described in goldfish from North America . Related cases of systemic inflammatory granulomatous lesions involving amoebae-like organisms in this host were also reported in Europe and Israel in the early 1990s . However, some authors suggested a Dermocystidium-like organism as the etiological agent of goldfish GSD . Extensive work to study this condition was conducted in , whose authors reported a high prevalence (59%) of compatible granulomatous lesions in goldfish from different local sources and provided a comprehensive pathological and ultrastructural description of the causative agent. However, their findings were not conclusive due to a lack of unambiguous morphological features allowing a diagnosis beyond an "amoeba-like" organisms. Similar, and possibly related pathological conditions have also been reported in other freshwater fish such as dwarf gourami (Colisa lalia) , European catfish (Silurus glanis) and tench (Tinca tinca) (reviewed in ). It was unforeseen that the causative agent of the emerging GSD syndrome in culture sole (Solea senegalensis) was identified as an amitochondriate Archamoebae representing a new piscine Endolimax clade . In this work, the characterization of the parasite causing goldfish GSD was tackled from the hypothesis that it could related to Endolimax piscium, on the basis of noted similarities in the morphology and pathogeny of Senegalese sole and goldfish GSD. The results of the histopathological and ultrastructural analyses in this work are largely in agreement with previous studies on this condition in goldfish and other freshwater fish. In all cases, the lesions consisted of inflammatory granulomas, most often present in kidney but also in other organs such as liver, spleen or even brain, associated with very small amoeba-like organisms which are usually present (albeit often in small numbers and difficult to detect) at the peripheral layers of inflammatory cells. This could be particularly well observed using ISH staining. The low numbers of parasites could be indicative of the chronicity of the disease . There are, however, some differences in pathogeny between goldfish GSD and the lesions caused by E. piscium in Senegalese sole. On the one hand, macroscopic lesions are much larger and more extensive in Senegalese sole, presenting a completely liquefied core characteristic of an abscess in most of the cases , and contrasting with the small nodules present in goldfish (only a few millimeters) and other freshwater species (up to 10 mm in tench). In addition, large numbers of parasites are usually involved in the lesions suggesting a more acute course of development. On the other hand, while lesions in goldfish (and, seemingly, in tench ) seem to be more restricted to the kidney, in Senegalese sole they mainly affect skeletal muscle. Histologically, in addition to the granulomatous lesions present in both species, E. piscium has also been associated with extensive necrosis and diffuse inflammation in different organs. A progression of the lesions in goldfish GSD was suggested in , starting from the kidney (early stages of the disease) to spleen, liver, mesenteries and even to the heart (late infections). However, histopathological analyses and specific studies with E. piscium using molecular diagnostic techniques revealed the parasites within the intestinal epithelium in asymptomatic sole, indicating the intestinal mucosa as the main route of invasion in the pathogenesis of sole GSD, from which it can spread to other organs . This location of E. piscium in the host also suggest that the parasites can remain as endocommensal or with a minimally invasive role at the intestinal epithelium. This possibility has not been confirmed in freshwater fish yet, but the detection of Endolimax via qPCR in a large proportion of the goldfish samples tested in this study (i.e., excluding the kidney) leaves open this possibility. Ultrastructural observations conducted in this work are also in agreement with previous data from goldfish GSD and they are virtually identical to those from tench . The parasites were always detected intracellularly, within a parasitophorous vacuoles inside macrophages. Expelled material, probably trophic, was often present filling the peripheral space of the parasitophorous vacuole. The absence of Golgi apparatus and mitochondria in their trophozoites is also remarkable, like the absence of mitosomes or hidrogenosomes typical of amitochondriate eukaryotes. In contrast, E. piscium can be detected extracellularly and without trophic material outside the amoebae. In addition, dictyosome-like structures resembling Golgi apparatus and mitosomes have been described in this parasite . The minute size of the trophozoites of both Endolimax species from fish ( and the present study) is also remarkable, being even smaller than E. nana . The characterization of the SSU rDNA sequence retrieved from goldfish kidney displaying granulomatous lesions identified the causative agent as a new Archamoeba, with Endolimax piscium as the closest matching genotype. Both fish sequences shared 87.5% pairwise identity along their overlapping region, which covers almost the complete gene sequence. Minor (<0.1%) intraspecific genetic diversity has been found at this locus in E. piscium , but the products sequenced from goldfish in this study derived from a single fish and no heterogeneity was detected. The qPCR test routinely used for the diagnostics of E. piscium in sole amplified 24/29 goldfish tested and these amplicons could be consistently discriminated in the melting curves from both hosts suggesting that the SSU rDNA in the goldfish parasite presents limited variability as well. It should be highlighted that this cross-reaction will facilitate the diagnosis of goldfish GSD and discrimination from E. piscium, since published tests for the later species can be used directly or with little modification. Considering the high prevalence of this parasite in goldfish (more than 80% in the present work) and its worldwide distribution, having effective diagnostic tools is especially relevant for the experimental use of SPF goldfish. Although large variability has been found in the SSU rDNA sequences of terrestrial Endolimax and Iodamoeba in the current and in previous studies ; the biological characterization of the source isolates is nearly non-existent and the possibility that they correspond to hypervariable intraspecific genotypes vs. distinct phylogeographical or phyloecological lineages is currently unknown. In this work, the overall phylogenetic reconstruction of Endolimax and Iodamoeba main lineages within the Mastigamoebidae was in agreement with previous studies . However, we obtained good support for a monophyletic Endolimax clade including terrestrial and piscine genotypes, which contrasts with a recent study using a very similar dataset . We found that some of the support values were vulnerable to taxon sampling and, particularly, to the stringency of the alignment trimming criteria due to the existence of large expansion and hypervariable regions in the SSU rDNA sequences studied, which add uncertainty to the alignment even while using secondary structure criteria. In our final dataset, we included 1547 positions as the maximum reasonable number with a rather relaxed stringency criteria, while in previous studies we used mask variations between 1350 and 1493 positions with a similar dataset . This range is much lower than the 2067 positions used in , and these differences can result in variable levels of signal to noise ratio affecting the support for certain reconstructions . It is clear that piscine and terrestrial Endolimax genotypes have a long evolutionary history as separate subclades and their relations with Iodamoeba can be difficult to settle with the currently available genetic datasets. While some of the differences discussed above in the pathogenesis of GSD in sole compared to goldfish and tench could be attributed in part to differences in the hosts responses modulating parasite virulence, tissue distribution patterns and chronicity of the lesions, the combined evidence of: (i) distinct anatomopathological and ultrastructural findings; (ii) lack of ecological overlapping between goldfish and sole; and (iii) substantial genetic differences between both isolates; support them being different species and leads us to propose Endolimax carassius n. sp. as the causative agent of goldfish GSD. Members of Archamoeba are generally not well studied, probably due to their limited clinical importance and the difficulties associated with their culture in the laboratory and with the amplification and sequencing of their ribosomal genes . The genus Entamoeba is by far the most studied, mainly due to E. histolytica, the only true pathogenic species for humans. E. histolytica infections usually include an intestinal phase, asymptomatic or with severe invasive infections, and an extraintestinal phase that generally affects the liver, with possible progression to other organs such as the lungs, brain or heart through blood dissemination . However, species of the genus Endolimax or Iodamoeba from terrestrial hosts are restricted to the intestinal lumen, playing an endocommensal role. In this sense, the pathogenesis of fish Endolimax infections presents remarkable similarities with that of E. histolytica and extends the diversity of pathogenic archamoebae species of concern in veterinary medicine. Considering the overwhelming ecological and phylogenetic diversity of the hosts, systemic Endolimax in fish are likely a diverse and cryptic group of parasites with serious pathological potential, which has emerged so far only in a small number of captive-bred hosts. It is also noteworthy that most, if not all, cases of amoebic GSD reported in fish were detected in animals reared under closed water re-circulation systems. As it has already been suggested in sole, these conditions likely favour the survival and permanence of amoebae stages outside the host and the continuous exposure to an oral-faecal contagion route, facilitating the emergence of clinic GSD due to Endolimax spp., particularly in facilities with intensive culture conditions . The current trends of production scale-up and diversification of aquaculture species may be threatened by emerging parasitism due to Endolimax. 5. Conclusions The characterization of Endolimax carassius clarifies a long-standing debate on the aetiology of goldfish GSD and extends the diversity of pathogenic Endolimax spp. Existing diagnostic tests for E. piscium can be used for E. carassius directly or with minimum modifications, and both genotypes can be discriminated using amplicon melting curves. Given the high prevalence of E. carassius observed in this work and in previous reports, the use of these tests is particularly relevant in goldfish destined for certain experimental studies, in which pathogen free or specific pathogen free status may be of the utmost importance. The results of this study support the existence of a diverse clade of Endolimax spp. in fish, which can have high pathogenic potential and clinical relevance in both freshwater and marine aquaculture production. Acknowledgments The authors express their acknowledgment to R. del Pozo (IATS, CSIC) for technical assistance with qPCR diagnostics and F. Padros (UAB) for his help in fish sampling. Phylogenetic artwork was prepared by I. Castell (i.castell@hotmail.com). Author Contributions M.C. and O.P. contributed equally to this work. All authors have read and agreed to the published version of the manuscript. Institutional Review Board Statement Ethical review and approval were waived for this study due to being carried out with tissue samples from goldfish used for practical training at the UAB school of veterinary medicine, reared and euthanized under approved protocols in compliance with Guidelines of the European Union Council (Directive 2010/63/EU) and the Spanish RD 118/2021. Data Availability Statement Partial SSU rDNA sequence of Endolimax carassius (2934 bps) was deposited in GenBank with accession number ON646298. Conflicts of Interest The authors declare no conflict of interest. Figure 1 Lesions caused by Endolimax carassius in the kidney of goldfish (Carassius auratus): (a) macroscopic nodules (*) surrounded by a layer of clearer material; (b-c) granulomatous inflammatory reactions with a large necrotic core. Note the presence of calcified material in the core (*) and macrophage centres (arrows) at the periphery of the lesions; (d) detail showing two amoebae inside macrophages (arrows); (e-g) ISH-stained histological sections with amoebic lesions: parasite cells are stained purple within the inflammatory layer of the granuloma with variable, but generally low infection intensity. Some apparently extracellular parasites could also be detected outside the granuloma (arrow). Figure 2 Transmission electron micrographs of parasites in Carassius auratus kidney. (a,b) Ultrastructure of the amoebae trophozoites, located inside parasitophorous vacuoles within host macrophages (M). Note the vesicular nucleus with central nucleolus, rough endoplasmic reticulum (arrow), small glycogen granules (arrowheads), and vacuole-like structures with degraded material (*). Large amount of expelled trophic material (etm) is adjoining the amoeba filling the space inside the parasitophorous vacuole; (c) detail of a nucleus with nuclear pores (arrows) and peripheral chromatin (*); (d) amoeba with digestive vacuoles (*) containing withered materials at different degrees of degradation, myelin figures and strips of rough endoplasmic reticulum (arrow). Figure 3 Derivative melting curves of Endolimax piscium and the new goldfish genotype amplified via qPCR. (a) The theoretical curve predicted in silico using uMelt with the sequences of both genotypes; (b) experimental data obtained with DNA samples from amoebae-infected sole and goldfish, using the E. piscium qPCR test described in . Figure 4 Phylogenetic analysis of Endolimax carassius SSU rDNA and selected representative Mastigamoebidae sequences. The analysis used 1547 alignment positions. The topology was inferred using Bayesian inference with a gamma distribution (six categories) and a proportion of invariable sites. Numbers at nodes represent the posterior probabilities followed by the bootstrap support obtained in a maximum likelihood analysis using the same dataset and model. All branches are drawn to scale. The scale bar represents 0.03 substitutions per site. Well-supported subclades are highlighted in coloured boxes. Disclaimer/Publisher's Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

PMC10000154

Vibration emissions during the transport of boar semen for artificial insemination (AI) affect sperm quality. In the present study, the common influence of the following factors was investigated: vibrations (displacement index (Di) = 0.5 to 6.0), duration of transport (0 to 12 h) and storage time (days 1 to 4). Normospermic ejaculates were collected from 39 fertile Pietrain boars (aged 18.6 +- 4.5 months) and diluted in a one-step procedure with an isothermic (32 degC) BTS (Minitub) extender (n = 546 samples). Sperm concentration was adjusted to 22 x 106 sperm*mL-1. Extended semen (85 +- 1 mL) was filled into 95 mL QuickTip Flexitubes (Minitub). For transport simulation on day 0, a laboratory shaker IKA MTS 4 was used. Total sperm motility (TSM) was evaluated on days 1 to 4. Thermo-resistance test (TRT), mitochondrial activity (MITO) and plasma membrane integrity (PMI) were assessed on day 4. Sperm quality dropped with increasing vibration intensity and transport duration, and the effect was enhanced by a longer storage time. A linear regression was performed using a mixed model, accounting for the boar as a random effect. The interaction between Di and transport duration significantly (p < 0.001) explained data for TSM (-0.30 +- 0.03%), TRT (-0.39 +- 0.06%), MITO (-0.45 +- 0.06%) and PMI (-0.43 +- 0.05%). Additionally, TSM decreased by 0.66 +- 0.08% with each day of storage (p < 0.001). It can be concluded that boar semen extended in BTS should be transported carefully. If this is not possible or the semen doses are transported a long way, the storage time should be reduced to a minimum. artificial insemination boar semen boar sperm quality shipping vibrations duration German Federal Ministry for Economic Affairs and Climate ActionIQ-TranS: 16KN077341 This research was funded by the German Federal Ministry for Economic Affairs and Climate Action (BMWK) on the basis of a decision by the German Bundestag. It is part of the Central Innovation Program for small and medium-sized enterprises (IQ-TranS: 16KN077341). pmc1. Introduction The purpose of pig breeding farms is to produce boar semen for the artificial insemination (AI) of sows. This perishable product must endure transport over long distances while maintaining its quality . The implementation of quality assurance programs helps to ensure the high quality of AI doses is maintained throughout production; however, the targeted monitoring of boar semen ends at the production line. There are currently no international standards for the transport of boar semen. Over the past decade, the average total sperm number per dose from 28 European AI centers has been reduced by about one-fifth, while sperm quality has steadily increased . Strategies such as post-cervical or fixed-time AI have contributed to a further reduction in sperm count per sow with comparable fertilization success. This has not only resulted in economic benefits but also contributed to environmental protection by reducing the number of animals needed in each AI center. For these reasons, the implementation of measures to maintain sperm quality from the point of production to insemination has become a topic of interest for several research groups. Studies have found that a number of factors have an impact on sperm quality during transport. These include transport temperature and type of semen extender as well as the intensity and duration of exposure to vibration. Taking these previous findings into consideration, the present study aims to fill a major knowledge gap by investigating the relationship between the duration and intensity of vibration emissions to find a cut-off value for harmful vibrations. A mobile sensing app able to measure vibrations during transport has been developed , and Schulze et al. showed the impact on boar semen quality after simulating these vibrations for six hours. This system was enhanced with an external measuring device and the displacement index (Di) to quantify the intensity of vibration emissions within transport boxes during boar semen deliveries under everyday production conditions . In the present study, these findings were simulated under laboratory conditions. In this case, the intensity of vibrations and duration of transport were not considered to be separate factors, and their interaction was examined in a wide range. For this purpose, transport times from 30 min up to 12 h, road type and speed-dependent low and high vibrations were taken into account . The impact of these factors on boar semen quality was evaluated using an extended spectrum of methods with the aim of providing new and comprehensive insights into the impact of vibration emissions on the quality of boar semen. This information can then be used to develop recommendations for implementation in the field. 2. Materials and Methods 2.1. Chemicals All chemicals used in this study were of analytical grade. Unless stated otherwise, they were purchased from Merck (Darmstadt, Germany) and Roth (Karlsruhe, Germany). Fluorescein isothiocyanate-conjugated peanut agglutinin (FITC-PNA), Pisum sativum agglutinin (FITC-PSA) and rhodamine 123 (R123) were obtained from Sigma-Aldrich (Steinheim, Germany), whereas propidium iodide (PI) was purchased from Thermo Fisher Scientific Inc. (Darmstadt, Germany). 2.2. Semen Processing All AI doses examined in this study (n = 546) were produced in a commercial AI center in Germany. Ejaculates were randomly collected from 39 fertile Pietrain boars (aged 18.6 +- 4.5 months) using the double gloved-hand method. After quality control to ensure they fulfilled minimum requirements for commercial use in AI, the semen was diluted in a one-step procedure with an isothermic (32 degC) Beltsville Thawing Solution (BTS) extender (Minitub GmbH, Tiefenbach, Germany). Semen was placed in 95 mL QuickTip Flexitubes(r) (Minitub GmbH) with a sperm concentration adjusted to 22 x 106 sperm*mL-1 and a filling volume of 85 +- 1 mL. Compliance with the minimum requirements of the German Livestock Associations standard for short-term extender (total sperm motility >= 70%, morphologically abnormal sperm <= 25%, sperm with cytoplasmic droplets <= 15%) was confirmed at the reference laboratory for spermatology at the Institute for Reproduction of Farm Animals Schonow (IFN). 2.3. Simulation of Transport Vibration Emissions The experimental design is based on an analysis of the transport conditions during the shipment of boar semen according to Hafemeister et al. . An orbital shaker IKA MTS 4 (Laborgerate Munchen, Germany) with circular horizontal shaking movements was found to be able to best simulate the vibrations from the field. However, when comparing different devices of the same model, different Di values were found, although the same settings were used. In order to eliminate this possible source of error, the system was modified to directly display the real-time Di values generated by the vibrations. The AI doses were placed in a polystyrene box equipped with the mobile measuring device. To simulate otherwise optimal transport conditions, the room in which the orbital shaker was kept was dark and maintained at 17 degC. The box with the AI doses was strapped to the orbital shaker, and the displacement index (Di +- 0.1) was set by the rotation speed of the shaker. One sample of each ejaculate was removed from the shaker every hour over a simulation period of 12 h. In order to simulate short distances, an additional sample was removed after 0.5 h of treatment. Thus, together with the unshaken control sample (duration = 0 h), 14 AI doses were analyzed per ejaculate. Furthermore, three ejaculates could be treated simultaneously (n = 42) for each vibration intensity. Slight deviations in the desired displacement index were caused by weight changes as a result of the hourly removal of samples; this was identified through real-time monitoring and corrected accordingly. After treatment and in accordance with best practices, all samples were stored motionless in a dark temperature-controlled cabinet (17 degC) while awaiting further investigation. The day of treatment was defined as day 0 (d0). To obtain an overview of the response of different vibration intensities on sperm quality, only total sperm motility was determined in a first preliminary round of experiments (n = 210 samples, Table 1). In the main round, the samples were examined using an extended spectrum of methods (n = 336), which are described below. 2.4. Assessing Sperm Quality 2.4.1. Total Sperm Motility After careful resuspension, an aliquot of 1.5 mL was incubated for 10 min at 38 degC, and total sperm motility (TSM) was assessed daily on day 1 (d1) through day 4 (d4) using the computer-assisted semen analysis (CASA) system AndroVision(r) (Minitub, Germany). An aliquot of 3.0 mL was placed on a preheated four-chamber slide (Leja products B.V., Nieuw-Vennep, The Netherlands), and at least 1000 sperm cells were analyzed. Sperm were defined as motile when showing an amplitude of lateral head displacement (ALH) > 1.0 mm and a velocity curved line (VCL) > 24.0 mm x s-1. Additionally, on d4, a thermo-resistance test (TRT) was performed. For this purpose, a 10 mL sample was incubated for 300 min at 38 degC with exposure to air; afterwards, the motility was determined as described above. 2.4.2. Mitochondrial Activity and Plasma Membrane/Acrosome Integrity To examine molecular processes in sperm cells, two flow cytometric assays were performed on d4. In a double staining with R123/PI, the percentage of viable spermatozoa with active mitochondria (MITO) was recorded (R123 pos., PI neg.), as described previously . A triple staining technique using PI, PNA and PSA was performed to determine the plasma membrane and acrosome integrity (PMI) of the spermatozoa according to Schulze et al. . PMI reports the percentage of spermatozoa with intact acrosomal membranes (PI neg., PNA neg. and PSA neg.). 2.5. Data Processing and Statistical Analysis Data processing and statistical analysis were performed with R , extended by the packages lmerTest for linear mixed models and ggplot2 for graphical representation. To compensate for the slightly divergent initial qualities of the individual ejaculates, the difference between each treatment sample and the unshaken control sample on the same examination day was calculated using Equation (1):(1) D[Parameter]=[Parameter]Treatment-[Parameter]Control The assignment of treatment and control parameters results in negative values representing a decreasing parameter due to the treatment. The results for each combination of Di and duration are reported as mean with standard deviation (mean +- SD) and mean difference with standard error (mean-D +- SE), respectively. To investigate the relationship between Di, duration and sperm quality characteristics, linear regression was performed. Based on the assumption that the ejaculates of different boars respond differently to vibration emissions, a mixed model was calculated that included the boar as a random effect. The interaction of Di and duration was used as a fixed effect explaining the measured sperm quality characteristics. For total sperm motility, the examination day (d1-4) was included in the model. This represents different storage times after shipping in order to reflect the influence of transport on storability of semen. Several model variants with different combinations of predictors were tested. A p-value < 0.05 was considered a significant predictor, and only significant predictors were used to explain the data. For further selection, the model variants were compared in an ANOVA, and the model with the lowest Akaike information criterion (AIC) was selected. 3. Results Within the following 13 experimental weeks, 546 AI doses from 39 different boars were treated and observed with 8 different vibration intensities (Table 1). The TSM of the ejaculates at d0 was determined from the unshaken control samples and averaged 79.4 +- 6.2% (mean +- SD), with morphological abnormalities < 20%. To obtain an overview of all subgroups formed by the combination of simulation duration and Di, only the data from three different durations are summarized in Table 2, representing short (1 h), middle (6 h) and long (12 h) transport conditions. The detailed results of all durations can be found in Supplementary Tables S1-S4. In order to comprehensively describe the relationship of the measured values, a mixed linear regression model was constructed for each sperm quality parameter. The predictors Di and duration applied individually did not show a significant impact on the data (p > 0.05). Only the storage time for TSM and the interaction term Di x duration had a significant effect (p < 0.001) on the data and were used as fixed effects. The different boars were included as a random effect on the intercept and slope. The correlation between fixed and random effects is low (|r| <= 0.32), which justifies the use of the mixed model. The regression analysis showed that with each increase of the product (Di x duration) by 1, sperm quality parameters decreased by 0.30-0.45% (Table 3). The TSM additionally decreased by 0.65% with every further storage day. Figure 1a shows the summarized data of TSM on d4, including the mean absolute and mean difference values (mean-D TSM) as well as the regression line of the calculated mixed model. Similarly, the data for TRT, MITO and PMI are shown in Figure 1b and Figure 2a,b, respectively. 4. Discussion The aim of this study was to determine the influence of different vibration intensities during transport on BTS-extended boar semen. The results demonstrate a significant negative impact caused by the interaction of the intensity and duration of vibration emissions. Our measurements also show that even low vibration intensities impair sperm quality if AI doses are subjected to them for a long time. Setting a cut-off value for harmful vibrations, as originally intended by this study, is, therefore, not very useful. Rather, it should generally be considered important to minimize the time BTS-extended boar semen is exposed to even low-vibration intensities during transport. As of 2023, genetically valuable boars are housed in a few AI centers. Their diluted ejaculates are shipped to sow farms across long distances using a wide variety of vehicles. Transport times up of to 12 h and distances up to 600 and 1500 km or more are reported. As a negative influence of rotation of AI doses during storage on sperm quality has been previously demonstrated , vibrations during the transport of boar semen have become a relevant topic and the newfound focus of research groups. Schulze et al. conducted an experiment in which vibrations within a vehicle, caused by varying road conditions, were detected by a mobile sensing app. They were then able to simulate these findings in a laboratory setting by using a shaker and adjusting the rotation speeds (revolutions per minute, rpm) in the laboratory . Later studies used these rpm values as a reference for their experimental designs . Although, until now, only Tamanini et al. have adjusted rpm for a different shaker model through calculations, the comparison of the rotation speeds is limited because varying loading affects the resulting vibrations . Furthermore, in the previous experiments, only two shaking frequencies with one duration , one frequency with varying durations or only one duration were studied , and other factors were added to the experimental designs. A comprehensive consideration of multiple intensities with different exposure times has been lacking, and our study aimed to elucidate this gap in knowledge. Total sperm motility is the parameter most commonly used in AI centers to assess the quality of ejaculates . Although high values do not guarantee high fertility capacity, low values are indeed associated with small litter sizes . Figure 1 shows that total sperm motility in the AI doses decreases the longer vibrations are applied, and the effect becomes greater the stronger the vibrations are. Regression analysis suggests that short heavy vibrations affect TSM in the same way as long mild ones. This confirms the results from the studies that have separately examined different levels of intensity or duration. Although in Schulze et al. the comparison between mild vibrations (100 rpm) and the control group was not significant, a trend could already be identified. Thus, it can be postulated that a shaking time longer than 6 h would result in a significant decrease in total sperm motility. Tamanini et al. , who simulated mild vibration emissions, were able to show a linear relationship between the interaction of duration and extender on motility loss, but the reduction in short-term extender (BTS) was not significant. In contrast, our regression model also explains low motility losses, which arise from mild vibrations. In addition, although the measured values show that motility decreases with each storage day, no relationship with the vibration treatment could be determined with the regression model. Therefore, it can be assumed that the observed slight decrease in total sperm motility corresponds to the normal loss rate during storage . Slight variations in the fertilization capacity of an ejaculate, which may remain hidden from common motility assessment, can be revealed through the application of a thermo-resistance test . Overall, the results show the same pattern as for TSM, but the drop in the values calculated by the regression model does not behave as assumed, as the regression lines at Di = 4.0 and 5.0 are flatter than expected. This could be explained by the boar-specific effect , which is influenced by the individual composition of the seminal plasma and the sperm cell membranes . It has been demonstrated that seminal plasma influences sperm cryotolerance . Thus, variable tolerance to vibration emissions is also conceivable and should be the subject of further research. A limitation of this study is the use of BTS semen extender. BTS is intended for use as a short-term extender; therefore, extended AI doses should be inseminated within 3-4 days. The extender contains a simple buffering system based on bicarbonate . Schulze et al. postulated that vibration emissions result in the loss of CO2, leading to an increase in pH and corresponding decrease in sperm quality . A relevant change in pH was also identified in a more complex and robust buffering system by Tamanini et al. when examining the long-term extender Androstar(r) Plus. However, Paschoal et al. were able to show a vibration effect without a significant pH change, suggesting that a pH change is not the only reason for an impairment in sperm quality due to vibration emissions. Positive rheotaxis is characterized by hyperactivated motility against a fluid flow to guide sperm cells through the female genital tract and has also been detected in liquid-preserved boar semen . Vibration-induced movement in the fluid of the AI dose could trigger rheotactical behavior of spermatozoa and reduce the quality of the insemination portion through the early consumption of energy. The treatment-dependent decrease in mitochondrial activity would support this assumption. Although not using the exact inducing pathways, hyperactivation and capacitation of spermatozoa are closely related . A capacitation process that has already occurred could explain the decreasing percentage of spermatozoa with intact plasma and acrosomal membranes. Another explanation, previously discussed by other authors , are reactive oxygen species (ROS). The damage to the cell membranes occurs due to the shearing forces of oxidative stress. Our results will help future investigations elucidate the underlying molecular mechanisms. As our study design was intended to describe the impact of vibration and duration across a broad spectrum of these, extreme situations were also investigated, for example, through the simulation of a transport duration of 12 h. While such transport lengths have been described in practice, it is unlikely that the transported AI doses would be continuously exposed to strong vibrations, as demonstrated by our experiment. Nevertheless, if it is assumed that the effects of short, violent vibrations as well as long, milder vibrations will accumulate during a shipping tour, then our findings remain a valid reflection of these conditions. Regardless, this cumulative effect has yet to be demonstrated with a specific experimental design. 5. Conclusions Liquid-preserved boar semen is a perishable product and should be transported carefully. As not only the intensity but also the duration of vibration emissions is decisive for the influence on sperm quality, a shipping route to the customer with as little vibration as possible should be selected, especially for longer transport times and BTS-extended semen. If there is no alternative to a route with rough road conditions, the driving behavior should be adjusted by reducing speed . A real-time monitoring system could evaluate the success of these and other compensatory measures. Acknowledgments The authors want to thank Katja Koenneker for the English proofreading of the manuscript and her assistance with grammar and editing. Additionally, the excellent technical support of Anita Retzlaff is gratefully acknowledged. Supplementary Materials The following are available online at Tables S1-S4: Summary of total sperm motility data on collected days (1-4) after treatment. Click here for additional data file. Author Contributions Conceptualization, M.S.; methodology, T.H., P.S., F.F.-K. and M.S.; software, P.S.; validation, T.H. and M.S.; formal analysis, T.H.; investigation, T.H.; resources, F.F.-K. and M.S.; data curation, T.H. and M.S.; writing--original draft preparation, T.H.; writing--review and editing, P.S., C.S., M.J., F.F.-K. and M.S.; visualization, T.H.; supervision, M.S.; project administration, C.S., F.F.-K. and M.S.; funding acquisition, C.S., M.J., F.F.-K. and M.S. All authors have read and agreed to the published version of the manuscript. Institutional Review Board Statement Not applicable. Informed Consent Statement Not applicable. Data Availability Statement The data presented in this study are available on request from the corresponding author. Conflicts of Interest The authors declare no conflict of interests. Figure 1 Summary of CASA measurements on day 4 after treatment: (a) total sperm motility after 10 min incubation at 38 degC (TSM, n = 546 samples); (b) total sperm motility after 300 min incubation at 38 degC (thermo-resistance test TRT, n = 336 samples). Mean absolute values, grouped by intensity (Di) and duration (h) of vibration treatment, are shown as bars with standard deviation (SD) as the error bar. The color of the bars represents the mean difference (D) to the control group (duration = 0 h). The colored line shows the values of the applied regression model, and the dashed line in (a) stands for the minimum requirement of 65% motile spermatozoa on the expiration day. Figure 2 Summary of flow cytometric measurements (n = 336 samples) on day 4 after treatment: (a) mitochondrial activity (MITO); (b) plasma membrane and acrosome integrity (PMI). Mean absolute values, grouped by intensity (Di) and duration (h) of vibration treatment, are shown as bars with standard deviation (SD) as the error bar. The color of the bars represents the mean difference (D) to the control group (duration = 0 h). The colored line shows the values of the applied regression model. animals-13-00952-t001_Table 1 Table 1 Experimental design: every examined ejaculate was split into 14 subsamples (n = 546 in total) and treated for 13 different durations (0.5 to 12 h) with rising vibration intensities (Di). The unshaken sample served as a control (duration = 0 h). Displacement Index (Di) Corresponding Road Type 1 Ejaculate Numbers (n) Examined with CASA 0.5 Smooth asphalt 3 1.0 3 1.5 Rough asphalt 6 2.0 6 3.0 Cobblestone 6 4.0 6 5.0 3 6.0 6 1 According to Hafemeister et al. . animals-13-00952-t002_Table 2 Table 2 Summarized impact of different vibration intensities (Di +- 0.1) on different sperm quality characteristics on d4 of semen storage, representing short (1 h), middle (6 h) and long (12 h) transport simulation. Displacement Index (Di) Duration (h) D-TSM (%) D-TRT (%) D-MITO (%) D-PMI (%) Control (n = 39, absolute values) 0 (non-shaken) 77.33 +- 6.59 63.73 +- 7.96 76.99 +- 5.83 83.25 +- 2.23 0.5 1 -1.5 +- 0.3 0.2 +- 1.2 -0.4 +- 1.1 -1.4 +- 0.5 6 0.4 +- 1.3 3.4 +- 2.2 -2.9 +- 2 -2.4 +- 1 12 0 +- 0.7 -2.1 +- 1.2 -4.9 +- 1.3 -5.5 +- 0.9 1.0 1 0.5 +- 0.2 -2.6 +- 0.9 -1.5 +- 0.8 -0.6 +- 1.4 6 -5.4 +- 0.7 -11.4 +- 3.9 -9.4 +- 0.8 -8.1 +- 1.2 12 -7.8 +- 1 -12.4 +- 2.7 -16.2 +- 0.9 -12.5 +- 1.1 1.5 1 -1.1 +- 2.2 0.3 +- 1.2 -2.1 +- 0.7 -2.7 +- 0.7 6 -3.4 +- 2.7 -5 +- 0.8 -7.9 +- 2 -8.9 +- 1.3 12 -3.8 +- 2.7 -12.2 +- 4.3 -9.9 +- 2.9 -10.5 +- 2.6 2.0 1 -0.8 +- 1.6 0.6 +- 2.5 -3.3 +- 1.4 -3.9 +- 0.7 6 -5 +- 1.9 -7.5 +- 3.7 -15.5 +- 1.1 -14.3 +- 0.9 12 -10.2 +- 2.8 -11.6 +- 2.3 -19.4 +- 3.2 -17.7 +- 3.7 3.0 1 -1 +- 2.1 -1.2 +- 5.1 -2.7 +- 1.6 -3.8 +- 0.8 6 -4.2 +- 1.5 0.8 +- 2.2 -5.2 +- 0.6 -9.9 +- 0.4 12 -11.7 +- 2.5 -15.3 +- 5.5 -12.5 +- 0.7 -12.6 +- 1.5 4.0 1 1.2 +- 1.3 0.1 +- 1.9 -1.4 +- 0.4 -2.6 +- 0.4 6 -3.9 +- 1.5 -0.5 +- 3.3 -6 +- 1.1 -9.1 +- 0.7 12 -11.6 +- 1.9 -5.7 +- 3.6 -15.6 +- 1.6 -15 +- 2.3 5.0 1 -3.2 +- 0.6 -2.3 +- 4.9 0 +- 0.6 -3.1 +- 1.6 6 -6.9 +- 2.4 -5.9 +- 3.5 -4.4 +- 1.5 -6 +- 0.5 12 -16.4 +- 7.1 -11.4 +- 6.1 -13.6 +- 5.2 -11.9 +- 3.9 6.0 1 -1.3 +- 2.3 -5.5 +- 0.7 -5 +- 1.4 -1.4 +- 0.3 6 -10.9 +- 2.9 -10.1 +- 4.1 -7.5 +- 1.2 -8.3 +- 1.6 12 -20.3 +- 8.9 -23.9 +- 2.1 -18.3 +- 3.9 -12.8 +- 0.8 TSM--total sperm motility, TRT--thermo-resistance test, MITO--spermatozoa with active mitochondria, PMI--spermatozoa with intact acrosomal membranes; D: difference between treatment and control of the same ejaculate; values are presented as mean +- standard error. animals-13-00952-t003_Table 3 Table 3 The results of the regression analysis are shown, explaining the impact of vibrations on sperm quality characteristics during transport. The linear mixed model includes the interaction of vibration intensity (Di) and duration of simulation as fixed effects and the boar as a random effect on intercept and slope. In addition, the storage days (d1-4) were considered for total sperm motility. Dependent Variable Independent Variable Estimates SE df t-Value p-Value Total sperm motility (Intercept) 79.79 0.99 41 80.42 Storage day -0.66 0.08 2104 -8.10 <0.001 Di x duration -0.30 0.03 32 -11.75 <0.001 Thermo-resistance test (Intercept) 64.46 2.08 23 30.98 Di x duration -0.39 0.06 16 -6.95 <0.001 Mitochondrial activity (Intercept) 75.48 1.62 23 46.74 Di x duration -0.45 0.06 17 -7.59 <0.001 Plasma membrane integrity (Intercept) 81.74 0.66 23 123.41 Di x duration -0.43 0.05 20 -7.93 <0.001 Di x duration--interaction between displacement index and duration, SE--standard error, df--degrees of freedom. Disclaimer/Publisher's Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

PMC10000155

(1) Idiopathic epilepsy (IE) is thought to have a genetic cause in several dog breeds. However, only two causal variants have been identified to date, and few risk loci are known. No genetic studies have been conducted on IE in the Dutch partridge dog (DPD), and little has been reported on the epileptic phenotype in this breed. (2) Owner-filled questionnaires and diagnostic investigations were used to characterize IE in the DPD. A genome-wide association study (GWAS) involving 16 cases and 43 controls was performed, followed by sequencing of the coding sequence and splice site regions of a candidate gene within the associated region. Subsequent whole-exome sequencing (WES) of one family (including one IE-affected dog, both parents, and an IE-free sibling) was performed. (3) IE in the DPD has a broad range in terms of age at onset, frequency, and duration of epileptic seizures. Most dogs showed focal epileptic seizures evolving into generalized seizures. A new risk locus on chromosome 12 (BICF2G630119560; praw = 4.4 x 10-7; padj = 0.043) was identified through GWAS. Sequencing of the GRIK2 candidate gene revealed no variants of interest. No WES variants were located within the associated GWAS region. However, a variant in CCDC85A (chromosome 10; XM_038680630.1: c.689C > T) was discovered, and dogs homozygous for the variant (T/T) had an increased risk of developing IE (OR: 6.0; 95% CI: 1.6-22.6). This variant was identified as likely pathogenic according to ACMG guidelines. (4) Further research is necessary before the risk locus or CCDC85A variant can be used for breeding decisions. canine genome-wide association study whole-exome sequencing GRIK2 This research received no external funding. pmc1. Introduction Epilepsy is one of the most common neurological diseases in dogs, with an estimated prevalence of 0.62-0.82% in the general dog population . It is caused by sudden, excessive neuronal activity in the brain, leading to epileptic seizures characterized by motor, autonomic, and/or behavioral signs. According to the current recommendations of the International Veterinary Epilepsy Task Force (IVETF), canine epilepsy can be classified by etiology as reactive, structural, or idiopathic . Idiopathic epilepsy (IE) can be subclassified into IE with a genetic cause, IE with a suspected genetic cause, and IE of unknown cause. A high breed prevalence is indicative of a genetic cause and has been reported in multiple predisposed breeds such as the Labrador retriever, petit basset griffon vendees, Belgian shepherd, Irish wolfhound, pug, boxer, basset hound, border terrier, and border collie . Although a genetic cause is suspected in many breeds, only three causal variants of epilepsy have been identified to date: a dodecamer repeat in the NHLRC1 gene that causes progressive myoclonic epilepsy (Lafora disease) first reported in the wirehaired dachshund (OMIA 000690-9615) , a nonsense variant in LGI2 that causes benign familial juvenile epilepsy in the Lagotto Romagnolo (OMIA 001596-9615) , and a deletion in the DIRAS1 gene that is the causal variant for juvenile myoclonic epilepsy with photosensitivity in the Rhodesian ridgeback (OMIA 002095-9615) . Whereas the two latter diseases are classified as idiopathic epilepsy, Lafora disease is not since it has a structurally metabolic etiology . A deletion in PITRM1 causes a neurodegenerative syndrome in the Parson Russel terrier, with severe epileptic seizures as the main clinical sign (OMIA 002324-9615) . As with Lafora disease, these epileptic seizures have a metabolic etiology. One of the many breeds without an identified causal variant for IE is the Dutch partridge dog (DPD), a Dutch hunting breed. Epilepsy has been reported in this breed since 1986, and several findings suggest a genetic component. A study published in 1986 demonstrated a familial predisposition to epilepsy . Secondly, IE in the DPD has a high prevalence; the 1986 study estimated a prevalence of at least 1.4% , and another study reported an incidence of 2.2% between 2006 and 2011 . Prevalences exceeding 2% support a genetic basis according to the IVETF . Finally, the study published in 1986 found a narrow sense heritability (h2) between 0.33 and 0.47 for IE . No genomic studies have been conducted to date on IE in the DPD. Therefore, this study aimed to identify IE risk loci and putative causal variants in this breed. First, IE was phenotypically characterized through the use of owner-filled questionnaires and diagnostic investigations performed by a veterinarian. Next, two parallel genomic methods were used. First, a traditional genome-wide association study (GWAS) was performed to identify a candidate region associated with IE in the DPD. Secondly, a heuristic approach was applied, using whole-exome sequencing (WES) of a single family to identify family-specific variants. 2. Materials and Methods 2.1. Study Cohort DPDs with and without epilepsy were recruited through a call to Belgian and Dutch breeder organizations and a team of independent breeders. Blood was collected in EDTA-laced tubes for all 292 dogs (between November 2015 and February 2018) and stored at -20 degC. Signed informed consents were collected from the dogs' owners, and the sample collection was approved by the Ethics Committee of the Faculties of Veterinary Medicine and Bioscience Engineering at Ghent University (EC2017-86). The approval date is 5 February 2018. 2.1.1. Healthy Controls Dogs were included as controls were at least 6 years old, healthy, and had never shown signs of epileptic seizures during their life. 2.1.2. Epileptic Dogs To assess whether dogs had IE, questionnaires were sent to the owners of 46 epileptic dogs (translated questionnaire, Supplementary File S1), and telephone interviews were conducted to clarify or supplement answers provided in the questionnaires. The questionnaires gathered information about the dogs' signalment; characteristics of the ictal, pre-, and post-ictal phases; (epileptic) seizure frequency, duration, and severity; age of onset of (epileptic) seizures; possible trigger factors; diagnostic investigations performed; and antiseizure medication (ASM) that was being administered. When available, videos of the seizures were evaluated by a trained neurologist. Dogs were considered to suffer from IE (hereafter referred to as cases) when no evidence of a reactive seizure or structural epilepsy was identified and when history, as well as clinical and neurological signs, agreed with 6 inclusion criteria: (1) the presence of muscle contractions (tonic and/or clinical, orofacial, or any other body part), (2) age of onset between 6 months and 6 years, (3) at least 2 autonomous symptoms (salivation, urination, defecation, or loss of consciousness), (4) presence of a post-ictal phase, (5) absence of neurological symptoms in between epileptic seizures, and (6) absence of head trauma. 2.1.3. Statistical Analysis A Fisher exact test was performed to look for a sex predisposition. 2.2. Genome-Wide Association Analysis Samples of 200-500 mL EDTA blood of 16 cases and 43 controls (i.e., the samples available at the time of the GWAS analysis) were sent to the GIGA-Genomics platform, University of Liege. The samples were genotyped for 172115 SNVs using an Illumina CanineHD BeadChip according to the manufacturer's instructions (Illumina, Inc., San Diego, CA, USA). Raw genotype data were subjected to a 4-step quality control process using PLINK v1.9 . (1) SNVs with quality scores <70% were removed (based on the GenTrainScore in GenomeStudio); (2) SNVs and individuals with levels of missingness exceeding 2% were consecutively filtered out (--geno and --mind filters in PLINK); (3) SNVs with a minor allele frequency below 5% were removed (--maf filter in PLINK); (4) SNVs with a significant difference in missingness between cases and controls were removed (--test-missing in PLINK). GWAS analysis was performed with the remaining 99424 SNVs and samples using Fisher's exact testing for allelic association in PLINK. To evaluate population stratification, a QQ plot was created in R v3.6.3 using the qqman package . To correct for the observed population stratification, a principal component analysis (PCA) was performed with a subset of 33779 genotyped SNVs. In order to retain the most informative SNVs, this subset only contained SNVs with squared allele count correlations (r2) < 0.5 within a 200 kb window using a sliding window approach (with 5 bp steps) (--indep-pairwise 200 kb 5 0.5 in PLINK). The first principal component was included in a univariate linear mixed model association test using GEMMA software . Bonferroni-adjusted p-values (i.e., p-value multiplied by the number of tests ) were used to identify chromosome-wide significant SNVs. Odds ratios (ORs, see Table S1, alleles for calculations) and 95% confidence intervals (95% CIs) were calculated for significant SNVs. Manhattan plots were created using the qqman package . 2.3. GWAS Candidate Gene The GRIK2 gene is an interesting candidate gene within a GWAS-established region; its coding sequence (including splice sites) was further examined using Sanger sequencing in six cases and one control sample. Genomic DNA was extracted from all samples as previously described . Primers flanking the coding exons of the gene were designed with NCBI Primer-BLAST based on the latest canine reference assembly, ROS_Cfam_1.0 (Acc. No. GCF_014441545.1; GRIK2 location: NC_051816.1: g.59766364_60827294), taking into account secondary structures (mFold ), known SNVs (employing SNV handling in Primer-BLAST ), and repeat sequences (repeat filter set to automatic)). Subsequently, a PCR reaction was performed for each primer pair, after which 2 mL of PCR product was loaded on agarose gel electrophoresis, while the remaining 8 mL was used to perform Sanger sequencing (using both primers), as previously described . GRIK2 exon information can be viewed in Table S2, primer sequences and amplicon lengths are presented in Table S3, and details of PCR/sequencing mixes and programs can be viewed in Table S4. Five known SNVs and one new variant were detected in the cases. 2.4. Whole-Exome Sequencing While GWAS was used to detect IE-associated regions and possible common genes, we performed an additional WES on a nuclear family to screen for putative family-specific variants as a parallel method. This family included a case, both seizure-free parents, and a seizure-free sibling. In humans, generalized epilepsies have displayed a Mendelian inheritance pattern in some cases, and several rare, family-specific deleterious variants have been identified using whole-exome sequencing . Blood was extracted from all four samples using a DNeasy blood and tissue kit (Qiagen, Hilden, Germany) according to the spin column protocol (the elution step was performed twice with 50 mL molecular-grade water instead of 200 mL buffer EA), and WES was performed as described by Broeckx et al. . Reads were aligned to the CanFam3.1 reference genome using BWA v0.7.17 , and duplicate reads were marked with Picard v2.21.6. Variants were called using GATK's HaplotypeCaller in GVCF mode according to the GATK Best Practices (GATK v4.1.2.0) . Subsequently, all 403562 variants were filtered with GATK VariantFiltration according to the "hard filtering" described in the GATK Best Practices. Using custom R scripts (R v3.6.3) , the remaining variants were only retained if they were absent in the variant database of the Animal Genetics Lab (Sequence Read Archive (SRA) accession number PRJNA891496) and followed an autosomal recessive mode of inheritance (which is the inheritance pattern that was detected for this family). Based on annotation according to the online variant effect predictor (VEP) tool , non-coding and synonymous variants were removed. For the 80 remaining variants, the function of the genes in which they were located was inspected for a potential link to epilepsy. Using the R script provided by Broeckx et al. , a threshold value (Tv) was calculated to filter out variants with a minor allele frequency (MAF) > Tv in the European Variation Archive (EVA) database . The disease prevalence (Pd) was set to 0.8%, coverage was set to 0.95, data sampling was set to "a", and an autosomal recessive inheritance pattern was presumed. The database population size was altered to correspond to the size for each variant. A total of 5 variants were retained. 2.5. Variant Analysis and ACMG Classification Candidate variants identified in the GRIK2 gene and by WES were further examined for their pathogenicity. The effects at the protein level of the newly discovered variants in the GRIK2 gene (absent in the reference sequence (ROS_Cfam_1.0) and the control sample) and of the remaining WES variants were evaluated with the PROVEAN online prediction tool , PolyPhen-2 , and MutPred2 (in case of indels, MutPred-Indel ). Only variants for which at least one tool indicated a deleterious effect were considered for further investigation. The remaining potentially deleterious variants (in ENAH, CCDC85A, VPS54, SPAST, and BRINP3) were subsequently genotyped in all 18 cases and 18 controls (individuals other than the family members of the WES case). The ENAH variant was genotyped using PCR followed by gel electrophoresis on a 3% agarose gel, while the other variants were genotyped using Sanger sequencing, as described above for GRIK2 sequencing. Primer sequences, amplicon lengths, and the primers used for sequencing are shown in Table S5. The variant allele frequencies were compared between cases and controls, and ORs and 95% CIs were calculated for each variant (calculations according to Table S1, alleles). Variants for which an OR above 5 was found were further examined, in agreement with the standards and guidelines for the interpretation of sequence variants by the American College of Medical Genetics and Genomics (ACMG) . The two remaining variants in the CCDC85A and SPAST genes were genotyped in all dogs included in the study, and the variant allele frequency (Vt%) was calculated for the entire population, as well as for the selected cases and controls. The number of homozygous variant (Vt/Vt) dogs was compared to the number of homozygous wild-type and heterozygous dogs (Wt/Wt + Wt/Vt) between selected cases and controls with a Fisher exact test, and the OR (calculations according to Table S1, genotypes) and 95% CI was calculated again. The presence of a homologous variant was examined in human variant databases. Variant classification was performed using the aforementioned ACMG guidelines. 3. Results 3.1. Study Cohort A total of 292 DPDs entered this study, including 45 dogs displaying epileptic seizures according to their owner and 247 healthy dogs. Most dogs were from the Netherlands (201/292 = 69%) or Belgium (84/292 = 29%), and some were from Germany and Denmark (4/292 and 3/292, respectively; both = 1%). The sample represents dogs from many different families, with some closely related individuals. For 52 litters, two or more full siblings were available, and for 18 of the dogs displaying epileptic seizures, a normal littermate was available. The sire was available for 25 litters (14 sires in total), and the dam was available for 32 litters (22 dams in total). In total, 135 dogs were male, and 149 were female (sex was unknown for 8 dogs). 3.1.1. Healthy Controls In total, 100 of the 247 seizure-free dogs were included as healthy controls, while the 147 remaining healthy dogs did not meet the age requirement. Of these 100 dogs, 82 were over 10 years old, 9 were between 8 and 10 years old, and 9 were between 6 and 8 years old. Several of the control dogs were included in the GWAS analysis (see below). 3.1.2. Diagnostic Examination A blood examination was performed for 24 epileptic dogs, and results for 10 dogs were available for inspection by the authors. The minimum database blood test (as recommended by the IVETF ) was performed for seven of these dogs, while complete blood serum biochemistry was missing for the three remaining dogs. Urinalysis was performed on seven dogs and included sediment cytology, protein, glucose, and pH for five dogs. Specific gravity was not determined in one dog, and for another dog, no details were available. No significant abnormalities were detected in the blood or urine tests. Thirteen dogs were examined neurologically by their veterinarian, with no abnormalities detected, except in two dogs that were examined shortly after an epileptic seizure. A magnetic resonance imaging (MRI) scan of the brain and cerebrospinal fluid (CSF) analysis was available for one dog, and a CT of the brain was available for another dog; neither showed significant abnormalities. In total, five dogs were diagnosed with IE at a tier I/II confidence level as described by the IVETF . 3.1.3. Epileptic Cases The questionnaire was completed for 42 out of 45 dogs suffering from epilepsy, and additional video material was available for 7 of them. All videos were analyzed by a veterinary neurologist (Dr. Sofie F.M. Bhatti), and one was included as a supplementary file as an example (Video S1). Two dogs only displayed one epileptic seizure and were excluded from further analysis. Seven more dogs were excluded because the age of onset of seizures was too low (2 months for one dog) or too high (six dogs; age of onset up to 10 years old). Fourteen dogs were not included because they did not display at least two autonomous symptoms, and another dog was excluded because it had no muscle contractions. The remaining 18 dogs met all inclusion criteria and were included as cases in this study. No significant sex predispositions were found (p > 0.05). The mean age of onset of epileptic seizures was 3.5 years old (s.d. = 1.4; ranging from 6 months to 5 years old). Two-thirds of the dogs had focal epileptic seizures evolving into generalized epileptic seizures, and the rest had generalized tonic-clonic epileptic seizures. Epileptic seizures could be predicted by the owner in 67% of the cases. A wide range of seizure frequency was reported by the owners (from more than once per week to less than once per year), and 39% of the dogs showed cluster seizures (two or more epileptic seizures within 24 h). Epileptic seizures never lasted longer than 15 min, and most lasted between 2 and 5 min (50%) or between 1 and 2 min (28%). Status epilepticus (an epileptic seizure longer than 5 min) was reported in three dogs (17%). Almost all dogs (83%) received ASM, and 80% of this group received a combination of multiple drugs. In 11 dogs (73%), the owners noticed an improvement in seizure frequency, severity, and/or duration after starting medical treatment, with seizure freedom in 2 dogs. A complete overview of seizure characteristics and medical treatment can be found in Table 1. 3.2. Genome-Wide Association Analysis In total, 16 of the 18 cases and 43 of the 100 healthy controls (all more than 10 years of age) were available at the time of the GWAS analysis. After quality control filtering, all dogs and 99424 variants remained. Allelic association testing using a Fisher exact test with Bonferroni correction revealed one genome-wide significantly associated SNV (BICF2G630119560; praw = 2.6 x 10-7; padj = 0.026) on chromosome 12. To delimit a region on this chromosome, chromosome-wide significance was inspected among the SNVs on chromosome 12 using Bonferroni correction, revealing nine other SNVs that reached chromosome-wide significance using this method. Manhattan and QQ plots are shown in Figures S1 and S2A, respectively. As the QQ plot with the p-values of the Fisher exact test did show a clear deviation , 33779 variants were used to perform a PCA . Association testing was performed with a univariate linear mixed model, including the first principle component as a covariate. After Bonferroni correction, the same SNV remained significant genome-wide (BICF2G630119560; praw = 4.4 x 10-7; padj = 0.043). This time, only two other SNVs reached chromosome-wide significance on chromosome 12, identifying a 0.5 Mb candidate region. An overview of the associated SNVs on chromosome 12 with ORs and 95% CIs can be found in Table S6. Manhattan plots are displayed in Figure 1a,b, and the QQ plot is shown in Figure S3B. The 0.5 Mb associated region only includes one gene on the Ensembl 104 annotation , GRIK2 (NCBI Gene ID 481938), which is an interesting candidate gene. Therefore, the coding and splice site regions of GRIK2 were sequenced in six cases and compared to the latest reference genome (ROS_Cfam_1.0), as well as to a control dog. Five known SNVs (rs851724710, rs8745274, rs8745273, rs850859789, and rs9151294) and one new variant were identified in the cases. Of the known SNVs, three are synonymous variants, and two are intronic variants. For both intronic variants, a high variant allele frequency is reported in the EVA database (47% and 32% for rs8745274 and rs850859789, respectively). Therefore, none of the known SNVs were investigated further. The new variant was absent in the control sample (NC_051816.1 (XM_038684247.1): c.589C > T (p.(Leu197Phe))) and therefore further examined (see below, variant analysis and ACMG classification) (Table 2). 3.3. Whole-Exome Sequencing After WES of one case, both seizure-free parents, and a seizure-free sibling, 403562 variants (SNVs and indels) were called. Following hard filtering and only retaining variants that were not present in the variant database of the Animal Genetics Lab (Ghent University, SRA PRJNA891496) and that followed an autosomal recessive mode of inheritance, 2592 variants remained. Of these, 80 were protein-coding and not synonymous. None of these variants was located within the GWAS candidate region on chromosome 12. For the variants located in genes with a function potentially linked to epilepsy, the maximum expected allelic frequency in the variant database (i.e., "the Tv threshold") was calculated to be 10% (lowest Tv = 10.1% and highest Tv = 10.3%). Five variants with an MAF below this threshold in the EVA database were retained in this manner (Table 2). 3.4. Variant Analysis and ACMG Classification Three prediction tools were used to estimate the effect of the GRIK2 variant and the five remaining WES variants on the produced protein. Only the GRIK2 variant was not predicted as deleterious by any of the prediction tools (Table 3). The 5 WES variants were genotyped in all 18 cases and in 18 controls (Table S7), and a significant difference in variant allele frequency was found for the CCDC85A (NCBI gene ID: 481377; OR: 8.5; 95% CI: 1.7-41.5) and SPAST (NCBI gene ID: 608582; OR: 7.1; 95% CI: 1.2-42.9) variants. According to the ACMG standards and guidelines, we endeavored to classify these two variants as (likely) pathogenic, (likely) benign, or of uncertain significance . Subsequently, all 292 samples were genotyped for both variants. Unfortunately, it was not possible to gather enough family material to investigate variant segregation in affected family members in favor of or against pathogenicity (PP1 or BS4, respectively) for either variant in this study. Genotyping results for the case group, control group, and total population, as well as Vt%, are displayed in Table 3. A significant difference (p = 0.012) was found for the CCDC85A variant when comparing the number of Vt/Vt dogs to the number of dogs that were either Wt/Wt or Wt/Vt between cases and controls, with an OR of 6.0 (95% CI: 1.6-22.6), providing strong evidence of pathogenicity (PS4). Additional moderate evidence (PM2) was provided by the very low occurrence of the variant in population databases. The Vt% is 0.5% in dogs in the EVA database. Since the same CCDC85A missense variant is described for humans in the dbSNP database (rs1052182749), its allele frequency was verified in human populations. The Vt% of the homologous variant in humans (rs1052182749) is much lower than in dogs (0.0016% in gnomAD exomes, 0.0032% in gnomAD genomes, and 0.0026 in TopMed). Computational evidence could not be used as supporting evidence (PP3/BP4) in the variant classification, as two algorithms predicted a deleterious effect at the protein level, whereas one other predicted a neutral effect (see Table 2). Little is also known about the potential role of CCDC85A in epilepsy pathogenesis, and few functional studies have been conducted (PS3/BS3). In summary, two relevant criteria were fulfilled (one strong (PS4) and one moderate (PM2)), which is sufficient to classify this variant as likely pathogenic (Table 4). No strong evidence of pathogenicity was found for the SPAST variant. Although a significant difference (p = 0.049) was found when comparing the allele frequencies between cases and controls, with an OR of 2.6 (95% CI: 1.0-6.4), the OR was below 5, which does not fulfill the PS4 criterion. A homologous variant in SPAST is also described in humans in the dbSNP database (rs1268625226), and low MAF values are reported in multiple studies (0.0007% in gnomAD genomes, 0.0008% in TopMed, and not detected in NCBI ALFA). These very low frequencies in human databases and the relatively low frequency in the EVA database (Vt% = 1.6%) provided moderate evidence for pathogenicity (PM2). As with CCDC85A, little is known about SPAST's role in epilepsy pathogenesis, and the PS3 criterion is not fulfilled. Supportive evidence is provided by the three algorithms, predicting a deleterious effect at the protein level (PP3), and by missense variants in SPAST (including in the microtubule interacting and transport (MIT) domain), which is a common mechanism for disease (PP2) . Since only one moderate (PM2) and two supportive criteria are met (PP2 and PP3), this variant is of uncertain significance according to the ACMG guidelines. 4. Discussion This study faced the standard complexity associated with epilepsy studies. The intricate inheritance of epilepsy is well known in human genetics, and the genetic understanding of the disease is limited . Genetic epilepsies range from simple monogenic to complex polygenic with considerable interaction of environmental factors, leading to a broad phenotypic spectrum . Although dogs generally provide a good model for GWAS because of the extensive occurrence of linkage disequilibrium (up to 100 times more extensive than in humans) , genetic epilepsy studies deal with similar problems as seen in human studies. While a genetic etiology is suspected in many dog breeds, very few causal variants and risk loci have been identified for IE to date, probably because of the genetic complexity of this disease . Moreover, correct phenotyping of IE, the crucial step in any genetic study represents another hurdle. The diagnosis is exclusion-based, and many diagnostic tests are required to differentiate epilepsy from other similar neurological diseases and to rule out metabolic or structural causes of epilepsy. The aforementioned difficulties make a final diagnosis of IE with a suspected genetic cause quite challenging. The aim of this study was to find a causal variant for IE in the DPD, a breed in which the prevalence of IE is high, with evidence pointing towards a genetic cause, and to phenotypically characterize the disease. To attempt correct IE phenotyping following the IVETF guidelines, information was gathered regarding phenotypical characteristics and diagnostics. While phenotyping based on an owner-filled questionnaire is arguably a limitation of this study, the questionnaire was very extensive to make the best possible estimate of the patients' phenotype. Moreover, any diagnostic information determined by the dogs' veterinarians was collected, and video material was collected for seven dogs. This led to sufficient information to diagnose five dogs with IE at a tier I/II confidence level . Strict inclusion criteria were used in this study, limiting the erroneous inclusion of dogs as cases. As a result of the questionnaires, the phenotype of IE in DPDs was described. Notably, several dogs were excluded because the age of onset was too low or too old. One dog even started showing epileptic seizures for the first time at 10 years of age. The fact that epilepsy in this breed can develop at a later age than usual has been reported before . While a higher age limit to include dogs as cases could have been used in this study, we decided to use a more stringent age of onset of between 6 months and 6 years old , as this improves the likelihood of correct phenotyping, and a uniform phenotype is of great importance in genetic studies . In the epileptic dog population, status epilepticus was observed in 21% of the dogs. Cluster seizures occurred in 23% of dogs, which greatly exceeds the frequency of <10% reported by Mandigers (2017) . However, that study reported the occurrence of three or more seizures within one time unit, while an occurrence of two or more was used here, as proposed by the IVETF , possibly explaining this difference. Very similar to what was reported in that study, seizures occur more often in males than females (58% male vs. 42% female). When only looking at the selected cases, the occurrence is slightly higher in females (44% male vs. 56% female). However, contrary to the great Swiss mountain dog (GSMD), in which males are more likely to be affected than females , no significant sex predisposition was found for the DPD in the current study, nor the study by Mandigers . About two-thirds of the epileptic cases in this study displayed generalized seizures evolving from focal seizures, similar to what was described for the GSMD , in contrast to the border collie, in the vast majority of which have generalized seizures and only 4% of which display a combination of both seizure types . Similar to what is reported in the border collie and GSMD , the majority of the dogs (83%) received medical treatment. The number of DPDs receiving single and combination drug therapy is comparable to the border collie, whereas the opposite is true for the GSMD. The lack of identified causal variants and susceptibility loci point to complex inheritance patterns of canine IE, as is also seen in human IE . To date, only two studies have identified a causal variant for IE in dogs , and one study identified a risk locus in the ADAM23 gene for the Belgian shepherd . Later, a common 28 kb risk haplotype in the same gene was established in the Belgian shepherd, as well as the schipperke, Finnish spitz, and beagle . The same risk haplotype was subsequently found to be associated with IE in four other breeds (Australian shepherd, Kromfohrlander, Labrador retriever, and whippet), proposing ADAM23 as a common risk gene for epilepsy with low penetrance . A recent study aimed to identify additional risk loci in the Belgian shepherd. The authors found a significant association between IE and a locus on chromosome 14, while a suggestive association was found adjacent to the previously described ADAM23 locus . Yet another study in the Belgian shepherd again validated the chromosome 14 and 37 risk haplotypes and identified an insertion in the RAPGEF5 gene adjacent to the chromosome 14 haplotype . While some studies have been successful, the majority have not. Here, a new risk locus was identified on chromosome 12. A GWAS analysis of 16 cases, 43 controls, and 99424 SNVs using a univariate linear mixed model test and Bonferroni correction revealed one SNV that was significantly associated with IE in the DPD genome-wide (BICF2G630119560; praw = 4.4 x 10-7; padj = 0.043). An OR of 10.4 was calculated for the associated allele (95% CI: 4.1-26.5). To the best of our knowledge, this is the first time that this locus has been identified as a susceptibility locus for IE in dogs. A 0.5 Mb candidate region was delineated through the location of two additional chromosome-wide associated SNVs on the same chromosome. Inspection of that region identified an interesting candidate gene, GRIK2, located 0.3 Mb upstream of BICF2G630119560. The GRIK2 gene encodes the kainite receptor (KAR) subunit GluK2 (previously known as GluR6). KARs are ionotropic glutamate receptors widely expressed in the central nervous system and are tetramers that can be formed from subunits GluK1-5 . They are key players in glutaminergic synaptic transmission in the hippocampus and probably other brain structures. KARs can be activated by glutamate, after which gamma-aminobutyric acid (GABA) release is depressed and the excitability of neuronal cells is increased . Epilepsies have been linked to KARs, including GluK2 , and Grik2 knockout mice have reduced susceptibility to seizures induced by kainate, a high-affinity KAR agonist . Furthermore, overexpression of the Grik2 kainate receptor in the hippocampus induces seizures in rats . In humans, post-translational modifications of GRIK2 were shown to cause/influence epileptic phenotypes, as was seen in mesial and lateral temporal lobe epilepsy patients . Furthermore, multiple variants were linked to epilepsy susceptibility in a Chinese population and a cohort of Chinese children , to a disorder including epilepsy , and to the presence of severe epilepsy in patients with neurodevelopmental disorders . In this study, one new variant, i.e., NC_051816.1 (XM_038684247.1): c.589C > T (p.(Leu197Phe)), was found among the six cases for which the coding DNA of GRIK2 and the regions surrounding the coding exons were sequenced. All prediction tools estimated that this variant would have a neutral effect on protein function. Therefore, the variant was not examined further. As we only sequenced the coding DNA and splice site regions, the presence of a causal intronic variant cannot be excluded. Furthermore, no brain tissue was available to investigate potential changes in GRIK2 expression in epileptic dogs. In humans, a downregulation of GRIK2 has already been shown in patients with epilepsy . Such experiments could provide useful information in the future. Since the candidate gene within the GWAS region revealed no interesting variants and family-specific variants have been identified in human epilepsy , the study cohort was inspected for nuclear families including a case, both seizure-free parents, and a seizure-free sibling to perform WES. Samples were available for two such families, but the DNA concentration for some samples of one family was insufficient. Therefore, WES was performed on the remaining nuclear family to search for family-specific variants in these dogs and as a validation of the GWAS results. Unfortunately, the analysis revealed no variants within the GWAS candidate region. The presence of structural variants was not investigated, as WES is a poor tool to detect these kinds of variants. Future investigations might include high-throughput whole-genome sequencing or long-read sequencing to search for structural variants. Another explanation for the lack of variants in the GWAS candidate region might be the family's genotype for the risk allele. While the case and mother fit the expected genotype (homozygous and heterozygous for the risk allele, respectively), the healthy father (still seizure-free at 10 years old) and sibling (still seizure-free at 7 years old) were also homozygous for the risk allele. This deviation from the expected phenotype could explain why no variants were found in the candidate region for this family. Nonetheless, WES could still be used as a parallel approach to reveal family-specific variants. Variants of interest outside of the GWAS candidate region were selected based on gene function, MAF in the EVA database (<10%), and an estimated deleterious effect by at least one prediction tool. After preliminary genotyping of a limited number of samples, the variants NC_051814.1 (XM_038680630.1): c.689C > T (p.(Pro230Leu)) in CCDC85A (chromosome 10) and NC_051821.1 (XM_038691182.1): c.493G > A (p.(Glu165Lys)) in SPAST (chromosome 17) appeared to be the most promising. According to the ACMG standards and guidelines, these variants were classified as (likely) pathogenic, (likely) benign, or of uncertain significance . Amongst others, we looked at the MAF in population databases. As IE follows a complex inheritance pattern, a (very) low MAF can be expected in these databases. Indeed, the Vt% values were 0.5% and 1.6% for the CCDC85A and SPAST variants in the EVA database, respectively. Therefore, we accepted these relatively low frequencies as moderate evidence for pathogenicity (PM2). While little is known about the potential roles of CCDC85A and SPAST in epilepsy pathogenesis and few functional studies have been conducted, they remain interesting candidate genes. CCDC85A's expression is enhanced in the brain (Human Protein Atlas, proteinatlas.org ). Furthermore, CCDC85A, B, and C are delta-interacting protein A (DIPA) family members and potentially participate in N-cadherin-mediated neuronal development through interaction with p120-1, a neuron-specific catenin . N-cadherin regulates the presynaptic function at glutamatergic synapses and controls presynaptic vesicle clustering. It probably has a critical role in maintaining synaptic homeostasis and plasticity . Additional functional studies of CCDC85A might further improve the classification of the variant in this study. As for SPAST, there is some evidence that it might have a role in the pathogenesis of epilepsy (PS3). This gene encodes spastin, a microtubule-severing enzyme, and the variant is situated in the MT-interacting and trafficking (MIT) domain. It has been established that variants in the MIT domain disrupt lysosomal function . Interestingly, a defective autophagy (in which lysosomes fulfill a vital function) is often associated with neurodevelopmental disorders associated with epilepsy . Furthermore, variants in SPAST are generally known to cause autosomal dominant spastic paraplegia 4 in humans, which is also increasingly associated with additional neurological symptoms such as epilepsy . However, the current knowledge on spastin's role in epilepsy pathogenesis is insufficient to fulfill ACMG's PS3 criterion. Two relevant criteria were fulfilled for the CCDC85A variant (one strong (PS4) and one moderate (PM2)), which is sufficient to classify this variant as likely pathogenic. Based on the ACMG guidelines, this variant can be used in human medicine in clinical decision making when combined with other evidence of IE. However, as genetic diversity in dogs tends to be low, we prefer to err on the safe side. Before including this variant, we recommend (1) independent validation of this association, with emphasis on detailed phenotyping, and (2) the collection of family material over multiple generations. Future, additional evidence might change this classification, either in favor or against pathogenicity. Since only one moderate (PM2) and two supportive criteria are met for the SPAST variant (PP2 and PP3), this variant is of uncertain significance according to the ACMG guidelines and should not be used for clinical decision making. 5. Conclusions Although no clear causal variant was found for IE in the DPD, this study is the first to report a new significantly associated SNV for IE on chromosome 12. Further investigations of the GRIK2 gene or other genes near this locus might reveal more candidate genes for the disease. Furthermore, WES revealed a potentially pathogenic variant in CCDC85A. Dogs homozygous for this variant have an increased risk of developing epilepsy. However, we do not recommend the use of either of these two variants as a tool to select against IE before more evidence is gathered. Acknowledgments We thank Linda Impe, Dominique Vander Donckt, Carolien Rogiers, and Ruben Van Gansbeke for excellent technical assistance. We also thank the breeder associations, breeders, and owners who collaborated on this study. Supplementary Materials The following supporting information can be downloaded at: Supplementary File S1: Questionnaire; Video S1: Video of a Dutch partridge dog displaying an epileptic seizure; Table S1: Odds ratio calculations; Table S2: Overview of the GRIK2 exons; Table S3: GRIK2 primer information; Table S4: PCR and sequencing information; Table S5: Candidate variant genotyping--primer information; Table S6: Associated SNPs on chromosome 12 based on the univariate linear mixed model; Table S7: Preliminary genotyping results for the candidate variants; Figure S1: PCA plot of the 57 dogs retained in the GWAS analysis; Figure S2: Manhattan plots after Fisher exact testing; Figure S3: QQ plots of the expected and observed -log(p) values. Click here for additional data file. Author Contributions Conceptualization, E.B., L.P. and B.J.G.B.; methodology, E.B.; software, E.B., M.V.P. and F.F.; validation, E.B.; formal analysis, E.B. and M.V.P.; investigation, E.B.; resources, E.B., S.F.M.B., P.M. and L.V.H.; data curation, E.B. and F.V.N.; writing--original draft preparation, E.B.; writing--review and editing, E.B., S.F.M.B., M.V.P., I.P., F.F., F.V.N., P.M., L.V.H., L.P. and B.J.G.B.; visualization, E.B.; supervision, L.P. and B.J.G.B.; project administration, L.P. and B.J.G.B. All authors have read and agreed to the published version of the manuscript. Institutional Review Board Statement The animal study protocol was approved by the Ethics Committee of the Faculties of Veterinary Medicine and Bioscience Engineering at Ghent University (EC2017-86). The approval date is 5 February 2018. Informed Consent Statement Informed consent was obtained from the owners of all subjects involved in the study. Data Availability Statement The data generated during this study can be found within the published article and its supporting information. The variant data for this study have been deposited in the European Variation Archive (EVA) at EMBL-EBI under accession number PRJEB56315. The raw WES data have been submitted to the Sequence Read Archive (SRA) under accession number PRJNA891496, samples SAMN31329656--SAMN31329659. Conflicts of Interest The authors declare no conflict of interest. Figure 1 Manhattan plots following univariate linear mixed model testing and genes in the candidate region. (a) Manhattan plot with red line indicating the genome-wide threshold. (b) Manhattan plot zoomed in on chromosome 12. The red line indicates the genome-wide threshold (threshold = 5.03 x 10-7), and the blue line indicates the chromosome-wide threshold (threshold = 1.48 x 10-5). (c) Genes in the GWAS candidate region according to Ensembl release 104. animals-13-00810-t001_Table 1 Table 1 Epileptic seizure characterization and medical treatment. Overview of the phenotypical characterization and medical treatment for the selected cases included in this study. Absolute % Dogs Total number of dogs 18 - Sex Male 8 44% Female 10 56% Age of onset (years) Mean +- s.d. 3.5 +- 1.4 - Median 4 - Maximum 5 - Minimum 0.6 - Epileptic seizure frequency Dogs with a known epileptic seizure frequency 17 - <=1 per year 3 18% 1-2 per year 2 12% 3-5 per year 2 12% 6-11 per year 2 12% 1-2 per month 4 24% 2-4 per month 3 18% >=1 per week 1 6% Clusters Occurrence of cluster seizures 7 39% Prediction Owner can predict the epileptic seizure 12 67% Triggers Epileptic seizures can be provoked 6 33% Stress 4 67% Sounds 1 17% Excitation 2 33% Getting startled 1 17% Meal 1 17% Exercise 0 0% Light (flashes/TV/sun) 0 0% Epileptic seizure duration <1 min 1 6% 1-2 min 5 28% 2-5 min 9 50% 5-10 min 2 11% 10-15 min 1 6% >15 min 0 0% Autonomous symptoms Dog displays at least one autonomous symptom 18 100% Salivating 17 94% Urinating 13 72% Defecation 3 17% Loss of consciousness 9 50% Loss of consciousness possible/unsure 6 33% Semiology Cycling movements 16 89% Generalized epileptic seizure evolved from focal 12 67% Generalized epileptic seizure 6 33% Focal epileptic seizure limited to legs/head 0 0% Unknown epileptic seizure type 0 0% Treatment Dogs not receiving antiseizure medication 2 11% Dogs receiving antiseizure medication 15 83% Dogs responding to treatment 11 73% Single drug 3 20% Phenobarbital 3 100% Imepitoin 0 0% KBr 0 0% Combination 12 80% Phenobarbital, KBr 4 33% Phenobarbital, imepitoin 1 8% Phenobarbital, cannabidiol 2 17% Imepitoin, KBr 1 8% Phenobarbital, KBr, levetiracetam 1 8% Phenobarbital, KBr, cannabidiol 3 25% Phenobarbital, KBr, imepitoin, cannabidiol 0 0% animals-13-00810-t002_Table 2 Table 2 Variants and predicted effects. The variant description is provided at the chromosomal (Chromosome), coding sequence (CDS), and protein (Protein) levels. The rs number (rs) and minor allele frequency in the EVA database (MAF) are provided when available. The outcomes of different online prediction tools are displayed, and deleterious/damaging predictions are indicated in bold. Variant no. Chromosome CDS Protein Gene Rs MAF PROVEAN PolyPhen-2 MutPred2/ Indel 1 * NC_006589.4: g.38710992_38711006del XM_022421182.1: c.662_676del p.(Glu229_Arg233del) ENAH rs851038082 6.8% -4.714 / 0.271 2 NC_051814.1: g.57941870C > T XM_038680630.1: c.689C > T p.(Pro230Leu) CCDC85A rs852050632 0.5% -3.165 1 0.086 3 NC_051814.1: g.64662153A > T XM_038680741.1: c.51T > A p.(Asp17Glu) VPS54 / / -0.470 0.993 0.09 4 ** NC_051816.1: g.60469829C > T XM_038684247.1: c.589C > T p.(Leu197Phe) GRIK2 / / -1.308 0.201 0.292 5 NC_051821.1: g.25973635G > A XM_038691182.1: c.493G > A p.(Glu165Lys) SPAST rs850566951 1.6% -3.165 0.997 0.643 6 NC_051842.1: g.8412424G > C XM_038448399.1: c.578G > C p.(Arg193Pro) BRINP3 rs852865827 0.7% -4.929 0.999 0.813 * Variant cannot be mapped to ROS_Cfam_1.0 and is displayed here according to CanFam3.1. ** Variant found in the GWAS region of interest. animals-13-00810-t003_Table 3 Table 3 Genotyping results for the CCDC85A and SPAST variants. The number of homozygous wild-type (Wt/Wt), heterozygous (Wt/Vt), and homozygous variant (Vt/Vt) dogs, as well as the variant allele frequency (Vt%), is shown for the case group (Cases), the control group (Controls), and the total population (Tot_pop). CCDC85A SPAST Wt/Wt Wt/Vt Vt/Vt Vt% Wt/Wt Wt/Vt Vt/Vt Vt% Cases 11 2 5 33.3% 11 6 1 22.2% Controls 67 27 6 19.5% 82 16 2 10.0% Tot_pop 186 82 24 22.3% 242 47 3 9.1% animals-13-00810-t004_Table 4 Table 4 Variant classification of the XM_038680630.1: c.689C > T variant. Criterion numbers are shown as provided by the American College of Medical Genetics. Results, remarks, and conclusions for each criterion are listed. Criterion Result Remarks Conclusion Significant OR > 5 (PS4) OR: 6.0; 95% CI: 1.6-22.6 PS4 fulfilled Low MAF in population databases (PM2) 0.5% in EVA database Homologous variant in humans has an much lower frequency in multiple population studies PM2 fulfilled Multiple lines of computational evidence (PP3/BP3) 2/4 programs predicted a deleterious effect, and 2/4 predicted a neutral effect. When in silico predictions disagree, this evidence should not be used to classify a variant PP3/BP3 cannot be used Variant segregation (PP1/BS4) N/A Insufficient family material available to investigate cosegregation in multiple affected family members PP1/BS4 cannot be used Functional studies show deleterious (PS3) or no deleterious (BS3) effect Enhanced expression in the brain; possible interaction with neuron-specific catenin p120-1, which probably plays a critical role in synaptic homeostasis and plasticity Too few functional studies exist to fulfill these criteria PS3/PS3 not fulfilled Disclaimer/Publisher's Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

PMC10000156

Precision livestock farming has a crucial function as farming grows in significance. It will help farmers make better decisions, alter their roles and perspectives as farmers and managers, and allow for the tracking and monitoring of product quality and animal welfare as mandated by the government and industry. Farmers can improve productivity, sustainability, and animal care by gaining a deeper understanding of their farm systems as a result of the increased use of data generated by smart farming equipment. Automation and robots in agriculture have the potential to play a significant role in helping society fulfill its future demands for food supply. These technologies have already enabled significant cost reductions in production, as well as reductions in the amount of intensive manual labor, improvements in product quality, and enhancements in environmental management. Wearable sensors can monitor eating, rumination, rumen pH, rumen temperature, body temperature, laying behavior, animal activity, and animal position or placement. Detachable or imprinted biosensors that are adaptable and enable remote data transfer might be highly important in this quickly growing industry. There are already multiple gadgets to evaluate illnesses such as ketosis or mastitis in cattle. The objective evaluation of sensor methods and systems employed on the farm is one of the difficulties presented by the implementation of modern technologies on dairy farms. The availability of sensors and high-precision technology for real-time monitoring of cattle raises the question of how to objectively evaluate the contribution of these technologies to the long-term viability of farms (productivity, health monitoring, welfare evaluation, and environmental effects). This review focuses on biosensing technologies that have the potential to change early illness diagnosis, management, and operations for livestock. biosensors precision dairy farming sensors technology dairy cattle early diagnosis This research received no external funding. pmc1. Introduction The future of animal farming will be guided by the principles of precision, sustainability, and intelligence. Accurate cattle production can only be attained with the rapid spread of intelligent technology for early warning of illnesses, feeding precision, and remote diagnosis . In recent years, the dairy sector has developed and deployed a number of different technologies in order to automatically monitor a variety of behavioral and physiological indicators . Collecting large amounts of data is made possible by the use of sensors and technology, and these data must be analyzed with sophisticated statistical methods before any conclusions can be drawn about the animals' behavior, health, or welfare. Innovations and information technologies (ITs) are essential for achieving sustainable operations because they enable early and rapid disease detection, help measure environmental emissions, and optimize production . Traditional diagnostic procedures are notoriously labor-intensive, time-consuming, and technical, necessitating the expertise of skilled specialists using specialized equipment. To overcome these obstacles and create quick-response, low-cost, and highly reliable biosensor devices, new technologies are being employed . The use of automated remote monitoring and detection of animal well-being, suggesting factors for body and body weight problems, might enhance biological metrics in cattle. This might be performed by analyzing sounds, photographs, videos, and other data in real time. Data from remote sensors such as microphones, cameras, accelerometers, and thermometers can be used to gather reliable information when combined with animal IDs and other observations and put into algorithms . Different sensors, such as radio frequency identification (RFID), accelerometers, load cells, and webcams, can be used to detect sudden changes in the activity, eating and drinking, physical condition, and health of animals . Devices with the potential to measure physiological, immunological, and behavioral responses in livestock and various animal species are referred to as biosensors . Biosensors are compact, portable, highly sensitive, fast, and can be extremely specific with a low chance of a false positive. They function in a variety of ways, including detecting changes in pH, ion concentrations, mass through particular hybridization, enzymatic reaction, functional loss, electrical potential change, color change, temperature, and so on. Novel biosensors have considerable advantages and uses in the management of livestock, including disease isolation and detection, reproductive cycle detection, health monitoring, and monitoring an animal's physiological wellness through the examination of its surroundings . The application of biosensors and wearable technology in animal health management is becoming increasingly important. These instruments can facilitate the early detection of diseases in animals, decreasing economic losses. A variety of sensors for animal health management are in various stages of commercialization across the world. Some approaches for properly detecting health status and illness diagnosis are solely relevant to humans, with only minor alterations or testing in animal models . The current state of medical technology makes early illness detection difficult and laboratory testing for animals expensive . There is a need for detection technologies that can anticipate when and in what group an incident is likely to occur, inform diagnosis and treatment options, and forecast potential repercussions on a specific community . These tools can speed up the monitoring process and are not only accurate and sensitive for the parameters being analyzed, but they also can be dependable and simple to use. General farm monitoring may be made easier and more reliable by employing portable devices instead of conventional techniques such as taking notes, keeping a farm diary, or using simple equipment without data-sharing features. Many solutions have been developed for portable devices to reduce the labor associated with manually recording data . Every year, new diseases that endanger the health of animals appear in the modern world. There are currently no viable, cost-effective diagnostic techniques for early disease identification in farmed livestock animals. Biosensing technologies have the ability to address these issues by providing novel diagnostic tools for the early detection of major health hazards in the agri-food animal sector . Breeding is an essential component of animal husbandry. The ovulation cycle in cattle must be monitored in order to estimate the temporal frame for artificial insemination. Mastitis (both clinical and subclinical), metabolic disorders such as ketosis and acidosis, the animal's reproductive status, and projected calving may all now be diagnosed using various technologies. As is known, automated milking systems (AMSs) pose various issues and possibilities for dairy producers. AMSs allow for the monitoring of milking frequency at the cow level, as well as quarter-level production and milk quality, which can contribute to the development of disease detection systems. Biomarkers such as plasma-hydroxybutyrate (BHB) and body condition score (BCS) might be beneficial not only in diagnosing illnesses, but also in evaluating cow pregnancy and re-production success . BCS is a valuable approach for monitoring the relationships between nutritional management, reproduction, and metabolic diseases, and it facilitates farm management decisions . BHB is one of the most useful indicators to diagnose ketosis . It was found that BHB can be useful not only as an indicator for metabolic disorders, but also that the concentration of BHB in the blood has a negative phenotypic connection with the pregnancy rate at the first service . Elevated BHB levels have been associated with uterine problems and delayed luteal activity . Because it is simple, inexpensive, and has acceptable sensitivity and specificity, measuring inline lactate dehydrogenase (LDH) activity in milk in robots is a reliable indicator for the detection of subclinical mastitis . As an inflammatory indication of mastitis, the milk enzyme LDH performed similarly to acute-phase proteins and somatic cell count, according to studies . Using accurate diagnostic technology can enhance our understanding of the elements influencing the reproductive physiology of dairy cows . In addition to assisting with reproductive control, the method enables the evaluation of luteal activity and its association with fertility through the analysis of frequent progesterone (mP4) data . The focus of this review is to analyze emerging biosensing technologies that have the potential to impact livestock early disease diagnostics, management, and relevant procedures. 2. Importance of Early Diagnostic in Dairy Farming Animal health management with biosensors is a new field that is gaining traction worldwide. Biosensors are increasingly being utilized in dairy farms to better monitor animal health and detect illnesses early on . Making a correct diagnosis is a crucial intermediate step between identifying a disease's root cause and treating it . Continuous observation of behavioral and physiological indicators may make it possible to spot subtle alterations before they manifest as overt clinical symptoms. Cattle may benefit from early sickness detection by preventing disease development and improving treatment response . Farm managers, veterinarians, and farmers themselves can use sensors to monitor animal movements, food intake, sleep cycles, and even shelter air quality. Big data-capable computers store and process raw data . Proficient estrus identification is a constant issue for efficient reproductive performance in dairy herds, especially on farms that use artificial insemination (AI) . In preparation for calving and milk production, dairy cattle experience many changes to their metabolism and bodies as they move from late gestation to early lactation. These profound alterations cause an increase in the likelihood of developing a wide variety of health problems, such as metritis, mastitis, ketosis, and displaced abomasum . The absence of sickness is an important aspect of general animal health and well-being. Disease, lameness, and limb problems are already posing substantial challenges to the dairy industry. Lameness is uncomfortable, and animals in pain usually change their activity, stride, food, posture, and appearance from their regular behavior. The economic expenses associated with treatment, decreased milk output, decreased fertility, mortality, or removal from the herd may be lowered if a disease is diagnosed earlier . The earlier adoption of intervention methods can be made possible by the earlier identification of cows at risk for health issues . Devices that can be implanted in an animal's body (thermography, pedometers) remain in the stomach (bolus) and provide owners with valuable information on the animal's behavior and medical conditions. Using various devices prevents farm managers and veterinarians from spending unnecessary time on evaluating and inspecting animals. Veterinarians can catch diseases early on with wearable sensors (pedometers, GPS, milk analyzers, body condition scoring), which keeps animals from getting subclinically sick, clinically sick, or even dying. Owners can sort out sick animals in time to keep diseases from spreading through whole herds . For instance, a decline in activity might be a sign of illness, and a decrease in time spent lying down could be a sign of discomfort or pain (pedometer, accelerometer) . 3. Innovative Tools in Farm Animals' Early Disease Diagnosis Farm management is being significantly influenced by technological breakthroughs, which are reducing physical labor, expenditures, and waste while increasing yields and profits. As a result of agricultural technological breakthroughs, a new farming method called "precision agriculture" has evolved . Monitoring real-time autonomic responses (e.g., respiration rate, heart rate variability and heart rate, blood pressure, changes in peripheral blood flow) and defense-related reflexes with innovative biosensor equipment can aid in understanding how housing, nutrition, and genotype influence animals' resilience to stressors. These sensors can contribute to the knowledge of factors that influence animal welfare and the creation of remedies (for example, husbandry techniques, genotype selection) that increase the welfare of livestock and companion animals. Wearable sensors can track feeding behavior, rumination behavior, rumen pH, rumen temperature, body temperature, laying behavior, animal activity, and animal location or placement . Wearable or imprinted biosensors that allow remote data transfer could be significant in this rapidly evolving field . There are many gadgets that can measure body temperature, behavior, and movement of the animal . Sensors and wearable technology can be inserted into animals to detect the components of their body fluids such as sweat . Cows are also fitted with commercially available biosensor collars to monitor the estrous cycle . Figure 1 demonstrates examples of various technologies applied to cows. 3.1. Milk Analyzers The use of technologies to evaluate physiological, behavioral, and production markers on individual animals in order to identify events of interest is included in the process of precision dairy monitoring . Milk analytes can be used as biomarkers for diseases or reproductive status detection. For example, DeLaval Herd Navigator (DeLaval Inc., Tumba, Sweden) detects the amount of progesterone in milk. The program also recommends the ideal timing for insemination, names animals that need to be confirmed as pregnant, flags early abortions, and lists cows at risk for cysts and protracted anestrus. Other milking robots such as Lely Astronaut A4 (Lely Campus, Cornelis van der Lely an 1, 3147, PB, Maassluis, The Netherlands) can determine milk analytes and milk electrical conductivity. Blood biomarkers are useful indicators of animal health, although they have limited economic use. They might provide a great deal of information, especially because biomarkers can detect subclinical stages of illnesses even when the cow appears fully healthy and shows no visible indications of illness. An alternative to blood biomarkers can be milk biomarkers . Sensor devices for determining the fat and protein content of milk are widely utilized on farms nowadays. According to each farm's milking system, a unique sensor system is utilized. These sensors provide information on the health and fertility of cattle. Using milk analyzers, the estrous cycle and reproductive performance in dairy cows can be tracked . There are various analyzers which may operate on different principles, some using chemical indicators and others based on spectroscopy . 3.1.1. Somatic Cell Count Mastitis-related milk production losses in dairy animals are economically significant . Monitoring somatic cell count (SCC) concentrations in milk is the most widely used method for detecting mastitis, especially in its subclinical forms. When SCC values surpass the limit, the milk's value plummets drastically. As a result, experts feel that SCC level is a significant parameter for evaluating udder health . Despite the absence of clinical signs, subclinical mastitis is distinguished by an increase in SCC in milk. SCC is a useful udder health indicator since it counts the quantity of somatic cells (mainly desquamated epithelial cells, macrophages, and neutrophils) in milk. Aside from being used as a criterion for selecting dairy cows that are less susceptible to mastitis, the presence of SCC in bovine milk is a well-established indicator of mammary gland inflammation, which is strongly linked to the presence of a mammary infection. SCC has proven to be a useful indicator of decreased milk supply due to subclinical mastitis, which is present when the cell count is greater than 200,000 per milliliter . Using cell staining methods and microscopy, the SCC concentration can be evaluated at the laboratory level. However, these approaches are time-consuming and call for specialized equipment and personnel . Modern techniques for SCC in milk detection are much less laborious and time-consuming . Automatic mastitis detection devices are examples of novel diagnostic processes that are field-adaptable, simple, and can rapidly provide results. There are many types of equipment such as the milk checker, Fossomatic meter (Hillerod, Denmark), Dramiski mastitis detector/Wykrywacz mastitis detector (Olsztyn, Poland), DeLaval cell counter (Tumba, Sweden), Afimilk mastitis detector (Kibbutz Afikim, Izrael), UdderCheck(r) test (Moorestown, USA), and PortaSCC(r) test (Moorestown, USA). They rely on either detecting physicochemical-biological alterations in milk or the udder or assessing biomarkers in body fluids (milk, serum) linked with mastitis . It has been demonstrated that the diagnostic capacity of infrared thermography (IRT) is comparable to that of the California mastitis test, and it also distinguishes instances of clinical mastitis from those of subclinical mastitis. As a result, IRT has the potential to develop into a diagnostic tool that is both convenient and portable . 3.1.2. Milk Progesterone Fertility control needs close coordination between farmers and veterinarians, systematic examination of farm records, and reliable clinical data. Furthermore, low fertility might be considered a sign of poor health and well-being . Breeding is an essential element of cattle husbandry. Detecting the ovulation phase in cattle is crucial for determining the best window for artificial insemination . The presence of progesterone implies the existence of a functioning corpus luteum. As a result, it has been utilized for decades as a biomarker of reproductive efficiency. Milk progesterone is a potential non-invasive indicator for reproductive status in dairy cows since progesterone is transported from blood to milk . Therefore, progesterone sensors could be useful sensor systems, despite the fact that little research has been carried out on the performance of such systems . The Herd Navigation(r) system (Tumba, Sweden), which integrates five sensing systems, including progesterone in milk, was developed for commercial usage in 2008. It detects progesterone levels in milk and recommends insemination times, animals for final pregnancy confirmation, early termination, and cows at risk for cysts and extended anestrus. In Denmark, estrus detection rates of 95-97% have been recorded, with much higher pregnancy rates (up to 42-50%) than traditional approaches . Endocrine regulation is crucial for optimal fertilization throughout the follicular period. Because progesterone production is strongly related to embryonic development from the early stages of pregnancy, adequate monitoring of both pre-ovulatory decline and post-insemination elevation in milk progesterone can be used to detect animals with reduced fertility. Reduced milk progesterone concentrations, for example, around days 4-7 following insemination are related to low fertility and an increased chance of embryonic loss . According to various studies, pregnant cows had higher quantities of milk progesterone in their milk samples for the first week following insemination . Infrared spectroscopy is a fast, inexpensive, and user-friendly technology that can be used for research as well as online and offline milk analyses . Near-infrared spectroscopy (NIRS) has long been used to measure the amount of milk components such as fat, protein, and lactose . Numerous studies demonstrate that a near-infrared spectroscopy approach is a viable method for assessing individual milk progesterone levels. In fact, monitoring progesterone levels in milk is an efficient and cost-effective method for determining a cow's reproductive status, detecting heat, and diagnosing pregnancy. Cows are milked twice or three times per day under standard dairy practices, implying that milk samples provide information on the current health of the herd/individual and may be collected and tested on a frequent basis without severely compromising the animal's daily living . In addition, it has been demonstrated that the physiological status of the animal affects the molecular structure of the water in milk; therefore, milk spectra can provide useful information regarding animal health and sickness . 3.2. Breath, Sweat and Saliva Analysis Researchers have long been interested in disease detection by the identification of volatile organic compounds (VOCs), representing a non-invasive technique. Animals and humans both exhale and excrete VOCs in their secretions with breath, blood, feces, skin, urine, and vaginal discharges . Gases such as hydrogen (H2) and methane (CH4), as well as volatile organic molecules such as fatty acids, which can serve as biomarkers for metabolic and pathologic processes, are examples of metabolites found in the breath. VOCs such as ketone bodies, ethanol, methanol, and exogenous compounds are commonly associated with blood glucose levels . VOC analysis has been used to study bovine respiratory sickness, brucellosis, bovine tuberculosis, Johne's disease, ketoacidosis, and normal rumen physiology in cattle . The majority of sweat metabolite analysis biosensors were created with the intention of monitoring human health. These have been put to use to measure lactate levels and salt concentrations, and they have also been made portable (in belt form) to measure sweat . Additionally, this sensor may be modified for use in measuring animal perspiration, particularly as an indicator of physical stress in animals . Saliva collection for disease and other biochemical markers of physiological health is an appealing non-invasive alternative to blood sampling . Because saliva contains both local and systemic components, it is a useful source of information regarding systemic processes occurring in the body, allowing for the evaluation of the organism's physiological or pathological status. However, the use of saliva in the diagnosis of animal illnesses necessitates a detailed examination of its protein composition under various situations . The technique is especially helpful for monitoring animals and diagnosing diseases because drawing blood from animals is thought to be a stressor and can affect the biochemical parameters being measured. Salivary biomarkers can be useful for several purposes, including disease early detection and diagnosis, decision support for managing animals, and disease progression monitoring . There are other diagnostic applications for saliva, such as biomarkers from saliva being investigated for the detection of oral cancer . According Mojsym et al., saliva can be a valuable diagnostic sample comprising possible indications of physiological and pathological conditions such as pregnancy status of cattle. Moreover, it can be useful for developing rapid tests from saliva . In buffalo, there are attempts to determine estrous time using saliva biomarkers for more precise insemination planning . Saliva analytes were explored for their relationships with lameness in another investigation. It was also anticipated that cows can change certain particles in their saliva, and some of these may reflect improvements in lameness after therapy . Table 1 shows a summary of milk and other body fluid analysis and its benefits. 3.3. Wearable Devices for Animals Portable electronic monitoring devices have the potential to transform intensive, large-scale dairy production by monitoring and managing cows on an individual basis. There has been a notable increase in the number of published research looking into the application of wearable electronic monitoring devices for use in commercial farming environments since the early 2000s . 3.3.1. Head/Muzzle and Noseband Sensors Three distinct kinds of biosensors can be employed to recognize the jaw movements that characterize cattle grazing behavior. These are electromyography sensors, mechanical/pressure sensors, and acoustic sensors . Cattle grazing behavior necessitates close observation of each cow based on three crucial factors: the cow's position, an analysis of its posture, and the cow's motions, particularly its gait and jaw movement . The amount of time an animal spends with its head in a downward posture is added to the sensor's recorded data to calculate grazing time . For instance, continuous observation of jaw motions can reveal information about diurnal grazing habits, animal health disorders, and forage deficiencies . Animals adjust their behavior in response to stresses, social changes, and environmental changes, and these can cause illnesses. Because of the labor necessary for the continuous monitoring of big groups of animals, tracking this behavior on a large scale becomes impossible. When combined with proper output interpretations, wearable sensor technologies enable the simultaneous measurement of real-time physiological parameters in a herd on a large scale. As a result, wearable sensor technologies offer an advantage over traditional herd-based systems since data from wearable sensors can be evaluated instantly, allowing for a short reaction time . For example, RumiWatch (RWS; ITIN + HOCH GmbH, Liestal, Switzerland), a noseband sensor that monitors feeding and rumination activity in dairy cows, was designed and tested as an effective scientific monitoring device for automated measurements of rumination behavior and activities. The correlations between direct observations and sensor readings reveal that the RumiWatch noseband sensor was effectively designed and validated as a scientific monitoring device for the automated detection of rumination and eating behaviors in stable-fed dairy cows . Other examples of commercial sensors and their detected analytes are presented in Table 2. According to the findings of the study, the rumination and feeding activity monitoring system is an effective tool for predicting calving time under farm conditions. It was found that in primiparous and multiparous cows, lying bouts increased but rumination chews declined while predicting calving. Logistic regression and ROC analysis were used to assess the sensitivity (Se) and specificity (Sp) for predicting the commencement of calving within 3 h (Se = 88.9%, 85% and Sp = 93.3%, 74% for multiparous and primiparous cows, respectively) . 3.3.2. Motion, Movement, and Behavior Sensors Activity can be reduced in cattle afflicted with lameness or diseases such as bovine respiratory diseases (BRD). Energy conservation for immune system metabolic costs and indirect effects of fever and inflammatory responses to infection were proposed as biological underpinnings for reduced activity in ill animals . There are plenty of technologies for motion, movement, and behavior analysis, such as accelerometer, pedometers, and global positioning system (GPS). Accelerometers have been used in dairy farming systems for the detection of diseases such as mastitis and to detect estrus and locomotion problems . Accelerometer reading changes can also be used to generate a benchmark level of activity, which can subsequently be recorded as calculated step counts or other movement indices such as activity ratios. Accelerometers have acquired popularity in beef cattle research because they allow for the continuous and long-term study of an animal's mobility and behavior . Moreover, ear-mounted sensors accurately identified grazing, standing, and walking in sheep with 94%, 95%, and 99% accuracy, respectively . Commercially available accelerometer devices include the IceTag and IceQube products manufactured by IceRobotics, Ltd. (Edinburgh, Scotland, UK), designed and validated for use in cattle. Other commercial accelerometer products designed for use in cattle include CowScout (GEA Group, Dusseldorf, Germany), SCR (Allflex, Madison, WI, USA), Pedometer Plus (Madero Dairy Systems, Houston, TX, USA), GYUHO SaaS (Fujitsu, Fukuoka, Japan), and GP1 SENSR (Reference LLC, Elkader, IA, USA). Another accelerometer device that has been successfully used to quantify cattle behavior, the HOBO Pendant G (Onset Computer Corp., Bourne, MA, USA), requires the user to build a method of leg attachment, and data management is more complicated . When compared to the accuracy reached with these devices (more than 90%), the accuracy of visual human observation is significantly lower . Understanding how grazing animals migrate throughout pastures and what they do in each region is essential for developing management plans that will maximize the potential productivity of grazing systems and limit their negative impacts on the environment (nutrient losses to water and gaseous emissions). Using real-time global positioning system (GPS) tracking and biologging technologies, it is possible to perform remote monitoring of animals to look for any indications of illness or concerns regarding their well-being . Animals on livestock farms can be monitored for their activity levels, which can provide useful information about the animals' overall health and degree of care . The global positioning system, radio tracking, and wireless local area network are now the most important technologies for monitoring livestock in the field; however, there are a few more tools (such as Bluetooth and ultrasound) that can be employed for indoor monitoring . GPS collars equipped with activity sensors enable the distinction between foraging sites and those used for other activities such as sleeping or travelling . Diverse studies conducted over the past decade have proved the utility of GPS telemetry devices for analyzing the behavior of cattle when combined with other devices/sensors. Combining GPS collars with activity sensors results in an effective method for tracking the whereabouts of grazing animals and determining animal behavior simultaneously. Real-time location systems have been created to pinpoint the position of an object within a particular area . Although little research has been conducted on the behavior and movement of lame dairy cows on pasture, GPS technology may be useful in enhancing pasture-based systems' automatic lameness diagnosis. According to Riaboff et al., severely lame cows spend 4.5 times less time grazing and nearly twice as much time resting in the laying position than their sound counterparts . Additionally, GPS is used to forecast the behavior of ruminants and locate their location in pastures. This geolocation technique appears promising for identifying animals with a high frequency and a low mistake rate, despite the GPS's inadequacy for forecasting behavior in a robust manner. Geolocated behaviors are especially intriguing for investigating changes in behavior connected to demanding events. The usage of data from accelerometers and GPS devices together is an approach that could prove to be both intriguing and beneficial in the process of researching how cows interact with their surroundings. These challenging scenarios can include heat stress, physical stress, resource depletion, restricted access to pasture, and other similar situations . These characteristics could be used to improve the efficacy of existing lameness detection sensors in pasture-based systems . Table 3 gives a brief summary of wearable sensors. There are GPS systems that allow users to track and confine animals. Special attention has been paid to a solar-powered GPS collar-based virtual fence system (NoFence, Beatnfjordsra, Norway). The GPS position data collected by the collar are shown on an app that the farmer uses to create a grazing border map for cattle, sheep, or goats. When the animal gets close to the virtual boundary, the collar will begin to emit warning audio stimuli at a volume of forty decibels and a rising frequency of two to four thousand hertz. This will give the animal the time to change course to avoid such stimuli. When an animal passes the virtual barrier, it is considered to have "escaped", and the auditory and the electric shock are turned off. Additionally, a push notification is delivered to the farmer's mobile app when the animal has crossed the boundary . GPS also can be promising in heat detection. Sheep estrus can be detected with global navigation satellite systems (GNSS) by monitoring a surge in activity levels followed by a return to "normal" patterns of behavior . Pedometer In animals, a pedometer (step counter) is a proven recording method for determining movement activity. Previously, it was mostly utilized for estrus detection . Pedometers objectively measure an animal's total number of steps and total distance traveled using an algorithm that calculates the steps. While pedometers are relatively simple to deploy and operate, the number of steps taken by each ruminant varies significantly depending on the day and ambient conditions. There could be a link between cattle distance traveled and stressful and unpleasant procedures; one study found that calves took fewer steps for four days after castration, while another found a link between calves' stress and the number of steps they took after castration . Other observations are made while recording active or lying behavior. The method is useful for the early identification of lameness in dairy herds. In one investigation, pedometers were used to detect lame calves before clinical indications appeared. Individual cows were examined and monitored, and it was shown that 92% of the cows acquired obvious lameness. When cattle were monitored using a pedometer, their hoof activity was reduced by at least 15% several days before the start of clinical lameness. The researchers concluded that pedometers are a beneficial tool for the early detection and treatment of the vast majority of cases of developing lameness . 3.4. Other Analyzers: BCS Camera, Infrared Thermography, Sensors of Bolus 3.4.1. Infrared Thermography Infrared thermography (IRT) is method for diagnosing and assessing pain since it indicates physiological changes . Thermography can be used to identify and determine thermal abnormalities in animals by identifying a rise or decrease in the surface temperature of the skin . IRT is a technique that is frequently utilized in the field of veterinary medicine to diagnose conditions such as infection, lameness in horses, and mastitis in both sheep and cattle, and to determine scrotal temperature in buffalo . IR thermography is a noninvasive method that monitors emitted infrared radiation and displays the data as a thermogram, which is a visual representation of an object's surface temperature. Each pixel in the thermogram represents the recorded surface temperature of an object. The data can be displayed in grayscale or color. The warmest areas are displayed in white or red on a color scale, while the coldest areas are shown in black or blue. When an animal is stressed, the hypothalamic-pituitary-adrenocortical axis is engaged, and heat is created as a result of increased catecholamine and cortisol concentrations, as well as blood blow reactions, resulting in changes in heat generation and heat loss from the animal. As a result, this technique may be beneficial as a general stress indicator . Changes in surface temperature patterns, particularly those generated by changes in blood flow, can be utilized to detect inflammation or damage associated with illnesses such as foot lesions. Thermal (color) variations represent thermal gradients, which represent changes in skin temperature induced by underlying illnesses. Thermography has the advantage of measuring heat emissions without having direct physical contact with the surface. Because of its high sensitivity, it is useful when paired with other exact data (such as pedometer, accelerometer activity). In general, thermography works best in concert with other modalities rather than as a replacement for them. Thermography frequently detects physiological changes before they emerge as clinical symptoms, allowing for early diagnosis and treatment . According research, there was no statistically significant difference in foot temperature amongst different illnesses, but there was a difference between sick and healthy cows . To reduce economic losses caused by lameness, preventive interventions and early diagnosis of lesions are required . The use of infrared thermography to detect lameness in cattle has increased in recent years, owing to its non-invasive qualities, ease of automation, and continued cost savings . Lameness in dairy cows has negative effects not only on dairy cow welfare and milk production, but also on reproductive ability and death rates . Early diagnosis of a foot lesion can help reduce the negative impact of lameness and boost treatment success, and is likely to be useful in preventing future pathological development . Thermography can also be employed in research of various diseases such as musculoskeletal affections and ocular temperature assessment in calves for early illness diagnosis. This instrument has also been used to identify mammary gland inflammation . Infrared thermography has been shown to be useful in assessing udder health and detecting quarters with subclinical mastitis . Mastitis raises the warmth of the udder before clinical symptoms appear. Furthermore, after inoculating lactating cows with Escherichia coli in various locations of the udder, Pezeshki et al. noticed a 2-3 degC rise in udder surface temperature . Berry et al. demonstrated that thermography of the udder can be a useful diagnostic tool for detecting mastitis in dairy cattle. Because the temperature rises considerably three days before ovulation, using thermography to identify cow estrus can increase pregnancy chances in regular or quiet estrus . An Australian study involving Holstein cattle found that a fall in vulva and muzzle temperature 48 h before ovulation correlates to corpus luteum regression, and an increase in temperature 24 h before ovulation corresponds to the period of estrus . Thermography could also be used to detect estrus in sheep and goats . Zaninelli et al. investigated the potential of IRT in the diagnosis of mastitis in their 2018 study and discovered that it corresponds well with the somatic cell count . Thermography has been demonstrated to distinguish between clinical and subclinical mastitis in both large and small ruminants, with diagnostic sensitivity and specificity comparable to the California mastitis test (CMT). As a result, with further adjustments and advancements, farmer-friendly and non-invasive infrared thermography has the potential to become a useful and practical instrument for use on farms in the future. Individual determinations for each mammary compartment can be made, with an increase in local temperature suggesting the presence of inflammation, if it exists . In cases of stress, fertility, welfare, metabolism, health, and illness detection, the animal's surface temperature can be used as an indicator trait to accurately measure an animal's physiological state . Thermal infrared sensors have shown that there is a strong link between changes in the temperature around the eyes and changes in the temperature of the body's core. Temperature readings from different parts of an animal's body give information about its health and allow for quick decisions about its welfare (e.g., isolating animals with higher body temperature or managing the internal temperatures of animal housing units) . Salles et al.'s study shows that among the body regions tested, IRT frontal temperature had the strongest correlation with rectal temperature, while the temperature-humidity index is substantially related with forehead and right and left flank temperature . Another investigation found out that setting the illness threshold for filthy feet at 27 degC correctly detected 80% of feet with lesions and 73% of feet without lesions . Although this method is sensitive in identifying changes in the temperature patterns of animals, it may not be adequate to pinpoint their causes . As the livestock industry's reliance on automated systems grows, technologies that can be implemented into these systems to monitor animal health and welfare must be developed. Infrared thermography (IRT) is one such technology that has been utilized for monitoring animal health and welfare and has the potential to be incorporated into automated agricultural systems through automation . 3.4.2. Bolus Sensors Bolus sensors are primarily intended to detect variations in ruminal temperature, which can indicate a change in animal physiological states . Ruminal temperature decreases in response to drinking and eating events, and it rises in response to increased body temperature. Monitoring changes in ruminal temperature and activity can help spot anomalous behavior, the estrous cycle, and infections early . Metabolic illnesses (rumen acidosis, hypocalcemia) and other diseases that cause fever and pain have an effect on the amplitude and frequency of ruminal contractions in cattle . Wireless intraruminal bolus sensors have been devised to monitor the temperature and pH levels of the rumen and reticulum via the esophagus. The availability of boluses for measuring reticuloruminal pH for purchase has simplified the evaluation process greatly. These boluses wirelessly transfer pH information to a central processing location on a regular basis, simplifying the evaluation procedure. An example of bolus sensors can be seen in Table 2. The pH level of the ruminal fluid is one of the most straightforward indicators that ruminal acidosis is present. The ruminal pH can be measured in real time by wireless pH probes that are put in boluses within the rumen. In addition to the pH of the rumen, various markers for either ruminal acidosis or subacute ruminal acidosis can be determined in blood, urine, feces, or milk. These can be used to diagnose either condition . Other studies, while using bolus sensors, have shown that cows with higher rumen pH (6.22-6.42) can emit 46.18% more methane than cows with lower ruminal pH . Cantor et al.'s study revealed that reticulorumen temperature is an accurate predictor of well-being factors in cows such as daily herd water intake and inflammation . 3.4.3. Body Condition Score Body condition score (BCS) is an evaluation of the cow's fat stores and indicates the cow's overall energy balance. The amount of fat mobilization in cows with a higher BCS is higher . There is a substantial correlation between body condition score (BCS) and many reproductive and lactational performances in small ruminants, making BCS an important indication of their well-being . BCS at calving and its fluctuations during lactation have been found to have an impact on the health and fertility of high-yielding dairy cows. When a cow's condition deviates from acceptable BCS standards, the occurrence of metabolic disorders, infertility, and lameness increases . Having a high or low BCS can have unfavorable effects on milk production, illness, and reproduction . However, because of its subjectivity and slowness, manual BCS evaluation is rarely used outside of experimental settings or large farms with many ruminants . The BCS scale typically consists of a five-point scale with increments of either 0.25 or 0.5 points . Reduced early lactation dry matter intake and milk production, as well as an increased risk of metabolic problems, are all connected with a calving BCS of 3.5 or below on a five-point scale. On a scale from 0 to 5, a BCS of 3.0 to 3.25 is considered ideal for calving. When it comes to production and reproduction, lower calving BCS is connected with lower rates, while greater calving BCS is associated with an increased risk of metabolic diseases . Consequently, measuring and regulating the body condition and obtaining optimal BCS at various phases of lactation are crucial for maintaining or enhancing the performance and welfare of cattle, especially dairy cows . Anatomical points from the rear end of the dairy cow (such as the hooks and tail-head area), which are located in an area of the cow where changes in subcutaneous body reserves are visually more evident, are visually evaluated in order to assign a score according to a standardized scale in the process of visual body condition scoring . In the past decade, numerous proposals for agricultural three-dimensional (3D) vision systems that are based on optical two-dimensional (2-D) and 3D sensors were developed . Recent years have seen an increase in the use of 3D sensors in the applications of BCS. These sensors bring more information about the body's surface than 2D-based or thermal image-based systems . Three-dimensional cameras have recently advanced in technology and could provide novel feed management solutions for dairy farms. Portable ASUS Xtion Pro sensors (ASUSTeK Computer Inc.) can be used to capture 3D photos of cows' rear ends . There is also a commercially available automatic BCS camera for use on dairy cows' heads (DeLaval Body Condition Scoring, BCS DeLaval International AB, Tumba, Sweden) . This camera system uses a radio frequency identification reader to individually identify cows that have been fitted with transponders. It also enables multiple BCS measurements to be taken within a single day. The camera software records individual daily BCS values for each scoring session and reports a daily trimmed, 7-day rolling average of BCS . The BCS camera system produces accurate BCS ratings between 3.00 and 3.75; however, the magnitude of low and high BCS scores is typically miscalculated. Time of flight (ToF) cameras are quickly becoming one of the most common types of sensors used to acquire 3D data. ToF systems use either visible or near-infrared (NIR) light, and the reflected light is received by smart pixel sensors, which then measure the amount of time it takes for the light to return. It was demonstrated that the characteristics extracted from the dorsal and posterior sections achieved 100% accuracy of the projected BCS within a 0.5 point deviation of the actual BCS) . Studies have shown that BCS can not only be a metabolic disease marker, but can also be associated with production, health, and reproduction. Antanaitis et al. found that BCS is associated with pregnancy success because the BCS (+0.29 score) and mP4 (10.93 ng/mL) of the pregnant cows were higher compared to the group of non-pregnant cows . 3.4.4. Animal Surveillance through Video and Imaging Livestock illness prediction and abnormal behavior management both benefit greatly from automated tracking systems. Animals can be monitored in a number of ways, some of which are manual, some of which include wearable devices, and some of which involve computerized tracking systems. However, relying on human observers to keep tabs on animals is labor-intensive and not always reliable. The level of intelligent livestock management can be greatly improved with the help of computer vision technology, which provides a non-contact and low-cost technique to track cattle. Researchers have put much time and effort into determining how to achieve the level of autonomous monitoring of animals shown in the video. Face recognition is another emerging technology that is sweeping the globe. One study compares three loss functions used in human face recognition paired with RetinaFace-mobilenet, and the results suggest that the proposed CattleFaceNet beats others with an identification accuracy of 91.3% and a processing time of 24 frames per second (FPS) . Li et al. suggested a method for recognizing individual dairy cows based on an image of the animal's tail and head. This approach retrieved form characteristics from the tail head image's region of interest using Zernike moments and categorized them using four classifiers . Gaber et al. proposed a muzzle-based livestock recognition strategy that employed the Weber local descriptor and the Adaboost classifier for each head photo to identify the head of cattle . In other studies to evaluate cow lameness, color edge detection and bilateral projection were utilized to detect the region of the cow's knees. For cow identification and recognition, these approaches rely on color and contour characteristics. However, the strong reliance on hand-crafted characteristics precludes these systems from being applied to complicated settings. Deep learning approaches and Convolutional Neural Network models (e.g., Fast R-CNN, Faster R-CNN, YOLO, SSD, Mask R-CNN, AlexNet, VGGNet, and ResNet) have recently achieved substantial advancements in image identification and recognition through end-to-end feature learning . With the release of a new large-scale cow dataset with about 50,000 annotated cow face detection data points and approximately 18,000 cow recognition data points, cow face detection and recognition have been improved. There is also a framework for cow face recognition that combines the detection and recognition models to improve recognition performance. The experimental results show that the proposed technique is superior. The detection accuracy is 98.3%, while the accuracy of cow facial recognition is up to 94.1% . A sheep study showed results suggesting that the method proposed in that study outperforms the others, with a recognition precision of 89.12%, and it was discovered that incorporating the biometrics of the sheep face can significantly boost the network's recognition capacity . In Kumar et al.'s study, experimental findings on a muzzle point picture database were described. Their solution achieved 93.87% identification accuracy, demonstrating its superiority over other existing machine-learning-based recognition systems . Su et al. conducted a study with the Dairy Goat Dataset (DG-dataset) containing 200 dairy goat motion videos with a total of 161,000 frames of images randomly collected from the farm. The study shows that their algorithm was successful in locating and tracking a single dairy goat . Video tracking of animals helps to recognize ill animals or strange behavior in animals without human contact. 3.4.5. Electronic Nose for Estrus Detection The MENT-EGAS prototype used electronic nose (EN) technology (ten unspecified chemical metal-oxide sensors) to detect estrus by direct sampling of odor from the perineal headspace. In cycling cows, principal component analysis (PCA) revealed effective discrimination between proestrus and estrus, as well as estrus and metestrus. Based on these findings, it was demonstrated for the first time that direct sampling of the perineal headspace using an EN device during milking may correctly detect estrus in dairy cattle . In this study, the MENT-EGAS prototype (Patent No. WO2010099800A2) provided by AIRSENSE ANALYTICS GmbH (Schwerin, Germany) was used to detect emanated odor changes from the perineal headspace of the cows. Another study described an effective electronic nose system created using polyaniline-based sensors doped with various acids for determining estrus in female cattle. Disposable swabs for material gathered from the perigenital area and vagina of the animals were employed in the suggested olfactory technology, eliminating rectal manipulation, as well as disposable sensors, providing simplicity of handling and health safety for the estrus determination procedure. The results demonstrated that the olfactory system reliably detected estrus, optimal AI moment (12 h after estrus detection), and diestrus (corpus luteum phase) in cows, and that this information could be utilized to efficiently suggest the ideal AI time in calves . Thermography and its measured analytes and other electronic devices are reviewed on Table 4. 4. Innovations for Common Procedures At present, innovations are becoming inseparable from animal husbandry. Mastitis (both clinical and subclinical), metabolic diseases such as ketosis and acidosis, the reproductive state of the animal, and predicted calving now can be determined by various technologies. Two features of milk quality--the somatic cell count (SCC) and the appearance of obviously abnormal milk in cases of clinical mastitis--are used to detect mastitis in milk . Some technologies and their functions in disease and behavior diagnostics are shown in Table 5. The real-time detection of beta-hydroxybutyrate from blood or milk is used to determine the animals' energy balance and can be greatly aided by nano-biosensors. One of the metabolic illnesses, subclinical ketosis, raises the chance of developing clinical ketosis, lowers milk production, impairs reproductive ability, and has a negative energy balance during the transition phase, all of which have an adverse economic impact . 5. Conclusions and Future Directions The primary goal of precision livestock farming is to generate reliable data using biosensors and run it through intelligent software systems to create value for the farmer, the environment, and the animals in the form of improved animal health and welfare, increased productivity and yields, and lower costs while minimizing environmental impact. Sensor-based technology has made important contributions to lowering animal stress, improving animal well-being, and thereby eliminating economic losses. By forecasting future disease outbreaks, the early identification of physiological reactions can assist farmers in taking targeted interventions to reduce pressure on their animals, increase animal welfare, and avert performance losses. Future technological advancements will lead to the identification of biomarkers for specific health and welfare concerns at a far earlier stage. Precision livestock farming aims to create a management system based on automatic, continuous, real-time monitoring and control of all aspects of livestock management, including reproduction, animal health and welfare, and the environmental impact of livestock production. Monitoring animal behavior without upsetting the animal is another component of management. Farmers can employ wearable sensors to detect illness early, reducing animal deaths. Farmers and veterinarians can also destroy sick animals before they spread disease to the entire herd of cattle, if they plan ahead. Through satellites and smartphones, the incorporation of revolutionary diagnostic and disease detection systems utilizing biosensors would keep cattle and the agricultural business one step ahead of unseen diseases. When biosensors are used to detect diseases early, the epidemiological curve can be moved to the left because quick action can be taken to stop the spread of the disease and its negative effects on production, society, and the economy. An early warning system for more effective livestock health management can be created by shortening the amount of time it takes to receive findings when diagnosing infectious disease biomarkers on a farm in a manner that is in real time. Author Contributions K.D., D.B. and R.A. wrote the paper; R.A. edited the paper. All authors have read and agreed to the published version of the manuscript. Institutional Review Board Statement Not applicable. Informed Consent Statement Not applicable. Data Availability Statement Not applicable. Conflicts of Interest The authors declare no conflict of interest. Figure 1 Innovative technologies applied to cows. animals-13-00780-t001_Table 1 Table 1 Body fluid analysis and its benefits. Technology Benefits of Use Reference Milk progesterone Milk progesterone is a potential non-invasive indicator of reproductive status in dairy cows Somatic cell count SCC has proven to be a useful, non-invasive indicator of subclinical mastitis Breath, Sweat and Saliva analysis Biomarkers for metabolic and pathologic processes are examples of metabolites found in the breath. VOCs such as ketone bodies, ethanol, methanol, and exogenous compounds are commonly associated with blood glucose levels. Saliva collection is a non-invasive alternative to blood sampling animals-13-00780-t002_Table 2 Table 2 Information about some of the commercial sensors. Sensor Method Detected Analytes Reference RumiWatch (Itin + Hoch GmbH, Liestal, Switzerland) The RW system comes with software for controlling the sensor (RW Manager) and studying unprocessed data (RW Converter). The RW sensors, which include a noseband pressure sensor, a three-axis accelerometer to track three-dimensional head motions, and a data logger, are built into a halter that fits the head of each particular animal. The noseband pressure sensor, which is mounted in a belt on the animal's nose bridge, is connected to a tube filled with propylene glycol to detect jaw movements. As the animal moves its jaw, pressure within the tube varies, and this information is recorded with a 10 Hz resolution. Approximately 100 days of raw data logging were covered by the battery life. Different pressure signatures of jaw motions, which are then detected and categorized into prehension bites, mastication chews, and rumination chews Ear tag-based accelerometer system (Smartbow GmbH, Weibern, Austria) The ear tag has an acceleration sensor, a radio chip, and a temperature sensor for calibration and it can monitor rumination and detect estrus and localization. Rumination, estrus, and current localization MoonSyst (Moonsyst International Ltd.: P.O. Box 1329, Kinsale, Co., Cork, Republic of Ireland) System captures rumen data in real time. The bolus is meant to be readily ingested and will remain in the rumen (particularly the reticulum) throughout the animal's life. System sends data from the animal to specialized cloud-based servers via a communication gateway. Farmers may use the Mooncloud software application to view information from anywhere, anytime. The bolus can be used on animals weighing more than 350 kg. Once implanted, the bolus interacts with a gateway over a large geographical region. Heats, monitor health conditions, activity, rumen temperature and movement SmaXtec (SmaXtec animal care GmbH, Graz, Austria) The rumen bolus accurately monitors direct, informative values inside cows' reticulum. The boluses are given once and require no further maintenance. The data from the boluses are read out by the readout devices with an integrated Internet connection and promptly transferred to the cloud. The pH and temperature variation data are gathered with an analogue-to-digital converter (A/D converter) and stored in an external memory chip. This indwelling system may be simply orally supplied to an adult cow due to its dimensions (length: 12 cm, width: 3.5 cm, weight: 210 g), and its particular construction makes it shock-proof and resistant to rumen fluid. pH, ruminal temperature, cow activity, drinking, eating, rumen behavior Body Condition Score Camera (DeLaval, International AB, Tumba, Sweden) Body condition score system is based on a 3D camera that records certain areas of the animal: from above, the rear part of the back from the short ribs to the tail end. When a cow moves in front of the camera, the system recognizes the movement and records photographs of the cow; it then selects the best image of the cow from the video clip. The 3D camera employs light coding technology to project a pattern of infrared ray dots on the cow's back. Following that, the distances between these specific dots are measured; the company claims that a 3D picture of the back is created, and an algorithm translates the image information into a body condition score. BCS CattleEye (Cattle Eye Ltd., Belfast, UK) Camera is above the exit gate of a milking parlor. It records video of each cow as it exits the milking parlor. If a sort of gate or RFID system provides ID information, use it. Artificial intelligence systems in the cloud analyze video to uniquely identify the cows and track their wellness, among other things. System allows tracking the health and performance of cows in real time. It includes a dashboard that monitors and visualizes a variety of vital indicators at the herd and cow levels. Cow identification Cainthus ((c) 2022 Ever.Ag, Frisco, TX, USA) Smart camera system that monitors animal behavior and farm activities 24 h a day, seven days a week, 365 days a year. It is artificial intelligence that converts visual input from cameras into real-time insights. These insights are provided daily on any farm device, phone, tablet, or computer. The information provided is accurate and unbiased. This technique is easily scalable, does not require any hardware on the cows, and requires extremely minimal maintenance in comparison to other solutions. Animal behavior BROLIS Herdline (Vilnius, Lithuania) The analyzer examines the composition of each cow's milk during each milking. This "mini-spectroscope" is installed in the milking stalls or milking robot in the milk line and does not use additional reagents and does not require special maintenance. The analysis of protein, fat, lactose, and electrical conductivity provides a proper evaluation of the health, productivity, and economic efficiency of dairy cattle. The data collected during milking are processed in real time and can be viewed using the BROLIS HerdLine application. Milk fat, protein, lactose, milk electrical conductivity HeatWatch (HeatWatch(r) DDx, Inc., Denver, CO, USA) A tiny radionic transmitter is linked to a pressure sensor in a stiff plastic box implanted in a nylon packaging that is glued to the cow's tail hair in the sacral region. The device is activated by the weight of the mounting animal for a minimum of 2 seconds, after which the transmitter sends the breeding approval signal to the system along with the animal's identification. In general, this device's assessed performance ranges from 37% to 94%. Heat detection animals-13-00780-t003_Table 3 Table 3 Wearable sensors and their benefits. Technology Benefits of Use Reference Head/muzzle and noseband sensors Noseband sensor was designed and validated as a scientific monitoring device for the automated detection of rumination and eating behaviors. It can be executed without contact with the animal. Motion, movement, and behavior sensors Accelerometers, pedometers, and GPS tracking all can be used to monitor animal behavior. Active time can predict heat; prolonged laying time can signal diseases such as mastitis, ketosis, and lameness. GPS helps to locate animals on the farm. animals-13-00780-t004_Table 4 Table 4 Other sensors and their benefits. Technology Benefits of Use Reference Infrared Thermography Determine thermal abnormalities in animals by identifying a rise or fall in the surface temperature of skin. Infrared thermography is a noninvasive method that monitors infrared radiation emitted from the body. Inflammation, stress, calving, and heat can be evaluated. Thermography can detect physiological changes before they emerge as clinical symptoms. Bolus Sensors Wireless intraruminal boluses without constant contact, can measure and analyze ruminal and eating behavior, examine ruminal pH. Body Condition Score Cameras Tracking BCS can help reduce postpartum disease percent; it helps to notice obese or poor health animals. When it comes to production and reproduction, lower calving BCS is connected with lower rates, while greater calving BCS is associated with an increased risk of metabolic diseases Cattle Face Recognition Face analysis can help to identify pain, unwell animals, locate, identify, and select animals on the farm. Electronic Nose for Estrus Detection Can detect estrus by direct sampling of odor from the perineal headspace. animals-13-00780-t005_Table 5 Table 5 Technologies for animal status determination. Disease/Status of Cow Technology for Diagnosis Analytes Reference Mastitis Image processing, spectroscopy, electrical conductivity, biosensors, SCC sensors, tri-axial accelerometers, pedometers, spectroscopy Temperature, lying behavior, eating behavior, milk analytes (fat, protein, electrical conductivity), rumination time, somatic cell count (SCC), milk pH, milk yield Metritis/Endometritis Tri-axial accelerometer, electronic feeding system Eating, drinking time, rumination, activity, laying time, Ketosis 3D cameras, spectroscopy, milking robots, accelerometers body condition score, BHB, milk analytes (fat, protein), milk yield, activity, rumination behavior Acidosis Three-axis accelerometers, angular velocity sensors, pH meter, milking robots Milk yield, milk analytes (fat, protein) activity, rumination behavior, walking behavior, feeding behavior Lameness Tri-axial accelerometers, pedometers, video observations, accelerometers, rumination sensor Walking behavior, feeding behavior, rumination, activity, and laying time have been linked to lameness Heat Tri-axial accelerometers, pedometers, video observations, accelerometers, spectroscopy, chemical analysis, electronic nose, acoustic sensors Activity, milk analytes (progesterone), odor from the perineal headspace, pression, friction, rumen movement Pregnancy Milking robots, radioimmunoassay, enzyme immunoassay, accelerometers, pedometers Milk progesterone, activity, temperature Calving, dystocia Intravaginal thermometer, tri-axial accelerometers, pedometers, video observations, accelerometers, rumination sensor, infrared thermometry (IRT imaging) Body temperature, activity, rumination time, laying time Disclaimer/Publisher's Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

PMC10000157

The dust level is included in the animal welfare legislation of the European Union, implying assessment of dust levels during veterinary welfare inspections. This study aimed to develop a valid and feasible method for measuring dust levels in poultry barns. Dust levels were assessed in 11 layer barns using six methods: light scattering measurement, the dust sheet test with durations of 1 h and 2-3 h, respectively, visibility assessment, deposition assessment, and a tape test. As a reference, gravimetric measurements were obtained - a method known to be accurate but unsuitable for veterinary inspection. The dust sheet test 2-3 h showed the highest correlation with the reference method with the data points scattered closely around the regression line and the slope being highly significant (p = 0.00003). In addition, the dust sheet test 2-3 h had the highest adjusted R2 (0.9192) and the lowest RMSE (0.3553), indicating a high capability of predicting the true concentration value of dust in layer barns. Thus, the dust sheet test with a test duration of 2-3 h is a valid method for assessing dust levels. A major challenge is the test duration as 2-3 h is longer than most veterinary inspections. Nevertheless, results showed that potentially, with some modifications to the scoring scale, the dust sheet test may be reduced to 1 h without losing validity. dust assessment dust sheet test laying hens validation EURCAW-Poultry-SFAEuropean CommissionSANTE/EURC/2021-2022/SI2.871763 This research was funded by the EURCAW-Poultry-SFA. European Commission Grant number: SANTE/EURC/2021-2022/SI2.871763. pmc1. Introduction Three terms are used to refer to what is commonly known as dust: aerosols, particulate matter (PM), and dust . The term aerosol refers to "airborne particles, in either solid or liquid state, that are usually stable in a gas for at least a few seconds". The term aerosol is synonymous with PM, whereas the term dust more specifically refers to 'solid particles formed by mechanical disintegration of a material'. The Council Directive 98/58/EC of 20 July 1998 concerning the protection of animals kept for farming purposes states that "dust levels [...] must be kept within limits which are not harmful to the animals" . Thus, in order to comply with animal welfare legislation, it is necessary to be able to measure dust levels in poultry houses. However, there is currently a gap of knowledge in regard to valid methods for measuring dust levels during poultry welfare inspections. Recently, at a workshop involving the competent authorities of the majority of the EU Member States, the participants were asked which requirements in the laying hen and broiler welfare legislation they thought were lacking a validated assessment method. The legal requirement on dust level came out in first place for both the laying hens and broilers . Follow-up interviews with official veterinarian inspectors confirmed that the methods currently available are considered insufficiently validated and/or insufficiently standardised to be reliable . In this respect, validity is the extent to which an indicator and its method of assessment is meaningful in terms of providing accurate information on a legal requirement concerning an animal or a group of animals, and reliability is the extent to which results are consistently the same between two or more observers or when the same observer repeats assessments after receiving reasonable training . Currently, the method used most frequently by inspectors is a sensorial assessment where the dust level is assessed based on visibility in the barn and the sensation of dust in the inspector's respiratory system . It may involve (1) a visual assessment of the dust level in the air of the poultry house, (2) an inspectors' sensation of irritation (assumed to be caused by dust) in their own eyes, throat, and lungs, and (3) signs of irritation of the birds' eyes, throat and lungs, assumed to be caused by dust. However, the method is considered to be of limited validity and reliability by the inspectors themselves as it relies on high levels of subjectivity . Most inspectors are also aware of the dust sheet test: a method described in the Welfare Quality Protocols for laying hens and broilers and tested in the present study. Thus, the test can be carried out in a standardised way, but the validity of the test has not previously been examined. Furthermore, the test duration is 3 h, which is considered a challenge as most veterinary inspections are of shorter duration. Dust levels may also be measured using dust measuring devices that mainly have two measuring principles: gravimetry and light scattering. When using a gravimetric device, the air to be assessed is drawn through an impactor or cyclone, and particles with a chosen diameter are collected on filters, which are weighed before and after loading to determine the mass of particles sampled . Gravimetry is the gold standard for dust measurement, and devices exploiting this principle are therefore typically used as reference methods . However, sampling dust by use of gravimetric devices is time-consuming, involving procedures both before and after the sampling. In contrast, light scattering devices determine the concentration of PM by directing the air stream to be sampled into a light scattering chamber. A beam of light is then directed onto the particles in the air, and a sensor measures the amount of light scattered by the particles . The device uses a relationship between the scattered light and the mass concentration of the particles, which is usually pre-set at the factory using a standard type of dust . Light scattering devices are portable, easy to use, and provide real-time data at a relatively low cost. The aim of the present study was to investigate the validity of existing and new methods of measuring dust levels, which are feasible for use during veterinary inspections of animal welfare. As a reference method, the dust level was collected by the use of a gravimetric measuring device. 2. Materials and Methods The study took place from May 2021 to August 2021 and involved 11 different layer barns located all over Denmark. The farms were selected from those suggested by the Danish egg companies and with the agreement of the farmers. 2.1. Animals Barns were visited when the birds were 22 (n = 4), 23 (n = 1), 32 (n = 2), 55 (n = 3), and 74 (n = 1) weeks old. All barns had a multitier housing system with two levels of slatted platforms as well as perches available for the hens. The floor was fully covered with litter, consisting of either wood pellets, straw pellets, or wood shavings. The number of hens per barn varied from 20,000 to 30,000, divided into four to five sections of 4000 to 7500 hens by wire mesh . Both ends of the barns contained corridors that were divided from the adjacent sections by wire mesh. The number of entrances from the corridors into the sections was 3 or 4 at each end of the barn. The stocking density was nine birds/m2 of usable floor area. The barns had forced ventilation systems and provided no access to a veranda or outdoor area. 2.2. Methodology In each barn, the dust level was assessed using six methods (explained in detail in the following sections): light scattering measurement, dust sheet test with a duration of 1 h, dust sheet test with a duration of 2-3 h, tape test, visibility assessment, and deposition assessment. These were compared to a reference method, i.e., gravimetric measurement. One observer collected all the data during the barn visits. The spots in the barns, where the different types of assessments took place, are indicated in Figure 1. 2.2.1. Gravimetric Measurement--The Reference Method As the reference method or gold standard, a gravimetric device was chosen: the Atmospheric Dust Captor device (Capteur Atmospherique de Poussiere, CAP 10; Arelco, Fontenay-Sous-Bois, France). Before each measurement, an unloaded filter was placed in a proofer at 40 degC for 12 h and then desiccated for 30 min. The filter was then weighed two times using a precise balance (A&D HR-120, Japan; readability: 0.0001 g). The average value was calculated as the filter weight. The device was cleaned and disinfected before each barn visit. The device was switched on as soon as entering the barn and placed on a tripod right at approximately 90 cm above floor level . The working period of the device was noted. This was determined by the duration of the barn visit and ranged between 2 and 3 h. Following sampling, a similar drying procedure as the one prior to the visit was performed before the loaded filter was weighed. The weight difference between the loaded and unloaded filter equaled the amount of collected dust. The dust concentration was calculated as the mass of collected dust divided by the operating time of the device. The total dust concentration was presented as milligrams per cubic metre of air. This concentration was considered the "true" dust concentration in the barn. 2.2.2. Light Scattering Measurement The concentration of PM10 was measured with a device using the light scattering principle (Dylos Corporation, DC1700-PM PM2.5 / PM10, Riverside, CA, USA). The result was given in milligrams per cubic metre of air, i.e., similar to the gravimetric measurement. Measurements were obtained approximately 50 cm above the floor and were carried out once in front of each entrance . The observer waited for a minute after arrival at the measuring spots before switching on the device to allow dust whirled up by the arrival of the observer to settle. The PM10 concentration was displayed 10 s after switching on the device. The device was cleaned and disinfected before each visit. However, the inside of the device cannot be fully cleaned, meaning that air passing through the inside of the device may potentially be contaminated by particles from previous farm visits. Therefore, it was chosen to tape a 100 L airtight plastic bag to the bottom opening of the device before each visit to avoid bringing in particles from previous farm visits. Once the measurements were completed, the device was placed in another airtight bag as once the device, and hence the fan inside is switched off, the air stored in the plastic bag taped to the bottom may escape through the top inlet. 2.2.3. Dust Sheet Tests The dust sheet tests were conducted as described in the Welfare Quality Protocol for laying hens . Two black A5-size papers were placed horizontally on the closed feed chains just outside each entrance . The papers were placed immediately after entering the barn. One paper for each spot was assessed and removed 1 h later ('dust sheet test 1 h'), whereas the second paper was left for 2 to 3 h ('dust sheet test 2-3 h'), depending on the duration of the farm visit. Finger strokes on the paper were used to assess the amount of dust accumulated on the paper while comparing it with clean paper. The scoring scale :Score 0 = No or minimal evidence of dust (sheet has same colour as clean sheet); Score 1 = Isolated specks or a thin layer of dust on sheet is detectable (without comparing with a clean sheet, the test sheet still appears black but there is a slight colour difference between the two sheets); Score 2 = Dust covers the sheet; even without comparing with a clean sheet, it is clear that the test sheet is no longer black (i.e., there is a clear difference in colour between clean and test sheets). 2.2.4. Tape Test To overcome the challenge of being back at a particular spot at an exact time point (as in the dust sheet test with 1 h duration), a tape test was developed using the principle of the dust sheet test. The upper and most horizontal part of the right shoulder of the assessor was covered with black duct tape (approx. 10 cm x 5 cm). The tape was placed when entering the barn, and the level of dust deposited on it was assessed 1 h after entering the barn. The cloth of the coverall was gently pulled away from the neck in order to be able to see the tape without having to pull it off the coverall. The scoring scale used for the dust sheet test was applied, and the tape on the shoulder was compared with a clean piece of tape. 2.2.5. Visibility Assessment The level of dust in the air was assessed in the visibility test where the observer stood approximately 20 m from the end wall within each area between the multi-tier systems, facing the wall . The scoring scale used was developed based on the information gained during interviews with veterinary inspectors describing how they performed assessments of dust levels in poultry barns:Score 0: No or limited dust: No visible dust specks in the air; Score 1: Moderate dust level: Isolated dust specks are detectable, but visibility has only slightly or not notably decreased - details of the wall are easily recognisable; Score 2: High dust level: Many dust specks are detectable, and visibility has notably decreased - details of the wall are not easily recognisable. 2.2.6. Deposition Assessment The level of dust was assessed based on the amount of dust accumulated on four structures in the barn. The structures selected were not easily accessible to the birds but still in a position where dust could accumulate, e.g., the pipe above the drinker line. As far as possible, it was attempted to use similar structures in all barns. The test was performed near the spots used for the visibility assessments . The scoring scale used to assess the level of dust deposited on each structure was as follows:Score 0 = No or minimal evidence of dust (structure has same colour as a spot nearby that has been wiped clean); Score 1 = Isolated specks or a thin layer of dust (<=1 mm) is detectable (when comparing with a cleaned part of the structure; a slight colour difference appears between the two parts of the structure); Score 2 = Dust covers the structure (clear difference in colour between clean and test parts). Dust layer is >1 mm but <5 mm; Score 3 = As in score 2, but the dust layer is >=5 mm. 2.3. Statistical Analysis Data were analysed using R Studio Software , and a significance level of 0.05 was used for all statistical tests. Gravimetric measurements (i.e., the reference method) were obtained from only nine of the 11 farm visits due to maloperation of aparatus. In addition, due to restrictions posed as a consequence of COVID-19 and some unexpected events during the visits, some data from methods examined were not obtained. Therefore, the number of pairs between the reference method and each method was: light scattering measurement (n = 7), dust sheet test 1 h (n = 9), dust sheet test 2-3 h (n = 9), tape test (n = 9), visibility assessment (n = 8) and deposition assessment (n = 8). Prior to analysis, the data from the reference method were log-transformed to obtain data normality. For the methods where the data were collected multiple times per barn visit, an average was calculated (i.e., all, except the tape test). A linear regression analysis was conducted separately for each method tested to assess whether the degree to which the dust level obtained with the different methods was in agreement with the concentration given by the reference method. The dust concentration measured by the reference method was used as the dependent variable, whereas the dust level obtained using the different methods under examination was used as the independent variable, i.e., we tested whether the actual dust level, given by the reference method, could be predicted from the one given by the method examined:Log (reference method) = m + a * [dust concentration from test i](1) where a is the slope, m is the intercept, and i one of the six methods examined. Before analysing the results of the linear regressions, the three assumptions of a linear model were checked. The assumption of normality of the residuals was checked using the Shapiro test and visually by the QQ-plot. The hypothesis of the homogeneity of variances was checked using the Breusch-Pagan test and visually by plotting the residuals against the model's predicted values for the observed values of the predictive variable (i.e., "residuals vs. fitted plot"). Finally, the hypothesis of non-correlation of the residuals was verified using the test. To determine the significance of the correlation between the reference method and each of the methods under examination, it was chosen to focus on the p-value of the slope of the regression line instead of the slope of the regression line between the dust concentration measured by the reference method and the methods under examination. The reason for this was that a slope of the regression line equal to 1 was not representative of identical concentrations between the reference method and the methods under examination because the latter express dust concentrations in different units (except for the light scattering measurements). A significant p-value (p < 0.05) of the slope of the regression line meant that the slope was different from 0, i.e., the values obtained by the reference method and the method under examination were associated. The adjusted coefficient of determination (adjusted R2) was used to describe the correlation of the measured dust levels between the reference method and the method under examination. The closer the adjusted R2 is to 1, the better the model is in terms of predicting the true concentration value. The Root-Mean-Square Error (RMSE) of the models was used to provide information about the performance of a model by allowing a term-by-term comparison of the actual difference between the estimated and the measured value. The lower the value of RMSE, the better the performance of the model. 3. Results The dust concentration measured using the gravimetric principle varied between 0.3-20.3 mg/m3 in the layer barns visited . The regression lines for all the methods under examination are shown in Figure 3A-F, and the p-values for the slopes of the regression lines, adjusted R2 and the RMSE are listed in Table 1. The dust sheet test 2-3 h showed the highest correlation with the reference method as the data points were scattered closely around the regression line , and the slope was highly significant (Table 1). In addition, the adjusted R2 for the dust sheet test 2-3 h also had the highest value of the methods examined (Table 1), being close to one, further indicating a strong correlation between the dust level given by the dust sheet test 2-3 h and the reference method. Concerning the RMSE, the dust sheet test 2-3 h had the lowest value (Table 1), again indicating that the dust sheet test 2-3 h was the method providing the best prediction of the true value of dust concentration in the layer barns. In contrast, the slope of the regression line for the deposition assessment was not significant , i.e., the dust level given by this method was not correlated to that of the reference method. In line with this, the slope was negative, i.e., the higher the concentration measured using the reference method, the lower the level of dust accumulated. In addition, the method received the lowest and thus the poorest adjusted coefficient of determination. Although the slopes of the regression lines were significant for the tape test and the visibility assessment , the distributions of the data points around the regression lines clearly showed that these methods performed poorly at predicting the dust level. The light scattering device and the dust sheet test 1 h performed intermediate in regard to the distributions of the data points around the regression lines. Both methods had a significant slope, although at a lower significance level as for the dust sheet test 2-3 h. However, the adjusted R2 and the RMSE were relatively low (only for dust sheet 1 h) and high, respectively, i.e., showing a weaker correlation between dust levels collected using these methods and the reference method than those collected using the dust sheet test 2-3 h. The plot of the residuals against the dust concentration values predicted by the model for the dust sheet test 1 h showed increased variance with increasing concentration of dust . This indicates that the validity of the dust sheet test 1 h may be improved if the scale is further developed into a more detailed protocol with additional categories, e.g., five levels instead of the current three levels. 4. Discussion Methods for measuring dust levels in poultry barns are vital for several reasons. Firstly, without knowledge of the dust levels in poultry barns, initiatives focusing on reducing harmful levels of dust are at risk of not being prioritised. Dust from animal houses is an indoor pollutant with negative impacts on animal performance, health, and welfare , as well as on the health and welfare of animal caretakers . In addition, dust is released outside animal houses by ventilation systems and is thus an outdoor pollutant. Poultry houses are, compared to other livestock houses, among those with the highest dust concentrations . Intensive poultry production releases considerable amounts of dust to the atmosphere, accounting for around 55% of dust emissions from agriculture in Europe . Dust contributes to the spread of diseases to other poultry houses in the vicinity and even to neighbouring human populations and is therefore a public health concern . Secondly, legal requirements need to be measurable for the veterinary inspectors to be able to assess compliance with the legislation. In regard to the current legal requirement on dust, an open norm is used, i.e., no exact threshold value is given. However, since it states that the dust level must be kept within limits, which are not harmful to the animals , knowledge is needed both on threshold values for when the dust becomes harmful and on methods applicable for the assessment of dust levels. In addition, the methods applied need to be valid, reliable, and feasible for animal welfare assessments to be fruitful . Six methods, considered relatively feasible for dust assessment during veterinary inspections, were examined in the present study for their validity. Although the number of farm visits was reduced compared to the original plan due to the COVID-19 health crisis and an avian influenza outbreak, the study revealed that the methods differed in potential. The most promising method was the dust sheet test, which is simple to perform and requires limited training, preparation, and investment as the only thing needed is pieces of black paper that can be discarded after each inspection. Based on the scattering of the data points closely around the regression line and good performance of all the statistical measures applied, i.e., significant p-value of the slope, high adjusted R2, and low RMSE, the dust sheet test with a test duration of 2-3 h was shown to be a valid method of assessing dust levels in poultry barns. The validity of the dust sheet test has not previously been examined even though the test is included in the Welfare Quality(r) assessment protocols for both laying hens and broilers , and therefore is applied in multiple studies of animal welfare (e.g., ). However, the long test duration is a challenge for its use during an official veterinary inspection, which typically only lasts approximately 1 h . Therefore, continued development of the protocol for the dust sheet test is needed with the aim of reducing the duration without losing the validity of the test. Although an association between the dust sheet test 1 h and the reference method was found (i.e., significant p-value of the slope), the relatively low adjusted R2 and high RMSE showed that a test duration of 1 h is insufficient time to obtain results as satisfactory as those obtained after 2-3 h, but the increased variation with increasing dust concentration indicates that more steps on the scoring scale may be a solution to improve the validity of the method when shorter test durations are applied. More research is needed to examine this hypothesis. Furthermore, in the present study, we discuss the feasibility and examined the validity of the methods, whereas no data on reliability were obtained. The dust sheet test is prone to subjectivity as it is based on the observers' visual assessment. Future studies should address the reliability of the dust sheet test within and among observers. Light scattering measurements appeared initially to be a feasible solution for the assessment of dust levels in poultry barns. The method is widely used for scientific dust studies and air quality control (e.g., ), but not yet for veterinary animal welfare inspections. However, the specific device used in the present study turned out to be unsuitable for use in poultry barns due to the lack of possibility of sufficiently cleaning the inner chamber, posing a biosecurity risk. We solved this with the attachment of a plastic bag, but this was an impractical solution that limited the number of measurements that could be obtained. In terms of validity, the results were intermediate. Some of the measures supported validity, i.e., the significant p-value of the slope and low adjusted R2. However, from the regression line of the log-transformed dust concentration from the reference method as a function of the dust level obtained by light scattering measurements, it appears that the data obtained using this method did not follow a linear pattern but perhaps more a hyperbolic curve. To test this hypothesis, and hence further examine the validation of the method, more data points, i.e., a larger sample size, are needed. With the continued developments in technology, the idea of light scattering devices for measuring dust levels in poultry barns should be further pursued. The other methods examined, i.e., the tape test, visibility assessment, and deposition assessment cannot be recommended. For the tape test and visibility assessment, it is clear from the regression analyses that the linear models were not well-adapted, including poor distribution of data points around the regression line, low adjusted R2 (tape test), and high RMSE (visibility test). However, due to the low number of data points, it was not possible to add additional parameters for a better model fit to be obtained. In any case, it is questionable whether extra data points will add more information, as the very low variability in the scores given when using these tests poorly reflects the variation of dust level concentration measured by the reference method in the barns. The idea of the tape test was to develop a method that allows the time of exposure to be consistent without the need for the observer or veterinary inspector to return to the specific spots where the dust sheets were placed at a specific time. The poor performance may have been due to the movements of the observer obstructing dust accumulation as compared to what is seen on stationary dust sheets. Regarding the visibility assessment, the protocol was an attempt to standardise the sensorial assessment currently performed by most veterinary inspectors . Obviously, the test is very subjective as it highly depends on the vision of the observer and the observer's interpretation of the sight. The idea of the deposition test also originated from interviews with veterinarian inspectors, where some informed us that they occasionally use the dust deposition on barn structures as an indicator of the dust level . None of the measures obtained for the deposition test indicated the validity of the test, i.e., negative slope, non-significant p-value of the slope, low R2, and high RMSE, and clearly, this method presents many challenges. Firstly, the barns are usually completely cleaned before new flocks are placed, so depending on the age of the birds at the time of the inspection there is more or less dust accumulated, which does not necessarily indicate the current airborne dust level. Secondly, it is difficult to standardise the spots for the assessment between barns as the housing systems and additional structures differ greatly between barns. Thirdly, it is difficult to find spots that are suitable for the assessment, as spots should be untouched by the birds and not influenced by a draft from the ventilation or air puffs from the birds, while at the same time allowing unhindered accumulation of dust. 5. Conclusions Currently, veterinarians conducting animal welfare inspections lack validated methods for measuring or assessing dust levels in poultry barns. In the present study, we developed new methods or refined existing methods for dust assessment in layer barns. Of the six methods, the dust sheet test with a duration of 2-3 h was found to be the most promising method, showing a high validity. However, a test duration of 2-3 his longer than most veterinarian inspections. A solution may be modifications of the scoring scale as the results indicated that with more steps on the scoring scale, it may potentially be possible to further reduce the duration of the dust sheet test without losing the validity. More research is needed to examine this hypothesis as well as the reliability that was not addressed in the present study. Acknowledgments We would like to thank the farmers for participating in this research study and senior researcher Leslie Foldager for his guidance during the statistical analyses. Author Contributions Conceptualization, F.M. and A.B.R.; Data curation, S.M.; Formal analysis, S.M.; Funding acquisition, A.B.R.; Investigation, S.M.; Methodology, F.M. and A.B.R.; Project administration, A.B.R.; Supervision, A.B.R.; Writing - original draft, S.M. and A.B.R.; Writing - review and editing, F.M., S.M. and A.B.R.. All authors have read and agreed to the published version of the manuscript. Institutional Review Board Statement Ethical review and approval were waived for this study due to the absence of invasive acts susceptible to causing harm or suffering to the animals. Informed Consent Statement Not applicable. Data Availability Statement Data is contained in the article. Conflicts of Interest The authors declare no conflict of interest. Figure 1 Spots of assessments or measurements in a common barn design. Figure 2 The total dust concentration (mg/m3) measured during the farm visits using the gravimetric principle (i.e., the reference method). No data were obtained during visits to Farms 1 and 8 due to failure of the device. Figure 3 The regression lines (in red) for the log-transformed dust concentration from the reference method as a function of the dust level obtained using the methods under examination: (A) light scattering measurement, (B) dust sheet test 1 h, (C) dust sheet test 2-3 h, (D) tape test, (E) visibility assessment and (F) deposition assessment. The intercept (Int) and regression coefficient (slope) of each linear regression are given above the corresponding graph. Figure 4 Plot of the residuals against the dust concentration values predicted by the model for the dust sheet test 1 h. animals-13-00783-t001_Table 1 Table 1 Results on the significance of the slopes of the regression lines for the log-transformed dust concentration of the reference method as a function of the dust concentrations of the methods under examination. The adjusted coefficient of determination (adjusted R2) and the Root-Mean-Square Error (RMSE) of each model are also given. Method Examined p-Value of Slope a Significance b Adjusted R2 RMSE Light scattering measurements 0.02174 * 0.6206 0.7567 Dust sheet test 1 h 0.03118 * 0.4376 0.7987 Dust sheet test 2-3 h 0.00003 *** 0.9192 0.3553 Tape test 0.04541 * 0.3801 0.4088 Visibility assessment 0.00994 ** 0.6462 1.1112 Deposition assessment 0.58756 n.s. -0.1062 0.6546 a Each slope was tested against the nullhypothesis (i.e., being different from 0). b p-values of the slopes indicated as n.s. were not significantly different from 0, whereas star symbols indicate rejection of the null hypothesis at a significance level of 0.05 (*), 0.01 (**), or 0.001 (***). Disclaimer/Publisher's Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

PMC10000158

Post-processing operations of extruded pet food kibbles involve coating the product with fats and flavorings. These processes increase the risk for cross-contamination with food-borne pathogens such as Salmonella and Shiga toxin-producing Escherichia coli (STEC), and mycotoxin-producing molds such as Aspergillus spp. after the thermal kill step. In this study, the antimicrobial effects of two types of organic acid mixtures containing 2-hydroxy-4-(methylthio) butanoic acid (HMTBa), Activate DATM and Activate US WD-MAXTM, against Salmonella enterica, STEC and Aspergillus flavus when used as a coating on pet food kibbles were evaluated. Using canola oil and dry dog digest as fat and flavor coatings, the efficacy of Activate DA (HMTBa + fumaric acid + benzoic acid) at 0%, 1% and 2%, and Activate US WD-MAX (HMTBa + lactic acid + phosphoric acid) at 0%, 0.5% and 1% was tested on kibbles inoculated with a cocktail of S. enterica serovars (Enteritidis, Heidelberg and Typhimurium) or Shiga toxin-producing E. coli (STEC) serovars (O121, and O26) at 37 degC for 0, 12, 24, 48, 72 h, 30 and 60 days. Similarly, their efficacy was tested against A. flavus at 25 degC for 0, 3, 7, 14, 21, 28 and 35 days. Activate DA at 2% and Activate US WD-MAX at 1% reduced Salmonella counts by ~3 logs after 12 h and 4-4.6 logs after 24 h. Similarly, STEC counts were reduced by ~2 logs and 3 logs after 12 h and 24 h, respectively. Levels of A. flavus did not vary up to 7 days, and afterwards started to decline by >2 logs in 14 days, and up to 3.8-log reduction in 28 days for Activate DA and Activate US WD-MAX at 2% and 1%, respectively. The results suggest that the use of these organic acid mixtures containing HMTBa during kibble coating may mitigate post-processing enteric pathogen and mold contamination in pet food kibbles, with Activate US WD-MAX being effective at a lower concentration (0.5-1%) compared to Activate DA. pet food kibble antimicrobial HMTBa Salmonella Escherichia coli STEC Aspergillus Novus InternationalThis research was carried out with funding from Novus International, Inc., St. Charles, Missouri, United States. pmc1. Introduction A large percentage of pet owners prefer feeding dry commercial pet foods to their pets due to the convenience and nutritional benefits. Dry pet food constitutes the most commonly sold type of pet food in the world, forming 75.2% of dog food and 53.9% of cat food categories . These foods contain a variety of plant and animal ingredients as raw materials. The animal proteins are processed by different methods, predominantly in rendering plants, where there is a risk of contamination with various pathogens such as Salmonella . The gastrointestinal tract of pets that consume these contaminated foods may get colonized with these bacteria, and yet they may not show any clinical symptoms . These occurrences confirm that pathogens such as Salmonella can be transmitted to humans through the handling of dry pet foods . Numerous cases of human salmonellosis have been linked to contaminated dry pet food such as dog and cat food kibbles , and animal feed . Although extruded pet food like kibbles go through a thermal kill step during processing, they can become cross-contaminated with pathogenic bacteria during subsequent processing steps such as coating with fats and flavors . By handling or consuming these foods, they may get transmitted to the pet which can serve as a carrier to humans. For instance, from 2006 to 2008, an outbreak of Salmonella enterica serotype Schwarzengrund, included 79 illnesses over 21 states, resulted in the recall of 105 brands of dry pet food and permanently closed a manufacturing plant in Pennsylvania . The process validation in the facility included a specified time-temperature combination to kill Salmonella, and then the food was subsequently moved to coating and flavoring steps where it was sprayed with fat and palatants. An epidemiological investigation led to the isolation of the bacterial strain related to the outbreak from the flavoring room, meaning that the Salmonella contamination occurred during that step . In 2012, there was also an outbreak of human Salmonella enterica serotype Infantis infections related to exposure to dry dog food . Escherichia coli are some of the most prevalent enteric bacteria in animals and humans, and are also important zoonotic agents, which can be implicated in animal and human infectious diseases . A recent study showed that antibiotic-resistant E. coli could easily spread between humans and their pets . Shiga toxin-producing strains of E. coli O157:H7 have also caused numerous deaths following the consumption of contaminated foods . Many studies on the prevalence of antimicrobial resistance in E. coli isolates from farm animals and pets have been performed in other countries beyond the United States . In 2016, the U.S. Food and Drug Administration (FDA) along with the Centers for Disease Control and Prevention (CDC) investigated a multi-state outbreak of Shiga toxin-producing E. coli (STEC) O121 and O26 infections. Sixty-three people infected with the outbreak strains of STEC O121 or O26 were reported from 24 states, and it was traced back to contaminated wheat flour from a General Mills facility in Kansas City, MO. Although pet food recalls due to STEC contamination have not been reported yet, recalls involving STEC-contaminated wheat flours have increased compared to previous years with 13 recalls occurring in 2019. Wheat flour is commonly used as an ingredient in pet foods, so the potential for STEC contamination exists. Previous research studies have investigated physical (e.g., vacuum steam) and chemical (e.g., sodium bisulfate) intervention strategies in wheat flour milling to mitigate STEC contamination . Another risk factor for human and animal food safety is the presence of fungi and the potential for mycotoxins that they may produce. Hazardous mycotoxins can occur in cereal grains due to stress during toxigenic fungal growth, can be compounded with improper storage and the process of cooking does not reduce their content . During pet food manufacturing, the foods can be contaminated with mold spores especially when cereal grains are ground, and the foods are pelleted or formed . Pet food kibbles with water activity of 0.50 are not very conducive for mold growth; however, mold spores prevalent in the environment can contaminate packaged foods that are opened by the consumer and can amplify during food storage, especially in a humid environment with >75% RH. Moldy foods reduce the nutritional value due to spoilage and under certain conditions may produce toxic metabolites called mycotoxins. These toxins with chemically diverse structures have been involved in disease outbreaks which have affected both animal and human health . A study by Beuno et al. identified commonly occurring molds in 21 pet foods including dog food kibbles across eight commercial brands produced in Argentina, and were comprised mainly of Aspergillus, followed by Rhizopus and Mucor spp. Fungal contamination can lead to economic losses associated with nutrient and palatability reduction. Furthermore, the presence of mycotoxins also affects both the animal's and human's health . Aspergillus flavus, being the most reported in pet food, is responsible for the production of aflatoxins. Dogs are extremely sensitive to this group of toxins, with the liver being their main target . In the U.S. in 2005, aflatoxicosis-related illnesses in dogs and A. flavus-contaminated dog food recalls were reported. More recently, in 2020, 28 deaths and eight illnesses were reported in dogs that consumed the recalled SportmixTM pet food product that was contaminated with aflatoxin. Pet food recalls by Sunshine Mills also happened in 2020, due to aflatoxin contamination from corn that was used as an ingredient in the pet food . Thus, there is a need to control or reduce toxigenic A. flavus contamination. The amino acid methionine is identified as a limiting amino acid in high forage cattle diets and has a positive impact on the health and performance of the animal. Methionine participates in a wide variety of metabolic pathways and serves as a precursor to other amino acids, such as cysteine. If free amino acids are fed directly to beef cattle, the rumen microbes destroy them before they even leave the rumen. Because amino acids must be presented to the small intestine in required amounts for the animal to synthesize protein, the amino acid must be protected or modified to avoid rumen degradation. Supplementing animal feed rations with a 'methionine hydroxy analogue' is an economical way to supply methionine. HMTBa (2-hyroxy-4-(methlythio) butanoic acid) is an organic acid and a methionine hydroxy analogue. It has been used as a methionine precursor in animal feed due to its unique chemical structure that allows protection from some of the microbial degradation in the rumen gut. HMTBa also provides acidifying effects of organic acids. These acidifying effects subsequently provide gut health advantages to the animal by mitigating pathogen growth in the gut . Methionine hydroxy analogue has also been shown to reduce nitrogen excretions , support animal performance during heat stress and offer antioxidant capacity . Other than these health benefits and its use as a methionine precursor in animal feed supplements, its potential role in enhancing food safety of processed foods or its application in pet foods has not been investigated so far. HMTBa is one of the main components of Activate DATM and Activate US WD-MAXTM, which are proprietary blends of organic acids from Novus International (St. Charles, MO, USA). According to the company's product information, research conducted by the CCL Institute in the Netherlands using HMTBa demonstrated its effectiveness in reducing bacterial populations such as Salmonella, E. coli and Campylobacter, all of which can be found in the drinking water of poultry. The combination of organic acids in Activate DA and Activate US WD-MAX effectively reduces the pH of the gastrointestinal tract, promotes the establishment of a desirable and more balanced intestinal flora and aids in digestion, providing more nutrients from feed and improving the performance of the animal. Activate DA (HMTBa + fumaric acid + benzoic acid + silica + mineral oil) is a granular mixture applied to premixes and finished feeds. Activate US WD-MAX (HMTBa + lactic acid + phosphoric acid) is used for the acidification of drinking water for poultry, making the drinking water a less favorable environment for pathogen growth, and it is shown to play an important role in the destruction of harmful microorganisms in the gut that could affect the birds' performance. Parker et al. evaluated the organic acid mixture Activate WDTM containing HMTBa at 0.04% and 0.08% in drinking water for poultry and found a reduction in the horizontal transmission of Salmonella in the broiler chickens. Guo-zheng et al. evaluated Activate WD against Staphylococcus aureus, Escherichia coli, Salmonella pullorum and Campylobacter jejuni in nutrient broth and found it to be effective at 0.6%. Deliephan evaluated Activate DA and Activate US WD-MAX as sanitizers on food contact surfaces to inhibit Salmonella. Very few studies had evaluated chemical additive coating in pet food kibbles using organic acids like 3-hydroxy-3-methylbutyrate (HMB) and medium chain fatty acids (caproic, caprylic, and capric) to mitigate post-extrusion cross-contamination from Salmonella. There is limited knowledge on the application of organic acid mixtures containing HMTBa in dry pet food kibbles to enhance food safety. The objectives of this research study were as follows: (i) to determine the efficacy of organic acid mixtures, Activate DA and Activate US WD-MAX, coated on extruded pet food kibble on the survival of Salmonella enterica, Escherichia coli (STEC) and Aspergillus flavus; and (ii) to evaluate the residual antimicrobial effect of the organic acid mixtures Activate DA and Activate US WD-MAX coated on extruded pet food kibble on Salmonella enterica, to maintain Salmonella-free kibble despite repeated exposure to recontamination over time. 2. Materials and Methods 2.1. Materials Uncoated dry pet food kibbles were custom manufactured at Extru-Tech Inc. (Manhattan, KS, USA). The final moisture content of the kibble was 5.6% with water activity (aw) of 0.50. The composition of the kibble is presented in Table 1. The organic acid mixtures evaluated in this study included Activate DA (dry formula) and Activate US WD-MAX (liquid formula) that were provided by the study sponsor Novus International (St. Charles, MO, USA). Activate DA was a mixture of HMTBa, fumaric acid, benzoic acid, silica and mineral oil. Activate US WD-MAX was a mixture of HMTBa, lactic acid and phosphoric acid. Activate DA, which was in granular form, was further ground to reduce its particle size using a laboratory hammer mill (Verder Scientific, Inc., Newtown, PA, USA) for use in this study. 2.2. Inoculum Preparation Salmonella enterica serovars Enteritidis (ATCC 4931), Heidelberg (ATCC 8326) and Typhimurium (ATCC 14028) were procured from the American Type Culture Collection (ATCC, Manassas, VA, USA) and maintained in tryptic soy broth (TSB)-glycerol (7:3) at -80 degC. Before use, the frozen cultures were streaked onto tryptic soy agar (TSA; BD Difco, Sparks, MD, USA) plates and incubated at 37 degC for 24 h. A single colony of each Salmonella strain was inoculated into 10 mL of TSB (BD Difco, Sparks, MD, USA) and incubated at 37 degC for 18 to 24 h. The cultures of each Salmonella serotype thus obtained were centrifuged for 10 min at 5000x g (Thermo Scientific, Waltham, MA, USA) at room temperature. The pellets were resuspended in pre-sterilized 0.1% peptone water (Difco Laboratories, Sparks, MD, USA), and an equal volume of each serotype was mixed to obtain the cocktail (~8 log CFU/mL). Shiga toxin-producing E. coli (STEC) serovars O121 (ATCC 2219) and O26 (ATCC 2196) were maintained in tryptic soy broth (TSB)-glycerol (7:3) at -80 degC. The same procedure explained earlier for the preparation of the Salmonella cocktail was followed for the STEC cocktail preparation. The final STEC cocktail inoculum had a concentration of ~8 log CFU/mL. The fungal culture of aflatoxin-producing Aspergillus flavus (ATCC 15548) was maintained in potato dextrose broth (PDB)-glycerol (7:3) at -80 degC. Before use, the frozen cultures were streaked onto potato dextrose agar (PDA) with incubation at 25 degC for 72 h. The fungal spores were collected from the grown culture on PDA by adding 5 mL of 0.1% peptone water to the surface of the dish. The spores were then dislodged from the solid medium using an L-shaped plastic rod. The spore suspension in peptone water was then collected and stored at 4 degC and was used as the fungal inoculum (~4 log CFU/mL). 2.3. MIC, MBC and MFC assays The minimum inhibitory concentrations (MIC) of the organic acid mixtures were determined by the broth macro-dilution assay according to the antimicrobial susceptibility testing methods described by the Clinical and Laboratory Standards Institute . To determine MIC, a 200 mL volume of Activate DA or Activate US WD-MAX consisting of twice the desired final concentration was dispensed in the first well of a 96-well microtiter plate (triplicate wells) and 100 mL of sterile water in the rest of the wells. A serial two-fold dilution of the organic acid mixtures was performed starting from 50% to 0.05% v/v concentration. A 100 mL aliquot of bacterial or fungal (Salmonella or STEC or A. flavus) culture (6 log CFU/mL for bacteria or 4 log CFU/mL for fungi) was added to each well of the plate already containing the 100 mL of decreasing concentrations of organic acid solutions to make a final volume of 200 mL per well. A positive control consisted of bacterial or fungal inoculum only (no treatment), and a negative control consisted of tryptic soy broth (TSB) or potato dextrose broth (PDB) only. The MIC was determined as the lowest concentration of organic acid mixture that inhibited visible growth of the target microorganism in the microtiter plate after 24 h (for bacteria) or 72 h (for fungi) of incubation at 37 degC or 28 degC, respectively. The inhibition of growth of the microorganisms was determined qualitatively by visual observation of turbidity in the growth media. To determine minimum bactericidal concentration (MBC) and minimum fungicidal concentration (MFC), 100 mL of the sample from each well from the MIC experiment was plated onto xylose lysine deoxycholate (XLD) agar for Salmonella, potato dextrose agar (PDA) for A. flavus or 1 mL onto Petrifilm (3M, Minneapolis, MN, USA) plates for E. coli counts, for the enumeration of Salmonella, A. flavus and STEC colonies at each concentration of organic acid mixture after incubation at 37 degC for 24 h (for Salmonella) or 37 degC for 48 h (for STEC) or 28 degC for 72 h (for A. flavus). The colonies were enumerated manually. The MBC and MFC were determined as the lowest concentration of the organic acid mixture that caused an absence (<=1 colony) of bacterial or fungal growth on the plates or Petrifilm. The study was replicated three times. 2.4. Coating of Kibbles, Inoculation and Microbiological Analysis To determine the antimicrobial efficacy of the organic acid mixtures, a 180 g aliquot of pet food kibbles for each treatment was transferred to a plastic container and autoclave sterilized. For each of the organic acid treatments (Activate US WD-MAX or Activate DA), for each pathogen tested (Salmonella, STEC or A. flavus), a total of six containers were maintained: four for each of the organic acid treatment concentrations (0.5% or 1% w/w of Activate US WD-MAX, and 1% or 2% w/w of Activate DA), one for the untreated and one for the negative control, for each pathogen tested. The organic acid mixtures were suspended in canola oil and applied to the kibbles using a pipette and mixing to coat them thoroughly to make a final weight of 200 g in each container. The final oil percentage on the kibbles was maintained at ~7-8%. The concentrations of the organic acid mixtures used were based on the MICs. The 'untreated' was without any organic acid treatment and inoculated with only the bacterial or fungal inoculum in TSB or PDB. The negative control was canola oil-coated kibble, without any organic acid treatment and without inoculation with bacteria or fungi. After 30 min of the organic acid treatment (coating), a bulk-harvested Salmonella culture or STEC culture cocktail (~8 log) was spot inoculated on the kibbles using a pipette. The initial moisture content of the kibbles was 5.6% and the final moisture content post-inoculation was maintained at ~8-9% dry basis. For example, for 0.5% Activate US WD-MAX treatment, 180 g kibbles + 14 g canola oil + 1 g organic acid (Activate US WD-MAX) + 5 mL inoculum was used to make a total weight of 200 g in the container. After uniform mixing of the kibbles, the containers were incubated at 37 degC. Similarly, for the fungal inoculation, A. flavus culture inoculum in PDB (~4 log) was spot inoculated on the kibbles. After uniform mixing of the kibbles, the containers were incubated at 28 degC. Microbiological analyses for Salmonella and STEC were conducted for each of the containers at various pre-determined time intervals: 2, 12, 24, 48, 72 h, 30 and 60 days, and the fungal analyses for A. flavus were conducted at 1, 3, 7, 14, 21, 28 and 35 days. From each treatment, a 25 g subsample was collected in sterile Whirl-Pak bags (Nasco, Ft. Atkinson, WI) and was mixed in 225 mL of buffered peptone water (Difco Laboratories, Sparks, MD, USA) and stomached for 2 min. The mixtures were serially diluted in 0.1% peptone water and plated on XLD agar (for Salmonella), Petrifilm (for STEC) and PDA (for A. flavus). The XLD agar, PDA plates and Petrifilms were incubated at 37 degC, 25 degC and 37 degC for 24 h, 72 h and 48 h, respectively, and then colonies were counted. The experiments were replicated three times. To evaluate the residual antimicrobial effect of the organic acid mixtures on Salmonella, uncoated pet food kibbles as described earlier were used. A cocktail culture containing Salmonella enterica serovars Enteritidis (ATCC 4931), Heidelberg (ATCC 8326) and Typhimurium (ATCC 14028) was prepared and used as the inoculum (~8 log CFU/mL). The minimum levels of organic acid mixtures Activate DA (1% and 2%) and Activate US WD-MAX at 0.5% and 1%) determined to be effective against Salmonella in pet food kibbles were used in this experiment. Salmonella-negative kibbles were coated with the minimum effective levels of organic acid mixtures Activate DA (1% and 2%) and Activate US WD-MAX (0.5% and 1%) and divided into one of three challenge groups (for day 1, day 30 and day 90). Organic acid mixture-coated kibble was challenged with Salmonella after 1, 30 and 90 days of storage to investigate the residual effect of the organic acid mixtures in the kibble on storage at 25 degC. At each time period, a freshly prepared Salmonella inoculum in TSB was spot inoculated on the kibbles using a pipette and mixed thoroughly. The final moisture content of the kibbles post-inoculation was maintained at ~8-9%. After the introduction of the challenge Salmonella, the kibble was incubated at 37 degC for 24 h and then analyzed for Salmonella counts and enumerated on XLD agar plates. The untreated sample consisted of kibble with no organic acid coating, and the negative control consisted of canola oil-coated kibble (no organic acid and no Salmonella inoculation). The experiment was replicated three times. 2.5. Confirmative Test for Salmonella A confirmative test for Salmonella for the experiments mentioned above was conducted according to the FDA-BAM method (Bacteriological Analytical Manual). In short, buffered peptone water from the pre-enrichment of each treatment sample, 1.0 mL and 0.1 mL, was transferred to 10 mL of Rappaport-Vassiliadis (RV; BD Difco, Sparks, MD, USA) and tetrathionate (TT; BD Difco, Sparks, MD, USA) broths, respectively, and incubated at 42 degC for 24 h for selective enrichment of Salmonella. From each RV and TT broth tubes, one loopful was streaked onto xylose lysine deoxycholate (XLD) agar plates in duplicate. Inverted plates were incubated at 37 degC for 24 h. Presumptive positive Salmonella colonies appeared as pink colonies with or without black centers, with the most positive Salmonella-producing colonies with large, glossy black centers or almost completely black. Presumptive Salmonella-positive colonies from XLD plates were then inoculated into triple sugar iron agar (TSI; BD Difco, Sparks, MD, USA) slants by streaking the slant and stabbing the butt, and lysine iron agar (LIA; BD Difco, Sparks, MD, USA) slants by stabbing the butt twice and then streaking the slant. The TSI and LIA slants were incubated at 37 degC for 24 h. Presumptive Salmonella-positive TSI reactions had alkaline (red) slants and acid (yellow) butts, while LIA reactions had alkaline (purple) butts with acidic (yellow) reactions negative for Salmonella. All cultures with an alkaline butt in LIA, regardless of TSI reaction, were retained as potential Salmonella isolates. Presumed-positive TSI and LIA slant cultures were inoculated into TSB and incubated at 37 degC for 24 h, from which cells were harvested, DNA extracted and confirmed as Salmonella based on a molecular analysis . 2.6. Statistical Analysis For the challenge study for each of the three pathogens tested (Salmonella, STEC and A. flavus), the mean log reductions at each sampling time period for the treatments were subjected to a two-way analysis of variance (ANOVA) using the GLIMMIX procedure of statistical software SAS (version 9.3), and the treatment means were separated using Tukey's post-hoc test when the F-test of the ANOVA per treatment was significant at p < 0.05 . The linear model y = a + bx was fit to a logarithmic reduction in bacterial counts over time (hours) for the treatments, where a is the intercept and b is the slope. The decimal reduction time or D-value (time taken for 1-log reduction of bacterial counts) was calculated as the negative-inverse of slope . Similarly, for the determination of the residual antimicrobial effect, the mean log reductions of Salmonella at each time period (day 1, day 30, day 90) for the treatments were subjected to a two-way ANOVA, and the treatment means were separated using Tukey's test (p < 0.05) . 3. Results The MICs and MBCs of Activate DA against Salmonella and STEC ranged from 0.5% to 1% and Activate US WD-MAX ranged from 0.4% to 0.5%. The MIC and MFC of both Activate DA and Activate US WD-MAX against A. flavus were 2% (Table 2). Based on the MIC, MBC and MFC results, the treatment concentration levels of Activate DA at 1% and 2% and Activate US WD-MAX at 0.5% and 1% were used subsequently in this study. For the challenge study against Salmonella, the initial load of Salmonella in the inoculum was 8 log CFU/mL. A reduction in Salmonella counts (p < 0.05) was observed over time (2 h to 60 days) due to the inclusion of Activate DA and Activate US WD-MAX as the coating on kibbles as shown in Figure 2 and Table 3. The untreated (no organic acid coating) showed a constant mean Salmonella load of 6.9 log CFU/mL until 24 h. By 48 h, the counts decreased to 4.7 log CFU/mL; by 72 h, the counts further reduced to 1.7 log CFU/mL; and by 60 days, the counts declined to 1 log CFU/mL. On an average, a log reduction of 1.1 logs (from an initial load of 8 logs) was observed for the untreated samples until 24 h, 3.3 logs by 48 h, 6.3 logs by 72 h and 7 logs by day 60. The treatments Activate DA and Activate US WD-MAX reduced Salmonella counts over time (p < 0.05) when compared to the untreated (Table 3). The inclusion of Activate DA at 1% decreased Salmonella counts resulting in a population reduction of 5.1 logs (from an initial inoculum load of 8 logs) by 24 h and 7 logs by 48 h. Activate DA at 2% also followed a similar trend decreasing Salmonella counts by 5 logs at 24 h and 7 logs at 48 h. However, at 12 h Activate DA at 2% reduced Salmonella counts by 5.2 logs whereas at 1% the log reduction was only 4.2 logs at the same time point. Activate US WD-MAX at 0.5% led to a log reduction of 6.5 logs at 24 h, and 7 logs at 48 h. A similar result was observed at 1% with a log reduction of 6.3 logs at 24 h and 7 logs at 48 h. At 12 h, Activate US WD-MAX at 1% reduced Salmonella counts by 5.4 logs, whereas at 0.5%, the log reduction was 4.3 logs at the same time point. Overall, there was no difference among the two treatments Activate DA and Activate US WD-MAX across the different concentrations tested (Table 3). Considering the log reductions of Salmonella over time, Activate US WD-MAX was effective at a lower concentration (0.5%) than Activate DA. Similarly, a reduction in STEC counts was observed over time (2 h to 60 days) due to the inclusion of Activate DA and Activate US WD-MAX as a coating on pet food kibbles . An average population reduction of 1.1 logs (from an initial load of 8 logs) was observed for the untreated samples until 24 h and then a reduction of 3.2 logs by 48 h, 5.4 logs by 72 h and 7 logs by day 60. The treatments Activate DA and Activate US WD-MAX reduced STEC counts over time (p < 0.05) when compared to the untreated (Table 4). The inclusion of Activate DA at 1% decreased STEC counts resulting in a population reduction of 5.1 logs (from an initial load of 8 logs) by 24 h and 5.7 logs by 48 h. Activate DA at 2% also followed a similar trend decreasing STEC counts by 5 logs at 24 h and 6.1 logs at 48 h. However, at 12 h, Activate DA at 2% reduced STEC counts by 4.2 logs, whereas at 1% the population reduction was only 3.2 logs at the same time point. Activate US WD-MAX at 0.5% resulted in a reduction of 5.4 logs at 24 h followed by 6.5 logs at 48 h. At 1%, a similar trend was also observed with a reduction of 5.3 logs at 24 h and 6.5 logs at 48 h. At 12 h, Activate US WD-MAX at 1% reduced STEC counts by 4.4 logs, whereas at 0.5% the log reduction was only 3.6 logs at the same time point. Overall, there was no difference among the two treatments Activate DA and Activate US WD-MAX across the different concentrations tested (Table 4). Considering the log reductions over time, Activate US WD-MAX was effective at a lower dose (0.5%) than the other treatments against STEC. For the challenge study against A. flavus, log reductions were not observed consistently over time (1 to 35 days) with the inclusion of Activate DA or Activate US WD-MAX as an oil-based coating on kibbles . An average increase of 0.7 logs (from an initial load of 4 logs) was observed for the untreated through day 21 and then a reduction of 0.5 logs by day 35. Overall, the treatments Activate DA and Activate US WD-MAX reduced A. flavus counts over time (p < 0.05) when compared to the untreated (Table 5). The inclusion of Activate DA at 1% decreased A. flavus counts by 0.4-0.9 logs by day 7 and 0.5 logs by day 35. Activate DA at 2% also decreased A. flavus counts by 0.9 logs at day 7 and 14, and 0.4 logs at day 35. The inclusion of Activate US WD-MAX at 0.5% resulted in a reduction of 1.4 logs at day 7 followed by an increase of 0.5 logs at day 28 and then a 0.4 log reduction at day 35. At 1%, Activate US WD-MAX showed a reduction of 1.5 logs at day 7 followed by an increase of 0.6 logs at day 28 and then a 0.4 log reduction at day 35. Overall, there was no difference among the two treatments Activate DA and Activate US WD-MAX across the different concentrations tested against A. flavus (Table 5). Consistent log reductions of A. flavus were not observed over time; however, the increase in mold counts was retarded due to the organic acid treatments compared to the untreated. Activate DA and Activate US WD-MAX exhibited a fungistatic effect during kibble storage but not a fungicidal effect. The decimal reduction time known as D-value is the time (in hours) required to achieve 1-log reduction in bacterial counts. For Salmonella, Activate DA had D-values ranging from 1.01 to 1.05 h (61-63 min) and Activate US WD-MAX had D-values from 1.03 to 1.08 h (62-65 min) (Table 6). However, there was no difference between the two treatments (p > 0.05). Similarly, for STEC, Activate DA had D-values ranging from 0.98 to 1.02 h (59-62 min) and Activate US WD-MAX had D-values from 1.02 to 1.05 h (62-63 min), and there was no difference between the two treatments (p > 0.05). D-values were not calculated for the effect of organic acid treatments against A. flavus as it did not show consistent log reduction of mold counts. Hence, Activate US WD-MAX at the lowest concentration of 0.5% can be considered effective against Salmonella and STEC on dry pet food kibbles. The untreated and treated (Activate DA and US WD-MAX) kibbles were inoculated with Salmonella (~8 log CFU/mL) on day 1, day 30 and day 90 to investigate the residual effect of the treatments over storage time . The untreated samples had an initial load of ~7 log CFU/mL of Salmonella. Activate US WD-MAX at 0.5% and 1% had a residual antimicrobial effect in the kibbles over time. After inoculation on day 30, analyzing it for Salmonella survival resulted in reductions of Salmonella by 4.3-5.4 logs. Similarly, Activate DA at 1% and 2% had a residual antimicrobial effect in the kibbles over time (on day 30) and resulted in reductions of Salmonella by 3.9-5.2 logs. Inoculating the treated kibbles with Salmonella on day 90 resulted in a similar decrease in bacterial counts by 4-5 logs for both Activate DA and Activate US WD-MAX. Compared to the untreated samples, the organic acid treatments reduced Salmonella counts (p < 0.05); however, there was no difference between the two treatments Activate DA and Activate US WD-MAX (p > 0.05) (Table 7). Therefore, both organic acid mixtures can be considered as having a residual antimicrobial effect for 90 days at concentrations 0.5-2% to mitigate the repeated post-processing kibble exposure to Salmonella. 4. Discussion The MIC, MBC and MFC tests are in vitro antimicrobial/antifungal susceptibility tests that are usually performed to evaluate the sensitivity of an organism to an antimicrobial or antifungal agent such as an antibiotic or chemical preservative. There have been only a few studies that investigated the antimicrobial properties of organic acid mixtures including HMTBa. Guo-zheng et al. evaluated Activate WD against Staphylococcus aureus, Escherichia coli, Salmonella pullorum and Campylobacter jejuni and determined the minimum inhibitory concentration to be 0.3% and minimum bactericidal concentration to be 0.6%. Parker et al. evaluated Activate WD at 0.04% and 0.08% in drinking water for poultry and found a reduction in the horizontal transmission of Salmonella in the broiler chickens. These studies show that the organic acid mixtures containing HMTBa have potential antimicrobial effects in feed. From the MIC, MBC and MFC assays in this study, Activate DA (dry formula) and Activate US WD-MAX (wet formula) were both found to be effective against Salmonella, STEC and A. flavus in nutrient broth (Table 2). From Table 2, Activate US WD-MAX had consistently lower MIC, MBC and MFC results when compared to Activate DA, and hence was slightly more antibacterial against Salmonella and STEC and more antimycotic against A. flavus than Activate DA. The solubility of an antimicrobial/chemical in the MIC assay plays an important role in reacting with the target organism and its inhibition of growth in the assay. Activate DA is a dry powder which has some solubility issues. Our work was limited by a solubility of 1% in water at 25 degC. There were concerns during the conduct of this research regarding the uniformity in water solution at the various concentrations tested for MIC. It was a powder with a fine particle size after laboratory hammer milling to facilitate this study. The MIC, MBC and MFC of organic acid mixtures against microorganisms depend on several variables including composition and concentration of their components, physical and chemical properties and the culture conditions for the test microorganisms, and thus a comparison of the results with those of other studies involving organic acids is not simple. Iba et al. and Franco et al. reported that the acidification of animal feeds by adding organic acids and organic salts can help control the growth of bacteria and fungi that reduce feed quality and produce toxins. The reduction in bacterial numbers in the animal's gut and improvement in the balance of gut microflora can potentially have an important prophylactic effect, reducing the opportunity for infection associated with the proliferation of dangerous pathogens in the gut . Certain feed additive organic acid mixtures include propionic, formic and butyric acid, which have been used in the past for their antimicrobial properties. These additives have previously been used as a method to control pathogens such as Salmonella and E. coli in poultry feed and other matrices . Of the organic acid mixtures evaluated in this study, Activate DA is a mixture of HMTBa, fumaric acid and benzoic acid, and Activate US WD-MAX is a mixture of HMTBa, lactic acid and phosphoric acid. This combination of acids should have an antimicrobial effect, potentially, as the individual acids in these mixtures were already known to have preservative effects. It is to be noted that the pH of Activate DA is slightly higher (pH 3) than Activate US WD-MAX (pH 2), and so Activate US WD-MAX has a slightly better acidification property than Activate DA. Activate US WD-MAX, being a liquid formula, is also very miscible in water at all concentrations. Comparing the composition of the mixtures tested, both primarily contain HMTBa, but the secondary constituents differ: fumaric acid and benzoic acid for Activate DA, and lactic acid and phosphoric acid for Activate US WD-MAX. Lactic and phosphoric acids have stronger acidification effects than fumaric acid, which also has solubility issues, and these could be the reasons why Activate US WD-MAX has slightly better antimicrobial properties and hence lower MIC, MBC and MFC than Activate DA. The MICs/MBCs of the organic acid mixtures were similar for Salmonella and STEC, while their MIC/MFC against A. flavus was higher by 1%. This is because molds are more robust organisms than bacteria, and the Aspergillus species was found to be more tolerant to pH and fluctuations in growth conditions . So, these molds require greater antimicrobial effects possibly at a higher concentration of the antimicrobial to inhibit their growth. According to Pankey and Sabath , when the ratio of MBC to MIC is less than or equal to 4, the antimicrobial can be considered bactericidal. Since the MBC and MFC to MIC ratios of organic acid mixtures in this study were in the range of 1 to 2, they can be considered as antimicrobial and should be effective against Salmonella, STEC and A. flavus when applied in food systems . The concentrations of antimicrobials tested effectively in a nutrient broth may not be adequate to be tested in a food substrate. This is because several components in the food system such as mineral salts can buffer the effects of antimicrobials. Therefore, it is common practice to test slightly higher concentrations of the antimicrobial in food systems. As the MIC/MBCs of Activate DA were 0.5-1%, we decided to test 1% and 2% of Activate DA for the food substrate kibble challenge study in the challenge studies. Similarly, Activate US WD-MAX had MIC/MBCs of 0.4-0.5% in the broth assay, so we tested it at 0.5% and 1% in kibbles for the challenge studies. In another study we found that Activate DA at 2% and Activate US WD-MAX at 1% were effective at reducing Salmonella counts on food contact surfaces by up to 3.2 logs and 3.5 logs respectively. In this study, for the Salmonella and STEC challenge studies, the initial load of bacteria inoculated to the kibbles was ~8 logs. For the untreated samples, while enumerating bacterial loads at the first time point after inoculation, i.e., 2 h, a reduction of about 1 log was observed . This was due to the limitation in recovery of bacterial loads from the food substrate during analysis. The bacterial loads remained consistent until about 48 h and then started to decline and decreased to about 1 log CFU/mL by 60 days. This was because kibbles are low water activity foods and incubation at 37 degC over an extended period can cause drying of the food substrate which inhibits bacterial growth by desiccation. At 2 h, the organic acid mixtures caused reductions of 2-2.5 logs for Salmonella and STEC, and by 12 h, the reductions ranged from 3.6 to 5.4 logs. By 24 h, in the treated kibbles, the organic acid mixtures reduced Salmonella loads by 5-6.5 logs and STEC loads by 5-5.4 logs. The organic acid mixture treatments greatly increased the bacterial count reductions within a short time (12 h) compared to the untreated samples. The use of multiple serovars of Salmonella and STEC as 'cocktail inoculums' in this study further corroborated the strong antimicrobial activity of the two organic acid mixtures. One might argue that even for the untreated samples there was consistent log reduction in bacterial counts (up to 7 logs) over the 60-day period due to reasons mentioned earlier, and packaged pet food kibbles would normally reach consumers only after several days or weeks. However, we believe that the rapid log reductions of bacterial counts due to the organic acid mixtures help in mitigating cross-contamination during processing and human handling in the processing facility and make the product safer. Also, during changes in storage conditions of the pet food products, such as humid environment, the untreated products may have pathogenic bacterial growth and treatment with these organic acid mixtures can be an effective mitigation strategy. For the A. flavus challenge study, pronounced log reductions of mold counts were not observed in the case of Activate DA and Activate US WD-MAX used as a coating on pet food kibbles . While the untreated sample had a slight increase in mold counts by 0.5 logs by 28 days, the organic acid-treated samples had a slight decrease in mold counts by 1 log by 7 days and then an increase of 0.6 logs in mold counts. As the treated samples differed from the untreated (p < 0.05) due to the fact that the increase in mold counts (as seen in the untreated samples) was slightly reduced by the presence of Activate DA and Activate US WD-MAX in the treated samples, we believe the organic acid mixtures had a fungistatic effect on A. flavus in the kibbles. Another reason for the static log counts of A. flavus over the 35-day period could be because the blank kibbles used in this study already contained 2% vinegar (acetic acid) in their formula (Table 1). Vinegar is typically added as a clean-label preservative ingredient in pet foods for microbial shelf-life stability . It also acts as a mold-inhibiting ingredient, similar to phosphoric acid which is used as a mold inhibitor in semi-moist pet foods. We propose conducting a mold challenge study with Activate DA and Activate US WD-MAX as the coating on kibbles that were manufactured without the inclusion of acids such as vinegar or phosphoric acid to test the effects of these mixtures exclusively on A. flavus, with no interference or synergism from other acids. Furthermore, in this challenge study, A. flavus counts on the untreated sample did not increase continuously during the 35-day incubation period. There was only a 0.5-log increase by day 35 despite no addition of organic acid mixtures. This was because pet food kibbles are low water activity foods (0.50 aw) and incubation at 25 degC for 35 days can result in drying of the food substrate, and hence prevent a mold count increase. Additionally, as it had been mold-challenged with a 4-log inoculum, there can be a competition of nutrients for the growth of mold colonies, which slows their growth. The D-values (Table 6) for Salmonella and STEC exposed to Activate DA and Activate US WD-MAX represent the times required for a 10-fold (90% or 1 log) destruction of the initial viable population of the pathogen. The linear regression model y = a + bx was best fit for the treatments with higher R2 values (>0.70) due to pronounced log reductions of the bacterial counts. The D-values for Salmonella and STEC were about 1 h in this study and there was no difference between the treatments Activate DA and Activate US WD-MAX (p > 0.05). Because of the absence of a fungicidal effect or a steady log reduction of mold counts in the case of A. flavus, the linear model y = a + bx was not fit to the regression over time, and hence D-values were not calculated for A. flavus. The residual antimicrobial effect of Activate DA and Activate US WD-MAX coated on pet food kibbles during a storage time from day 1 to day 90 was evaluated , and the treated kibbles when exposed to Salmonella on day 30 resulted in a 3.9-5.4 log reduction of the bacteria. On day 90, the Salmonella counts were reduced by 3-4.6 logs due to the organic acid treatment's residual effect during storage. Although not significant, the log reductions on day 90 were slightly lower when compared to day 30, probably because of the buffering effect of other ingredients in the kibble on the organic acids. The mechanism of the antibacterial activity of organic acids against Gram-negative bacteria such as Salmonella has been described in previous research studies . Organic acids in their undissociated and uncharged state are capable of bypassing bacterial cell membranes due to their lipophilic nature. HMTBa has a pKa value of 3.53 (with one active functioning group, i.e., carboxyl group) and being an organic acid remains undissociated in a low pH range (3-5 pH) and thereby able to diffuse into cell membranes of bacteria. Upon entering the more alkaline interior of a bacterium, the anion and proton from organic acids may have deleterious effects on the bacterium by increasing osmotic stress and disrupting important biomolecule synthesis, which finally cause bacterial death. One shortcoming in the method that we realized later in the study was not measuring the water activity after coating the kibbles with the oil plus organic acid mixtures. As it was not a semi-moist pet food product but a dry kibble product with low initial aw (0.50), the coating was performed at a time later than the manufacturing of kibbles that were otherwise stable. We propose measuring water activity following kibble coating in future experiments to better understand if the antimicrobial agents dissolve in these dry products. While the two organic acid mixtures Activate DA and Activate US WD-MAX showed promising antibacterial properties against Salmonella and STEC when applied as a coating on pet food kibbles and have a strong residual antimicrobial effect over extended storage times on the kibbles, their acceptability to pets needs to be evaluated. Even though these have been in use as supplements in animal feed, we speculate that coating them on kibbles may have a strong effect of smell/flavor to the pets, which may lead to differences in acceptability. Dhakal and Aldrich reported that coating dog food kibbles with a combination of medium chain fatty acids (caproic, caprylic and capric) was effective in mitigating Salmonella but reduced the acceptability of the kibbles due to strong aroma of the organic acids. Therefore, for this study, we propose conducting palatability tests, such as a two-bowl forced choice evaluation test , to determine the acceptability of these organic acid-coated kibbles by pets. In case of any changes in acceptability of these kibbles, a palatant may be necessary as a further coating to mask the aroma or flavor of the organic acid mixtures when applied to kibbles. Recently, combinations of organic acids and medium chain fatty acids (e.g., lauric acid) have also demonstrated synergistic benefits on animal intestinal health due to their antibacterial properties . For future work, we propose investigating the synergistic effects of HMTBa with medium chain fatty acids as coating on pet food kibbles to control pathogen recontamination. Furthermore, it is more likely for pathogens such as Salmonella to be reintroduced to kibbles post-processing via dust, flies or employee-handling as opposed to a liquid contamination source. Therefore, we propose conducting challenge studies using a dry inoculum of Salmonella as water-based inoculum can have aversion to the oil-based surface coating on the kibbles. Additionally, it would be interesting to measure the production of aflatoxins in future challenge studies with A. flavus to study whether the mold is more sensitive to organic acids inhibiting mycotoxin production than growth. 5. Conclusions The results of this study indicate that the use of organic acid mixtures containing HMTBa, Activate DA and Activate US WD-MAX, as a coating ingredient on dry pet food kibbles showed a promising effect as a food-safe ingredient to mitigate post-processing Salmonella and STEC contamination. Being effective at a low concentration among the treatments tested and for ease of application with no solubility issues, we believe that Activate US WD-MAX at 0.5% or 1% was the most effective to be used as a kibble coating to control Salmonella and STEC contamination. Acknowledgments The authors would like to thank Novus International and the Department of Grain Science and Industry (Kansas State University, Manhattan, KS) for their support in this study. Author Contributions A.D.--conceptualization, data gathering, analysis, writing--original draft preparation; J.D.--conceptualization, supervision, funding acquisition; B.S.--conceptualization, supervision, writing--review and editing of original draft; C.G.A.--conceptualization, supervision, funding acquisition, writing--review and editing of original draft. All authors have read and agreed to the published version of the manuscript. Institutional Review Board Statement Not applicable. Informed Consent Statement Not applicable. Data Availability Statement Data will be provided upon reasonable request by author Aiswariya Deliephan. Conflicts of Interest The authors declare no conflict of interest. Figure 1 Comparison of chemical structures of HMTBa (methionine hydroxy analogue) and amino acid methionine. Figure 2 Mean logarithmic counts (log CFU/mL) of Salmonella in pet food kibble treatments with inclusion of organic acid mixtures Activate DA at 1% and 2% and Activate US WD-MAX at 0.5% and 1% concentrations as coating in comparison with untreated (0% organic acid). Furthermore, 0 h denotes initial load of Salmonella in inoculum before inoculation to the kibble. Salmonella counts at day 30 and day 60 are 1 log CFU/mL (not shown). A negative control consisted of canola oil-coated kibble (no organic acid, no inoculation). Limit of detection is 1 log CFU/mL for this study. Figure 3 Mean logarithmic counts (log CFU/mL) of E. coli (STEC) in pet food kibble treatments with inclusion of organic acid mixtures Activate DA at 1% and 2% and Activate US WD-MAX at 0.5% and 1% concentrations as coating in comparison with untreated (0% organic acid). Furthermore, 0 h denotes initial load of STEC in inoculum before inoculation to the kibble. STEC counts at day 30 and day 60 are 1 log CFU/mL (not shown). A negative control consisted of canola oil-coated kibble (no organic acid, no inoculation). Limit of detection is 1 log CFU/mL for this study. Figure 4 Mean logarithmic counts (log CFU/mL) of A. flavus in pet food kibble treatments with inclusion of organic acid mixtures Activate DA at 1% and 2% and Activate US WD-MAX at 0.5% and 1% concentrations as coating in comparison with untreated (0% organic acid). Furthermore, 0 h denotes initial load of A. flavus in inoculum before inoculation to the kibble. A negative control consisted of canola oil-coated kibble (no organic acid, no inoculation). Limit of detection is 1 log CFU/mL for this study. Figure 5 Mean logarithmic counts of Salmonella in pet food kibble treatments with inclusion of organic acid mixtures Activate DA at 1% and 2% and Activate US WD-MAX at 0.5% and 1% concentrations as coating, in comparison with untreated (0% organic acid) to investigate residual effect of treatments over 1, 30 and 90 days after repeated exposure to Salmonella. A negative control consisted of canola oil-coated kibble (no organic acid, no inoculation). Limit of detection is 1 log CFU/mL for this study. animals-13-00877-t001_Table 1 Table 1 Formulation of uncoated dry pet food kibbles. Ingredient Percent w/w Chicken meal 42.6 Corn meal 17.7 Wheat flour 17.7 Rice flour 17.7 Vinegar 2.0 Salt 0.5 Potassium chloride 0.3 Choline chloride 0.3 Dicalcium phosphate 0.3 Calcium carbonate 0.3 Trace mineral premix 0.2 Vitamin premix 0.1 Fish oil 0.1 Taurine 0.1 Natural antioxidant 0.1 Total 100.0 animals-13-00877-t002_Table 2 Table 2 Minimum inhibitory concentrations (MICs), minimum bactericidal concentrations (MBCs) and minimum fungicidal concentrations (MFCs) of organic acid mixtures Activate DA and Activate US WD-MAX in nutrient broth (TSB or PDB) against Salmonella, Escherichia coli (STEC) and Aspergillus flavus. A positive control consisted of bacterial or fungal inoculum only (no treatment), and a negative control consisted of tryptic soy broth (TSB) or potato dextrose broth (PDB) only (no treatment, no inoculation). Treatment Salmonella E. coli (STEC) A. flavus MIC 1 (%) MBC 1 (%) MIC 1 (%) MBC 1 (%) MIC 1 (%) MFC 1 (%) Activate DA 0.5 1.0 1.0 1.0 2.0 2.0 Activate US WD-MAX 0.4 0.5 0.4 0.5 1.0 1.0 1 The most recurring value of three replicates is reported as MIC, MBC or MFC. animals-13-00877-t003_Table 3 Table 3 Mean logarithmic reduction of Salmonella counts in pet food kibble treatments with inclusion of organic acid mixtures Activate DA at 1% and 2% and Activate US WD-MAX at 0.5% and 1% concentrations as coating, in comparison with untreated (0% organic acid). The logarithmic reductions were calculated as the difference between Salmonella counts in the inoculum (8 log CFU/mL) and on the kibbles (untreated and treated). A negative control consisted of canola oil-coated kibble (no organic acid, no inoculation). Time Salmonella Log Reduction (log CFU/mL) (Mean +- SE) 1,2,3 Untreated Activate US WD-MAX Activate DA 0.0% 0.5% 1.0% 1.0% 2.0% 0 h 0.0 +- 0.0 a,A 0.0 +- 0.0 b,A 0.0 +- 0.0 b,A 0.0 +- 0.0 b,A 0.0 +- 0.0 b,A 2 h 1.1 +- 0.3 a,B 2.2 +- 0.3 b,B 2.2 +- 0.4 b,B 2.2 +- 0.4 b,B 2.2 +- 0.6 b,B 12 h 1.1 +- 0.4 a,C 4.3 +- 0.3 b,C 5.4 +- 0.3 b,C 4.2 +- 0.4 b,C 5.2 +- 0.4 b,C 24 h 1.1 +- 0.7 a,C 6.5 +- 0.3 b,C 6.3 +- 0.5 b,C 5.1 +- 0.1 b,C 5.0 +- 0.5 b,C 48 h 3.3 +- 0.0 a,D 7.0 +- 0.7 b,D 7.0 +- 0.5 b,D 7.0 +- 0.5 b,D 7.0 +- 0.1 b,D 72 h 6.3 +- 0.3 a,D 7.0 +- 0.3 b,D 7.0 +- 0.1 b,D 7.0 +- 0.3 b,D 7.0 +- 0.3 b,D 30 d 7.0 +- 0.5 a,D 7.0 +- 0.1 b,D 7.0 +- 0.7 b,D 7.0 +- 0.6 b,D 7.0 +- 0.4 b,D 60 d 7.0 +- 0.5 a,D 7.0 +- 0.4 b,D 7.0 +- 0.6 b,D 7.0 +- 0.7 b,D 7.0 +- 0.6 b,D 1 Each mean is based on n = 3 replications. 2 Means among the treatments across concentrations followed by different letters in lower case are significantly different (p < 0.05, Tukey's test). 3 Within each treatment, means among different times followed by different letters in upper case are significantly different (p < 0.05, Tukey's test). animals-13-00877-t004_Table 4 Table 4 Mean logarithmic reduction of E. coli (STEC) counts in pet food kibble treatments with inclusion of organic acid mixtures Activate DA at 1% and 2% and Activate US WD-MAX at 0.5% and 1% concentrations as coating, in comparison with untreated (0% organic acid). The logarithmic reductions were calculated as the difference between E. coli counts in the inoculum (8 log CFU/mL) and on the kibbles (untreated and treated). A negative control consisted of canola oil-coated kibble (no organic acid, no inoculation). Time E. coli (STEC) Log Reduction (log CFU/mL) (Mean +- SE) 1,2,3 Untreated Activate US WD-MAX Activate DA 0.0% 0.5% 1.0% 1.0% 2.0% 0 h 0.0 +- 0.0 a,A 0.0 +- 0.0 b,A 0.0 +- 0.0 b,A 0.0 +- 0.0 b,A 0.0 +- 0.0 b,A 2 h 1.1 +- 0.2 a,B 2.6 +- 0.6 b,B 2.7 +- 0.6 b,B 2.1 +- 0.1 b,B 2.2 +- 0.4 b,B 12 h 1.1 +- 0.4 a,C 3.6 +- 0.5 b,C 4.4 +- 0.6 b,C 3.2 +- 0.1 b,C 4.2 +- 0.4 b,C 24 h 1.3 +- 0.2 a,D 5.4 +- 0.3 b,D 5.3 +- 0.4 b,D 5.1 +- 0.1 b,D 5.0 +- 0.2 b,D 48 h 3.2 +- 0.4 a,E 6.5 +- 0.3 b,E 6.5 +- 0.4 b,E 5.7 +- 0.3 b,E 6.1 +- 0.6 b,E 72 h 5.4 +- 0.5 a,F 7.0 +- 0.8 b,F 7.0 +- 0.2 b,F 7.0 +- 0.1 b,F 7.0 +- 0.5 b,F 30 d 7.0 +- 0.5 a,F 7.0 +- 0.3 b,F 7.0 +- 0.5 b,F 7.0 +- 0.2 b,F 7.0 +- 0.4 b,F 60 d 7.0 +- 0.2 a,F 7.0 +- 0.2 b,F 7.0 +- 0.7 b,F 7.0 +- 0.4 b,F 7.0 +- 0.7 b,F 1 Each mean is based on n = 3 replications. 2 Means among the treatments across concentrations followed by different letters in lower case are significantly different (p < 0.05, Tukey's test). 3 Within each treatment, means among different times followed by different letters in upper case are significantly different (p < 0.05, Tukey's test). animals-13-00877-t005_Table 5 Table 5 Mean logarithmic reduction of Aspergillus flavus counts in pet food kibble treatments with inclusion of organic acid mixtures Activate DA at 1% and 2% and Activate US WD-MAX at 0.5% and 1% concentrations as coating, in comparison with untreated (0% organic acid). The logarithmic reductions were calculated as the difference between A. flavus counts in the inoculum (4 log CFU/mL) and on the kibbles (untreated and treated). A negative control consisted of canola oil-coated kibble (no organic acid, no inoculation). Time A. flavus Log Reduction (log CFU/mL) (Mean +- SE) 1,2,3 Untreated Activate US WD-MAX Activate DA 0.0% 0.5% 1.0% 1.0% 2.0% 0 h 0.0 +- 0.0 a,B 0.0 +- 0.0 b,B 0.0 +- 0.0 b,B 0.0 +- 0.0 b,B 0.0 +- 0.0 b,B 1 d 0.4 +- 0.1 a,C 0.4 +- 0.6 b,C 0.4 +- 0.4 b,C 0.4 +- 0.4 b,C 0.4 +- 0.2 b,C 3 d 0.5 +- 0.4 a,C 0.5 +- 0.4 b,C 0.4 +- 0.2 b,C 0.4 +- 0.2 b,C 0.4 +- 0.7 b,C 7 d 0.5 +- 0.6 a,D 1.4 +- 0.5 b,D 1.5 +- 0.1 b,D 0.9 +- 0.5 b,D 0.9 +- 0.6 b,D 14 d -0.1 +- 0.7 a,C 0.6 +- 0.4 b,C 0.6 +- 0.3 b,C 0.6 +- 0.4 b,C 0.9 +- 0.4 b,C 21 d -0.7 +- 0.3 a,A -0.6 +- 0.6 b,A -0.6 +- 0.3 b,A -0.6 +- 0.7 b,A -0.4 +- 0.3 b,A 28 d -0.4 +- 0.2 a,B -0.5 +- 0.7 b,B -0.6 +- 0.5 b,B 0.4 +- 0.3 b,B 0.5 +- 0.6 b,B 35 d 0.5 +- 0.2 a,C 0.4 +- 0.8 b,C 0.5 +- 0.5 b,C 0.5 +- 0.4 b,C 0.4 +- 0.7 b,C 1 Each mean is based on n = 3 replications. 2 Means among the treatments across concentrations followed by different letters in lower case are significantly different (p < 0.05, Tukey's test). 3 Within each treatment, means among different times followed by different letters in upper case are significantly different (p < 0.05, Tukey's test). animals-13-00877-t006_Table 6 Table 6 Linear regression (linear model y = a + bx) parameters of logarithmic reduction of Salmonella and E. coli (STEC) counts in pet food kibble treatments with inclusion of organic acid mixtures Activate DA at 1% and 2% and Activate US WD-MAX at 0.5% and 1% concentrations as coating. Treatment Concentration (%) Linear Regression Parameters a b R 2 D-Value 1,2 (h) Salmonella: Activate US WD-MAX 0.5 7.24 -0.97 0.77 1.03 1.0 6.97 -0.93 0.73 1.08 Activate DA 1.0 7.50 -0.99 0.83 1.01 2.0 7.22 -0.95 0.78 1.05 E. coli (STEC): Activate US WD-MAX 0.5 7.51 -0.98 0.86 1.02 1.0 7.28 -0.95 0.84 1.05 Activate DA 1.0 7.93 -1.02 0.91 0.98 2.0 7.58 -0.98 0.87 1.02 a and b are linear regression parameters; a = intercept; b = slope. 1 D-value (-1/b) shows the decimal reduction time (in hours) for 1-log reduction of Salmonella and E. coli counts. 2 D-values were not calculated for treatments showing no log reduction of A. flavus counts over time. animals-13-00877-t007_Table 7 Table 7 Mean logarithmic reduction of Salmonella counts in pet food kibble treatments with inclusion of organic acid mixtures Activate DA at 1% and 2% and Activate US WD-MAX at 0.5% and 1% concentrations as coating, in comparison with untreated (0% organic acid), showing residual effect of treatments over 1, 30 and 90 days during storage. The logarithmic reductions were calculated as the difference between Salmonella counts in the inoculum (8 log CFU/mL) and on the kibbles (untreated and treated). A negative control consisted of canola oil-coated kibble (no organic acid, no inoculation). Time Salmonella Log Reduction (log CFU/mL) (Mean +- SE) 1,2,3 Untreated Activate US WD-MAX Activate DA 0.0% 0.5% 1.0% 1.0% 2.0% 0 h 0.0 +- 0.0 a,A 0.0 +- 0.0 b,A 0.0 +- 0.0 b,A 0.0 +- 0.0 b,A 0.0 +- 0.0 b,A 1 d 0.9 +- 0.8 a,B 4.6 +- 0.7 b,B 5.3 +- 0.7 b,B 4.2 +- 0.9 b,B 5.2 +- 0.5 b,B 30 d 1.0 +- 0.9 a,B 4.3 +- 1.0 b,B 5.4 +- 0.5 b,B 3.9 +- 0.4 b,B 5.2 +- 0.7 b,B 90 d 1.0 +- 0.8 a,B 3.0 +- 0.6 b,B 3.8 +- 0.4 b,B 3.2 +- 0.7 b,B 4.6 +- 0.4 b,B 1 Each mean is based on n = 3 replications. 2 Means among the treatments across concentrations followed by different letters in lower case are significantly different (p < 0.05, Tukey's test). 3 Within each treatment, means among different times followed by different letters in upper case are significantly different (p < 0.05, Tukey's test). Disclaimer/Publisher's Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). 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PMC10000159

Prosociality occurs in many species and is likely to be a crucial factor for the survival of group-living animals. Social feedback is an important mechanism for the coordination of group decisions. Since group-living animals with specific personality axes, i.e., boldness, are known to provide certain benefits for their group, bold actions might receive more prosocial feedback than other actions. Our case study aims to determine whether bold behaviour, i.e., novel object interaction (Nobj), might be answered more frequently with prosocial behaviours. We investigated the differences in the frequency of occurrence in prosocial behaviours after three different individual actions in two groups of grey wolves. We aim to outline the development of a social reward behavioural category as part of social feedback mechanisms. We used Markov chain models for probability analyses, and a non-parametric ANOVA to test for differences between the influences of individual behaviours on the probability of a prosocial behaviour chain. We additionally tested for the potential influences of age, sex and personality on the frequency of Nobj. Our results suggest that bold interactions are more often responded to with prosocial behaviour. Bold behaviour might be more often socially rewarded because of its benefits for group-living animals. More research is needed to investigate whether bold behaviour is more frequently responded to prosocially, and to investigate the social reward phenomenon. social reward wolves prosociality mammals social behaviour Canis lupus lupus novel object behavioural chains German Research Foundation512648189 Open Access Publication Fund of the Thuringian UniversityState Library JenaWe acknowledge support by the German Research Foundation Project-Nr. 512648189 and the Open Access Publication Fund of the Thuringian University and State Library Jena. pmc1. Introduction Personality traits, which are consistent inter-individual differences in behaviour within populations, are a widespread phenomenon across various animal species . Studies have shown that mammals , birds , fish , arthropods , amphibians and cephalopods exhibit consistent individual differences in behavioural traits, which are expressed in specific situations . Extraversion and boldness are two of the most well-known phenomena in animal personality research. Boldness describes a consistent difference between individuals in their response to perceived risk . Boldness is considered to be part of a major 'proactive-reactive' axis of personality variation, where boldness is one of a suite of behaviours which includes exploration, activity and aggression, which correlate positively with each other . When presented with a startling stimulus or a novel object, individuals may differ consistently in their responses over repeated observations, a behavioural complex that might influence species dispersal as well as the approach to obtaining food in a task involving a novel object. Bolder individuals might have greater food intake when foraging , or take more risks to acquire food, investigate novel objects, or explore new environments. Previous research has shown that bolder individuals may be more likely to lead , whereas shy individuals may be more likely to group and to respond to the decisions of their bolder conspecifics . As bolder individuals are more prone to be neophilic and to take risks, they potentially are beneficial for the rest of their group, as they might serve as foragers, investigators, guardians or warners, since each individual profits from such division of labour. Across taxa, individuals show the cognitive ability to use their observations of the social interactions of others to inform their own behaviours, including several primate species , ravens , hyenas and fish . While it has been shown that individuals observe each other and react to these observations, we do not currently understand how individuals integrate this information on the outcomes of their own interactions with observations of others' interactions, on their future actions. Social feedback, the tendency of a group to answer prosocially or agonistically to another group member's individual behaviour, is an important mechanism for the coordination of group decisions. We suggest that there might be a special type of social feedback: social reward. We define social reward as prosocial behaviours that are shown in direct response to a desirable and group-beneficial behaviour, i.e., bold behaviour, and aim to encourage the behaviour. This reward mechanism could be partly intended in order to increase the frequency of the occurrence of the displayed behaviour, or to encourage other group members to engage in the behaviour. Bold behaviours may be risky for individuals, but they may also prove to be beneficial to the group, and might therefore be supported by social feedback. In distinction to prosocial behaviour, which in addition to confirmation, has various other functions such as reproduction, rank verification, group cohesion, togetherness etc., we define social reward behaviour as a behavioural category that exclusively serves positive social feedback and is thus part of the social feedback mechanism. Social reward, as prosocial behaviours sent by the group towards an individual of the group to give feedback on a previous action, could be a special tool to encourage the actions of a group member that are in the interest of the group but that cannot be carried out by other group members, such as actions that require boldness. Social feedback or social reward might be useful within groups to engage in group-benefiting actions. Emotional contagion, the transmission of emotional states from one animal to another, might also be involved in social feedback processes and group decision-making. Most studies on emotional contagion in non-human animals have focused on the transmission of negative emotional states, e.g., fear, but observations on farm animals suggest that animals may transmit both negative and positive emotions: respectively, pleasant and unpleasant emotions such as joy or fear . Pigs, for example, responded differently to being reunited with group mates who had experienced either a negative or a positive treatment. They exhibited decreased activity and exploration when group mates received a negative treatment, and increased social contact and exploration when group mates received a positive treatment . Since bolder animals, such as fast-bold explorers, show a reduced stress-induced glucocorticoid release compared to slow-shy , prosocial acts towards bold-behaving group members could also be influenced by mechanisms of emotional contagion. Here, we take a look at the relationship between bold actions and social feedback mechanisms in two groups of European grey wolves (Canis lupus lupus), to address the question of how bold behaviours can influence the frequency of prosocial interactions towards the bold individual. We observed two groups of European grey wolves in two different zoological facilities in Germany. The observations were part of a large-scale project on cooperative behaviour in canids, which also included Arctic wolves, Hudson Bay wolves and Timber wolves, as well as various other canids. 2. Materials and Methods 2.1. Study Animals Our case study included two different wolf groups, with 5 individuals per group, at Zoo Wingst and Schwarze Berge Wildlife Park (see Table 1). 2.2. Novel Object An apparatus of 1.80 m x 0.60 m x 1.20 m was installed in front of the enclosure . The purpose of the apparatus was to study foraging cooperation in Canidae; therefore, it was equipped with food tubes, ropes for pulling and food flaps that were connected to the ropes . The European grey wolf groups did not use the apparatus communally, and no cooperative food procurement was shown. 2.3. Observational Methodology Behavioural observations were made in total for 192 h from June to August 2020, and from May to June 2021, via means of a rare event ("all-occurrence") sampling , which led to 242 behavioural observations and the record of 140 behaviours of interest in total. We used the ethogram of Goodmann et al. (2002) to place the behaviours that we observed to a behavioural category (see Table 2). For a detailed version of the ethogram, see . The behaviours of interest are categorised under submissive behaviour, care-giving behaviour, locomotion/exploratory behaviour and feeding . Definitions of Behaviours of Interest We define behaviours of interest, referring to those behaviours as initial behaviours (see Table 3) that have led to a social reward behaviour chain (see Table 4). The social reward behaviour chain was recorded as such if a subsequent element directly (<15 s) followed the previous behaviour. Initial behaviours commonly led to a complete or partially performed behavioural chain, which included the behavioural states listed in Table 4. Behavioural states that were performed with the same frequency and that fell under the same behavioural category in our ethogram , were classified as a combined state. 2.4. Markov Chain Modelling Markov chains quantify the dependence of an event on preceding events or an on initial state. There are several degrees of dependence. If sequencing events are independent, they are described by a zero-order Markov chain. In the case where an event depends only on the immediately preceding one, it fits a first-order Markov chain. If an event depends on the two most preceding events, it is a second-order Markov chain, and so on. We decided to assess the difference in transition from one event to another, and the probability to reach the succeeding state of the behavioural chain, depending on the initial state of the observed sequence. To simplify the analytical design, we concentrated only on a first-order Markov chain model. Transition probabilities (from initial to preceding to succeeding behaviours) were determined in all three chains using:(1) pij=aijj=16aij,j=16pij=1 where i is the initial behaviour, j is the succeeding behaviour (i and j range from 1 to 6, because there are six behavioural states in the chain), aij is the number of transitions observed from behaviour i to j, and pij is the transition probability from i to j in the Markov chain. Since the initial state influenced the succeeding state, we used an initial state vector of . We then calculated the steady-state vector (v) for our transition matrix. To determine the state of the system after one step (A1), the following applies: A1 = P - A0. P is a transfer matrix; it can be used to describe how a system changes over time from an initial-state vector A0. To determine the state of the system after one step (A1), the following applies: A1 = P - A0. Thus, to calculate any step t + 1, it only needs the transfer matrix P and the vector in the previous step At + 1. To be exact: At + 1 = P - At. Using Python version 2022.2.4 (PyCharm Community Edition), we have calculated any number of steps by multiplying the vector of the current step by the transfer matrix P to reach the next step. At some point of Pn , the vector no longer changes with the steps, and thus remains the same after each step, and so v = P - v is true. 2.5. Statistical Evaluation of Markov Chain Probabilities The results of the Markov chains were used to determine whether there were differences in the influence of the initial state on the success of the behavioural chain. We used a non-parametric ANOVA, the Kruskal-Wallis test, to determine whether differences between initial states existed. The influence on the initial state of a successful behavioural chain (reaching the succeeding state) were calculated using the R version 4.2.2. (R Development Core Team, R Foundation for Statistical Computing, Vienna, Austria), with the "ggpubr" package for Windows. Normality was assessed using the Kolmogorov-Smirnov test. The relationship between the initial state and the success score was estimated using the Kruskal-Wallis test. To compare variables with significant inter-group variability, the Wilcoxon rank-sum was used for data with non-normal normal distribution, and the Dunn's test for pairwise comparisons to test for significant means. The returned p-values were adjusted using Bonferroni corrections for multiple comparisons. The results were presented as a median. The level of statistical significance was preset to p < 0.05. 2.6. Influence Factors on the Frequency of Novel Object Interaction Factors such as age, sex and personality traits are known to influence behaviour. Therefore, we tested the influences of age, sex and the personality traits of the individuals on the frequency of novel object interaction. To determine whether there was a statistically significant mean difference in the frequency of novel object interaction between female and male European grey wolves, we ran an independent samples t-test using package "ggpubr" in R. To estimate the relationship between the age of the individuals and the frequency of novel object interaction, a Wilcoxon rank sum test was performed using the package "coin". We estimated the data of 5 female and 5 male European grey wolves from 2 different groups. For age differences in novel object interaction, we compared the age data of the same groups, creating two different age groups (mean 2.5 | 9). In order to determine the influence of personality factor on the frequency of novel object interaction, the DOGS questionnaire and the Monash Canine Personality Questionnaire--Revised (MCPQ-R) were given to the animal keepers of each group. We adapted the DOGS questionnaire and the MCPQ-R for canids living in zoological institutions, and modified the questions accordingly (see Supplementary Materials). The DOGS personality questionnaire includes the factors of trainability, sociability, extraversion and calmness. The MCPQ-R contains the factors extraversion, self-assuredness/motivation, training focus, amicability and neuroticism. Since the personality factors of the questionnaires are similar, we grouped the factors extraversion/extraversion and sociability/amicability into personality dimensions. Since the trainability factor contains items of playfulness and intelligence, and is comparable to the Big Five dimension of Openness , we split this factor into two factors: Curiosity/Openness and Playfulness. Curiosity refers to an interest in new things, perceptiveness, learnability and cognitive flexibility. Playfulness refers to social play with conspecifics, playful interest in people and object-related play (see Table 5). We also calculated a David's Score (DS) as an alternative method to access dominance rank. DS is a type of cardinal rank (dominance rank is an ordinal type of ranking) that is calculated from an individual's proportion of wins and losses in relation to the wins and losses of its opponents; ranging from -3 to 3 for triads, where -3 represents a maximum proportion of losses and 3 represents a maximum proportion of wins . The DS was combined with the personality traits as a possible influencing factor on the frequency of novel object interaction. As per Ley et al. and Turcsan et al. , the raw scores for each adjective within each personality factor subscale were summed and divided by the maximum score possible for the subscale. The result was converted to a percentage, thereby creating a percentage score for each of the five personality factors for every individual. To evaluate the influence of the personality factors and DS on the frequency of novel object interaction, a correlation matrix was created, and a multiple correlation analysis was performed. The multiple correlation analysis was carried out using the package "Hmsic". A linear regression analysis was then performed using the packages "dplyr", "broom" and ggpubr". 3. Results 3.1. Observations Despite the animals' neophobia and object-related fear, four individual group members showed repeated approaches and exploratory behaviours towards the novel object (max. 16, min. 1). Some group members--two in one group, three, partially, in another--independently approached the novel object directly and interacted with it by sniffing on it or pulling the rope located in the enclosure. There was only one animal at a time approaching the apparatus while the others kept distance, with a maximum of 14 interactions of a single individual in one group, and 16 interactions of another individual in the other group. After pulling, the animals immediately left the vicinity of the novel object and returned to their group. When returning to the group, the animals that interacted directly with the apparatus were welcomed by the group, while the animals that were in the close-up range as observers were not greeting, but they contributed to group formation. The "interactors'" muzzles were licked, they were greeted with a tail wag, the muzzle was licked by every other group member, and submissive behaviour was shown, after which the group retreated in a body. This behavioural chain was shown after each direct approach to the novel object, and was uniform in sequence in both wolf groups. This behavioural chain reinforced and increased the approach behaviour to the novel object, but did not reduce object-related fear sufficiently enough for observations of foraging cooperation. In one group, these behaviours seemed to result in another animal occasionally approaching the apparatus independently, which was then greeted in the same way, but were ultimately prevented from doing so by the "first interactor". Behavioural chain: Initial (A0) - group formation (A1) - greet. and sub. gest. (A2, A3, A4, A5) - group leaving together (A6). 3.2. Markov Probabilities of Initial Behaviours on the Succeeding Behaviours When the behavioural chain started with the initial state Nobj (Novel object interaction), the probability to reach the succeeding state was 56% after seven steps, which was the same as after 24 steps (vector [0.56, 0.44]). Starting with individual behaviours, the calculated probability of reaching the succeeding state was 08% for Indbhv1 and 01% for Indbhv2 after seven steps , and remaining steady after 8 and 24 steps, respectively. Success (A6) was defined as the completion of the behavioural chain, while failure (fail) was defined as the probability of the termination or non-completion of the behavioural chain; for example, if the chain stops at an any state before (A6). The relationship between the initial state and the success score was 0.63 for Nobj (Novel object interaction), 0.264 for Indbhv1 (run), and 0.266 for Indbhv2 (forage) (see Table 6). Since the data consist of a behavioural chain where states can merge into each other, and since states can be omitted, but succeeding states cannot merge into preceding states, it is represented as a probability chain and not a probability matrix. The difference between the initial states were only statistically significant for Nobj and Indhbv2; the statistical significance was p = 0.015 before and p = 0.04 after Bonferroni correction; for Indbhv1 and Indbhv2 = p = 1, for Nobj and Indbhv1 = p = 0.20 after Bonferroni corrections . 3.3. Influence of Sex, Age and Personality Traits on the Frequency of Novel Object Interaction The results of the relationship between sex and novel object interaction were (0.75 +- 0.957) for female wolves compared to the male group (6.60 +- 7.797). There was no statistically relevant difference between the frequency of novel object interaction in male and female wolves (t = 1.04, df = 8, p = 0.18) . For age differences in novel object interaction, we compared the age data of the same groups, creating two different age groups (mean 2.5 | 9). We found no effect of age on frequency of novel object interaction (W = 11, Z = 1.22, p = 0.33) . Personality factor means were relatively low with high standard derivation, suggesting that wolves may generally have lower scores in behavioural traits such as playfulness or motivation, or they may score lower in these categories (see Table 7). In particular, the scores for playfulness can be explained, as wolves do not exhibit paedomorphism. We performed a multiple t-test to test for the influence of sex and age on each personality dimension, no statistically relevant results were found. All personality traits showed a positive correlation with the frequency of novel object interaction, but this effect was only statistically significant for the factor "curiosity/openness" (CRS) (r = 0.73, p = 0.02). All other dimensions or factors showed a positive trend, which was marginal. The other two factors with a higher correlation to frequency of novel object interaction (NOBJ) were "playfulness" (PLAY) (r = 0.52, p = 0.15) and "motivation" (MOTV) (r = 0.54, p = 0.12). The lowest correlation was found in David's Score, the "Dominance Score" (DS) (r = 0.25, p = 0.51) . Some dimensions and factors also showed positive correlations with each other. For example, the two traits "Curiosity" and "Playfulness", which can be assigned to the factor "Trainability", are positively correlated (r = 0.72, p = 0.28). "Motivation" and "Playfulness" also show a significant positive correlation (r = 0.87, p = 0.02). "Extraversion" (EXTR) is positively correlated to "Playfulness" (r = 0.74, p = 0.02), "Motivation" (r = 0.86, p = 0.003) and "Dominance Score" (r = 0.82, p = 0.006). "Sociability" was most positively correlated to "playfulness" (r = 0.64, p = 0.06) . Due to the statistically significant positive correlation between "curiosity" and the frequency of novel object interaction (Nobj), we also conducted a linear regression analysis . The results show a moderate correlation between the personality factor "curiosity" and the frequency of novel object interaction (R2 = 0.48, df = 8, p = 0.02). 4. Discussion The application of transition matrix analyses to the study of behaviour provided more information than standard techniques would have; therefore, it proved to be useful as a tool for the probability analysis of behavioural chains. As animals are not infinite-state automata, Markov analyses might have limitations. We have to take into account that with a transition matrix, where every state can change into any other state of the matrix, different probabilities would probably have been obtained than with our behaviour chain, in which transitions only run in one direction. In addition, we excluded other possible initial behaviours that never led to completion: for example, success, of the behavioural chain, in order not to potentially drive up the significance level of the results. As we started collecting the data based on the observed chain of behaviour, a bias can be assumed. Behavioural states are generally difficult to sample adequately in the field without observer bias; therefore, we categorised the observed behaviours to established categories in the reliable ethogram to avoid possible miscategorisation or strong assumptions about the functions of the observed behaviours. Since many of the behaviours we observed are part of the ethogram, we consider categorisation errors and interpretation errors to be, for the most part, unlikely. Our results are mainly not statistically significant, which may be due to the sample size, as well as the excluded data. Nevertheless, our results show that the influence of bold behaviour, such as an interaction with a novel object, more often led to a behavioural chain of prosocial behaviours, whereas other individual behaviours that are also attention-grabbing, such as running around or foraging, did not regularly lead to the success of such a behavioural chain. Despite the low statistical power of the results, we would like to point out the relevance of the findings, as social reward is an unmentioned concept so far, but our results provide first indications that this behavioural category might exist as a subcategory of social feedback. Wolves are known to be one of the most cooperative canine species. Likely, the cooperative propensity is derived from the fact that each individual needs its other group members for survival. The group functions as a unit in which each individual collaborates in territory defence, hunting and the rearing of offspring . One example for bold behaviour benefiting the group is cooperation during intergroup conflicts. Individuals actively dealing with conflicts for the benefit of the group, regardless of possible disadvantages for themselves, provide an example of a cooperative behaviour that is costly to participants, because it involves a considerable expenditure of energy and risk of injury, and that often results in benefits to both the cooperating and noncooperating group members, in terms of increased access to contested resources . Individuals with more affiliative partners are more likely to act boldly in social contexts . It could be assumed that not only the already existing number of social affiliations, but also the expected feedback in terms of social prestige might play a role in decision-making and action processes. Expected social prestige could promote risky, bold behaviour. In addition, pleasant emotional states might result from successful risk taking, e.g., caused by a dopaminergic reward response. The likeliness of the social reward-chain after novel object interaction could also be partly explained by social support, which plays a role when group members are exposed to stress . Social support, which can express through seeking contact or rapprochement, as well as affiliative behaviour, can have a stress-reducing effect . Therefore, calming mechanisms may also have played a role as an answer to the novel object interaction, given that such was potentially stressful for generally neophobic animals such as wolves. However, due to the lower stress response in risky or novel situations that bold individuals generally show , emotional contagion may also have played a role, in that group members who felt stressed by the sight of the new object, but then perceived the approach of a bold group member, felt less stressed and thus also showed a positive response to the approach. This could also have been a factor in the social reward response. Thus, a combination of different social behaviours and mechanisms may well play a role; both the reinforcement of bold behaviour and the reduction in stress on the individual and group levels. Yet, it is known that boldness has several advantages on the group-level , but also some on the individual level, including that bold individuals often have a comparatively high status in the group, as aggressiveness, exploratory behaviour and boldness are positively correlated across individuals . In our results, extraversion and dominance score were positively correlated. Since extraversion is associated with boldness, as the shyness-boldness continuum shares traits with the extraversion-introversion axis , it is not surprising that individuals that scored higher in extraversion also had the higher status within their group, such as the highest success in conflicts. This also supports the hypothesis that boldness is positively related to status. Although boldness is generally age-dependent--as has been shown in dogs (Canis lupus familiaris) --as well as sex-dependent , our results did not show a significant influence of age or sex on bold behaviour, i.e., the frequency of novel object interaction. Possible explanations are that wolves might be less variable in their personality or behavioural traits, as with dingoes , but the results are likely also influenced by the small sample size, as well as the large age gaps between the wolves. It is difficult to observe prosocial behaviours that follow bold individual behaviours in observations of wild animals, or even in observations of the social behaviours of captive animals, without a special experimental set-up. Therefore, it is likely that social reward is more frequently exhibited in wolves, as well as mammals in general, but it may be categorised as prosocial individual behaviours. It is likely that social reward does not only occur as a result of bold actions, but refers to diverse behaviours that can be beneficial to the group, such as cooperative behaviours. To better understand animal behaviour, it is important to be able to identify it as accurately and as precisely as possible. Therefore, we suggest that the phenomenon of social feedback should be investigated in more detail, and potential subcategories such as social reward should be explored. If we can identify reward mechanisms in group processes, this may prove helpful in assessing the animals' individual and group-oriented decisions, their social ecology, and possibly also the development of group dynamics. This can be useful, for example, in assessing which animal might take which future rank in captive animal groups, to detect conflicts between captive group-living animals at an early stage, or to contribute to the understanding of the processes and the adaptive mechanisms of wild animals. By identifying behaviour that is worth rewarding, it might even be possible to draw conclusions about evolutionary drivers. As social reward and social-reward chains are not an established concept, we suggest that this potential phenomenon should be addressed, and that social reward is worth considering as part of prosociality. Due to our small sample size, more research is needed, with larger group sizes and in wider contexts, to investigate social reward. However, we hypothesise that social reward mechanisms exist among animals, or wolves, and that they can contribute to behavioural reinforcement, particularly in the area of group decision-making and the benefits of individual actions to groups or populations. As our findings are part of a case study, the data are limited. Until more research is conducted, our results should be treated as tentative. 5. Conclusions Our findings suggest that there may be specific behavioural chains as part of social feedback in grey wolves. The prosocial behaviours of those behavioural chains could have reward functions, which is potentially due to the benefits of an individual's bold actions for the group. Our results may be interpreted to indicate that bold behaviour in social mammals, such as wolves, is responded to with social reward and prosocial behaviour, which is potentially via reason of the benefits that individual bold actions can implicate for the group. Due to the small number of groups and the potential bias in the observation, our results should be interpreted with caution. Further research is needed to investigate social feedback mechanisms in wolves and other social animals. Acknowledgments We would like to thank the Schwarze Berge Wildlife Park; in particular Manuel Martens, and the Zoo Wings, for making it possible to carry out the research. My special thanks goes to Ole Meyer for his technical support. Supplementary Materials The following supporting information can be downloaded at: Figure S1: Technical drawing of the apparatus and its function; Figure S2: Photo of the apparatus in use on another wolf group. Click here for additional data file. Author Contributions Conceptualisation, H.T. and U.G.; methodology, H.T. and U.G.; formal analysis, H.T.; investigation, H.T.; data curation, H.T.; writing--original draft preparation, H.T.; writing--review and editing, H.T. and U.G.; visualisation, H.T.; supervision, U.G.; project administration, H.T. All authors have read and agreed to the published version of the manuscript. Institutional Review Board Statement Ethical review and approval were waived for this study due to not involving manipulation, medical testing, laboratory animals or practices that could affect or harm animals. As we only conducted behavioural observations in zoological institutions without animal contact, there is no requirement for ethical review and approval. Informed Consent Statement Informed consent was obtained from all subjects involved in the study. Data Availability Statement The data obtained during the case study are available upon request. Conflicts of Interest The authors declare no conflict of interest. Figure 1 Exemplary presentation of the novel object and the described experimental set-up, adapted from Stefan_Alphonso. Stock Illustration ID:1397963840. Figure 2 Modified version of on wolf behaviour. The asterisk indicates the categorisation of "behaviours of interest" into behavioural categories. Figure 3 Markov chains representing the probabilities of transition in behavioural state: (a) behavioural chain after novel object interaction; (b) behavioural chain after individual behaviour "run"; (c) behavioural chain after individual behaviour "forage". Only transitions within the behavioural chain are represented; values are percentages. Behavioural states are defined in Table 4. Initial behaviours are defined in Table 3. Figure 4 Effect of initial state interactions on transitions in behavioural state of the behavioural chain, based on differences in transition probabilities (pij(Nobj) - pij(Indbhv1) - pij(Indbhv2). The star symbol indicates the statistically significant values, the dot symbol indicates the outliers. Figure 5 Influence of sex on frequency of novel object interaction. Figure 6 Influence of age on frequency of novel object interaction. The dot symbol indicates the outliers. Figure 7 Correlation matrix of personality traits, DS and frequency of novel object interaction. (a) Correlation plot pairs for multiple correlations, (b) Correlation plot matrix. Figure 8 Linear regression model of influence of curiosity on frequency of novel object interaction with the grey area as 95% confidence level. The dots indicate the respective data points. Data points outside the grey confidence level are outliers. animals-13-00872-t001_Table 1 Table 1 Animals included in the study. Name Abbreviation Sex Birth Date Facility Runa EU1 Female 2018 Schwarze Berge Yuuki EU2 Male 2018 Schwarze Berge Dunja EU3 Female 2013 Schwarze Berge Django EU4 Male 2014 Schwarze Berge Skadi EU5 Female 2017 Schwarze Berge Wolfgang EUW1 Male 2011 Zoo Wingst Rudolf EUW2 Male 2011 Zoo Wingst Anfa EUW3 Female 2011 Zoo Wingst Wolle EUW4 Male 2013 Zoo Wingst Andra EUW5 Female 2011 Zoo Wingst animals-13-00872-t002_Table 2 Table 2 Modified ethogram . Behaviours and categories added to the ethogram are written in bold. Behavioural Category Original Category Subcategories Behaviours in the Social Reward Chain Agonistic behaviours Agonistic behaviours Elicited Aggression Food-related Aggression Sex-related Aggression All-Out Attack Defence and Submission Offensive Threat Ritualised Attack, Counterattack, Fight None Play behaviours Play behaviours Agonistic Play Social Play Solitary Play None Caregiving Caregiving, Care Solicitation None Approach Sniffle Face Wipe Follow Hold Out Face, Airplane Ears Submissive behaviours Not existent None Expose Belly Down and Lick Lick Snout Low Posture Follow Feeding (Eating) Feeding (EAT) None Approach Cache Carry Object Drag Eat Forage Grab Lick Mouth Paw Tug Greeting Greeting None Airplane Ears Approach Body Rub Ears Back Ears Pricked Greet Group Formation/Group Together Grin Hug Hum Lick Leave Together Parallel Gait Parallel Walk Sniff Noses Tail Wag Locomotion/exploratory Locomotion None Amble Approach Avoid Follow Observation Object Interaction Observation Jump Run Scent-Marking, Elimination Scent-Marking, Elimination None None Other Other None Explore Indirect Approach Orient Wander Predation, Hunting Predation, Hunting None None Resting Resting None None animals-13-00872-t003_Table 3 Table 3 Definitions of initial behaviours. Initial Behaviours Description of Observed Behaviours Novel object interaction (Nobj) Approaching the novel object (apparatus) up to maximum 1 m distance, and sniffing on the rope or interacting with it by trying to pull the rope/pulling the rope Forage Walking around the enclosure, searching for leftovers of food or dead animals, eating, or hiding food Run Running around individually in the enclosure from one area to another animals-13-00872-t004_Table 4 Table 4 Definitions of the social reward behaviour states in a behavioural chain. State Description of Observed Behaviours Group formation (GrpFrmt) Individuals grouping together, observing the individual performing an "initial behaviour" Greeting (Greet) Tail wagging, body rubbing and face/body sniffing of group formation towards "active performer" Submissive behaviour (LickSn) Snout licking, lowered body posture, whimpering, lying on back/exposing belly towards the individual performing the "initial behaviour" Combined submissive behaviours (C.SubmBhv) Body rubbing, ears back, opening group formation and including the individual performing the "initial behaviour" to the group Leaving together (LeaveTgh) Leaving proximity of "initial behaviour performance location" as a united group animals-13-00872-t005_Table 5 Table 5 Questions and items related to personality factors curiosity and playfulness in . Factor Questions and Items to Determine Factor Values Curiosity/Openness "is inventive and resourceful when it comes to finding or reaching hidden food or toys" "does not have many interests apart from eating and sleeping, e.g., is unexplorative, uncurious, show limited interest in new objects, humans or animals" "has a good grasp of things and learns quickly" "attentive" "intelligent" "clever" Playfulness "is enthusiastic and encourages his peers to play" "is easy to get excited about new play ideas" "often does not understand what is being asked of him in play situations" animals-13-00872-t006_Table 6 Table 6 Initial state and success score of Markov chain calculation (n = 24). Initial State Success Score (Mean +- SD) Nobj (novel object interaction) 0.63 * +- 0.091 Indhbhv1 ("run") 0.318 +- 0.394 Indbhv2 ("forage") 0.279 +- 0.362 * = statistically significant value. animals-13-00872-t007_Table 7 Table 7 Mean values for personality factors of the two European wolf groups. Personality Trait/Factor Result (Mean +- SD) Playfulness 31.66 +- 15.81 Extraversion 42.22 +- 24.25 Curiosity 42.77 +- 18.72 Sociability 40.75 +- 14.38 Disclaimer/Publisher's Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

PMC10000160

In this scientometric review, we employ the Web of Science Core Collection to assess current publications and research trends regarding coral reefs in relation to climate change. Thirty-seven keywords for climate change and seven keywords for coral reefs were used in the analysis of 7743 articles on coral reefs and climate change. The field entered an accelerated uptrend phase in 2016, and it is anticipated that this phase will last for the next 5 to 10 years of research publication and citation. The United States and Australia have produced the greatest number of publications in this field. A cluster (i.e., focused issue) analysis showed that coral bleaching dominated the literature from 2000 to 2010, ocean acidification from 2010 to 2020, and sea-level rise, as well as the central Red Sea (Africa/Asia), in 2021. Three different types of keywords appear in the analysis based on which are the (i) most recent (2021), (ii) most influential (highly cited), and (iii) mostly used (frequently used keywords in the article) in the field. The Great Barrier Reef, which is found in the waters of Australia, is thought to be the subject of current coral reef and climate change research. Interestingly, climate-induced temperature changes in "ocean warming" and "sea surface temperature" are the most recent significant and dominant keywords in the coral reef and climate change area. coral bleaching coral disease coral growth elevated carbon dioxide phase shift ocean acidification sea level rise Symbiodinium surface temperature future scenario Ministry of Higher EducationLRGS/1/2020/UMT/01/1; LRGS UMT Vot No. 56040 PASIFIC program GeoReco project funding from the European Union's Horizon 2020 Research and Innovation programme, under the Marie Sklodowska-Curie847639 Ministry of Education and ScienceThe present study was supported by the Department of Higher Education, Ministry of Higher Education Malaysia, under the LRGS program (LRGS/1/2020/UMT/01/1; LRGS UMT Vot No. 56040) entitled "Ocean Climate Change: Potential Risk, Impact and Adaptation Towards Marine and Coastal Ecosystem Services in Malaysia'. The work was also supported by PASIFIC program GeoReco project funding from the European Union's Horizon 2020 Research and Innovation programme, under the Marie Sklodowska-Curie grant agreement No. 847639, and from the Ministry of Education and Science. pmc1. Introduction Scleractinians, or stony corals, emerged during the Cambrian period and constructed the earliest reefs, dating to approximately 410 million years ago . Five major coral extinctions have occurred since then, all of which have been linked to rising temperatures and higher levels of carbon dioxide in the atmosphere . While coral reef research commenced more than 100 years ago, concern over the state of coral reefs is relatively recent, occurring only in the last four decades. In 1981, at the 4th International Coral Reef Symposium, Edgardo Gomez initiated the conversation on threats to coral reefs by presenting his concerns to the scientific community . Coral reefs are considered an important marine resource for coastal communities, and the conference participants were mainly focused on coral reef management and environmental impacts and related fisheries activities . In addition to the natural stresses that have always existed on coral reefs, such as storms, freshwater inundation, and seismic and volcanic events, there is growing evidence of new emerging threats potentially causing global damage to coral reefs . At current extinction rates, it is estimated that we are commencing the sixth mass extinction event , with individual extinctions occurring approximately 1000-fold faster than the expected background extinction rate. Theoretically, species extinctions occur at a rate proportional to the rate of speciation or the creation of new species . Current extinction rates are much higher than speciation rates. This is largely due to the fact of anthropogenic factors , such as habitat destruction , deforestation , pollution , ocean acidification, climate change resulting from greenhouse gas emissions, and overexploitation of ecological resources . It is estimated that 75% of species will go extinct unless human pressures on the environment are scaled back soon . Further management efforts are required to reduce the impacts of climate change and human anthropogenic stress towards coral reef communities . Anthropogenic pressures on reefs have been the dominant factor damaging coral reefs through a range of stresses . Unsustainable land-based human activities, such as deforestation, poorly regulated agriculture, and urban/industrial development, are major contributors to the release of excessive sediments and nutrients into the environment . Increases in human-caused greenhouse gas emissions are the primary factor in the current climatic shift around the world . The ocean acts as a massive sink that absorbs carbon dioxide, resulting in the acidification of the oceans . Coral bleaching and widespread damage to the coral reef ecosystem have become increasingly common because of thermal stress brought on by rising ocean temperatures . Lack of food to sustain the coral reef ecosystem and disruption of larvae dispersal are both attributable to altered currents, upwelling, and/or vertical mixing brought on by changing currents and winds . Storms and cyclones are "agents of mortality" on coral reefs and can have a direct impact on the structure and local distribution of coral reef assemblages, especially through the large waves they produce . Reduced salinity caused by heavy rainfall and enhanced surface run-off onto nearshore reefs during cyclones, storms, and heavy precipitation rates can lead to algal blooms and other devastating results . The rise in sea level, caused by thermal expansion and the melting of ice on land, has varied across different regions of the world over the past century, with an average increase of approximately 20 cm . The sedimentary mechanisms triggered by the rising sea levels have the potential to intensify and jeopardize crucial physiological reef processes, such as photosynthesis, feeding, and recruitment, thereby posing a severe threat to coral reefs and related ecosystems, such as seagrass meadows and mangrove forests . This threat, coupled with increasing carbon dioxide (CO2) emissions, can negatively impact these vital ecosystems. Without effective local measures and a concerted effort to reduce carbon dioxide emissions, these effects are projected to intensify, leading to an unprecedented degradation of marine biodiversity and ecological balance . Given the length of time that scientists have been studying climate change , the sheer volume of published research in the field can make it difficult for scientists to develop an overview of the topic . Bibliometric analysis can be used to provide an overview of voluminous scientific literature . Research output in each field can be charted in terms of its characteristics and evolution through quantitative examination of publication data . Performance and research patterns of authors, journals, countries, and institutions can all be evaluated with the help of bibliometric methods, and patterns of collaboration between these entities can be identified and quantified . A research domain's multidisciplinary nature and the variety of journals publishing on a given topic can be inferred from the subject categories assigned to publications and the number of journals publishing on a given topic. The most recent developments, research directions, and top-of-mind issues in a particular field can be gleaned from bibliometrics . In addition, bibliometrics can be a useful tool for guiding scientific policy. Findings from bibliometric analyses not only inform researchers and policymakers but also aid in the distribution of funds for scientific investigation . 2. Scientometric Analysis Software programs, such as VOSviewer (Centre for Science and Technology Studies (CWTS), Leiden University, Leiden, The Netherlands), Pajek (University of Ljubljana, Ljubljana, Slovenia), and CiteSpace (Drexel University, Philadelphia, PA, USA) can be used to create scientometric visualizations. CiteSpace is a scientometric-based analysis tool that provides two main outputs for researchers. Firstly, it includes three central concepts: burst detection, betweenness centrality, and heterogeneous networks. Secondly, CiteSpace addresses three practical issues: identifying the nature of a research front, labeling specialties, and detecting emerging trends and abrupt changes in a timely manner. Identifying these outputs involves six main procedures: time slicing, thresholding, modelling, pruning, merging, and mapping. CiteSpace's functionalities, such as dual map overlay, burst detection, cluster explorer, and timeline view, are particularly helpful in identifying the current research trends of a specific field. Researchers can use this information to gain insights into the overall state of research in a given field, the pace of advancement, and the most prominent areas of interest . Briefly, the dual map overlay graphically identifies both original documents and cocited networks and expresses their relationships by connecting them with lines. Burst detection refers to a frequency surge of a specific knowledge domain. A cluster is defined as the frequency of citations of cited references. A timeline arranges papers in chronological rows, with each cluster represented by one row and papers represented as nodes . 3. Objective of the Review This review addresses the question, "How have coral reefs been affected by climate change and their interactions using bibliometrics?". Specific objectives were to assess the literature in terms of (i) the annual number of articles published, (ii) countries/regions involved in the field, (iii) research topics, (iv) cocited networks (i.e., frequency of two different documents are cited together in other documents), (v) cluster networks, (vi) research topic (i.e., keywords) burstiness, (vii) dual map overlay, and (viii) the future trends of the knowledge domain (i.e., coral reef and climate change). The main article structure diagram is shared in Figure 2. 4. Systematic Data Collection This study was analyzed based on the Web of Science database Core Collection (WOSCC) on 16 November 2022. Keywords used for data collection comprised terms related to climate change and coral reefs . CiteSpace, 6.1.4, version 64-bit for Windows, was used to visualize current trends. The search string are as follows: CLIMATE CHANGE: ("climat* chang*") OR ("global warm*") OR ("seasonal* variat*") OR ("extrem* event*") OR ("environment* variab*") OR ("anthropogenic effect*") OR ("greenhouse effect*") OR ("sea level ris*") OR (erosio*) OR ("agricult* run-off") OR ("weather* variab*") OR ("weather* extrem*") OR ("extreme* climat*") OR ("environment* impact*") OR ("environment* chang*") OR ("anthropogenic stres*") OR ("temperature ris*") OR ("temperature effect*") OR ("warm* ocean") OR ("sea surface* temperat*") OR (heatwav*) OR (acidific*) OR (hurrican*) OR ("el nino") OR ("el-nino") OR ("la nina") OR (la-nina) OR (drought*) OR (flood*) OR ("high precipit*") OR ("heavy rainfall*") OR ("CO2 concentrat*") OR ("melt* of the glacier*") OR ("melt* ice*") OR ("therm* stress*") OR ("drought") OR ("hypoxia") AND CORAL REEF: ("coral reef*") OR ("barrier reef*") OR ("atolls") OR ("fring* reef*") OR ("coral island*") OR ("atoll lagoon*") OR ("biogenic deposit*"). 5. Evolution of the Literature 5.1. Global Publication The coral reef and climate change field showed an increase in published articles, indicating that the research is in rising momentum . Since 2006, studies on coral reef and climate change have been published, amounting to approximately 1.5% to almost 10% of total articles on the WoS platform in 2021, especially in the WoS Core Collection (WOSCC) database. The field entered an accelerated uptrend phase in 2016, and it is anticipated that this phase will last for the next 5 to 10 years for research publications and citations. This could be because the WOSCC added a new edition of the Emerging Sources Citation Index (ESCI) database in 2015 . The leading countries in coral reef and climate change research are shown in Figure 4. The USA and Australia produced the most publications, with 2940 and 2933 articles, respectively, more than triple the output of third-ranked England, with approximately 719 articles. The USA and Australia contributed more than 75% of all publications. The study also found that there is a lack of studies conducted in the African regions. As expected, countries without coastal areas, such as Kazakhstan and Mongolia, showed no articles published in the coral reef and climate change fields. Additionally, most island nations, such as Japan, New Zealand, Australia, or Cuba, contributed to the knowledge of the coral reef and climate change. 5.2. Leading Institutions, Funding, and Authorship Distribution Ellegard and Wallin opined that the distribution of research institutions is a useful indicator of academic support for a discipline. The network of institutions generated 948 nodes (i.e., group of entities) and 2666 collaborative links among institutions conducting research . The 4159 documented affiliations reflect the importance of this field in academia and the intensity of the investigations. Institutions in this collaborative network (a collaborative affiliation appeared in the article) that have contributed the most to this field are shown in Figure 5 and Table 1. An analysis of 7743 publications related to coral reefs and climate change identified a total of 29,890 affiliations. The top 20 institutions contributed to 7231 affiliations, which accounted for nearly 24% of all publications. Specifically, James Cook University was found to have contributed the highest number of publications (1119, 14% of the total), followed by the Australian Institute of Marine Science and the University of Queensland. These findings suggest that a small number of institutions have played a significant role in the research on the impact of climate change on coral reefs. Table 2 lists the major funding agencies cited by publications in this field. The Australian Research Council funded 1001 publications, or nearly 13% of the total. Australian institutions have been at the forefront of climate change research partly because they have had greater access to funding. For instance, the Great Barrier Reef Foundation (GBRF) was awarded USD 443.3 million for the Reef Trust project between 2018 and 2019, the largest investment in reef protection to date. In 2019 and 2020, the budget allocated USD twenty-three million dollars for water quality projects, USD 4.33 million for Crown-of-Thorns (COT) control, USD 16.3 million for reef protection aimed at supporting traditional owners, USD 2.6 million for community reef protection, and USD 1.5 million for integrated monitoring and reporting. This funding allocation was based on the 2019 Great Barrier Reef Foundation's budget plan (GBRF, 2019). In addition to these investments, the Australian Government also dedicated USD 6 million in 2018 to support the Reef Restoration and Adaptation Program's concept feasibility phase. This program aimed to investigate the most effective science and technology options for restoring the reef, including methods for cooling and shading reef structures, coral reproduction and recruitment, biocontrol, field treatments, and coral plantation initiatives . The ARC Centre of Excellence for Coral Reef Studies, located at James Cook University, conducts cutting-edge research on coral reefs. With strong collaborative ties to 24 other institutions in nine countries, it is Australia's top contributor to coral reef sciences. Five of the ten most prominent scientists identified by CiteSpace analysis are associated with the ARC Centre of Excellence for Coral Reef Studies. The network of authors generated in CiteSpace is a valuable tool for understanding collaborations and identifying potential future collaborations within a given field. In this study, the network of authors in the field of coral reefs and climate change generated 1680 nodes and 6931 links, where nodes represent authors with the number of publications, and links between nodes represent collaborations between authors. As a general rule, a higher number of nodes generated from CiteSpace indicates a greater degree of collaboration between authors. The top authors in Table 3 are crucial indicators of further scientific collaboration and advancement within the field of coral reefs and climate change. However, it is important to note that while first authorship is typically assigned to the individual who made the most significant contribution to the research, this does not mean that coauthors' contributions are any less significant or valuable. Coauthors can provide specialized expertise, contribute to data analysis and interpretation, or support the project through funding, logistical support, or other means. The authorship order and the number of authors on a publication may vary based on the specific circumstances of the research project and the agreed-upon conventions of the discipline. Ultimately, understanding the network of authors and their collaborations can help identify potential areas of future research and scientific collaboration within the coral reefs and climate change. Emeritus Professor Terry Hughes of James Cook University, who served as the ARC Centre of Excellence for Coral Reef Studies Director from 2005 to 2020, topped the list. In 2016, Nature named Hughes one of the "10 people who mattered this year" for addressing the widespread coral bleaching event brought on by climate change. Hughes's research has led to practical solutions to improve marine environmental management . His work on the effects of climate change on coral reefs has been widely cited, especially his paper on the resistance of some coral reefs to climate change and anthropogenic factors . Second on the list was Professor Ove Hoegh-Guldberg from the University of Queensland (UQ), Australia. He serves as Director of the Global Change Institute at UQ and also as a Chief Investigator at the ARC Centre of Excellence for Coral Reef Studies . Dr. Katharina Fabricus is a coral reef ecologist and a Senior Principal Research Scientist at the Australian Institute of Marine Science . Many of her highly cited publications are on topics related to ocean acidification , the impacts of water quality on coral reefs and understanding the effects of terrestrial run-off on coral reefs . Dr. Peter W. Glynn, from the National Center for Coral Reef Research, University of Miami, was among the pioneers in analyzing and reporting the impacts of the 1982-1983 El Nino warming event on Eastern Pacific coral reefs . This was followed by Tim McClanahan, a senior conservation zoologist at the Wildlife Conservation Society and also an associate at the ARC Centre of Excellence for Coral Reef Studies . His global study of more than 2500 reefs produced a Bayesian hierarchical model to predict how reef fish biomass is related to 18 socioeconomic drivers and environmental conditions . Dr. Peter Mumby is a coral reef biologist from the University of Queensland and also a Chief Investigator at the ARC Centre of Excellence for Coral Reef Studies . He collaborated with Professor Ove Hoegh-Guldberg to publish "Coral Reefs under Rapid Climate Change and Ocean Acidification", which is one of the most cited papers in the field (see Table 4) . Another study published in Nature reported the resilience of Caribbean coral reefs against moderate hurricanes . Dr. David Bellwood, an Australian Laureate Fellow and Distinguished Professor at James Cook University , has reported on the effects of climate change on coral reef ecosystems, even though his primary research interests are in biology and the evolution of reef fish . Dr. John Pandolfi from the University of Queensland is a paleoecologist and a Chief investigator at ARC. His research integrates long-term ecological and environmental time series data to discover past and future influences of natural variability, human impact, and climate change on coral reef resilience. Among his highly cited works is a projection of the future of coral reefs under global warming and ocean acidification . Dr. Kenneth RN Anthony, an associate scientist at the Australian Institute of Marine Science and director of Environmental Strategies ES5, has published widely on ocean acidification . Dr. John Bruno, from the University of North Carolina, is a marine ecologist focusing on the impacts of climate change on marine ecosystems, particularly coral reef ecology. His publication with Dr. Ove Hoegh-Guldberg, on the effects of climate change on global marine ecosystems is one of his most cited works . Next on the list is Emeritus Professor Barbara E. Brown from Newcastle University, who conducted extensive research on coral bleaching, specifically on the role of zooxanthellae . Next on the list is Professor Andrew C. Baker, a marine biologist at the University of Miami, who studies coral reefs and climate change. He leads the Coral Reef Futures Lab and focuses on developing and testing methods to increase coral reef resilience . Glenn De'ath and Ray Berkelmans, both from The Australian Institute of Marine Science, are also highly cited for their research on coral reefs. De'ath's work involves statistics and ecology, specifically on the Great Barrier Reef coral cover decline , while Berkelmans' research focuses on thermal stress, adaptation to climate warming, the resilience of reef communities, and upwelling . Joan Kleypas, a Senior Scientist from the National Center for Atmospheric Research, is also on the list, and her highly cited works revolve around the impact of ocean acidification on coral reefs . Next is Professor Nick Graham from Lancaster University, who assesses the impacts of climate-induced coral bleaching on coral reef fish assemblages, fisheries, and ecosystem stability . Emeritus Professor Michael Lesser from the University of New Hampshire is also highly cited for his work on climate change-related stressors' biochemical and physiological impacts on coral reefs . Professor Joseph Loya from Tel Aviv University quantifies changes in biodiversity and assesses reef health , while Professor Peter Edmunds focuses on the physiological ecology of tropical coral reefs . Lastly, Toby Gardner is a Senior Research Fellow from Stockholm Environment Institute, known for his extensive work on Caribbean corals. He co-leads SEI's Initiative on producer-to-consumer sustainability and the transparency for sustainable economies platform. His long-term observations revealed that the coral cover of the Caribbean basin declined by 80% in just thirty years . 5.3. Emerging Research Disciplines CiteSpace's "Category" node type was used to generate a visual map showing research disciplinary categories represented by papers addressing issues related to climate change's impact on coral reefs. The centrality of a network (i.e., the center of collaborative activities) comprising 135 nodes and 336 links was computed after the data were simplified and merged (i.e., automatically generated from the CiteSpace algorithm and programming) . The five disciplines with the most publications in descending order were marine and freshwater biology, environmental sciences, ecology, oceanography, and geosciences. The study of coral reefs is a multifaceted research topic that includes many fields of study, as demonstrated by the distribution map. Disciplines in related subjects such as biodiversity conservation, geography, physical sciences, biology, evolutionary biology, geology, paleontology, and water resources, show strong connections, represented by the sizes of the nodes. The number of published papers is comparably low in some research disciplines, such as toxicology, biotechnology and applied microbiology, green and sustainable science and technology, and biochemistry and molecular biology. However, the relatively high betweenness centrality values of these fields suggest their significant contribution to interdisciplinary research, signifying their pivotal position in the scientific network. This centrality may also hint at their potential for future development and advancement in the field. 5.4. Research Cluster Analysis Cluster analysis is a popular method of statistical data analysis and knowledge discovery because of its ability to uncover latent semantic themes in textual data . Cluster analysis can divide a large body of research data into various units based on the relative degree of term correlation, making it easier to identify the research themes, trends, and connections within a given field of study . A cluster's homogeneity can be quantified using an index called the mean silhouette, with values ranging from -1 to 1. The average silhouette value for each cluster was determined using CiteSpace. The higher the value, the more similar the cluster's members are to one another . The network showed 24 clusters in the context of the scientometric analysis mapping the link between climate change and coral reefs . The largest cluster (#0) has 291 members (i.e., number of publications) and a silhouette value of 0.863 and is labeled as "coral reef." The most cited article of this cluster is by Gilmour et al. . They monitored and assessed the impacts of the 2016 heat stress event on Western Australian coral reefs. They found that mass bleaching in 2016 reduced coral cover by 70% at Scott Reef and caused widespread mortality (>30%) at Christmas Island, Ashmore Reef, and inshore reefs in southern Kimberley. A coral phase shift is characterized by a rapid decline in coral abundance or cover and an accompanying rise in non-reef-building organisms, like algae and soft corals . The second largest cluster (#1) has 247 members and a silhouette value of 0.876, labeled as "phase shift." This publication by Brodie et al. , entitled "Terrestrial pollutant run-off to the Great Barrier Reef: An update of issues, priorities and management responses", is the most cited article in this cluster. They addressed findings from studies of problems caused by surface run-off of pollutants like nitrates from fertilizers, herbicides from crops, etc. Within the cluster of the study, there are three different types of management generated automatically and have mentioned (i) Reef Plant 2009, (ii) Reef Rescue, and the (iii) Reef Protection Package in the analysis. These topics are just some of the initiatives set up to continuously monitor and report on levels of discharges into the Great Barrier Reef. Multiple observations of specific facets of the topic have been published; Hughes ; McManus and Polsenberg ; Idjadi et al. ; Norstrom et al. ; Graham et al. ; Crisp et al. . Many anthropogenic stressors have been linked to this phenomenon . Nutrients play a pivotal role in conceptual models of how coral reef communities form. These studies show that corals have a competitive advantage over macroalgae in low nutrient conditions but that the advantage shifts to macroalgae in higher nutrient conditions . Siltation, resulting in mud-bacterial complexes, collectively known as "marine snow," is another factor that hinders coral growth. In addition, excess nutrients resulting in plankton blooms reduce light, thereby inhibiting coral growth . The fourth largest cluster (#3), "ocean acidification", has 233 members and a silhouette value of 0.959. The most cited article of this cluster is Bates , which reported twenty years (1996 to 2016) of marine carbon cycle observations at Devils Hole, Bermuda. Her findings shed light on the dynamic nature of biogeochemical processes like primary production, respiration, calcification, and CaCO3 deposition in the Bermuda reef system. During this period, neither warming nor cooling of any significance was observed. However, increases in inorganic carbon in onshore waters were primarily due to increased salinity (45%), uptake of anthropogenic CO2 (25%), and changes in Bermuda reef biogeochemical processes (30%). Increases in atmospheric carbon dioxide concentrations result in the absorption of more carbon dioxide by oceans, which in turn causes a decrease in pH . The majority of research on ocean acidification has focused on the impact of changes in ocean chemistry towards suboptimal states of aragonite and calcite saturation on the calcification processes of pelagic and benthic organisms . However, it is likely that ocean acidification also has an effect on other physiological processes, such as growth and reproduction in significant reef-building species . The Indo-Pacific region's Coral Triangle, the world's epicenter of marine biodiversity , is predicted to become a "marginal" coral habitat between 2020 and 2050 unless CO2 emissions are reduced . In addition to reducing coral diversity, acidification also results in a decline in shellfish and fish species due to the loss of reef structure, which provides habitat for these other species and reduces the reefs' capacity to mitigate the effects of storm waves and erosion . Ocean acidification has a devastating impact on the economies of ocean-dependent sectors of the global economy. Previous studies have provided estimates of the economic impact of ocean acidification on marine mollusk and shellfish production, as well as the bioeconomic costs associated with coral reef damage . These studies have shed light on the detrimental effects of ocean acidification on marine ecosystems, which in turn, can have severe economic implications. Estimating these costs can aid in developing policies aimed at reducing the negative effects of ocean acidification and promoting the sustainable use of marine resources. For instance, according to a study by Narita et al. , the global annual loss of mollusk production due to the fact of ocean acidification could amount to between USD 6 billion and USD 100 billion. Commercially valuable finfish populations will suffer as a result of global ocean changes that reduce coral reef coverage, resulting in a loss of habitat, reduced availability of prey, and increased predation . The scientometric analysis has identified four prominent clusters, also referred to as topics, which represent distinct research areas based on their geographic location. These clusters include the "central red sea" (#3), the "eastern pacific" (#5), the "great barrier reef" (#8), and the "Dominican Republic" (#18). These geographic regions are frequently cited in scientific research as they represent the study location of many relevant studies. The most cited article of the "Red Sea" cluster is by Osman et al. , which mapped coral microbiome composition along the northern Red Sea. The Red Sea is a distinctive body of water that is an evaporative basin with a high salinity above 38 ppt . It is home to some of the world's most thriving and productive coral reef ecosystems . Osman et al. research offered a fresh understanding of the coral microbiome's exclusive and endemic characteristics along the northern Red Sea refugia. They looked into the surface mucus layer (SML) for bacterial communities from six dominant coral species and discovered five novel algal endosymbionts. Over the past four decades, the average annual sea surface temperature in most of the world's tropics and subtropics has risen between 0.4 degC and 1 degC. However, in the central Red Sea, where reef growth and scleractinian coral diversity are abundant, warming is more extensive than the observed mean tropical temperature increase . The 2010 "Thuwal bleaching" in the central Red Sea was caused by a temperature rise of 10-11 degC, the largest coral bleaching event ever recorded. Furby et al. conducted a survey and found that the "Thuwal bleaching" event caused more severe bleaching of inshore reefs (74% of hard corals were bleached) than offshore reefs (14% of hard corals were bleached). One mechanism that can lead to higher tolerance is repeated exposure to thermal stress . Based on current knowledge, it is hypothesized that the reefs in the Red Sea will be relatively resistant to bleaching as sea temperatures rise, as noted in a study by Grimsditch and Salm . However, reports indicate that bleaching is beginning to occur in the Red Sea, as documented by Kleinhaus et al. . For instance, Rich et al. reported a winter bleaching event in the central Red Sea in January 2020 due to sea surface temperatures (SSTs) falling below 18 degC. Additionally, inshore bleaching events in the central Arabian Red Sea were observed during the "3rd global coral bleaching event" in 2015, as reported by Monroe et al. . The Eastern Tropical Pacific (ETP) comprises the ocean basin extending from the Gulf of California in Mexico to Peru and includes areas of the continental shelf and offshore islands (Coco Island, the Galapagos Islands, the Revillagigedo Archipelago and Clipperton Atoll). The most cited article of the cluster "Eastern Pacific" is Spencer , which discussed potentialities, uncertainties and complexities in the response of coral reefs to future sea-level rise of reef islands in the Pacific Ocean and the Caribbean Sea. Throughout the Holocene, sea levels rose without being stabilized, and reefs in the Caribbean grew in tandem with these elevation changes . The once structurally complex coral reefs in the Caribbean have suffered a dramatic decline since the 1970s, with only a minority of reefs maintaining a mean live coral cover of 10% or more . A strong hurricane season brought on by unusually warm waters in the tropical Atlantic, and the Caribbean in 2005 caused the worst bleaching event ever observed in the basin . There was a 60% decline in coral cover on reefs in the US Virgin Islands due to the fact of a severe disease outbreak brought on by the 2005 bleaching events in the Caribbean region, as reported by Miller et al. . The Great Barrier Reef is the largest coral reef ecosystem, with over 348,000 km2 of coverage consisting of 2900 individual reefs and 900 islands stretching over 2300 kilometers . Three major coral bleaching events within a span of five years (2016, 2017, and 2020) along with the effects of severe tropical cyclones, poor water quality from catchment run-off, population growth and urbanization, overexploitation of marine resources, and habitat loss have all been the factors towards the degradation of coral reefs in the Great Barrier Reefs . Cluster #4, which is the fifth largest cluster, contains 176 publications and has a silhouette value of 0.9. This cluster is strongly associated with cluster #6, "symbiotic dinoflagellate," and cluster #12, "coral disease". The high silhouette value of 1.0 indicates that there is a focused field of study in the context of coral reefs and climate change. The most cited article in cluster #4 is by Reaser et al. on scientific findings and policy recommendations for coral bleaching and global climate change. Coral bleaching, which is the ability of animals with a symbiotic relationship with Symbiodinium to turn white, is an important issue associated with climate change-based literature. According to Douglas , all animals that have a symbiotic association with the dinoflagellate algae of the Symbiodinium genus, which are also referred to as zooxanthellae, have the ability to undergo bleaching. Symbiodinium have been reported to form extracellular symbioses with giant clams and intracellular symbioses with various organisms, including corals, anemones, jellyfish, nudibranchs, ciliophora, foraminifera, zoanthids, and sponges . Fujise et al. reported that the expulsion mechanisms of Symbiodinium were temperature-dependent; however, under non-thermal stress conditions, the expulsions of this algae were part of a regulatory mechanism to maintain a constant Symbiodinium density. In response to moderate thermal stress, Symbiodinium becomes damaged, and corals either selectively digest or expel the damaged cells. During extended periods of thermal stress, damaged Symbiodinium may accumulate in coral tissues, resulting in coral bleaching. Multiple factors have been shown to cause bleaching, including high oxidative stress , intense light , high temperature , low salinity , sedimentation , pollutants , decreased seawater temperature , diseases , bacterial infection , and ENSO-related marine heatwave events . Degree heating weeks (DHW), defined as 1degC above the long-term climate level for the warmest month at a given locality, have become a common global predictor of bleaching . Severe bleaching is typical at 8 DHW and above . A global analysis report of coral bleaching from 1998 to 2017 found that coral bleaching was most prevalent in regions with high-intensity and high-frequency thermal-stress anomalies. In areas where sea-surface temperature (SST) anomalies varied greatly, such as the Gulf of Aqaba region , the Caribbean Sea , and the Indo-Pacific , coral communities were significantly less susceptible to coral bleaching . Globally, coral reefs have been threatened by coral disease, which is now recognized as one of the biggest threats to these ecosystems . Similar to bleaching, coral disease was not considered a severe threat to coral reefs until recently , despite its first documentation in 1965 . Since their initial descriptions, both the variety of coral diseases and the number of reported cases have skyrocketed . Approximately 76% of all coral diseases described worldwide are found within this relatively small basin, leading experts to label the Caribbean a "hot spot" for disease . For example, two dominant Acropora species in the Caribbean have been replaced by low-encrusting Agaricia due to the fact of coral disease . Common coral diseases include Black band disease, which is caused by increased seawater temperature and anthropogenic factors, ciliates cause the Brown band disease, Cyanobacteria cause the Red band disease, and the White plague is caused by a bacterial infection . 5.5. Timeline Co-citation Analysis The timeline for the document co-citation analysis is an important indicator to explain the period when the study got the attention of the researcher worldwide . From 2010 to 2021, there have been bursts in citations for research clusters on (#0) "coral reefs", (#2) "ocean acidification", (#3) "central sea", (#11) "sea level rise", and (#5) "eastern pacific". When taken as a whole, these studies shed light on the growing interest in studying the effects of ocean acidification and sea level rise on coral reefs, with particular attention paid to the plight of these ecosystems within the eastern pacific area, such as in the central Red Sea and the Dominican Republic. 5.6. Highly Cited Articles in the Field CiteSpace's visualized analysis of 7743 publications yielded a co-citation network (frequency of two different documents are cited together in other documents) with 2525 cited documents (nodes) and 5440 links or connections indicating co-citations between nodes . The larger the node, the more often a document is cited, demonstrating its impact on coral reef and climate change research. Document co-citation analysis locates essential literature. The given references (in the article) were the most cited among 7743 Web of Science references. Table 4 presents the twenty most cited references based on co-citation analysis along with their frequency, burstiness, and centrality indices. An increase in citations reflects increased interest in that topic. "Citation bursts" demonstrate correlations between publications and sudden increases in citations. When comparing clusters, the centrality index indicates how well they are connected (i.e., coral reef and climate change). An elevated centrality score indicates that the publication is located between two or more sizable subclusters . Dr. Terry Hughes's research was widely cited, with three of his Nature and one of his Science publications ranking among the top five most-cited references. The publication entitled Global warming and recurrent mass bleaching of corals topped the list with a frequency index of 546, a burst index of 157 and a centrality index of 0.7. The burst in citation period of this publication was from 2018 to 2021. The findings were based on the third global-scale pan-tropical coral bleaching episode that occurred between 2015 and 2016. The reef ecosystem of eastern and western Australia was studied using aerial and underwater surveys along with sea surface temperatures obtained from satellites. According to their findings, the devastating bleaching event in 2016 was only slightly impacted by water quality and fishing pressure, indicating that local reef protection offers little to no protection against extreme heat. The second paper on the list, with a frequency index of 359 and burst index of 125, was a global study analyzing the bleaching records of 100 globally distributed reefs from 1980 to 2016 . According to their findings, mass coral bleaching events happen every year regardless of the presence or absence of El Nino. They forecast that the intervals between recurrent events will eventually become too short to permit a complete recovery of mature coral assemblages, typically taking 10 to 15 years to reach the fastest-growing species. They warned that if temperatures rise by 1.5 or 2 degrees Celsius above preindustrial levels, it will exacerbate the already severe decline of coral reefs around the world. Similar findings were found in another study of his that also appeared on the list of the most-cited research. Research into the effects of climate change on coral reef ecosystems, with a special emphasis on the Great Barrier Reef, ranked fifth . They found that the Great Barrier Reef's 2016 record-breaking heatwave had caused widespread loss of functionally diverse corals across the reef's most remote and pristine regions. Ranked third on the list was the study by Hoegh-Guldberg et al. , which investigated the effects of climate change and ocean acidification on coral reefs. This study was closely linked to the 3rd (#2) research cluster, also known as "ocean acidification". The research review presented future scenarios for coral reefs, which suggested increasingly detrimental impacts on various sectors, including tourism, coastal protection, and the fisheries industry. These predictions were based on the assumption that global temperatures would rise by at least 2 degC between 2050 and 2100, coupled with atmospheric carbon dioxide concentrations exceeding 500 ppm. The findings of this study emphasize the urgent need for effective measures to mitigate climate change and ocean acidification to ensure the long-term survival and sustainability of coral reefs and the associated ecosystems. The article by LaJeunesse et al. on coral endosymbionts has garnered significant attention, with a citation frequency of 134, a burst index of 68, and a burst period spanning from 2013 to 2017. This publication is associated with cluster (3), also known as "symbiotic dinoflagellate". The article describes Symbiodinium clades and proposes that the divergent evolutionary Symbiodinium "clades" correspond to genera within the Symbiodiniaceae family. The study affirms that the long evolutionary history of the Symbiodiniaceae family is appropriately acknowledged within the suggested framework. The findings of this study provide valuable insights into the evolutionary relationships and ecological functions of these endosymbionts, highlighting the critical role they play in the health and survival of coral reefs. The list of 11 to 20 top-cited articles on climate change on coral reefs cover a wide range of topics, including ocean acidification, declining coral cover, Symbiodinium diversity, and coral reef resilience. Several studies indicate that warming trends and bleaching stress are increasing, and coral bleaching protection mechanisms are becoming less effective, ultimately leading to significant declines in coral populations. Research on the impacts of ocean acidification and warming on marine organisms, as well as the interactions between these factors, has also shed light on the mechanisms underlying the sensitivity of coral reefs to climate change . Studies on the diversity, distribution and stability of Symbiodinium have provided insights into the potential for coral resilience, while research on the decline of coral cover in the Indo-Pacific region has highlighted the extent and timing of this phenomenon . These studies demonstrate that collaborative research efforts are essential to understanding the impacts of climate change on coral reefs and developing more efficient conservation and management strategies. 5.7. Distribution of Keywords Using the co-cited keyword analysis performed in CiteSpace, 963 unique keywords were generated. In order to better understand the connections between these terms, a clustering tool was used to categorize them into groups . This generated seven major clusters consisting of "sea surface temperature", "Symbiodinium", "coral reef fish", "marine protected area", "water quality", "ocean acidification", and "hydrocorals". Each of these clusters can be analyzed independently to determine which descriptors are most applicable. The major keywords used to discuss "sea surface temperature"--record, Indian ocean, and reef; "Symbiodinium"--scleractinian coral, diversity, zooxanthellae, population, nutrient enrichment, elevated pressure, and oxidative stress; "coral reef fish"--phase shift, ecosystem, disturbance, fish, dynamics, community, recruitment, abundance, thermal tolerance, Stylophora pistillata, and ecology; "marine protected area"--management, assemblage, biodiversity, susceptibility, degradation, adaptation, response, and recovery; "water quality"--sea level, Great barrier reef, rate, transport, coral bleaching, French Polynesia, and fringing reef; "ocean acidification"--climate, El Nino, impact temperature, coral reef, calcification, and carbon; and "hydrocorals"--seawater and carbon dioxide. Table 5 displays the top 10 keywords with the strongest citation burst. With the exception of "ocean warming," all the most frequently cited keywords emerged in the early 1990s and experienced a citation burst that extended until the late 2000s. The keywords identified were "French Polynesia," "record," "El Nino," "Australia," "Indian Ocean," "Continental," "Shelf," "Sea level," "Sea surface," "temperature," and "Island." Notably, the keyword "ocean warming" only gained popularity in 2017, with citations peaking from 2018 to 2021. This demonstrates the significance of research on climate-related temperatures in the field of coral reefs and climate change. The keywords "Australia" and "Continental shelf" demonstrated citation bursts lasting over 15 years. In contrast, "French Polynesia" had the highest frequency of citations during a relatively shorter period, commencing in 1992 and concluding in 2006. French Polynesia, situated in the westernmost region of the South Pacific, comprises 118 islands and atolls, classified into five main clusters: the Marquesas, Society, Tuamotu, Gambier, and Austral islands . These regions exhibit a north-south gradient for variables such as sea surface temperature (SST), solar insolation, evaporation, and humidity. The Millennium Coral Reef Mapping Project (MCRMP) has successfully mapped the Austral, Gambier, Society, and Tuamotu islands and atolls; however, significant research remains to be undertaken in this extensive region, which accounts for the enduring citation burst for this keyword. The findings of this study highlight the scientific interest and importance of French Polynesia as a unique and diverse region for further research and conservation efforts. 5.8. Dual Map Overlay Figure 10 illustrates the dual-map overlay of the number of articles pertaining to the type or focus of the journal. The map labels represent the research subjects covered by the journals, with the citing journals displayed on the left side and the cited journals on the right. The trajectory of the citation links provides valuable insights into inter-specialty relationships. A shift in trajectory from one region to another would indicate the influence of articles from another discipline on a specific field. In the domain of coral reef and climate change interaction, the dominant fields were found to be "ecology, earth, and marine". The most influential discipline was "plant, ecology, and zoology", with a z-score of 7.66, followed by "earth, geology, and geophysics", with a z-score of 4.99 and, lastly, "molecular, biology, and genetics", with a z-score of 2.80. These findings provide a valuable understanding of the interdisciplinary relationships within the field of coral reef and climate change research, highlighting the influence of various disciplines in shaping the current research landscape. 6. General Discussions Coral reefs are a vital marine ecosystem service, providing high biodiversity and supporting the livelihoods of coastal communities. However, ocean warming and temperature are the largest threats to corals from anthropogenic climate change . Between 1997 and 2018, the global average percentage of coral cover was approximately 32%, but by 2100, RCP 8.5 predicts a global decline in coral cover of 5 and 15%, equating to a relative global decline of more than 40% . This decline is due to the fact that sea surface temperatures (SSTs) are projected to increase by more than 3 degC by the turn of the century . These declines could have significant ecological and socioeconomic impacts, particularly in coastal communities that rely on coral reefs for food, tourism, and other ecosystem services. For example, The Republic of Palau, a small Micronesian nation, has already experienced significant losses in coral reef cover . Over 87% of Palau's households are linked to coral reef-associated activities, which are critical to the country's economic and social well-being. While tourism, particularly ecotourism, is a significant contributor to GDP growth, tax revenue, and employment, climate change-related stressors have caused a steady decline in coral reef cover. This decline has indirectly caused a major decline in tourism, threatening the country's economic sustainability . According to Barnett , climate change is a significant threat to food security for people in Pacific SIDS, primarily due to the decline in fisheries output resulting from the impact of climate change on total coral cover. Apart from impacting the socioecological structure, the impact of climate change can have cascading effects on the entire reef ecosystem, affecting the abundance and diversity of other marine species that depend on corals for food and shelter. Up to 14% of species may be in imminent danger of extinction at a warming of 1.5 degC and up to 29% at a warming of 3 degC. This rise in ocean temperature will probably force coral to colonize higher latitudes that currently lack reefs . However, various factors, including the need for a suitable substrate , connectivity to other reefs , ocean acidification , and light intensity , may outweigh the advantages of reefs as they expand to high latitudes . Through the timeline co-citation analysis, we have observed a significant increase in research interest in the topic of climate change impacts on coral reefs between 2010 and 2022. The analysis identified several research clusters that gained traction in the scientific community, including those related to "coral reefs," "ocean acidification," "central red sea", "Great Barrier Reef", and "sea level rise". These clusters have evolved to become research hotspots under the overarching topic of climate change impacts on coral reefs. For example, the research clusters related to the central red sea and the Great Barrier Reef have emerged as prominent research areas, given their unique characteristics and ecological importance. Similarly, the impact of ocean acidification and sea level rise on coral reefs has gained significant research interest, given their severe consequences on the health and survival of coral reefs. To better understand the impact of these research clusters, our overall discussions have been designed to incorporate the subtopics "climate change threats to coral reefs" and "adaptive strategies for coral resistance and resilience". 6.1. The Threat of Climate Change to Coral Reefs: Investigating the Impacts of Temperature and Ocean Acidification Climate-induced changes in temperature are a major threat to coral reef ecosystems, and extensive research has highlighted several key areas for investigation , with marine heatwaves, solar radiation, heat tolerance, and thermal thresholds representing the most promising areas for future research. Marine heat waves have become increasingly prevalent and intense as a result of climate change. These extreme events, characterized by prolonged periods of elevated water temperatures, significantly impact coral reef ecosystems. For instance, the mass global coral bleaching event of 2016-2017 was the most extensive and long lasting on record, as documented by Eakin et al. . The event, which was associated with the El Nino Southern Oscillation (ENSO), had varying impacts on coral reefs worldwide , with some regions experiencing more severe bleaching than others, as reported by Kim et al. . Corals are thermophilic, but their thermal tolerance is narrowly defined . For instance, the rate of calcification increases with temperature up to a threshold level, beyond which it declines . Tropical corals live close to their upper thermal limits and are, therefore, highly sensitive to periods of elevated sea surface temperatures and ocean warming . Coral reefs in the Persian Gulf have been observed to have the highest upper-temperature thresholds of approximately 35-36 degC . However, it has also been noted that these corals remain highly vulnerable to thermal stress when temperatures surpass their local maximum summer temperatures . The escalating frequency and gravity of thermally induced mass bleaching events have sparked worldwide attention to the elevated temperature impacts on corals . As a result, research endeavors have focused on establishing maximum thermal tolerance thresholds and variations in diverse coral species and regions and exploring potential coral refugia to brace for future ocean warming . Corals rely on their symbiotic relationship with unicellular algae of the genus Symbiodinium for photosynthesis, and over 90% of their energy budget is needed for essential functions, such as calcification, tissue growth, and reproduction . This critical association is threatened when corals experience thermal stress, such as elevated sea surface temperatures (SST), resulting in coral bleaching, where the algal endosymbionts are expelled. The resulting impairment and expulsion of the algal symbionts are linked to reactive oxygen species (ROS) generation from the host, the algal symbiont, or both, triggering a host immune response . Protracted coral bleaching can lead to extensive coral mortality, severely affecting the ecosystem and associated reef fauna. Based on the timeline cocitation analysis, it was evident that the Red Sea (Cluster #3) and Great Barrier Reef (GBR) (cluster #8) are major research hotspots in terms of geographic regions. Although the Persian Gulf is a hot sea that supports coral reef ecosystems, the Red Sea harbors corals with greater thermal stress tolerance, with some coral genotypes capable of surviving temperatures over 5 degC above their summer maxima . Corals in the southern end of the Red Sea are more heat resistant, surviving prolonged high temperatures, while the northern Red Sea benefits from heat-resistant genotypes that have migrated from the south . The importance of broad latitudinal temperature gradients in promoting adaptation to high temperatures and exchanging heat-resistant genotypes across latitudes for genetic rescue in coral reefs is exemplified in the evolutionary history of coral reefs in the northern Red Sea . On the other hand, the GBR, known as the world's largest coral ecosystem, was severely impacted by the 2015-2016 climate change-amplified strong El Nino event that triggered the warmest temperatures on record. This resulted in a massive bleaching event affecting nearly 90% of reefs along the northern region, leading to a loss of approximately 30% of live coral cover in the following six months . Research has increasingly linked climate change to a rise in coral diseases. Bruno et al. used a high-resolution satellite dataset to investigate the relationship between temperature anomalies and coral disease on a large spatial scale of 1500 km in Australia's Great Barrier Reef. Their findings showed a significant positive correlation between warm temperature anomalies and the incidence of the white syndrome, an emergent disease in Pacific reef-building corals. In a similar vein, Tignat-Perrier et al. noted a decline in populations of two gorgonian species (Paramuricea clavata and Eunicella cavolini) found in the Mediterranean Sea due to the fact of microbial diseases during thermal stress events. These studies illustrate the growing concern that climate change is contributing to the increased incidence and severity of coral diseases, which could ultimately lead to a decline in the health of marine ecosystems. In the past, studies on the impact of climate change on coral reefs primarily centered on the thermal tolerance of corals and the consequences of massive, abrupt coral loss on organisms associated with reefs . However, research has recently shifted towards investigating the distinct and synergistic effects of ocean warming and ocean acidification resulting from increased atmospheric CO2 levels. The timeline co-citation analysis reveals that these emerging research fields are highly significant with recent citation bursts, as evidenced by their identification as Cluster #2 (Ocean acidification) and Cluster #10 (Elevated CO2), respectively. The escalation of atmospheric carbon dioxide (CO2) concentrations has resulted in ocean acidification, which is among the foremost threats to coral reef ecosystems. Forecasts for 2100 anticipate a rise in CO2 concentrations to between 540 and 970 ppm, leading to a global decrease in seawater pH by 0.14 to 0.35 units . As demonstrated by Fabricius et al. , ecological traits of coral reefs will gradually transform as seawater pH decreases to 7.8, and a decline below this level (at 750 ppm pCO2) would be catastrophic for these ecosystems. Ocean acidification reduces the availability of carbonate ions that corals require to form their calcium carbonate skeletons, ultimately leading to a decrease in coral calcification rates . Ocean acidification has also been shown to decrease the ability of coral larvae to settle and survive and increase their susceptibility to disease . Research has shown that even modest increases in ocean acidity can impact the physiological processes of corals. For example, exposure to high levels of CO2 reduces coral growth and calcification rates . In addition to the direct effects on coral physiology, ocean acidification can have cascading impacts on the entire coral reef ecosystem. For instance, reduced calcification by corals can reduce the complexity of the coral reef structure, potentially leading to the loss of important habitats for fish and other marine organisms . Furthermore, ocean acidification can impact the symbiotic relationship between corals and their algal symbionts, potentially leading to a decline in the productivity of the reef ecosystem as a whole . The combination of ocean warming and acidification is particularly concerning, as they act synergistically to exacerbate the negative impacts on coral reef ecosystems . With continuing increases in atmospheric CO2 levels, the effects of ocean acidification on coral reefs are expected to become even more pronounced, highlighting the need for urgent action to reduce greenhouse gas emissions and protect these valuable and vulnerable ecosystems. The rate of atmospheric CO2 increase continues to accelerate, with emission scenarios predicting CO2 concentrations of 540-970 ppm and a decline in seawater pH by 0.14-0.35 units globally for 2100 . Fabricius et al. demonstrated that many ecological properties in coral reefs will gradually change as pH declines to 7.8 and that it would be catastrophic for coral reefs if seawater pH dropped below 7.8 (at 750 ppm pCO2). 6.2. Adaptive Strategies for Enhancing Coral Resistance and Resilience in the Face of Climate Change Coral resistance and resilience are scientific constructs that pertain to the capacity of coral reefs to withstand and recuperate from various stressors. Coral resistance is defined as the ability of corals to endure or tolerate perturbations and stressors, such as variations in water temperature, ocean acidification, pollution, and physical injury. Corals that possess a greater resistance to these stressors exhibit a greater ability to sustain their structure and function despite disturbances and are less prone to suffering from coral bleaching, disease, or mortality . A myriad of studies has reported on the bleaching thresholds of corals inhabiting the Persian Gulf, despite conditions at least 2 degC higher than other coral reef ecosystems worldwide . Additionally, corals from the Indo-Pacific and Caribbean regions have been found to maintain calcification rates even in low aragonite saturation states, present in naturally acidified locales . The eastern Pacific region of Palau has revealed the thriving of reefs in waters with natural acidification, resulting from biological processes and reef system circulation patterns . However, it is noteworthy that coral communities in Palau's relatively acidic reef zones developed over thousands of years, fostering an inherent resistance that differs from coral communities in regions affected by higher anthropogenic interventions. Coral resilience, in contrast, refers to the ability of coral reefs to recover from disturbances and stressors. Corals that exhibit higher resilience can reproduce, regenerate, and rebuild their structural complexity after experiencing bleaching . These mechanisms are attributable to genetic diversity within coral populations and their symbiotic association with Symbiodinium algae, which are critical to their health and survival . Genetic adaptation in corals is mediated through various factors, including the activation of heat-shock proteins, oxidoreductase enzymes, and microsporine-like amino acids. The coral surface micro-layer that absorbs UV radiation has also been identified as a significant mechanism for adaptation . In-depth research on corals that thrive in the warm waters of the Persian Gulf has demonstrated their capacity for resilience, attributable to metabolic trade-offs, unique physiological characteristics, and specific genetic signatures, including a heat-specialist algal endosymbiont, Symbiodinium thermophilum . S. thermophilum can thrive in high-temperature and high-salinity environments, allowing the coral to develop a temperature-stress-resistant phenotype . Symbiodinium, a diverse group of dinoflagellates, is classified into nine clades (A-I) based on their phylogenetic characteristics . Among these clades, Symbiodinium clade D has garnered attention for its exceptional thermal resilience ability, despite its relatively low representation (less than 10%) in the endosymbiotic community of coral hosts . Various coral species, including fast-growing branching types, such as Acropora, Stylophora, and Pocillopora, as well as slow-growing massive, encrusting, and solitary corals, have been associated with Symbiodinium clade D . The prevalence of clade D Symbiodinium in corals from the Persian Gulf has been linked to their higher thermal tolerance, particularly in comparison to corals associated with clade C, which is the dominant lineage in corals from the Great Barrier Reef and other Pacific coral reef ecosystems , and clade B in corals from the Atlantic . These findings highlight the significance of Symbiodinium diversity in understanding the thermal resilience of coral reefs and the potential mechanisms underlying their adaptation to changing environmental conditions. McCulloch et al. explored the ability of coral species to withstand the adverse impacts of ocean acidification and global warming on coral reefs. Their study revealed that some coral species (i.e., Stylophora pistillata and Porites spp.) exhibit the capacity to increase pH levels within their calcifying fluid, crucial for the deposition of calcium carbonate and maintenance of the coral structure, even in the face of declining seawater pH levels. The study demonstrated the significance of acid-base regulation mechanisms for corals' resilience to the effects of ocean acidification, allowing them to maintain or increase their calcification rates despite rising ocean acidification. Moreover, the study indicated that corals could acclimate to extended acidification, which enables them to maintain or increase their calcification rates by upregulating their internal pH levels, thus providing insight into potential strategies for mitigating the effects of climate change on coral reefs. A similar adaptation resilience strategy against ocean acidification was observed in cold-water scleractinian corals (i.e., Caryophyllia smithii, Desmophyllum dianthus, Enallopsammia rostrata, Lophelia pertusa, and Madrepora oculate) . Oceanographic processes, such as upwelling and tidal currents, also play a significant role in helping corals avoid bleaching. In areas where upwelling events mix deeper, cooler water with shallow warmer water, thermal stress is reduced ; for example, in northern Galapagos during the 2015/16 ENSO and Nanwan Bay, southern Taiwan, during summer . Similarly, a coral reef's ability to resist bleaching is bolstered by the elimination of potentially damaging oxygen radicals due to the swift water flow associated with tidal currents . Therefore, in summary, the scientific community has identified various adaptive strategies that could enhance the resilience and resistance of coral reefs to these challenges. Going forward, it is crucial to continue ongoing research efforts to better understand the mechanisms underlying coral resilience and resistance, identify research gaps, and develop new management strategies for protecting these vital ecosystems. This can be achieved through a multidisciplinary approach that combines laboratory-based experimentation, field research, and community engagement. In addition, collaborations between the scientific community and policymakers can facilitate the implementation of evidence-based management practices that promote the resilience and resistance of coral reefs to climate change and other stressors. 7. Conclusions The scientometric analysis that is presented in this article demonstrates that research on coral reefs in relation to climate change has emerged as one of the potential fields, with interest in this topic has grown steadily since the 2000s. The increasing global temperatures are posing a significant threat to coral reefs, leading to widespread coral bleaching and mortality. Moreover, changes in ocean chemistry brought on by an increase in carbon dioxide levels lead to ocean acidification, which can worsen the effects of rising temperatures on corals . In addition, sea level rise and coastal development are transforming the physical structure of coral reef ecosystems, exacerbating the negative effects of the other stressors . These changes are harmful not only to the coral reefs but also to the plethora of species that rely on them for survival and the communities that rely on them for livelihoods and for protecting the coast. Future challenges for developing countries like those within the coral reef triangle initiative (i.e., Indonesia, Malaysia, the Philippines, Papua New Guinea, Timor Leste, and the Solomon Islands) will center on access to funding for conservation-restoration efforts and continued monitoring studies. There are several ways that ongoing research and coordinated action can help coral reefs cope with the effects of climate change:Monitoring: Regular coral reef monitoring can reveal vital details about the well-being and state of the reefs as well as the effects of climate change. These data can be used to pinpoint especially vulnerable regions and monitor long-term changes. Scientists and environmentalists can detect early warning signs of coral bleaching and other detrimental effects by monitoring coral reefs, which enables them to take action before it is too late; Research: Collaborative research efforts can contribute to a better understanding of the impacts of climate change on coral reefs and the mechanisms underlying these impacts and can also aid in developing and rigorously testing intervention and restoration techniques for coral reefs. Given that the preservation of coral reef ecosystems requires a range of interventions, including biological, ecological, and social strategies for mitigation and adaptation , such research can help create more efficient restoration, conservation and management strategies; Conservation and management: Collaborative conservation and management efforts can assist in mitigating the effects of climate change on coral reefs. Protected areas and marine reserves, for instance, can aid in mitigating the effects of overfishing and pollution, thereby making coral reefs more resilient to the effects of climate change; Mitigation: joint efforts can also aid in lowering atmospheric greenhouse gas concentrations, which are primarily responsible for climate change, for instance, by collaborating with regional organizations and authorities to advance sustainable development and lower carbon emissions; Public education and awareness: Raising public understanding of the effects of climate change on coral reefs can encourage support for management and conservation initiatives. Author Contributions Conceptualization, C.S.T. and M.N.A.; methodology, M.N.A.; software, C.S.T.; validation, J.B., Z.V.-G. and V.R.; formal analysis, F.L.; investigation, G.S.; resources, I.G.; data curation, J.B.; writing--original draft preparation, C.S.T.; writing--review and editing, M.N.A.; visualization, F.L., G.S., I.G., V.R. and Z.V.-G.; supervision, M.N.A.; project administration, G.S.; funding acquisition, J.B., Z.V.-G., V.R. and I.G. All authors have read and agreed to the published version of the manuscript. Institutional Review Board Statement Not applicable. Informed Consent Statement Not applicable. Data Availability Statement No data was generated from the study. Conflicts of Interest The authors declare no conflict of interest. Figure 1 Threats to coral reefs posed by factors related to climate change. Figure 2 Article structure diagram. Figure 3 Number of original research articles on the impact of climate change on coral reefs published annually from 1977 until 2021. Figure 4 Nations publishing the most research on coral reefs and climate change generated from the freely editable map chart website (accessed on 10 January 2023)). Figure 5 Collaborative network of institutions researching coral reefs and climate change from 1976 to 2021. Figure 6 Network of linked research disciplines. The sizes of the modes are proportional to the frequency of the subject category cooccurrence. The thickness of the lines between the two nodes is proportional to the strength of the linkages between the two research disciplines. Figure 7 The reference co-citation research cluster network. Based on a one-year interval, a 24-cluster network of document co-citation with burst detection from 1976 to 2021. Node sizes are proportional to the frequency of the publications' co-citations. Figure 8 A timeline co-citation analysis. Nodes represent references, whereas lines represent connections between those references. Larger nodes indicate higher frequencies of citations. References with strong citation bursts are shown as red circles, whereas references with high centrality are shown as yellow circles. Longer line segments indicate longer time spans. Figure 9 Distribution of co-cited keywords in the field of coral reef and climate change. Figure 10 Dual-map overlay on the impact of climate change on coral reefs research. animals-13-00949-t001_Table 1 Table 1 Top 20 institutions in the field of coral reef and climate change. Institution Record Count James Cook University (Australia) 1119 Australian Institute of Marine Science (Australia) 727 University of Queensland (Australia) 643 University of California System (USA) 449 Centre National de la Recherche Scientifique, CNRS (France) 443 UDICE French Research Universities (France) 400 National Oceanic Atmospheric Admin NOAA (USA) 379 University of Hawaii System (USA) 345 University of Western Australia (Australia) 335 Commonwealth Scientific Industrial Research Organization CSIRO (Australia) 312 Institut de Recherche pour le Developpement (France) 279 State University System of Florida (USA) 270 University of Miami (USA) 242 United States Department of the Interior (USA) 199 University of Hawaii Manoa (USA) 191 King Abdullah University of Science Technology (Saudi Arabia) 185 Smithsonian Institution (USA) 182 United States Geological Survey (USA) 178 University of California San Diego (USA) 177 Australian National University (Australia) 176 animals-13-00949-t002_Table 2 Table 2 Top 20 funding agencies related to coral reef and climate-change-related studies. Funding Agency Record Count Australian Research Council (Australia) 1001 National Science Foundation (USA) 807 Australian Government (Australia) 307 National Oceanic and Atmospheric Administration (USA) 258 UK Research Innovation (United Kingdom) 248 Natural Environment Research Council (United Kingdom) 231 National Natural Science Foundation (China) 201 European Commission of the European Union (EU) 187 National Science Foundation (NSF) Directorate for Geosciences (USA) 177 Ministry of Education Culture, Sports Science, and Technology (Japan) 161 Japan Society for The Promotion of Science (Japan) 142 Australian Institute of Marine Science (Australia) 134 CGIAR (United Nations) 126 German Research Foundation (Germany) 124 Consejo Nacional de Ciencia y Tecnologia (Mexico) 117 National Council for Scientific and Technological Development (Brazil) 114 Natural Sciences and Engineering Research Council of Canada (Canada) 102 King Abdullah University of Science Technology (Saudi Arabia) 92 Agence Nationale de la Recherche (France) 84 Grants-in-Aid for Scientific Research (Kakenhi) (Japan) 78 animals-13-00949-t003_Table 3 Table 3 Top 20 authors in the field of coral reef and climate change. Institution Affiliation Citation Count Terry Hughes James Cook University ARC Centre of Excellence for Coral Reef Studies 2605 Ove Hoegh-Guldberg The University of Queensland ARC Centre of Excellence for Coral Reef Studies 2441 Katharina Fabricius Australian Institute of Marine Science 1108 Peter W. Glynn University of Miami 979 Tim McClanahan The Wildlife Conservation Society ARC Centre of Excellence for Coral Reef Studies 960 Peter Mumby The University of Queensland ARC Centre of Excellence for Coral Reef Studies 907 David R. Bellwood James Cook University 891 John Pandolfi The University of Queensland ARC Centre of Excellence for Coral Reef Studies 866 Kenneth R.N. Anthony Australian Institute of Marine Science 841 John Bruno The University of North Carolina 781 Barbara E. Brown Newcastle University 773 Andrew C. Baker University of Miami 749 Glenn De'ath The Australian Institute of Marine Science 746 Joan Kleypas National Center for Atmospheric Research 729 Ray Berkelmans Australian Institute of Marine Science 705 Nick Graham Lancaster University 687 Michael Lesser The University of New Hampshire 661 Joseph Loya Tel Aviv University 636 Peter J. Edmunds California State University 596 Toby Gardner Stockholm Environment Institute 570 animals-13-00949-t004_Table 4 Table 4 The most highly cited references about coral reefs and climate change. Reference Citations Burst Index * Burst Period Journal Centrality ** Cluster Hughes et al. 546 157.67 2018-2021 Nature 0.7 #0, #4 Hughes et al. 359 125.52 2018-2021 Science 0 #4 Hoegh-Guldberg et al. 351 166.14 2008-2012 Science 0.3 #0, #2 Hughes et al. 252 87.69 2018-2021 Nature 0.4 #0, #7 Hughes et al. 246 94.42 2019-2021 Nature 0.01 #8 De'Ath et al. 204 79.65 2013-2017 PNAS 0.6 #7, #8 Pandolfi et al. 175 68.19 2012-2016 Science 0.4 #0, #2 Fabricius et al. 162 63.09 2012-2016 Nature Climate Change 0.05 #2 LaJeunesse et al. 134 68.39 2013-2017 Current Biology 0 #6 Hughes et al. 122 48.71 2011-2015 Trends in Ecology & Evolution 0.01 #0 Heron et al. 118 33.85 2018-2021 Scientific Reports 0.6 #4 Ainsworth et al. 117 28.75 2017-2021 Science 0.4 #0, #4 Palumbi et al. 116 39.87 2015-2019 Science 0.05 #0 Baker et al. 108 50.7 2010-2013 CETP 0.04 #5, #6 Anthony et al. 105 45.6 2009-2013 Global Change Biology 0.4 #10 Kroeker et al. 104 39.22 2014-2018 Global Change Biology 0.4 #2, #10 Zeebe and Wolf-Gladrow 97 39.97 2010-2014 Gulf Professional Publishing 0.6 #4, #10 Jackson et al. 96 34.08 2016-2019 Global Coral Reef Monitoring Network 0.4 #7, #18 Bruno and Selig 95 44.45 2008-2012 PLoS one 0.05 #0, #7 * Burst index: the value generated from the CiteSpace indicates the level of importance of each article in the field. ** Centrality: the main focused article between cited references in the publications. animals-13-00949-t005_Table 5 Table 5 Top 10 keywords with the strongest citation bursts. Keyword Year Strength Begin End 1976-2021 French Polynesia 1992 24.51 1992 2006 ############################################## Record 1992 24.36 1992 2005 ############################################## El Nino 1995 22.08 1995 2006 ############################################## Australia 1992 21.33 1992 2007 ############################################## Indian Ocean 1990 17.72 1998 2010 ############################################## Continental shelf 1993 17.14 1993 2010 ############################################## Sea level 1991 16.43 1991 2005 ############################################## Sea surface temperature 1996 15.91 1996 2006 ############################################## Ocean warming 2017 15.09 2018 2021 ############################################## Island 1991 14.65 1991 2005 ############################################## Disclaimer/Publisher's Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). 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PMC10000161

Naked mole-rats (Heterocephalus glaber) are very unusual among subterranean mammals in that they live in large colonies and are extremely social, spending large amounts of time gathered together in underground nests more than a meter below the surface. Many respiring individuals resting in deep, poorly ventilated nests deplete the oxygen supply and increase the concentration of carbon dioxide. Consistent with living in that atmosphere, naked mole-rats tolerate levels of low oxygen and high carbon dioxide that are deadly to most surface-dwelling mammals. Naked mole-rats appear to have evolved a number of remarkable adaptations to be able to thrive in this harsh atmosphere. In order to successfully survive low oxygen atmospheres, they conserve energy utilization by reducing the physiological activity of all organs, manifest by reduced heart rate and brain activity. Amazingly, they resort to the anaerobic metabolism of fructose rather than glucose as a fuel to generate energy when challenged by anoxia. Similarly, high carbon dioxide atmospheres normally cause tissue acidosis, while naked mole-rats have a genetic mutation preventing both acid-induced pain and pulmonary edema. Together, these putative adaptations and the tolerances they provide make the naked mole-rat an important model for studying a host of biomedical challenges. naked mole-rat hypoxia anoxia pain NaV1.7 HIF-1a VEGF Substance P KCC2 National Science Foundation2201647 The writing of this review article was supported by the National Science Foundation (2201647). pmc1. Introduction Naked mole-rats are small rodents endemic to the arid and semi-arid regions of the sub-Saharan northeastern horn of Africa. Here, they live in a complex maze of underground burrows reaching more than 3000 m in length and reaching depths of more than 1.8 m . They primarily forage and disperse through excavating tunnels, an energetically costly activity, exacerbated by food being sparse and patchily distributed . Naked mole-rats meet all their energy and water requirements through the foods they consume and the metabolic water they generate . In addition to having to contend with the daunting problems associated with foraging below ground, they may also encounter numerous physiological challenges imposed by underground atmospheric conditions. Both gas and heat exchange are dependent on the diffusion and porosity properties of the soil and may be impaired. As fossil evidence suggests they have occupied this subterranean niche since the early Miocene , it is not surprising that they have evolved a fascinating montage of biological traits well suited to the harsh conditions they encounter in their hot, humid, and poorly ventilated sealed maze of burrows (for a review, see ). These include having a lower body temperature (32 degC) than most mammals and a reduced basal metabolic rate for their body size. Not surprisingly, when they are housed outside of the warm and humid confines of these equatorial burrows, and lacking an insulatory pelage, naked mole-rats are poor thermoregulators, unable to counter the high rates of heat loss with effective endothermic mechanisms and rather rely on communal huddling to keep warm . Naked mole-rats live in large groups of up to 295 individuals . Reproduction is confined to a single breeding female and 1-3 breeding males within the colony, and there is a high reproductive skew such that less than 1% of all individuals within the colony get the opportunity to breed over their lifetime, contributing to their description as one of only two eusocial mammals . Living in large colonies, they have distinct, culturally transmitted vocal dialects , despite having a non-functional cochlear amplifier and associated poor auditory thresholds . Many of their other adaptive traits to this environmental niche may also protect against numerous disease states, including cancer, neurodegeneration, and cardiovascular disease, contributing to prolonged healthy aging and extreme longevity . Naked mole-rats are extremely long-lived for their body size, living approximately six times longer than predicted allometrically. Intriguingly, unlike every other mammalian species studied to date, in which after the age of sexual maturity there is an exponential increase in risk of dying, naked mole-rats do not. Rather, death is random, with similar likelihoods of dying at 2 and 20 years of age . In other words, they do not show an increased risk of dying with advancing age, defying Gompertzian fundamental laws of mortality. This stochastic pattern of age-independent risk of dying is accompanied by a negligible senescence phenotype, maintaining physiological function well into their fourth decade , as well as retaining many youthful paedomorphic features, including high oxygen affinity fetal hemoglobin in adult life . These and many other unusual qualities make the naked mole-rat an interesting model species with an abundance of recent genomic and epigenetic aging clock data available, facilitating the creation of explanatory models . In this review, we will cover significant adaptations that the naked mole-rat has evolved, likely due to living in their unique subterranean niche . Their communal lifestyle, while facilitating better foraging success, is not without problems. The animals rest and respire together in deep nests where diffusion through the soil is poor, creating an atmosphere that is both hypoxic and hypercapnic. Naked mole-rats are very tolerant of these environmental challenges , and here we report on some of the underlying mechanisms that have been identified to date . 2. Tolerance to Oxygen Deprivation Naked mole-rats demonstrate a profound tolerance to oxygen deprivation when compared to mice . This includes resilience against exposure to low oxygen (hypoxia) atmospheres as well as no oxygen (anoxia). While all mice can survive a 30 s exposure to anoxia, none survive a 1 min exposure . In contrast, all naked mole-rats survived an 18 min exposure to anoxia, although no naked mole-rats survived a 30 min exposure. During anoxia, naked mole-rats drastically reduced physiological activities, presumably to conserve energy expenditure . Within 30 s of exposure to anoxia, animals lose consciousness, and their respiration rate is drastically depressed , suggestive of a lower metabolic rate . The red line below the X-axis indicates the time course of anoxia exposure. After exposure to anoxia and a return to normoxic room air, the respiration rate increased toward baseline levels. During anoxic exposure, the heart rate of naked mole-rats decreased from about 200 beats per minute to about 50 beats per minute and then recovered to baseline levels of activity under normoxic recovery conditions . This is consistent with data collected by Ilacqua et al. , showing that under acute hypoxia and anoxia, naked mole-rats reduce metabolic rates by decreasing locomotion and body temperature. For mice, the electrical activity in mouse hearts was not detectable in the electrocardiogram after 6 min of exposure to anoxia. Another physiological metric is brain activity as measured by electroencephalography (EEG) of the caudate putamen. It was found that brain activity in naked mole-rats was dramatically reduced during a 10 min exposure to anoxia . The EEG revealed that within 3-5 min, brain activity had declined and remained at these low levels for the duration of anoxic exposure. EEG recordings were acquired using a four-channel Pinnacle Technology EEG system, and electrodes were positioned stereotaxically. It is important to note that naked mole-rats in the wild are likely to encounter hypoxia, not full anoxia. Park et al. showed that naked mole-rats tolerate 5% O2 for at least 5 h, whereas mice cannot survive for more than about 12 min. Additionally, Ilacqua et al. tested naked mole-rats with 9%, 7%, 5%, and 3% O2 and showed an incremental decline in activity with increasing severity of hypoxia. Additional studies using electrophysiological assessments of hippocampal brain slices in an interface recording chamber reported intrinsic brain tolerance to anoxia in naked mole-rats , see Figure 3A. Evoked potential data for one slice from a mouse and one slice from a naked mole-rat are illustrated in Figure 3B, wherein the slices were stimulated alternately every 10 s. After reaching a stable baseline, the slices were exposed to anoxia until both slices lost function (shaded area). In these examples, the slice from a naked mole-rat took significantly longer to experience anoxic depolarization (AD) and loss of detectable functionality. After reoxygenation, naked mole-rat slices showed recovery of synaptic transmission, while mouse slices did not. Given that naked mole-rats and mice have disparate body temperatures (32 degC and 37 degC, respectively), studies were undertaken at waterbath temperatures approximating their divergent body temperatures . While waterbath temperature greatly affects the time to loss of synaptic function and transmission, it should be noted that at both temperatures, the naked mole-rats showed a significantly longer time to loss of detectable functionality. Naked mole-rat brain cells show a much lower accumulation of intracellular calcium during hypoxia compared to cells from mice . One detrimental effect of hypoxia is that it triggers an increase in intracellular calcium concentration, resulting in the activation of several toxic downstream signaling cascades . Unlike the brain slices from mice that show a significant increase in calcium influx during hypoxic challenge, that observed in mole-rat brain slices is considerably attenuated . One of the major avenues for neuronal calcium entry is the NMDA receptor (NMDAR). The NMDA receptor is a fundamental building block of neuronal processing, being a central mediator of Hebbian learning and integral to the induction of Long-term Potentiation (LTP) and Long-term Depression (LTD) of neurons. Once the NMDAR is opened, it is notable for its large calcium current that activates second messenger pathways within the neuron. Although calcium entry is necessary for many forms of synaptic plasticity and physiological behavior of the NMDAR, it is highly regulated in the cell, and errant entry during times of overactivation can quickly lead to excitotoxicity . The canonical NMDAR is composed of several subunits, with each receptor being a tetramer that is composed of two requisite NR1 (NMDA receptor, subunit 1) subunits and two subunits of a combination of GluN2A, GluN2B, GluN2C, or GluN2D. Bickler et al. found that the GluN2D subunit reduces its mean open time during hypoxia, reducing hypoxia-induced calcium accumulation . It was demonstrated that calcium accumulation and NMDAR currents were reduced under hypoxia in NMDARs composed of the GluN2D subunit. This alteration in the opening of these channels likely provides a mechanistic contribution to the greater tolerance of hypoxic conditions. NMDAR subunit composition is region-dependent in most mammals, but globally the proportion of GluN2B and GluN2D decreases with maturity . Adult mice retain only about 10% of the hypoxia-resistant GluN2D subunit compared to neonate mice . On the other hand, adult naked mole-rats retain about 66% of these receptors with GluN2D subunits that close under hypoxia compared to neonate naked mole-rats . It is believed that this neotenous feature of the naked mole-rat is an adaptation to the hypoxic environment described here, where evolution has favored the trade-off of kinetic characteristics idiosyncratic to the GluN2D subunit, such as lower conductance for a diminished entry of calcium, as more optimal at their point on the fitness landscape. Figure 4 Reduced calcium accumulation from hypoxia in naked mole-rat hippocampal cells (A) and greater retention of GluN2D in naked mole-rats compared to mice (B) **** p < 0.0001. (A) is from , and (B) is from . Unique molecular adaptations to hypoxia have also been discovered in the naked mole-rat. These include findings by Xiao et al. that Hypoxia Inducible Factor 1 alpha (HIF-1a) and Vascular Endothelial Growth Factor A (VEGFA) are significantly upregulated in naked mole-rat tissues compared to ICR (Institute of Cancer Research) control mice . Since the discovery in 1995 of HIF-1a by Semenza and its functional role as a molecular sensor of oxygen tension, HIF-1a has played a central mechanistic role in hypoxia research. HIF is hydroxylated in an oxygen-dependent reaction on conserved prolines, allowing for its recognition by Von Hippel-Lindau (VHL), a substrate binder component of the E3 ubiquitin ligase, and thereby targeted for the rapid proteasomal destruction of HIF under normoxic conditions . Under hypoxic conditions, this hydroxylation cannot occur, and HIF levels remain stable and translocate into the nucleus. This oxygen-dependent transcriptional activator regulates the expression of over 100 genes and thereby provides a central hub for deploying the appropriate hypoxic response in the various organ systems . In this manner, HIF regulates biochemical responses to oxygen deprivation by modulating the expression of genes involved in glucose uptake, energy metabolism, as well as cell proliferation and cell death . Xiao et al. have found that even under the normoxic conditions found in the laboratory, naked mole-rat expression of HIF-1a is significantly elevated in several tissues, with concomitant increases in VEGFA, which controls angiogenesis and oxygen delivery . These responses are undoubtedly of benefit to the naked mole-rat, which experiences bouts of hypoxia in their crowded nest environments and perhaps other burrow segments in the wild. Genetic analysis of the naked mole-rat genome by Kim et al. uncovered two intriguing mutations hardwired into the HIF-VHL system . Within the HIF-1 sequence at location 407, the naked mole-rat has exchanged threonine for isoleucine (T407I). This point mutation was proposed to be significant as, within the 3D structure of HIF-1, it lies within the VHL-binding domain , altering substrate binding for ubiquitination. In addition, it was found that naked mole-rat VHL contains a mutation at residue 166 (V166I). This site is functionally important since other mutations found here are correlated with a pathological outcome, Von Hippel-Lindau disease, due to a weakened interaction in the HIF-VHL complex resulting in reduced HIF1a degradation, a longer HIF-1 half-life, and concomitantly higher constitutive levels . These mutations may contribute significantly to the higher constitutive levels observed by Xiao et al. and are consistent with an evolutionary adaptation to tolerate hypoxic conditions as described in this review. Naked mole-rats have another unusual systemic adaptation for dealing with hypoxia and anoxia that involves undergoing drastic metabolic rewiring, which enables the anaerobic glycolysis of fructose . In this study, it was demonstrated that naked mole-rats express GLUT5 (solute carrier family 2 member 5), the fructose transporter, at significantly higher levels (>10-fold) in their brain and heart cells compared to mice . Naked mole-rats also showed a substantial and significant increase in fructose in the blood during anoxia, whereas mice did not. Levels of ketohexokinase (KHK), which phosphorylates fructose into fructose-1-phosphate (F1P), were significantly elevated in the naked mole-rat. Additionally, in brain slices exposed to hypoxia, a metabolic flux analysis demonstrated that fructose-derived carbons accumulated at a significantly higher level within many glycolytic intermediates in slices from naked mole-rats compared to slices from mice, indicating the metabolism and incorporation of fructose into naked mole-rat metabolism. Finally, in a demonstration of the physiological utility of this metabolic rewiring, brain slices and isolated hearts from naked mole-rats and mice were tested by switching glucose to fructose in the bath, and their functionality was measured. In both the brain and heart preparations, naked mole-rats retained significantly better performance with fructose compared to mice. A model was proposed in which glucose metabolism is blocked under near-anoxia by inhibition at the rate-limiting step of phosphofructokinase (PFK). This feedback inhibition is induced by protons, ATP binding, or downstream intermediates such as citrate . The authors put forward that the naked mole-rat has evolved to efficiently interface with and make use of fructose under near-anoxic conditions to bypass the metabolic block at PFK that glucose suffers from and provide for a continued, albeit less efficient, energetic source under these trying periods. Another detrimental effect of hypoxia in most mammals is a rapid buildup of fluid in the lungs known as pulmonary edema. This is caused by altered sympathetic tone, vasoconstriction, and pulmonary hypertension, as well as altered alveolar endothelial and epithelial permeability to water and macromolecules, causing an increased permeability across the alveolar barrier and the capillaries to leak into the alveolar sacs and interstitial spaces that overwhelm the alveolar reabsorption capacity . This is manifested by a change in the wet/dry weight of excised lung tissue. Naked mole-rats appear to be completely immune to this effect. Park et al. exposed naked mole-rats and mice to normoxia (20% O2) or hypoxia (10%, 7.5%, or 5% O2) for 15 min. Mice showed severe pulmonary edema for hypoxic atmospheres of 7.5% and 5% O2, while naked mole-rats showed no edema at either of these low O2 concentrations. . Currently, the mechanisms facilitating the lack of hypoxia-induced edema in naked mole-rats are unknown. 3. Tolerance to High Concentrations of CO2 Room air contains about 0.03% CO2. Breathing higher concentrations of CO2 causes acidification of the tissues, with a variety of associated behavioral and physiological responses. For example, humans and laboratory mice find elevated CO2 concentrations to be painful to the upper respiratory tract . On the other hand, naked mole-rats are less averse to elevated CO2 concentrations compared to mice. In an avoidance test, naked mole-rats and mice were placed in a rectangular arena where CO2 was infused at one end and room air at the other . In that study, mice avoided all three of the CO2 concentrations tested: 2.5%, 5%, and 10% , whereas naked mole-rats only avoided the highest concentration of 10% CO2 , implying that the naked mole-rats did not find 2.5% or 5% CO2 to be aversive. Elevated CO2 concentrations also trigger an increase in respiration rate . This can be observed in mice for 5% and 10% CO2 . Naked mole-rats only showed an increase in respiration rate of 10% and greater concentrations of CO2 . Johansen et al. showed that naked mole-rat blood can buffer acid better than mouse blood. Consistent with this observation, Park et al. showed that naked mole-rats do not show systemic acidification from breathing CO2 concentrations below 10%, whereas mice show acidification from breathing even 1% CO2 . Zions et al. demonstrated that captive naked mole-rat habitats have a high degree of anisotropy in carbon dioxide levels that are tightly coupled to the location of the nest chamber . The previously described eusocial nature of this species lends itself to individuals spending a higher fractional time respiring in the nest chamber than the surrounding area. This is reflected in a higher carbon dioxide load in the nest chambers and the surrounding areas. Figure 9A shows a drawing of one of the colony caging systems used in their study. The nest chamber, indicated by the hashtag symbol, was the area showing consistently the highest concentration of CO2 in the system and is the chamber in which the naked mole-rats prefer to crowd together, despite the high concentration of CO2 as shown in Figure 9B. Hypercapnia has been demonstrated to suppress neuronal activity . Zions et al. report that the neuronal potassium-chloride cotransporter 2 (KCC2) has a loss-of-function point mutation at position 952 where a highly conserved arginine has been mutated to histidine in the naked mole-rat (R952H) . This mutation results in less chloride being extruded from the neuron, altered chloride homeostasis, and ultimately a reduction in inhibitory currents through GABAergic ion channels (gamma-aminobutyric acid receptors). Zions et al., therefore, propose an elegant hypothesis in which hypercapnia's ability to suppress neural activity acts to shift the excitation-inhibition balance in naked mole-rats' neuronal circuits and allows them to conserve energy by reducing the expenditure necessary to drive KCC2 . This hypothesis is supported by their finding of a similar mutation (R952C) in the eusocial relative of the naked mole-rat, the Damaraland mole-rat. In another study, LaVinka and Park showed that naked mole-rats do not avoid fumes from 10% ammonia (household cleaning ammonia) or 20% acetic acid . In contrast, laboratory rats, mice, and Damaraland mole-rats significantly avoided these irritants. Breathing high concentrations of CO2 also induces rapid and life-threatening pulmonary edema. CO2 induces changes in the alveolar permeability and reabsorption properties of the lung, and pulmonary edema happens when CO2 acidifies the tissues of the lungs, activating acid-sensitive sensory nerves in the tissues that trigger neurogenic inflammation and edema. Park et al. measured pulmonary edema from CO2 in naked mole-rats and mice . Naked mole-rats showed no edema even at the highest concentration of CO2 tested (50%) . In contrast, mice showed significant edema at 15% and robust edema at 20-50% CO2 concentrations. A mutation in the voltage-gated sodium channel NaV1.7 likely contributes to the lack of CO2-induced pulmonary edema in naked mole-rats . Unlike the positive charge of a highly conserved loop motif (the KKV motif in humans) in domain IV observed in most species, this motif in naked mole-rats has a net negative charge . This mutation of residues 1718 and 1720 imparts an altered charge landscape in this region through the placement of two mutated glutamate (E) residues flanking a conserved lysine (the EKE motif). As a result, the sensory nerves that normally respond to acid become significantly less sensitive, blocking the presence of acid-induced neural signaling . The authors suggest that there is a greater level of proton inhibition in naked mole-rat NaV1.7 that alters the excitation dynamics of these neurons under acidic conditions. This in turn serves to balance depolarization from TRPV1 (transient receptor potential cation channel subfamily V member 1) and ASIC (acid-sensing ion channel) receptors and inhibit the propagation of this signal via NaV1.7. Ultimately, this mutation likely reduces the level of signaling, for under acidic conditions, ASIC receptors will be continually activated by protons. This mutation is likely an evolutionary adaptation to living in an underground hypercapnic niche. 4. Insensitivity to Chemical Pain Consistent with a gene mutation in the nerves that sense acid, naked mole-rats are completely insensitive to acid pain in their skin . Figure 13A shows the behavioral response of mice and naked mole-rats that received an injection of acidic saline in the skin of one hind paw. Mice responded with robust licking of the injection site, whereas naked mole-rats had virtually no response. Consistent with acid insensitivity being associated with a gene mutation, naked mole-rat pain-sensing neurons also showed no response to an acidic bath solution . Naked mole-rats were also behaviorally insensitive to the injection of capsaicin, the spicy substance found in "hot" chili peppers . However, unexpectedly, pain-sensing neurons from naked mole-rats demonstrated robust responses to capsaicin in the bath solution . Because the mutation in NaV1.7 is specific for sensing acid pain, there must be a different mechanism associated with insensitivity to capsaicin. Park et al. showed that naked mole-rats lack the neuropeptide transmitters Substance P and Calcitonin Gene-Related Peptide from their pain-sensing peripheral nerves. In a subsequent study, Park et al. showed that introducing exogenous Substance P alone was capable of rescuing pain behaviors from capsaicin . The researchers used two techniques to introduce Substance P: the application of a transgenic herpes virus expressing the preprotachykinin (PPT1/TAC1) gene for Substance P and the intrathecal injection of Substance P directly into the spinal cord . Figure 14B shows pain behavior for the virus-treated foot and the untreated foot . In this test, the naked mole-rats were lightly anesthetized, and the feet were heated with a calibrated heat source. The first three data points show foot withdrawal latencies from the heat source of about 12 s for both feet. The remaining data points show withdrawal latencies after topical application of capsaicin to both feet. The untreated foot shows withdrawal latencies that are similar to the latencies prior to capsaicin application of about 12 s. However, the virus-treated foot shows robust sensitization to capsaicin in the form of significantly faster withdrawal latencies of about 6 s. Figure 14C shows the results of introducing Substance P via intrathecal injection. The two bars on the left show the results of testing with capsaicin. In this test, one hind paw was injected with a capsaicin solution prior to Substance P application . As noted previously, there was virtually no response. However, following the introduction of Substance P, the naked mole-rats spent a significant amount of time licking the injection site, supporting the theory that exogenous Substance P could rescue capsaicin pain. The two bars on the right show the results for acid pain. In this case, the application of Substance P had no effect on acid pain insensitivity, supporting the hypothesis that acid insensitivity was mediated by another mechanism, namely the mutation in NaV1.7. It is noteworthy that naked mole-rats respond to acute mechanical pain (pinch) and acute thermal pain (heat) in the same way as mice . Both naked mole-rats and mice react to a pinch with a tail clip in about 6 s , and both naked mole-rats and mice react to heat pain in about 12 s . The naked mole-rat was the first mammal identified to lack acid and capsaicin pain. However, a subsequent study found that some other African subterranean rodents also showed pain insensitivities . Figure 16 shows the results of behavioral tests on eight African mole-rat species, the East African root rat, and the laboratory mouse. Animals were tested with diluted solutions of capsaicin , acid (the lemon drawing), and allyl isothiocyanate (AITC), the active ingredient in mustard oil and wasabi root (the wasabi root drawing). Pain insensitivity is indicated by an "X" over the drawings. In addition to the naked mole-rat, there was one other species, the Natal mole-rat, that was insensitive to capsaicin, and two other species that were insensitive to acid: the Cape mole-rat and the East African root rat. An interesting finding was that the Highveld mole-rat, while sensitive to acid burn, was insensitive to AITC, which activates TRPA1 (transient receptor potential cation channel, subfamily A, member 1) receptors . The Highveld mole-rat shares its burrows with the Natal droptail ant, whose normally painful sting is known to activate TRPA1 receptors, likely suggesting a co-evolutionary history. Eigenbrod, et al. showed that, in addition to being insensitive to AITC, the Highveld mole-rat was insensitive to the sting of the Natal droptail ant. Other species tested in the same study showed pain behaviors from the sting. Additional experiments showed that the Highveld mole-rat overexpresses the leak channel NALCN (sodium leak channel, nonselective), which could act to dampen excitation in pain nerves after TRPA1 activation, thus rendering Highveld mole-rats insensitive to the ant's sting . 5. Sensory Vibrissae Mediate Orientation to Touch In addition to pain insensitivities, the somatosensory system of the naked mole-rat has another interesting feature. Although naked mole-rats lack fur, their bodies have 10 rows of sensitive vibrissae that form a sensory array . One row of vibrissae can be seen in the photograph in Figure 17. Crish et al. found that deflecting a single vibrissa triggered an accurate and repeatable orientation of the snout to the position of the deflected vibrissa. Interestingly, simultaneously deflecting two vibrissae on the same side of the body triggered an orientation response to a position that is the average of the two stimulated vibrissae. For example, stimulating a vibrissa at the shoulder and hip triggered an orientation of the snout to a position halfway between the shoulder and the hip . This result indicates that the naked mole-rat nervous system is capable of making a computation about touch location. However, this is not the case for simultaneously deflecting two vibrissae on opposite sides of the body, which triggers an orientation to either one or the other deflected vibrissa in a winner-take-all strategy . 6. Conclusions Naked mole-rats have evolved under extraordinary environmental challenges, resulting in the evolution of a suite of exceptional adaptations to deal with those challenges. The current review article highlights some of those adaptations related to the naked mole-rat's tolerance to hypoxia, hypercapnia, and pain, including some of the underlying mechanisms. Although, there is still much to be learned about naked mole-rat adaptations to the atmospheric conditions under which they have evolved, elucidating the protective mechanisms they employ may highlight new directions for the treatment of human pathological conditions such as inflammation and concomitant pain, as well as ischemia/reperfusion injury as occurs in cardiac infarctions and stroke. Acknowledgments We are grateful to Rochelle Buffenstein for a critical reading of an early draft of this manuscript. We acknowledge funding from the National Science Foundation (T.J.P.), the UIC Liberal Arts and Sciences Undergraduate Initiative (A.Z.), and the UIC Honors College Research Grant (A.Z.). Author Contributions Conceptualization, V.G.A. and T.J.P.; writing--original draft preparation, V.G.A. and T.J.P.; writing--review and editing, V.G.A., A.Z., I.V. and T.J.P.; project administration, T.J.P. All authors have read and agreed to the published version of the manuscript. Conflicts of Interest The authors declare no conflict of interest. Figure 1 Naked mole-rats survive much longer than mice in 0% oxygen. The graph shows the percent of animals able to survive a given duration of anoxia. Different animals were tested for each time point. NMR = naked mole-rat. These data are from . Figure 2 Naked mole-rats show reduced physiological activity during anoxia. Average respiration rate (A) and heart rate (B) of naked mole-rats during an 18 min anoxia exposure (red line below the X-axis) followed by normoxia. These data are from . (C) Top, an example electroencephalography (EEG) trace from a naked mole-rat before, during, and after a 10 min of exposure to anoxia. Bottom: average brain activity for naked mole-rats before, during, and after a 10 min exposure to anoxia. This data is from . N = 3 for panels (A-C). Figure 3 Hippocampal brain slices from naked mole-rats maintain functionality during anoxia much longer than slices from mice. (A) This photograph shows the interface recording chamber where slices were tested (photo by Thomas Park). (B) The graph shows evoked potential amplitude before, during, and after exposure to anoxia for one slice from a mouse and one slice from a naked mole-rat. The shaded region corresponds to when anoxia was applied; at other times, the brain slices were exposed to normoxia. (C) Mean time to anoxic depolarization for slices from mice (MSE), n = 19, and naked mole-rats (NMR), n = 13. * p < 0.01. These data are from . Figure 5 Quantified levels of HIF-1a mRNA in naked mole-rats compared to ICR mice are shown in (A), where a significant increase in expression was seen in all six measured tissues. Significant increases were also seen in VEGFA mRNA expression in the naked mole-rat across the same tissues as shown in (B). * p < 0.05, ** p < 0.01. These data are from . Figure 6 Naked mole-rats have significantly higher levels of the fructose transporter, Glut5, in most organs compared to mice. * p < 0.05, ** p < 0.01, *** p < 0.001. This data is from . Figure 7 Lack of hypoxia-induced pulmonary edema in naked mole-rats. The curves show the percent change in lung wet--to--dry ratio as a function of the percent oxygen in the atmosphere. Animals were exposed to a given atmosphere for 15 min and then anesthetized with isoflurane and euthanized by rapid decapitation prior to removing and weighing the lungs. This figure is from . Figure 8 Responses of naked mole-rats and mice to elevated CO2 concentrations. Aversion behaviors are shown in (A,B). Respiration rates are shown in (C,D). Systemic acidosis is shown in (E). * p < 0.05, ** p < 0.01, *** p < 0.001. These graphs are from . Figure 9 Measured CO2 levels of a naked mole-rat cage system as employed by Zions et al. are shown in (A). # = location of the nest. The high density of respiring individuals can be seen in a typical nest environment, as seen in a colony housed at the University of Illinois Chicago (B), photo by Thomas Park. Figure 10 Naked mole-rats (NMR) do not avoid regions containing sponges saturated with noxious irritants creating ammonia or acetic acid fumes, whereas laboratory rats, mice, and Damaraland mole-rats (DMR) significantly avoid these irritants. * p< 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. This graph is from . Figure 11 Lack of CO2-induced pulmonary edema in naked mole-rats. The curves show the percent change in lung wet--to-dry ratio as a function of the percent CO2 in the atmosphere. Animals were exposed to a given atmosphere for 15 min prior to removing and determining both the wet and dry weight of lung tissue. This figure is from . Figure 12 Sequence alignment demonstrating a variant in naked mole-rat NaV1.7 that changes the pH sensitivity of the channel. The motif of interest is indicated by a yellow star. The associated charges are indicated at the far right in a green box. The orange square denotes the outer carboxylate ring. This figure is from . This mutation would alter sensitivity to proton accumulation. Figure 13 Naked mole-rats are behaviorally (A) and physiologically (B) insensitive to acid pain. They are behaviorally insensitive to capsaicin pain (C), although they showed a physiological response to capsaicin (D). The behavioral tests involved injecting an acidic saline or capsaicin solution into the skin of one hind paw. The physiological data were collected from pain-sensing C fibers from the saphenous nerve, using the in vitro skin nerve preparation as described in . These data are from . Figure 14 Rescue of capsaicin pain in naked mole-rats from the introduction of exogenous Substance P. (A) Application of a transgenic herpes virus expressing the preprotachykinin (PPT/TAC1) gene for Substance P (top) and intrathecal injection of Substance P (bottom). (B) This graph is associated with the animals treated with the virus. The graph shows foot withdrawal latency to heat before and after the topical application of capsaicin. The virus-treated foot (PPT Virus) shows sensitization from capsaicin, whereas the "No Virus" foot does not. (C) This graph shows the amount of time spent licking the injection site for capsaicin or acid injection before and after an intrathecal application of Substance P. * p < 0.05. These data are from . Figure 15 Naked mole-rats show normal (mouse-like) responses to acute mechanical pain (pinch) and acute thermal pain (heat). The bar graphs show latency to respond to a 450 g pinch from a clip at the base of the tail (the clip in the illustration is similar to the one used) or foot withdrawal from a calibrated heat source. These data are from . Figure 16 In addition to naked mole-rats, several other African subterranean rodents display insensitivity to capsaicin, acid, or AITC. On the left is a phylogenetic tree for the 10 species tested. Insensitivity is indicated by an "X" over the drawings of a chili pepper, a lemon, or a wasabi root. On the right are photographs of four of the species. This data is from . Figure 17 Sensory vibrissae mediate orientation to touch in naked mole-rats. One row of sensory vibrissae can be seen in the photograph of a naked mole-rat against a dark background (photo: Emily Vice). (A) Circular histograms show the turn vectors corresponding to orientation responses triggered by deflecting one or two vibrissae on the same side of the animal. (B) Circular histograms show the turn vectors corresponding to orientation responses triggered by deflecting one or two vibrissae on opposite sides of the animal. These graphs are from . Disclaimer/Publisher's Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). 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PMC10000162

Exosomes are biological vesicles secreted and released by cells that act as mediators of intercellular communication and play a unique role in virus infection, antigen presentation, and suppression/promotion of body immunity. Porcine reproductive and respiratory syndrome virus (PRRSV) is one of the most damaging pathogens in the pig industry and can cause reproductive disorders in sows, respiratory diseases in pigs, reduced growth performance, and other diseases leading to pig mortality. In this study, we used the PRRSV NADC30-like CHsx1401 strain to artificially infect 42-day-old pigs and isolate serum exosomes. Based on high-throughput sequencing technology, 305 miRNAs were identified in serum exosomes before and after infection, among which 33 miRNAs were significantly differentially expressed between groups (13 relatively upregulated and 20 relatively downregulated). Sequence conservation analysis of the CHsx1401 genome identified 8 conserved regions, of which a total of 16 differentially expressed (DE) miRNAs were predicted to bind to the conserved region closest to the 3' UTR of the CHsx1401 genome, including 5 DE miRNAs capable of binding to the CHsx1401 3' UTR (ssc-miR-34c, ssc-miR-375, ssc-miR-378, ssc-miR-486, ssc-miR-6529). Further analysis revealed that the target genes of differentially expressed miRNAs were widely involved in exosomal function-related and innate immunity-related signaling pathways, and 18 DE miRNAs (ssc-miR-4331-3p, ssc-miR-744, ssc-miR-320, ssc-miR-10b, ssc-miR-124a, ssc-miR-128, etc.) associated with PRRSV infection and immunity were screened as potential functional molecules involved in the regulation of PRRSV virus infection by exosomes. PRRSV serum exosome miRNAs National Natural Science Foundation of China81860150 Special Project on Innovation Driven Development of GuangxiGuikeAA18118051 National Modern Agricultural Industrial Technology Systemnycytxgxcxtd-15-01 This work was supported by grants from the National Natural Science Foundation of China (81860150), Special Project on Innovation Driven Development of Guangxi (GuikeAA18118051), and National Modern Agricultural Industrial Technology System (nycytxgxcxtd-15-01). pmc1. Introduction Porcine reproductive and respiratory syndrome virus (PRRSV) is a single-stranded positive-strand RNA virus with an envelope structure belonging to the order Nidovirales, family Arteriviridae, genus Betaarterivirus . It is spherical or ellipsoidal with a diameter of 50-65 nm under a freezing electron microscope . The PRRSV genome is about 15 kb in length with a 5' cap and a 3' polyA-tail and contains at least 10 open reading frames (ORFs) flanked by untranslated regions (UTRs) at both the 5' and 3' termini , and is wrapped by nucleocapsid protein, with lipid double-layer coating to form virus particles. Exosomes belong to vesicles with monolayer membrane structures and have the same topological structure as cells . The shape is "cup-shaped" or "disc-shaped" under an electron microscope . Exosomes can exist in the circulatory system for a long time, and substances in exosomes can be absorbed by adjacent cells or distant receptor cells and then regulate the receptor cells to participate in the exchange of genetic materials between cells . They are mainly composed of membrane surface substances and carried contents, including cell surface receptors, membrane proteins, soluble proteins, lipids, RNA (mRNA, miRNA, lncRNA, and viral RNA, etc.), genomic DNA, mitochondrial DNA . MicroRNAs (miRNAs) are a class of 18-25 nucleotides (nt) evolutionarily conserved endogenous non-coding single-stranded small RNAs, which inhibit the translation process by inducing the degradation of target mRNA or by binding with 3' UTR of target mRNA, leading to post-transcriptional gene silencing, then regulating the gene expression at the post-transcriptional level . It is estimated that miRNAs regulate more than 60% of mammalian genes post-transcriptionally . MiRNAs play an important role in intercellular communication and can also be used as a potential functional molecule for disease and virus infection, transmission, and defense . A growing number of studies have shown that miRNAs can be present in body fluids, such as saliva, urine, breast milk, and blood, and act through the body's fluid circulatory system . Exosomal miRNAs are considered to be endogenous regulators of gene expression and metabolism and can indicate various pathological conditions . Over the past two decades, it has been shown that miRNAs have crucial roles in the regulation of immune cell development, innate immune responses, and acquired immune responses. Some other miRNAs are reported to impair PRRSV infection through the following ways, directly target the PRRSV genome or PRRSV receptor, or play a role by regulating the host's innate immune response. The miR-26 family can significantly damage virus replication, and miR-26a can inhibit the replication of type 1 and type 2 PRRSV strains in porcine alveolar macrophages (PAMs) by regulating the type I interferon (IFN) pathway, which is more efficient than miR-26b . miR-30c and miR-125b are identified to modulate host innate immune response by targeting the type I IFN pathway and NF-kB pathway, respectively . MiR-23, miR-378, and miR-505 are antiviral host factors targeting PRRSV and have conservative target sites in type 2 PRRSV strains . At the same time, host miR-506 has been identified to inhibit PRRSV replication by directly targeting PRRSV receptor CD151 in MARC-145 cells . miR-181 also can indirectly inhibit PRRSV replication by down-regulating PRRSV receptor CD163 in blood monocytes and PAMs . In addition, miRNAs can promote PRRSV replication by interfering with basic cell physiology. MiR-24-3p and miR-22 directly target 3'UTR of HO-1 during PRRSV infection to escape the inhibition of heme oxygenase-1 (HO-1), a heat shock protein (also known as HSP32) on PRRSV . Pigs are known to be more susceptible to PRRSV and less able to defend themselves against the entry of this pathogen into the organism . In the present study, the innate immunity and acquired immunity of pigs infected with this virus were studied at the molecular level using a strain prevalent in the field. A serum exosome isolation kit, transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA), and Western blot (WB) were used to isolate and identify serum exosomes before and after infection with PRRSV, followed by small RNA sequencing analysis, identification, and analysis of differential expression results using bioinformatics methods to obtain a number of PRRSV-associated serum exosome miRNAs, followed by identification of data results using quantitative real-time PCR (qRT-PCR). 2. Materials and Methods 2.1. Animal Experiments Six PRRSV antigen and antibody double-negative healthy 42-day-old large white pigs were placed in the pig clean feeding system for isolation, healthcare, and environmental adaptation. All pigs were free to eat and drink without restrictions. When they were familiar with the conditions in the isolator, the pigs were nasally inoculated with 2 mL 105 TCID50/mL PRRSV NADC30-like CHsx1401, which was mentioned by predecessors . The blood of the pigs before (control group, n = 6) and 7 days after (treatment group, n = 6) virus inoculation was collected from the anterior vena cava for serum isolation. The cellular debris in the serum was removed by centrifugation at 3000 g for 15 min. All animal experiments in our study were approved by the Animal Ethics Committee of the Institute of Animal Science, Chinese Academy of Agricultural Sciences (CAAS) (Beijing, China), IAS2022-130. 2.2. Isolation and Purification of Serum Exosomes Exosome isolation and purification were carried out using the exoEasy Maxi kit (QIAGEN, Hilden, Germany, cat. no. 76064) according to the manufacturer's protocol. 2.3. Transmission Electron Microscopy (TEM) Extracted exosome suspensions were spotted onto the formvar carbo-coated copper mesh, and the exosomes were rinsed with PBS and subjected to standard uranyl acetate staining for 3 min at room temperature. After drying for several minutes at room temperature, the grid was visualized and photographed at 100 kV by transmission electron microscope (HT-7700, Hitachi-High Tech, Tokyo, Japan). 2.4. Nanoparticle Tracking Analysis (NTA) Extracted exosomes were diluted with 1 x PBS by changing the volume from 10 to 30 mL. After the sample was tested, the concentration and size of serum exosomes were analyzed by an N30E flow nano-analyzer following the manufacturer's instructions (NanoFCM, Xiamen, China). 2.5. Western Blot The extracted exosome samples were added to RIPA lysate mixed with protease inhibitor (Invitrogen, Waltham, MA, USA) and phenylmethylsulfonyl fluoride (PMSF) to extract the exosome protein, which was lysed on ice for 30 min. Then, according to the instructions of the Bradford kit, we quantified the concentration of serum exosome protein. Exosome proteins underwent thermal denaturation. The same amount of protein was separated on 12% SDS-PAGE gel and then transferred to a polyvinylidene fluoride (PVDF) membrane (Millipore, Burlington, MA, USA). It was soaked in TBST containing 5% skimmed milk powder and sealed for 1 h at room temperature. We soaked the membrane in the diluted primary antibody (anti-CD9 antibody, Abcam, Boston, MA, USA, #ab92726; anti-CD81 antibody, Abcam, Boston, MA, USA, #ab109201) overnight at 4 degC, and recovered the primary antibody. We soaked the membrane in the diluted secondary antibody, incubated it at room temperature for 1 h, and recovered the secondary antibody. We laid the washed film of PBST on the fresh-keeping film, added equal volume mixed ECL a/b chromogenic solution, and placed it in the chemiluminescence imager. 2.6. Exosomal Small RNA Sequencing and Data Analyses Total RNA from the exosomes was extracted with Trizol according to the manufacturer's instructions. We then detected the RNA concentration and optical density (OD) value and detected the degradation and purity of RNA with 1% agarose gel electrophoresis. Meanwhile, Agilent Bioanalyzer 2100 was used to detect the integrity of RNA. We used the total RNA of exosomes after quality inspection. According to the manufacturer's instructions, we used NEB NEXT multiplex small RNA library prep set for Illumina(r) (Illumina, San Diego, CA, USA). The kit prepared a small RNA cDNA library and sequenced it to produce 50 nt single-end reads by the Illumina Novaseq 6000 platform. All the procedures for small RNA library preparation were accomplished by Novogene (Beijing, China). The data after quality control were aligned to the porcine reference genome (Sus scrofa 11.1) using bowtie. Known miRNAs were identified by the miRbase (v22.0) database accessed on 14 January 2022), miRdeep2 (v0.0.5) , and miRevo (v1.1) and were used to predict new miRNAs. At the same time, the differential expression analysis for miRNAs was performed by DESeq (v1.24.0) , requiring |fold change| > 1.6 and p < 0.05. Alignment was performed using MEGA (V11) followed by single base scoring using PHAST (v1.6.9) and evaluation of the most conserved regions of 10 virus genes, including WUH3 (GenBank accession no. HM853973), VR2332 (GenBank accession no. U87392), JXA1 (GenBank accession no. EF112445), CH-1a (GenBank accession no. AY032626), NADC30 (GenBank accession no. HN654459), HUN4 (GenBank accession no. EF635006), HLJZD22-1812 (GenBank accession no. MN648450), SC/DJY (GenBank accession no. MT075480), and Lelystad (GenBank accession no. M96262.2). RNAhybrid (V2.0) was used to predict the binding of the identified miRNA sequence to the 3' UTR of the CHsx1401 virus genome. MiRanda (v3.3a) and RNAhybrid were used to target gene prediction. The clusterProfiler R package was used for GO (Gene Ontology) functional enrichment analysis of target genes and KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway enrichment analysis. 2.7. Validation of miRNA Expression by RT-qPCR Total RNA was isolated from serum exosomes using Trizol (Invitrogen, Shanghai, China) according to the manufacturer's protocol. The isolated RNA was verified by RT-qPCR on samples (n = 6 per group). cDNA was synthesized according to the instructions of miRNA 1st strand cDNA synthesis (by stem-loop) kit (Vazyme, Nanjing, China), and the fluorescence quantification was performed using ABI 7500 according to the instructions of miRNA universal SYBR qPCR master mix (Vazyme, Nanjing, China). The thermal cycle parameters used were as follows: the first stage: 95 degC for 30 s; Stage 2: 95 degC for 5 s, 60 degC for 34 s, and 40 cycles; Stage 3: 95 degC for 15 s, 60 degC for 1 min, and 95 degC for 15 s. Primer sequences of miRNAs, the U6 gene, were used as a reference and listed in Supplementary Table S1. All qRT-PCR verifications were performed using three biological replicates and with three replicates for each sample. The relative abundance of transcripts was calculated by the 2-DDCt method, and SPSS (v22.0) and GraphPad Prism (v8.0) were used for data analysis and mapping, respectively. p < 0.05 means the difference is statistically significant. 3. Results 3.1. Relative Value of Antigen and Antibody after Virus Inoculation The results of PRRSV antigen and antibody tests before (day 0) and after the (day 7) challenge are shown in Table 1. The serological detection of the PRRSV antigen and antibody before the challenge was negative, and the antigen was positive after the challenge, indicating that the pigs were successfully infected with CHsx1401. 3.2. Isolation and Identification of Serum Exosomes The vesicles isolated from serum were discovered by TEM. Most vesicles can clearly see the concave disc-shaped exosomes in the middle. The membrane edge of exosomes is clearly visible, and the morphology is relatively complete . The nanoparticle tracking analysis showed that 95.73% of the exosomes had a diameter of 30-150 nm, mainly around 72.25 nm, with an average diameter of 76.22 nm, which was consistent with the size characteristics of exosomes . This size range was similar to that detected by TEM and further confirmed the identity of these vesicles as exosomes. Western blot analysis showed that the vesicles isolated from the serum samples were positive for CD9 and CD81 proteins . The above characteristics conform to the exosome identification standards formulated by the international society for extracellular vesicles (ISEV) in MISEV2018 . 3.3. Small RNA Sequencing of Serum Exosomes For each sample, the clean data reached 0.5 Gb, and the Q30 base percentage was above 96.20%. The clean reads of each sample were aligned with the pig reference genome. Among the 12 samples, the control group obtained 10,920,887, 10,248,696, 10,109,117, 10,655,494, 9,217,285, and 9,782,523 reads, respectively. The treatment group obtained 11,889,518, 10,593,504, 10,593,504, 12,846,080, 10,105,325, 11,729,451, and 9,789,542 reads, respectively. On average, 77.96% of the total clean reads comprised 19-22 nucleotides (nt) in length . The reads after quality control accounted for more than 92.59% of the total reads. The processed clean reads were aligned to the porcine reference genome, and the mapped rate of 12 libraries on the genome was more than 92.30%, and the mapped rate was 94.98% . It indicated that the constructed serum exosomal miRNA library was of high quality and suitable for further analysis. Details are listed in Supplementary Table S2. 3.4. Differentially Expression Analysis of miRNAs After quantitative analysis of the identified miRNA expression, miRNAs were screened by the thresholds described previously in Section 2.6. A total of 305 miRNAs were obtained before and after inoculation of the CHsx1401 strain (control, n = 6; treatment, n = 6). A total of 33 differentially expressed (DE) miRNAs were identified between the two groups, 13 DE miRNAs were upregulated, and 20 DE miRNAs were downregulated in the treatment group . 3.5. Functional Enrichment Analysis of miRNA Target Genes A total of 7283 target genes were predicted by 33 DE miRNAs, and the functions of target genes were mainly concentrated in the positive regulation of MAPK cascade, lipid metabolism process, regulation of intracellular signal transduction, ERK1 and ERK2 cascade, etc. . In terms of molecular functions, the differentially expressed miRNAs target genes mainly focus on GTP-enzyme regulatory activity, kinase activity, nucleoside triphosphatase regulatory activity, and other functions related to signal transduction and energy metabolism . In addition, among the cell components, the target genes mainly participate in the biological functions of supramolecular polymers, Golgi, autophagosomes, cell surface, early endosomes, etc. . The functions of these components are closely related to the formation of exosomes, which also explains the accuracy of the sequencing. KEGG pathway enrichment analysis showed that the target genes were significantly enriched in endocytosis, the MAPK signaling pathway, the Rap1 signaling pathway, the sphingolipid signaling pathway, and the PI3K Akt signaling pathway (p < 0.05) . At the same time, the enriched pathways were classified and analyzed. The results showed that the KEGG pathway of the target gene was mainly enriched in environmental information processing, human diseases, and biological systems . 3.6. Targeting Prediction of Serum Exosomal miRNA and PRRSV CHsx1401 Genome According to the phastCons score of a single base after alignment by PHAST, a total of eight most conserved segments (black bands above the peak map) were obtained among the viral genomes . A total of 31 DE miRNAs were found to bind to the conserved segment by predicting the miRNAs bound to the conserved segment. Among them, in the conserved region (14,644-15,020 nt) closest to the 3' UTR (14,870-15,020) of CHsx1401 genome, 16 DE miRNAs are predicted to bind to it, including 5 miRNAs (ssc-miR-34c, ssc-miR-375, ssc-miR-378, ssc-miR-486, and ssc-miR-6529) that can bind to the 3' UTR of CHsx1401. Among these miRNAs, only ssc-miR-223 was upregulated after infection, and other miRNAs were downregulated after infection. See Supplementary Table S4 for details. 3.7. Screening DE miRNAs Related to Exosome Function and PRRSV A variety of differentially expressed miRNAs related to the function of exosomes and PRRSV were found by functional enrichment analysis of target genes. Among them, 11 DE miRNAs such as ssc-miR-4331-3p, ssc-miR-744, and ssc-miR-320 are involved in exosome uptake, and their target genes are mainly concentrated in the Ras gene family, annexin family, and ADP ribosylation gene family. Eighteen DE miRNAs, including ssc-miR-10b, ssc-miR-124a, and ssc-miR-128, participate in immune-related pathways, and their target genes are mainly concentrated in the MAPK gene family, PIK3 gene family, and protein phosphatase gene family. While 11 DE miRNAs are involved in virus invasion, the related target genes are mainly concentrated in the MAPK gene family and protein phosphatase gene family. Furthermore, multiple differentially expressed miRNAs, such as novel_102. Six DE miRNAs, including ssc-miR-320, ssc-miR-423-5p, ssc-miR-4331-3p, ssc-miR-7137-3p, and ssc-miR-744, are co-expressed in exosome function, PRRSV virus invasion, and immune-related pathways, as shown in Figure 7. Details are shown in Supplementary Table S5. 3.8. QRT-PCR Assay of DE miRNAs between the Two Groups Five DE miRNAs were randomly selected for verification. According to the qRT-PCR results, the expression of ssc-miR-19a and ssc-miR-32 increased in the treatment group, while ssc-miR-124a, ssc-miR-375, and ssc-miR-34c showed higher expression in the control group, consistent with the sequencing data . 4. Discussion PRRSV is still a stubborn pathogen in the global pig industry, causing huge economic losses in the world. At present, vaccination is mainly used to prevent and control PRRSV, among which the modified live (MLV) virus vaccine is the most widely used . Although this vaccine was effective in reducing PRRS outbreaks and incidence, it also greatly increased genetic variation and diversity of the virus and led to viral recombination between wild and live vaccine viruses in the field . In recent years, the spread and prevalence of the recombinant virus NADC30-like PRRSV strain have caused multiple outbreaks of porcine reproductive and respiratory syndrome in China. The similarity between CHsx1401 and NADC30 used in this study remained at 92.2-99.1%. Since then, it has become an epidemic strain in China. Exosomes, as mediators of cell communication, are widely found in various body fluids and have unique advantages in disease diagnosis and treatment . According to previous reports, exosomes play an important communication role in antigen presentation , immune response , virus replication , cancer , neurodegenerative diseases , angiogenesis , tumor cell migration and invasion , and have high research value. In this study, high-throughput sequencing technology was used to construct the miRNA expression profile of serum exosomes, and 33 DE miRNAs were identified. As we all know, the host-encoded miRNA can bind with the viral genome and then regulate the replication, synthesis, and release of the virus to limit infection and affect the pathological process . Studies of miRNAs targeting the viral genome have also been repeatedly reported in animals. gga-miR-454 and gga-miR-130b in chicken infectious bursal disease can target the viral genome to inhibit viral replication, while gga-miR-21 directly targets the viral protein VP1 to inhibit viral protein translation . In PRRSV studies, ssc-miR-181 specifically binds to a highly conserved region downstream of the viral genome ORF4 and strongly inhibits PRRSV replication . In this study, the expression difference of ssc-miR-181 between the two groups did not reach a significant level. In our study, the genomes of nine different PRRSV viruses were compared with those of the CHsx1401 strain, and the eight most conserved segments were identified. It was predicted that 31 DE miRNAs could bind to the 8 most conserved segments of CHsx1401, and 16 DE miRNAs could bind to the conserved sequences close to the 3' UTR of CHsx1401. Among them, 5 DE miRNAs (ssc-miR-34c, ssc-miR-375, ssc-miR-378, ssc-miR-486, and ssc-miR-6529) can simultaneously bind to the CHsx1401 3' UTR. In addition, the upregulated expression of ssc-miR-223 was predicted to bind to the 3'UTR target of the PRRSV genome. The results showed that the conserved sequences of the virus genome might play a key role in its pathogenicity, and the miRNAs that can bind to the conserved sequences between the genomes of different PRRSV strains may have important significance in controlling the pathogenicity of the virus. Some differentially expressed miRNAs have been proven to be related to PRRSV by previous studies and even directly involved in the regulation of PRRSV, including ssc-miR-10b , ssc-miR-378 , ssc-miR-124a , let-7f-5p , ssc-miR-744 , and ssc-miR-19a . PRRSV can evade host defense by interfering with innate immune response. This process is regulated by many signaling pathways, including the MAPK signaling pathway, PI3K Akt signaling pathway, autophagy, chemokine, and TNF signaling pathway. At present, the MAPK signaling pathway includes three main pathways: ERK1/2, JNK, and p38 pathway. Activation of the MAPK cascade can promote host cell apoptosis, assist the virus in escaping the host immune defense response and promote PRRSV replication . Moreover, the activation of c-Jun N-terminal kinases (JNKs) and p38 can also promote the release of the inflammatory factor IL-10 and enhance the inflammatory effect. In addition to inducing apoptosis, PRRSV can also induce autophagy, which can promote PRRSV replication. The activation of PI3K/Akt is necessary for virus entry and promotion of virus replication, and PRRSV-activated Akt inhibits host cell apoptosis by negatively regulating the JNK pathway . TNFa It can play an important role in the induction and regulation of inflammatory response together with other inflammatory factors, but TNF a Expression is affected by the negative regulation of PRRSV replication . In the present study, miRNAs (ssc-miR-10b, ssc-miR-122-5p, ssc-miR-124a, ssc-miR-128, ssc-miR-129a-5p, etc.) enriched in these pathways are involved in PRRSV-induced apoptosis, autophagy, and inflammation and are closely associated with viral immune response, immune evasion, and replication. The cell plasma membrane is rich in a variety of lipid rafts, and cholesterol-rich in sphingolipids (sphingomyelin and glycosphingolipids) are key molecules of lipid rafts. The recognition of lipids by some proteins of the virus may be a necessary condition for the entry of the virus . Envelope viruses insert viral envelope glycoproteins into lipid rafts at the stage of virus entry, interact with receptors located in lipid rafts, or change from their natural state to activated form to initiate or promote viral internalization/fusion, such as HSV, SARS coronavirus, and piglet epidemic diarrhea virus . Previous studies found that the removal of cholesterol from the surface of MARC-145 cells significantly reduced PRRSV infection, demonstrating that inhibition of PRRSV infection was specifically mediated by the removal of cellular cholesterol. Depletion of cell membrane cholesterol significantly inhibited virus entry, particularly virus attachment, and release . Obviously, sphingolipid metabolism can regulate membrane structure and adhesion, which is of great significance in PRRSV virus invasion. Endocytosis was the most significant enrichment in this study. Endocytosis is an important mechanism of exosome uptake by target cells. Previous studies have shown that exosome uptake is an energy-demanding and cytoskeleton-dependent process, which highlights the potential role of endocytosis in this process . It has been proved that there are several pathways that can mediate this process, including phagocytosis, macropinocytosis, clathrin, etc. , which led to different classifications and roles of endocytosed substances. The enrichment of differentially expressed exosomal miRNAs in this pathway indicates that exosomes play an important role in PRRSV infection, and the regulation of content transport and uptake in exosomes may lead to pathophysiological changes in target cells and organs. 5. Conclusions Through the identification and bioinformatics analysis of serum exosomal miRNAs from PRRSV-infected pigs, a variety of PRRSV-related pathways and differentially expressed miRNAs were obtained in this study, such as ssc-miR-4331-3p, ssc-miR-744, ssc-miR-320, ssc-miR-10b, ssc-miR-124a, ssc-miR-128, etc., which play potential functional roles in PRRSV-induced immune response, invasion, and exosome uptake. In addition, because a single miRNA can target multiple genes and a single gene is also regulated by multiple miRNAs, there are a number of miRNAs that perform multiple functions in the above pathways. Some miRNAs have been verified to regulate PRRSV infection by acting on key receptors or directly targeting the virus genome, such as ssc-miR-10b, ssc-miR-378, miR-124a, let-7f-5p, ssc-miR-744, ssc-miR-19a, etc. Meanwhile, the present study also predicted a variety of miRNAs that can bind to the most conserved fragment of the 3' UTR of the CHX1401 virus genome, including ssc-miR-34c, ssc-miR-375, ssc-miR-378, ssc-miR-486, and ssc-miR-6529, which may be important for regulating viral pathogenicity. Acknowledgments We sincerely thank personnel from the animal genetics and breeding laboratory of Guangxi University. Supplementary Materials The following supporting information can be downloaded at: Table S1: Primer sequence of miRNA for qRT-PCR; Table S2: Length distribution of read counts; Table S3: DE miRNAs in between control and treatment group; Table S4: Predicted binding of differentially expressed miRNA to CHsx1401 genome; Table S5: DE miRNAs related to exosome function and PRRSV. Click here for additional data file. Author Contributions L.W. (Ligang Wang) and J.L. conceived and designed the experiment. F.C., L.Z. and H.Y. designed and performed the animal experiments. F.C. and H.W. performed the exosomes and miRNA analysis. F.C., L.W. (Lixan Wang) and G.L. performed the statistical analysis. H.W., F.C. and J.L. wrote the first draft of the manuscript. All authors have read and agreed to the published version of the manuscript. Institutional Review Board Statement Not applicable. Informed Consent Statement Not applicable. Data Availability Statement The data presented in this study are openly available in NCBI Sequence Read Archive under accession number PRJNA938232. Conflicts of Interest The authors declare no conflict of interest. Figure 1 Main characteristics of serum exosomes. (A,B) show the morphological characteristics of vesicles by TEM. Scale bars are 500 nm and 100 nm, respectively. (C) NTA shows the diameter and concentration of most vesicles. (D) Western blot showed the presence of exosome markers CD81 and CD9 in serum exosomes. Note: Mix in WB results is the sample mixed suspension isolated by the exoEasy Maxi kit. Figure 2 Overview of the small RNA transcriptome data. (A) Length distribution of read counts of serum exosome samples (nt = nucleotides); (B) rate of 12 samples mapped to the reference genome. Figure 3 Differential expression of miRNAs in exosomes. (A) Volcano plot of miRNAs between control and treatment groups; (B) hierarchical clustering heatmap of DE miRNAs between control and treatment groups. Figure 4 GO function enrichment analysis of DE miRNAs target genes. (A) Biological process of DE miRNAs target genes; (B) molecular functions of DE miRNAs target genes; (C) cellular components of DE miRNAs target genes. Figure 5 KEGG pathway enrichment analysis of target genes. (A) Significantly enriched KEGG pathway with target genes of DE miRNAs; (B) classification of significantly enriched KEGG pathways. Figure 6 Conserved segments in the genome of CHsx1401 strain predicted by PHAST. Figure 7 DE miRNAs related to exosome uptake, PRRSV invasion, and immunity. Figure 8 Five DE miRNAs validated by qRT-PCR. animals-13-00876-t001_Table 1 Table 1 Antigen and antibody of (day 0) and (day 7) with challenge virus. Time Control/before Inoculation (Day 0) Treatment/after Inoculation (Day 7) Antigen (Ct) / 26.05 / 25.31 / 20.69 / 24.70 / 25.01 / 22.55 Antibody (S/P) 0.03 / 0.12 / 0.15 / 0.06 / 0.25 / 0.00 / Note: antigen "/" means negative, antibody < 0.4 means negative, Ct means cycle threshold, S/P value = (sample value - negative control mean value)/(positive control mean value - negative control mean value). Disclaimer/Publisher's Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). 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PMC10000163

To investigate the effect of poultry by-product meal (PBM) replacing fish meal on the growth and intestinal health of Chinese soft-shelled turtle (Pelodiscus sinensis). Four experimental diets were prepared. Fish meal was replaced by 0 (control group, PBM0), 5% (PBM5), 10% (PBM10), and 15% (PBM15) PBM. Compared to the control group, final body weight, weight gain, and specific growth rate were significantly increased, while feed conversion rate decreased significantly in the PBM10 group (p < 0.05). The PBM15 group significantly increased the moisture content and significantly decreased the ash content of the turtles (p < 0.05). The PBM5 and PBM15 groups significantly decreased the whole-body crude lipid (p < 0.05). The serum glucose content increased significantly in the PBM10 group (p < 0.05). The liver malonaldehyde content significantly decreased in the PBM5 group and in the PBM10 group (p < 0.05). Liver glutamic-oxalacetic transaminase and intestinal pepsin activity were increased significantly in the PBM15 group (p < 0.05). The expression of the intestinal interleukin 10 (IL-10) gene was significantly down-regulated in the PBM10 group and the PBM15 group (p < 0.05), the expression of the intestinal interferon-g (IFN-g), interleukin-8 (IL-8), and liver toll-like receptor 4 (TLR4) and toll-like receptor 5 (TLR5) genes were significantly up-regulated in the PBM5 group (p < 0.05). In summary, poultry by-product meal can be used as a protein source to replace fish meal in turtle feed. Based on quadratic regression analysis, the optimal replacement ratio is 7.39%. poultry by-product meal growth immunity antioxidation intestinal health Pelodiscus sinensis National Natural Science Foundation of China31972800 This work was supported by the fund of the National Natural Science Foundation of China (31972800). pmc1. Introduction Fish meal has always been regarded as the highest quality protein material for aquaculture feed due to its high protein content, good palatability, low anti-nutritional factors, and easy digestion . In recent years, fish meal production has decreased. With aquaculture expansion, the demand for fish meal has also increased , and the price has increased, seriously restricting the sustainable development of aquaculture . The search for protein resources that can replace fish meal has become an inevitable trend in the development of aquaculture feed. Currently, the substitution of fish meal can be roughly divided into three directions: animal protein source , plant protein source , and mixed protein source . The type of protein in feed plays an important role in the growth and development of fish. However, the presence of a large number of anti-nutritional factors in plant protein feed , amino acid imbalance, and low utilization dramatically limits the use of plant protein in aquatic animal feed . Poultry by-product meal (PBM) is a new type of animal protein feed raw material processed from the remaining part of the breast meat of chickens removed from the shelf and white-striped chickens. The overall nutritional index of poultry by-product meal is comparable to that of fish meal. The content of protein, fat, and some vitamins is slightly higher than that of high-quality fish meal, and the freshness is better controlled than fish meal . In recent years, it has been widely used in the United States and Southeast Asia. Studies have shown that an appropriate amount of PBM instead of fish meal has positive results in feeds such as white shrimp (Litopenaeus vannamei) , snakehead (Channa argus) , black sea bream (Acanthoparus schlegelii) , and nile tilapia (Oreochromis niloticus) . PBM has a high content of saturated fatty acids and ash, affecting fish digestion and absorption . Meanwhile, the high proportion of replacement of fish meal will affect the growth performance of aquatic animals . Goda et al. showed that replacing 75% and 100% of fish meal with PBM affected the growth performance of African catfish (Clarias gariepinus). Jr et al. found that replacing fish meal with PBM led to a decrease in the growth performance of pompano (Trachinotus carolinus). Turkey et al. found that the optimal replacement ratio of fish meal by PBM in a turbot diet was 25%. The effects of PBM replacing fish meal on the growth performance of aquatic animals were different in different species. The Chinese soft-shelled turtle is widely cultured because of its high nutritional value and high breeding efficiency. However, the high dependence on fish meal of the Chinese soft-shelled turtle has seriously restricted the development of its breeding industry. It is urgent to develop a new protein source to replace fish meal, so this experiment intended to use PBM instead of fish meal on the growth, immunity, and intestinal health of Chinese soft-shelled turtles and provided theoretical support for low fish meal feed. 2. Materials and Methods 2.1. Experimental Animals, Diets, and Feeding Trial The experiment was carried out in the greenhouse breeding system of Zhuji Jindadi Agriculture Co., Ltd., Shaoxing City, Zhejiang Province. 480 turtles with an average weight of 3.48 +- 0.01 g were randomly distributed in 16 cages (0.6 m x 0.6 m x 0.6 m) with 30 tails per cage. Each dietary group had four replicates, and 30 turtles were stocked in each replicate cage. The turtles were fed three times a day (6:00, 12:00, and 18:00 h) and the feeding amount was set at 4% of the body weight. The chemical composition of fish meal and the poultry by-product meal used in the present study is shown in Table S1. PBM was used to replace fish meal in a ratio of 0 (PBM0 (control group), 5% (PBM5), 10% (PBM10), and 15% (PBM15), and the basic feed is shown in Table S2. The amino acid composition of each group is shown in Table S3. All ingredients were finely ground and thoroughly mixed in a blender. The 2.2 mm expanded particles were made by an expanded granulator and dried by an air dryer. The feeding trial lasted eight weeks. During the experiment, the water temperature was 30 +- 1 degC, the dissolved oxygen was kept at more than 5 mg/L, the ammonia nitrogen was less than 0.8 mg/L, and the nitrite was less than 0.05 mg/L. The turtles in each cage were weighed at the start (initial body weight) and end (final body weight) of the 56-day feeding experiment. The survival rate, weight gain, and feed coefficient of each treatment group were recorded and analyzed. These parameters were calculated as follows:Survival rate (%) = (Nf/Ni) x 100; Weight gain (WG, %) = (Wf - Wi)/Wi x 100; SGR (specific growth rate, %/day) = ((ln Wf - ln Wi)/days) x 100; Feed conversion ratio (FCR) = Feed consumed (g)/(Wf - Wi); where Wf and Wi are the initial and final body weight, Nf and Ni are the final and initial numbers of turtles. 2.2. Sample Collection At the end of the feeding trial, three turtles were randomly selected from each replicate and used for whole-body composition analysis . The moisture content was determined by the 105 degC constant temperature drying method. The crude protein content was determined by the Kjeldahl nitrometer. The crude lipid was determined by soxhlet extraction. Ash content was determined by Muffle furnace combustion at 550 degC. Three turtles per cage were decapitated, and blood was collected in a 2 mL centrifuge tube, placed at 4 degC for 6 h, and centrifuged at 3000 rpm for 10 min to obtain serum samples. The turtle was then dissected, and the liver and intestine were separated. The tissue was mixed with a 0.9% sodium chloride solution at a ratio of 1:9 (mass/volume), homogenized with a homogenizer, then centrifuged at 2500 rpm for 10 min, and the supernatant was stored in a refrigerator at -80 degC for further analysis. Finally, the collected samples were mixed in pairs, and 6 samples were obtained from each treatment group. The midguts were taken and placed in Bouin's fixative solution to make histological sections. 2.3. Biochemical Analysis Serum total cholesterol (T-CHO), blood urea nitrogen (BUN), glucose (GLU), triglyceride (TG), high-density lipoprotein cholesterol (HDL-C), and low-density lipoprotein cholesterol (LDL-C) were measured. The contents of superoxide dismutase (SOD), reduced glutathione (GSH), catalase (CAT), malondialdehyde (MDA), and aspartate aminotransferase (GOT) in the liver were determined. The activities of intestinal trypsin, pepsin, lipase, and amylase were determined. Kits produced by the Nanjing Jiancheng Institute of Bioengineering were used for determination and analysis. 2.4. Intestinal Histological Analysis The midgut tissue was fixed in Bouin's fixative solution for 48 h, which was dehydrated with ethanol, transparentized with xylene, embedded in paraffin, sectioned, and stained with hematoxylin-eosin (HE). And finally, the morphological characteristics of the intestinal villi were observed under a light microscope; images were taken; and villi height, width, and thickness of the muscular layer were measured with a micrometer ruler. 2.5. Analysis of Intestinal and Liver Gene Expression Total RNA was extracted from turtle intestinal and liver tissue samples according to the RNA rapid extraction kit (RN28-EASYspin Plus), and cDNA was synthesized by the reverse transcription kit (MonScript TM 5 x RTIIII All-in-One Mix, Mona, Suzhou, China). The antibody dye quantitative PCR master mix (Mon Amp TM SYBR(r) Green q PCR Mix) was used to perform RT-qPCR on the real-time quantitative PCR instrument (ABI 7500), and specific operation steps were carried out according to the instructions. The reaction system was 20 mL, including 10 mL MonAmp TM SYBR(r) Green qPCR Mix (Mona, Suzhou, China), 0.4 mL forward and reverse primers (10 mmol/L), 1.5 mL template cDNA, 7.7 mL nuclease-free water. The thermal cycle program of qRT-PCR was as follows: 95 degC for 5 min, then 40 cycles at 95 degC for 10 s, 60 degC for 30 s, and 72 degC for 30 s. The melting curve was drawn to determine the correctness of the amplification product. By making a gradient dilution concentration standard curve, it was found that the amplification efficiency of the target gene and the internal reference gene was close to 1 , which was consistent with the target gene 2-DDCt method. The primer sequences of genes related to intestinal and liver health-related genes in Chinese soft-shelled turtles are shown in Table S4. 2.6. Statistical Analysis The experimental data were analyzed by one-way analysis of variance (ANOVA) using SPSS 26.0 software, and the data for each group were compared using the Turkey method for multiple comparisons, and p < 0.05 was considered a significant difference. Experimental data are expressed as the mean +- standard error (mean +- SE). 3. Results 3.1. Growth Performance There was no significant difference in IBW and SR among all groups (p > 0.05). Compared to the control group, the PBM10 group significantly increased FBW, WG, and SGR (p < 0.05) while significantly decreased turtle FCR (p < 0.05) (Table 1). Based on the second-regression analysis model between WG and dietary PBM levels, the PBM optimal substitution level for fish meal was 7.39% . 3.2. whole-body Composition Compared to the control group, the PBM15 group significantly increased the moisture content and significantly decreased the ash content of the turtles (p < 0.05). The PBM5 and PBM15 groups significantly decreased the whole-body crude lipid (p < 0.05). The dietary groups PBM5, PBM10, and PBM15 had no significant effect on the crude protein of the turtles (p > 0.05) (Table 2). 3.3. Serum Biochemical Indicators There were no significant differences in serum levels of T-CHO, BUN, TG, HDL-C, and LDL-C among all groups (p > 0.05). Compared to the control group, the PBM10 group significantly increased serum GLU content (p < 0.05) . 3.4. Antioxidant Parameters of the Liver There were no significant differences in liver SOD, CAT activity, and GSH content among all groups (p > 0.05). Compared to the control group, the PBM5 group and the PBM10 group significantly decreased the liver MDA content (p < 0.05), and the PBM15 group significantly increased liver GOT activity (p < 0.05) . 3.5. Intestinal Digestive Enzyme Activity There were no significant differences in trypsin, lipase, and amylase activities among all groups (p > 0.05). Compared to the control group, the PBM5 group and the PBM10 group significantly increased intestinal pepsin activity (p < 0.05) . 3.6. Histological Study of the Intestine There were no significant differences in intestinal villi length and width among all groups (p > 0.05). As the proportion of PBM that replaced fish meal was increased, the thickness of the muscle layer gradually was decreased, and compared to the PBM5 group, the thickness of the muscle layer was significantly decreased in the PBM15 group (p < 0.05) . 3.7. Expression of Intestinal and Liver Genes There were no significant differences in the expression levels of intestinal IL-1b and IL-15 genes among all groups (p > 0.05). Compared to the control group, the PBM10 and PBM15 groups significantly down-regulated the intestinal IL-10 gene expression (p < 0.05), and the PBM5 group significantly up-regulated the intestinal IFN-g and IL-8 gene expressions (p < 0.05) . There were no significant differences in the expression levels of IL-1b, IGF-1, and TLR8 in the liver (p > 0.05). The expressions of the TLR4 and TLR5 genes in the liver were significantly up-regulated in the PBM5 group (p < 0.05) . 4. Discussion This study shows that PBM can be used as a protein source instead of fish meal in Chinese soft-shelled turtle feed. The turtle had a good tolerance to PBM, and the growth performance and feed utilization rate were the best when the fish meal replacement ratio was 7.39%. Substituting fish meal with PBM in the diet can improve turtle growth performance, which is consistent with previous findings in various species, including rainbow trout (Oncorhynchus mykiss) , flounder (Paralichthys olivaceus) , and black sea bream (Acanthopagrus schlegelii) . These studies have shown that PBM meets the nutritional and energy needs of aquatic animals and promotes their growth. According to Sayed et al. and Wang et al. , increasing the proportion of PBM instead of fish meal had a negative impact on the growth performance of aquatic animals. In this study, the replacement of 15% fish meal with PBM did not affect turtle growth performance but showed a decreasing trend, which is consistent with the results of Ji et al. . This result may be because turtles are typical carnivores and prefer diets with a higher proportion of fish meal . Poultry by-product meal altered the body composition of turtles. When 15% fish meal was replaced by poultry by-product meal, the moisture content of Pelodiscus sinensis increased and the ash content decreased. When 5% and 15% fish meal were replaced by poultry by-product meal, the crude fat content of p. sinensis decreased. The same poultry by-product meal also affects the body composition of turbot . Studies have shown that the replacement of fish meal with PBM has no significant effect on the body composition of crucian carp and tilapia , but Zhou et al. found that the replacement of fish meal with PBM significantly decreased the protein content of juvenile cobia (Rachycentron canadum) . This result may be due to different species, and the replacement of fish meal with PBM has other effects on the whole-body composition, and the specific mechanism needs to be further explored. Serum indicators reflect the health status, physiological function, and nutritional level of the body . Serum levels of T-CHO and TG reflect the body's lipid metabolism , and serum levels of HDL-C and LDL-C represent the breakdown and transport of body lipids . The serum content of BUN reflects the protein metabolism and amino acid balance of the body . In this experiment, the replacement of fish meal by PBM did not affect the serum levels of T-CHO, TG, BUN, HDL-C, and LDL-C of turtles, which is consistent with the results of Sugita et al. in milkfish (Chanos chanos). The serum GLU content of the PBM10 group increased, indicating that the body's energy level was higher , and the growth rate was accelerated, indicating that the replacement of fish meal with PBM could affect the energy metabolism of the Chinese soft-shelled turtle. As an important immune organ of aquatic animals, the liver has the functions of immune metabolism, detoxification, and anti-oxidation . SOD, CAT, and GSH play important roles in maintaining the balance of oxidation and anti-oxidation in the body and avoiding oxidative damage . MDA is the main peroxidation-decomposing substance in the body, whose concentration directly reflects the degree of peroxidation in the body and the degree of damage to cells . In this study, the replacement of fish meal with PBM did not affect SOD, CAT, or GSH in the liver of turtles, which was consistent with the results of Zhou et al. . Replacement of 5% and 10% fish meal with PBM could reduce the content of MDA in the liver, indicating that the appropriate proportion of PBM instead of fish meal would be beneficial to the liver health of Chinese soft-shelled turtles. GOT is an important amino acid metabolic enzyme in the liver of aquatic animals . The increase in GOT activity indicates that proteins are used to enhance energy metabolism . In this study, GOT activity in the liver increased with increased levels of PBM replacement, which is consistent with the results of Lu et al. on rainbow trout. This result may be due to the increase in the amino acid imbalance in the feed with increasing PBM, which leads to an improvement in amino acid metabolism in the liver of turtles and an improvement in GOT activity to adapt to the increase in PBM content in the feed. Enhancement of digestive enzyme activity can promote food digestion and nutrient absorption in the body, thereby promoting fish growth. In this study, different proportions of PBM replacing fish meal had no significant effect on trypsin, lipase, and amylase activities. Pepsin activity first increased and then decreased with increasing PBM replacement ratios, which is consistent with the results in sea bass . The above studies showed that properly replacing fish meal with PBM could increase pepsin activity in the intestinal tract of turtles and then improve growth performance. Intestinal villus length, width, and muscle thickness can help predict the absorption and digestion mechanisms in aquatic animals . In this study, the replacement of PBM with fish meal did not affect the length and width of the intestinal villi. But with the increase in the PBM replacement ratio, the thickness of the muscle layer gradually decreased. Muscle thickness can reflect the contractile capacity of the intestine . The results showed that when the high proportion of fish meal was replaced by PBM, it had a negative impact on the intestinal contractile capacity, which in turn affected the intestinal absorption capacity of turtles. Aquatic animals can regulate the immune response by enhancing or inhibiting the production of cytokines . Cytokines in aquatic animals can be divided into anti-inflammatory (such as IL-10 and TGF-b1) and pro-inflammatory (such as TNF-a, IL-1b, IL-15, and IL-8) . Inflammatory injury can be measured by the expression levels of pro-inflammatory factors and anti-inflammatory factors . Yang et al. found that replacing 25%, 50%, 75%, and 100% fish meal with PBM did not show obvious enteritis symptoms in red swamp crayfish (Procambarus clarkii). Basilio et al. found that PBM completely replaced plant protein sources in gilthead seabream (Sparus aurata) feed, and the response rate to inflammation was decreased. In this study, we found that the expression of the IL-10 gene in the intestinal tract of turtles decreased with an increasing proportion of PBM. IFN-g and IL-8 genes were significantly up-regulated when the proportion of PBM replacing fish meal was 5%. However, when PBM replaced fish meal at 10% and 15%, the expressions of the inflammatory factors IFN-g and IL-8 genes were not up-regulated. The specific mechanism needs to be explored further. These results indicate that PBM does not have a negative effect on the intestinal health of turtles when replacing 10% and 15% of fish meal. toll-like receptors are mediators that connect specific and non-specific immunity and can control cytokine synthesis and release to regulate inflammatory responses . The TLR4, TLR5, and TLR8 molecules belong to MyD88-dependent signaling pathways in TLRs, and their expression levels can reflect the immune system's strength . In this study, PBM replacing 5% fish meal could up-regulate the expression of the TLR4 and TLR5 genes in the liver. This study showed that PBM might activate turtle liver cells through the TLR4 and TLR5 pathways, cause an immune response, and improve immunity. 5. Conclusions In summary, this study showed that replacing fish meal with poultry by-product meal did not affect growth performance, feed utilization, immunity, antioxidant capacity, or intestinal health of turtles. Based on the quadratic regression analysis between WG and the level of poultry by-product meal in feed, the optimal ratio of poultry by-product meal replacing fish meal was 7.39%. Acknowledgments We thank the students for their help and the experimental base provided by Zhejiang Jindi Agricultural Co., Ltd. Supplementary Materials The following supporting information can be downloaded at: Table S1. Chemical composition of the fish meal and poultry by-product meal; Table S2. Ingredients composition of the experimental diets (%); Table S3. Chemical and amino acid composition and fatty acid profile of the test diets; Table S4. Primer sequences of the intestinal and liver genes of the Chinese soft-shelled turtle. Click here for additional data file. Author Contributions Z.Q. conceptualization, formal analysis, investigation and writing--original draft; Q.X. methodology, project administration, writing--review and editing and funding acquisition; D.X. and J.Z. (Jiantao Zhao) resources and supervision; F.Y.Y. writing--review and editing; H.X. resources; J.Z. (Jianhua Zhao) methodology, supervision and project administration. The first draft of the manuscript was written by Z.Q. All authors commented on previous versions of the manuscript. All authors have read and agreed to the published version of the manuscript. Institutional Review Board Statement The study was conducted in accordance with the Declaration of Helsinki, and approved by the medical ethics committee of Huzhou University (20220916). Informed Consent Statement Not applicable. Data Availability Statement All data presented this study are available from the corresponding author, upon responsible request. Conflicts of Interest The authors declare no conflict of interest in this manuscript. Figure 1 The relationship between WG and dietary PBM replacement level in Chinese soft-shelled turtle. Figure 2 Effect of dietary replacement of fish meal with poultry by-product meal on the serum biochemical indicators of Chinese soft-shelled turtle. (A) T-CHO, total cholesterol; (B) GLU, glucose; (C) BUN, blood urea nitrogen; (D) TG, triglyceride; (E) HDL-C, high-density lipoprotein cholesterol; (F) LDL-C, low-density lipoprotein cholesterol; Error bars was represented the mean +- standard error (n = 6). Different letters indicated significant differences between groups (p < 0.05). Figure 3 Effect of dietary replacement of fish meal with poultry by-product meal on the liver antioxidant of Chinese soft-shelled turtle. (A) Sod, superoxide dismutase; (B) MDA, malonaldehyde; (C) CAT, catalase; (D) GSH, glutathione; (E) GOT, glutamic-oxalacetic transaminase. The error bars represented the mean +- standard error (n = 6). Different letters indicated significant differences between groups (p < 0.05). Figure 4 Effect of dietary replacement of fish meal with poultry by-product meal on the intestinal digestive enzyme activity of Chinese soft-shelled turtle. (A) Trypsin; (B) pepsin; (C) lipase; (D) amylase; Error bars was represented the mean +- standard error (n = 6). Different letters indicated significant differences between groups (p < 0.05). Figure 5 Histological sections of the intestines of Chinese soft-shelled turtle. Magnification times are 100x. (A) Turtle fed with Con diet. (B) Turtle fed with PBM5 diet. (C) Turtle fed with PBM10 diet. (D) Turtle fed with PBM15 diet. The scale bar was 200 mm. Figure 6 Effects of dietary replacement of fish meal with poultry by-product meal on the intestinal gene expression of Chinese soft-shelled turtle. Error bars was represented the mean +- standard error (n = 6). Different letters indicated significant differences between groups (p < 0.05). Figure 7 Effects of dietary replacement of fish meal with poultry by-product meal on the liver gene expression of Chinese soft-shelled turtle. Error bars was represented the mean +- standard error (n = 6). Different letters indicated significant differences between groups (p < 0.05). animals-13-00865-t001_Table 1 Table 1 Effect of dietary replacement of fish meal with poultry by-product meal on the growth performance of Chinese soft-shelled turtle. Groups IBW (g) FBW (g) SR (%) WG (%) SGR (%/d) FCR PBM0 3.47 +- 0.00 31.89 +- 0.06 b 91.67 +- 2.15 818.93 +- 1.27 b 3.96 +- 0.00 b 0.81 +- 0.02 ab PBM5 3.48 +- 0.00 34.10 +- 0.48 ab 92.50 +- 2.85 880.71 +- 13.90 ab 4.08 +- 0.03 ab 0.80 +- 0.01 b PBM10 3.47 +- 0.00 34.93 +- 1.30 a 91.67 +- 3.19 906.13 +- 37.20 a 4.12 +- 0.07 a 0.77 +- 0.03 b PBM15 3.48 +- 0.00 31.81 +- 0.30 b 92.50 +- 2.85 813.99 +- 8.92 b 3.95 +- 0.02 b 0.88 +- 0.02 a p-Value 0.31 0.02 0.99 0.02 0.02 0.02 Note: IBW: initial body weight; FBW: final body weight; WG: weight gain; SR: survival rate; FCR: feed conversion rate; SGR: specific growth rate. Values are presented as means +- standard error (n = 4). Mean values in the same row with different superscripts are significant different (p < 0.05). animals-13-00865-t002_Table 2 Table 2 Effect of dietary replacement of fish meal with poultry by-product meal on the whole-body proximate composition of Chinese soft-shelled turtle. Groups Moisture (%) Crude Protein (%) Crude Lipid (%) Ash (%) PBM0 69.46 +- 0.76 bc 19.59 +- 0.58 ab 4.24 +- 0.15 a 5.24 +- 0.08 a PBM5 71.39 +- 0.61 ab 18.25 +- 0.49 ab 3.54 +- 0.10 b 4.80 +- 0.09 ab PBM10 68.67 +- 0.69 c 20.46 +- 0.60 a 4.16 +- 0.17 a 5.10 +- 0.07 a PBM15 73.90 +- 0.43 a 17.55 +- 0.51 b 3.42 +- 0.10 b 4.53 +- 0.20 b p-Value <0.01 0.01 <0.01 <0.01 Note: Values are presented as means +- standard error (n = 4). Mean values in the same row with different superscripts are significant different (p < 0.05). animals-13-00865-t003_Table 3 Table 3 Effects of dietary replacement of fish meal with poultry by-product meal on the intestinal villus height, villus width, and muscle layer thickness of Chinese soft-shelled turtle. Groups Villus Height (mm) Villus Width (mm) Muscle Layer Thickness (mm) PBM0 373.62 +- 9.11 109.21 +- 4.87 72.98 +- 2.58 ab PBM5 414.65 +- 18.98 111.64 +- 6.17 80.39 +- 8.99 a PBM10 409.46 +- 16.65 110.40 +- 6.62 71.84 +- 5.36 ab PBM15 354.38 +- 19.19 97.52 +- 6.95 55.17 +- 2.92 b p-Value 0.05 0.37 0.03 Note: Values are presented as means +- standard error (n = 6). Mean values in the same row with different superscripts are significant different (p < 0.05). Disclaimer/Publisher's Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

PMC10000164

Mullets (Osteichthyes: Mugilidae) are a euryhaline species widely distributed all over the world, thus representing an excellent study model for host-parasite interactions. From March to June 2022, 150 mullets, belonging to Chelon labrosus (n = 99), Chelon auratus (n = 37), and Oedalechilus labeo (n = 14) species, were caught to identify the helminth parasite fauna of the different mullet species present in the Ganzirri Lagoon (Messina, Sicily, Italy). A parasitological evaluation of the gastrointestinal tract (GIT) was carried out with a total worm count technique (TWC) to detect helminth presence. All collected parasites were stored in 70% ethanol until morphological evaluation, and frozen at -80 degC for subsequent molecular analysis, using 28S, ITS-2, 18S primers. The morphological evaluation allowed for the identification Acanthocephalan parasites (Neoechinorhynchus agilis) from two C. labrosus specimens. Sixty-six samples were positive for adult digenean trematodes (C. labrosus, 49.5 %; C. auratus, 27%, and O. labeo, 50%), molecularly identified as Haploporus benedeni. This study represents the first survey of helminthic parasite fauna of mullets from the south of Italy. The presence of Hydrobia sp. in the stomach contents of mullets allowed us to infer the H. benedeni life cycle in the Ganzirri lagoon. Acanthocephalan trematode Chelon labrosus Chelon auratus Oedalechilus labeo PO FEAMP 2014-2020 2.56 FISH PATH NETG47B18000130009 Research funds were provided by PO FEAMP 2014-2020 2.56 FISH PATH NET grant no. G47B18000130009, Project code SIPA 01/MS/18. pmc1. Introduction Fish are increasingly becoming the subjects of research studies, above all for their importance as a valuable source of protein. However, severe parasitic infections can cause functional disturbances, such as growth reduction and sensitivity to many secondary bacterial infections, as well as giving fish an unsightly appearance, thereby reducing their economic value. Fish populations play a key role in aquatic biological communities; moreover, they regulate the dynamics of the food chain, maintaining the balance of nutrients . Thanks to the study of diet and migratory movements, fish fauna provide important data on anthropogenic action on all aquatic ecosystems , including reliable information on the possibility of accumulating pathogen organisms, such as bacteria, viruses, and parasites, as well as pollutants that pose a risk to human health . Mugilidae, also known as mullets, are a large teleost family, divided into seventeen genera that include a high number of species . They belong to the Actinopterygii and are characterized by a significant presence in shallow costal brackish waters, such as lagoons, rivers, estuaries, and lakes, between temperate, sub-tropical, and tropical areas, in both hemispheres . They are also considered a secondary consumer, occupying an intermediate position in the food chain due to their eating habits . In the Mediterranean Sea, four mullet genera (Chelon, Liza, Mugil, Oedalechilus) and six species (Chelon labrosus, C. auratus, L. ramada, L. saliens, Mugil cephalus, O. labeo) have been described . Ganzirri Lagoon (Messina, Sicily, South Italy) is connected by several canals to the northern part of the Strait of Messina, forming two small ecosystems characterized by brackish water and high biodiversity levels in comparison with the other areas in the Strait, making it suitable for the exploitation of biological resources and for shellfish farming, an activity that has been practiced for several centuries. The lake is approximately 338,000 m2, 1.7 km long and 250 m wide, with a maximum depth of approximately 7 m and an average temperature that varies between 11deg in January and 30deg in August . Mullet parasites associated with large-scale freshwater and seawater polyculture have been described worldwide since 1970 . Mullets are economically important teleosts and are often farmed or caught for human consumption. However, like many species of fish, mullet can also be affected by parasites. Among the protozoan parasites that affect the Mugilidae, Cryptocaryon irritans , Trichodina sp. . Trichodinella sp. , Tetrahymena pyriformis, and T. pediculus have been reported globally. Dinoflagellata, Amyloodinium ocellatum, agent of the "velvet disease", represents one of the most dangerous parasites in both wild and cultured mullets, causing high mortality rates . Zoonotic Heterophidae metacercariae attributable to Ascocotyle sp., Heterophyes heterophyes and Stictodora sp., have been isolated from the muscle tissue of C. labrosus caught off the island of Sardinia and then morphologically and molecularly identified . The same parasite species have been isolated in M. cephalus from the Atlantic and Pacific Oceans and in L. ramada from the Black Sea . Monogenean flukes, such as Microcotyle mugilis, Ligophorus sp., and Ergenstrema sp. were found in M. cephalus sampled from the Marmara Sea (Turkey) and L. ramada gills from Cabras Lagoon (Sardinia, Italy) . Adult digenean trematodes, such as Saccocoelium cephali, S. obesum, S. tensum, and Haploporus benedeni were molecularly identified from C. auratus and M. cephalus gastrointestinal tracts caught along the Spanish Mediterranean coasts and L. ramada (p = 36%) and C. labrosus (p = 67%) , where the Haploporidae was the most retrieved parasite family in all investigated species. Neoechinorhynchus agilis (Acanthocephala: Neoechinorhynchidae), which attaches to the intestinal submucosa layer, has mainly been observed in M. cephalus from the island of Sardinia (p = 91%) , and in other areas worldwide , as well as in other species such as C. labrosus and C. auratus . Nematode infections in mullets are represented mainly by Anisakidae . Contracaecum sp. larvae and Capillaria sp. have been described in Mugilids (C. labrosus and C. auratus) encysted in the celomic organ serosa and inside the gastrointestinal lumen , in the Mediterranean Sea, and particularly from the island of Sardinia. The lack of information on the Mugilid gastrointestinal parasitic fauna in the study area is strictly related to the complexity of the environmental characteristics. Due to many variables, such as water movement and the prey present, it is difficult to improve current data on the control of parasites in wild fish. Thus, the aim of the current study is to characterize the gastrointestinal helminth parasite fauna of mullets from Ganzirri Lagoon (southern Italy). 2. Materials and Methods 2.1. Fish Sampling From March to June 2021, during two different sampling trips in the same sampling site, a total of 150 mullet specimens were caught by a throwing net, also known as sparrow hawk, in the Nature Reserve of Ganzirri Lagoon (38deg15'41'' N, 15deg37'35'' E) (Messina, Sicily, Italy). After the sampling, all specimens were stored at +4 degC and transferred to the Experimental Fish Pathology Centre of Sicily (CISS), at the Department of Veterinary Sciences, University of Messina, Italy. All specimens were morphologically identified according to the key suggested by . Fish were sexed, measured (total length, TL), and weighed (body weight, BW) (PBA220, Mettler Toledo, Columbus, OH, USA, accuracy of 1 g). All biometric data were recorded and subsequently reported as main values (mean length, ML; mean weight, MW). The specimens included in this manuscript were not part of any experiment and were commissioned by fish farmers for diagnostic purposes, specifically for fish disease control. For this reason, no ethical committee approval was needed, but all animal handling was, in any case, performed under European and Italian guidelines on animal welfare. All the sampling activities were authorized by the Regional Agency for the Protection of the Environment (ARPA Sicilia authorization n.1138/A of 15.03.2021). 2.2. Parasitological Examination The evaluation of organs was carried out according to , modified according to requirements. The gastrointestinal (GI) tract was opened using scissors and the gastric and intestinal mucosa were gently scraped with the aid of a microscope slide; pyloric caeca were considered as part of the intestine; the organs were analyzed separately. GI contents and mucous were transferred into 1l graduated conical beakers and filled with saline solution for the subsequent sedimentation phase; the supernatant was replaced hourly to clarify the sediment. After 2 or 3 water changes, depending on supernatant clarity, the sediment was transferred to a Petri dish and monitored with the aid of a stereomicroscope (SteREO Discovery.V12 Zeiss, Jena, Germany). The collected parasite specimens were preserved in 70% ethanol until morphological identification. Other specimens were stored at -80 degC for subsequent molecular analyses. All retrieved specimens were diaphanized by immersion in glycerin for 24h, mounted and then identified with morphological keys . Due to the unclear trematode identification by the morphological keys used, molecular analysis was performed only for these parasites. The morphological evaluation was performed under a light microscope (Axioskop 2 plus, Zeiss, Jena, Germany) and all pictures were taken using a digital camera (Axiocam Mrc, Zeiss, Jena, Germany) supported by a digital system (Axiovision, Zeiss, Jena, Germany). 2.3. Molecular Analysis Only the trematode specimens were used for molecular identification. After morphological evaluation and confirmation of the morphological characteristics, the samples were collected in three different pools and placed in 1.5 mL (Eppendorf, Hamburg, Germany); three series of 20 min washes were performed with a phosphate buffer saline (PBS) buffer solution. DNA was extracted using a Wizard SV Genomic DNA Purification System (Promega, Madison, WI, USA). Concentration and purity were verified using a Nanodrop spectrophotometer (Thermo Fisher Scientific, Waltham, MA, United States). The amplification of different rDNA regions was performed using a GoTaq(r) Colorless Master Mix (Promega, Madison, WI, United States) and different primers for trematode identification: 28S, ITS-2 and 18S: forward primer 5' GTCCGATAGCGAACAAGTACCGT 3' and reverse primer 5' AGCATAGTTCACCATCTTTCGGGTCTCAA 3' for 28S; forward primer 5' GTCGTAACAAGGTAGCTGTA 3' and reverse primer 5' TATGCTTAAGTTCAGCGGGT 3' for the partial ITS-2 , while for the oligonucleotide primers, C-For ATGGCTCATTAAATCAGCTAT and A-Rev TGCTTTGAGCACTCAAATTTG were used for 18S amplification . The thermal cycling conditions were the following: initial denaturation for 30 s at 94 degC, 35 cycles of denaturation for 30 s at 94 degC, annealing at 58 degC for 30 s, elongation for 60 s at 72 degC with final extension of 10 min at 72 degC for 28S rDNA; initial denaturation for 30 s at 94 degC, 35 cycles of denaturation for 30 s at 94 degC, annealing at 56 degC for 90 s, elongation for 90 s at 72 degC with final extension of 10 min at 72 degC for ITS 2; initial denaturation at 94 degC for 2 min, denaturation at 94 degC for 30 s, annealing at 56 degC for 30 s, elongation at 72 degC for 60 s repeated 30 cycles, followed by a final 60 s elongation at 72 degC for 18 s . Positivity was assessed on 1% (w/v) agarose gel. Once the presence of bands had been ascertained, a gel extraction was carried out using the Promega kit Wizard(r) SV Gel and PCR Clean-Up System. The concentration and purity were verified using the Nanodrop spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA) and samples were sent to Genechron Biotech for sequencing in both forward and reverse directions using the same primers used for amplification. 2.4. Statistical Analysis Epidemiological infection indices of prevalence (p, %), mean abundance (MA), and mean intensity (MI) were calculated according to . All data are presented as the mean +- standard deviation (SD). A chi-squared test was performed to assess parasite occurrence among investigated Mugilid species. The non-parametric Kruskal-Wallis's test, followed by Dunn's multiple comparisons test, were performed to highlight any significant difference in parasite abundance between districts and investigated Mugilid species. Furthermore, a Spearman correlation analysis was performed to detect any possible correlation between parasite abundance and Mugilid morphological features. The level of significance was set at p values < 0.05. A statistical analysis was performed using the software Prism V.9.0.0 (Graphpad Software Ldt., La Jolla, CA, USA). 3. Results A total of 150 mullets were examined. Identification of Mugilidae sampled species revealed a heterogeneous group composed mainly of C. labrosus (99/150), followed by C. auratus (37/150) and O. labeo (14/150). All biometric ML and MW data on the three retrieved species are reported in Table 1. 3.1. Parasitological Findings Of the 150 sampled fish GI tracts, 67 (44.7%) presented at least one parasite species. Sixty-six (44%) specimens were affected by adult digenean trematodes, both in stomach and gut. Of these, 2 C. labrosus specimens (1.3%) were affected by a total number of 16 Acanthocephala in the gut, identified according to as Neochinorhynchus agilis (Neoechinorhynchidae, Rudolphi, 1819) . Trematode loads per species are reported in Table 2. The results of the chi-squared test did not show any significant difference in parasite occurrence between the three Mugilidae species (kh2 = 5.7; df = 2; p = 0.056). A different result was observed for the abundance data, which varied significantly between C. auratus and C. labrosus (p <0.05), as shown in Table 3 and Figure 2. No significant differences were found between districts (p > 0.05). Finally, Spearman's correlation was performed to detect any possible correlation between parasite abundance and morphometric fish features, i.e., total length (TL) and body weight (BW). The results did not show any significant correlation between parasite abundance detected in CA and CL with these parameters. However, OL parasite abundance was negatively correlated both to the TL (r = -0.68; p = 0.009) and BW (r = -0.64; p = 0.015) of the specimens. 3.2. Molecular Analysis The nucleotide sequences of the amplified products were identical among biological replicates. The sequences obtained for 18S (727 bp), ITS-2 (401 bp), and 28S (871 bp) were analyzed by BLAST similarity search against the National Center for Biotechnology Information (NCBI; (accessed on 14 July 2022) database to calculate the statistical significance of the matches found. In particular, BLAST analysis of the sequences obtained from ITS-2 showed a similarity of 99.5% (E-value 0.0; query cover 100%) with the relative sequence from Haploporus benedeni (accession number FJ211247.1) while there was a similarity of 100% for 28S (Accession number FJ211237.1) and 99.86% for 18S (accession number FJ211228); Haploporus benedeni (Stossich, 1887) was found with an E-value of 0.0 and a Query cover of 100% for both sequences. Amplification of the 28S rDNA gene included the D2 domain, the most robust domain, since it could provide a higher resolution of the species. The representative DNA sequence for 18S, ITS-2, and 28S was submitted to GenBank (accession numbers OQ283825, OQ283827, OQ283826, respectively). 4. Discussion The present study represents the first survey on mullet helminth parasite fauna from a Special Protection Area (SPA) in the central Mediterranean Sea. Digenean parasites in mullets from different areas of the Mediterranean Sea have been reported . Several digenean parasites were collected in the intestinal mucosa , as reported in L. saliens and L. ramada . In the present study, the most abundant collected species was C. labrosus; nevertheless, the most parasitized species was O. labeo, although it is the least representative collected species and the high prevalence is probably related to a specific ecological characteristic of O. labeo to host the trematode herein described, localized mainly in the intestine, rather than the stomach. Another relevant aspect of the investigated area, characterized by regular movement of water, is the difference in the salinity rate in comparison with the nearby sea. Salinity is strictly related to mollusk and crustacean evolution, regulating both growth and intermediate host vulnerability to the predators . The likelihood that the sampling spot is distant from the infection point, even by a couple of kilometers, in an unknown area, characterized by different environmental conditions, should be highlighted . The parasitological examination and subsequent molecular confirmation allowed us to identify the trematode Haploporus benedeni (Haploporidae, Stossich 1887) using the ITS2, 18S, and 28S ribosomal RNA genes. Despite the high number of reports of H. benedeni in most Mugilid species from the Mediterranean basin , few studies provide a molecular identification of this species ; therefore, our study improves the current knowledge about the molecular description of this digenean trematodes. The H. benedeni life cycle is characterized by the free-swimming miracidium and an intermediate host belonging to the genus Hydrobia, as reported by . In particular, Hydrobia sp. is a small aquatic gastropod mollusk belonging to the family Hydrobiidae. The presence of Hydrobia sp. inside the mullet stomach could be associated to the H. benedeni prevalence in each of the three studied species, which also indicates a high distribution of snails belonging to the Hydrobia sp. in the investigated area. The H. benedeni prevalence reported by in C. labrosus (67%) was higher than the present study, where the most infected species was O. labeo. Neoechinorhynchus agilis is a widespread parasite of grey mullets worldwide . In , N. agilis specimens attached to the intestinal mucosal and submucosal layers of L. saliens and L. ramada (41.6%) at a higher prevalence than in the present study (1.3%). This Acanthocephalan has also usually been considered as a M. cephalus parasite . C. labrosus was found in the eastern Atlantic Ocean and Mediterranean Sea as one of the most infected species (up to 54%) by N. agilis , a prevalence higher than our study (1.3%), which is probably related to the difference in fish size. The identified parasites, H. benedeni and N. agilis, previously reported from the Mistras Lagoon (Sardinia--Italy--western Mediterranean Sea) , were reported for the first time in the central Mediterranean Sea, showing an equal distribution in two different Mediterranean areas, as well as in Libya and Egypt . This ecological evaluation is also supported by the current information on the diversity and distribution of parasites, such as H. benedeni and N. agilis in mullets. 5. Conclusions In conclusion, this study improves the current knowledge on mullet helminth parasitic fauna from the Ganzirri Lagoon in the central Mediterranean Sea. Mullet, while not characterized by a high commercial value, are increasingly being appreciated both for their greater production in aquaculture and for their use in traditional food preparation in the Mediterranean basin. Hydrobia spp., a common mollusk found in Ganzirri Lake, and considered a host for a wide range of trematode species that can infect several marine species such as mullet, can be used as a biological tag for further studies in the same area. Moreover, even if mullets are considered a host of parasites which are potentially dangerous for human health, the parasitic species identified in the examined GIT are not considered zoonotic agents. Author Contributions Conceptualization, G.G., A.G. and C.I.; methodology, G.D.B., K.R. and M.A.; validation, G.G., A.G. and C.I.; formal analysis, S.N. and C.G.; investigation, G.D.B., F.C. and C.I.; data curation, A.G., S.S. and S.N.; writing--original draft preparation, G.D.B. and F.C.; writing--review and editing, G.D.B., F.C., S.S. and C.I.; supervision, G.G., A.G. and C.I. All authors have read and agreed to the published version of the manuscript. Institutional Review Board Statement No procedures on live animals were carried out and, thus, the requirement of ethical approval is not applicable. Informed Consent Statement Not applicable. Data Availability Statement Not applicable. Conflicts of Interest The authors declare no conflict of interest. Figure 1 Neoechinorhynchus agilis cranial and posterior end: (A) proboscis (P), proboscis receptacle (R), lemnisci (L1, L2); (B) proboscis (P), proboscis receptacle (R), hook systems (H); (C) N. agilis male posterior end: seminal vesicle (SV), Saefftigen's pouch (SP), bursa (BU), calotte (CA), genital pore (GP). Figure 2 Mean abundance of Haploporus benedeni found in all three mullet species: CL: Chelon labrosus; CA: Chelon auratus; OL: Oedalechilus labeo. Each datum is shown as mean +- SD. Letters are only present in the case of significant statistical differences. Different letters refer to significant differences between different species. Differences were considered significant when p < 0.05. animals-13-00847-t001_Table 1 Table 1 Mean lengths, mean weights, and standard deviations (SD) of the three species. Species Mean Length (cm) +- SD Mean Weight (g) +- SD Chelon labrosus 20.18 +- 3.99 91.66 +- 139.67 Chelon auratus 19.75 +- 1.9 72.08 +- 22.19 Oedalechilus labeo 18.75 +- 2.74 65.28 +- 23.97 animals-13-00847-t002_Table 2 Table 2 Prevalence, intensity, and abundance of Haploporus benedeni in the three species studied. S--Stomach; I--Intestine; GIT--Gastro-intestinal tract. Chelon labrosus S I GIT Prevalence % 12.12 14.14 24.24 Mean intensity 6.16 7.78 346.3 Mean abundance 0.74 1.1 83.9 Chelon auratus S I GIT Prevalence % 2.7 16.2 8.1 Mean intensity 2 7 57.6 Mean abundance 0.05 1.13 4.67 Oedalechilus labeo S I GIT Prevalence % 50 - 50 Mean intensity 8.66 - 6.5 Mean abundance 1.85 - 1.85 animals-13-00847-t003_Table 3 Table 3 Results of the Kruskal-Wallis test and Dunn's multiple comparison test of the Haploporus benedeni abundances in the three mullet species. Kruskal-Wallis Test p value 0.0374 p value summary * Do the medians vary signif. (p < 0.05)? Yes Number of groups 3 Kruskal-Wallis statistic 6.573 Dunn's multiple comparisons test Mean rank diff. Summary Adjusted p Value H. benedeni -CA vs. H. benedeni -CL -19.29 * 0.0343 H. benedeni -CA vs. H. benedeni -OL -18.69 ns 0.397 H. benedeni -CL vs. H. benedeni -OL 0.5988 ns >0.9999 * p value <= 0.05 Disclaimer/Publisher's Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

PMC10000165

The impact of cyclic heat stress (CHS) and turning the lights on and off on pig feeding behavior (FB) was investigated. The FB of 90 gilts was recorded in real-time under two ambient temperatures (AT): thermoneutrality (TN, 22 degC) or CHS (22/35 degC). The day was divided into four periods: PI (06-08 h); PII (08-18 h); PIII (18-20 h); and PIV (20-06 h). Automatic and Intelligent Precision Feeders recorded each feed event for each pig. An estimated meal criterion (49 min) was used to calculate the FB variables. Feed behavior in both ATs followed a circadian pattern. The CHS reduced the feed intake by 6.9%. The pigs prioritized feed intake during the coolest hours of the day; however, nocturnal cooling did not allow the pigs to compensate for the reduced meal size due to CHS. The highest meal size and most of the meals were observed during the lighting-on period. The pigs reduced their interval between meals during PII and PIII. The lighting program increased the meal size when the lights were switched on and reduced the meal size when the lights were switched off. Thus, the dynamics of the FB were largely influenced by AT, whereas the meal size was affected by the lighting program. circadian rhythm feed pattern light program meal pattern precision feeding swine Sao Paulo Research Foundation2018/15559-7 National Council for Scientific and Technological Development142555/2019-3 CAPES-PrInt88881.310312/2018-01 This research was funded by the Sao Paulo Research Foundation (FAPESP), grant 2018/15559-7. This study was possible thanks to the scholarship granted from the Brazilian National Council for Scientific and Technological Development (CNPq), grant 142555/2019-3, in the scope of the Program CAPES-PrInt (process number 88881.310312/2018-01). pmc1. Introduction The improper thermal environment of housing facilities and lighting programs are detrimental factors that affect pig growth performance. Ambient temperature (AT) is related to the regulation of voluntary feed intake (VFI), triggering changes in feed behavior (FB). Finishing pigs exposed to high AT in cyclic heat stress (CHS) conditions reduce their VFI by 7.3% and up to 50% in constant heat stress (HS) of 35 degC . Reduction of VFI is a pig's adaptive response in an attempt to decrease the metabolic heat production inherent to digestive and metabolic processes . In addition to the impact on VFI, the AT may change the FB based on the type of heat challenge. The FB dynamics of pigs reared in constant HS differ from those of pigs reared in CHS . In a CHS challenge, when the AT returns to the thermoneutrality (TN) zone, pigs increase VFI rapidly to similar levels to that of unchallenged pigs, or the VFI can temporarily exceed the normal level . Regarding the behaviors that underlie feed intake, meal size, ingestion time per day, and occupation time of the feeding station decrease as AT increases from 19 to 29 degC, whereas the daily number of meals remains constant . However, the impact on VFI is smaller for daily CHS compared to constant HS. This is because pigs show specific behavioral adaptations, such as reducing VFI during the warm periods of the day and increasing it during fresh periods, especially in the nocturnal period . Because pigs are sensitive to their environment , turning the lights on and off, henceforth referred to as 'light events', may also alter the feeding pattern of pigs. Light events may modulate or, in some cases, block outright the circadian rhythm via a process referred to as 'masking' . However, to our knowledge, no data regarding the effects after lighting events on FB of CHS pigs are available. A better understanding of pigs' FB during CHS and light events could provide key information for improving productivity and animal well-being, especially in tropical climate areas where pigs are usually exposed to cyclic variation in AT . Accordingly, a strategy to evaluate the effects of AT and light events on FB is to compare pigs with similar body weight (BW), genetics, and sex that were raised in similar facilities, management systems, and diets but in differing AT. The aforementioned factors affect FB in different ways. Even when all these influences are kept constant, individual differences still exist , making it difficult to compare studies that do not have the same overall experimental conditions. Thus, considering that only AT differs in the current study, we hypothesized that: (I) pigs raised in CHS conditions prioritize their feed intake in the coolest hours of the day; (II) pigs overconsume during the TN period of the CHS challenge, thus having a higher meal size than their counterpart raised in TN conditions; and (III) light events modulate the meal size of pigs according to AT. Therefore, the objectives of this study were to: (i) investigate the effects of CHS compared to TN conditions on FB and (ii) evaluate the impact of light events on FB in both AT conditions of finishing pigs. 2. Materials and Methods 2.1. Data Source The animals, facilities, and experimental procedures used in this study were previously described . In this study, the feeding behavior of 90 crossbred (Landrace x Large White) finishing gilts was recorded in real-time with the aid of 10 automatic precision feeders (Automatic and Intelligent Precision Feeder--AIPF; University of Lleida, Lleida, Spain). A detailed description of the feeders is available in . Pigs were allotted at the Swine Research Facilities of Sao Paulo State University (Unesp), Jaboticabal, SP, Brazil with 24.5 +- 2.9 kg of BW and group-housed in two similar rooms (45 pigs/room) on a full concrete floor, with a single 95 m2 pen each, equipped with fans and an evaporative pad cooling system (Big Dutchman, Araraquara, SP, Brazil). Pigs had free access to water via low-pressure nipple drinkers and were fed ad libitum, individually, and with diets formulated to meet or exceed their nutritional requirements according to NRC recommendations. The lighting program was fixed to obtain 12 h of light as the sum of natural sunlight and LED light source (1800 Lumens). Artificial light was controlled by a timer switch from 06 to 18 h. All pigs were fitted with an ear tag with an electronic chip (passive transponders of radio frequency identification) in the right ear, with the electronic chip number being previously registered in the feeder station system. The electronic chip provided an exclusive identification to each pig, which enabled all animals to be housed in the same pen and allowed each individual pig to be an experimental unit. Therefore, pigs had free access to any of the feeders in the room and had their individual feed intake (FI) recorded during the trial. For feed delivery, when each pig introduced its head into the feeder, the electronic chip was identified, and when the pig triggered a push button to demand feed, the AIPF delivered a service to each request. With the aim of maintaining the services per pig per day at less than 150 requests (following the manufacturer's recommendation), the size of the service was adjusted. The service was set to deliver the feed according to the experimental phase (EP), with 20 g during EP1 (0-20 days, 68 to 93 kg of BW) and 23 g during EP2 (21-38 days, 93 to 111 kg of BW). To avoid feed waste and ensure that pigs consumed each serving before requesting a new one, a time lag of 30 s was imposed between services. Total FI in each visit was the sum of several requests. Normally, the pigs empty the feeder after each visit, but sometimes they leave the feeder hopper with small amounts of feed (less than the service size). However, it is not significant when considering the total FI in the day. Regardless of the facility, during the growing phase (25 to 67 kg of BW), pigs were kept in similar conditions of AT (thermoneutrality), feeding management, and diets until achieving a BW of 67.7 +- 6.2 kg when the trial started (finishing phase). Pigs remained in the experiment for 45 days, which consisted of a 7-day adaptation period to experimental diets and CHS and a subsequent 38-day experimental phase, which was divided into EP1 (Days 0 to 20) and EP2 (Days 21 to 38). During the experimental period, in the facility with TN conditions, the AT was maintained at 22 degC, whereas in the CHS conditions, a diurnal cyclic variation in AT was set and ranged from 22 degC (20-08 h) to 35 degC (8-20 h). Thermostat-controlled heaters (Furio, Sao Paulo, SP, Brazil) were used to increase the AT in CHS conditions. To cool down the AT until thermoneutrality, fans, and evaporative pad cooling systems (Big Dutchman, Araraquara, SP, Brazil) were used in both conditions. An air humidifier (Hidrogiro, Ribeirao Preto, SP, Brazil) was used to increase the relative humidity of the air in the CHS condition (8-20 h) to decrease the evaporation rate and exacerbate the effect of HS. These temperatures and time periods aimed to mimic diurnal variation in ambient temperature based on studies with cyclic heat stress . The AT and relative humidity were recorded daily at 30 min intervals throughout the experiment using a thermohygrometer (Instrutherm Mod. HT-70, Sao Paulo, Brazil). 2.2. Data Collection and Management The visit of each pig to the AIPF was registered by a monitoring device that continuously and automatically recorded the start and end time of each visit (day, hour, minute, and second), the number of requests made by the pig, and the total amount of feed served in each visit. Collected information was used to build the initial database, which was initially managed by using a Microsoft Excel spreadsheet. Feeding information collected on the days that pigs were handled (i.e., blood collection, dual-energy X-ray absorptiometry scanning, as described in ) was removed from the dataset (3 days). In the beginning, the initial dataset had 25,704 records, which were then analyzed using R Statistical Software (version 2.14.0; R Foundation, Vienna, Austria). Initially, the dataset was evaluated by searching for the presence of outliers. Using graphical analysis, only 1 observation was removed from the dataset during the experimental period (visit longer than 3 h), which was considered a technical failure of the system. Observations of zero FI (0.06%) were excluded based on the assumption that this visit may be associated with animal-feeder interactions, in which pigs quickly introduce and remove their head from the feeder and which, hence, are recognized and recorded by the AIPF software as a visit. 2.3. Definition of the Meal Criterion A visit represents every time a pig introduces its head into the feeder. However, it does not imply a meal. A meal may be defined as an accumulation of sequential visits that were separated by an interval below a calculated meal criterion. The interval represents short breaks mainly for feeder change and water intake , along with social interactions that can occur during breaks within a meal. Thus, expressing FB data in terms of meals is considered a more appropriate unit. The meal criterion used in this study was based on the analysis of the starting probability. This method defines a meal based on statistical measures rather than behavioral measures, which were previously described and recommended for situations in which neither the the between-meal interval distributions are known . For this purpose, the probability of the pigs starting a new feeding event within the next minute (pstart) was calculated over the day (all dataset) and for the TN period, as well as for the HS period, and plotted against the interval between visits to the feeder. The meal criterion was estimated as the first point that minimizes the pstart curve as previously described and had been recently used in this type of dataset . To reduce the effect of random variation in the pstart function, a simple moving average over 21 min intervals was used. 2.4. Calculations and Statistical Analysis Using the defined meal criterion (see pstart and meal criterion section), a new database was created for group visits that consisted of the same meal, resulting in a database with 13,834 records. In the new database, intervals between meals longer than 1440 min were arbitrarily excluded and assumed to be physiologically noncompatible, but this exclusion represents 0.8% of observations from the initial database, which is a percentage that does not compromise the final analysis. Based on the data grouped into meals, the number of meals (n/pig), meal size (feed intake, g/meal), meal duration (feeder occupancy, min/meal), feed intake rate (defined as the feed intake for each minute spent feeding over a meal, g/min/meal), and the interval between meals (min) of each animal were calculated. It should be noted that pig FB changes throughout a 24 h day occur regardless of AT . In addition, the circadian rhythm may also change FB . Thus, for a more adequate approach, the day was divided into four periods: PI (06-08 h), PII (08-18 h), PIII (18-20 h), and PIV (20-06 h). PI (06-08 h) and PII (08-18 h) together represent the diurnal period of the day, whereas PIII (18-20 h) and PIV (20-06 h) are the nocturnal period. PII (08-18 h) and PIII (18-20 h) were defined as the HS period of the day, whereas PI (06-08 h) and PIV (20-06 h) were the TN period. It was assumed that PI (06-08 h) and PIII (18-20 h) represented the short-time effects after lighting-on and -off events, respectively. All the data were analyzed using the MIXED procedure in SAS software (version 9.4; SAS Inst. Inc., Cary, NC, USA). The presence of outliers was evaluated through the residual analysis of data and by daily records of anomalies. The normality of studentized residuals was verified using the Cramer-von Mises test. Meal duration and interval between meals were log-transformed to adjust for lack of normality. If the data were transformed for analysis, least squares means were back-transformed to the original scale for reporting purposes. Each pig was considered an experimental unit. The model included the fixed effects of temperature (T), period of the day (P), experimental phase (EP), and their interactions (T x P, T x EP, P x EP, and EP x T x P) as follows:Yijmk = m + Ti + Pj + EPm + (T x P)ij + (T x EP)im + (P x EP)jm + (T x P x EP)ijm + eijmk(1) where Yijmk is the observed variable (observation for the kth pig from the (i,j,m)th cell), m is the overall mean, Ti is the effect of the ith level of the T factor, Pj is the effect of the jth level of the P factor, EPm is the effect of the mth level of the EP factor, and their interactions (that is, ith level of T with the jth level of P, (T x P)ij; ith level of T with the mth level of EP, (T x EP)im; jth level of P with the mth level of the EP, (P x EP)jm; and ith level of T with the jth level of P with the mth level of the EP, (T x P x EP)ijm). The eijmk was considered the random error of the kth observation from (i,j,m)th. The repeated measurement option was used with a compound symmetry covariance structure to account for the animal effect over sampling time, and the initial body weight was included as a covariate. Preplanned contrast comparisons were performed to evaluate the effects of light-on (C1, PI (06-08 h) x [PIII (18-20 h) + PIV (20-06 h)]) and light-off events (C2, PIII (18-20 h) x [PI (06-08 h) + PII (08-18 h)]). A third contrast (C3, [PI (06-08 h) + PII (08-18 h)] x [PIII (18-20 h) + PIV (20-06 h)]) was performed to compare the diurnal and nocturnal periods. Differences were considered significant if p <= 0.05. When there was a P x EP x P interaction effect (p < 0.05), adjusted means of P were compared in each T within each EP using the Tukey-Kramer test. When there was a T x P interaction effect (p < 0.05), adjusted means of P were compared in each T using the Tukey-Kramer test. Likewise, when there was a P x EP interaction effect (p < 0.05), adjusted means of P were compared in each EP. Finally, a t-test was used to compare the AT and air relative humidity differences between rooms. 3. Results 3.1. General Observations The average AT and air relative humidity experienced by the animals inside each room (TN and CHS) during the experiment are shown in Figure 1. The AT differed between rooms (p < 0.01) on a 24 h average basis. Overall, the AT in TN conditions averaged 21.9 +- 0.4 degC, whereas in CHS, the AT ranged from 22.3 +- 0.2 degC (TN period) to 33.8 +- 0.3 degC (HS period). The relative humidity also differed between rooms (p < 0.01) and averaged 87 +- 1.7% and 79.6 +- 2.7%, for TN and CHS rooms, respectively. Detailed information on growth performance and carcass composition was reported in our previous study , and individual pig behavior profiles showing the heterogeneity in FB response are available in Supplementary Figure S1. 3.2. pstart and Meal Criterion A total of 24,128 visit records were used to calculate the pstart function. There was no difference in determining the meal criterion using the pstart methodology for exposure to different temperatures. Thus, the probability of pigs starting a new feeding event within the next minute after the last visit--over the course of the day, during exposure to CHS (35 degC) or TN (22 degC)--was 49 min . 3.3. Feeding Behavior Responses Overall, the feed intake (meal size x number of meals) of pigs throughout the experiment on a 24 h basis was 2416 g and 2268 g for the TN and CHS conditions, respectively, indicating that CHS reduced the FI by 6.13%. The meal size continuously recorded in TN and CHS conditions followed a circadian pattern with typical diurnal FB . Interestingly, the meal size of the pigs decreased during the first hour after the light had been switched on, independent of the AT. In the TN condition, the hourly meal size peaked twice over the diurnal period, whereas in the CHS condition, it peaked once. Under TN conditions, the first peak took place 5 h after the light had been switched on (11 h), whereas the second peak took place before the end of the diurnal period (18 h). In the CHS condition, the diurnal peak took place 3 h after the light had been switched on (9 h). When the lights were switched off, pigs in the TN condition started to decrease the meal size until 23 h, whereas in the CHS condition, an increase in meal size was observed between 19 and 21 h. The meal duration had a similar trend to that of meal size . Most feed intake, 77.2% and 73.2%, and meals, 71.6% and 72.2%, were observed between 6 and 18 h in the TN and CHS conditions, respectively, which corresponded to the light-on period . Irrespective of AT, the interval between meals started to decrease during the diurnal period, right after the light had been switched on . Overall, the feed intake rate was quite similar throughout the 24 h day . Feeding behavior responses of the pigs are presented in Table 1. Residuals of feed intake rate, meal size, and number of meals were normally distributed, whereas the residuals of meal duration and the interval between meals were log-transformed to adjust for lack of normality. Initial BW as a covariate was significant for all studied variables (p < 0.01). Overall, responses were affected by P and EP, except the number of meals (p < 0.01). Interactions between P x T were detected for feed intake rate (Table 1). In the CHS condition, pigs had the highest feed intake rate during PIII (HS, 18-20 h), the lowest feed intake rate during the diurnal period, from 06 to 18 h (PI (TN, 06-08 h) + PII (HS, 08-18 h)), and an intermediate value during PIV (TN, 20-06 h) (p < 0.01, P x T). However, there was no difference in the feed intake rate in the TN condition during the periods of the day. A P x EP interaction was detected for feed intake rate (Table 2). During EP1, pigs had a greater feed intake rate during PIII (18-20 h) than during the diurnal periods, whereas no effect of P was observed in EP2 (p = 0.01, P x EP). In contrast, as expected, regardless of the period of the day, pigs during EP2 had a greater feed intake rate than those during EP1 (p = 0.01, data not shown). The interaction between EP x T x P was significant for meal size, meal duration, and the interval between meals (Table 3). In terms of period effects within each temperature, in EP1 (Days 0 to 20), in TN conditions, pigs had the highest meal size and duration in PII (08-18 h) whereas in the other period, similar values were observed (p < 0.01). Under CHS conditions, pigs had a decrease in meal size in PIII (18-20 h), whereas in the remaining period, similar values were observed (p < 0.01). The highest meal duration was observed in PII (08-18 h), and the lowest meal duration was observed in PIII (18-20 h) (p < 0.01). It should be noted that for both ATs, pigs had a decrease (p < 0.05) in meal size and duration from PII (08-18 h) to PIII (18-20 h) (after turning off the light). However, only for the CHS condition did pigs show an increased (p < 0.05) meal size and duration from PIII (HS, 18-20 h) to PIV (TN, 20-06 h). In both conditions, pigs had a lower interval between meals from 08 to 20 h (PII (08-18 h) + PIII (18-20 h)) (p < 0.01). In EP2 (Days 21 to 38), pigs in TN conditions had the highest and lowest meal size and duration in PII (08-18 h) and PIII (18-20 h), respectively (p < 0.01). The highest interval between meals was observed in PIV (20-06 h), the lowest in PI (06-08 h), and intermediate values from 08 to 20 h (PII (08-18 h) + PIII (18-20 h)) (p < 0.01). Under CHS conditions, the lowest meal size and duration were observed in PIII (HS, 18-20 h). Meal duration in the remaining periods was similar (p > 0.05). For both AT, pigs had a decrease (p < 0.05) in meal size and duration from PII (08-18 h) to PIII (18-20 h) (after turning off the light) and an increase (p < 0.05) in meal size and duration from PIII (18-20 h) to PIV (20-06 h). The highest interval between meals was observed in PIV (20-06 h), whereas the lowest was observed in PII (HS, 08-18 h) (p < 0.01). Regarding temperature conditions (TN x CHS) within each period, several changes were observed. Overall, the CHS temperature reduced the values for all analyzed variables in all periods (p < 0.01), except for the interval between meals in PIV (20-06 h) during EP1 and in PIII (18-20 h) during EP2 (p > 0.05). Switching on the lights stimulated the feed intake of CHS pigs by 12.9% (p < 0.01) in comparison to the entire light-off period (C1, Table 4), whereas no effect was observed for TN pigs (4.2%, p > 0.05). Alternatively, switching off the lights induced a reduction in the feed intake of pigs, and hence in meal size by 17.8% for TN and 19.1% for CHS conditions related to the entire light-on period (C2, p < 0.01). During the entire light-on period, the meal size was 16.8% and 9.3% greater in TN and CHS, respectively, compared to the light-off period (C3, p < 0.01). 4. Discussion The current literature evaluating the effects of CHS and pig circadian rhythm on FB traits is scarce. Understanding the impact of AT variation and light events may aid in explaining the physiology and growth response of pigs under CHS conditions. Therefore, we hypothesized that pigs reared in CHS conditions prioritize feed intake in the coolest hours of the day, whereas light events alter the FB of pigs under cyclic AT variation over the day. Our major and original finding was that even when adapted, pigs in CHS conditions had a different FB when compared to TN pigs. Therefore, this FB response may help to explain differences in growth performance and body composition for CHS pigs, as previously reported . Feeding behavior may be affected by many factors such as sex , breed , body weight , circadian rhythm , ambient temperature , type of heat challenge (warm constant x cyclic temperatures; ), origin, age , diet, and handling. Therefore, a single experiment with pigs which have similar characteristics and differ only in the AT condition, as described in the present study, seems to be more suitable for evaluating the impact of AT on FB. In the current study, it was possible to measure the feed intake over different periods of the day. Period II (08-18 h) and PIV (20-06 h) represented more than 80% of the diurnal and nocturnal period of a 24 h day. Thus, we used both periods to evaluate the dynamic impact of HS on feed intake. Alternatively, PI (06-08 h) and PIII (18-20 h) represented the period after switching the lights on and off, respectively, and herein, these periods were used to compare changes that occurred in the FB due to light events. Feed intake rate increases over time because body size and oral capacity become larger, which allows pigs to take larger bites and eat more feed in a shorter time . This supports the feed intake rate observed in the current study. Although changes in feed intake rate were observed between periods of the day in EP1, no differences were detected during EP2. We believe that changes in feed intake rate in EP1 may be more closely associated with HS impact once a reduced feed intake is observed in pigs during the warmer period of the day. Because pigs under CHS conditions were in an adaptation process during EP1, the higher feed intake rate for CHS pigs in some specific periods of the day may have contributed to the overall mean, which could have triggered the interaction effects. This concept is supported by the higher feed intake rate observed during the nocturnal period (the coolest part of the day). Alternatively, as long as pigs became more adapted to the CHS condition, they normalized their feed intake rate over the day, which resulted in a lack of period effect in the EP2. Our outcomes agree with those of Fraga et al. , who also did not observe changes in feed intake rate throughout the day for pigs (22 kg to 105 kg of body weight) raised under a similar CHS protocol. To maintain a constant feed intake rate throughout the day under CHS conditions, pigs increase the meal size, which implies an increase in meal duration . Moreover, an interaction between P x T was observed for feed intake rate for pigs in different AT conditions. In our study, as expected for pigs reared under unrestricted conditions , feed intake rate was kept relatively constant throughout the day for TN pigs. However, it should be noted that for pigs under CHS conditions, feed intake rate increased when the lights were switched off, whereas for TN, no changes were observed. Such behavior highlights the fact that the light events modulated pigs' FB according to the AT. As widely described in the literature , pigs in HS conditions reduce physical activity, which probably disrupted the normal feed intake rate of pigs in the current study. The increase in feed intake rate associated with the light-off event seems to be an attempt of pigs to overcome the lower feed intake rate during the HS diurnal period. Considering that a light-off event would be expected to lower the feed intake (positively correlated with feed intake rate, ), the opposite behavior observed herein might be understood as a feeding motivation for pigs to try to compensate for the HS diurnal impact. In other words, the light-off event may have triggered some competition for feeder access, because group-housed pigs in restricted conditions increase feed intake rate as a mechanism to maintain meal size levels . The current study showed that meal size, duration, and interval between meals were affected by AT and P, which varied according to the EP. These variables are expected to be correlated because when the meal size increases, the meal duration increases simultaneously because the feed intake rate is relatively constant throughout the day for pigs under unrestricted conditions . Irrespective of AT, as long as pigs increase the meal size and duration while keeping a constant number of daily meals, the interval between meals decreases . According to the concept of satiety, when a pig is satiated at the end of a meal, the probability of starting a new meal soon is low but increases over time . This finding supports the reduction in meal size and duration and the increased interval between meals observed for CHS pigs during the nocturnal compared to the diurnal period, irrespective of EP. Regarding the P effect within each EP, the pattern of meal size, meal duration, and the interval between meals for CHS pigs changed in comparison to TN pigs. During EP1, pigs in CHS conditions had similar meal sizes in the diurnal and nocturnal periods. Interestingly, during EP2, the meal size during the nocturnal period exceeded that observed for the diurnal period, whereas no changes in meal duration were observed. Because the animals had a constant number of daily meals, pigs increased the meal size in the same meal duration, which implied a higher feed intake. Taken together, these points reinforce the concept that pigs raised under CHS use mechanisms associated with changes in the FB to adapt to AT conditions and to maintain a constant number of meals throughout the day. Irrespective of the EP, our study demonstrated that pigs raised in TN reduced their meal size during the nocturnal period. In contrast, pigs raised in CHS had a similar meal size during the diurnal and nocturnal periods in EP1, whereas it was greater during the nocturnal period in EP2. Thus, our data support the hypothesis that the pigs tried to compensate for the impact of CHS by eating more during the TN period of challenge. These results agree with previous studies that also evaluated pigs exposed to CHS . However, it should be noted that the compensatory response was not enough to allow a similar meal size compared to the pigs under TN conditions. Although pigs exhibit adaptive FB, the time required for feed intake recovery after exposure to HS to reach a similar level to that of TN pigs can vary between 2 and 5 days . However, factors related to the physical capacity of the pigs digestive tract and the impact of the consecutive daily heat challenge might not have allowed a full recovery of feed consumption in the current study. Thus, changes in the FB of pigs under CHS observed in the current study may be associated with the adaptation process to the cyclical AT condition over the day. This adaptation response may explain the differences in growth performance, as well as body composition, which was previously reported . Pigs predominantly exhibit a diurnal feeding consumption, with most meals (73% on average) consumed between 06 and 18 h . Similar values were observed in our experiment during the diurnal period (71.6% for TN and 72.2% for CHS). In the TN condition, the hourly meal size peaked twice over the diurnal period (at approximately 11 and 18 h), suggesting that this response may be related to the lighting program used . In contrast, the meal size of pigs under CHS conditions, in addition to being lower, ended up in just a single peak at the beginning of the HS period (at approximately 9 h). This response may be an adaptation mechanism of pigs to HS in an attempt to decrease heat production and, consequently, the amount of heat that needs to be dissipated into the environment . According to our results, an interesting finding is that the meal size of CHS pigs decreased during the first hour after the light-off event and started to increase shortly thereafter. In contrast, when the light was switched off, a huge drop in meal size was observed for pigs under TN conditions, and this response was maintained until the end of the day. This behavior confirms the strong influence of lighting events on circadian rhythm, which drive an increased activity at a period of the day and a decrease in activity at another for TN pigs. In contrast, for CHS pigs, after the first hour of darkness, the meal size continuously increased for 2 h. This indicates that although the nocturnal period had begun, the pigs continued to ingest feed, which suggests that CHS conditions affected the pigs circadian rhythm. This response coincides also with a higher observed feed intake rate. Our data showed that the light events change the meal size of pigs. However, the changes were different in each AT condition. Pigs in TN conditions increased the meal size by 4.2% and reduced it by 17.8% during the light-on and -off events, whereas pigs in CHS conditions increased the meal size by 12.9% and reduced it by 19.1% during the light-on and -off events, respectively. The higher improvement (in %) in meal size for CHS than for TN pigs during a light-on event may be associated with the reduced feed intake during the light-off period. The calorie restriction might increase motor activity . In addition, a change in FB is also part of the metabolic cues associated with calorie restriction, which is reported to affect the suprachiasmatic nuclei of the hypothalamus and, hence, the synchronization to light . Thus, the FB of CHS was modulated by light events when compared to TN pigs. 5. Conclusions In conclusion, the outcomes showed that pigs raised under CHS changed their FB to maintain a constant number of meals throughout the day. However, despite our initial hypothesis that pigs could prioritize their feed intake in the coolest hours of the day, our results demonstrate that pigs did not fully compensate for feed intake depression caused by the HS. Despite both AT being affected by light events, CHS pigs had a greater increase in feeding activity than TN pigs when the light was switched on. The understanding of the pig responses to temperature variation is of the utmost importance in establishing strategies to improve pig production under warm climates. Because our results showed that light events affect feeding behavior under daily cyclic high-ambient-temperature conditions, research on diet modulation over the day is encouraged. Acknowledgments The authors would like to thank Seara Alimentos and Evonik Nutrition and Care GmbH for the donation in kind of animals and raw materials for the production of feed at the trial used to collect the data. Supplementary Materials The following supporting information can be downloaded at: Figure S1: Individual pig behavior profiles of pigs during the experimental period throughout 24 h day according to ambient temperature (Thermoneutral vs. Cyclic heat stress) and periods of the day: PI (06-08 h); PII (08-18 h); PIII (18-20 h); PIV (20-06 h). Click here for additional data file. Author Contributions Conceptualization, M.J.K.d.O. and L.H.; methodology, M.V. and I.A.; formal analysis, M.J.K.d.O. and M.V.; investigation, M.J.K.d.O., A.D.B.M., D.A.M., C.A.S., G.A.d.C.V., P.R.A. and J.P.R.G.; resources, L.H.; data curation, M.J.K.d.O. and M.V.; writing--original draft preparation, M.J.K.d.O.; writing--review and editing, M.J.K.d.O., M.V., A.D.B.M., D.A.M., C.A.S., G.A.d.C.V., P.R.A., J.P.R.G., I.A. and L.H.; visualization, M.J.K.d.O., A.D.B.M., D.A.M. and L.H.; supervision, I.A. and L.H.; project administration, L.H.; funding acquisition, L.H. All authors have read and agreed to the published version of the manuscript. Institutional Review Board Statement Ethical review and approval were waived for this study because this study had been developed using only database analysis. The original data were collected in a previously published study, which has the experimental procedures reviewed and approved by the Institutional Animal Care and Use Committee at Sao Paulo State University, SP, Brazil (protocol No. 3380/20). Data Availability Statement The data supporting the conclusions of this article will be made available by the authors upon request. Conflicts of Interest The authors declare no conflict of interest. Figure 1 Average ambient temperature (a) and air relative humidity (b) were measured in thermoneutral (TN) and cyclic heat stress (CHS) conditions, with the aid of a thermohygrometer, at 30 min intervals, during the experimental days. Each vertical bar represents the standard error of the mean. Figure 2 Probability of pigs starting a new feeding event (pstart) within the next minute since the last visit over the course of the day, or during exposure to cyclic high ambient temperature (35 degC, 0800-2000 h) or thermoneutrality (22 degC, 2000-0800 h). Figure 3 Circadian variation in (a) meal size, (b) meal duration, (c) number of meals, (d) interval between meals, and (e) feed intake rate for finishing pigs reared in thermoneutrality (TN, 24 h at 22 degC) or cyclic heat stress (CHS, 12 h at 22 degC, 20-08 h, and 12 h at 35 degC, 08-20 h) conditions for 38 days of the experimental period. Periods of the day: PI (06-08 h); PII (08-18 h); PIII (18-20 h); PIV (20-06 h). Average data of 45 pigs per AT condition throughout 24 h day, regardless of the experimental phase. The lighting program was fixed to 12 h of artificial light (06 to 18 h). animals-13-00908-t001_Table 1 Table 1 Feeding behavior of finishing pigs exposed to thermoneutrality or cyclic heat stress conditions, according to the period of the day 1,2. Temperature (T) Thermoneutrality Cyclic Heat Stress ANOVA p-Value 4 Period of the Day (P) PI (06-08 h) PII (08-18 h) PIII (18-20 h) PIV (20-06 h) PI (TN, 06-08 h) PII (HS, 08-18 h) PIII (HS, 18-20 h) PIV (TN, 20-06 h) P x T P x EP T x EP P x EP x T Feed intake rate, g/min 5 27.4 (0.40) 27.9 (0.32) 27.4 (0.45) 27.4 (0.35) 27.6 b (0.39) 27.5 b (0.32) 28.6 a (0.41) 27.9 ab (0.34) <0.01 0.01 0.89 0.30 Meal size 6, g/meal 605 (15) 751 (12) 557 (18) 604 (14) 516 (15) 483 (12) 404 (16) 510 (13) <0.01 <0.01 0.83 <0.01 Meal duration 3,6, min/meal 25.9 (0.87) 31.5 (0.59) 22.4 (1.03) 26.0 (0.72) 23.3 (0.83) 22.0 (0.56) 16.4 (0.91) 21.5 (0.66) <0.01 0.02 0.50 <0.01 Interval between meals 3,6, min 507 (14) 310 (11) 300 (15) 504 (12) 350 (13) 224 (11) 219 (14) 421 (12) <0.01 <0.01 0.73 0.01 Number of meals, n/pig 0.23 (0.01) 0.19 (0.01) 0.15 (0.01) 0.09 (0.01) 0.27 (0.01) 0.25 (0.01) 0.21 (0.01) 0.12 (0.01) 0.17 0.71 0.82 0.81 1 Data after application of meal criterion defined as 49 min and expressed per hour of the day since each periods of the day had a different duration. The thermoneutrality condition was 24 h at 22 degC, whereas the cyclic heat stress condition was set at 12 h at 22 degC, 20-08 h, and 12 h at 35 degC, 8-20 h. 2 Least squares means and standard error (SE). 3 Meal duration and the interval between meals were log-transformed for statistical analysis and back-transformed to the original scale for reporting purposes. 4 Probability of periods of the day (n = 4; P), temperature (n = 2; T), experimental phase (n = 2; EP), and interactions between P x T, P x EP, T x EP, and P x EP x T. For variables whose interaction was significant, average values were analyzed by Tukey's test. Initial BW as a covariate was significant for all variables; p < 0.01. 5 The P x EP interaction for feed intake rate is presented in Table 2. 6 The P x EP x T interaction for feed intake, meal duration, and the interval between meals are presented in Table 3. a,b Values within a row and temperature, with different superscripts, differed during the period of the day (P x T, p < 0.01). animals-13-00908-t002_Table 2 Table 2 Feed intake rate of finishing pigs according to the period of the day in each experimental phase 1,2. Period of the Day (P) ANOVA Experimental Phase (EP) PI (06-08 h) PII (08-18 h) PIII (18-20 h) PIV (20-06 h) p-Value 3 Experimental phase 1 (0-20 days) Feed intake rate, g/min 25.6 b (0.40) 25.7 b (0.32) 26.9 a (0.42) 26.2 ab (0.34) <0.01 Experimental phase 2 (21-38 days) Feed intake rate, g/min 29.3 (0.40) 29.7 (0.32) 29.3 (0.44) 29.2 (0.35) 0.22 1 Data after application of meal criterion defined as 49 min and expressed per hour of the day since each periods of the day had a different duration. 2 Least squares means and standard error (SE), represent the average feed intake rate obtained from pigs raised under thermoneutrality (24 h at 22 degC) and cyclic heat stress conditions (12 h at 22 degC, 20-08 h, and 12 h at 35 degC, 8-20 h). 3 Statistical analyses between periods of the day in each EP. a,b Values within a row with different superscripts differed between periods of the day in each EP (p < 0.05) according to Tukey's test. animals-13-00908-t003_Table 3 Table 3 Feeding behavior of finishing pigs exposed to thermoneutrality conditions or cyclic heat stress conditions, according to the period of the day in each experimental phase 1,2. Temperature (T) Thermoneutrality Cyclic Heat Stress ANOVA Period of the Day (P) PI (06-08 h) PII (08-18 h) PIII (18-20 h) PIV (20-06 h) PI (TN, 06-08 h) PII (HS, 08-18 h) PIII (HS, 18-20 h) PIV (TN, 20-06 h) p-Value 4 Experimental phase 1 (0-20 days) Meal size, g/meal 587 b (22) 686 a (17) 581 b (24) 569 b (19) 484 a (21) 470 a (17) 387 b (22) 460 a (18) <0.01 Meal duration 3, min/meal 27.0 b (1.22) 31.6 a (0.82) 24.1 b (1.43) 24.7 b (0.96) 23.7 ab (1.15) 23.1 a (0.79) 16.4 c (1.24) 20.7 b (0.93) <0.01 Interval between meals 3, min 507 a (19) 289 b (15) 303 b (21) 432 a (17) 381 b (18) 222 c (15) 204 c (19) 393 a (16) <0.01 Experimental phase 2 (21-38 days) Meal size, g/meal 624 b (22) 816 a (17) 534 c (25) 640 b (20) 547 a (21) 496 b (17) 420 c (23) 559 a (18) <0.01 Meal duration, min/meal 24.7 b (1.25) 31.5 a (0.84) 20.7 c (1.50) 27.2 b (1.07) 23.0 a (1.19) 20.8 a (0.80) 16.5 b (1.33) 22.3 a (0.95) <0.01 Interval between meals, min 507 b (19) 331 c (15) 297 c (22) 576 a (18) 319 b (19) 226 c (15) 234 bc (20) 448 a (16) <0.01 1 Data after application of meal criterion defined as 49 min and expressed per hour of the day since each periods of the day had a different duration. The thermoneutrality condition was 24 h at 22 degC, whereas the cyclic heat stress condition was set at 12 h at 22 degC, 20-08 h, and 12 h at 35 degC, 8-20 h. 2 Least squares means and standard error (SE). 3 Meal duration and the interval between meals were log-transformed for statistical analysis and back-transformed to the original scale for reporting purposes. 4 Statistical analyses between periods of the day in each experimental phase for control and challenged barn. a-c Values within a row with different superscripts differed between periods of the day in each barn (p < 0.05) according to Tukey's test. animals-13-00908-t004_Table 4 Table 4 Meal size of finishing pigs exposed to thermoneutrality conditions or cyclic heat stress conditions, according to the period of the day 1,2. Period of the Day (P) ANOVA p-Value 3 Temperature (T) PI (06-08 h) PII (08-18 h) PIII (18-20 h) PIV (20-06 h) C1 C2 C3 Thermoneutrality Meal size, g/meal 605 (15) 751 (12) 557 (18) 604 (14) 0.15 <0.01 <0.01 Cyclic heat stress Meal size, g/meal 516 (15) 483 (12) 404 (16) 510 (13) <0.01 <0.01 <0.01 1 Data after application of meal criterion defined as 49 min and expressed per hour of the day since each periods of the day had a different duration. 2 Least squares means and standard error (SE) represent the average meal size obtained from pigs raised under thermoneutrality (24 h at 22 degC) and cyclic heat stress conditions (12 h at 22 degC, 20-08 h, and 12 h at 35 degC, 8-20 h). 3 C1, PI (06-08 h) x [PIII (18-20 h) + PIV (20-06 h)]; C2, PIII (18-20 h) x [PI (06-08 h) + PII (08-18 h)]; C3, [PI (06-08 h) + PII (08-18 h)] x [PIII (18-20 h) + PIV (20-06 h)]. 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PMC10000166

We monitored liver damage in cattle (cows, heifers, fattening bulls, and calves culled from the herd), pigs (sows, finishing pigs, and piglets culled from the farm), sheep (ewes and lambs), goats (does and kids), rabbits, and poultry (end-of-lay hens, broiler chickens, turkeys, domestic ducks, and domestic geese) in the period from 2010 to 2021. All animals (n = 1,425,710,143) reared on Czech farms and slaughtered at slaughterhouses in the Czech Republic were included in the analysis. We determined the total number of damaged livers for individual categories of animals and also analyzed separately the incidence of damage of acute, chronic, parasitic, and other origin. The overall incidence of liver damage was higher in adult animals compared to fattening animals in all species. In cattle and pigs, the incidence was also higher in young animals culled from the herd compared to fattening animals. When comparing adult animals by species, the incidence of liver damage was highest in cows (46.38%), followed by sows (17.51%), ewes (12.97%), and does (4.26%). When comparing fattening animals by species, the incidence was highest in heifers (14.17%) and fattening bulls (7.97 %), followed by finishing pigs (11.26%), lambs (4.73%), and kids (0.59%). When comparing young culled from the herd by species, it was higher in piglets (32.39%) than in calves (17.6 %), and when poultry and rabbits were compared, the incidence was highest in turkeys (3.38%), followed by ducks (2.20%), geese (1.09%), broiler chickens (0.08%), and rabbits (0.04%). The results indicate that fattening animals have a better liver condition than mature animals and that culled young have a worse liver condition than older fattening animals. Chronic lesions represented the dominant proportion of pathological findings. Parasitic lesions occurred, first and foremost, in animals grazed on meadows with likely parasitic invasion, i.e., in ewes (7.51%), lambs (3.51%), and heifers (1.31%), and in animals in which antiparasitic protection is limited in view of the protection of meat from antiparasitic residues, i.e., finishing pigs (3.68%). Parasitic damage to the liver was rarely detected in rabbits and poultry. The results obtained represent a body of knowledge for measures to improve the health and condition of the liver in food animals. livestock poultry liver veterinary inspection pathological findings Internal Creative Agency of the University of Veterinary Sciences Brno2021ITA22 This research was funded by the Internal Creative Agency of the University of Veterinary Sciences Brno, project number 2021ITA22. pmc1. Introduction The liver is the principal organ involved in the metabolism of fat, carbohydrate, protein, vitamins, and minerals and in the immunity of animals , constituting their lifeline system. In contrast, liver damage results in a deterioration of animal health and, thereby, their well-being. A good understanding of all the factors that affect the function of the liver is important in view of the fact that the liver is the core of many digestive and metabolic processes. In cases of dietary imbalance or too-rapid feed transitions, the liver can be considerably affected and it could lead to the accumulation of waste and toxins. The influence of individual aspects of nutrition (the nutritional composition of the feed ration, feed restriction, anti-nutritional factors, the structural composition of the feed, nutritional supplements, etc.) on the size, development, and function of the liver must be thoroughly investigated. Research has shown that the composition of the prepartum diet also has an impact on lipid metabolism, liver function, and the overall health of the cow postpartum . Petit et al. have demonstrated that feeding saturated fatty acids increased the risk of fatty liver, while feeding with sources of unsaturated fatty acids, e.g., flaxseed (3.3% and 11.0% of the dry matter in prepartum and postpartum diets, respectively) during the transition period could increase liver concentrations of glycogen and decrease liver triglycerides after calving. A recent study has demonstrated that sheep can develop hepatic steatosis due not only to negative but also to positive energy balance based on excess carbohydrate energy . Conversely, in swine, although they develop metabolic-syndrome-like features on a high-fructose diet, a substantial dietary fat is required to develop hepatic steatosis . The weight of the liver appears to increase or decrease proportionally to the nutritional plan and across physiological stages . It has been reported that in cattle and sheep, an increase in the functional workload of the liver through dietary manipulation resulted in an increase in the liver weight associated with hepatocyte hypertrophy , unlike as observed in rats, in which a decrease in liver metabolism due to the surgical manipulation caused hepatocyte hypertrophy . Attention must also be focused on feed-processing technology (such as heat treatment, pelleting, particle size, and the modification of whole-grain products) in terms of its impact on the state of the liver tissue . For example, the use of amoniation-fermentation (amofer) technology for a complete feed formula has shown a significant benefit on livestock, such as feed efficiency, feed conversion, body weight increment, animal productivity, and liver function . Liver damage generally occurs when the metabolism is disrupted, most often due to deficiencies in animal nutrition, or when the liver is damaged as a result of parasitic invasion as a target organ or an organ of the migration of parasites as part of the standard or random migration path in the animal organism. The liver may also be damaged as a result of an infectious process or by intoxication of the organism (by heavy metals or mould toxins, for example) . In addition, a housing system can predispose animals to certain lesions. For example, fatty liver syndromes are mainly found in caged hens, as reported by Keutgen et al. , who investigated pathologic changes in laying hens in various housing systems. The results of an extensive Lithuanian study show that pathological lesions are detected most frequently in the respiratory system of livestock animals (specifically, in 35.61% of all slaughtered animals), closely followed by lesions on the liver (recorded in 34.19% of all slaughtered animals), during postmortem veterinary examination at Lithuanian slaughterhouses. Pathological findings in the livers of slaughtered animals are mentioned in a number of studies that investigated the reasons for the condemnation of livestock carcasses during postmortem veterinary examination. The majority of these studies have, however, focused only on a specific species, e.g., cattle , pigs , rabbits , broiler chickens , laying hens , and turkeys . The aim of this study was to compare the incidence of liver damage in different species and categories of animals slaughtered at slaughterhouses. This study also aimed to distinguish the incidence of damage of acute, chronic, and parasitic origin. 2. Materials and Methods Liver damage was monitored in animals slaughtered at slaughterhouses as part of the veterinary supervision of the health safety of the organs of slaughtered animals performed during post-slaughter veterinary inspection as required by the relevant European Union legislation . Data for analysis were obtained retrospectively from the information system of the State Veterinary Administration of the Czech Republic, which contains information on the number of slaughtered animals and the results of veterinary inspections recorded by official veterinary inspectors of the Regional Veterinary Administrations of the State Veterinary Administration operating at slaughterhouses in the Czech Republic. For the purposes of the study, data on all animals reared on Czech farms and slaughtered for human consumption at any slaughterhouse in the Czech Republic between 2010 and 2021 were collected. A total of 1,425,710,143 animals were included in the analysis, namely 1,348,393 cows, 315,406 heifers, 1,214,298 fattening bulls, 120,238 calves, 683,912 sows, 29,628,524 finishing pigs, 152,088 piglets, 26,026 ewes, 132,553 lambs, 1680 does, 7690 kids, 2,403,779 rabbits, 23,116,811 end-of-lay hens, 1,328,693,065 broiler chickens, 1,516,365 turkeys, 52,360 geese, and 36,296,955 ducks. The animals were transported to the slaughterhouse in accordance with the requirements of the Council Regulation (EC) No 1/2005 and slaughtered in accordance with the requirements of the Council Regulation (EC) No 1099/2009 . The veterinary examination of slaughtered animals was conducted by official veterinarians who distinguished between livers without damage and livers with damage and recorded the nature of any liver damage found, distinguishing between acute damage, chronic damage, damage of parasitic origin, and other damage (usually icteric changes). The classification of findings was based on the methodology for postmortem inspection and was performed in a uniform way by slaughterhouse veterinary inspectors, i.e., specialized official veterinarians who received a specific training and obtained a certificate of competence. Pathological changes caused by an inflammatory process lasting a short period before slaughter were classified as acute damage. These changes included, e.g., marked hyperemia, liver fragility, the presence of hemorrhages, swelling, increased organ size, and liver abscesses. Pathological changes caused by an inflammatory process lasting for a prolonged period before slaughter were classed as chronic damage. These changes included changes to the original structure of the tissue parenchyma involving penetration of the connective tissue, the formation of connective tissue scarring, and the appearance of adhesions. The chronic damage was also indicated by a reduction in the size of the liver and its stiffness, lighter color, smooth or wrinkled surface, the presence of cysts or calcifying abscesses, etc. A specific case of chronic changes is fatty degeneration manifested by enlargement of the liver, with the edges of the liver rounded and the liver being fragile and having a yellowish-brown color. Changes that pointed to the invasion and migration of parasites and pathological processes caused by parasites in the host organism were classed as parasitic damage. Parasitic changes were not included in acute or chronic changes but were recorded separately in order to determine the extent of parasitic invasions. Parasitic damage included changes typical of parasitic invasion of the liver, i.e., focal changes characterized by small white spots or already-white scars on the surface of the liver (e.g., ascariasis in pigs--findings of scars in the liver after invasion of ascarids; coccidiosis in rabbits--findings of scars in the liver after invasion of coccidia), or directly with the finding of parasites in the bile ducts or in the liver tissue (e.g., Fasciola magna, Fasciola hepatica, etc.). The incidence of liver damage was monitored in cattle, namely in cows (females after the first calving), heifers (females from 6 months of age to the first calving), fattening bulls (males over 6 months of age), and calves (up to 6 months of age, culled from the herd prematurely); in pigs, namely in sows (females after the first farrowing), finishing pigs (pigs that have reached the market weight), and piglets (from 10 weeks to premature slaughter (before reaching market weight); in sheep, namely in ewes (mature females) and lambs (around 4-6 months after reaching market weight); in goats, namely in does (mature females) and kids (around 3-6 months after reaching market weight); in rabbits; and in poultry, namely in end-of-lay hens, broiler chickens, turkeys, ducks, and geese. The number of slaughtered animals and the number of damaged livers broken down into damage of chronic, acute, parasitic, and other origin were determined for each category of slaughtered animal over the entire monitored period of 12 years. The incidence of liver damage was expressed as a percentage. The overall incidence of liver damage as well as the incidence of chronic, acute, and parasitic lesions were compared between individual species and categories of slaughtered animals. Furthermore, the incidence of liver damage was compared between fattening animals (fattening bulls, heifers, finishing pigs, lambs, kids), adult animals (cows, sows, ewes, does) and young animals culled from the herd for inadequate condition or health (calves, piglets). The results were evaluated statistically using the program Unistat 6.5 for Excel. For the purposes of statistical comparison of the frequency of pathological findings in individual categories, a chi-square test was used to assess the statistical significance in a 2 x 2 contingency table . Yates' correction was used on frequencies exceeding 5, while Fisher's exact test was used at frequencies lower than 5. Spearman's rank test was used to assess the trend in the number of pathological findings . The result of testing was the determination of Spearman's rank correlation coefficients (rSp) used to assess a positive or negative trend in the numbers of findings. A value of p < 0.05 was considered statistically significant. 3. Results The incidence of liver damage in individual categories of slaughtered animals is shown in Figure 1. The statistical comparison revealed significant (p < 0.05) differences between individual species and categories, with the exception of the comparison of goats and lambs (p = 0.10) and sows and calves (p = 0.36). The results show that the incidence of liver damage was in all species higher in adult animals compared to fattening animals. It was higher in cattle and pigs in young culled from the herd compared to fattening animals. When individual species of adult animals were compared, it was highest in cows, followed by sows, sheep, and goats; when individual species of fattening animals were compared it was highest in heifers and fattening bulls, followed by finishing pigs, lambs, and kids; and when poultry and rabbits were compared it was highest in turkeys, followed by ducks, geese, broiler chickens, and rabbits. The numbers of slaughtered animals and the occurrence of liver damage broken down into acute, chronic, parasitic, and other changes are shown in Table 1. Chronic changes were found to be more frequent (p < 0.05) than acute or parasitic changes in most species and categories of animals, with the exception of piglets and ducks, in which the most frequent changes were acute changes, and of sheep and lambs, in which the most frequent changes were parasitic changes. The occurrence of chronic and acute liver damage shows a similar structure to that of the overall changes. In contrast, the incidence of liver damage of parasitic origin was higher in adult animals compared to fattening animals only in sheep and goats, with the opposite being true in cattle and pigs. In cattle and pigs, the incidence of parasitic damage as higher in fattening animals in comparison with young animals culled from the herd. When adult animals were compared by species, the incidence of parasitic damage was highest in ewes, followed by does, cows, and sows, and when animals for fattening were compared by species, it was highest in finishing pigs, followed by lambs, heifers, bulls, and kids. Parasitic changes were found to be rare in poultry and rabbits. The results of the Spearman rank correlations of the 12-year data within an animal category showed a significantly decreasing trend over the years for heifers, fattening bulls, ewes, lambs, kids, and end-of-lay hens and a significantly increasing trend over the years for calves, piglets, rabbits, and turkeys (Table 2). 4. Discussion The occurrence of liver damage is significantly higher in adult animals compared to fattening animals. This is the case in cattle (cows 46.38% vs. fattening bulls 7.97% and heifers 14.17%), pigs (sows 17.51% vs. finishing pigs 11.26%), sheep (ewes 12.97% vs. lambs 4.73%), goats (does 4.26% vs. kids 0.59%), and domestic chicken (end-of-lay hens 20.32% vs. broiler chickens 0.08%). This finding is evidence of the fact that fattening animals are organisms with better liver health than adult animals. The burden on the liver is shorter-lasting in young animals in view of their age, and they also have better regenerative capacity, which is manifested in a lower number of abnormalities in the liver. With respect to the liver, young animals are for this reason in better condition than adult animals whose livers are burdened by long-term intensive metabolism resulting from long-term intensive production. Diets that are high in grain may increase liver abscesses in fed cattle ; however, this was not the case in animals monitored in our study. Whereas feedlot cattle in North America are typically fed high-grain diets (a ration is usually 70 to 90% grain and protein concentrates), in the Czech Republic, fattening bulls are typically fed a maize-silage-based diet containing only 30 to 35% of grain. Dairy cows, in particular, though also suckler cows, remain on farms significantly longer than other categories of livestock animals. The average length of the productive life of dairy cows on farms ranges between 4.5 and 6 years. This period may be up to twice as long on average in the case of suckler cows . In dairy cows in particular, a long rearing period connected with intensive milk production has an effect on the state of health and on the possible occurrence of production diseases that are associated with characteristic findings in the organs, including the liver. Chronic liver lesions are common in old cows . Consequently, Dupuy et al. documented the influence of age on the rate of condemnations at slaughterhouses and found the highest condemnation rate in animals slaughtered at the age of 5-10 years (37.37%). As far as farmed species of birds are concerned, a relatively long lifespan (compared to broiler chickens or fattening turkeys, for example) combined with intensive egg production is typical of laying hens in particular. The rearing period, the housing system (cage housing still continues to prevail in the Czech Republic), and the use of laying hens are significantly different from other slaughtered poultry. While broiler chickens, geese, ducks, and turkeys are slaughtered after a few weeks or months of fattening (and are therefore usually young birds in good condition), laying hens are sent to slaughter after around a year of intensive egg production. In addition, it has also been shown that cage housing (compared to housing on litter or free-range housing) is one of the factors that contribute to the development of fatty liver syndrome in laying hens . Our study confirmed these assumptions, with liver lesions recorded in 20.32% of all slaughtered laying hens, which is the highest proportion by far among poultry and the third-highest overall (after cows and piglets). The probable cause of the unsatisfactory condition of the liver in end-of-lay hens is nutrition that does not correspond to the intensity of their production (250-320 eggs per laying period, average egg weight 60-63 g). Laying hens may experience liver and muscle proteolysis, adipose tissue lipolysis, and bone resorption if they do not receive sufficient nutrients relative to the level of egg production . The effect of age and the method and period of rearing is particularly evident in the difference between the number of liver lesions in end-of-lay hens (chronic 17.76%, acute 2.56%) and broiler chickens (chronic 0.04%, acute 0.03%), i.e., different categories within the same species of animal (the domestic chicken). The results of this study also show that the incidence of liver damage is significantly higher in young culled from the herd (calves, piglets) compared to fattening animals (fattening bulls, heifers, finishing pigs). This is the case in cattle (calves 17.67% vs. fattening bulls 7.97% and heifers 14.17%) and in pigs (piglets 32.39% vs. finishing pigs 11.26%). This finding is evidence of the fact that young animals culled from the herd due to poor condition or poor health also have a lower level of liver health than animals kept in the herd, i.e., fattening animals (even when they are older than the culled young). When adult cattle, pigs, sheep, and goats are compared, the incidence of liver damage is highest in cows (46.38%), followed by sows (17.51%), ewes (12.97%), and does (4.26%). This finding is evidence of the fact that, from the perspective of the liver, the greatest burden is seen in cows, probably as a result of the greatest difference between nutrients provided in the diet and metabolic needs, which leads to an enormous burden on the liver resulting in chronic or acute changes to the liver tissue. During early lactation, high-yielding dairy cows often develop a severe negative energy balance due to insufficient feed intake. Consequently, fat depots are mobilized in order to provide NEFA (non-esterified fatty acids) as an energy fuel. As a consequence of fat mobilization, the hepatic fat stores increase in the postpartum period. Excessive NEFA concentrations and a high liver fat content can lead to metabolic imbalances (i.e., ketosis and fatty liver syndrome) . The effect of productivity on the condemnation rate at slaughterhouses in Switzerland was mentioned by Vial et al. , with this being considerably higher in dairy cattle (79%) compared to beef cattle (21%). Januskeviciene et al. and Ceccarelli et al. , who did not differentiate between individual categories of cattle in their research, reported pathological findings (detected during postmortem veterinary examination) in cattle livers in 25.09% and 7.87% of all the examined animals, respectively. Ceccarelli et al. reported distomatosis, followed by liver abscesses and hydatidosis (Echinococcus granulosus), as the commonest findings in the livers of cattle. The impact on the liver is significantly lower in sows, likely due to the nature of nutrition based on feed mixtures with better control of nutrients in the diet. Nevertheless, pigs are a species of livestock animal in which a relatively large number of liver abnormalities were recorded in our study (piglets 32.39%, sows 17.51%, finishing pigs 11.26%). A high rate of condemnation in pigs due to liver lesions (9.85%, 31.79%, and 17.20%, respectively) was also reported by Ceccarelli et al. , Januskeviciene et al. , and Vecerek et al. . Januskeviciene et al. and Ceccarelli et al. , in agreement with our study, found the largest number of pathological lesions in the livers of cattle and pigs, though in contrast to our study they recorded a relatively larger number of lesions in pigs than in cattle. The impact on the liver is even lower in sheep whose nutrition is provided by grazing in the vegetation season, when the organism regulates the supply of nutrients with regard to its metabolism by choosing its feed itself. In goats, nutrition is provided in stalls and, to a limited extent, also by grazing, and special attention is devoted to goat nutrition with a view to their general sensitivity to the level of nutrition and changes in nutrition, which is reflected in a low number of pathological changes in the liver. The generally better state of health of sheep and goats compared to cattle and pigs is confirmed by the studies by Januskeviciene et al. and Ceccarelli et al. , who detected pathological lesions in the livers of 2.87% of all slaughtered sheep and goats, which corresponds quantitatively to our findings. According to the findings reported by Ceccarelli et al. , the liver was the organ most frequently affected pathologically in sheep (the liver accounted for 77.15% of all condemned organs in sheep). Sheep are reared largely under extensive rearing conditions in the Czech Republic. Sheep are reared all year round or for most of the year on pasture, which may be associated with a risk of the occurrence of parasites . Unlike sheep, goats are not typical grazing animals and it is natural for them to feed selectively primarily on the leaves of bushes and trees. Unlike sheep, they also avoid soiled sites . The risk of infection by parasitic life cycle stages may therefore be lower in goats, which may also be a reason for the better level of liver health seen in goats compared to sheep . The occurrence of liver damage significantly differs also when individual species of fattening animals are compared, ranking in the order cattle, pigs, sheep, and goats, i.e., heifers (14.17%) and fattening bulls (7.97%), finishing pigs (11.26%), lambs (4.73%), and kids (0.59%). The same order of species in fattening animals as in adult animals is also evidence of a similar disproportion between the actual nutrition and metabolic needs in fattening animals and in adult animals. The occurrence of liver damage also significantly differs when individual species of fattening poultry and rabbits are compared. The highest incidence is in turkeys (3.38%), followed by ducks (2.20%), geese (1.09%), broiler chickens (0.08%), and rabbits (0.04%). The results corroborate the higher sensitivity of turkeys to the correct content of nutrients in the diet, while in broiler chickens and rabbits, the low incidence of liver damage is evidence of the appropriate composition of feed mixtures with a view to the metabolic needs of broiler chickens and rabbits at the individual stages of their fattening. The rabbits reaching the slaughterhouse are animals in the category of fattening rabbits, which means that they are individuals in good condition. Liver lesions were not among the frequent reasons for the condemnation of the carcasses or organs of rabbits at Polish slaughterhouses . Hepatic coccidiosis, for example, was detected in 0.015% of all slaughtered rabbits during postmortem veterinary examinations performed in the period from 2010 to 2018 . In the years between 2007 and 2011, meanwhile, hepatic coccidiosis was detected in 3.34% of rabbits slaughtered in Poland , which indicates a favourable falling trend in the incidence of this parasitic liver damage. Rampin et al. , who studied pathological findings in rabbits slaughtered at slaughterhouses in Italy, found liver damage in 0.23% of all slaughtered rabbits, with hepatic coccidiosis, steatosis, and necrotising hepatitis being the most frequent causes of liver tissue damage. Conficoni et al. , who considered pathological lesions found during postmortem veterinary examinations of rabbits at slaughterhouses in Italy in the years 2003 to 2017, did not report liver lesions among the findings recorded at slaughterhouses at all. A markedly lower number of liver lesions was found when species of fattening poultry were compared with end-of-lay hens. The findings also show the higher sensitivity of turkeys to the correct content of nutrients in the diet compared to other species of fattening poultry. Turkeys, in general, have a much higher protein requirement than other poultry, especially when they are young and growing. Furthermore, the amino acid composition of the diet affects the occurrence of neurodegenerative changes in the brain and liver of turkeys . Pathological changes in the liver were detected in 3.38% of all slaughtered turkeys, and these were almost exclusively chronic changes. Similarly, Januskeviciene et al. found that turkeys showed the largest number of pathological lesions of all studied species of poultry (broilers, turkeys, ducks). Liver lesions were the second-most frequent (45.68% of all slaughtered turkeys) after lesions on the limbs (85.79% of all slaughtered turkeys). Broiler chickens, in contrast, generally show the lowest number of pathological changes of all studied species and categories of animals , which is associated with the fact that this is also the category slaughtered at the lowest age. In agreement with the results of our study, neither Jakob et al. nor Salines et al. mention liver lesions as a reason for the condemnation of broiler carcasses. Buzdugan et al. reported perihepatitis as the third-commonest cause of the condemnation of broiler carcasses in England. A Canadian study reflects an increased condemnation rate of broiler carcasses as a consequence of pathological lesions in the liver (necrotic hepatitis, perihepatitis, and haemorrhage). Immunosuppression caused by infectious bursal disease (IBD) has been identified as the primary cause of these liver lesions. A frequency of liver lesions only slightly higher than in broiler chickens was recorded in geese (1.09%). A probable explanation for this is the fact that this is a relatively undemanding and adaptable species that is also often reared for commercial purposes in extensive farming systems (an enclosure with pasture and access to water) that take the natural needs of this species into consideration . Hepatic necrosis, which may be caused by inflammation or intoxication, is rarely detected in geese . A relatively low number of liver damage was also detected in ducks (2.20%). It is, however, interesting that ducks show the highest frequency of acute changes (1.12%) of all fattening poultry, which may be caused by infection (for example, hepatitis B and reoviruses) or intoxication resulting from grazing (heavy metals, aflatoxicosis) . Chronic liver damage in ducks may include steatosis or even liver cancer (caused by the hepatitis virus or aflatoxicosis) . When comparing the incidence of chronic, acute, and parasitic changes in the liver, chronic lesions are significantly more frequent than acute or parasitic lesions in the majority of species and categories of animal species, with the exception of piglets and ducks, in which acute lesions are the most frequent, and ewes and lambs, in which parasitic lesions are the most frequent. When the incidence of chronic lesions is compared, it is higher in adult animals than in fattening animals, and it is higher in young animals culled from the herd than in fattening animals. When individual species of adult animals are compared in terms of the occurrence of chronic lesions, they rank in the order cows, sows, ewes, and does. When individual species of fattening animals are compared they rank in the order heifers and fattening bulls, finishing pigs, lambs, and kids, and when individual species of fattening poultry and rabbits are compared they rank in the order turkeys, ducks, geese, broilers, and rabbits. Chronic lesions represent the dominant proportion of liver damage, and the explanations for chronic changes are similar to those for overall damage, though chronic lesions are attributed more to the long-term effects of unbalanced nutrition than to sudden changes in animal nutrition. A high production performance makes animals prone to suffering from hepatic stress, making the organ more susceptible to different challenges linked to inadequate feed management and nutritional imbalances, such as diets with an inadequate protein-energy ratio that cause the fatty liver syndrome . Steatosis is a typical example of a chronic liver disease in livestock animals. The liver is the place where fat synthesis takes place, though excess fat can be stored directly in the liver under certain circumstances and this may cause health problems . Severe fatty liver syndrome occurs in poultry, the development of which involves a number of factors (environmental, hormonal, genetic), some of which have not yet been fully elucidated. Laying hens and turkeys are most frequently affected by this syndrome . Acute lesions correspond less to the overall incidence of liver damage than chronic lesions. Of all the species and categories of livestock animals studied, the largest number of acute changes was detected in piglets (15.97%), with a considerably smaller number found in calves (5.95%) and cows (5.23%). The largest proportion of acute lesions to the overall number of liver abnormalities was determined in piglets, broilers, and calves, i.e., in young animals in which chronic processes had not yet had time to develop fully. Acute lesions are attributed more to changes in nutrition than to the long-term action of nutritional effects. In addition to dietary causes, acute pathological processes in the liver tissue may also develop as a result of infection or intoxication. In poultry, e.g., acute lesions in the liver are found as a result of salmonellosis, campylobacteriosis, bird flu, and Marek's disease, while intoxication is often recorded in the form of aflatoxicosis . The frequency of parasitic damage is affected by species' susceptibility to various species of parasites and the age of the animals, and these factors lead to a structure of findings different to that seen in the case of chronic and acute changes. When the number of parasitic lesions in adult animals was compared with the number in fattening animals, a significantly higher incidence was found in heifers (1.31%) compared to cows (0.69%) and fattening bulls (0.51%) and in finishing pigs (3.68%) compared to sows (0.58%), while in contrast a significantly larger number of lesions was found in ewes (7.51%) compared to lambs (3.51%) and in does (0.74%) compared to kids (0.18%). These findings correspond to the feeding of different species and categories of animals in relation to the possibilities of parasitic invasion. In cattle, heifers are, first and foremost, pasture-fed, for which reason the level of parasitic invasion in heifers is higher than that seen in cows and fattening bulls, which are fed in stalls with feed mixtures, silages, and haylage. Pig nutrition is based on feed mixtures, though in view of the danger of invasion by roundworms, better parasite hygiene care on farms is devoted to sows due to their value and reproductive benefit than to finishing pigs, in which parasitic damage to the liver (ascariasis) is recorded most often (3.68%) of all categories of pigs. Infestation of pig farms with roundworms was also mentioned by Ceccarelli et al. , who reported parasitic hepatitis as the cause of condemnation in 96.2% of all condemned pig livers. In sheep, grazing on pasture leads to parasitic invasion, the impact of which is higher in ewes, in which it is more frequent and longer-lasting than it is in lambs in view of their age. Sheep (ewes and lambs) are the only species in which liver lesions of parasitic origin predominated. This finding is also confirmed by Ceccarelli et al. , who found that as many as 96.2% of all liver lesions found in sheep were of parasitic origin. Torina et al. draw attention to the fact that as much as 80% of pathology in sheep and goats is associated with the presence of parasites. Besides pasturing, differences in the antibody isotype responses of sheep and cattle to fasciolosis may play an important role in the frequency of liver lesions detected. Phiri et al. documented a difference in the IgG2 isotype response in cattle (resistant) compared to sheep (susceptible). Does are reared both in stalls and on pasture, for which reason the frequency of parasitic lesions is low, though higher than in kids in view of the longer period of possible infection in adult animals. In contrast to our study, Ceccarelli et al. recorded a significant quantity of pathological changes of parasitic origin in the liver (primarily ascariasis and distomatosis) in practically all livestock species studied. When the number of parasitic lesions in young culled from the herd was compared with that in fattening animals, a significantly higher incidence was found in heifers (1.31%) and fattening bulls (0.51%) compared to calves (0.13%). A higher incidence was also found in finishing pigs (3.68%) compared to piglets (1.21%). The impact of parasitic invasion is likely higher in heifers and fattening bulls than in calves due to their older age and, therefore, the longer period of time for possible parasitic invasion than in calves. The same is also true when finishing pigs and piglets are compared. When adult animals of individual species are compared, the number of parasitic lesions is significantly higher in ewes (7.51%) than in does (0.74%), cows (0.69%), and sows (0.58%). This ranking of parasitic lesions in adult animals by species likely corresponds to the method of feeding associated with the possibility of parasitic invasion, with the number being high in ewes, which graze over the long term in the open with a large possibility of parasitic infection. Cows and sows are primarily housed in stalls, while does are reared both in stalls and on the pasture, with limited possibilities for parasitic invasion, for which reason there are significantly fewer parasitic lesions found in the liver in these species compared with ewes. When fattening animals are compared by individual species, a significantly higher incidence of parasitic lesions is found in finishing pigs (3.68%) and lambs (3.51%) in comparison with heifers (1.31%), fattening bulls (0.51%), and kids (0.18%). This ranking of parasitic damage in fattening animals by species corresponds to the method of feeding associated with the possibilities of parasitic invasion, and is higher in lambs, which graze in the open with ewes and are exposed to considerable opportunities for parasitic infection. Heifers are also often kept on pasture, though considering the nature of these pastures in the Czech Republic (generally not pastures in damp and waterlogged areas) they are characterized by lower opportunities for parasitic invasion. The occurrence of parasitic invasion with roundworms is found in finishing pigs and this is treated only to an extremely limited extent in view of the period of fattening and protection of the meat against residual antiparasitics, for which reason the incidence of parasitic lesions in the liver of finishing pigs is recorded more often. Kids are generally fed in stalls rather than by grazing in view of the period of their fattening, for which reason the possibilities for parasitic invasion are limited. Parasitic lesions in rabbits are extremely rare in view of their feeding with granules. They may occur in sporadic cases in connection with hepatic coccidiosis in animals kept on small farms where feed is also provided in the form of grass and hay. Parasitic findings are rare in poultry. 5. Conclusions Varying proportions of animals suffering from liver lesions were found in different animal species and categories slaughtered for human consumption at the slaughterhouse. The results form a body of knowledge for measures to improve the health and liver condition of food animals. Besides the species-specific differences, the effect of age, rearing method, housing system, and nutrition should be considered when adopting measures aimed to improve the liver health of farm animals. Such measures should be adopted namely in calves, piglets, and turkeys, in which the incidence of liver damage showed a significantly increasing trend during the monitored period. Author Contributions Conceptualization, V.V.; Methodology, L.V., S.N. and V.V.; Formal analysis, V.V., L.V., S.N. and E.V.; Investigation, V.V., S.N. and E.V.; Writing--original draft preparation, V.V., S.N. and E.V.; Writing--review and editing, L.V. and A.P.; Supervision, V.V.; Project administration, E.V. and V.V.; Funding acquisition, E.V. All authors have read and agreed to the published version of the manuscript. Institutional Review Board Statement Not applicable. Informed Consent Statement Not applicable. Data Availability Statement Data for analysis were obtained from the information system of the State Veterinary Administration of the Czech Republic. The datasets generated and analyzed during the current study are available from the corresponding author on reasonable request. Conflicts of Interest The authors declare no conflict of interest. Figure 1 The overall occurrence of liver damage in individual categories of slaughtered animals. a-o values with different letters are significantly different at p < 0.05. animals-13-00839-t001_Table 1 Table 1 Numbers of slaughtered animals and the occurrence of liver damage broken down into acute, chronic, parasitic, and other damage for individual categories of slaughtered animals. Origin of Damage Species/Category of Animals (Number of Slaughtered Animals) Cows (1,348,393) Heifers (315,406) Fattening bulls (1,214,298) Calves (120,238) Sows (683,912) Finishing pigs (29,628,524) Piglets (152,088) Ewes (26,026) Lambs (132,553) Does (1680) Kids (7690) Occurrence of Liver Damage (%) Acute 5.23 b 1.35 b 0.40 c 5.95 b 1.00 b 0.22 c 15.97 a 0.09 c 0.04 c 0.00 c 0.00 c Chronic 39.13 a 11.41 a 7.04 a 11.49 a 15.93 a 7.35 a 15.21 b 5.35 b 1.18 b 3.42 a 0.41 a Parasitic 0.69 d 1.31 b 0.51 b 0.13 c 0.58 c 3.68 b 1.21 c 7.51 a 3.51 a 0.74 b 0.18 b Other 1.33 c 0.10 c 0.01 d 0.10 d 0.01 d 0.00 d 0.00 d 0.02 d 0.00 d 0.09 c 0.00 c Origin of Damage Species/Category of Animals (Number of Slaughtered Animals) Rabbits (2,403,779) End-of-lay hens (23,116,811) Broiler chickens (1,328,693,065) Turkeys (1,516,365) Geese (52,360) Ducks (36,296,955) Occurrence of Liver Damage (%) Acute 0.00 c 2.55 b 0.03 b 0.00 d 0.00 c 1.12 a Chronic 0.04 a 17.76 a 0.04 a 2.75 a 1.05 a 1.08 b Parasitic 0.00 b 0.00 d 0.00 d 0.01 c 0.03 b 0.00 c Other 0.00 c 0.01 c 0.00 c 0.62 b 0.00 c 0.00 d a-d values in columns within the same species/category of animals with different letters of superscripts are significantly different at p < 0.05. animals-13-00839-t002_Table 2 Table 2 Spearman rank correlation coefficients of the 12-year data within the animal category. Species/Category of Animals Correlation Coefficient Significance (p) Species/Category of Animals Correlation Coefficient Significance (p) Cows -0.4825 0.1121 Does 0.1399 0.6646 Heifers -0.8462 0.0005 Kids -0.7042 0.0106 Fattening bulls -0.6923 0.0126 Rabbits 0.6643 0.0185 Calves 0.8951 0.0001 End-of-lay hens -0.9021 0.0001 Sows -0.0350 0.9141 Broiler chickens -0.2657 0.4038 Finishing pigs 0.0699 0.8290 Turkeys 0.6014 0.0386 Piglets 0.9161 0.0000 Geese 0.2867 0.3663 Ewes -0.7552 0.0045 Ducks 0.3986 0.1993 Lambs -0.7902 0.0022 Disclaimer/Publisher's Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

PMC10000167

Integrating warm-season grasses into cool-season equine grazing systems can increase pasture availability during summer months. The objective of this study was to evaluate effects of this management strategy on the fecal microbiome and relationships between fecal microbiota, forage nutrients, and metabolic responses of grazing horses. Fecal samples were collected from 8 mares after grazing cool-season pasture in spring, warm-season pasture in summer, and cool-season pasture in fall as well as after adaptation to standardized hay diets prior to spring grazing and at the end of the grazing season. Random forest classification was able to predict forage type based on microbial composition (accuracy: 0.90 +- 0.09); regression predicted forage crude protein (CP) and non-structural carbohydrate (NSC) concentrations (p < 0.0001). Akkermansia and Clostridium butyricum were enriched in horses grazing warm-season pasture and were positively correlated with CP and negatively with NSC; Clostridum butyricum was negatively correlated with peak plasma glucose concentrations following oral sugar tests (p <= 0.05). These results indicate that distinct shifts in the equine fecal microbiota occur in response different forages. Based on relationships identified between the microbiota, forage nutrients, and metabolic responses, further research should focus on the roles of Akkermansia spp. and Clostridium butyricum within the equine hindgut. equine microbiome glycemic response non-structural carbohydrates rotational grazing USDA National Institute of Food and Agriculture1003557 New Jersey Agricultural Experiment StationNJ06260 SDA Northeast Sustainable Agriculture, Research, and Education projectgne17-162 Rutgers University Equine Science CenterThis research was funded by the USDA National Institute of Food and Agriculture, Hatch project 1003557, through the New Jersey Agricultural Experiment Station, Hatch project NJ06260, and the USDA Northeast Sustainable Agriculture, Research, and Education project, gne17-162. Additional funding was provided by the Rutgers University Equine Science Center (57 US 1, New Brunswick, NJ 08901). pmc1. Introduction Integrating warm-season grasses into traditional cool-season grass rotational grazing systems can provide productive pasture for grazing horses during the hot, dry months of the "summer slump" period . Despite reported benefits for pasture yield the impacts of this practice on equine metabolic health and the hindgut microbiome have not been previously investigated. cool-season grasses have different mechanisms for storage of soluble carbohydrates , with non-structural carbohydrate (NSC = sugars + starch + fructans) concentrations in cool-season grasses typically greater than that of warm-season grasses . These differences in NSC content are of interest in equine management as current feeding recommendations for horses with existing metabolic dysfunction include limiting dietary NSC concentrations . Thus, lower-NSC warm-season grasses have been suggested as an alternative forage source . Feeding supplemental concentrate higher in NSC has been shown to lower insulin sensitivity in horses , but over the relatively smaller range of NSC concentrations observed in forages, potential benefits of limiting NSC intake are less clear . However, glycemic and insulinemic responses of horses grazing low-NSC warm-season grasses have not been extensively evaluated. In addition to the potential metabolic effects, transitioning horses between forage types may also have implications for equine gastrointestinal health. Diet has been identified as a dominant factor shaping the community structure of the gut microbiota both in humans and across animal species including horses . However, prior studies on the influence of diet on the equine hindgut microbiome have focused primarily on concentrate vs. concentrate or concentrate vs. forage diets . Recent studies have demonstrated that type of hay (alfalfa vs. grass) and transitions between hay and pasture grass can impact equine cecal or fecal microbial community composition . Overall, few studies have evaluated the hindgut microbiome of grazing horses and only one previous study has been conducted in horses grazing . warm-season pasture grasses . A number of studies have begun to explore associations between the equine hindgut microbiota and metabolic health , but there is a lack of information on the interplay between forage nutrients, the hindgut microbiome, and metabolism of grazing horses. Biddle et al. explored relationships between abundance profiles of fecal microbial taxa, feed types (pasture, hay, or hay supplemented with concentrate), and blood analytes including circulating glucose and insulin, finding negative correlations between insulin and over 50 taxa. Studies conducted in mouse models have conclusively demonstrated that changes in diet influence host metabolism in a microbiome-dependent manner . Thus, there is a need for future research to better understand potential roles of the gut microbiota in modulating metabolism of grazing horses and the influence of specific forage nutrients on these interactions. While shifts in hindgut microbial communities have been documented in pastured horses over time, little is known regarding relationships between these changes in bacterial composition and forage nutrient profiles or metabolic responses of grazing horses. Therefore, the aims of this study were to characterize shifts in glucose metabolism and the fecal microbiota of horses adapted to different forage types and to explore relationships between forage nutrients, microbial composition, fecal metabolites, and metabolic responses of grazing horses. 2. Materials and Methods Research was conducted in 2018 at the Ryders Lane Environmental Best Management Practices Demonstration Horse Farm (Rutgers, The State University of New Jersey; New Brunswick, New Jersey, NJ, USA). Weather data for the study period and historical averages are presented in Table S1 . 2.1. Grazing Systems Two separate 1.5 ha integrated cool-season rotational grazing systems were utilized in this study. Grazing system design and management were as published in previous companion studies . In brief, warm-season grass pasture sections contained either Wrangler bermudagrass [BER; Cynodon dactylon (L.) Pers.; Johnston Seed Company, Enid, OK, USA] or Quick-N-Big crabgrass [CRB; Digitaria sanguinalis (L.) Scop.; Dalrymple Farms, Thomas, OK, USA]. Mixed cool-season grass sections contained Inavale orchardgrass [Dactylis glomerata (L.)], Tower tall fescue (endophyte-free) [Lolium arundinaceum (Schreb.) Darbysh.], and Argyle Kentucky bluegrass [Poa pratensis (L.)] (DLF Pickseed, Halsey, OR, USA). Grazing management was guided by established best management practices . 2.2. Animal and Grazing Management Use of animals in this study was approved by the Rutgers University Institutional Animal Care and Use Committee protocol #PROTO201800013. Eight adult Standardbred mares (age: 18 +- 0.71 yr; body weight (BW): 537 +- 17 kg; body condition score (BCS): 5-7)) were used in this study, with horses grouped by BW and BCS (Henneke Body Condition Score scale . Horses were then randomly assigned to each system (n = 4 horses system-1). Prior to the study, oral sugar tests (OST) were administered to screen horses for impaired insulin sensitivity . Insulinemic responses of all horses were found to be normal (peak insulin <=45 mIU/L) . Animal care and management have been previously detailed in companion studies . Horse condition measures over the course of the study can be found in Table S2. 2.3. Fecal and Blood Sample Collection Manual grab fecal samples were collected (0800 h) rectally from horses after 21 d adaptation to the initial hay diet in the spring (HAY-SP), cool-season grass pasture in the spring (CSG-SP), warm-season grass (WSG)--either BER or CRB during the "summer slump" period, and again following a return to cool-season grass in the fall (CSG-FA) and a final cool-season grass hay at the end of the grazing season (HAY-FA). See Figure S1 for a diagram of experimental design and sampling protocol. Due to delayed establishment and grazing of BER, only 17 days of grazing were possible prior to sample collection. Upon collection, samples were immediately placed on ice, transported to the laboratory, and then stored at -80 degC. To determine impact of forage type within integrated systems on glycemic and insulinemic responses of grazing horses, OST were conducted following adaptation to CSG-SP, WSG, and HAY-FA. On the evening prior to each collection period, horses were confined to dry lots at 2000 h with no access to either hay or pasture. Following the overnight fast, horses were moved into the barn facility at 0800 h. Two consecutive baseline blood samples were collected (15 min apart) by venipuncture at 0800 and an oral dose of Karo Syrup (0.25 mL/kg BW; modified dosing as utilized by Jacob et al. ) was immediately administered after the second baseline sample. Subsequent blood samples were collected at 30, 60, 90, 120, 180, and 240 min following Karo Syrup administration. Whole blood was collected into sodium heparin Vacutainer tubes, which were inverted several times before being placed on ice. Due to logistics of distance and travel time (~10 min from the barn to the laboratory), samples were transported to the laboratory after the 120, 180, and 240 min samples. In the laboratory, whole blood was centrifuged at 3700 rpm for 7 min. Following centrifugation, plasma was harvested from the Vacutainer tubes and aliquoted into microcentrifuge tubes, which were then stored at -80 degC. 2.4. Forage Sampling Representative hand-clipped forage samples were also collected (0800-1000 h) on three days per period for analysis of nutrient composition. Pasture samples were collected according to previously published procedures and dried (60 degC; 36 h minimum) in a Thelco oven (Precision Scientific, Chicago, IL, USA). Samples were ground (1 mm) and submitted to a commercial laboratory (Equi-Analytical Laboratories, Ithaca, NY, USA) for analysis by near-infrared spectroscopy. The mean nutrient composition of hay diets and pasture forages are shown in Table 1. 2.5. Analysis of Plasma Glucose and Insulin Plasma glucose was analyzed by colorimetric assay (Glucose C-2, Wako Chemicals, Richmond, VA, USA), with the commercial kit adapted for microplate assay following manufacturer instructions. Plasma insulin was evaluated using an enzyme-linked immunoassay (Mercodia Equine Insulin ELISA, Mercodia, Winston-Salem, NC, USA) previously validated in horses . Inter-assay and intra-assay coefficients of variation for glucose were 4.0% and 2.9%, respectively. Inter-assay and intra-assay coefficients of variation for insulin were 7.8% and 3.4%, respectively. 2.6. Fecal Sample Analyses Fecal pH was measured in duplicate with a handheld Accumet pH meter (Fisher Scientific; Waltham, MA, USA) using a previously published protocol for preparation of fecal slurries . Short-chain and branched-chain fatty acid (SCFA and BCFA, respectively) concentrations were determined by GC-MS analysis of fecal samples. The SCFA analyzed included acetate, propionate, butyrate, and valerate. The BCFA analyzed included isobutyrate, isovalerate and isocaproate. Sample preparation was performed according to a previously published protocol . In brief, frozen fecal samples were weighed and deposited in bead tubes over dry ice. Feces were then resuspended in 1 mL of 0.5% phosphoric acid per 0.1 g of sample and tubes were beaten for 5 min at 22.5 rpm in a cold case. Samples were again frozen at -80 degC until analyzed by Gas Chromatography and Mass Spectrometry system GC-MS (Agilent Technologies, Santa Clara, CA, USA). Prior to GC-MS analyses, thawed fecal suspensions were re-homogenized and centrifuged (10 min at 17,949x g), with the aqueous phase extracted using diethyl ether in a 1:1 volume to volume ratio. Before analysis, 2-methyl hexanoic acid (Thermo Fisher Scientific, Dallas, TX, USA) was added to the organic phase extract as an internal standard. Samples were analyzed in duplicate, with independent extractions for each replicate. The specifications of the GC-MS system and analyses as well as data acquisition and procedures for SCFA/BCFA quantitation have been previously described by Honarbakhsh et al. and Garcia-Villalba et al. . Quick-DNA Fecal/Soil Microbe Kits (Zymo Research; Irvine, CA, USA) were used for DNA extraction (in triplicate). The highest yielding replicate (quantified with a Qubit 2.0 Flourometer [Invitrogen; Carlsbad, CA, USA]) was submitted to a commercial laboratory (RTL Genomics; Lubbock, TX, USA). Amplification of the V4-V5 region of the 16S rRNA gene was conducted using region specific primers (515F/926R) ; sequencing was conducted by Illumina MiSeq. 2.7. Sequence and Statistical Analysis Sequence and statistical analyses were performed in QIIME 2 (Quantitative Insights into Microbial Ecology, v. 2020.8) and R (v. 4.0.2) . Network mapping was conducted in Cytoscape (v. 3.8.0) . Animal was considered the experimental unit. Quality and chimera filtering of forward reads was conducted using DADA2 (read length = 185) . Mafft and FastTree (q2-phylogeny plugin) were used to create trees for diversity analyses . The lower quartile of Amplicon Sequence Variants (ASV) based on absolute abundance were removed (minimum frequency = 16; minimum samples = 4). The feature table was rarefied to a minimum sampling depth of 10,600 prior to b-diversity analyses. The a-diversity metrics were analyzed by Kruskal-Wallis tests . The b-diversity metrics analyzed included Weighted and Unweighted UniFrac by permutational ANOVA (PERMANOVA) . Benjamini and Hochberg FDR adjustments for multiple pairwise comparisons were applied for all diversity analyses. Permutational multivariate analysis of dispersion (PERMDISP) was used to test homogeneity of dispersion . To further explore differential abundances, ASV were then grouped into bacterial co-abundance groups (BCG) based on abundance profiles using Sparce Cooccurrence Network Investigation for Compositional Data (SCNIC) . A random forest classifier with nested cross validation was applied to determine if forage type could be predicted based on BCG composition . Features (BCG) were removed from the model based on importance scores generated by the random forest classifier in an iterative process (serial reduction with increments of 5) until the point at which model accuracy began to decline . Relative abundances of remaining BCG and any uncorrelated ASV retained in the model following feature reduction processes were then analyzed by linear discriminant analysis effect size (LEfSe) to identify BCG specific to each forage type, with significance set at an LDA score >2.0 . Taxonomy was then assigned using the latest SILVA database (SSU 138) . Glucose and insulin response variables as well as SCFA/BCFA concentrations and fecal pH were analyzed by mixed model ANOVA in R, with grazing system, forage type and their interactions set as fixed factors and horse as the random factor in the initial model. The area under the curve (AUC) was calculated based on the trapezoid rule as the positive incremental AUC utilizing a published macro in SAS (v.9.4 SAS Institute, Cary, NC, USA). Proxies for insulin sensitivity (fasting glucose-to-insulin ratio (FGIR); reciprocal of the square root of insulin (RISQI)) and insulin secretory response (modified insulin-to-glucose ratio (MIRG)) were calculated using baseline (fasting) values as previously described . The relationship between forage nutrients and SCFA/BCFA fermentation metabolites (as well as pH) and fecal microbial community composition was then explored using random forest regression with nested cross validation to determine if nutrient concentrations and/or metabolite concentrations could be predicted based on bacterial abundance profiles. Spearman correlations between BCG and forage nutrients and fecal metabolites were analyzed in R. Relationships between forage nutrients/fermentation metabolites and BCG were then visualized in Cytoscape. Spearman correlations between metabolic variables and the BCG, as well as between forage nutrients and metabolic responses were also evaluated in R. For all variables analyzed by mixed model, model residuals were analyzed for normality using the Shapiro-Wilk test. Log, square root, or inverse data transformations were applied where appropriate for non-normal data. Means were separated using Tukey's method. When analyzing pairwise comparisons of transformed data, means and standard errors were back-transformed to the original variable scale following application of Tukey's method with the delta method. For all analyses which generated p-values, results were considered significant at p <= 0.05, with trends considered at p <= 0.10. Data for variables analyzed by mixed model are presented as means +- SEM. Overall analysis of microbiome, fecal metabolite, and glucose/insulin data did not reveal differences by grazing system. Therefore, grazing system was removed from models and results for combined data are presented with n = 8. 3. Results 3.1. Initial 16s rRNA Sequence Analysis A summary of 16S rRNA gene sequencing reads before and after quality and chimera filtering as well as after filtering of low abundance features in the 40 samples analyzed for this study is shown in Table 2. There were 1264 distinct ASV in the final dataset (taxonomy can be found in Table S3). 3.2. Diversity Analyses All a-diversity metrics evaluated, including the Shannon Diversity Index, Faith's Phylogenetic Diversity, Pielou's Evenness, and Observed ASVs, differed by forage . Shannon Diversity was greater when horses were adapted to WSG than CSG-SP (p < 0.05), and there was a trend for greater diversity when adapted to HAY-SP compared to CSG-SP (p < 0.08) and WSG vs. CSG-FA (p = 0.09). However, the only differences in evenness (Pielou's Evenness) were trends for greater evenness in WSG and CSG-SP than in CSG-FA (p < 0.06). Conversely, richness (Observed ASVs) was greater in horses adapted to WSG, CSG-FA, and HAY-FA than CSG-SP (p < 0.02), but did not differ between horses adapted to HAY-SP and CSG-SP. Similarly, phylogenetic differences (Faith's Phylogenetic Diversity) were also found between CSG-SP and subsequent forages (WSG, CSG-FA, HAY-FA; p < 0.02). Principal coordinate analysis of b-diversity metrics including Weighted and Unweighted UniFrac did not reveal distinct clustering by forage . However, statistical analysis by PERMANOVA (with Benjamini and Hochberg FDR adjustments) found significant differences in these measures (p <= 0.02). Subsequent PERMDISP analysis confirmed that these differences were not due to differences of variance or dispersion within groups. Unweighted UniFrac differed for all pairwise comparisons of forages (p < 0.02), with the exception of WSG vs. CSG-FA and WSG vs. HAY-FA for which there were trends for differences (p < 0.10). In contrast, there were no significant differences in any pairwise comparisons of forages for Weighted UniFrac. The percent of variation explained by PC1 was almost 3 times greater for Weighted than for Unweighted UniFrac, indicating an influence of abundance profiles in addition to phylogenetic differences. 3.3. Differential Abundance Application of SCNIC identified 333 BCG, with 224 individual ASV remaining ungrouped. Iterative reduction of features (BCG) based on random forest model importance scores revealed that model accuracy increased through reduction to the top 65 features (0.90 +- 0.09). Further feature reduction to the top 25 features (based on importance scores) did not impact random forest model accuracy (0.90 +- 0.09 with the top 25 features retained). All retained features were BCG; no ungrouped ASV remained in the reduced feature set. These 25 BCG were retained for further analysis, with 11.54% of the total microbial community abundance represented by the reduced 25 BCG feature set. The strength of the random forest model accuracy score indicated that forage type could be predicted based on bacterial composition, and that, conversely, bacterial community composition was influenced by forage. Subsequent Linear discriminant analysis Effect Size (LEfSe) analysis conducted on features retained from the random forest classification modelling identified 6 BCG as markers of HAY-SP, 3 BCG enriched in CSG-SP, 5 BCG for WSG, 5 BCG for CSG-FA, and 5 BCG for HAY-FA (LDA > 4.0; p < 0.03). Forage-specific BCG markers as well as taxonomic classifications of subsequent linear discriminant analysis Effect Size analysis conducted on features retained from the random forest classification modelling identified 6 BCG as markers of HAY-SP, 3 BCG enriched in CSG-SP, 5 BCG for WSG, 5 BCG for CSG-FA, and 5 BCG for HAY-FA (LDA > 4.0; p < 0.03). Forage-specific BCG markers as well as taxonomic classifications of individual ASV within each BCG are presented in Table 3, Table 4 and Table 5. Numerous taxa were represented in BCG markers for multiple forages. At the family level, the BCG markers for HAY-SP contained twice as many ASV mapped to the Lachnospiraceae family as any other forage (CSG-SP: 0; WSG: 2; CSG-FA: 3; HAY-FA: 2 ASV). The family Oscillospiraceae also had ASV members of BCG markers for all forages but CSG-SP (HAY-SP: 3; WSG: 3; CSG-FA: 2; HAY-FA: 2 ASV). At the genus level, ASV assigned to Christensenellaceae R-7 group were present in BCG markers of both hay diets (HAY-SP: 2; HAY-FA:1 ASV) and WSG (2 ASV); ASV assigned to the NK4A214 group of Oscillospiraceae were among members of BCG identified as markers of HAY-SP (2 ASV), WSG (1 ASV), and CSG-FA (1 ASV). The BCG markers of both hay diets and CSG-FA each included an ASV mapped to Rikenellaceae RC9 gut group, and BCG markers of HAY-SP and WSG each contained ASV assigned to Fibrobacter and Papillibacter. The BCG markers of HAY-SP and CSG-FA contained ASV within Catenisphaera and Lachnospiraceae UCG-009. Amplicon sequence variants mapped to the genus Clostridium sensu stricto 1 were found in BCG markers of CSG-SP and WSG. The BCG markers of WSG and CSG-FA included ASV assigned to Bacteriodales RF16 group and the Family XIII AD3011 group of Anaerovoracaceae. The BCG markers of CSG-FA and HAY-FA each contained an ASV assigned to Coprostanoligenes group, Lachnospiraceae XPB1014 group and Marvinbryantia, and ASV assigned to Treponema were assigned to BCG markers of WSG and HAY-FA. Amplicon sequence variants assigned to Ruminococcus, Pseudobutyvibrio, Anaerovibrio, probable genus 10 of Lachnospiraceae, and the p-251-o5 genus and family of the order Bacteroidales were only present in BCG markers identified for HAY-SP; no other ASV assigned to these taxa were found in BCG markers of other forages. An ASV within Bacteroidales BS11 gut group was the only taxa specific to BCG markers of CSG-SP. Amplicon sequence variants mapped to the genera Akkermansia, Mogibacterium, and the Hallii group of Lachnospiraceae as well as UCG-005 metagenome of Oscillospiraceae and Clostridium butyricum were only found in BCG markers of WSG. The BCG markers of CSG-FA contained ASV assigned to Alloprevotella, Erysipelatoclostridium, Bacteroidales UCG-001, the WCHB1-41 genus, family and class within the class Kiritimatiellae, and Denitrobacterium detoxificans; these taxa were not identified in BCG markers of other forages. Taxa specific to only BCG markers of HAY-FA included ASV from the Incertae Sedis genus of Ethanoligenenaceae as well as Streptococcus. 3.4. Fecal pH and Fermentation Metabolites Fecal pH differed by forage type (mixed model ANOVA with Tukey's post hoc adjustment; p < 0.0001). Fecal pH was greater in horses adapted to WSG (7.56 +- 0.18) and HAY-FA (7.57 +- 0.18) than in HAY-SP (6.73 +- 0.18), CSG-SP (6.58 +- 0.18), or CSG-FA . Fecal concentrations of short-chain fatty acids (SCFA) and branched-chain fatty acids (BCFA) also differed by forage (mixed model ANOVA with Tukey's post hoc adjustment; p < 0.001; Table 6). Unlike fecal pH, differences in SCFA and BCFA were primarily between pasture forages and the hay diets, with horses adapted to WSG often intermediate (numerically) between cool-season pasture and hay diets. Total BCFA were greater in CSG-SP, WSG, and CSG-FA than for either HAY-SP or HAY-FA (p < 0.05). Total SCFA were greater in CSG-SP and CSG-FA than HAY-SP or HAY-FA; WSG did not differ from CSG-SP and CSG-FA or HAY-SP, but was greater in comparison to HAY-FA (p < 0.002). Similarly, acetate was greater in CSG-FA than HAY-SP or HAY-FA, while WSG only differed from HAY-FA (p < 0.02). Acetate concentrations for CSG-FA also differed with HAY-FA (p = 0.0003), but there was only a trend for a difference between CSG-SP and HAY-SP (p = 0.09). Fecal butyrate was lower for HAY-FA than when horses were adapted to any of the pasture forages (p < 0.03), but there was no difference between HAY-SP and any other forage. Fecal propionate was lowest in horses adapted to HAY-FA (p < 0.002), but propionate concentrations were also lower for HAY-SP than either CSG-SP or CSG-FA (p < 0.03). Propionate did not differ between horses adapted to WSG and all other forages. Fecal valerate was lower for horses adapted to HAY-FA than CSG-SP or CSG-FA (p < 0.03) but did not differ from WSG or HAY-SP. Horses adapted to HAY-SP also had fecal valerate concentrations lower than CSG-SP or CSG-FA (p <= 0.0002). Isobutyrate was greater for all pasture forages in comparison to both HAY-SP and HAY-FA (p < 0.02). Isovalerate was greater in CSG-SP and CSG-FA vs. HAY-SP and HAY-FA (p <= 0.002), while concentrations in horses adapted to WSG were once again intermediate. Isocaproate was detected in all fecal samples, but concentrations were negligible and below the limit of quantification (<1.0 ug g feces-1). 3.5. Relationships between Fecal Microbiota and Metabolites Random forest regressors were applied to determine if fecal pH and metabolite concentrations could be predicted based on microbial composition. Random forest regression did not support a strong influence of bacterial composition of the full microbial community on these fecal variables. Relatively weak predictive accuracy was found for isovalerate, valerate, hexanoate, and heptanoate (model R2 and p-values are shown in Table 7). Major SCFA including acetate, butyrate, and propionate could not be predicted based on BCG abundance profiles. However, when random forest regressors were applied to only the top 25 BCG identified as most predictive of forage type, model accuracies improved for all fecal variables with the exception of valerate (Table 7). While predictive accuracy was still relatively weak, all metabolites (and pH) could be predicted with statistically significant accuracy (p <= 0.001). Individual correlations were found between 19 of these BCG and at least one fecal metabolite or pH , with most BCG correlated with multiple fecal variables. There were positive correlations between BCG_300 and total SCFA and total BCFA as well as with acetate, butyrate, propionate, valerate, isobutyrate, and isovalerate, while this BCG was negatively correlated with hexanoate, heptanoate, and pH. The ASV members of this BCG were assigned to the genera Lachnospiraceae XPB1014 and Streptococcus. There were also positive correlations between both BCG_9 and BCG_259 and total SCFA and BCFA in addition to acetate, butyrate, propionate, valerate, isobutyrate, and isovalerate; these BCG were negatively correlated with heptoanate. These BCG included multiple ASV assigned to Lachnospiraceae as well as ASV within the genera Christensenellaceae R-7 group, Rikenellaceae RC9 gut group, Papillibacter, and the NK4214 group of Oscillospiraceae. Positive correlations were also found between BCG_173 and BCG_201 and total SCFA and BCFA, acetate, propionate, valerate, isobutyrate, and isovalerate, with negative correlations between these BCG and both hexoanate and heptoanate. These BCG included ASV assigned to Treponema, Rikenellaceae RC9 gut group, the NKA214 group of Oscillospiraceae, and the p-251-o5 and F082 genera and families of Bacteroidales. Total SCFA and BCFA as well as acetate, butyrate, propionate, isobutyrate, and isovalerate were positively correlated with BCG_113, while hexanoate was negatively correlated. This BCG contained ASV assigned to taxa including Synergistaceae, Bacteroidales RF16 group, Papillibacter, Christensenellaceae R-7 Group, Mogibacterium, and Clostridium butyricum. Seven BCG were positively correlated with total BCFA and some combination of propionate, valerate, isobutyrate, and isovalerate, while negative correlations were found between these BCG and hexanoate and/or heptanoate. These BCG included ASV members assigned to the family level for Anaerovoracaceae, Oscillospiraceae, and Lachnospiraceae. Other ASV within these BCG were assigned to Lactobacillus equigenerosi and the genera Akkermansia, Fibrobacter, Treponema, Sphaerocheata, Phasolarctobacterium, Christensenellaceae R-7 group, Lachnoclostridium, Cellulosilyticum, Marvinbryantia, Coprostanoligenes group, Lachnospiraceae XPB1014, Lachnospiraceae UCG-009, Erysipelatoclostridium, Clostridium sensu stricto 1, and Bacteroidales BS11 gut group as well as Family XIII AD3011 group of Anaerovoracaceae, the UCG-004 genus within Erysipelatoclostridiaceae, the UCG-002 and UCG-005 genuses of Oscillospiraceae, and the UCG-010 genus and family of Oscillospirales. Fecal pH was positively correlated with five BCG (in addition to BCG_300) (rs >= |0.30|; p <= 0.05), which contained ASV from taxa including Anaerovoracaceae, the Family XIII AD3011 group of Anaerovoracaceae, Akkermansia, Christensenellaceae R-7 group, Fibrobacter, Treponema, Catenisphaera, Alloprevotella, Bacteroidales RF16 group, Marvinbryantia, Coprostanoligenes group, Bacteroidales UCG-001, the NK4A214 group of Oscillospiraceae, and the WCHB1-41 genus, family, and order within Kiritimatiellae. Fecal pH was negatively correlated with BCG_94. This BCG included ASV assigned to Prevotella, Sarcina, and Clostridium sensu stricto 1. 3.6. Relationships between Fecal Microbiota and Forage Nutrients Application of random forest regressors demonstrated that forage nutrient concentrations could be predicted based on bacterial community composition (BCG composition of the full microbial community), including water-soluble carbohydrate (WSC) and NSC at a predictive accuracy > R2 = 0.50 and crude protein (CP) with a predictive accuracy of R2 = 0.41 (p < 0.0001; Table 7). Weaker, but still statistically significant, predictive accuracy was found for digestible energy (DE), acid detergent fiber (ADF), neutral detergent fiber (NDF), ethanol-soluble (ESC), and starch (p < 0.04). Similar to results for fecal metabolites, when random forest regressors were applied only to the top 25 BCG identified as most predictive of forage type, model accuracies improved (Table 7). Forage CP, NSC, and WSC remained as the nutrients most accurately predicted by the bacterial composition of this subset of BCG, all with R2 > 0.60 (p < 0.0001). Model predictive capacity with the reduced feature set was also above R2 = 0.40 for starch; weaker, but statistically significant, predictive accuracy was found all other nutrients (p < 0.0001). Subsequent correlation analysis found 23 BCG (of the 25 in the reduced feature set) were correlated with at least one forage nutrient . Nine BCG were correlated with ADF, NDF, CP, and DE. In all cases, opposite correlations were seen between ADF/NDF and CP/DE (i.e., if ADF and NDF were positively correlated with a BCG, CP and DE were negatively correlated). There were distinct ASV from the taxa Christensenellaceae R-7 group and NK4A214 group of Oscillospiraceae that were members of separate BCG which were positively and negatively responding to these nutrients (i.e., these taxonomic classifications were found across all positive and negative responder groups). The BCG negatively correlated with ADF/NDF, and thus positively correlated with CP/DE included ASV members assigned to Clostridium butyricum and genera Papillibacter, Lachnoclostridium, Cellulosilyticum, Fibrobacter, Treponema, Catenisphaera, Bacteroidales RF16 group, Synergistaceae, Mogibacterium, Rikenellaceae RC9 gut group, the Hallii group of Lachnospiraceae, the p-251-o5 genus and family of Oscillospiraceae, and the F082 genus and family of Bacteroidales, as well as multiple ASV assigned only to the family level for Lachnospiraceae. The BCG positively correlated with ADF/NDF and negatively correlated with CP/DE included ASV within Sphaerochaeta, Phascolarctobacterium, Bacteroidales UCG-001, Rikenellaceae RC9 gut group, Coprostanoligenes group, the UCG-002 genus within Oscillospiraceae, and the UCG-010 genus and family within Oscillospirales. Four additional BCG were correlated with some combination of ADF/NDF and CP/DE. The BCG negatively correlated with ADF/NDF, and thus positively correlated with CP/ DE contained ASV members assigned to additional taxa including Ruminococcus, Anaerovibrio, Pseudobutyvibrio, Lachnospiraceae UCG-009, probable genus 10 of Lachnospiraceae, and the WCHB1-41 genus, family and order of Kiritimatiellae. The BCG positively correlated with ADF/NDF, and negatively correlated with CP/DE included ASV within Bacteroidales BS11 gut group and Clostridium sensu stricto 1. Forage ADF and NDF were also negatively correlated with BCG_300 (Streptococcus and Lachnospiraceae XPB1014), but this BCG was not correlated with either CP or DE. Forage CP was also positively correlated with BCG_35, for which there was no relationship with ADF, NDF, or DE, but for which there was also a negative correlation with NSC and WSC. The ASV members of BCG_35 were assigned to taxa including Akkermansia, Fibrobacter, Treponema, Christensenellaceae R-7 group, and Family XIII AD3011 within Anaerovoracaceae. Forage NSC and WSC were correlated with 13 and 12 BCG, respectively (rs >= |0.30|; p <= 0.05). In addition to the above-mentioned taxa from ASV within BCG_35, ASV within BCG negatively correlated with both NSC and WSC were assigned to Lactobacillus equigenerosi and genera including Lachnoclostridium, Cellulosilyticum, Catenisphaera, Marvinbryantia, Erysipelatoclostridium, Mogibacterium, Lachnospiraceae XPB1014 group, Lachnospiraceae UCG-009, Coprostanoligenes group, UCG-005 metagenome within Oscillospiraceae, the Hallii group within Lachnospiraceae, the NK4A214 group of Oscillospiraceae, the Incertae Sedis genus of Ethanoligenenaceae, the WCHB1-41 genus, family, and order within Kiritimatiellae, as well as Denitrobacterium detoxificans. Additional ASV were assigned only to the family level for Synergistaceae, Lachnospiraceae, Oscillospiraceae, and Anaerovoracaceae. Forage NSC and WSC were positively correlated with BCG_94 (Clostridium sensu stricto 1, Prevotella, and Sarcina). Forage ESC was correlated with 9 of the same BCG as NSC and WSC, but also had a negative correlation with BCG_173, which contained multiple ASV assigned to Lachnospiraceae, probable genus 10 within Lachnospiraceae, Pseudobutyvibrio as well as to the p-251 genus and family of Bacteroidales. Starch was positively correlated with BCG_113 and BCG_300 and negatively correlated with two BCG, BCG_43 and BCG_48, containing ASV from Sphaerochaeta, Phascolarctobacterium, Christensenellaceae R-7 group, Coprostanoligenes group, Bacteroidales UCG-001, Rikenellaceae RC9 gut group, the NK4A214 group of Oscillospiraceae, the UCG-002 genus within Oscillospiraceae, and the UCG-010 genus and family within Oscillospirales; these BCG were also among those positively correlated with ADF/NDF and negatively correlated with CP/DE. Relationships between fecal variables and forage nutrients were also evaluated through correlation analysis . Acetate, butyrate, propionate, valerate, isobutyrate, isovalerate and total SCFA and BCFA were positively correlated with DE (Spearman correlation; rs >= 0.62) and CP (rs >= 0.41), but were negatively correlated with NDF (rs <= -0.48) and ADF (rs <= -0.57; p <= 0.008). There was also a weaker positive correlation between these fecal metabolites and starch (rs >= 0.39; p <= 0.01). Total BCFA, isobutyrate, and isovalerate were negatively correlated with NSC, WSC, and ESC (rs <= -0.37; p <= 0.02), with a weaker negative correlation between valerate and NSC and WSC (rs <= -0.34; p <= 0.03). Hexanoate and heptanoate were negatively correlated with DE, CP, and starch (rs <= -0.39; p <= 0.01), but were positively correlated with NDF, ADF, NSC, WSC, and ESC (rs >= 0.37; p <= 0.02). Conversely, fecal pH was positively correlated with ADF (rs = 0.47; p = 0.002), but negatively correlated with DE, NSC, WSC, ESC, and starch (rs <= -0.32; p <= 0.04). 3.7. Blood Samples and Glycemic/Insulinemic Responses Plasma glucose responses to OST administration differed by forage (mixed model ANOVA with Tukey's post hoc adjustment; p <= 0.01). The AUC for glucose was lowest when horses were adapted to WSG (42.4 +- 6.8 mg/dL*h) vs. CSG-SP (70.0 +- 6.8 mg/dL*h) and HAY-FA . Peak plasma glucose was also lower for WSG (110 +- 3 mg/dL) in comparison to HAY-FA (123 +- 3 mg/dL; p = 0.01), and there was a trend for lower peak plasma glucose for WSG than for CSG-SP . Forage did not, however, impact OST insulin responses. Neither AUC (CSG-SP: 36.1; WSG: 29.1; HAY-FA: 32.7 +- 6.4 mIU/L*h) or peak plasma insulin (CSG-SP: 28.8; WSG: 25.5; HAY-FA: 26.6 +- 3.3 mIU/L) varied by forage . Fasting plasma insulin (CSG-SP: 3.69; WSG: 5.04; HAY-FA: 5.20 +- 0.64 mIU/L) and glucose (CSG-SP: 81.4; WSG: 78.7; HAY-FA: 81.3 +- 1.4 mg/dL) also did not differ by forage . There were trends for differences by forage in proxies for insulin sensitivity including the fasting glucose-to-insulin ratio (FGIR) and reciprocal of the square root of insulin . Differences in FGIR and RISQI were limited to WSG (FGIR: 25.2 +- 16.4; RISQI: 0.58 +- 0.04) vs. HAY (FGIR: 16.4 +- 2.1; RISQI: 0.46 +- 0.04; p = 0.08). However, the modified insulin-to-glucose ratio (MIRG), a proxy for the insulin secretory response, did not vary by forage . 3.8. Relationships between Glucose Metabolism and the Fecal Microbiota Glucose and insulin dynamics were only assessed after three of the forages (CSG-SP, WSG, and HAY-FA), and this smaller dataset precluded the use of random forest regression modelling for these variables. As the primary metabolic differences in grazing horse metabolism were AUC and peak plasma glucose in response to OST administration, correlation analysis was conducted to explore relationships between these metabolic variables and BCG. Only one BCG (of the 25 BCG in the reduced feature set) was correlated with AUC (rs = -0.48; p = 0.04), with members of BCG_124 including ASV within Papillibacter, Christensenellaceae R-7 group, and Synergistaceae. Four additional BCG were correlated with peak plasma glucose. Positive correlations were found between peak glucose and BCG_51 and BCG_305 (rs >= 0.43; p <= 0.04). These BCG included ASV mapped to genera including Lachnospiraceae XPB1014, Lachnospiraceae UCG-009, Erysipelatoclostridium, Catenisphaera, and Fibrobacter as well to the family level for Anaerovoracaceae. Conversely, BCG_113 (Clostridium butyricum, Papillibacter, Bacteroidales RF16 group) and BCG_259 were negatively correlated with peak glucose (rs = -0.44; p = 0.03). Members of BCG_259 included ASV assigned at the family level to Lachnospiraceae in addition to the Hallii group within Lachnospiraceae. While relationships between AUC and peak plasma glucose and forage nutrients were identified through correlation analysis , these metabolic variables were not correlated with any fecal variables including SCFA, BCFA, and pH. Forage NSC, WSC, and ESC were positively correlated with AUC (rs >= 0.53; p <= 0.007) and peak glucose (rs >= 0.41; p <= 0.04). There was also a negative correlation between CP and glucose responses to OST administration (rs <= -0.50; p <= 0.01). Forage DE, NDF, ADF, and starch, however, were not correlated with AUC or peak glucose. 4. Discussion 4.1. Forage Type and the Microbiome Warm-season grasses can be utilized to bridge the "summer slump" forage gap in cool-season grass grazing systems , and differences in NSC between these forage types could have implications for equine metabolic health . However, there is limited information on the impacts of grazing warm-season grasses on the equine hindgut microbiome as well as potential associations between forage nutrients, the hindgut microbiome, and equine metabolic responses. Therefore, this study aimed of to characterize the fecal microbiota of horses adapted to different forage types and to explore relationships between forage nutrients, microbial composition, fecal metabolites, and glycemic responses of grazing horses. Results of this study clearly demonstrated that shifts in fecal microbiome structure and species composition occur as horses are adapted to different forages within an integrated cool-season grass rotational grazing system. This was supported by statistical differences in both b-diversity. Furthermore, random forest classification modelling was able to predict forage type based on microbial community composition, indicating the influence of forage type on the fecal microbiome. Finally, forage-specific BCG were identified through LEfSe analysis, but the 25 BCG identified as most predictive of forage type through random forest modeling and subsequently analyzed by LEfSe represented only ~10% of the total fecal microbiota across all forages. This indicates that distinct and identifiable shifts in microbial composition do occur as horses adapt to different forage types. However, the majority of the microbiome (~90% in the current study) is resistant and/or resilient to potential perturbations induced by transitioning among forage types with different physical and chemical properties, including between cool-season and warm-season grass pastures. The BCG identified as markers specific to HAY-SP included multiple ASV assigned to taxa to which fibrolytic and butyrate-producing functions have been previously ascribed . The BCG specific to this forage contained a heavy representation of ASV within the Lachnospiraceae family as well as Fibrobacter, Ruminococcus, and Pseudobutyvibrio. Prior studies have also reported increased prevalence of Lachnospiraceae in horses fed hay vs. pasture . Conversely, ASV assigned to Anaerovibrio were also identified in BCG markers of HAY-SP. Increases in this genus in response to abrupt inclusion of dietary starch have been documented as well as in temporal proximity to oral administration of oligofructose in experimental laminitis induction models . The co-occurrence of bacteria in these taxonomic groups could potentially be reflective of the nutrient composition of HAY-SP, which was the highest in both fiber and NSC of all forages. Co-occurrence of these bacteria may also reflect cross-feeding relationships between bacteria and/or metabolic plasticity of bacterial populations. These factors may also account for unexpected associations revealed by analysis in the current study, such as ASV assigned to Streptococcus within BCG markers of HAY-FA, despite the relatively low NSC content of this forage. However, Zhu et al. also reported a lower abundance of species within Streptococcaceae in horses maintained on pasture vs. horses fed hay or silage . Overall, the fecal microbiota of horses adapted to cool-season pasture was characterized by bacteria capable of utilizing rapidly fermentable fibers, which could explain the greater fecal SCFA concentrations for CSG-SP and CSG-FA. Capacity for hemicellulose fermentation and SCFA production have been previously documented in the Bacteroidales BS11 gut group , which was identified only in BCG markers of CSG-SP. Equine studies have found increased relative abundance of Bacteroidales BS11 gut group in horses fed barley vs. a hay diet and in horses presenting with colic . For CSG-FA, BCG markers included genera such as Alloprevotella and Erysipelatoclostridium, which are also associated with degradation of fermentable fibers . The BCG markers of CSG-FA also included ASV assigned WCHB1-41 clade within Kiritimatiellae. Prior studies have found enrichment of the phylum Kiritimatiellaeota in the fecal microbiota of horses with equine metabolic syndrome and insulin dysregulation . In contrast to results of the present study, Fitzgerald et al. reported increased abundance of this phylum in response to a change from pasture to a hay diet in both healthy and insulin dysregulated ponies, and Ericsson et al. similarly found increased abundance of the class Kiritimatiellae when horses with equine metabolic syndrome were transitioned from pasture to a hay diet. These conflicting results reinforce that nutritional composition of specific forages, rather than form of forage alone, need to be considered when evaluating interstudy effects of diet on the equine microbiome. A number of interesting relationships were found between BCG identified as markers specific to WSG and fecal variables, forage nutrients, and horse glucose metabolism. Warm-season grasses are characteristically lower in soluble carbohydrates than cool-season grasses , and of all forages evaluated in the current study, NSC and WSC were lowest in WSG. Four of the five BCG markers of WSG were negatively correlated with forage NSC and WSC, including BCG_35, which contained ASV assigned Akkermansia as well as Fibrobacter, Treponema, Christensenellaceae R-7 group, and the Family XIII AD3011 group of Anaerovoracaceae. Akkermansia has been linked to metabolic health via regulation of inflammatory responses and has also been explored for probiotic use in animal species . Lindenberg et al. recently demonstrated immune (and inflammatory) modulation by specific hindgut microbiota in horses, including Akkermansia spp. Furthermore, supplementation with mannanoligosaccharides and fructooligosaccharides led to increased abundance of Akkermansia muciniphilia in a subsequent study , and improvements in insulin sensitivity have been previously documented in horses supplemented with low doses of fructooligosaccharide . Markers of WSG also included BCG_113, which contained an ASV assigned to Clostridium butyricum. Clostridium butyricum is a prolific butyrate producer that has also been investigated for probiotic use due to its capacity to promote anti-inflammatory responses and improve gut barrier function and metabolic health . BCG_35 was positively correlated with CP as well as isobutyrate and total BCFA, which reflects the proteolytic capacity of this BCG. BCG_113 was also positively correlated with total SCFA and all three major SCFA (acetate, butyrate, and propionate) in addition to isobutyrate, isovalerate, and total BCFA. This BCG was also negatively correlated with peak plasma glucose. These findings suggest that this bacterial group including Clostridium butyricum could play a role in modulation of equine metabolic health. While limited connections between the gut microbiota and metabolic responses were observed in the current study, three of the five BCG correlated with AUC and peak plasma glucose were WSG-specific BCG markers. Further research is necessary to determine if other WSG species or varieties would produce similar effects as those observed in the current study. While Akkermansia and Clostridium butyricum have been investigated in mice and other species, comparatively little research has been conducted to understand the function of these bacteria in the equine hindgut. The relationships found between ASV in these taxa and forage nutrients, fecal metabolites, and equine glycemic responses in addition to associations with low-NSC warm-season grasses in the current study support further research to determine the role of these bacteria, factors including dietary interventions that can promote prevalence in the hindgut, and potential probiotic applications. 4.2. Forage Nutrients and the Microbiome Results of this study also confirmed the strong influence of dietary nutrients on the equine microbiome and provided insights into the complex relationships between forage nutrients and the gut microbiota. The concentrations of several nutrients could be predicted based on microbial community composition. Furthermore, regression model accuracy improved when the reduced feature set identified through prior forage classification modeling was utilized for prediction of nutrient concentrations. This indicates that differences in forage nutrients were driving the shifts in equine fecal microbial communities as horses adapted to various forages within the integrated grazing system. Results of this study revealed the influence of soluble carbohydrates including NSC and WSC on fecal microbial community composition. In contrast, fiber was not identified as a key nutrient shaping differences in microbial communities. Forage NSC and WSC concentrations could be predicted based on microbial community composition with an accuracy of R2 = 0.61 and R2 = 0.67, respectively, while predictive accuracy for ADF and NDF was < 0.40 (R2). Random forest modeling also revealed that in addition to soluble carbohydrates, the gut microbiota were also influenced by CP (R2 = 0.62). The impact of CP on the gut microbial community was interesting, but unsurprising, as two-thirds to three-fourths of total tract nitrogen digestion and absorption occurs in the equine hindgut , and many microbial species are capable of proteolytic fermentation . Prior studies in horses have found distinct effects of high-fiber vs. lower-fiber diets on the hindgut microbial community, with benefits of increased fiber including greater microbial diversity , reduction of lactic-acid producing bacteria , and less-acidic hindgut pH . However, these previous studies have been primarily conducted in horses fed concentrate vs. forage-based diets in which there was broad variation in fiber concentrations between treatments. In the current study, horses were maintained on a forage-only diet throughout, and there were relatively high concentrations of NDF and ADF found across all forages. It is possible that only minimal shifts occur in bacterial populations most responsive to fiber above a certain concentration threshold, and that these populations remained relatively stable throughout the study (and thus fiber concentration was not able to be predicted with strong accuracy by random forest modeling). However, it should be noted that the concentrations of NSC, WSC, and CP in forages is low in comparison to that of fiber. Additionally, in contrast to fiber, these nutrients are all subject to digestion in the equine foregut, and only a fraction of the total soluble carbohydrates and CP ingested would be available for bacterial fermentation in the hindgut. Thus, only a small amount of these nutrients were capable of exerting influence on gut microbial communities. Digestibility was not evaluated in the current study, however, and a more in-depth analysis of total digestibility and digestibility of specific nutrients would be necessary to fully understand the interactions between forage nutrients and the gut microbiota. 4.3. Fecal pH and Fermentation Metabolites Fecal pH is commonly utilized as a marker of microbial activity in the equine hindgut . Differences in fecal pH in the current study indicated that functional changes occur in the equine gut microbiota, in addition to shifts in microbiome structure and composition, as horses adapt to different forages. Fecal pH was highest in horses adapted to WSG and HAY-FA, which were lower in NSC than CSG-SP, CSG-FA. Accordingly, fecal pH was negatively correlated with forage NSC. Lower fecal pH is broadly associated with hindgut dysfunction in horses , and thus the higher fecal pH in horses adapted to WSG in comparison to cool-season pasture could suggest some benefit to grazing horses on warm-season grass pastures. However, mean pH was above 6.5 for CSG-SP, CSG-FA, and HAY-SP, which is within ranges previously documented in healthy forage-fed horses . Therefore, while statistically significant, the difference in fecal pH between horses adapted to WSG vs. cool-season pasture may not be of physiological relevance. Differences in fermentation metabolites across forages did not mirror results for fecal pH, as differences between forages for SCFA and BCFA were primarily between pasture forages and the hay diets. Numerically, SCFA and BCFA concentrations in horses adapted to WSG were intermediate between the cool-season pasture (greatest) and hay (lowest), but these differences were not statistically significant. Furthermore, while statistically significant, the predictive accuracy of random forest regressors for fecal metabolites and pH were lower in comparison to those found for forage nutrients. Numerous studies have noted that while the gastrointestinal tract harbors a phylogenetically diverse community, many unrelated microorganisms are capable of performing similar functions . The relatively poor predictive accuracy of regressors for fecal metabolites in the current study is likely due to this functional redundancy within the equine microbial community. Functional redundancy has been previously suggested as a contributing factor in stability of the gut microbial communities including within the equine microbiome during transitions between cool-season grasses . 4.4. Glucose and Insulin Metabolism While there were distinct shifts in the fecal microbiota across forages in the integrated system, forage type exerted minimal effects on glucose metabolism. A large body of research, primarily conducted in mouse models, has established that diet (and dietary intervention) modulates metabolic health in a microbiome-dependent manner . However, shifts in gut microbial structure and function may precede changes in metabolic outcomes . The adaptation period utilized in the present study was of a similar duration as used in prior studies evaluating the microbiome of forage-fed horses , and is considered sufficient for stabilization of microbial communities . However, longer treatment periods are often required to assess metabolic adaptations to diet . The lower AUC and peak plasma glucose in horses adapted to WSG in the current study does suggest that glucose may be cleared more rapidly in horses adapted to this forage, but a longer adaptation period would be necessary to confirm these results. Regardless, glucose and insulin responses to the OST for all forages evaluated in the current study were within normal and previously reported ranges , indicating that substantial metabolic changes are unlikely within the context of integrated rotational grazing management even with the observed changes in the fecal microbiota, fecal metabolites, and pH. Additionally, the lack of correlation between any fermentation metabolites and glucose responses, as well as the relatively small number of BCG correlated with AUC and peak plasma glucose, suggest that any difference in glycemic responses of horses in the current study may not have been heavily dependent on shifts in the hindgut microbiome. 4.5. Additional Considerations Seasonal and environmental factors should be considered when interpreting results of this study. Prior studies have reported seasonal changes in microbial diversity and species composition but also lacked a true seasonal control, and thus, the effect of seasonality on the equine hindgut microbiome requires further investigation. Seasonal variance in insulin sensitivity has been documented in grazing horses , but when fed controlled diets without nutrient fluctuations seen in pasture forages, minimal seasonal differences in circulating glucose and insulin and insulin sensitivity have been found in healthy horses . Environment and management factors beyond season can also impact pasture forage nutrient composition. Characterizing the microbiome of a larger number of grazing horses over multiple years and in multiple locations/regions would allow a more robust evaluation of the impacts of warm-season grasses on the equine hindgut microbiome. Finally, it should be noted that the analytical approach in this study differs from more conventional taxon-based analysis. Rather, this study implemented a guild-based approach, grouping individual microbial ASV by co-abundance to subsequent analysis of abundance profiles . This strategy has been previously utilized as an alternative to grouping bacteria by taxonomy . Substantial genetic variation is possible within taxa, even at the species level, and therefore bacteria with similar taxonomic assignments may not represent a functionally homologous group . Results of the current study also illustrate this concept, as ASV with the same assigned taxonomy were found in BCG characteristic of different forages as well as in separate BCG that positively and negatively responded to forage nutrients and with both positive and negative relationships with fecal metabolites and grazing horse metabolism. 5. Conclusions In conclusion, distinct shifts in equine fecal microbial community structure and composition occur as horses adapt to different forages within an integrated cool-season grass rotational pasture system, but a substantial impact of this management practice on glucose metabolism in healthy adult grazing horses is unlikely. Forage NSC, WSC, and CP were the most influential nutrients driving these shifts in microbial composition. Results of this study underscore the potential for relatively small amounts of NSC to influence hindgut microbial composition and also that protein utilization may be an important ecological niche within the microbiome of forage-fed horses. Fecal BCFA and SCFA concentrations were higher in horses adapted to all pasture forages versus hay, but in comparison to forage nutrients, bacterial community composition did not have as strong an impact on fermentation metabolites, likely reflecting functional redundancy of the microbial community. The guild-based analytical approach utilized in this study also identified key relationships between specific bacterial groups associated with adaptation to warm-season grass pasture and forage nutrients, fecal metabolites, and equine glycemic responses to administration of oral sugar tests. Relationships identified in this study revealed new insights and targets for future research necessary to better understand the function of Akkermansia spp. and Clostridium butyricum in the hindgut microbiome of grazing horses, as these bacteria may play a role in modulation of equine metabolic health. Acknowledgments We thank DLF Pickseed (Halsey, OR) and Dalrymple Farms (Thomas, OK) for donations of grass seed used to establish the pastures grazed in this study. We also thank the Rutgers Equine Science Center for providing office space and computers utilized for data analyses. SCFA analyses were carried out at the Rutgers New Jersey Institute for Food, Nutrition, and Health Lipidomics Core. We thank Deeptha Kumaraswamy for technical assistance with SCFA analyses. Supplementary Materials The following are available online at Figure S1: Diagram of experimental design and sampling protocol, Figure S2: Insulinemic responses of horses to administration of oral sugar tests, Figure S3: Fasting plasma glucose and insulin prior to administration of oral sugar tests, Figure S4: Proxies for insulin sensitivity and the insulin secretory response in horses administered oral sugar tests, Table S1: Monthly average temperatures and precipitation totals and historical averages, Table S2: Horse condition measures following adaptation to hay diets and pasture forages, Table S3: Taxonomic classification of amplicon sequence variants (ASV) after quality filtering, chimera checking, and filtering low-abundance ASV. Click here for additional data file. Author Contributions Conceptualization, J.R.W.-N. and C.A.W.; methodology, J.R.W.-N., A.S.B., H.S. and C.A.W.; formal analysis, J.R.W.-N. and A.S.B.; investigation, J.R.W.-N. and C.A.W.; resources, C.A.W.; data curation, J.R.W.-N.; writing--original draft; J.R.W.-N. and H.S.; writing--review and editing, J.R.W.-N., A.S.B., H.S. and C.A.W.; visualization, J.R.W.-N.; supervision, C.A.W.; project administration, J.R.W.-N. and C.A.W.; funding acquisition--J.R.W.-N. and C.A.W. All authors have read and agreed to the published version of the manuscript. Institutional Review Board Statement The study was approved by the Institutional Animal Care and Use Committee of RUTGERS, THE STATE UNIVERSITY OF NEW JERSEY (protocol # PROTO201800013). Data Availability Statement The datasets generated and analyzed during the current study are available in the NCBI Sequence Read Archive at (accessed on 31 July 2022), Bioproject: PRJNA864051, Accession numbers: SAMN30072246-SAMN30072285. Conflicts of Interest The authors declare no conflict of interest. Figure 1 Fecal microbiota a-diversity across forages within an integrated rotational grazing system including: HAY-SP: initial standardized cool-season grass hay diet in the spring; CSG-SP: cool-season grass pasture in the spring; WSG: warm-season grass, either crabgrass or bermudagrass, in the summer slump period; CSG-FA: cool-season pasture in the fall; HAY-FA: final standardized cool-season grass hay diet in the fall. Shannon Diversity Index (a), Faith's Phylogenetic Diversity (b), Pielou's Evenness (c) and Observed ASV (d) were analyzed by Kruskal-Wallis tests with Benjamini and Hochberg FDR adjustments for multiple pairwise comparisons. A "" indicates a statistical outlier in the data. A single asterisk indicates differences between forages at p <= 0.05. A double asterisk indicates a trend for differences between forages at p <= 0.10. Figure 2 Fecal microbiota b-diversity across forages within an integrated rotational grazing system including: HAY-SP: initial standardized cool-season grass hay diet in the spring; CSG-SP: cool-season grass pasture in the spring; WSG: warm-season grass, either crabgrass or bermudagrass, in the summer slump period; CSG-FA: cool-season pasture in the fall; HAY-FA: final standardized cool-season grass hay diet in the fall. Weighted UniFrac (a) and Unweighted UniFrac (b) differed by forage based on analysis by Permutational ANOVA (PERMANOVA) (p <= 0.05). Figure 3 Fecal pH across forages within an integrated rotational grazing system including: HAY-SP: initial standardized cool-season grass hay diet in the spring; CSG-SP: cool-season grass pasture in the spring; WSG: warm-season grass, either crabgrass or bermudagrass, in the summer slump period; CSG-FA: cool-season pasture in the fall; HAY-FA: final standardized cool-season grass hay diet in the fall. Data are presented as least squares means +- SEM. A single asterisk indicates differences between forages at p <= 0.05 (mixed model ANOVA with Tukey's post hoc adjustment). Figure 4 Network map of forage nutrients and bacterial co-abundance groups (BCG). Nutrients included digestible energy (DE), crude protein (CP) acid detergent fiber (ADF), neutral detergent fiber (NDF), non-structural carbohydrate (NSC), water-soluble carbohydrate (WSC) and ethanol-soluble carbohydrate (ESC). Correlations were evaluated between nutrient concentrations and relative abundance of BCG most predictive of forage type (based on random forest modeling of BCG composition). In addition to node color, node size was also mapped to BCG relative abundance. Line width is mapped to correlation strength, with wider lines indicating stronger correlations between nutrients and BCG. Significant Spearman correlations (rs >= |0.30|; p <= 0.05) were included in the network map. Figure 5 Network map of bacterial co-abundance groups (BCG) and fermentation metabolites as well as fecal pH. Correlations were evaluated between BCA most predictive of forage type (based on random forest modeling of BCG composition) and fecal pH, short-chain fatty acids (SCFA) and branched-chain fatty acids (BCFA). In addition to node color, node size was also mapped to BCG relative abundance. Line width is mapped to correlation strength, with wider lines indicating stronger correlations between nutrients and BCG. Significant Spearman correlations (rs >= |0.30|; p <= 0.05) were included in the network map. Figure 6 Correlation of forage nutrients and fermentation metabolites. Nutrients included digestible energy (DE), crude protein (CP) acid detergent fiber (ADF), neutral detergent fiber (NDF), non-structural carbohydrate (NSC), water-soluble carbohydrate (WSC) and ethanol-soluble carbohydrate (ESC). Significance of Spearman correlations was set at rs >= |0.30| (p <= 0.05). Cells with an "X" indicate non-significant correlations. Color and size of circles within cells are mapped to direction and strength of correlation. Larger, darker blue circles indicate a stronger positive correlation (closer to rs = 1.0); larger, lighter yellow circles indicate a stronger negative correlation (closer to rs = -1.0). Figure 7 Plasma glucose responses to oral sugar tests and relationship with forage nutrients and fermentation metabolites. Oral sugar tests were administered to horses adapted to spring cool-season grass pasture (CSG-SP) and warm-season grass in the summer slump period (WSG) in addition to a standardized hay diet in the fall at the end of the grazing season (HAY-FA). Glycemic responses including the positive incremental area under the curve (a) and peak plasma glucose (b) are presented as least squares means +- SEM. A single asterisk indicates differences between forages at p <= 0.05 (mixed model ANOVA with Tukey's post hoc adjustment). A double asterisk indicates a trend for differences at p <= 0.10. (c) Correlated nutrients included digestible energy (DE), crude protein (CP) acid detergent fiber (ADF), neutral detergent fiber (NDF), non-structural carbohydrate (NSC), water-soluble carbohydrate (WSC) and ethanol-soluble carbohydrate (ESC). Fecal metabolites included branched-chain fatty acids. Significance of Spearman correlations was set at rs >= |0.30| (p <= 0.05). An "X" indicates non-significant correlations. Color and size of circles within cells are mapped to direction and strength of correlation. Larger, darker blue circles indicate a stronger positive correlation (closer to rs = 1.0); larger, lighter yellow circles indicate a stronger negative correlation (closer to rs = -1.0). animals-13-00790-t001_Table 1 Table 1 Nutrient composition 1 of hay diets and pasture forages. Nutrients 2,3 Forage 4 HAY-SP CSG-SP WSG CSG-FA HAY-FA BRS CRS BRS CRS BRS CRS DE, Mcal/kg 2.02 2.18 2.06 2.15 2.03 2.34 2.22 2.00 CP, % 5.6 13.1 16.5 21.4 22.7 23.7 21.5 10.2 ADF, % 40.9 35.3 36.4 31.4 36.9 30.5 30.0 42.2 NDF, % 66.1 59.4 62.1 58.7 61.8 50.5 55.4 65.8 NSC, % 18.4 12.4 8.5 7.1 2.6 11.3 11.5 9.1 WSC, % 18.0 10.3 7.4 4.8 2.3 10.2 10.2 8.1 ESC, % 7.0 6.6 3.7 4.0 1.9 8.3 6.9 6.9 Starch, % 0.40 2.07 1.17 2.30 1.37 1.13 1.23 1.00 1 Determined by near-infrared spectroscopy (Equi-Analytical Laboratories, Ithaca, NY, USA). 2 Nutrient concentrations are reported on a dry-matter basis as means of three representative samples collected per forage. 3 Abbreviations: digestible energy (DE), crude protein (CP), neutral detergent fiber (NDF), acid detergent fiber (ADF), non-structural carbohydrates (NSC), water-soluble carbohydrates (WSC), ethanol-soluble carbohydrates (ESC). 4 HAY-SP: initial standardized cool-season grass hay diet in the spring; CSG-SP: cool-season grass pasture in the spring; WSG: warm-season grass, either crabgrass or bermudagrass, in the summer slump period; CSG-FA: cool-season pasture in the fall; HAY-FA: final standardized cool-season grass hay diet in the fall. animals-13-00790-t002_Table 2 Table 2 16S rRNA gene sequence read counts before and after filtering 1. Step Statistic Count Initial Total 881,189 Quality and Chimera Filtering 2 Total 730,065 Minimum sample-1 12,460 Maximum sample-1 30,170 Mean sample-1 18,252 Median sample-1 18,890 Removal of Low Abundance Features Total 648,797 Minimum sample-1 10,989 Maximum sample-1 26,948 Mean sample-1 16,220 Median sample-1 15,587 1 Filtering was conducted in Qiime 2 (v.2020.8) . 2 Filtering was conducted using DADA2 . animals-13-00790-t003_Table 3 Table 3 Bacterial co-abundance groups (BCG) 1 identified as markers 2 of standardized hay diets. Forage 2 BCG 3 LDA 4 ASV Taxonomic Lineage 5 HAY-SP BCG_9 4.55 Firmicutes | Clostridia | Oscillospirales | Oscillospiraceae | Papillibacter | uncultured_Clostridiales Bacteroidota | Bacteroidia | Bacteroidales | Rikenellaceae | Rikenellaceae_RC9_gut_group | uncultured_bacterium Firmicutes | Clostridia | Christensenellales | Christensenellaceae | Christensenellaceae_R-7_group | uncultured_Clostridia Firmicutes | Clostridia | Lachnospirales | Lachnospiraceae (2 ASV) Firmicutes | Clostridia | Oscillospirales | Oscillospiraceae | NK4A214_group | uncultured_rumen BCG_18 5.03 Firmicutes | Clostridia | Oscillospirales | Ruminococcaceae | Ruminococcus | Ruminococcus_sp. Firmicutes | Clostridia | Lachnospirales | Lachnospiraceae (3 ASV) Firmicutes | Clostridia | Lachnospirales | Lachnospiraceae | Lachnospiraceae_UCG-009 | uncultured_bacterium Firmicutes | Clostridia | Christensenellales | Christensenellaceae | Christensenellaceae_R-7_group | uncultured_prokaryote BCG_93 4.55 Bacteroidota | Bacteroidia | Bacteroidales | p-251-o5 | p-251-o5 | uncultured_bacterium Firmicutes | Clostridia | Lachnospirales | Lachnospiraceae Firmicutes | Clostridia | Lachnospirales | Lachnospiraceae | probable_genus_10 | uncultured_bacterium Firmicutes | Clostridia | Lachnospirales | Lachnospiraceae | Pseudobutyrivibrio BCG_116 4.52 Bacteroidota | Bacteroidia | Bacteroidales | p-251-o5 | p-251-o5 | uncultured_bacterium Firmicutes | Negativicutes | Veillonellales-Selenomonadales | Selenomonadaceae | Anaerovibrio | uncultured_bacterium Firmicutes | Clostridia | Oscillospirales | Ruminococcaceae | Ruminococcus BCG_201 4.28 Firmicutes | Clostridia | Oscillospirales | Oscillospiraceae | NK4A214_group Bacteroidota | Bacteroidia | Bacteroidales | p-251-o5 | p-251-o5 | uncultured_bacterium BCG_305 4.27 Firmicutes | Bacilli | Erysipelotrichales | Erysipelotrichaceae | Catenisphaera | uncultured_bacterium Fibrobacterota | Fibrobacteria | Fibrobacterales | Fibrobacteraceae | Fibrobacter | uncultured_bacterium HAY-FA BCG_22 4.67 Firmicutes | Clostridia | Lachnospirales | Lachnospiraceae Firmicutes | Bacilli | Lactobacillales | Lactobacillaceae | Lactobacillus | Lactobacillus_equigenerosi Firmicutes | Clostridia | Oscillospirales | [Eubacterium]_coprostanoligenes_group | [Eubacterium]_coprostanoligenes_group Firmicutes | Clostridia | Oscillospirales | Oscillospiraceae | uncultured BCG_118 4.56 Firmicutes | Bacilli | Erysipelotrichales | Erysipelatoclostridiaceae | UCG-004 | uncultured_rumen Firmicutes | Clostridia | Lachnospirales | Lachnospiraceae | Marvinbryantia Firmicutes | Clostridia | Oscillospirales | Oscillospiraceae | UCG-005 | uncultured_bacterium BCG_173 4.20 Spirochaetota | Spirochaetia | Spirochaetales | Spirochaetaceae | Treponema | uncultured_bacterium Bacteroidota | Bacteroidia | Bacteroidales | F082 | F082 | uncultured_bacterium Bacteroidota | Bacteroidia | Bacteroidales | Rikenellaceae | Rikenellaceae_RC9_gut_group | uncultured_bacterium BCG_300 4.87 Firmicutes | Clostridia | Lachnospirales | Lachnospiraceae | Lachnospiraceae_XPB1014_group | uncultured_bacterium Firmicutes | Bacilli | Lactobacillales | Streptococcaceae | Streptococcus BCG_309 4.25 Firmicutes | Clostridia | Oscillospirales | Ethanoligenenaceae | Incertae_Sedis | uncultured_bacterium Firmicutes | Clostridia | Christensenellales | Christensenellaceae | Christensenellaceae_R-7_group | uncultured_rumen 1 The BCG markers were identified by linear discriminant analysis effect size (LEfSe) of BCG remaining in the reduced and optimized random forest classification model for prediction of forage type. Features were retained in the model based on feature importance scores . 2 HAY-SP: initial standardized cool-season grass hay diet in the spring; HAY-FA: final standardized cool-season grass hay diet in the fall. 3 Amplicon sequence variants (ASV) were grouped into BCG using Sparse Cooccurrence Network Investigation for Compositional Data in Qiime 2 (v.2020.8) . 4 For LEfSe, significance was set at LDA >2.0; p <= 0.05. 5 Taxonomic assignment of ASV was conducted using the most recent SILVA database (SSU 138). animals-13-00790-t004_Table 4 Table 4 Bacterial co-abundance groups (BCG) identified as markers 1 of cool-season grass pasture. Forage 2 BCG 3 LDA 4 ASV Taxonomic Lineage 5 CSG-SP BCG_43 4.98 Spirochaetota | Spirochaetia | Spirochaetales | Spirochaetaceae | Sphaerochaeta | uncultured_rumen Firmicutes | Negativicutes | Acidaminococcales | Acidaminococcaceae | Phascolarctobacterium Firmicutes | Clostridia | Oscillospirales | Oscillospiraceae | UCG-002 | uncultured_bacterium Firmicutes | Clostridia | Oscillospirales | UCG-010 | UCG-010 | uncultured_bacterium Firmicutes | Clostridia | Christensenellales | Christensenellaceae | Christensenellaceae_R-7_group BCG_94 4.70 Bacteroidota | Bacteroidia | Bacteroidales | Prevotellaceae | Prevotella | uncultured_Prevotellaceae Firmicutes | Clostridia | Clostridiales | Clostridiaceae | Sarcina | uncultured_bacterium Firmicutes | Clostridia | Clostridiales | Clostridiaceae | Clostridium_sensu_stricto_1 BCG_233 4.80 Firmicutes | Clostridia | Clostridiales | Clostridiaceae | Clostridium_sensu_stricto_1 Bacteroidota | Bacteroidia | Bacteroidales | Bacteroidales_BS11_gut_group | Bacteroidales_BS11_gut_group | uncultured_bacterium CSG-FA BCG_45 4.35 Firmicutes | Clostridia | Peptostreptococcales-Tissierellales | Anaerovoracaceae | Family_XIII_AD3011_group | uncultured_bacterium Firmicutes | Bacilli | Erysipelotrichales | Erysipelotrichaceae | Catenisphaera | uncultured_bacterium Actinobacteriota | Coriobacteriia | Coriobacteriales | Eggerthellaceae | Denitrobacterium | Denitrobacterium_detoxificans Firmicutes | Clostridia | Lachnospirales | Lachnospiraceae | Marvinbryantia | uncultured_rumen Firmicutes | Clostridia | Peptostreptococcales-Tissierellales | Anaerovoracaceae BCG_48 4.79 Firmicutes | Clostridia | Oscillospirales | [Eubacterium]_coprostanoligenes_group | [Eubacterium]_coprostanoligenes_group | uncultured_bacterium Bacteroidota | Bacteroidia | Bacteroidales | Bacteroidales_UCG-001 | Bacteroidales_UCG-001 Bacteroidota | Bacteroidia | Bacteroidales | Rikenellaceae | Rikenellaceae_RC9_gut_group | uncultured_bacterium Firmicutes | Clostridia | Oscillospirales | Oscillospiraceae | NK4A214_group BCG_51 4.36 Firmicutes | Clostridia | Lachnospirales | Lachnospiraceae | Lachnospiraceae_XPB1014_group | uncultured_bacterium Firmicutes | Clostridia | Lachnospirales | Lachnospiraceae | Lachnospiraceae_UCG-009 | uncultured_bacterium Firmicutes | Clostridia | Peptostreptococcales-Tissierellales | Anaerovoracaceae | uncultured | uncultured_bacterium Firmicutes | Bacilli | Erysipelotrichales | Erysipelatoclostridiaceae | Erysipelatoclostridium | uncultured_bacterium BCG_219 4.55 Bacteroidota | Bacteroidia | Bacteroidales | Prevotellaceae | Alloprevotella | uncultured_bacterium Bacteroidota | Bacteroidia | Bacteroidales | Bacteroidales_RF16_group | Bacteroidales_RF16_group | uncultured_bacterium BCG_275 4.45 Firmicutes | Clostridia | Oscillospirales | Oscillospiraceae | NK4A214_group | uncultured_bacterium Verrucomicrobiota | Kiritimatiellae | WCHB1-41 | WCHB1-41 | WCHB1-41 | uncultured_bacterium 1 The BCG markers were identified by linear discriminant analysis effect size (LEfSe) of BCG remaining in the reduced and optimized random forest classification model for prediction of forage type. Features were retained in the model based on feature importance scores . 2 HAY-SP: initial standardized cool-season grass hay diet in the spring; HAY-FA: final standardized cool-season grass hay diet in the fall. 3 Amplicon sequence variants (ASV) were grouped into BCG using Sparse Cooccurrence Network Investigation for Compositional Data in Qiime 2 (v.2020.8) . 4 For LEfSe, significance was set at LDA >2.0; p <= 0.05. 5 Taxonomic assignment of ASV was conducted using the most recent SILVA database (SSU 138). animals-13-00790-t005_Table 5 Table 5 Bacterial co-abundance groups (BCG) identified as markers 1 of warm-season grass pasture. Forage 2 BCG 3 LDA 4 ASV Taxonomic Lineage 5 WSG BCG_35 4.68 Verrucomicrobiota | Verrucomicrobiae | Verrucomicrobiales | Akkermansiaceae | Akkermansia | uncultured_bacterium Firmicutes | Clostridia | Christensenellales | Christensenellaceae | Christensenellaceae_R-7_group Fibrobacterota | Fibrobacteria | Fibrobacterales | Fibrobacteraceae | Fibrobacter | Fibrobacter_sp. Spirochaetota | Spirochaetia | Spirochaetales | Spirochaetaceae | Treponema | uncultured_bacterium Firmicutes | Clostridia | Peptostreptococcales-Tissierellales | Anaerovoracaceae | Family_XIII_AD3011_group | uncultured_bacterium BCG_100 4.21 Firmicutes | Clostridia | Oscillospirales | Oscillospiraceae | NK4A214_group Spirochaetota | Spirochaetia | Spirochaetales | Spirochaetaceae | Treponema Firmicutes | Clostridia | Oscillospirales | Oscillospiraceae | UCG-005 | metagenome BCG_113 4.85 Bacteroidota | Bacteroidia | Bacteroidales | Bacteroidales_RF16_group | Bacteroidales_RF16_group | uncultured_bacterium Firmicutes | Clostridia | Clostridiales | Clostridiaceae | Clostridium_sensu_stricto_1 | Clostridium_butyricum Firmicutes | Clostridia | Oscillospirales | Oscillospiraceae | Papillibacter | uncultured_bacterium BCG_124 4.58 Firmicutes | Clostridia | Christensenellales | Christensenellaceae | Christensenellaceae_R-7_group Synergistota | Synergistia | Synergistales | Synergistaceae | uncultured | uncultured_bacterium Firmicutes | Clostridia | Peptostreptococcales-Tissierellales | Anaerovoracaceae | Mogibacterium BCG_259 4.46 Firmicutes | Clostridia | Lachnospirales | Lachnospiraceae Firmicutes | Clostridia | Lachnospirales | Lachnospiraceae | [Eubacterium]_hallii_group | uncultured_bacterium 1 The BCG markers were identified by linear discriminant analysis effect size (LEfSe) of BCG remaining in the reduced and optimized random forest classification model for prediction of forage type. Features were retained in the model based on feature importance scores . 2 HAY-SP: initial standardized cool-season grass hay diet in the spring; HAY-FA: final standardized cool-season grass hay diet in the fall. 3 Amplicon sequence variants (ASV) were grouped into BCG using Sparse Cooccurrence Network Investigation for Compositional Data in Qiime 2 (v.2020.8) . 4 For LEfSe, significance was set at LDA >2.0; p <= 0.05. 5 Taxonomic assignment of ASV was conducted using the most recent SILVA database (SSU 138). animals-13-00790-t006_Table 6 Table 6 Fecal metabolite concentration 1 of horses adapted to hay diets and pasture forages. Fecal Metabolites 2, ug g Feces -1 Forage 3 HAY-SP CSG-SP WSG CSG-FA HAY-FA Total SCFA 674 +- 76 ab 1071 +- 121 c 860 +- 98 ac 1134 +- 129 c 437 +- 50 b Total BCFA 48 +- 6 a 136 +- 16 b 113 +- 13 b 126 +- 15 b 70 +- 8 a Acetate 323 +- 34 ab 473 +- 50 ac 423 +- 45 ac 524 +- 55 c 229 +- 24 b Butyrate 101 +- 22 ab 174 +- 30 a 128 +- 22 a 170 +- 29 a 62 +- 11 b Propionate 217 +- 25 a 365 +- 42 b 274 +- 31 ab 390 +- 45 b 110 +- 13 c Valerate 12.0 +- 2.1 ab 40.4 +- 7.2 c 24.2 +- 4.3 bc 35.3 +- 6.3 c 18.1 +- 3.2 b Hexanoate 14.2 +- 3.3 a 3.4 +- 0.8 b 5.2 +- 1.2 b 3.6 +- 0.8 b 11.6 +- 2.7 a Heptanoate 2.8 +- 0.5 a 0.8 +- 0.1 b 1.2 +- 0.2 b 0.8 +- 0.1 b 1.7 +- 0.3 ab Isobutyrate 34.4 +- 3.5 a 81.9 +- 8.3 b 76.9 +- 7.8 b 77.7 +- 7.9 b 47.7 +- 4.8 a Isovalerate 13.5 +- 2.1 a 51.2 +- 7.9 b 35.0 +- 5.4 bc 47.1 +- 7.3 b 21.6 +- 3.4 ac 1 Concentrations were determined by gas chromatography--mass spectroscopy (GC-MS). Data are presented as means +- SEM. 2 SCFA: short-chain fatty acids; BCFA: branched chain-fatty acids. 3 HAY-SP: initial standardized cool-season grass hay diet in the spring; CSG-SP: cool-season grass pasture in the spring; WSG: warm-season grass, either crabgrass or bermudagrass, in the summer slump period; CSG-FA: cool-season pasture in the fall; HAY-FA: final standardized cool-season grass hay diet in the fall. a,b,c Indicates significant differences between forages within rows (mixed model ANOVA with Tukey's post hoc adjustment; p <= 0.05). animals-13-00790-t007_Table 7 Table 7 Random forest regression accuracy 1 for predicting fecal variables and forage nutrients based on microbial composition. Model Accuracy 2 Full BCG Dataset 2 Reduced BCG Dataset 3 R2 p-Value R2 p-Value Fecal Variables 4 Total SCFA 0.06 0.13 0.24 0.001 Total BCFA 0.08 0.07 0.28 0.0004 Acetate 0.05 0.19 0.29 0.0003 Butyrate 0.04 0.20 0.26 0.002 Propionate 0.03 0.31 0.17 0.008 Valerate 0.24 0.49 0.15 0.01 Hexanoate 0.26 0.0007 0.48 <0.0001 Heptanoate 0.11 0.03 0.24 0.001 Isobutyrate 0.01 0.54 0.27 0.0006 Isovalerate 0.26 0.0007 0.26 0.0007 Fecal pH 0.06 0.13 0.17 0.008 Forage Nutrients Digestible Energy 0.26 0.0008 0.34 <0.0001 Crude Protein 0.41 <0.0001 0.62 <0.0001 Acid Detergent Fiber 0.19 0.005 0.39 <0.0001 Neutral Detergent Fiber 0.27 0.0005 0.35 <0.0001 Non-Structural Carbohydrate 0.53 <0.0001 0.61 <0.0001 Water-Soluble Carbohydrate 0.52 <0.0001 0.67 <0.0001 Ethanol-Soluble Carbohydrate 0.10 0.04 0.18 0.006 Starch 0.21 0.003 0.40 <0.0001 1 Random forest regression modeling with nested cross-validation was conducted in Qiime2 (v.2020.8) . 2 Initial model accuracies for random forest regressors were determined using the full feature set of bacterial co-abundance groups (BCG) and ungrouped amplicon sequence variants generated using Sparse Co-Occurrence Network Investigation for Compositional Data . 3 Random forest regressors were then applied to a reduced BCG dataset (iterative reduction to 25 features most predictive of forage type [based on model importance scores]) . 4 SCFA: short-chain fatty acids; BCFA: branched chain-fatty acids. 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PMC10000168

Increasing human-bear conflicts are a growing concern, and managers often assume bears in developed areas are food-conditioned. We examined the relationship between human-bear conflicts and food conditioning by analyzing isotopic values of hair from black bears (Ursus americanus floridanus) involved in research (n = 34) and conflicts (n = 45). We separated research bears into wild and developed subgroups based on the impervious surface within their home ranges and separated conflict bears based on observations of human food consumption (anthropogenic = observations; management = no observations). We initially assumed wild bears were not food conditioned and anthropogenic bears were. However, using isotopic values, we classified 79% of anthropogenic bears and 8% of wild bears as food conditioned. Next, we assigned these bears to the appropriate food conditioned category and used the categorizations as a training set to classify developed and management bears. We estimated that 53% of management bears and 20% of developed bears were food conditioned. Only 60% of bears captured within or using developed areas showed evidence of food conditioning. We also found that d13C values were a better predictor of anthropogenic foods in a bear's diet than d15N values. Our results indicate that bears in developed areas are not necessarily food conditioned and caution against management actions based on limited observations of bear behavior. stable isotopes Ursus americanus floridanus food conditioning wildlife conflict the FWC's Bear research program, Fish and Wildlife Foundation of FloridaCWT 1617-01 University of Florida's Institute for Food and Agricultural ScienceThis project received funding from the FWC's Bear research program, Fish and Wildlife Foundation of Florida, grant # CWT 1617-01 and University of Florida's Institute for Food and Agricultural Science. pmc1. Introduction Growth in human populations and development over the last half century, as well as increases in some generalist wildlife populations, have increased interactions between humans and wildlife . Certain wildlife species have been able to adapt to newly human-dominated landscapes where they use anthropogenic food sources (e.g., garbage, pet food, crops, livestock, wildlife feeders ) and share space with humans . For example, bears (Ursus spp.) often traverse human-dominated landscapes and can exploit human resources , but they typically avoid areas with more human development that they likely perceive as risky . However, competition-based dispersal, availability of seasonal foods, and physiological periods with high-energy requirements (such as hyperphagia ) may contribute to the use of developed areas despite the risks . In recent decades, wildlife managers have reported increased human-bear interactions across the human-dominated landscapes of North America . The dominant driver behind these interactions appears to be the availability and consumption of anthropogenic foods by bears . Bears that use anthropogenic foods often alter their movement and foraging behaviors to find these calorie-rich foods . The American black bear (Ursus americanus) is the most common bear species in North America, and the majority of human-bear interactions involve this species. Black bears are intelligent, behaviorally plastic, have broad tolerances for food and vegetation types, and can move over large areas . These traits can make them susceptible to habituation and food conditioning , learned behaviors that form when the association between developed areas and risk is reduced and an animal equates human development with food . Naive bears presumably enter developed areas for the first time during exploratory movements , then, over repeated visits, they become habituated and/or food-conditioned . The degree of a bear's food conditioning is influenced by the availability of natural and anthropogenic food sources , tolerance of humans , and reproductive status , with highly food-conditioned bears more likely to have negative human interactions (e.g., cause property damage and injuries to humans ). Traditionally, any incident of a bear occurring in or near human developments would be classified as a conflict due to the potential for injuries to people or damage to property , with the view that repeat offenders should be translocated or euthanized . A common underlying assumption behind the classification of a conflict animal is that a bear in a developed area is either food-conditioned or is likely to become food-conditioned. In contrast, Elfstrom et al. found that limited observations of any particular bear were insufficient to discriminate between food-conditioned and non-food-conditioned brown bears (U. arctos); however, a bear's movements associated with human development might be more predictive . Additionally, other factors, such as the time of year, abundance of natural foods, and the density of bears, can influence a bear's movements around human development . For example, a bear may periodically access human-dominated areas that it would normally avoid during dispersal , hyperphagia , years of low natural food availability , or when natural foods occur near development is abundant . Accordingly, management agencies recognize a need for a more adaptive, contextual, and data-driven approach in their response to bears in and around human development . Stable isotope analysis is a powerful but coarse measurement that can be used to understand animal diet and potentially inform management policies . Carbon and nitrogen stable isotopes in animal tissue, reported as d13C and d15N values, respectively, are assimilated via dietary foods and reflect the resources consumed . Hair can be collected invasively and non-invasively , and stable isotope analysis of hair can be used to determine if bears had consistently relied on human-sourced foods, which we interpret as an indication of food conditioning. Bears with elevated d13C values in areas without naturally occurring C4 plants likely have a diet subsidized by corn or sugar cane via anthropogenic food sources . Higher d15N values are indicative of trophic levels and could identify the consumption of animal-protein-based foods ; such values are well suited for identifying anthropogenic foods in areas where black bears have a predominantly plant-based diet . As black bears continue to increase in abundance and expand their range, it is essential that wildlife managers find additional tools to interpret bear behavior and assess risk of human-bear conflicts. Accordingly, our objective for this study was to examine the relationship between human-bear conflicts and food conditioning in bears by direct but limited observations during human-bear conflicts and seasonal use of developed areas to develop a predictive model of food conditioning. We predicted that direct but periodic observations of bear behavior would be of limited value when predicting isotopic values indicative of food conditioning. Alternatively, longer-term information on a bear's presence around human development is likely to be more predictive . 2. Materials and Methods 2.1. Study Area We conducted our study on the Tate's Hell State Forest and neighboring private lands in the eastern panhandle of Florida , a region that supports the second largest subpopulation (1060 individuals) of black bears in Florida . Tate's Hell State Forest is the second largest contiguous piece of state land in the panhandle of Florida and is composed of 819 km2 of contiguous wet flatwoods, wet prairie, sandhill, upland hardwood, floodplain swamp, scrub, and titi thickets. The forest is heavily ditched, a remnant of past commercial timber production, and it is characterized by poorly drained soils. The forest sustains a high level of recreational activity and is open to off-road vehicles all year. The towns of Carrabelle (pop. 2778), Eastpoint (pop. 2337), and the smaller, unincorporated Lanark Village are on US road 98 along the Gulf of Mexico, which formed the southern border of the study area; the Apalachicola National Forest is adjacent along the entire northern border. Except for rural residents, much of the private lands are managed for commercial timber and leased for hunting (currently, bear cannot be legally hunted in Florida). The local climate consists of hot, humid summers, and short, cool winters with average daily temperatures ranging from 8 degC to 18 degC in January and 24 degC to 33 degC in July. Precipitation in the area is highly variable and primarily falls in the summer and early fall . 2.2. Overview We collected hair from bears from 2015 to 2017 to determine if food conditioning, assessed by stable isotopes in hair, could be predicted by proximity to developed areas or a direct observation of bears eating anthropogenic foods. We categorized bears into different groups and subgroups based on the data available for each individual . We classified the radio-tagged research bears, captured only during summer months, as wild or developed based on their home range. We classified conflict bears removed from the population by managers (killed or translocated during any month of the year) as anthropogenic if observed consuming anthropogenic foods or management if removed without being observed consuming anthropogenic foods. 2.3. Classification of Bears 2.3.1. Capture and Monitoring of Research Bears Research bears used in this study were part of a larger project conducted by the Florida Fish and Wildlife Conservation Commission (FWC) to study the demographics of the Apalachicola bear subpopulation, for which only female bears were radiocollared. For that study, we captured bears between May and July from 2016 and 2017 using cage traps, culvert traps, and spring-activated Aldrich foot snares (Margo Supplies Ltd., High River, Alberta, Canada), following Johnson and Pelton , as modified by Scheick et al. . All traps were set on public lands or nearby commercial timberlands. We immobilized bears with a 1:1 mixture of tiletamine hydrochloride and zolazepam hydrochloride (Telazol) administered at 4 mg/kg of estimated body weight (dosage ranged 2.0-10.5 mg/kg) via a CO2 powered dart gun (Telinject, Inc., Arleta, CA, USA). We processed each individual and collected hair samples from the dorsum. We fitted 37 subadult and adult female bears with iridium GPS satellite-tracking collars (Lotek Wireless Inc., New Market, ON, Canada) programmed to collect fixes at 2-h intervals for two years post-capture. 2.3.2. Research Bears To categorize our research bears as wild or developed, we used impervious surfaces (e.g., roofs, paved roads, parking lots, compacted soils) that prevent water from entering soils, as a common measure of human development . Specifically, we calculated the proportion of impervious surface across the home range area of each individual during the months associated with hyperphagia in Florida (1 September-30 November) in 2016 and 2017. This physiological state represents the period of greatest bear activity and therefore increased the likelihood that these bears would enter human development for food resources. Our dataset of research bears included 34 individuals after we removed three individuals that did not have locations within our study area during the majority of the three-month period. We also removed all GPS fixes that were not three-dimensionally validated (four or more satellites) to maintain data quality. We estimated fall home range areas by 95% minimum convex polygon (MCP) using the adehabitatHR package in program R . To calculate the amount of impervious surface, we extracted the values from an impervious surface raster file and quantified the mean impervious surface within the range of each bear using the raster package on the R platform . The mean impervious surface across all home ranges was 0.99%, with a standard deviation of 1.1 . We selected an impervious surface level just above the mean (1%) as the cutoff and classified bears with <=1% impervious surface in their range as wild and those >1% as developed (Table S1). We chose this cutoff based on observations that bears with >1% of their MCP containing impervious surface altered their behaviors to human approach (unpublished data). To ensure that MCP was an appropriate measure of bear's exposure to human development, we compared the amount of impervious surface within each bear's 95% MCP to the proportion of its GPS locations with an impervious surface > 1% divided by locations with an impervious surface < 1% (to estimate the amount of time spent there). Finding both measurements highly correlated (0.76; p-value <= 0.01), we proceeded using MCP as a measure of exposure to development. 2.3.3. Conflict Bears We used hair samples and data collected from 45 bears captured by FWC staff, from across Florida, in response to human-bear conflicts between July 2015 and November 2017. These bears were captured using cage or culvert traps similar to those used for the research bears; however, they were set in specific areas where conflict behavior had been reported. We used written reports that summarized the direct observations by the complainant and the responding FWC biologists to classify these conflict bears as anthropogenic or management. We classified 28 bears as anthropogenic because reports for each of these bears indicated they were observed eating anthropogenic food, usually garbage, at least once. The stated capture reason for most of these bears was getting into garbage, but eight were also considered a human safety risk based on the bear's behavior (not for repeatedly consuming garbage). Two family groups were included (Bear ID 17355 was the mother of 11-month-old cubs 17356 and 17357; Bear ID 17523 was the mother of a dependent yearling 17522), and 46% were cubs or subadults (aged <= 2.9 years). We classified 17 bears as management bears because they were captured and removed for conflict behavior (one attacked a human, seven caused property damage, and nine were considered a human safety risk), but without direct observation that they ate anthropogenic food. Fifty-nine percent of the management bears were juveniles. 2.4. Stable Isotope Analysis We used stable isotope analysis to determine the d13C and d15N values of each individual to establish its dietary status. We assumed that all anthropogenic bears were food conditioned (FC) because they were seen eating human food and that all wild bears were not food conditioned (NFC) because they were rarely present in the human-dominated landscape. There were no commercial agricultural fields in our study area, but automated corn feeders for deer were prevalent on private lands, and small amounts of corn could be available from residential gardens. We tested these assumptions by comparing the isotopic values of the two groups. We then used these results to reclassify bears more appropriately as FC or NFC and establish a training dataset capable of predicting the FC or NFC classification. Next, we used our training dataset to test the prediction that bears with movements that overlapped the human-dominated landscape (developed research bears) or observed in the human-dominated areas but with no direct observation of anthropogenic food consumption (management conflict bears) would display isotopic values consistent with FC classifications. A bear's hair grows in roughly linear fashion over the period when they are active . During periods of growth, hair integrates assimilated protein, carbohydrates, and fats in metabolically inert keratin, thereby preserving the isotopic information indefinitely . The ability to determine average diet during the period of growth (i.e., whole growth analysis) makes hair useful in stable isotope analysis . Bears undergo a single molt per year that occurs between early May and fall, depending on the quantity and quality of available forage . Therefore, we assumed the full-length guard hairs collected during summer research prior to molting represented the isotopic composition of the diet from the previous year's molt until the time of capture in the current year. Similarly, full-length hair collected from conflict bears in or prior to August was assumed to represent the previous year's diet, while hair collected in or after September represented the capture year's diet (August to time of capture). We cleaned hair samples with a 2:1 chloroform-methanol solution to remove surface oils before air-drying them . We cut hairs at the base and weighed samples in tin capsules and sealed them for isotopic analysis. Values of d13C and d15N were determined using continuous flow isotope ratio mass spectrometry using a Thermo-Scientific DeltaV (Waltham, MA) Advantage Isotope Ratio Mass Spectrometer connected by a ConFlo II interface to a Thermo-Scientific Carlo Erba NA 1500 CNHS elemental analyzer at the Stable Isotope Mass Spectrometry Lab at the University of Florida. Stable isotope values were expressed as per mil (%0) in standard delta (d) notation:(1) dX=(Rsample/Rstandard)-1 where dX is 13C or 15N, and R is the corresponding ratio of heavy-to-light isotopes (13C/12C or 15N/14N) in the sample. We reported results as ratios relative to international reference standards Vienna Peedee Belmnite (V-PDB) for carbon and to atmospheric N2 (AIR) for nitrogen. The precision for d13C and d15N values was +-0.06%0 and 0.11%0 respectively, based on 5 analyses of USGS40. 2.5. Statistical Analyses We used MANOVA to test if bears classified as anthropogenic bears based on reported human-sourced food use and wild bears based on impervious surfaces had different isotopic values. We then performed linear discriminant analysis (LDA) to assess our ability to distinguish anthropogenic bears and wild bears (Table S2) based on the d13C and d15N values of their hair . We used the posterior probability predicted by the LDA to reclassify the bears categorized as wild or anthropogenic into new groupings, FC or NFC, based on their respective isotopic values. We then used the reclassified FC bears and NFC bears to establish a training dataset capable of predicting the FC or NFC status of the developed and management bears. To evaluate the accuracy of the training data to predict group assignments we used leave-one-out cross-validation (LCV ). Next, we used the training data to assign developed and management bears to a category (FC or NFC) based on the highest posterior probability predicted by the LDA with LCV . We used the structure coefficients from the LDA to determine whether carbon or nitrogen was better at discriminating between FC and NFC bears. We performed all statistical analyses on the R platform . 3. Results We determined the isotopic composition of hair, based on the d13C and d15N values, from 79 individual bears. Samples collected from anthropogenic bears had a d13C mean = -20.33, SD = 2.02, d15N mean = 6.00, and SD = 1.38, and wild bears (n = 24, Table S2B) had a d13C mean = -23.57, SD = 1.46, d15N mean = 4.36, and SD = 0.96. Anthropogenic and wild bears together had a d13C mean = -21.90, SD = 2.35, and d15N mean = 5.21, SD = 1.44; management bears (n = 17) had d13C mean = -21.25, SD = 1.92; d15N mean = 4.99, SD = 1.25, Table S2); and developed bears (n = 10) had a d13C mean = -22.84, SD = 1.79; d15N mean = 4.83, SD = 0.91, Table S2). Using the results of the MANOVA, we established that anthropogenic bears and wild bears were significantly different groups (multivariate F (1,2) = 22.87, p < 0.001) prior to applying the discriminant analysis. We used LDA with LCV to determine that 79% (n = 22) of the anthropogenic bears (high d13C and d15N values) were classified as FC bears and the remaining 21% (n = 6; low d13C and d15N values) were reclassified as NFC bears . A larger proportion of wild bears, 92% (n = 22), were classified as NFC bears, with the remaining 8% (n = 2) reclassified as FC bears . The classification rate for our model was 85%, which suggests high predictive capacity using d13C and d 15N values, supporting their use as training data. The data on wild and anthropogenic bears, including those reclassified as FC or NFC, were used as training data to determine the FC and NFC status for the remaining two subgroups. We predicted that 53% of management bears were FC bears, and the remaining 47% were NFC bears , and we predicted that 20% of the developed bears were FC, and the remaining 80% were NFC . We used the structure coefficients from our LDA to determine that d13C values predicted anthropogenic foods in a bear's diet 96% better than d15N values, although FC bears were more likely to have higher values in both d13C and d15N than NFC bears . 4. Discussion Our results indicate that bears in developed areas are not necessarily food conditioned and, in fact, not all bears observed consuming anthropogenic foods had isotopic values indicative of food conditioning. Of the 55 bears that had been captured in or near developed areas, including those from the conflict group and the developed subgroup, 40% (22 of 55) lacked evidence of food conditioning. Even among the 28 anthropogenic bears that had been directly observed eating human-sourced foods, 21% (six) were NFC. For predictive power, <1% impervious surface as determined by telemetry data was a better predictor of food conditioning status than direct but limited observations (92% of wild bears were NFC vs. 79% of anthropogenic bears were FC). However, increased impervious surface area was not associated with increased food conditioning, as seen by NFC bears with 5% impervious surface (Table S1). We also determined that d13C values were a better predictor of food-conditioning status than d15N values for bears in Florida, although FC bears usually had both higher d13C and d15N values than NFC bears. A few NFC wild bears had high d15N values relative to others in the subgroup, and most d15N values from all subgroups were higher than that of terrestrial browsers, perhaps indicating some use of carrion . Such naturally occurring animal protein would reduce the ability of d15N values to identify FC status. The isotope values indicative of food conditioning would require repeated and consistent consumption of anthropogenic food sources over time during the diet year . Dispersal behavior and exploratory food searching would explain why bears accessing developed areas for brief periods might not consume anthropogenic foods or consume them consistently enough to display isotopic values indicative of food conditioning . Due to limited sample sizes, we were not able to further subset our data to compare conflict bears of different sexes and ages. However, more than half of the conflict bears analyzed in this study were subadults, supporting the hypothesis that exploratory movements bring naive bears into developments . Additionally, some NFC bears in our study that were in or near developed areas may have been new to developed areas, or they may have switched their foraging behavior between anthropogenic and natural foods. Johnson et al. found a negative correlation between availability of natural foods and reliance on anthropogenic foods and corresponding shifts in bears' use of developed areas. Isotope values indicated that only 53% of management bears were food-conditioned , but conflict bears in this study were removed for specific behaviors, not because of an assumption of food conditioning. For many years, the public in our study area has reported bears eating acorns in residential yards (FWC unpublished data), and our results suggest that, even when in developed areas, some bears rely on natural rather than anthropogenic foods. Whereas it was traditionally assumed the nuisance bears were food conditioned managers have pivoted to assessing the risk to human life and property based on season, availability of natural foods, and the bear's age, sex, and behavior (level of aggression, repeated conflicts, etc. ). However, information on a bear's history is rarely available, and it is hard to determine if recurring conflicts in the same areas were caused by the same bear. Seasonal or annual shifts in foraging areas may explain the 8% (two out of twenty-four) of wild bears in our model that were reclassified as FC. Although we do not know the movements of the two FC bears (F605 and F608; Tables S1 and S2B) during the year prior to their capture, the time period for which the isotopic values reflected their diet, we examined the movement data from 2016 to 2017 (unpublished FWC data). We determined that, in the fall, they had moved with their cubs into areas with scattered homes (but not developed according to our 1% impervious surface category) where they could have accessed anthropogenic food sources, stayed in that general area for the winter, and then returned to their normal summer home range area in spring. In fact, F605 had been photographed with her cubs at a feeder (FWC unpublished data). Such movements suggest that, while living farther from developments might reduce the odds that a bear is food conditioned, few bears in Florida live in areas where people or human-sourced foods are absent. These seasonal shifts may also explain why bears in family groups that were captured together during a human-bear conflict did not all show the same FC status. Bear ID 17355, captured in January, was not classified as food conditioned, but both of her nearly 11-month-old cubs were, so it seems likely that she brought her cubs to anthropogenic food sources that she had seldom or only recently used prior to her capture. In contrast, Bear ID 17253 and her dependent yearling were both food conditioned. Implementing proactive management strategies (i.e., garbage management and educational programs) are effective means of reducing human-bear conflicts . Johnson et al. found that 60% compliance with keeping trash secured reduced human-bear conflicts by 60%. Removing readily available trash should also reduce food conditioning , although that is more difficult to prove empirically. There appears to be a lag between reduced conflicts and increased tolerance of people for bears, even when that reduction was caused by active participation in making trash less accessible to bears . 5. Conclusions Expanding human development into more natural areas is expected to continue, increasing the likelihood of human-bear interactions, and exacerbating both real and perceived risks . The complexity of the issues surrounding human-bear interactions requires wildlife researchers and managers to develop a more comprehensive understanding of the factors shaping bears' diet, movement, and behaviors . Kirby et al. reported that each 1%0 increase in d13C increased the likelihood that a bear would cause human-bear conflicts by 60%. We recognize that managers cannot wait for stable isotope analysis to determine the most appropriate management actions. Still, our findings illustrate the benefit of stable isotope analysis before classifying bears as food conditioned and they validate the importance of not assuming all bears observed in neighborhoods, or even directly observed feeding on human-provided foods, are food conditioned. Our results show that a bear's presence in a developed area does not necessarily indicate it is food-conditioned. This should be taken into consideration when making management decisions and incorporated into educational materials. A public that has a better understanding of how anthropogenic foods affect the gradient of a bear's learned behavior would be a more helpful partner with management agencies in reducing human-bear conflicts. Acknowledgments We thank J. Curtis at the University of Florida for the stable isotope analysis. Special thanks to the McCleery Lab for providing useful feedback throughout the research process. We thank B. Bernhardt, A. Dyson, P. Grunwald, J. Johnson, A. Kornak, C. Kurz, H. Manninen, J. Morgan, S. Shiver, Z. Wardle, Z. Wesner, and E. Wildey for long hours of field work and B. Bankovich, M. Sconyers, D. Alix, P. Wharton, S. Chafin, D. Shoemaker, D. Gandy, and staff of the Tate's Hell State Forest for field support or technical assistance. We appreciate Florida State University's Coastal and Marine Laboratory for the use of their dorms. We express our appreciation to the Florida Fish and Wildlife Conservation Commission, Florida Wildlife Foundation, Florida Wildlife Federation, and the University of Florida for funding. M. Orlando D. Telesco and E. Braun de Torrez offered constructive criticisms on several drafts of this manuscript. Supplementary Materials The following supporting information can be downloaded at: Figure S1: Average percent of impervious surface available within the fall home range areas (1 September-30 November) for each of the 34 female bears in the research group, Table S1: Summary of each research bear's minimum convex polygon (MCP), mean impervious surface (% Imp. surf.) across the fall home range area, and the subgroup where each female was placed based on impervious surface. This subgroup determined the original assumption of food conditioning. The MCP values are reported in kilometers squared (km2) rounded to the nearest hundredth. Table S2: Individual, mean and SDs of stable isotope (d13C and d15N) values of anthropogenic (A) and management (C) bears sampled throughout Florida during 2015-2017 and wild (B) and developed (D) bears sampled in and around Tate's Hell State Forest, Florida, USA, 2016-2017. Subadult age class includes bears aged 1-2.9 years. Diet-year denotes the capture year or prior year for anthropogenic and management bears, depending on month of capture, and the year prior to capture for wild and developed bears. LDA classification denotes the two categories (not food conditioned [NFC] or food conditioned [FC]) based on the linear discriminant analysis with leave-one-out cross validation. P-FC is the probability of each individual being food conditioned. While all anthropogenic bears were initially assumed to be FC, and all wild bears were initially assumed to be NFC, some bears from each subgroup, in bold, were reclassified based on the LDA outputs. Click here for additional data file. Author Contributions This project was conceptualized by conceptualization by D.W.H.J., R.A.M. and J.W.M. Field work was conducted by D.W.H.J., B.K.S. and J.W.M. The formal analysis was conducted by D.W.H.J. and H.B.V.Z. The original draft of the manuscript was prepared by D.W.H.J. and all author contributed to writing, review and editing of the final product. All authors have read and agreed to the published version of the manuscript. Institutional Review Board Statement All field work was conducted by FWC personnel and research procedures followed policies of the FWC, the state agency with statutory authority to handle and conduct research on wildlife in Florida without IACUC review. These procedures followed American Society of Mammalogists (ASM) guidelines for research on live animals . Informed Consent Statement Not applicable. Data Availability Statement The data used in the paper are provided in Supplemental Tables S1 and S2. Conflicts of Interest The authors declare no conflict of interest. Figure 1 The study area consisted of Tate's Hell State Forest (darker shaded area) and neighboring commercial timberlands. The towns mentioned in text are shown as are major highways and Apalachicola National Forest boundaries (lighter shaded area). Figure 2 The conceptual framework for assessing the relationship between food conditioning and bears grouped by behavior or movements and the results of stable isotope and linear discriminant analysis (LDA) analyses. The development of groups and subgroups were based on movements in black bears as determined by the percent impervious surface within individual 95% MCP fall home ranges or bear behavior reported when human-bear conflicts were reported. The dotted box highlights the training data for the LDA analysis. Figure 3 Stable isotope values (d13C and d15N) for bear hair samples. Isotope values for bears captured throughout Florida from 2015 to 2017 comprise the anthropogenic bears (#) sampling subgroup and bears captured in and around Tate's Hell State Forest, Florida, USA from 2016-2017 comprise the wild bears (#) subgroup. Higher values indicate more human-sourced food. The original classification, plot (A), depicts classifications based on amount of impervious surface (wild bears) and observed conflict behavior (anthropogenic bears). The food conditioned classification plot (B) depicts the same individuals reclassified via a linear discriminant analysis with leave-one-out cross validation; six anthropogenic bears and two wild bears were reclassified to predict food conditioning FC () and not food conditioned NFC (). Figure 4 Stable isotope values (d13C and d15N) of management bears predicted by linear discriminant analysis as food conditioned (FC; ) or not food conditioned (NFC; ^) compared to the training data showing the training data that reclassified anthropogenic and wild bears as food conditioned FC () and not food conditioned NFC (). Higher values indicate more human-sourced food. Figure 5 Stable isotope values (d13C and d15N) of developed bears predicted by linear discriminant analysis as food conditioned (FC; ) or not food conditioned (NFC; ^) compared to the training data showing the training data that reclassified anthropogenic and wild bears as food conditioned FC () and not food conditioned NFC (). Higher values indicate more human-sourced food. Disclaimer/Publisher's Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

PMC10000169

The concept of psychological resilience is well-explored in the human literature and is often described as the ability to 'bounce back' following adversity. However, it remains a neglected research area in dogs despite observations that like humans, dogs vary in their ability to cope with stress. This study aimed to develop the first canine 'resilience' scale. An on-line survey was developed for owners. This covered demographics, medical/behavioural history of the dog, and 19 potential resilience items assessed using a 5-point Likert scale; 1084 complete responses were received during the survey period, with 329 respondents subsequently completing the questionnaire a second time, 6-8 weeks later. Intra-rater reliability was assessed, and only reliable items retained. A principal component analysis (PCA) with varimax rotation was then performed with components extracted on the basis of the inspection of scree plots and the Kaiser criterion. Items were retained if they loaded >0.4 onto one of the components but removed if they cross-loaded onto more than one component. This resulted in a 14-item, 2-component solution. One component appeared to describe "Adaptability/behavioural flexibility" and the other "Perseverance", which are described in the human literature on resilience. Predictive validity was established for expected correlates, such as problem behaviour. The resulting instrument was called the Lincoln Canine Adaptability and Resilience Scale (L-CARS) and is the first to be developed for the assessment of resilience in dogs. behaviour Lincoln canine adaptability and resilience scale L-CARS dog psychometric scale resilience trait This research received no external funding. pmc1. Introduction Psychological resilience is generally regarded as the ability to 'bounce back' following adversity or trauma . It is frequently discussed in the human literature in terms of exploring the different ways that individuals respond to difficult life events . Anecdotally, dogs show great variation between individuals in their coping abilities; some appear to recover rapidly from what we would perceive as quite significant trauma, whilst others struggle following subjectively milder disturbances. However, whilst of interest and importance , differences in resilience in dogs remain an under-researched topic. In humans, resilience is often considered within the context of the risk of mental health issues and psychiatric disturbance . Lower resilience scores have been found to correlate with neuroticism (a personality trait indicating tendencies towards negative emotions and emotional instability) and specific disorders, such as post-traumatic stress disorder (PTSD) and generalised anxiety . Those considered less resilient carry an increased risk of suffering from a range of negative psychological states, and it seems reasonable to suppose that the same might occur for dogs. Dogs face a variety of commonplace stressors in their lives, such as during veterinary visits , car travel and kennelling . Whilst various studies have explored risk factors for behavioural complaints involving negative effects , the literature lacks a focus on factors, such as resilience, which may protect against the initial development of such problems. There are numerous validated psychometric resilience scales for use in humans , and this may reflect a lack of consensus regarding the definition . Studies have generally described resilience from one or more of three perspectives: (a) as a set of inherent character traits (often referred to as 'trait resilience'); (b) as part of a dynamic process, of adapting and recovering from adverse events; or c) as an outcome, which evaluates resilience based on the favourability of the outcome itself . The importance of consistent terminology to the development of the scientific basis of clinical animal behaviour has recently been highlighted . Given the complex and clearly multi-faceted nature of psychological resilience as a construct, we chose to focus on 'trait resilience' in the current study. This assumes that some individuals carry personal characteristics that increase their ability to cope in the face of adversity and allow positive adaptation to new circumstances and was therefore deemed most likely to be measurable between individuals via a psychometric scale. Despite the inability of non-human animals to self-report, the development of psychometric scales using owners as proxy responders for the evaluation of temperament traits has already been applied successfully to develop valid scales for several other traits . Therefore, the current study aimed to use a similar approach to that used previously , to develop a psychometric scale for use in dogs to assess trait resilience and determine the evidence of validity. 2. Materials and Methods The questionnaire was created in Qualtrics(r)XM0722 as an on-line questionnaire, written in British English. The questionnaire comprised two sections. 2.1. Demographic Data Demographic data were collected relating to both the owner (country currently living in) and dog (age; breed; sex and neuter status; presence of any medical, behavioural or painful conditions; presence of an unusual gait or movement; whether any prescription medications, supplements/nutraceuticals or pheromone products are used). Some factors were included based on extrapolation from the human literature, such as age and gender . Other factors were included because it was hypothesised that they may impact canine resilience, such as the impact of pain on behaviour . 2.2. Resilience and Related Items An extensive review of the human resilience literature was performed to define trait resilience in a way that would be relevant to pet dogs (face validity). Our definition was based on the three domains described by Maltby and colleagues :Engineering resilience--an individual's ability to return to normal ('bounce back') following adversity; Ecological resilience--the capacity to tolerate/withstand disruptions, with the ability to maintain composure during challenges (focus on 'best efforts', perseverance); Adaptive capacity--the ability to handle and adapt to change, i.e., flexibility in strategies and even enjoyment of change. These domains were then used to create statements (potential items in the instrument) that might reflect the equivalent state as a form of trait resilience in dogs, which could be answered by a typical owner. For example, for the Engineering resilience domain there was the item: "My dog takes a long time to recover from set-backs in life..."; for the Ecological resilience domain, there was the item: "My dog will persevere even when they do not succeed in something straight away..." (to reflect 'best efforts' when faced with challenges); and for the Adaptive capacity domain, there was the item: "I would regard my dog to be very adaptable i.e., able to fit into any situation". An item was also incorporated to allow assessments of convergent validity with the construct of interest: "I believe my dog to be a resilient individual i.e., able to cope with, 'bounce back' from and/or adapt to adversity or change". Nineteen items were included in our preliminary questionnaire, which included 6 items for each of the three described domains and 1 item for the assessment of convergent validity [see Supplementary Material S1 for a full list of initial items]. A 5-point Likert scale was used to rate each of these items, ranging from "Strongly Agree" through "Mainly Agree", "Partly Agree, Partly Disagree", "Mainly Disagree" to "Strongly Disagree", with six items reverse scored to reduce the risk of response set acquiescence ; an "N/A or Don't Know" option was also included to avoid participants feeling forced to select a response. Similar rating systems have been used in other validated canine temperament scales . A random order generator function was used on Qualtrics(r)XM to minimise the risk of grouping items in relation to one of the three facets , which might result in an order bias. Finally, questions pertaining to the presence or absence of a range of commonly reported behaviour problems were included given the correlations between resilience and psychiatric disorders described in the human literature . 2.3. Questionnaire Piloting and Distribution The questionnaire was trialled with a pilot sample of 5 individual dog owners to assess comprehensibility and clarity. Minor amendments were made, and the questionnaire was subsequently made live to the public, under the heading: "Can resilience be measured as a trait in pet dogs?" [see Supplementary Material S2, for full questionnaire]. An advertisement containing an electronic link to the questionnaire was created. This was distributed via several social media platforms (Facebook, Instagram and Twitter), as well as via direct contacts. Both methods had the potential for snowballing sampling effects. The inclusion criteria required participants to be a minimum of 18 years of age and be able to provide informed consent. It was also specified that they currently owned the dog for whom they intended to complete the questionnaire. Participants owning multiple dogs were asked to complete it for only one dog and were requested to select the dog they had owned longest. Participants were requested to read the participant information sheet and complete an electronic consent form before they could take part in the study. The original questionnaire was made available from the 4th until 29th of July 2022. Participants were given the option to provide an email address to enable future intra-rater reliability testing. Respondents who provided an email address to the original circulation were contacted again a minimum of 6 weeks (maximum 7 weeks) after they had completed the first questionnaire. They were asked to complete the questionnaire again for the same dog so that the intra-rater reliability of the items could be assessed. Any questionnaires completed within 1 week from being contacted were included in our analysis, giving a minimum 6-week and maximum 8-week window between first and second questionnaires. The questionnaire items and layout were unchanged between surveys. 2.4. Statistical Analysis Statistical analyses were conducted using R 4.2.1. 2.4.1. Intra-Rater Reliability Assessment Percentage agreements were calculated for each item, i.e., how many respondents selected the same response between the first and second completion of the questionnaire. Following this, non-parametric Spearman's rank order correlations and Wilcoxon signed rank tests were performed on each of the items in turn, with any "N/A or Don't Know" responses excluded from this part of the analysis. Items were excluded on the basis of weak correlations (Spearman's rank order correlation <0.4) or significant differences (p < 0.05) between the first and second questionnaire (Wilcoxon signed rank test). Bonferroni adjustments were applied to p-values to correct for multiple testing. 2.4.2. Psychometric Evaluation Items were scored to reflect the hypothesis that higher scoring individuals would have higher resilience. The Likert scale responses were converted into numerical scores: typically, 5 for "Strongly Agree"; 4 for "Mainly Agree"; 3 for "Partly Agree, Partly Disagree; 2 for "Mainly Disagree" and 1 for "Strongly Disagree", but the negatively worded items were reverse scored [(R) in Supplementary Material S1] (i.e., "Strongly Agree" becomes 1 and "Strongly Disagree" becomes 5). Given the response rate and potential complications that can occur when imputing missing data , it was decided that records would be deleted for respondents who answered 3 or more of the 19 resilience items with an "N/A or Don't Know" response. The remaining data were then sorted into complete and incomplete datasets for a subsequent comparison of item scores between these two populations, using Wilcoxon rank sum tests. Bonferroni adjustments were applied for multiple testing. Using the complete datasets (i.e., any individual response sets with no "N/A or Don't Know" responses), a between-items correlation matrix was created for the resilience items. Any items suggesting singularity by lacking any correlations >0.2 with any other item or suggesting multicollinearity and redundancy due to correlations >0.8 with another item were removed . A principal component analysis (PCA) with varimax rotation was then performed on the remaining items . The aim of a PCA is to determine whether a large number of interrelated variables can be reduced into a smaller number of measures . A varimax rotation was selected to maximise the difference between factors . The number of components to be extracted was decided using the point of inflection on the scree plot along with the Kaiser Criterion, which proposes the retention of components with an Eigenvalue >1 . For interpretative purposes, items were deemed to load onto a given component if the loading was >0.4 . Items that did not load >0.4 onto any component or those that cross-loaded (i.e., loaded >0.4 onto more than one component) were removed from further analysis. The internal consistency of the remaining scale was measured using Cronbach's alpha . Relationships between the principal component scores and demographic data were explored using a multifactor linear model, with each principal component score as the dependent variable in turn. The independent variables were fitted into the same model and included the fixed factors country, age (presented in range categories), breed, sex, neuter status, presence of a medical condition, whether the dog takes any prescription medications, whether any supplements or nutraceuticals are taken, whether any pheromone products are used and finally the presence/absence of the 7 named problem behaviours asked in the original questionnaire (see Q10 in Supplementary Material S2). Where significant differences were found, post-hoc tests were performed using Bonferroni adjustments. In order to establish normal ranges for the instrument, scores were calculated for individuals for each of the principal components (PCs), using the combination of complete and incomplete datasets. These were calculated by dividing the sum of the scores for the items in the given component by the number of items scored multiplied by 5 (since 5 is the maximum score). Items answered "N/A or Don't Know" were excluded from the calculation, and so the related PC was divided by a smaller number. This produced a component score with a possible range of 0.2-1.0 which allowed for standardisation of the scores and a comparison between dogs. A 'normal range' for each principal component was then calculated based on individuals remaining after the removal of any factors found in the preceding linear models to significantly affect the PC score. 3. Results 3.1. Responses Here, 1441 responses were collected; 248 were excluded for being incomplete. The rate of "N/A or Don't Know" responses was generally low with the highest rate on Item 1 at 12.6%; 82 responses contained >=3 "N/A or Don't Know" answers to the potential resilience items, and so were deleted from the dataset. Missing potential resilience data remained in only 27 responses (2.3%). Missing rates were very low, with the highest rate on Item 7 at 0.5%. This left 1084 complete response sets. No evidence of response sets was identified among respondents. 3.2. Demographics Most respondents were from the United Kingdom (n = 659, 60.8%), followed by the USA (n = 159, 14.7%). Respondents from other countries were classified as "Other countries" for the purpose of analysis. This included 76 (7.0%) from Australia, 36 (3.3%) from Canada, 12 (1.1%) from Ireland, 3 (0.3%) from New Zealand, and 1 (0.1%) from Jersey; 77 respondents (7.1%) selected the "Other/Not on list" option, and 61 respondents (5.6%) did not provide a country. Whilst only a small number of dogs (n = 5, 0.5%) was observed in the "6 months old or less" category, data spanned all five age categories used in the questionnaire (see Supplementary Material S3). The majority of dogs (n = 667, 61.5%) were reported to be pure bred, with 109 breeds represented in the data. A total of 322 (29.7%) reported their dog to be a "Cross-breed" (defined as "a mix between two or more breeds"). Eleven breeds, which each accounted for >1% of the study population, were classified as individual breeds for the purpose of the analysis. All specific breeds comprising <1% were labelled "Other pure-breed" (n = 98) (see Supplementary Material S3 for details). A small number (n = 72, 6.6%) did not provide breed details. There was a relatively even sex split (male = 572, female = 512) with the majority of dogs reported to be neutered: male neutered (n = 427, 39.4%), female neutered (n = 422, 38.9%), male entire (n = 145, 13.4%) and female entire (n = 90, 8.3%). The percentage of respondents who reported their dog as having a pre-existing medical condition was 28.9% (n = 313), and 68.4% (n = 214) of these reported that the condition(s) caused pain. A total of 391 respondents (36.1%) reported an unusual gait or movement, and over one quarter of respondents (n = 309, 28.5%) reported their dog to be currently taking a prescription medication. Nearly three quarters of respondents (n = 785, 72.4%) reported their dog to have at least one problem behaviour. Table 1 details the prevalence of individual problem behaviours within our sample. 3.3. Intra-Rater Reliability (See Supplementary Material S4 for Full Data) Complete responses for the second questionnaire totalled 329. Of these, three provided an email address that could not be matched to the one in the original questionnaire, and nine did not provide an email address. This left 317 completed paired questionnaires available for this part of the analysis. All items other than item 5 and 13 displayed a percentage agreement greater than 40%, with the majority greater than 50%. Spearman's rank order correlations performed on each of the items revealed strong correlations (rho = 0.5-1.0) for 18 of the 19 items, with one item (Item 2) displaying a moderate correlation in the upper part of the range (rho = 0.3-0.49). Wilcoxon signed rank tests revealed significant differences in median test-retest scores for three items: Item 1, Item 3 and Item 13--these items were therefore removed from the questionnaire. 3.4. Inter-Item Correlations and Principal Component Analysis (PCA) (See Supplementary Material S5 for Supporting Details) The correlation matrix of the remaining 16 items revealed that all items satisfied the requirements of correlations >0.4 with at least one other item whilst having no correlations >0.8 with another item. A PCA with Varimax orthogonal rotation was performed on the remaining 16 reliable items, following a Kaiser-Meyer-Olkin (KMO) Measure of Sampling Adequacy and Bartlett's Test of Sphericity indicating the suitability for this type of analysis [KMO = 0.93; Bartlett's Test of Sphericity was highly significant kh2 (120) = 6259.14 (p < 0.001)]. The point of inflection on the scree plot suggested a possible two or three component solution; however, the Kaiser criterion supported the extraction of only two components. These two components explained 55.2% of the variance. Item 2 did not load >0.4 onto either component and was removed from subsequent analysis. Item 16 cross-loaded onto both components, so it was also removed. The remaining 14 items displayed sufficient loading (>0.4) onto only one of the components and so were retained. 3.5. Comparison of 'Complete' and 'Incomplete' Datasets There were no significant differences identified between medians in the complete and incomplete datasets for either of the principal components (PC1 and PC2) or for any of the remaining resilience items (Bonferroni-adjusted p > 0.05, see Supplementary Materials S6 for details). 3.6. Construct Validity of Components The remaining two components with their 14 items were assessed for face validity in relation to relevant resilience constructs. PC1 (11 items) appeared to reflect Adaptability/Behavioural Flexibility and PC2 (3 items), Perseverance (Table 2). The main construct item (Item 19: I believe my dog to be a resilient individual i.e., ...) loaded strongly (0.79) onto principal component 1 (PC1). The resulting instrument was called the Lincoln Canine Adaptability and Resilience Scale (L-CARS)--See Supplementary Material S7 for a copy of the final instrument. 3.7. Internal Consistency Overall Cronbach's alpha for the 14-item scale was = 0.896. Whilst the deletion of PC2 resulted in a very minor increase to the Cronbach's alpha score (=0.907, see Supplementary Materials S8), this was considered trivial, and so it was elected to retain both components in the Canine Adaptability and Resilience Scale. 3.8. Relationship between Principal Component Scores and Demographics The results of the multifactor linear models exploring the effect of demographic variables on the principal component scores (Adaptability/Behavioural Flexibility and Perseverance scores) are summarised below and in Table 3 (details of the content of the variables are included in Supplementary Materials S8). There was a significant relationship with the use of pheromone products on the Adaptability/Behavioural Flexibility Score with the use of pheromone products (diffuser, spray and/or collar) related to a lower PC1 Score (Linear Model: F1,889 = 5.660, Bonferroni-adjusted p = 0.035). There were also significant effects relating to the Adaptability/Behavioural Flexibility Score and the following behaviour problems identified by the owner: unfriendly or aggressive behaviour towards dogs (Linear Model: F1,889 = 75.109, Bonferroni-adjusted p < 0.001); fears or phobias, e.g., noise reactivity/fear, fear of car travel (Linear Model: F1,889 = 243.870, Bonferroni-adjusted p < 0.001); separation-related behaviour problem (Linear Model: F1,889 = 31.687, Bonferroni-adjusted p < 0.001); unfriendly or aggressive behaviour towards people (Linear Model: F1,889 = 59.557, Bonferroni-adjusted p < 0.001); other problem behaviour not listed (Linear Model: F1,889 = 32.975, Bonferroni-adjusted p < 0.001). In each case the PC Score was significantly lower when the behaviour problem was present. There were no other significant relationships with this PC. There was a significant effect of breed on the Perseverance score . Post-hoc analyses compared firstly all pure-breed to cross-breed dogs and then the 11 most common breeds with both "Other pure-breed" and "Cross-breed" dogs. Border Collies were found to have a significantly higher Perseverance score than cross-breeds (Linear Model: F1,889 = 10.083, Bonferroni-adjusted p = 0.037); Greyhounds were found to have a significantly lower Perseverance score than both 'other pure-breeds' (Linear Model: F1,889 = 51.941, Bonferroni-adjusted p < 0.001) and cross-breed dogs (Linear Model: F1,889 = 54.085, Bonferroni-adjusted p < 0.001). No significant differences were detected between pure-breeds and cross-breeds overall. Perseverance scores were also significantly lower for dogs with fears and phobias. All other comparisons were not statistically significant. 3.9. Principal Component Scores Principal component scores were calculated for each individual dog based on the remaining 14 items. This was performed on the entire dataset (n = 1084) as described in the statistical methods. The scores were found to range from 0.2 to 1.0 (see Supplementary Material S8 for full descriptive statistics). The 'normal ranges' for dogs for Adaptability/Behavioural Flexibility (PC1) and Perseverance (PC2) within L-CARS were calculated for individuals following the removal of those with non-permanent characteristics identified as resulting in a significant difference in the PC score. In other words, the Adaptability/Behavioural Flexibility 'normal range' was calculated from scores of individuals not using pheromones and who did not have any of the following problem behaviours: aggressive to dogs, fears or phobias, separation-related behaviour problems, aggressive to people, or another un-named problem behaviours. This gave a mean value +- standard deviation of 0.82 +- 0.10. The Perseverance 'normal range' was calculated based on all individuals other than those with reported fears or phobias and produced a normal reference range of 0.81 +- 0.17. Finally, ranges for each PC were calculated for each of the problem behaviours: aggressive to dogs (PC1 = 0.61 +- 0.15; PC2 = 0.79 +- 0.18); fears or phobias (PC1 = 0.59 +- 0.15; PC2 = 0.76 +- 0.19); separation related behaviour problem (PC1 = 0.63 +- 0.15; PC2 = 0.77 +- 0.19); hyperactivity/overexcitement (PC1 = 0.65 +- 0.16; PC2 = 0.82 +- 0.16); aggressive to people (PC1 = 0.56 +- 0.15; PC2 = 0.77 +- 0.18), repetitive behaviour (PC1 = 0.65 +- 0.17; PC2 = 0.75 +- 0.20); and unnamed problem behaviour (PC1 = 0.59 +- 0.18; PC2 = 0.77 +- 0.20). 4. Discussion We successfully developed a robust and concise psychometric instrument "The Lincoln Canine Adaptability Resilience Scale (L-CARS)", which provides a foundation for the future assessment of trait-level resilience in pet dogs. Conciseness is important to the practicality of the instrument. A scale that is too long is less likely to be used; however, removing too many items can come at a cost through the elimination of potentially meaningful items. A balance must also be struck between the retention and deletion of items in order to maximise specificity, whilst retaining sensitivity. We elected to remove the items that cross-loaded to increase the specificity of the constituent components. Furthermore, as these items did not relate to specific scenarios, their removal was unlikely to greatly reduce sensitivity. PC1 appeared to reflect "Adaptability/behavioural flexibility", and the constituent items seem to align with both the "Engineering Resilience" (ability to bounce back) and the "Adaptive Capacity" facets described by Maltby et al. , and this perhaps reflects a common understanding of resilience. It is therefore not surprising that the construct item specifically asking about resilience loaded strongly on this PC. A dog scoring highly in this domain may be expected to adapt well (and possibly even flourish) in response to change and be able to adjust their behaviour in accordance with differing contexts ('Behavioural Flexibility'). By contrast, PC2 appeared to reflect "Perseverance", which perhaps aligns most closely with the concept of "Ecological Resilience" described by these authors . In humans, this relates to maintaining best efforts and composure in the face of adversity, but in non-human animals may be recognised as an individual persisting in the face of disruption, which reflects the content of the items of the second PC. This is perhaps a less commonly appreciated aspect of resilience, and so the poor loading with the main construct item is not surprising. It is also interesting to note that the perseverance component was associated more with breed-related differences than problem behaviour, and so this might reflect a more general behavioural (rather than psychological) trait. Nonetheless, the two-component solution described here appears to cover the conceptual breadth of resilience described in humans , and so the scale has good face validity. Concurrent validity concerns the degree to which a measure agrees with an established indicator of the facet of interest assessed at the same time. In this regard, the occurrence of behaviour problems associated with fear and anxiety in dogs recorded within the survey may be of particular interest, given the associations identified between resilience and related issues in humans . It is notable that problems, such as fears/phobias, aggressive behaviour and separation-related issues, were strongly related to lower "Adaptability/Behavioural Flexibility" scores, while other problems, such as hyperactivity (which has been found to be unrelated to negative stress levels ) and repetitive behaviour problems (which are commonly related to frustration or play in dogs, e.g., ) were not. This reinforces the suggestion that this component is the main measure of resilience within L-CARS. However, the relationship between the "Perseverance" component and fears/phobias indicates that this second component may also be of importance, hence our decision to retain it within the final instrument. Future work should explore the relationship between these components and other measures of relevance to resilience, including physiological correlates , and further evaluate its discriminant validity. Both principal component scores displayed negatively skewed distributions with a 'normal' mean value around 0.8 out of a possible maximum score of 1.0 and a standard deviation of around 0.1. Although it is possible that these skews arise from a recruitment bias resulting in the attraction of enthusiastic, knowledgeable dog owners who are proud of their dog's purported 'resilience', the prevalence of problem behaviour in our population would suggest that this is not the case. We accept that this instrument assesses owner perception of their dogs' behaviour and it is possible that owners overestimate their dogs' resilience. The literature shows that without training, people can be poor interpreters of dog behaviour . For this reason, further investigation into the validity of this instrument needs to be undertaken. This should include the testing of inter-rater reliability, as well as an examination of convergent validity by investigating physiological and independent behavioural correlates, as has been done in the development of other psychometric scales . However, even if future work confirms a skew within the dog population, this is unlikely to be of great importance given the most probable application of the scale will be in relation to predictions based on those who are less resilient, rather than differentiation between 'normal' and exceptionally resilient individuals. In particular, the relationship between lower scores and the risk of developing a range of behaviour and welfare conditions of concern may be of particular value. At present, the relationships identified with problem behaviour are correlational and the direction of any causal association cannot be identified. It might be that animals with the problems described are seen to be less resilient or that less resilient individuals are at greater risk. In their regard, the association with the use of pheromone-related products is of interest. Whilst the same caveats regarding the causal nature of the relationship apply, it is worth noting that these products were developed in order to help individuals cope with a range of stressors . This scale may therefore provide a useful instrument for evaluating and monitoring the more general effects of these products and other measures aimed at increasing resilience. Although gender and age differences in resilience are widely reported in humans , our data suggest no relationship between resilience and gender, neuter status or age. The reasons for this deserve further investigation. There was also an absence of significant effects in relation to the presence of medical conditions, owner-perceived presence of pain or owner-perceived presence of an abnormal gait, despite the known impact these factors can have on behaviour in dogs . However, these findings are based on an owner-report, which is a common potential limitation of survey-based research. In this regard, it is worth noting that many medical conditions, such as osteoarthritis, are often unrecognised by dog owners , and some inherited disorders may be regarded as 'normal' for the breed , and so there may be many false negatives within the unaffected group. Furthermore, it is interesting that the number reporting an abnormal gait was higher than the number reporting a medical condition, supporting the notion that medical conditions may be under-reported here. Further work associated with more specific diagnostics is warranted to explore this further. 5. Conclusions Understanding the factors that promote a dog's ability to cope with adversity is important for our ability to promote positive welfare. Having a means of assessing resilience is a first step to future studies into this area. Overall, L-CARS showed good correlation with many expected resilience-related associations, and there were no significant relationships that could not be explained in relation to resilience (i.e., no evidence to falsify the validity of the scale). Although it is important that investigation is undertaken to further validate the scale, we believe that L-CARS has good construct validity and provides a useful foundation for future research into canine resilience. Supplementary Materials The following supporting information can be downloaded at: S1: Preliminary 'resilience' items used in Likert Scale and Scoring system; S2: Participant information sheet and full questionnaire; S3: Basic demographic information relating to initial survey; S4: Initial metrics on item quality; S5: Inter-item correlations and principal component analysis (PCA); S6: Metrics relating to comparison of 'complete' and 'incomplete' datasets for The Lincoln Canine Adaptability and Resilience Scale (L-CARS); S7: The Lincoln Canine Adaptability Resilience Scale (L-CARS); S8: Metrics relating to final questionnaire: The Lincoln Canine Adaptability and Resilience Scale (L-CARS). Click here for additional data file. Author Contributions Conceptualisation, E.L.M.M., H.Z. and D.S.M.; methodology, E.L.M.M., H.Z. and D.S.M.; formal analysis, E.L.M.M. and D.S.M.; data curation, E.L.M.M.; writing original draft preparation, E.L.M.M.; writing--review and editing, E.L.M.M., D.S.M. and H.Z.; supervision, H.Z. All authors have read and agreed to the published version of the manuscript. Institutional Review Board Statement The study was conducted in accordance with the Declaration of Helsinki and approved by the Institutional Review Board (or Ethics Committee) of The University of Lincoln (protocol code UoL2022_1069 on 28 June 2022). Informed Consent Statement Informed consent was obtained from all subjects involved in the study--a copy is provided in the Supplementary Materials. Data Availability Statement The data that support the findings of this study are available from the corresponding authors upon reasonable request. Conflicts of Interest The authors declare no conflict of interest. Figure 1 Relationship between breed and Perseverance Score (PC2) (significance codes: * = p < 0.05, *** = p< 0.001). Key: A. Shepherd = Australian Shepherd; B. Collie = Border Collie; E.C.Sp. = English Cocker Spaniel; E.Spr.Sp. = English Springer Spaniel; G.Retr. = Golden Retriever; GSD = German Shepherd Dog; JRT = Jack Russell Terrier; Lab.Retr. = Labrador Retriever; Min.Schnauzer = Miniature Schnauzer; SBT = Staffordshire Bull Terrier. animals-13-00859-t001_Table 1 Table 1 Prevalence of problem behaviours within the sample. Problem Behaviour Category Number Percentage (%) "Unfriendly or aggressive behaviour towards dogs" 407 37.5 "Fears or phobias e.g., noise reactivity/fear, fear of car travel" 355 32.7 "Separation related behaviour problem" 237 21.9 "General hyperactivity/overexcitement" 233 20.6 "Unfriendly or aggressive behaviour towards people" 188 17.3 "Repetitive behaviour including self-mutilation (e.g., compulsive licking/chewing), tail-chasing or shadow chasing" 108 10.0 "Other problem behaviour not listed" 96 8.9 animals-13-00859-t002_Table 2 Table 2 Final 14-item Lincoln Canine Adaptability and Resilience Scale (L-CARS), 2-component solution based on PCA with varimax rotation, with biological interpretations and variance explained by each component. PC Item Number Item (R) = Reverse Scored Variance Explained (%) PC1 Adaptability/ behavioural flexibility 4 If something frightened my dog, he/she would be nervous to return to that location for a long time afterwards (R) 42.61 5 If my dog were to have a bad experience with another individual (dog or person), he/she would forget about it quickly and not hold onto it 6 If something were to startle or frighten my dog, he/she would remain on edge for some time afterwards (R) 8 My dog does not get upset easily 9 My dog generally takes stressful situations in their stride 11 My dog will sometimes seem out of sorts for no apparent reason (R) 12 If another dog has a negative reaction to something, my dog is likely to become upset too (R) 14 My dog enjoys anything that is new or unusual, e.g., objects, animals or anything they have not seen before 15 I would regard my dog to be very adaptable, i.e., able to fit into any situation 18 If something unexpected were to happen and my life circumstances were to change, I know my dog could cope 19 I believe my dog to be a resilient individual, i.e., able to cope with, 'bounce back' from and/or adapt to, adversity or change PC2 Perseverance 7 My dog always tries his/her hardest even when the task is difficult 12.61 10 My dog will persevere even when they do not succeed in something straight away, e.g., when learning a new trick, when trying to solve a puzzle, etc. 17 My dog enjoys challenges, e.g., learning new tricks, finding hidden items, solving a difficult task animals-13-00859-t003_Table 3 Table 3 Details of outputs from linear models using the demographic variables listed, with PC1 and PC2 as the dependent variable (significance codes: * = p < 0.05, *** = p < 0.001). PC1 Adaptability/Behavioural Flexibility PC2 Perseverance Demographic Variable df F p df F p Country 2 3.179 0.084 2 1.338 0.526 Age 4 1.645 0.322 4 2.083 0.162 Breed 12 1.605 0.170 12 8.269 <0.001 *** Sex 1 0.263 1.000 1 0.918 0.677 Neuter Status 1 1.819 0.356 1 1.290 0.513 Pre-existing medical condition 1 1.595 0.414 1 0.298 1.000 Takes prescription medication 1 2.931 0.175 1 2.912 0.177 Takes supplements or nutraceuticals 1 0.312 1.000 1 1.828 0.353 Uses pheromone products 1 5.660 0.035 * 1 0.087 1.000 Problem Behaviour Category: * Aggressive to dogs 1 75.109 <0.001 *** 1 0.030 1.000 * Fears/phobias 1 243.870 <0.001 *** 1 9.749 0.026 * * Separation related behaviour problem 1 31.687 <0.001 *** 1 3.071 1.000 * General hyperactivity/overexcitement 1 1.995 1.000 1 5.037 0.350 * Aggressive to people 1 59.557 <0.001 *** 1 3.156 1.000 * Repetitive behaviour 1 2.930 1.000 1 8.366 0.055 * Other problem behaviour 1 32.975 <0.001 *** 1 3.102 1.000 Disclaimer/Publisher's Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

PMC10000170

The objective of this study was to characterize the dependence and spatial distribution of bedding attributes in an open compost-bedded pack barn (CBP) system with positive pressure ventilation during the winter period in Brazil. The study was conducted in July 2021, in the Zona da Mata region, Minas Gerais, Brazil. The bedding area (shavings and wood sawdust) was divided into a mesh with 44 equidistant points. At each point, the bedding temperature at the surface (tB-sur) and at a depth of 0.2 m (tB-20) and the air velocity at bedding level (vair,B) were measured, and bedding samples were collected. The bedding samples were used to determine the moisture level and pH at the surface (MB-sur e pHB-sur) and at a depth of 0.2 m (MB-20 and pHB-20). The spatial behavior of the variables was evaluated using geostatistics techniques. For all variables, the occurrence of strong spatial dependence was verified. Through the maps, it was observed that tB-sur, tB-20, MB-sur, MB-20, and vair,B showed high spatial variability, whereas pHB-sur and pHB-20 demonstrated low variation. On the surface, values of tB-sur < 20 degC and MB-sur > 60% were observed. At the subsurface, there was a predominance of tB-20 < 40 degC, MB-20 > 60%, and pH > 9, which are indications of low bedding composting activity. dairy cattle housing systems composting bed quality geostatistics CNPq, National Council for Scientific and Technological Development, Brazil422912/2018-2 This research was funded by the CNPq, National Council for Scientific and Technological Development, Brazil, process number 422912/2018-2. pmc1. Introduction The use of facilities free of stalls for dairy cattle housing, able to improve the thermal comfort conditions and ensure greater freedom of movement for the animals, has grown in Brazil in recent decades . A system that is becoming prevalent is the compost-bedded pack barn (CBP), which, in addition to enabling improvements in the comfort, productivity, health, and longevity of the herd, has a lower cost of implementation and greater environmental appeal, as it uses less water to carry waste and enables the formation of compost with good agronomic features . In CBPs, the cows are housed in facilities with a large covered area, with bedding composed of a soft and comfortable organic material substrate, which, under certain conditions, undergoes the decomposition process over time . In this system, the bedding management must ensure hygienic and comfortable surface conditions for the animals, at the same time as enabling the provision of subsurface conditions conducive to the development of aerobic decomposing microorganisms . To promote improvements in the bedding drying process, enhance the activity of aerobic decomposing microorganisms, and carry out the incorporation of waste, the material must be revolved and aerated daily, two to three times . Bedding composting in CBP systems is a biological process, influenced by factors such as temperature, humidity, hydrogenionic potential (pH), nutrient concentrations, and aeration of the bedding material . Thus, variables such as temperature, humidity, bedding pH, and air velocity at the bedding level must be monitored and evaluated frequently. These parameters can be used as indicators of the thermal and hygiene conditions, when evaluated on the bed surface, and of the development of the composting process, when evaluated in the aerobically active layer (0.15-0.30 m) . In fact, evaluating and monitoring the bed environment in CBP systems is important, as the results obtained can be used as a basis for adapting the management applied to the bedding, considered a key point for this system's success . However, few works with this objective were carried out in Brazilian climatic conditions, and this is a knowledge gap that has not yet been solved. For bedding evaluation and monitoring, innovative computational methods should be used, such as geostatistics, a tool that makes it possible to evaluate dependence and spatial distribution and interpret the results from the natural structuring of the data . The use of this tool has been shown to be satisfactory in mapping the thermal and the bedding environment in CBP systems, making it possible to analyze the spatial distribution of the parameters of interest more precisely . In view of the above, the objective of this study was to evaluate and characterize whether the variables temperature, humidity, and bedding pH, as well as air velocity at the bedding level, have dependence and spatial variability in relation to the internal area of one compost-bedded pack barn system with positive pressure ventilation, during the winter period in Brazil. 2. Materials and Methods This study was conducted during three consecutive weeks, in July 2021, the winter period in Brazil. At this time, extreme conditions are observed regarding the management of bedding material in compost-bedded pack barn (CBP) systems, due to excessive humidity and low temperatures. These conditions can compromise the quality of the composting process, cause increased animal soiling, and lead to a loss in milk quality. The study was approved by the Ethics Committee on Animal Use of the Federal University of Vicosa (Process 04/2021). All procedures described were conducted in accordance with the guidelines recommended by the committee. 2.1. Characterization of the Compost-Bedded Pack Barn Installation and Management Techniques To conduct this study, experimental data were collected in a confinement facility for lactating dairy cattle, in an open CBP system with positive pressure ventilation. The facility at which the study was conducted belongs to a commercial property located in the mesoregion of Zona da Mata, Minas Gerais, Brazil (latitude 20deg46'41'' S; longitude 42deg48'51'' W; and altitude 670 m). According to the Koppen climate classification, the climate in the region is Cwa: Mesothermal subtropical, with cold winters and hot and rainy summers . The barn was built in July 2019, was oriented in the southeast-northwest direction and had the following construction characteristics: 60.0 m length; 27.6 m width; 5.0 m of eave height, gabled roof, with structure and metal roof tiles; a central opening with 1.0 m overlap and 0.8 m eaves . The feeding alley was 4.2 m wide, located in the southwest region of the facility, and had four tipper drinkers (2.0 m length each) and a single feeder (60.0 m length). The facility had positive pressure ventilation, provided through six mechanical fans with low volume and high rotation (LVHS). The fans were installed along the facility length, specifically two three-propeller fans, with 1.52 m diameter, 1.5 hp and 86,000 m3*h-1 air flow, installed on the southeast face of the facility, and four six-propeller fans, with 1.53 m diameter, 2.0 hp and 55,000 m3*h-1 air flow, installed in pairs . Such equipment was installed at a 3.0 m height, with a 45deg inclination, and remained activated 24 h*day-1. The facility had a lighting system consisting of 100 W LED lamps (nine in the central region of the bedding and nine in the border region between the feeding alley and drive-through alley). The lighting system was activated only at night (06:00 p.m. to 06:00 a.m.). On the sides, as well as between the bedding area and the feeding alley, there was a small 0.2 m-high wall, with the functions of preventing bedding material loss, passage of bedding material to the feeding alley, and/or waste for the bedding area. In places where tipper drinkers were installed, taller walls (1.2 m) were built to contain the animals, preventing them from having access to water directly from the bedding area, which could cause wetting. In the feeding, service, and drive-through alleys, the floor was composed of beaded concrete. In the evaluated system, the "bedding" substrate consisted of a mixture of wood shavings (<8.0 mm) and sawdust (8.0-25.0 mm) from Eucalyptus, with a thickness of approximately 0.6 m and use time of four months (at the beginning of the study). For the bedding composition, a 0.3-m-thick dry sawdust was added, which, together with the waste deposited by the animals (feces and urine), started the composting process. Subsequently, dry materials (wood shavings and sawdust from Eucalyptus, with moisture content between 10 and 15%) were added whenever the bedding moisture was too high, causing increased dirtiness among animals, excessive compaction, and a consequent anaerobiosis situation in the compost. During the entire experimental period, only bedding replacement with dry material was performed (twice, approximately 8 tonnes each, when it was observed that the bedding was excessively wet). To rotate the bedding material, a hybrid device was used (bedding rototiller with cultivator, 2.0 m wide, five rods, 540 rpm rotational speed of the working element, 0.5 m maximum depth and 0.3 m of effective depth), coupled to a tractor (light line, 78 hp and 2400 rpm nominal rotation of the engine). Turning was conducted twice daily (09:00 a.m. and 04:00 p.m.), according to the farm's standard routine. During the experimental period, the animals housed inside the facility were distributed in two pens (established by the farm), according to milk productivity. The animals with the highest productivity were housed in Pen 1, which had a 518.4 m2 bedding area (36.0 x 14.4 m) and was located near the southeast face of the facility. The animals with lower productivity remained housed in Pen 2, which had a 345.6 m2 bedding area (24.0 x 14.4 m) and was located near the northwest face of the facility. During the experimental period, 80 Holstein cows (pure of origin, 600 kg average weight, in lactation) were housed inside the facility [45 in Pen 1 (11.52 m2*animal-1) and 35 in Pen 2 (9.87 m2*animal-1)]. The area available per cow met the welfare standards recommended by the Ethics Committee on Animal Use of the Federal University of Vicosa. The standard routine of activities in the system was maintained throughout the experimental period, with two milkings being performed and roughage provided twice daily. Milking started at 04:00 a.m. and at 04:00 p.m., with a 2 h 30 min average duration, and were performed in a 2 x 6 fishbone-type room, located in a facility attached to the CBP system, where a waiting area was also located. At all times, the cows had access to the feeding alley, where food (total mixed ration) and water were provided, both ad libitum. The feeding alley floor was washed once a day, in the morning, using a flushing system. For the cows to become familiar with the experiment presence, the experimental collections were performed only from the third experimental day. 2.2. Characterization of the Thermal Microenvironment To characterize the thermal microenvironment, data on the dry-bulb air temperature (tdb) and relative humidity (RH) inside the facility were collected every 5 min, 24 h*day-1, throughout the experimental period. The thermal environment data were used as background for consecutive inferences about the bedding attribute results. In the bedding facility area, the tdb and RH collections were performed using DHT22 sensors (model AM2302; temperature measurement range from -40 to 80 degC and accuracy of 0.5 degC; humidity measurement range from 0 to 100% and 2% accuracy; Aosong Electronics Co., Ltd., Guangzhou, China). The data collected by the sensors were processed and stored using collection stations, which consisted of an Arduino Uno R3 board (ATmega328 microcontroller; 5.0 V supply voltage; 16 MHz clock speed; Atmel Corporation, San Jose, CA, USA), connected to a Data Logger Shield with RTC and SD Reader (SD card slot, integrated real-time clock DS1307; FAT16 or FAT32 card formatting; 3.3 V supply voltage; Dallas Semiconductor, Dallas, TX, USA) and an LCD Display 16 x 2 (I2C Backlight Blue, 5.0 V supply voltage; 4 or 8 bits communication; Beijing Qingyuan Innovation and Technology Development Co., Ltd., Shenzhen, China), following a methodology adapted from Freitas et al. . The sensors were installed 2.5 m above the bedding level, to allow the passage of the tractor used for bedding turning. To characterize the air currents available for the drying of the bedding surface layer, air velocity data were collected at the bedding level (vair,B). The collections were always carried out in the morning (09:00 a.m.), twice in each experimental week, constituting six repetitions. Data were collected using a hot wire anemometer (model TAFR-190, with a scale between 0.1 and 25.0 m*s-1 and 5% accuracy, Instrutherm Instrumentos de Medicao Ltda., Sao Paulo, SP, Brazil). The collections were always carried out with the animal's presence. To collect the tdb, RH, and vair,B data, the bedding area was divided into a regular mesh (6.0 x 4.5 m), consisting of 44 equidistant points, distributed according to the system's construction characteristics . Data collection for the characterization of bedding attributes was performed using the same mesh. 2.3. Characterization of Bedding Material The characterization of the bedding material present in the system was performed by determining the variables of temperature, humidity, and bedding hydrogenionic potential (pH), on the surface and at a depth of 0.2 m. The bedding temperature collection and bedding material samples for the determination of moisture and pH were carried out during the three experimental weeks, twice a week, constituting six repetitions. Data and bedding samples were collected immediately before turning over the bed (08:00 a.m. to 09:30 a.m.), during which time the bed remained for between 16 and 18 h without being turned over. At each mesh point , bedding temperature data and bedding samples were collected at the surface and at a depth of 0.2 m. For data collection on the bedding surface temperature (tB-sur), a digital infrared thermometer was used (model 62 MAX, with a measurement capacity of between -30.0 and 500.0 degC and 1.5% accuracy, Fluke Corporation, Everett, Washington, WA, USA), with a 1.0 m focal length and with emissivity (E) adjusted to 0.9, as recommended by Silva et al. . For bedding temperature measurements at a depth of 0.2 m (tB-20), a rod thermometer was used (model TP101, with a scale of -50.0 to 300.0 degC and 2% accuracy, Pyromed Instrumentos de Medicao e Controle, Contagem, MG, Brazil), which was inserted into the bed for approximately one minute at each collection point, or until its temperature reading stabilized. To determine the moisture and pH values of the bedding material, samples were collected at each of the sampling points. The collections were carried out on the surface and at a depth of 0.2 m, using a post hole digger with marked depth values. The material was collected in the morning, immediately before turning over the bed, twice a week, during the three experimental weeks. The samples collected at each point were placed in hermetically sealed and duly identified plastic packages. After each collection, the samples were placed in isothermal boxes and sent to the Laboratory of Anaerobic Digestion of the Center for Research in Ambience and Engineering of Agroindustrial Systems at the Federal University of Vicosa, in Vicosa, Minas Gerais, Brazil. The pH of the bedding samples was measured in distilled water, using 10 g of the compound sample and 25 mL of distilled water (ratio 1.0:2.5), according to the methodology described by Zhao et al. . To perform the readings, a digital benchtop pH meter was used (model PH-2600, with a measurement scale between 0 and 14 and 0.01 precision, Instrutherm Instrumentos de Medicao Ltda., Sao Paulo, SP, Brazil), duly calibrated (Instrutherm Calibration Certificate Number 121239/21). The moisture of the bedding samples was determined using the standard oven method (gravimetric), following the methodology described by Teixeira et al. . Samples were weighed using an analytical balance (model AY220, with a 220.0 g measuring capacity and 0.0001 g accuracy, Shimadzu Corporation, Kyoto, Japan), duly calibrated, and drying was carried out in an oven at 105.0 +- 5.0 degC. The moisture, as a percentage, was calculated through the ratio between the mass of water removed and the mass of dry sample, multiplied by 100. 2.4. Statistical Analysis 2.4.1. Descriptive Statistics Analysis To characterize the thermal microenvironment inside the CBP system, the hourly mean value and standard deviation of the dry-bulb air temperature (tdb) and relative humidity (RH) variables were calculated. From the calculated data, daily behavior curves of these variables were generated. The variables of bedding temperature at the surface (tB-sur), bedding temperature at a depth of 0.2 m (tB-20), bedding moisture at the surface (MB-sur), bedding moisture at a depth of 0.2 m (MB-20), bedding pH at the surface (pHB-sur), bedding pH at a depth of 0.2 m (pHB-20), and air velocity at bedding level (vair,B) were initially analyzed using descriptive statistics, through the R Development Core Team computer system . Mean, median, minimum, maximum, standard deviation (SD), and coefficient of variation (CV) data were obtained. The variability of the experimental data was evaluated using the CV classification proposed by Warrick & Nielsen : CV < 0.12 = low dispersion; 0.12 <= CV < 0.24 = moderate dispersion; and CV >= 0.24 = high dispersion. 2.4.2. Geostatistics Analysis The spatial behavior of the attributes of interest (tB-sur, tB-20, MB-sur, MB-20, pHB-sur, pHB-20, and vair,B) was analyzed using geostatistics techniques, which made it possible to verify whether they had a dependency and visualized their spatial distribution. The analyses were performed using the R Development Core Team computer system , through the geoR library . Using Matheron estimator , semivariogram adjustments were performed (Equation (1)). (1) g^h=12Nhi=1NhZXi-ZXi+h2 where g^h is the semivariance, Nh is the number of pairs of experimental observations ZXi and ZXi+h, and h is the distance between the experimental observations. The experimental semivariograms were fitted using the ordinary least squares (OLS) and restricted maximum likelihood (REML) methods. The spherical, exponential, and gaussian models (Equations (2)-(4), respectively) were tested, as described by Vieira et al. . (2) g^h=C0+C1x1.5xha-0.5xha3, if h<=aor g^h=C0+C1, se h>a (3) g^h=C0+C1x1-e-3ha (4) g^h=C0+C1x1-e-3xha2 where C0 is the nugget effect, C1 is the contribution, and a is the range. The evaluation and choice of the adjusted semivariograms was carried out using cross-validation procedures, calculating the mean error (ME), mean-error standard deviation (SDM), reduced error (RE), and reduced-error standard deviation (SDR), as detailed by Ferraz et al. . For each variable, the semivariogram adjustment was chosen in which the ME and RE were closer to zero, lower SDM and SDR closer to one, as recommended by Isaaks and Srivastava . From the mathematical models g^h chosen, the coefficients of the theoretical semivariogram were obtained, referred to as the nugget effect (C0), contribution (C1), sill (C0 + C1), range (a), and practical range (a'). For spatial dependence analysis, the spatial dependence index (SDI) was used, obtained through the ratio between the nugget effect (C0) and the sill (C0 + C1). The SDI assessment was performed using the classification by Cambardella et al. : SDI <= 0.25 = strong spatial dependence; 0.25 < SDI <= 0.75 = moderate spatial dependence; and SDI > 0.75 = weak spatial dependence. Once the occurrence of spatial dependence was verified, interpolation was performed using ordinary kriging to obtain the levels of the variables in non-sampled locations in the bedding area. Based on the interpolated data, response surface maps were generated using the computational program ArqGIS(r), version 10.1, licensed for use by the Department of Agricultural Engineering of the Federal University of Vicosa (DEA/UFV). Seeking to obtain greater detail in the variables' spatial distribution, the percentages of area occupied by each interval were calculated and histograms generated. 3. Results and Discussion 3.1. Hourly Average Characterization of the Thermal Microenvironment Inside the Facility Figure 2 shows the hourly curves of the average data recorded during the winter experimental period for dry-bulb air temperature (tdb, in degC) and relative humidity (RH, in %), inside the open compost-bedded pack barn facility. As seen in Figure 2, the average values of tdb inside the facility ranged from 9.7 +- 2.5 degC (05:00 a.m.) to 22.8 +- 2.4 degC (03:00 p.m.). The lowest mean tdb values were recorded in the morning (00:00 a.m. to 06:00 a.m.) and night (06:00 p.m. to 00:00 a.m.) periods, when the mean tdb was less than 18.0 degC. The highest mean values were recorded in the afternoon period (12:00 a.m. to 6:00 p.m.), when the mean tdb was greater than 18.0 degC. For RH, it was observed that the hourly average values curve had behavior contrary to that observed for tdb, with the occurrence of high values in the night and dawn periods (06:00 p.m. to 06:00 a.m., RH > 80.0%) and decreasing values in the morning and afternoon periods (06:00 a.m. to 06:00 p.m., RH <= 80.0%). Even during the day, the mean RH values inside the facility remained high (>=60.0%). Further details on the thermal variations during the experimental period within the evaluated CBP system can be found in Oliveira et al. . 3.2. Characterization of the Spatial Variability of Bedding Attributes Table 1 lists the methods, models, and parameters estimated from the adjusted experimental semivariograms for the variables tB-sur, tB-20, MB-sur, MB-20, pHB-sur, pHB-20, and vair,B. For all variables, the best adjustments were obtained using the restricted maximum likelihood (REML) method (Table 1). This method has been used to adjust small data groups, such as those commonly found in the animal ambience area, as it results in less biased estimates . For the variables MB-sur, MB-20, pHB-sur, and pHB-20, the best fits of experimental semivariograms were obtained using the spherical model, while, for tB-sur, tB-20, and vair,B the Gaussian model returned better fits (Table 1). Both models are adequate to describe the spatial variability of these variables, since their functions are conditional positive, a condition that ensures that the calculated variances are also positive . The adequacy of the cited models to describe the spatial variability of the variables was reinforced by the data obtained by cross-validation, in which ME and RE values close to zero (<0.01) and SDR close to one were observed. These results corroborate those described by Andrade et al. , Silva et al. , Oliveira et al. , and Peixoto et al. , who, when studying the spatial distribution of bedding variables in CBP systems, also obtained better adjustments using the spherical and Gaussian models. According to Ferraz et al. , one of the main geostatistics parameters is the nugget effect (C0), which refers to unexplained variability, considering the distance between sampled points. For all variables, the C0 values were low (near or equal to zero), an indication that they had low unexplained variability, and that the adjusted semivariograms did not have discontinuity (Table 1). Only for the pHB-sur variable was the C0 value different from zero, but low, when evaluated in relation to the sill. The C0 can be attributed to several factors, such as collection and/or analysis errors, local variations, etc. As it is not possible to quantify the contribution of each factor directly and individually, it is important that other evaluation forms are used . One of the most common methods is through the spatial dependence index (SDI), which expresses the C0 in relation to the sill (C0 + C1), and makes comparisons possible, through the classification of Cambardella et al. . Using the SDI to evaluate the contribution of C0 in the sill composition, it is observed that the values obtained were lower than 0.25 (Table 1). Therefore, there was strong spatial dependence for all variables, and only for pHB-sur was an SDI value different from zero (0.2384) obtained, but still lower than 0.25. According to Curi et al. , the results obtained with the kriging interpolation techniques are better when low nugget effect contributions are obtained for the sill. Therefore, it can be concluded that the results achieved using the kriging techniques satisfactorily represented the studied attributes. These results also show that the bedding conditions were not homogeneous in the evaluated CBP system, as stated by Andrade et al. . In geostatistics, another parameter of great importance is the range (a), which is used to determine the spatial dependence limit, separating correlated samples from independent samples . As can be seen in Table 1, for all the attributes, values were obtained that were greater than the shortest distance between collection points (4.5 m), with the smallest being obtained for the variable vair,B (6.0831 m). The occurrence of values of a greater than the distance between collection points was an indication that the mesh used was adequate for the purposes proposed in this study. Through the models and parameters of the semivariograms adjusted for the variables (Table 1), it was verified that they did not have random distribution in space, since there was spatial dependence occurrence. Therefore, it was possible to use ordinary kriging to predict the variables' levels in non-sampled locations, and to obtain spatial distribution maps. Figure 3 shows the spatial distribution maps of the tB-sur and tB-20 variables. Through the models and parameters of the semivariograms adjusted for the variables (Table 1), it was verified that they did not have random distribution in space, since there was spatial dependence occurrence. Therefore, it was possible to use ordinary kriging to predict the variables levels in non-sampled locations, and to get spatial distribution maps. Figure 3 shows the spatial distribution maps of the tB-sur and tB-20 variables. For tB-sur and tB-20 , it can be observed that close mean (X ) and median (Med.) values were obtained, indicating that the collected data did not have accentuated asymmetry . Based on the CV classification proposed by Warrick and Nielsen , it can be observed that the dispersion of the tB-sur data was low (CV < 0.12), whereas that for the tB-20 data was moderate (0.12 <= CV < 0.24). According to Eckelkamp et al. , the bedding surface temperature in CBP systems is influenced by the environment and therefore increases or decreases according to the recorded dry-bulb air temperature levels. On the bedding surface, ventilation removes heat, through the evaporation of the water part present there, and causes tB-sur values to be observed close to the environment . Considering the authors' statement, the tB-sur can be evaluated based on the dry-bulb temperature of the air (tdb), whose hourly variation curve is illustrated in Figure 2. During the period of tB-sur and tB-20 collection (08:00 a.m. to 09:30 a.m.), mean dry-bulb air temperature values were between 13.0 +- 2.1 degC and 19.0 +- 2.2 degC. Therefore, by recording the average tB-sur values between 14.1 and 19.6 degC along the bedding area , this attribute was found to be directly related to tdb. The tB-sur results observed in this work corroborate those described by Andrade et al. , who evaluated a closed CBP air-conditioned system by negative pressure ventilation associated with adiabatic cooling, during the winter and summer periods, always in the morning. In the winter period, the authors reported that mean tB-sur values between 13.8 and 17.8 degC were recorded, with higher levels observed in the bedding's central region. According to Janni et al. , tB-sur is important for thermal comfort, given that the activity of decomposing microorganisms is non-existent or low on the bedding surface. For this reason, it is recommended that the tB-sur levels be kept within the thermoneutral range for lactating dairy cattle (4.0 <= tdb < 24.0 degC, according to Naas ), ensuring a thermally comfortable surface. Through Figure 3a, it is observed that the average tB-sur levels recorded during the winter experimental period (08:00 a.m. to 09:30 a.m.) were lower than the upper threshold of thermal comfort for lactating dairy cattle (24.0 degC). According to Almeida et al. , under conditions of thermal comfort, these animals spend approximately 50.0% of the time lying down. Therefore, it can be inferred that the tB-sur during the evaluated period (winter mornings) was adequate for the animals to remain lying down, a condition that makes it possible to reduce their energy expenditure to maintain their body's core temperature. In any case, we observed the occurrence of tB-sur spatial variability along the bedding area , with lower levels recorded in the peripheral region, especially close to the southeast face of the facility (tB-sur < 16.0 degC). In this region, there were two fan lines (levels 0 and 12 m, from southeast to northwest), which remained activated 24 h*day-1. Therefore, it can be inferred that the lowest tB-sur levels occurred due to the air flow generated by the fans, which make it possible to cool the bedding surface, as stated by Black et al. . On the other hand, in the central region and close to the northwest face of the bed area, higher tB-sur levels were recorded . It can be inferred that the higher tB-sur values observed in this region are due to the low vair,B recorded in these locations, which were not effective in reducing the heat on the bedding surface, as recommended by Llonch et al. . Despite the fans remaining in operation 24 h*day-1, their distribution along the bedding area of the facility was irregular, suggesting that it was not possible to ensure homogeneous tB-sur conditions . Near the southeast face of the facility, four fans were installed (0 and 12 m, from southeast to northwest), but in the rest of the bedding area, there were only two more fans (36 m, from southeast to northwest). Therefore, through the results illustrated in Figure 3a, it can be inferred that the number and arrangement of fans used in this facility were not satisfactory in promoting a reduction in the average tB-sur values in the entire bedding area, especially in more distant areas, such as near the northwest face of the facility. Through Figure 3b, it can be observed that there was high tB-20 spatial variability along the bedding area, where mean values between 17.8 and 49.6 degC were recorded. As expected, tB-20 values were higher than those observed on the bedding surface. This is, therefore, an indication that the bedding decomposition process was taking place in an active manner, since the decomposing microorganisms' activity results in heat production . According to Black et al. , the desired temperature range in the aerobically active layer of CBP systems (0.15 to 0.30 m) corresponds to the range of 43 and 65 degC, and below 40 degC, the material degradation is slow, while, above 55 degC, pathogens are eliminated. Based on this tB-20 range, it was found that in a huge portion of the bedding area, the average tB-20 values were lower than desired in the aerobically active layer . Along the bedding area, the lowest average tB-20 levels were recorded in peripheral regions (southeast faces--extremity; and southwest--between the bedding and the feeding alley) . Regarding the observation of low tB-20 values in these regions, two potential justifications can be mentioned. The first is the relative difficulty of turning over the bedding material in these places, due to the proximity to dividing walls. The second is the intense movement of the animals from the bedding area to the feeding alley, where they had access to food and water. In both cases, it is inferred that there was a trend toward the compaction of the subsurface bedding layer and, as a consequence, a reduction in the oxygen concentration, decomposition activity, and heat production was seen . In contrast, mean desired tB-20 levels (>40 degC) were recorded only in two specific regions of the central bedding area of the facility: between the first two fans' lines, and immediately below fan line three . In the first case, it is estimated that the highest ventilation rate provided (86,000 m3*h-1 of air flow--first fans' line) ensured the supply of the necessary aeration in the region. In the second case, it is assumed that in the region below fan line three, there was no formation of compacted subsurface structures, due to the presence of the electric dividing fence between pens 1 and 2, which prevented the animals' movement and ensured that the bedding was not compacted. Therefore, it is understood that in this region, the supply of adequate oxygen to the decomposing microorganisms was ensured, as evidenced by the highest tB-20 levels recorded . To obtain detailed numerical information about the fractions of area occupied by each attribute class, frequency distribution graphs of tB-sur and tB-20 were generated, as shown in Figure 4. In the entire bedding area, the mean tB-sur values recorded were lower than 24 degC , an indication that the surface temperature conditions of the bedding were favorable for the animals to remain lying down. However, given the similarity between the tB-sur values and ambient temperature (tdb) observed in this study and portrayed by other authors , it can be inferred that the tB-sur was not suitable for the animals to remain lying down at certain times of the day. In the period from 01:00 p.m. at 04:00 p.m., tdb values greater than 24 degC were recorded . Therefore, it is estimated that in this period, the tB-sur interval in some regions may have been higher than the thermal comfort threshold for lactating dairy cattle. In approximately 87% of the bedding area, the recorded tB-20 was below 40 degC , an indication that the bedding degradation process in this facility was occurring slowly, as well as that the compost obtained may not have had good agronomic features . The tB-20 results portrayed in this study corroborate those observed by other authors who evaluated the bedding quality in CBP systems in Brazil and observed the occurrence of tB-20 values below 40 degC . Figure 5 shows the spatial distribution maps of bedding moisture at the surface (MB-sur) and at a depth of 0.2 m (MB-20). As verified for tB-sur and tB-20 , it can be observed that for MB-sur and MB-20, close mean (X ) and median (Med.) values were obtained . Therefore, it can be inferred that the data distributions of these variables did not have accentuated asymmetry . Regarding data variability, in both cases, moderate dispersion was observed (0.12 <= CV < 0.24), according to the classification suggested by Warrick and Nielsen . Similar spatial distributions were obtained for MB-sur and MB-20 , but the variation range was greater at the bedding surface (67.1% and 55.0%, respectively). This fact occurred due to the spillage of water by the animals after ingestion and during the cleaning of the feeding alley, as well as the deposition of feces and urine in the bedding's superficial layer. The recording of MB-sur and MB-20 data with high variation is an indication that the ventilation system and the management applied to the bed were not able to ensure homogeneous conditions of humidity throughout the facility area. The high variation amplitudes of MB-sur and MB-20 depicted in this study corroborate the results described by Oliveira et al. , who evaluated a closed CBP system during the spring period and observed variation amplitudes close to 50% at the bedding surface and at 0.2 m depth. On the other hand, they differ from the results portrayed by Andrade et al. , who evaluated bedding moisture in a closed CBP system during the winter period and obtained spatial distributions of MB-sur and MB-20 with low variation amplitudes (approximately 8%). According to Black et al. , the moisture levels in the bedding must be kept within the range of 40 to 60%, to ensure the maintenance of aerobic conditions and the survival of decomposing microorganisms. As one of the objectives of CBP systems is to provide a comfortable and hygienic surface for housed dairy cattle, keeping the bedding moisture below 60% is also important for the animals' health and milk quality . In the present study, however, MB-sur and MB-20 values were greater than 60%, with higher levels always recorded near the northwest face and along the vicinity of the feeding alley . Close to the feeding alley, it is inferred that there was a tendency toward an increase in the bedding moisture levels, due to the spillage of water by the animals after ingestion, splashing of water during the cleaning of the feeding alley floor and greater deposition of feces and urine, as portrayed by Andrade et al. and Oliveira et al. . At the northwest extremity, in a region close to the feeding alley, MB-sur and MB-20 values were always higher than 80% . In this location, bedding wetting occurred due to the water overflow from the feeding alley to the bedding area during floor cleaning (flushing). When cleaning the floor, there was an overload of waste and water in the gutter, causing overflow. To correct this problem, the waste gutter could be renovated and expanded, and a slightly higher dividing wall could be built at the site (>0.2 m). Even if the greater deposition of feces and urine was observed in the region close to the feeding alley, the existing ventilation system and the bedding management should have been effective in promoting the removal and incorporation of the excess moisture in the bedding. In view of the results illustrated in Figure 5, it can be inferred that the ventilation system and bedding turning were not efficient in promoting the uniform drying of the bedding in the facility, given the elevated moisture levels observed. In the superficial layer (MB-sur), the bedding moisture is more important in relation to health, animal cleanliness, and milk quality. When it is excessively humid, as observed in approximately 37% of the bedding area , the material tends to adhere to the surfaces of the animals' bodies, increasing their dirtiness, mastitis risk and somatic cell count (SCC) . On the other hand, in peripheral regions of the facility , MB-sur and MB-20 values were low (<40%), which may have occurred due to excessive bedding drying in these areas, which received direct solar radiation at certain times of the day. According to Oliveira et al. , the registration of regions with low moisture values in the bedding surface layer is desirable, as it will provide better conditions of cleanliness and health for the animals. Keeping MB-20 at adequate levels is important for the composting process. In the subsurface layer, it was observed that in approximately 5.5% of the bedding area, the MB-20 values recorded were lower than the minimum level recommended for the maintenance of the bedding composting process (40%) . Although not very representative, it can be inferred that the decomposition activity was slower or nonexistent in this bedding area percentage, since MB-20 levels below 40% can lead to a reduction in the populations of decomposing microorganisms . According to Damasceno , when the bedding material is excessively humid (>60%), as observed in around 49% of the bedding subsurface area , the spaces available for oxygen circulation are filled by water. Consequently, there is a reduction in the oxygen concentration, causing an anaerobiosis condition. Therefore, considering the tB-20 and MB-20 values recorded in the bedding's subsurface layer , it is estimated that the material decomposition process occurred slowly in this facility. Furthermore, based on the low tB-20 levels and high MB-20 levels recorded in the bed, it can be inferred that the compost formed may not have good agronomic features, due to the potential nutrient leaching and non-elimination of pathogens . The high MB-20 values portrayed in this study differ from those reported by other authors . These authors evaluated the bedding moisture levels in the subsurface layer (0.15-0.20 m) of CBP systems in distinct locations in Brazil and observed that the MB-20 values were always lower than 70%. Regarding bedding management in this CBP system, two points must be considered. The first is related to the bedding's replacement with dry material, which was performed twice during the period, but the amount added was not sufficient to maintain the bedding moisture at adequate levels. The second refers to the turning of the bedding twice a day (09:00 a.m. and 4:00 p.m.), with a long interval between the second turning of one day and the first of the next day (16-18 h). Based on the MB-sur and MB-20 results obtained in this study , which reflected the bedding conditions after an extended period without turning, it is pertinent to consider performing one additional daily turning operation, preferably at the time of the first milking (04:00 a.m.). At the same time, it is recommended to reallocate the times of the other turnings to late morning (after 11:00 a.m.) and late afternoon (after 5:00 p.m.). In this way, it is expected that it will be possible to promote the homogenization of the material, incorporation of oxygen, and removal of excess moisture levels, considering that the bedding's adequate management is a primary factor for the CBP system's performance . Figure 7 illustrates the spatial distribution maps of pHB-sur and pHB-20 during the winter period. The mean and median values of pHB-sur and pHB-20 were close and, therefore, their distributions did not mark asymmetry . In both cases, the CV was less than 0.12 and characterized by low variability, according to the classification proposed by Warrick and Nielsen . In both bedding layers evaluated, the pH showed low spatial variability during the winter period , with variation amplitudes equal to 1.2 and 1.3, at the surface and at 0.2 m depth, respectively. The spatial distribution behaviors were similar on the surface and subsurface, an indication that the phenomena that govern the distribution of this variable in the bedding are similar between layers, and that the bedding turning ensured the relative homogenization of the material along the profile. Considering that the bedding decomposition process occurred in an analogous manner along the profile, it is pertinent to state that the compost generated at the end of the composting process may have slightly uniform agronomic features along the bedding depth, as portrayed by Oliveira et al. . According to Pereira Neto , the pH range considered ideal for most decomposing microorganisms is between 5.5 and 8.0. Other authors claim that the optimal range is wider, given that most enzymes are active in the range of 5.5 to 8.5 and, therefore, this would be the desired range during the composting process . In the present study, it was observed that the bedding pH values were above the recommended values in the entire bedding area, in both evaluated layers . pH values lower than 8.5 were obtained only in some peripheral regions of the bedding area (on the four faces). By means of joint spatial distribution map observations of temperature , humidity , and bedding pH , it was verified that the regions in which the lowest average pH values were obtained (<=8.5) coincided with the locations where lower temperatures (<30 degC) and/or higher moisture levels (>80%) were observed. Therefore, we can infer that the lower pH levels in these places were due to slow or nonexistent decomposition activity, due to the low temperatures and high moisture recorded in the bedding material, which can limit or inhibit the development of microorganisms acting in the composting process . On the other hand, pH values lower than 8.5 were not very representative in the system evaluated in this study, given that they were recorded in less than 1% of the bedding area . The average levels of pHB-sur and pHB-20 were mostly higher than 8.5 . Results such as those observed are common in advanced stages of the composting process, in which the complete consumption of the organic acids formed in the initial stage has already occurred, or when the carbon:nitrogen ratio (C:N) is low . The high pH values obtained are also evidence that there may have been losses of nitrogen (N) by volatilization, which are particularly high when the pH is greater than 7.0, according to Bernal et al. . The bedding pH results portrayed in this study corroborate those observed by other authors, in CPB systems in distinct locations and times of the year. Andrade et al. evaluated the bedding pH spatial distribution in a closed CBP system during the winter period and observed the occurrence of values greater than 8.7. Oliveira et al. evaluated the bedding pH in a closed CBP system and obtained pH spatial distributions with values mostly greater than 9.0. Oliveira et al. evaluated CBP systems in the south of the state of Minas Gerais and described the occurrence of mean pHB-20 values of 9.0 +- 0.8. Favero et al. carried out studies on CBP systems in the state of Sao Paulo and observed the occurrence of pHB-20 values greater than 8.5 in the winter period. Radavelli et al. carried out a characterization study of CBP systems in the west of Santa Catarina state and described the occurrence of mean pHB-20 values of 8.7 +- 0.5. According to Favero et al. , pH levels greater than 8.8 may inhibit the growth of environmental mastitis pathogens. Therefore, it can be inferred that the high pH levels observed in the present study may have been beneficial for the animals' health in the evaluated CBP system, as well as for the milk quality produced. In CBP systems, the bedding composting process generally occurs within a year, and it may be accelerated or delayed depending on the material used as a substrate, the animal stocking, the management employed and the frequency and volume of bedding replacement with dry material. The high pHB-sur and pHB-20 values observed in this study may be an indication that the C:N ratio of the bedding material present in this facility was low, despite this material having been changed only four months before the study started. With the intense decomposing microorganisms' activity, common in the initial composting stage, the carbon initially made available is usually consumed, making it necessary to initiate new additions via replacement with dry material. It is through bedding replacement that an adequate carbon supply is ensured, and pH values are maintained within the recommended range for decomposing microorganisms. As the frequency and amount of dry bedding added by replenishment at this facility was low, the pH levels were above the recommended values, and the bedding already showed low carbon availability. These conditions led to low decomposition activity and low heat production, as already portrayed through the tB-20 spatial distribution maps . In cases such as this, it is recommended that more bedding replacement operations be conducted with dry material, as well as that the volume added in each replacement be greater. The spatial distribution map and frequency distribution graph of the air velocity at bedding level (vair,B) are illustrated in Figure 9. Through the figure, it can be observed that the spatial distribution of vair,B was highly heterogeneous throughout the bedding area, given that average values between 0.2 and 4.0 ms-1 were recorded (variation equal to 3.8 ms-1). For CBP systems, it is recommended that the air velocity be kept close to 1.8 ms-1 throughout the animal-occupied zone (AOZ), to ensure bedding drying, removing gases, and favoring thermal exchanges . Considering 1.8 ms-1 as the minimum air velocity for cooling and bedding drying (MVCBD), it was found that in the bedding area, there were regions with vair,B values lower than desired . Along the bedding area, the largest vair,B magnitudes were recorded in the zones where the first two fans' lines operated, an indication that they were due to air currents generated by mechanical ventilation. However, we also noted the occurrence of zones with low vair,B in the regions below fan lines one and three, and in the northwest extremity of the facility. Fagundes et al. evaluated the airflow generated by mechanical fans with low volume and high rotation (LVHS) using computational fluid dynamics (CFD) techniques. The authors observed that in the region immediately below the first fan line, a zone with low air velocity occurred. Of course, as in the CBP facility studied, the fans were installed at a 3.0 m height and with 45deg inclination in relation to the horizontal; the airflow in the immediately anterior region was sucked in and directed by the equipment, forming an area with low vair,B below the first ventilation line . In this study, the vair,B spatial distribution along the bedding area is an indication that the number and arrangement of mechanical fans present in the facility was not adequate, given that regions with low vair,B also formed in the central and posterior regions of the bedding area. Regarding the central and posterior regions of the facility, it can be inferred that the low vair,B values recorded were due to the distance being greater than that recommended between fan lines (12.0 m, according to Damasceno ). As the distances between the second and third ventilation lines and the northwest end were 24.0 m, it was observed that the existing ventilation system was not sufficient to ensure homogeneous vair,B conditions along the entire bedding area . Only in the first 20.0 m of the bedding area, from the southeast to northwest of the facility, the mean vair,B values were equal to or higher than that recommended for drying the bed, removing gases, and favoring thermal exchange (1.8 ms-1). In the rest of the area, as well as in the southeast extremity (near the taller wall), the vair,B magnitude was lower than MVCBD. The vair,B results portrayed in this study corroborate those observed by Oliveira et al. , who evaluated the spatial distribution of thermal conditions in open CBP systems and with different ventilation types (natural, mechanical HVLS and mechanical LVHS) during the fall period. In the CBP system with LVHS ventilation, the authors observed that the vair,B distribution was not uniform, and that there was a predominance of vair,B magnitudes below 1.8 ms-1 in 58.4% of the bedding area. In the present study, it was observed that vair,B was lower than recommended in approximately 70% of the bedding area . Through this observation, the inference is reinforced that the number and distribution of LVHS fans along the evaluated system did not meet the need for ventilation required at the site. To improve the air velocity uniformity throughout the bedding area and, consequently, the levels of temperature and bedding moisture surface, it is recommended that the ventilation system used in the place be resized, changing the number and arrangement of fans present. 4. Conclusions Through the application of geostatistical techniques, it was possible to verify that the variables of bedding temperature at the surface (tB-sur) and at a depth of 0.2 m (tB-20), bedding moisture at the surface (MB-sur) and at a depth of 0.2 m (MB-20), bedding hydrogenionic potential at the surface (pHB-sur) and at a depth of 0.2 m (pHB-20), and air velocity at bedding level (vair,B) had spatial dependence. For all these variables, the occurrence of strong spatial dependence was verified, enabling the application of kriging interpolation techniques and the generation of spatial distribution maps. From the generated maps, it was possible to observe that the variables tB-sur, tB-20, MB-sur, MB-20, and vair,B showed high spatial variability along the bedding area of the facility, while the attributes pHB-sur and pHB-20 had low variation (8.2-9.7). In the superficial layer, the bedding temperature was below 20 degC (14.1 to 19.6 degC), indicating that it was comfortable for the animals to remain lying down. However, the average MB-sur values recorded in the region close to the feeding alley were higher than 60.0%, a condition that can generate health and milk quality problems. At 0.2 m depth, tB-20 levels predominantly below 40.0 degC (87.0% of the area) and MB-20 above 60.0% (49.0% of the area) were observed in some local areas (peripheral regions and close to the feeding alley), indications that the bedding material's decomposition process was taking place slowly, as reflected by the high pHB-sur and pHB-20 values (>9.0). Regarding vair,B, a non-uniform distribution and velocity magnitudes lower than 1.8 ms-1 were found in approximately 70.0% of the bedding area of the facility. Adequate vair,B, levels were observed only in the first 20 m of the facility, from southeast to northwest, because of the fan lines present in this region. Acknowledgments The authors would like to thank the Federal University of Vicosa (UFV), Federal University of Lavras (UFLA), and University of Firenze (UniFl), whose support is appreciated. This work was conducted with the support of the National Council for Scientific and Technological Development, Brazil (CNPq), Coordination of Superior Level Staff Improvement, Brazil (CAPES), and Research Supporting Foundation of Minas Gerais State, Brazil (FAPEMIG). Author Contributions Conceptualization, C.E.A.O., I.d.F.F.T., F.A.D., F.C.d.S. and M.B.; methodology, C.E.A.O., I.d.F.F.T., F.A.D. and F.C.d.S.; formal analysis, C.E.A.O.; investigation, C.E.A.O., I.d.F.F.T., V.C.d.O., P.H.d.M.R., L.F.d.S. and R.R.A.; data curation, C.E.A.O.; writing--original draft preparation, C.E.A.O.; writing--review and editing, C.E.A.O., I.d.F.F.T., F.A.D., F.C.d.S., M.B. and G.B.; supervision, I.d.F.F.T., F.A.D., F.C.d.S. and M.B.; project administration, I.d.F.F.T.; funding acquisition, I.d.F.F.T. All authors have read and agreed to the published version of the manuscript. Institutional Review Board Statement This study was conducted in accordance with the Declaration of Helsinki and approved by the Ethics Committee in Animal Use of the Federal University of Vicosa (protocol code 04/2021, with approval date 16 May 2022). Informed Consent Statement Not applicable. Data Availability Statement The data presented in this study are available upon request from the corresponding author. Conflicts of Interest The authors declare no conflict of interest. Figure 1 Schematic representation of the compost-bedded pack barn system where the data were collected: floor plan with collection points (a) and cross-sectional AA (b). AFD--air-flow direction; dimensions in meters (m); Source: Oliveira et al. . Figure 2 Curves of average hourly values, with standard deviation, of dry-bulb air temperature, and relative humidity throughout the day inside the CBP facility. Figure 3 Spatial distribution of bedding temperature (tB, in degC) at the surface--tB-sur (a) and at a depth of 0.2 m--tB-20 (b). AFD--air-flow direction; X --mean; Med.--median; Min.--minimum; Max.--maximum; SD--standard deviation; CV--coefficient of variation. Figure 4 Frequency distribution of bedding temperature at the surface--tB-sur (a) and at a depth of 0.2 m--tB-20 (b). Figure 5 Spatial distribution of bedding moisture (MB, in %) at the surface--MB-sur (a) and at a depth of 0.2 m--MB-20 (b). AFD--air-flow direction; X --mean; Med.--median; Min.--minimum; Max.--maximum; SD--standard deviation; CV--coefficient of variation. Figure 6 Frequency distribution of bedding moisture at the surface--MB-sur (a) and at a depth of 0.2 m--MB-20 (b). Figure 7 Spatial distribution of bedding hydrogenionic potential (pHB) at the surface--pHB-sur (a) and at a depth of 0.2 m--pHB-20 (b). AFD--air-flow direction; X --mean; Med.--median; Min.--minimum; Max.--maximum; SD--standard deviation; CV--coefficient of variation. Figure 8 Frequency distribution of bedding hydrogenionic potential at the surface--pHB-sur (a) and at a depth of 0.2 m--pHB-20 (b). Figure 9 Spatial (a) and frequency (b) distribution of air velocity at bedding level (vair,B). AFD--air-flow direction; X --mean; Med.--median; Min.--minimum; Max.--maximum; SD--standard deviation; CV--coefficient of variation. animals-13-00786-t001_Table 1 Table 1 Methods, models, and parameters estimated from the semivariograms adjusted for the mean values of bedding temperature at the surface (tB-sur) and at the depth of 0.2 m (tB-20), bedding moisture at the surface (MB-sur) and at the depth of 0.2 m (MB-20), bedding pH at the surface (pHB-sur) and at the depth of 0.2 m (pHB-20) and air velocity at bedding level (vair,B), during the experimental winter period. Variable Method Model C 0 C 1 C0 + C1 a a' SDI ME SDM RE SDR tB-sur REML Gaussian 0.0000 2.8010 2.8010 6.6460 11.5038 0.0000 -0.0774 0.8309 -0.0479 1.0218 tB-20 REML Gaussian 0.0000 76.6540 76.6540 6.4740 11.2061 0.0000 -0.4229 4.3183 -0.0477 0.9759 MB-sur REML Spherical 0.0000 274.2200 274.2200 10.2400 10.2387 0.0000 -0.2836 14.1767 -0.0104 0.9928 MB-20 REML Spherical 0.0000 248.1420 248.1420 9.9570 9.9571 0.0000 -0.1750 13.8022 -0.0066 1.0018 pHB-sur REML Spherical 0.0165 0.0527 0.0692 27.5256 27.5256 0.2384 -0.0053 0.2053 -0.0139 1.0618 pHB-20 REML Spherical 0.0000 0.1079 0.1079 8.6834 8.6834 0.0000 -0.0078 0.3130 -0.0130 1.0251 vair,B REML Gaussian 0.0000 0.6892 0.6892 6.0831 10.5288 0.0000 -0.0120 0.4273 -0.0116 0.8959 C0--nugget effect; C1--contribution; C0 + C1--sill; a--range; a'--practical range; SDI--spatial dependence index; ME--mean error; SDM--mean-error standard deviation; RE--reduced error; SDR--reduced-error standard deviation; and REML--restricted maximum likelihood. 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PMC10000171

Background: The Prostate Health Index (PHI) and Proclarix (PCLX) have been proposed as blood-based tests for prostate cancer (PCa). In this study, we evaluated the feasibility of an artificial neural network (ANN)-based approach to develop a combinatorial model including PHI and PCLX biomarkers to recognize clinically significant PCa (csPCa) at initial diagnosis. Methods: To this aim, we prospectively enrolled 344 men from two different centres. All patients underwent radical prostatectomy (RP). All men had a prostate-specific antigen (PSA) between 2 and 10 ng/mL. We used an artificial neural network to develop models that can identify csPCa efficiently. As inputs, the model uses [-2]proPSA, freePSA, total PSA, cathepsin D, thrombospondin, and age. Results: The output of the model is an estimate of the presence of a low or high Gleason score PCa defined at RP. After training on a dataset of up to 220 samples and optimization of the variables, the model achieved values as high as 78% for sensitivity and 62% for specificity for all-cancer detection compared with those of PHI and PCLX alone. For csPCa detection, the model showed 66% (95% CI 66-68%) for sensitivity and 68% (95% CI 66-68%) for specificity. These values were significantly different compared with those of PHI (p < 0.0001 and 0.0001, respectively) and PCLX (p = 0.0003 and 0.0006, respectively) alone. Conclusions: Our preliminary study suggests that combining PHI and PCLX biomarkers may help to estimate, with higher accuracy, the presence of csPCa at initial diagnosis, allowing a personalized treatment approach. Further studies training the model on larger datasets are strongly encouraged to support the efficiency of this approach. artificial neural network Phi PCLX prostate cancer tumor markers University of Naples Federico IIFinanziamento Ricerca di Ateneo FRA2020 line B AIRC25656 This proposal was funded by Finanziamento Ricerca di Ateneo FRA2020 line B of the University of Naples Federico II and by AIRC Grant Programme (grant no. 25656). pmc1. Introduction Widespread use of prostate specific antigen (PSA) for the early detection of prostate cancer (PCa) led to a high number of unnecessary biopsies and increased diagnosis of indolent PCa . This low specificity prompted the researchers to find new biomarkers not only for early PCa detection, but also for better discrimination of clinically significant PCa (csPCa, defined as GG >= 2) . In this context, several new tests have been proposed--one of the most promising is the prostate health index (PHI; Beckman Coulter). The PHI score is calculated combining the values of PSA, %fPSA, and [-2]proPSA. Several authors showed that PHI has a good ability to detect csPCa . More recently, the Proclarix (Proteomedix, Switzerland) blood test has been developed. The Proclarix score is obtained using thrombospondin-1 (THBS1), cathepsin D (CTSD), total PSA (tPSA), free PSA (fPSA), and patient age values . We previously demonstrated that the combination of PHI and Proclarix test results synergistically improved the diagnostic performance of the individual test . On this basis, we hypothesized that it could be valuable to explore whether a new model comprising the kallikrein markers included in PHI and the cancer-related markers of Proclarix may achieve higher values of sensitivity and specificity for the detection of both all cancers and csPCa than the tests alone. To address this issue, considering the complexity of the problem, we used an artificial neural network (ANN)-based approach. The number of potential variations that can be derived from a combination of five variables extracted by different subjects is very large. ANN is better than deterministic models to describe such extremely complex datasets and to reveal laws of the relationships among variables by analysing the output of the system . We developed a deep learning algorithm that can identify PCa and csPCa from a panel of five clinical variables plus the patient's age. The levels of (i) total PSA (tPSA), (ii) free PSA (fPSA), (iii) the isoform-2 of proPSA (p2PSA), (iv) thrombospondin-1 (THBS1), and (v) cathepsin D (CTSD) were determined in 344 patients using automated immunometric and ELISA assays. Based on our previous studies , a model containing those variables can potentially detect PCa and csPCa early with higher accuracy compared with PHI and Proclarix individual test results. As shown in a recently published systematic review , several studies demonstrated the potential of ANNs for the identification and validation of PCa biomarkers. PSA and the other biomarkers are commonly used for PCa diagnosis, prognosis, and follow-up. However, an AI-based approach can provide several advantages such as managing enormous data sets, identifying complex relationships, and using very few resources. Our data confirmed that such an approach is useful to identify new tools for clinical management of PCa. Moreover, according to other authors , we found that the ANN model has the highest accuracy to predict all cancers and csPCa. 2. Materials and Methods 2.1. Study Population Serum samples (n = 344) were collected at two clinical centres: 159 (97 with PCa) from the Department of Translational Medical Sciences, Federico II University, Naples, Italy, and 185 (91 with PCa) from the Martini-Klinik, University Hospital Hamburg-Eppendorf, Hamburg, Germany. All samples were obtained consecutively from May 2020 to July 2021 (Naples) and from 2013 to 2016 (Hamburg) before prostate biopsies. A systematic 12-core TRUS-guided biopsy was executed in Hamburg, while patients in Naples had mpMRI-guided biopsy. Primary and secondary Gleason scores (GS) were assigned by a genitourinary pathologist, according to the 2005 consensus conference of the International Society of Urological Pathology definitions . Men with total PSA values between 2 and 10 ng/mL were included and subjects receiving drugs (i.e., 5-alpha reductase inhibitors) were excluded from the study. Prostate volume was assessed by TRUS. The study was approved by the local ethics committees (prot. n. 118/20 for the institutional Ethics Committee of the University of Naples Federico II). Participants provided written approved consent. 2.2. Determination of Proclarix and PHI Patients had blood drawn before digital rectal exploration (DRE). Whole blood was allowed to clot before serum was separated by centrifugation. Serum aliquots were stored at -80 degC until samples were processed, according to Semjonow et al. . PSA, fPSA, and p2PSA were measured by Access2 Immunoassay System analyzer (Beckman Coulter, Brea, CA, USA) calibrated against the WHO standard for PSA and fPSA. The analytical performance of the measurements assessed with control materials (Beckman Coulter) showed values within the allowed recommended limits. Measurement of Proclarix was performed by CTSD and THBS1 ELISA from the Proclarix kit (Proteomedix), as described previously . Total PSA and free PSA values were determined using a Cobas system (Roche) for Hamburg samples and on an Access2 Immunoassay System analyzer (Beckman-Coulter) calibrated against the WHO standard for Naples samples. In both cases, the Proclarix score was calculated according to the instruction for use using the online risk-score calculator (www.proclarix.com/risk/calculator, accessed on 13 October 2021). PHI measurements were conducted at University Federico II (Naples, Italy) for all samples, according to the manufacturer's instructions for use. 2.3. Neural Networks Design Clinical and non-clinical variables of patients suspected of having PCa were processed by a deep neural network model. We used as input variables the values of total PSA, free PSA, p2 PSA, CTSD, and THBS1, determined following the procedure described above. In some tests and more sophisticated versions of the model, we used the age of the patients as an additional input variable. The deep neural network model that we have used in this work is composed of an input layer, an output layer, and four other layers in between. These extra intermediate layers are the functions that operate sequentially on the data, transforming an originating set of variables (input) into a diagnosis on cancer (output), and representing the kernel of the algorithm. In this study, after testing different combinations of neurons and layers, we found that the sequence of layers that optimizes performance is as follows: (1) linear layer, (2) batch normalization layer, (3) non-linear (hyperbolic tangent function) layer, and (4) linear layer. The output layer is represented by a 0/1 data type variable B, such that, if B = 1 (0), the patient is diagnosed (is not diagnosed) with cancer by the model template. The number of nodes in the input layer is 5 (6 when considering age), onto which values of the variables are passed. The number of nodes of the intermediate layers is 6 (7 when considering age). The neural network code was written in Mathematica. Layers of the network were concatenated using the function NetGraph(), then the function NetInitialize() was used to instantiate the neural network model. The algorithm was trained using datasets of varying sample size; elements in the training datasets were chosen randomly from an initial database (O) of 344 patients. For each training set (T), the corresponding validation (V) set was determined as the difference between two sets O and T: V = O - T. The values of total PSA, free PSA, p2 PSA, CTSD, and THBS1, plus age, were concatenated to create a single feature set conveyed to the model input. The training procedure was accomplished using an epoch number of 8000 samples and a batch size of 1024 elements. To test performance, for each neural network configuration, training, and validation sample size, we performed 100 different simulations. As elements in the training and validation sets were selected randomly, in each of these model runs, the composition of the sample was different from the others, causing, as a result, a different output of the neural network model under the same values of parameters and model conditions. In all cases, the neural network model achieved convergence and the error loss settled to within an error range of less than 1% of the steady state value. 2.4. Training and Validation of the Neural Network Model We used training datasets with variable sizes during the learning process to fit the model parameters of the classifier. For the classification task, we used supervised machine learning, i.e., we used labelled datasets to train the algorithm in a way that it may make the diagnosis of PCa accurate. Training samples were labelled using the GS of the patient, i.e., an external variable determined independently from the values of total PSA, free PSA, p2 PSA, CTSD, THBS1, and age, used as input in the neural network model. The GS is an external estimate of the state of a patient and is considered here as the ground truth diagnosis on cancer. The GS is a set of two numbers, the combination of which describes the severity of PCa. The higher the GS, the more likely that the cancer will grow and spread quickly. Cases with no reported GS are considered not clinically relevant. Here, we used two different training criteria based on the GS of data: (i) training without GS stratification, where we categorize samples as cancer-positive if they have GS, however high; and (ii) training with GS stratification, in which case we assumed that samples with a value of GS < 7 (>=7) are indolent (cancer). Parameters and weights of the neural network model were then tuned to match the GS prediction on cancer. To assess the performance of the model, we used validation or test datasets independent of the training data sets, i.e., never used during the training procedure. In doing so, we minimized overfitting. The model's performance was measured using sensitivity and specificity. The sensitivity (specificity) of a binary classifier is the proportion of positive (negative) results that are true positives (negative). 3. Results The ANN Model Performance Our results showed how the neural network (NN) model with the five variables of total PSA, free PSA, p2 PSA, CTSD, and THBS1, plus age, compares to simpler linear regression models of the sole PHI (LRPhi) or Proclarix variable (LRPro). The NN model was built to receive as input the clinical variables associated with patients suspected to have prostate cancer, and to generate as an output the diagnosis of cancer. To train and validate the model, predictions of the NN algorithm were compared to the true health status of the individuals determined through the conventional Gleason score grading system, as described in the methods. Models were trained on a dataset with a size varying from 130 to 260 elements and were validated on a complementary dataset of 215 to 85 elements, such that the original sample size is 344. Here, we present the results of an NN model of all clinical variables plus age. Moreover, in determining whether patients with known levels of total PSA, free PSA, p2 PSA, CTSD, and THBS1--and known age--have csPCa, we used information on the GS. Individuals with a value of GS >= 7 are diagnosed with csPCa. Individuals without or with GS < 7 are not. We measured the performance of the model using the sensitivity (true positive rate) and specificity (true negative rate) of the classification of the model, trained on a dataset of 180 elements, and evaluated on a validation dataset of 164 elements. Moreover, to improve the accuracy and statistical significance, we evaluated the model's performance against 100 different randomized training and validation datasets, sampled from an original population of 344 elements . The efficiency of the NN model was compared to the efficiency of a simple linear logistic regression (LR) model of the PHI and Proclarix variable. The average values of sensitivity (se) and specificity (sp) determined for the models are se = 0.672 and sp = 0.674 for the NN model, se = 0.595 and sp = 0.714 for the LRPhi model, and se = 0.628 and sp = 0.712 for the LRPro model, respectively . The results of the classification test indicate that the NN model provides an improvement over the LR model of PHI and Proclarix in terms of sensitivity, with a measured enhancement of ~13% with respect to PHI (p < 0.0001) and ~7% with respect to Proclarix (p = 0.0001). The results also indicate that the specificity of the classification of the NN model is lower than the specificity measured for the LR model of PHI (p = 0.0003) and Proclarix (p = 0.0006), with a decrease of ~6.8% and ~6%, respectively . An extended test campaign aimed at measuring the performance of the NN model as a function of sample size indicated that the values of sensitivity and specificity of the model showed moderate sensitivity to the number of elements in the training and validation set . For values of the training (validation) dataset dimension varying between 140 (205) and 260 (85), we found that the mean values of sensitivity and specificity of the model oscillated between 0.65 and 0.68 and 0.66 and 0.69, respectively. For each considered size, the distributions of sensitivity and specificity determined for 100 different randomized models are described by the box and whisker plots in Figure 2a (sensitivity) and Figure 2b (specificity). The results shown in Figure 1 and Figure 2 are relative to a model of all considered clinical variables plus age, which makes use of the complete consolidated knowledge on the Gleason score (model with GS stratification). However, to examine whether the model results are affected by the model design, database composition, and rules of inference, we performed several additional tests in which the working parameters of the tests were varied. Using different model templates, different values of the model parameters, and different combinations of the input variables, we generated many output results. Below, we report the results in the same diagrams to enable a direct comparison between models and to draw general conclusions. Figure 3a shows the box and whisker plots of the values of sensitivity, determined over 100 different tests as a function of the model design. The results were determined for the NN of the input variables total PSA, free PSA, p2 PSA, CTSD, and THBS1, with (NN2) or without (NN1) GS stratification, and for the neural networks of the same variables plus age, with (NN4) or without (NN3) GS stratification. These values of sensitivity were compared to those found for the linear logistic regression (LR) model of the PHI variable, with (PHI1) and without (PHI2) GS stratification, and to the values of sensitivity relative to a LR model of Proclarix, with (PRO1) and without (PRO2) GS stratification. The neural network model of the total PSA, free PSA, p2 PSA, CTSD, and THBS1 performed better than the others, with a mean value of sensitivity se ~0.78, either considering or not the additional variable age. In contrast, the sensitivity associated with the Proclarix variable was se ~0.77. The sensitivity of all other model templates was lower than se ~0.70. Figure 3b shows the box and whisker plots of the values of specificity, determined for the same model templates and training and validation datasets, used for determining sensitivity and described above. In contrast to sensitivity, the higher value of specificity was achieved by the LR model of PHI with no GS stratification (spe ~0.71), followed by the NN model of the complete set of variables and by the LR model of Proclarix, both with GS stratification (spe ~0.69). All other values of specificity ranged between 0.61 and 0.68. The results presented in this section are relative to a training (validation) sample size of 200 (145) elements. To verify whether performance was mainly due to the model design, we determined the values of sensitivity and specificity of the classification as a function of the model design and of the sample size, changed between 200 and 260. Density plots of the resulting values of sensitivity and specificity demonstrated that the model's performance showed a very high sensitivity to the model characteristics, and was only moderately influenced by sample size, at least for the range of dimensions considered in this study. Thus, the NN model without GS stratification achieved higher sensitivity than the Proclarix test and significantly higher sensitivity than the PHI test. Regarding specificity, there was not a model that performs significantly better than the others. The results presented in Figure 3 and Figure 4 also indicated that the variability of the NN model output was lower than the variability of the PHI or Proclarix test. As low levels of variation were associated with high accuracy, high precision, and elevated repeatability, collectively, the results suggested that the NN model of total, free PSA, and p2 PSA, as well as of CTSD and THBS1, may be a valuable tool to assess the diagnosis of PCa. 4. Discussion Overdiagnosis and overtreatment of indolent PCa are well-known consequences of PSA use . Several new blood-based tests as well as risk calculators showed promising results , but none are available in routine clinical practice. Thus, there is an urgent clinical need for new tools able to ensure, at the same time, early detection of PCa and no overtreatment. Men with low-risk cancer at initial diagnosis have the option of active surveillance (AS) and deferred treatment, avoiding invasive treatment (radical prostatectomy or radiation therapy) and the associated harmful side effects (81% patients develop erectile dysfunction, 17% urinary incontinence, and 12% bowel dysfunction) . At present, the D'Amico/NCCN and EAU risk stratification systems or Partin's table, developed more than a decade ago, are the gold standard to select patients for AS. More recently, several new biomarkers have been associated with csPCa , and the use of a non-invasive test to stratify the risk of aggressive PCa is an appealing strategy. In this study, we described the ability of an ANN-based model including total PSA, free PSA, p2 PSA, CTSD, and THBS1 to detect high-grade PCa in an initial biopsy cohort of men aged >=50 years. We selected the blood-based proteins of PHI and Proclarix tests as several authors showed that they were significantly associated with an increased risk for a positive biopsy and for a GS of >=7 at RP . Applying a deep learning strategy, we demonstrated that the addition of CTSD and THBS1 to the three different PSA molecular forms included in PHI calculation showed the highest accuracy and precision for the identification of all cancers and high-grade PCa (GS >= 7). The sensitivity and specificity obtained for the ANN model including age demonstrated comparable values to the model when age was excluded, suggesting that circulating biomarkers could be much stronger predictors than age. Accordingly, several new blood-based tests have been proposed in the last decades as tools able to identify csPCa at initial diagnosis . Combining the single biomarkers of PHI and Proclarix by the ANN approach rather than the final scores by logistic regression analysis, as in our previous work , we achieved an improvement in specificity (68% vs. 49%, respectively) for csPCa identification. We previously applied ANNs to predict csPCa, combining only PSA molecular forms with PSA density or PHI with mpMRI , showing encouraging results for this approach. A few other authors used this approach to explore the potential of circulating biomarkers to predict PCa grading and of ANN models to select men for biopsy . Computational intelligence models are near to ideal as tools to identify new strategies for PCa diagnosis and prognosis, as they can deal with the indeterminateness of biomarkers values. It is well recognized that, in every single patient, it is likely one will find different abnormal biomarker results, and their combination is a better way to assess PCa diagnosis and prognosis . For an AI tool to be clinically useful, two conditions must be addressed. First, it must provide the clinicians with information not available via the conventional clinical tools. Second, the tool must provide information affecting patients' clinical management. On this basis, there are several areas of potential clinical utility in the case of prostate cancer: (i) prediction of biopsy outcomes (benign prostatic hyperplasia vs. cancer); (ii) prediction of PCa grading at RP (low-grade vs. high-grade); (iii) prediction of response to conventional therapy (responders vs. non-responders); and (iv) prediction of PCa relapse after surgery (relapsers vs. non-relapsers). In all of these cases, it is not possible to assess the clinical outcome based on the initial data, but such an assessment is able to affect clinical management. So, the clinical utility of such a tool is not necessarily assured by high levels of sensitivity and specificity. Accuracy, sensitivity, and specificity do not necessarily need to be very high to impact clinical management . For example, when the clinical decision may evolve during the disease, even a mild improvement in prediction (e.g., from 50 to 70%) may be considered clinically useful. In addition, tools with the potential to impact the patient's perceptions and behaviors about their disease could also have some clinical utility. The results produced by our study will also provide researchers with data to bridge the gap between models and tools. If tested on a large amount of clinical data at multiple centers, clinical utility will be reinforced. To overcome the obstacles in the clinical translation of the AI model, robust clinical evaluation, via easy-to-use measurements of quality of care and patient outcomes, will be essential. Further investigation is required (1) to identify biases of algorithms and develop actions to address these, (2) to improve generalizability, and (3) to create methods able to facilitate the interpretability of machine learning results. If these goals will be achieved, the clinical utility for patients is likely to be guaranteed. In addition, ANN models showed performance comparable to logistic regression models in predicting csPCa , and such an approach associated with sensors could represent an innovative method for large-scale, rapid, and low-cost PCa screening, aiming to avoid overdiagnosis and overtreatment. Our study has some limitations. Firstly, our study includes data of two populations from two different clinical centres, thus we have no centralized pathology evaluation. Secondly, it includes only patients with tPSA between 2 and 10 ng/mL and we did not evaluate how the model performs in patients with tPSA values higher than 10 ng/mL, who are at higher risk of PCa. However, our present study highlighted the high potential of ANN models for the prediction of PCa aggressiveness. The proposed model combining PHI and Proclarix biomarkers, once prospectively validated on a large study population, could represent an easy-to-use and widely accessible tool to distinguish, at initial diagnosis, patients who harbour high-grade PCa and really need invasive treatment. Thus, it might be useful for a personalized clinical decision-making process. Despite that the combination of five biomarkers is expensive as a first-line test, it will lead to a reduction in the costs of the entire diagnostic-therapeutic pathway of PCa patients, in terms of biopsies, surgical interventions, and the avoidance of drugs for post-surgical complications. Large prospective validation is needed to assess whether our model is associated with overall survival, PCa-specific mortality, and progression-free survival. 5. Conclusions In conclusion, an ANN model including PSA, fPSA, p2PSA, CTSD, and THBS1 showed improved accuracy for the detection of high-grade cancer. Such a model may provide information on disease aggressiveness at initial diagnosis by a non-invasive method, favoring a personalized treatment choice. Thus, these findings strongly encourage to explore the use of ANN-based approaches to identify new tools for the management of PCa patients. Acknowledgments The authors would like to thank the laboratory staff for the support and continual help in sampling and storage of the blood samples. We thank Proteomedix and Beckman Coulter for the support. We are also grateful to all men who took part in this study. We thank Italian Association for Cancer Research (AIRC) for supporting our project. Author Contributions Conceptualization, F.G., D.T., T.S. and R.V.; methodology, F.G.; validation, F.G. and D.T.; formal analysis, F.G.; data curation, E.L.C., M.F., B.D.V., P.T. and F.C.; writing--original draft preparation, F.G. and D.T.; writing--review and editing, D.T.; visualization, D.B.; supervision, D.T.; funding acquisition, F.G. and D.T. All authors have read and agreed to the published version of the manuscript. Institutional Review Board Statement The study was conducted in accordance with the Declaration of Helsinki and approved by the institutional Ethics Committee of the University of Naples Federico II (118/20, approved 5 July 2020). Informed Consent Statement Participants provided written approved consent. Data Availability Statement The data that support the findings of this study are available upon request from the corresponding authors (F.G. and D.T.). Conflicts of Interest T.S. is an advisor to Proteomedix. T.S. is an inventor of the following patent application (WO2018011212). Figure 1 Performance of the neural network model in terms of sensitivity and specificity (a), compared to a linear logistic regression model of the PHI (b) and Proclarix variable (c). In these tests, the neural network model was built to receive, as an input, the pool of five variables: total PSA, free PSA, p2 PSA, CTSD, and THBS1, plus age. In determining performance, samples were stratified using information on the Gleason score. Patients with a value of GS < 7 were considered healthy. Mean values of sensitivity and specificity for the NN and LR models, evaluated over 100 simulations (d). Figure 2 Box and whisker plot of the sensitivity (a) and specificity (b) of the NN model in performing the diagnosis of patients with prostate cancer. In determining the values of sensitivity and specificity, we used information on the tumour Gleason score determined at RP. Patients with a value of GS < 7 were considered healthy. In tests, the neural network model was built to receive, as an input, the pool of five variables: total PSA, free PSA, p2 PSA, CTSD, and THBS1, plus age. Descriptive statistics plots were determined as a function of the training and validation sample size. The left and right boundary, in each box plot, corresponds to the 25% and 75% quartiles, respectively, of the distributions, while the thick white band marks the median value. Black points are the distribution outliers. Figure 3 Descriptive statistics plots illustrating the sensitivity (a) and specificity (b) of different neural networks and logistic regression models. The models were designed to receive as an input the levels of total/free/p2 PSA, the levels of CTSD and THBS1, and the age of patients (NN models); the PHI and Proclarix test (LR model). Moreover, models were conceived to either use or not information about the GS of the patients. Differences between model designs and conditions are reflected by different values of sensitivity and specificity of the classification of tumour cases in 140-element validation datasets. Figure 4 Density plots of sensitivity (a) and specificity (b) of different neural networks and logistic regression models as a function of the model design and size of the training and validation set. Disclaimer/Publisher's Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

PMC10000172

Colorectal cancer (CRC) is one of the most common cancers with a high mortality rate. Early diagnosis and therapies for CRC may reduce the mortality rate. However, so far, no researchers have yet investigated core genes (CGs) rigorously for early diagnosis, prognosis, and therapies of CRC. Therefore, an attempt was made in this study to explore CRC-related CGs for early diagnosis, prognosis, and therapies. At first, we identified 252 common differentially expressed genes (cDEGs) between CRC and control samples based on three gene-expression datasets. Then, we identified ten cDEGs (AURKA, TOP2A, CDK1, PTTG1, CDKN3, CDC20, MAD2L1, CKS2, MELK, and TPX2) as the CGs, highlighting their mechanisms in CRC progression. The enrichment analysis of CGs with GO terms and KEGG pathways revealed some crucial biological processes, molecular functions, and signaling pathways that are associated with CRC progression. The survival probability curves and box-plot analyses with the expressions of CGs in different stages of CRC indicated their strong prognostic performance from the earlier stage of the disease. Then, we detected CGs-guided seven candidate drugs (Manzamine A, Cardidigin, Staurosporine, Sitosterol, Benzo[a]pyrene, Nocardiopsis sp., and Riccardin D) by molecular docking. Finally, the binding stability of four top-ranked complexes (TPX2 vs. Manzamine A, CDC20 vs. Cardidigin, MELK vs. Staurosporine, and CDK1 vs. Riccardin D) was investigated by using 100 ns molecular dynamics simulation studies, and their stable performance was observed. Therefore, the output of this study may play a vital role in developing a proper treatment plan at the earlier stages of CRC. colorectal cancer gene expression profiles core genes early diagnosis prognosis therapies integrated statistics and bioinformatics approaches The authors declare that no funds, grants, or other support were received during this research work. pmc1. Introduction Cancer is a complex disease caused by multiple alterations at the genetic and epigenetic levels that increasingly lead to abnormal cell division and cellular transformation . Colorectal cancer (CRC) is the third most common solid malignancy and the second deadliest tumor worldwide . The CRC incidence is expected to rise by 60%, with 2.2 million new cases and 1.1 million deaths globally by 2030 . The number of new incidences and mortalities is increasing due to insufficient evidence about diagnostic biomarkers and the molecular mechanism of CRC . Early detection of CRC is associated with lower morbidity and mortality and a higher survival rate compared with late detection. For example, the five-year survival rate increases from 11% (late detection) to 90% at early detection of CRC . However, the survival rate significantly decreases, and the cost of treatment increases, whereas CRC is identified in later stages compared to earlier stages . Therefore, early diagnosis, prognosis, and therapies leads to reduce CRC-related mortality . Several factors, including excessive alcohol consumption, obesity, unhealthy dietary habits, and an abnormal lifestyle, are all considered non-causal risk factors for CRC development. However, these non-causal risk factors cannot be used for CRC detection at an earlier stage. Generally, differentially expressed genes (DEGs) between cancer and control samples are considered as the cancer-causing/stimulating genes. A gene may show a differential expression pattern between cancer and control samples for several reasons, including mutation, DNA methylation, and other epigenetic stimulations. The genes that are associated with the development of cancer, are known as oncogenes (upregulated DEGs) and tumour-suppressor genes (down-regulated DEGs) . Thus, cancer incidence, development, recurrence, and non-recurrence are associated with pathogenetic processes of DEGs . Several studies reported some dysregulating genes in the CRC cases compared to cases that are associated with CRC proliferation, differentiation, apoptosis, metastasis, recurrence, and lower survival . Many earlier transcriptomics studies explored the pathogenetic processes of CRC through DEGs . However, none of them discussed rigorously about early diagnosis, prognosis, and therapies for CRC. Patil et al. (2021) identified forty CRC-causing/stimulating core DEGs and recommended their application for the early diagnosis of CRC. Though the number 40 is much smaller than the whole genome size, it may also not be suitable for further investigation by the wet-lab researchers, since wet-lab experiments with 40 DEGs might be costly, time-consuming, and laborious. So, a smaller set of core DEGs might be required for further experimental investigation. On the other hand, this study did not provide any recommendations about suggested core DEGs guide any drug molecules for therapies for CRC. Therefore, the present study attempted to discover CRC-causing/stimulating core-DEGs, highlighting their pathogenetic processes for early prognosis, diagnosis, and therapies of CRC. The pipeline of this study is given in Figure 1. 2. Materials and Methods 2.1. Data Sources and Descriptions The necessary datasets that were analyzed in this study are described below. 2.1.1. Collection of Microarray Datasets to Explore CRC-Causing Core Genes We downloaded three microarray datasets of CRC using the accession IDs GSE106582, GSE110223, and GSE74602 from the NCBI Gene Expression Omnibus (GEO) database. The GPL10558 platform was used for the GSE106582 dataset, which contains 194 samples, including 77 cancer and 117 adjacent tissue samples. The GPL96 platform was used for the GSE110223 dataset, which contains 26 samples, including 13 cancer and 13 adjacent tissue samples. The GPL6104 platform was used for the GSE74602 dataset, which contains 60 samples, including 30 cancer and 30 adjacent tissue samples. 2.1.2. Collection of Drug Molecules Set for Drug Repositioning For identifying candidate repurposed drugs, we collected target receptor-guided drug molecules from the DSigDB database (Table S1). 2.2. Method for Identification of DEGs We used the GEO2R web tool based on the LIMMA (linear models for microarray data) approach to identify DEGs between cancer and adjacent tissue samples for each of the three datasets. The LIMMA method uses modified t-statistics to calculate p-values. We used the Benjamini-Hochberg (BH) approach to adjust the p-values. The log2 fold-change (Log2FC) and adjusted p-values were used to separate the down-regulated DEGs by the following cut-offs:(1) DEGsg=DEG Upregulated, if adj.p.value<0.05 and Log2FCg>+1.0DEG Downregulated, if adj.p.value<0.05 and Log2FCg<-1.0 We considered common DEGs (cDEGs) from three datasets to identify core CGs. All DEGs were visualized using a Venn diagram using FunRich 3.1.3 . 2.3. Protein-Protein Interaction (PPI) Network Analysis The protein-protein interaction (PPI) network was utilized to detect core genes (CGs) from cDEGs. We considered the STRING database with a median confidence score (MCS) of 0.4 to produce a PPI network of cDEGs and Cytoscape software for better visualization of the network . The CGs were selected from the PPI network using the CytoHubba plugin in Cytoscape . The present study used maximal clique centrality (MCC) topology analysis methods to identify the CGs. 2.4. Association of CGs with Different Stages of CRC Progression To investigate the association of CGs with the different stages of CRC based on independent databases, we performed box-plot analysis based on their expression levels in different CRC progression stages (Normal status, Stage 1, Stage 2, Stage 3, and Stage 4) through the UALCAN web tool with the TCGA-COAD and TCGA-READ databases . 2.5. Prognosis Power of CGs To investigate the prognosis power of CGs by multivariate Kaplan-Meier survival probability curves, we considered the SurvExpress web tool based on the TCGA-COAD and TCGA-READ databases accessed date: 2 January 2022) . The log-rank test was used in SurvExpress, and the risk group hazard ratio with a 95% confidence interval was included in the Kaplan-Meier survival plot . The p-value < 0.05 was used as the cut-off. 2.6. CGs-Set Enrichment Analysis CGs-set enrichment analysis (cGSEA) determines the classes of genes or proteins that are over-represented (enriched) in a predefined large set of genes or proteins that are associated with the terms of interest, including gene ontology (GO), pathways, diseases, chemicals, drugs, biomolecules (miRNA, TFs), and so on. To detect the significantly enriched terms of interest, let Ai be the predefined gene-set in the ith term of interest, and Mi denotes the number of CGs in Ai (i = 1, 2,..., r); T is considered as an enriched gene number that created a combined set A such that A=i=1rAi=AiAic and T<=i=1rMi; where Aic is considered as the complement-set of Ai. Again, suppose t represents the number of CGs and mi denotes the number of CGs subset of Ai. Table 1 summarizes these results. The Enrichr web tool was considered to investigate the association of CGs with terms of interest. This web tool uses the Fisher exact test to examine the significance of the association between CGs and ith term of interest. 2.7. Association of CGs with Different Diseases We considered the Enrichr web tool to verify the association of CGs with different diseases, including CRC, using the DisGeNET database, which was constructed based on 21,671 genes and 30,170 diseases . It measures the association of a disease with a group of CGs that are overlapped (common) with the reference gene set of that disease (see Table 1). To investigate the pan-cancer role of CGs, we performed the pan-cancer analysis of each CG by the TIMER 2.0 web tool with the TCGA database . In both cases, the p-value < 0.05 was selected as the cut-off for statistical significance. 2.8. Association of CGs with GO Terms and KEGG Pathway To disclose the pathogenetic processes of CGs, we performed CGs-set enrichment analysis with GO terms and pathways by using the Enrichr web tool . Biological process (BPs), molecular function (MFs), and cellular component (CCs) were investigated to explore potential GO terms and pathways based on the KEGG database as displayed in Table 1. A p-value < 0.001 was used as the cut-off for the statistical significance. 2.9. CGs Regulatory Network Analysis A gene regulatory network (GRN) provides information about molecular regulators that connect to regulate the gene expression level of mRNA. Transcription factors (TFs) and microRNAs (miRNAs) are considered the major regulators of gene expression. TFs proteins are regarded as the significant contributors to GRN because they bind to a particular region of DNA (enricher/promoter) and influence gene expression at the transcriptional level. A miRNA is a non-coding RNA considered a central post-transcriptional regulator of gene expression. The human genome contains up to 1600 TFs and 1900 miRNAs. A TFs vs. CGs network is considered an undirected graph, where nodes represent TFs or CGs and edges depict interactions between TFs and CGs, respectively. A TF-node is considered the major regulatory factor for CGs if it contains the largest number of interactions with CG nodes. We considered regulatory analysis of CGs (transcription factors (TFs) vs. CGs and micro-RNAs (miRNAs) vs. CGs) through Network Analyst platform-based JASPAR and TarBase databases, respectively, to detect the core transcriptional and post-transcriptional regulators of CGs. For better illustration, we used Cytoscape software . The core regulators were chosen by utilizing degree and betweenness scores. 2.10. Molecular Docking We conducted molecular docking studies of receptors and drug molecules to explore FDA-approved repurposable drugs for CRC. CGs-mediated proteins and related TFs proteins were considered drug target receptors (p = 14). The online database DSigDB was used to extract CGs-guided drug agents. The 3-dimensional (3D) structures of AURKA, TOP2A, CDK1, PTTG1, CDC20, MAD2L1, CKS2, MELK, TPX2, YY1, and SRF targets were retrieved from the Protein Data Bank (PDB) with IDs 6VPM, 5NNE, 5LQF, 7NJ1, 4GGC, 2V64, 5LQF, 5M5A, 6VPM, 4C5I, and 1HBX, respectively. The remaining targets, FOXC1, CDKN3, and NFIC, were retrieved from the SWISS-MODEL with the Uniport IDs Q12948, P08651, and Q16667, respectively. Using the PubChem database , we retrieve the 3D structures of all (q = 158) drug molecules. The visualization of the receptor proteins and co-crystal ligands were performed via the Discovery Studio Visualizer 2019 . The receptor proteins were processed using AutoDock tools and the Swiss PDB viewer by adding the structural charges and reducing the energy of receptors, respectively . The docked complexes were analyzed through Discovery Studio Visualizer 2019. Let Bij be the binding affinity (BA) score of ith receptors (i = 1, 2,..., p) and jth drugs (j = 1, 2,..., q). The receptors and drug molecules were sorted by the decreasing order of their average BA score for selecting the top-ordered few potential candidate repurposable drugs. 2.11. Molecular Dynamics (MD) Simulation We performed MD simulations of the top-orderedprotein-ligand complexes (TPX2-Manzamine A, CDC20-Cardidigin, MELK-Staurosporine, and CDK1-Riccardin D) through the YASARA software (Version: 22.8.22) based on the AMBER14 force field . Prior to simulation, the hydrogen-bonding network of each complex in a simulated cell was optimized using a TIP3P water model . The periodic limit conditions were kept constant at 0.997 gL-1 of solvent concentration. The primary energy was minimized in each simulation by considering the steepest gradient technique with 5000 cycles. The complexes "TPX2-Manzamine A", "CDC20-Cardidigin", "MELK-Staurosporine", and "CDK1-Riccardin D" consist of a total of 56,287, 35,859, 81,347, and 45,153 atoms, respectively. At the Berendsen thermostat and constant pressure, a 100 ns MD simulation was examined. Please see our previous publications for details about the MD simulation strategy . For subsequent analysis, we took snapshots of the trajectories every 250 ps, ran them via the built-in script of YASARA macro, and calculated the binding free energy of the MM-Poisson-Boltzmann surface area (MM-PBSA) by analyzing all the snapshots . To calculate binding-free energy, we used the following formula:(2) Binding free energy=EpotReceptor+EsolvReceptor+EpotLigand+EpotComplex-EsolvComplex More positive binding energy represents stronger binding. 3. Results 3.1. Identification of DEGs To identify DEGs from each of three microarray gene-expression datasets, we used the statistical LIMMA approach through the GEO2R web tool, with the cut-off at adjusted p-value < 0.05 and |log2(fold change)| > 1. In the GSE106582 dataset, we identified 594 DEGs, including 213 upregulated and 381 downregulated genes. In the GSE110223 dataset, we identified 625 DEGs that contain 260 upregulated and 365 downregulated genes. In the GSE74602 dataset, we identified 1674 DEGs, including 673 upregulated and 1001 downregulated genes. The Venn diagram in Figure 2 visualizes the common DEGs among the three datasets. The Venn diagram exhibits 252 cDEGs among the three datasets. 3.2. Identification of Core Genes (CGs) We construct the PPI network of cDEGs and visualize the PPI network to identify the potential genes most significantly associated with the development of CRC. The PPI network contains 216 nodes and 616 edges, with a confidence score of 0.40. Then, the MCC topology analysis method of CytoHubba was performed to calculate the top-ranked CGs within the network. We found the ten top-ranked (AURKA, TOP2A, CDK1, PTTG1, CDKN3, CDC20, MAD2L1, CKS2, MELK, and TPX2) genes . These top ten CGs were identified as major controllers of CRC and considered for subsequent analysis. 3.3. Association of CGs with Different Stages of CRC Progression The box-plot analysis based on an independent database represents a high difference between normal expression and every CRC progression stage (Stage 1, Stage 2, Stage 3, and Stage 4) expression of all CGs . So, our proposed CGs have strong prognostic power to identify CRC at an earlier development stage. 3.4. Prognosis Power of CGs A survival analysis was performed to examine the prognosis power of CGs. The multivariate Kaplan-Meier survival plot of CGs expressions using the TCGA-COAD and TCGA-READ databases represents a significant difference (p-value < 0.001) between lower-risk and higher-risk groups . 3.5. Association of CGs with Different Diseases The enrichment analysis of the CGs-set with different diseases based on the DisGeNET database showed that the CGs-set is significantly associated with various diseases (p-value < 0.05). Figure 3A and Table S2 show the top-ranked 20 diseases, all of which are different types of cancer, including CRC. We observed that 3 CGs (MELK, CKS2, CDC20) and 2 CGs (MELK, CDC20) do not overlap with the reference gene-sets of colon carcinoma and colorectal carcinoma, respectively . These results suggested a pan-cancer role for CGs . To investigate the pan-cancer role of CGs, we also performed pan-cancer analysis based on the TCGA database. We selected the top-ranked 20 cancers as displayed in Figure 5B. Figure 5A,B commonly showed that eight cancers (colon adenocarcinoma, bladder urothelial carcinoma, esophageal carcinoma, glioblastoma multiforme, liver hepatocellular carcinoma, lung adenocarcinoma, prostate adenocarcinoma, and stomach adenocarcinoma) are significantly associated with CGs. 3.6. Association of CGs with GO Terms and KEGG Pathway Enrichment analysis of the CGs was performed using the Enrichr web tool. Table 2 shows the annotated GO terms in three categories (BPs, CCs, and MFs). In the case of biological processes (BPs), CGs were mainly involved in mitotic cell-cycle phase transition (GO:0044772), anaphase-promoting complex-dependent catabolic process (GO:0031145), regulation of G2/M transition of mitotic cell cycle (GO:0010389), mitotic spindle organization (GO:0007052), regulation of mitotic cell cycle (GO:0007346). In molecular function (MFs), CGs were mainly involved in histone kinase activity (GO:0035173), RNA polymerase II CTD heptapeptide repeat kinase activity (GO:0008353), protein kinase binding (GO:0019901), CXCR chemokine receptor binding (GO:0045236), cyclin-dependent protein kinase activity (GO:0097472), etc. In cellular components (CCs), CGs were mainly involved in the spindle (GO:0005819), cyclin-dependent protein kinase holoenzyme complex (GO:0000307), serine/threonine-protein kinase complex (GO:1902554), intracellular non-membrane-bounded organelle (GO:0043232), mitotic spindle (GO:0072686), etc. The KEGG pathway enrichment analysis results for CGs were also shown in Table 2. The KEGG pathways of CGs were enriched in the cell cycle, bladder cancer, oocyte meiosis, human T-cell leukemia virus one infection, progesterone-mediated oocyte maturation, etc. 3.7. Identification of Regulatory Factors TFs proteins and miRNAs play a fundamental role in the modification of gene expression at the transcriptional and post-transcriptional levels, respectively. To explore the major transcriptional regulatory factors of CGs, we constructed a TFs vs. CGs interaction network where round nodes with red color represent the CGs and square nodes with green/purple color represent the TFs . TFs proteins vs. CGs regulatory analysis revealed four highest-ranking significant candidate TFs modifiers (NFIC, FOXC1, YY1, and GATA2) that may regulate the expression of CGs at the transcriptional level . Similarly, we constructed an undirected interaction network of miRNAs vs. CGs to reveal the post-transcriptional regulator of CGs, where red color nodes represent the CGs and green/blue color nodes illustrate the miRNAs . The miRNAs vs. CGs regulatory network analysis revealed six highly interacted non-coding RNAs (miRNAs) such as hsa-mir-147a, hsa-mir-129-2-3p, hsa-mir-124-3p, hsa-mir-34a-5p, hsa-mir-23b-3p, and hsa-mir-16-5p that act as gene expression regulators at the post-transcriptional level . So, those identified TFs and miRNAs may influence the gene expression of CGs at the transcriptional and post-transcriptional levels, respectively. 3.8. Drug Repurposing through Molecular Docking Studies We considered 10 CG-guided proteins and 4 TFs proteins as target-receptor proteins for molecular docking. A structural interaction was carried out between target-receptor proteins and 158 drug agents by molecular docking studies, which computed the receptor-drug binding affinities (BA) for each interaction . Manzamine A, Cardidigin, Staurosporine, Sitosterol, Benzo[a]pyrene, Nocardiopsis sp., and Riccardin D were shown to have strong BA against all of the target receptors, and their average BA lies between -9.20 and -8.2 (kcal mol-1). Among those drugs, Manzamine A was shown to have the highest BA against almost every target protein, with an average BA of -9.2 kcal mol-1. Therefore, we proposed those seven drugs (Manzamine A, Cardidigin, Staurosporine, Sitosterol, Benzo[a]pyrene, Nocardiopsis sp., and Riccardin D) as candidate drug agents and displayed them in red color in Figure 7. We also revealed the structural interaction profiles of four top-ordered receptor proteins and drug complexes in Table 3. 3.9. Molecular Dynamic (MD) Simulations TPX2-Manzamine A, CDC20-Cardidigin, MELK-Staurosporine, and CDK1-Riccardin D complexes have shown the highest BA in molecular docking analysis (Table 3). So, we considered those complexes for examining their binding stability through MD simulations. We observed that each protein-ligand complex (TPX2-Manzamine A, CDC20-Cardidigin, MELK-Staurosporine, and CDK1-Riccardin D) showed significant stability in a 100 ns MM-PSSA simulation . RMSD values corresponding to each complex were calculated . RMSD values showed lower flexibility around 1.5 A to 3.0 A for all four complexes. The average RMSD values for TPX2-Manzamine A, CDC20-Cardidigin, MELK-Staurosporine, and CDK1-Riccardin D complexes were 2.592 A, 1.724 A, 2.235 A, and 2.516 A, respectively. The CDC20-Cardigin complex showed a more substantial structural rigidity than the other three complexes, gained equilibrium at four ns, and displayed good stability after that. MELK-Staurosporine showed a gradual increase in RMSD before 22 ns, and after this time point, the RMSD score of the complex illustrated almost stable movement between 2.2 A and 2.50 A for 68 ns. After that, there were irregular fluctuations in the RMSD. On the contrary, CDK1-Riccardin D complexes exhibited instability, and the RMSD displayed an upward trend from 2.0 A to 3.2 A over time. Similarly, TPX2-Manzamine A showed irregular oscillation in RMSD between 1.7 A and 3.3 A. In addition, the MM-PBSA binding energy for four complexes was also computed. Figure 8B illustrates the binding energies of the complexes. On average, TPX2-Manzamine A, CDC20-Cardidigin, MELK-Staurosporine, and CDK1-Riccardin D complexes produced MM-PBSA binding energies of 84.39 KJ mol-1, -95.07 KJ mol-1, -235.86 KJ mol-1, and 154.39 KJ mol-1, respectively. 4. Discussion To explore core genomic biomarkers and their mechanisms in the CRC progression, firstly, we identified 252 cDEGs between CRC and control samples, out of around 40,000 genes in three gene expression datasets of the NCBI-GEO database with accession numbers GSE106582, GSE110223, and GSE74602. Though the number of DEGs is much smaller than the total number of genes, it may still be a large number for further investigation by wet-lab experiments since it would be laborious, time-consuming, and costly. Therefore, a smaller set of DEGs that are representative of all DEGs is required to reduce time, cost, and labor during further experiments by the wet-lab researchers. Though total DEGs are more informative than any smaller set of DEGs, a smaller representative set of DEGs would be more beneficial from the viewpoints of time, cost, and labor. Therefore, in this study, we proposed 10 top-ranked DEGs (AURKA, TOP2A, CDK1, PTTG1, CDKN3, CDC20, MAD2L1, CKS2, MELK, and TPX2) as the core genes (CGs) for early diagnosis, prognosis, and therapies of CRC. The survival probability curves and box-plot analyses with the expressions of CGs in different stages (control, Stage 1, Stage 2, Stage 3, and Stage 4) of CRC with the TCGA database indicated their strong prognostic performance from the earlier stages of the disease. It should be mentioned here that Patil et al. (2021) identified CRC-causing 40 CGs for early diagnosis, which might be a large number for further investigation by the wet-lab researchers since it would be laborious, time-consuming, and costly, as mentioned earlier. In our proposed 10 CGs, 9 genes (AURKA, TOP2A, CDK1, PTTG1, CDKN3, CDC20, MAD2L1, MELK, and TPX2) were overlapped/common with their 40 CGs. In addition, we also investigated the association of our proposed CGs with different diseases. We found they have a strong pan-cancer role, including CRC. The literature review also supported the association of our proposed CGs with CRC. For example, AURKA is considered an oncogene that significantly impacts the proliferation and progression of colorectal carcinoma from colorectal adenoma . Generally, AURKA is overexpressed and amplified in CRCs . TOP2A is highly expressed during tumor development and responds to drug therapy for CRC . CDK1 is also overexpressed and sensitive to apoptosis in CRC cells . CDKN3 is highly expressed in CRC tissues and remarkably related to patients' diagnoses . CKS2 overexpression is correlated with aggressive tumor development in CRC, meaning that CKS2 might function as a decent CRC biomarker . CKS2 is a promising biomarker contributing to CRC tumor development . CDC20, PTTG1, and MAD2L1 might be CRC stage-related genes . MELK might play a role as an effective therapeutic target for CRC . TPX2 is highly upregulated in CRC tissues . We identified the top-ranked five GO terms and KEGG pathways of CGs to reveal their molecular mechanisms in CRC progression. The identified GO term 'cell cycle' is one of the most important biological processes in the human body . It has four sequential phases. Arguably, the most important phases are the S phase (DNA replication occurs) and the M phase (cell divides into two daughter cells) . The fundamental task of the cell cycle is to ensure that DNA is faithfully replicated once during S phase and that identical chromosomal copies are distributed equally to two daughter cells during M phase. Usually, in adult tissue, there is a delicate balance between cell death and proliferation (cell division), producing a steady state. Disruption of this equilibrium by loss of cell cycle control may eventually lead to tumor development , including colorectal cancer , colon cancer , liver cancer , glioblastoma , breast cancer , lung cancer , gastric cancer , etc. So, the cell cycle is considered a vital cancer progression process. TOP2A is related to tumor development and poor survival outcomes by regulating cell proliferation and the CRC cell cycle . CDK1 controls the cell cycle and aids in the development of colorectal tumors via an iron-regulated signalling axis . In most CRCs, chromosomal variability resulting in an abnormal chromosome number, aneuploidy, was systematically related to a mitotic checkpoint's loss of function . The GO term 'mitotic spindle orientation' can influence tissue organization and control the placement of daughter cells within a tissue. Spindle misorientation greatly affects cancer development and progression , including CRC . The top-ranked five MFs (cyclin-dependent protein kinase activity, CXCR chemokine receptor binding, protein kinase binding, RNA polymerase II CTD heptapeptide repeat kinase activity, and histone kinase activity) play a vital role in CRC development and proliferation . Similarly, the enriched five CCs, including the spindle, mitotic spindle, cyclin-dependent protein kinase holoenzyme complex, intracellular non-membrane-bounded organelle, and serine/threonine-protein kinase complex, are strongly related to the progression of CRC . Chromosomal instability happens in 80%-85% of CRCs and is considered the most common subtype of CRC . Subsequent research showed that chromosomal instability is caused by mutations in the genes that govern the mitotic spindle checkpoint . The consecutive activation of a group of serine-threonine kinases controls eukaryotic cell-cycle checkpoints . We identified the top 5 enriched common KEGG pathways (cell cycle, bladder cancer , oocyte meiosis , human T-cell leukemia virus 1 infection, progesterone-mediated oocyte maturation) that are also reported by some other studies . We also identified key regulators of CGs, such as four TFs (NFIC, FOXC1, YY1, and GATA2) and six miRNAs that played a significant role in CRC development. FOXC1, a member of the forkhead box family, has been connected to the growth and progression of numerous diseases , particularly CRC . YY1 is a multipurpose TF protein that can stimulate or suppress gene expression and plays a significant role in CRC tumor growth . In CRC, the high-level expressions of GATA2 were linked with a poor prognosis and recurrence in solid tumors . Nuclear factor 1 C-type (NFIC) regulates PFKB3 in response to CRC . To find effective repurposable drugs against CRC, we performed molecular docking and computed binding scores among 158 CGs-associated drug agents and CGs-guided receptors. Then we proposed seven top-ordered drugs (Manzamine A, Cardidigin, Staurosporine, Benzo[a]pyren, Sitosterol, Nocardiopsis sp., and Riccardin D) based on binding affinities as the candidate repurposable drug. Manzamine A , Cardidigin , Staurosporine , Benzo[a]pyrene , Sitosterol , Nocardiopsis sp. , and Riccardin D also suggested by some other studies for the treatment of CRC. Finally, the binding stability of top-docked four complexes (TPX2 vs. Manzamine A, CDC20 vs. Cardidigin, MELK vs. Staurosporine, and CDK1 vs. Riccardin D) was investigated by molecular dynamics (MD)-based MM-PBSA simulation and found their performance to be stable . Thus, our findings may play a vital role in early diagnosis, prognosis, and therapies for CRC. 5. Conclusions The present study identified the 10 top-ranked DEGs (AURKA, TOP2A, CDK1, PTTG1, CDKN3, CDC20, MAD2L1, CKS2, MELK, and TPX2) as the core genes (CGs), which showed Strong prognostic performance in the earlier stages of CRC. The CGs-disease and pan-cancer analysis commonly showed that CGs have a robust pan-cancer role, including CRC, bladder urothelial carcinoma, esophageal carcinoma, glioblastoma multiforme, liver hepatocellular carcinoma, lung adenocarcinoma, prostate adenocarcinoma, and stomach adenocarcinoma. The CGs regulatory network analysis detected some essential TFs proteins (NFIC, FOXC1, YY1, and GATA2) and miRNAs (hsa-mir-147a, hsa-mir-129-2-3p, hsa-mir-124-3p, hsa-mir-34a-5p, hsa-mir-23b-3p, and hsa-mir-16-5p) as the transcriptional and post-transcriptional regulators of CGs. The enrichment analysis also revealed some important CRC-causing GO terms and signaling pathways. For example, cell cycle and mitotic spindle pathways have a significant association with CRC progression. Finally, we recommended our proposed CGs-guided 7 candidate drug molecules (Manzamine A, Cardidigin, Staurosporine, Sitosterol, Benzo[a]pyrene, Nocardiopsis sp., and Riccardin D) for the treatment against the CRC by molecular docking analysis. Thus, the findings of this study may be more useful compared to the previous computation study results for early diagnosis, prognosis, and therapies forf CRC. Acknowledgments We would like to thank all other members of Bioinformatics Lab, Department of Statistics, University of Rajshahi, Bangladesh, for their excellent cooperation during this work. Supplementary Materials The following supporting information can be downloaded at: Figure S1: Box plots for the expressions of KGs with different stages (Stage 1, Stage 2, Stage 3, and Stage 4) of READ (Rectal adenocarcinoma), including the control stage in the TCGA database. Figure S2: Core gene-set enrichment analysis with different diseases. Figure S3: Pan-cancer analysis of core genes (A) AURKA, (B) CDC20, (C) CDK1, (D) CDKN3, (E) CKS2, (F) MAD2L1, (G) MELK, (H) PTTG1, (I) TOP2A, and (J) TPX2. Table S1: Transcriptome-guided 158 meta-drug agents associated with CRC infections collected from the DSigDB online database. Table S2: The list of the top 20 significant (p-value < 0.05) comorbidities associated with CGs. Click here for additional data file. Author Contributions M.A.I. and M.N.H.M. conceived the idea of the study. M.A.I. and M.B.H. analyzed the microarray data and drafted the manuscript. M.A.I. and M.A.H. (Md. Abu Horaira) performed molecular docking for drug screening. M.A.I. and M.K.K. performed molecular dynamics simulation. M.A.H. (Md. Alim Hossen) and K.F.T. helped during the data preparation. M.S.R., M.O.F., F.K. and R.A.M. reviewed the manuscript and provided suggestions. M.N.H.M. edited the manuscript and supervised the project. All authors have read and agreed to the published version of the manuscript. Institutional Review Board Statement Not Applicable. Informed Consent Statement Not Applicable. Data Availability Statement All data revolved are describe in Section 2.1 (Data Sources and Descriptions). Conflicts of Interest The authors declare no conflict of interest. Figure 1 The pipeline of this study. Figure 2 Common differentially expressed genes among GSE110223, GSE106582, and GSE74602 datasets were visualized through a Venn diagram, and 252 genes were found as cDEGs from CRC patients. Figure 3 Protein-protein interaction network for cDEGs. Edges specify the interconnection between two proteins. The PPI network illustrates 216 nodes and 616 edges. Red color nodes (AURKA, TOP2A, CDK1, PTTG1, CDKN3, CDC20, MAD2L1, CKS2, MELK, and TPX2) represented the CGs. Figure 4 (A) Box plots for the expressions of CGs with various stages of colon adenocarcinoma (Stage 1, Stage 2, Stage 3, and Stage 4) using the COAD database and comparison with the control stage from the TCGA database. (B) The multivariate Kaplan-Meier survival probability plot of CRC patients with the CGs-expressions using the TCGA-COAD and TCGA-READ databases. Figure 5 Association of CRC-causing CGs with different diseases. (A) Top 20 diseases associated with CGS obtained by disease-CGs association studies based on the Enrichr web tool with the DisGeNET database, where red and green colors indicate presence and absence, respectively. The red colour text in the column represent CRC-related cancers. (B) Top 20 cancers associated with CGs obtained by pan-cancer analysis based on the TIMER2 web tool with the TCGA database, where red and green colors indicate significant (p-value < 0.05) and insignificant (p-value >= 0.05) pairwise gene-disease association, respectively. The red colors text in the column represents CRC-related cancers. Figure 6 Regulatory network analysis of CGs. (A) TFs-CGs regulatory network contains 72 nodes and 174 edges. The red node indicates the core genes, whereas purple color nodes represent TFs. Among the TFs, green nodes represent the core TFs. (B) miRNA-CGs interaction network contains 223 nodes and 450 edges. The red node indicates the core genes, whereas blue nodes represent miRNAs. Among the miRNAs, green color represents the core miRNA. Figure 7 Matrix of molecular docking analysis results. The target-receptor proteins are ordered in a row and drug agents are ordered in a column, where red colors represent strong binding affinity. The red colors text in the row represents proposed drug agents. Figure 8 MD simulations of top-ranked complexes. (A) Time evolution of RMSDs for each of top-ranked complexes. (B) Binding stability of top-ranked four complexes by MM-PBSA binding free energy (kJ mol-1) against each ns of simulation; greater positive values show strong binding. Complexes: red color CDC20-Cardidigin, green color MELK-Staurosporine, pink color TPX2-Manzamine A, and blue color CDK1-Riccardin-D. cancers-15-01369-t001_Table 1 Table 1 Contingency table. Predefined Gene-Set CGs (Proposed) Not CGs (Proposed) Marginal Total ith term of interest (Ai) mi Mi - mi Mi Complement of Ai (Aic) t - mi T - Mi - t + mi T - Mi Marginal total t T - t T (Grand total) cancers-15-01369-t002_Table 2 Table 2 List of the top five significantly (p-value < 0.001) annotated GO terms and KEGG pathways by CGs. GO ID GO Term p-Value Associated CGs Biological Process (BPs) GO:0044772 mitotic cell cycle phase transition 8.50 x 10-10 MELK;CDK4;MYC;CDK1;AURKA;CDC25B;CDKN3 GO:0031145 anaphase-promoting complex-dependent catabolic process 1.71 x 10-8 CDC20;PTTG1;CDK1;AURKA;MAD2L1 GO:0010389 regulation of G2/M transition of mitotic cell cycle 3.04 x 10-7 TPX2;CDK4;CDK1;AURKA;CDC25B GO:0007052 mitotic spindle organization 3.94 x 10-7 CDC20;TPX2;CENPN;AURKA;MAD2L1 GO:0007346 regulation of mitotic cell cycle 7.34 x 10-7 CDC20;CDK1;CKS2;CDC25B;MAD2L1 Molecular Function (MFs) GO:0035173 histone kinase activity 2.65 x 10-5 CDK1;AURKA GO:0008353 RNA polymerase II CTD heptapeptide repeat kinase activity 6.23 x 10-5 CDK4;CDK1 GO:0019901 protein kinase binding 1.15 x 10-4 TOP2A;TPX2;CKS2;AURKA;CDC25B GO:0045236 CXCR chemokine receptor binding 1.28 x 10-4 CXCL8;CXCL12 GO:0097472 cyclin-dependent protein kinase activity 2.37 x 10-4 CDK4;CDK1 Cellular Component (CCs) GO:0005819 Spindle 1.07 x 10-6 CDC20;TPX2;CDK1;AURKA;MAD2L1 GO:0000307 cyclin-dependent protein kinase holoenzyme complex 3.41 x 10-6 CDK4;CDK1;CKS2 GO:1902554 serine/threonine protein kinase complex 6.50 x 10-6 CDK4;CDK1;CKS2 GO:0043232 intracellular non-membrane-bounded organelle 8.52 x 10-5 TOP2A;CDC20;TPX2;CDK4;MYC;TRIP13;AURKA GO:0072686 mitotic spindle 2.23 x 10-4 TPX2;CDK1;MAD2L1 Pathways p-Value Associated CGs KEGG Pathway Cell cycle 2.15 x 10-11 CDC20;PTTG1;CDK4;MYC;CDK1;CDC25B;MAD2L1 Bladder cancer 7.19 x 10-8 CXCL8;MMP1;CDK4;MYC Oocyte meiosis 1.48 x 10-7 CDC20;PTTG1;CDK1;AURKA;MAD2L1 Human T-cell leukemia virus 1 infection 2.04 x 10-6 CDC20;PTTG1;CDK4;MYC;MAD2L1 Progesterone-mediated oocyte maturation 2.68 x 10-6 CDK1;AURKA;CDC25B;MAD2L1 cancers-15-01369-t003_Table 3 Table 3 The 1st, 2nd, 3rd column show potential targets, 2-dimentional(2d) structure of lead compounds, top ordered binding affinities (kcal mol-1), respectively. The 3-dimension(3d) view of top ranking drug-target complexes is shown in the 4th column. Finally, the last column shows key elements of interacting amino acids, including hydrogen bond, hydrophobic interactions, and electrostatic. Potential Targets Structure of Top Compounds Binding Affinity Score (kcal mol-1) 3D Structures of Complex with Interactions Interacting Amino Acids Hydrogen Bond Hydrophobic Interactions Electrostatic TPX2 Manzamine A -12.4 - VAL317 TRP313 HIS366 - CDC20 Cardidigin -11.0 TRP317 PRO319 VAL190 LEU449 PRO319 LYS236 - MELK Staurosporine -13.4 GLU87 GLU136 CYS89 ASP150 ILE17 VAL25 LEU139 LEU149 ALA38 ILE149 LYS40 - CDK1 Riccardin D -11.3 LEU83, ASP146, LYS33 VAL18 LEU135 ILE10 ALA31 VAL64 - Disclaimer/Publisher's Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

PMC10000173

Background: This study aimed to identify the important ancillary features (AFs) and determine the utilization of a machine-learning-based strategy for applying AFs for LI-RADS LR3/4 observations on gadoxetate disodium-enhanced MRI. Methods: We retrospectively analyzed MRI features of LR3/4 determined with only major features. multivariate analyses and random forest analysis were performed to identify AFs associated with HCC. A decision tree algorithm of applying AFs for LR3/4 was compared with other alternative strategies using McNemar's test. Results: We evaluated 246 observations from 165 patients. In multivariate analysis, restricted diffusion and mild-moderate T2 hyperintensity showed independent associations with HCC (odds ratios: 12.4 [p < 0.001] and 2.5 [p = 0.02]). In random forest analysis, restricted diffusion is the most important feature for HCC. Our decision tree algorithm showed higher AUC, sensitivity, and accuracy (0.84, 92.0%, and 84.5%) than the criteria of usage of restricted diffusion (0.78, 64.5%, and 76.4%; all p < 0.05); however, our decision tree algorithm showed lower specificity than the criterion of usage of restricted diffusion (71.1% vs. 91.3%; p < 0.001). Conclusion: Our decision tree algorithm of applying AFs for LR3/4 shows significantly increased AUC, sensitivity, and accuracy but reduced specificity. These appear to be more appropriate in certain circumstances in which there is an emphasis on the early detection of HCC. hepatoceullular carcinoma Liver Imaging Reporting and Data System MRI Ewha Womans University ResearchKorea government (MSIT)2022H1D8A303804012 Soonchunhyang UniversityThis work was supported by the Ewha Womans University Research Grant of 2021. Moreover, this work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT) (No. 2022H1D8A303804012) and Soonchunhyang University. pmc1. Introduction The Liver Imaging Reporting and Data System (LI-RADS) was released in 2011 by the American College of Radiology and was continuously updated until 2018 to improve diagnostic accuracy and promote communication between healthcare providers by standardizing the interpretation and categorization of liver observations. In the LI-RADS, liver observations are categorized as LR1 to LR5 according to the probability of benignity and hepatocellular carcinoma (HCC), though the algorithm includes the size and major features (MFs) such as nonrim arterial phase hyperenhancement (APHE), enhancing capsule, nonperipheral washout, and threshold growth. After allocation to a category based on the MFs, adjustment using ancillary features (AFs) is allowed . Observations can be upgraded by one category up to LR4 using AFs favoring malignancy, whereas they can be downgraded by one category using AFs favoring benignity. Based on a recent meta-analysis, the occurrence rate of HCC is 0% in LR1, 13% in LR2, 38% in LR3, 74% in LR4, and 94% in LR5 , although these rates in the lower categories may be inflated owing to selection bias for biopsied lesions. Once the LR category is assigned to each observation, cases with LR3 and LR4 observations are recommended for repeat or alternative diagnostic imaging in 3-6 months, multidisciplinary discussion, or biopsy. However, an invasive biopsy is a risky procedure and may result in biopsy failure because small lesions are classified as mainly LR3 or LR4. In addition, there is a risk of missing local treatment opportunities or a decrease in the number of treatment options due to increased lesion size or vascular invasion during follow-up without treatment. The LI-RADS does not provide specific guidelines for the application of Afs, and the utilization of AFs are at the discretion of the radiologist according to each case. Furthermore, variability of the proportion of the change in the LI-RADS category has been shown after the application of AFs among the studies, that is, from 18.1% to 56.4% . Studies have shown that some observations remained in the initial category even after the application of AFs; therefore, specific and appropriate instructions for the application of AFs are necessary to improve the accuracy and timeliness of diagnosis. Prior studies have reported the widely variable performance of AFs of 3~62% in sensitivity and 79~99% in specificity , and certain AFs, such as mild-moderate T2 hyperintensity or hepatobiliary hypointensity, showed stronger associations than other AFs. However, some studies were limited to the analysis of hepatic observations already categorized as LR5; thus, these studies involved unavoidable inflated sensitivity and unreliable significant association of AFs for diagnosing HCC. Those studies reported various rules for applying AFs for improving the diagnostic performance of LR3 and LR4 observations on gadoxetate disodium-enhanced MRI (using 2-4 or more AFs or specific combinations using independent features identified by multivariate analysis) . Recently, artificial intelligence has been actively utilized in many complex problems to facilitate the identification of complex patterns and relationships within various parameters. It has the potential to rapidly evolve into an applicable solution in the medical field to improve diagnostic accuracy, treatment strategy, and follow-up outcomes . However, to our knowledge, no study has examined the effect of machine learning algorithms on applying AFs in the LI-RADS. Therefore, this study aimed to identify the important features of AFs and determine the utilization of a machine-learning-based strategy for applying AFs to LR3 and LR4 observations on gadoxetate disodium-enhanced MRI. 2. Materials and Methods This retrospective study was conducted at a single center after approval was obtained from the institutional review board. The requirement for informed consent was waived due to the retrospective nature of the study. Interventional studies involving animals or humans and other studies that require ethical approval must list the authority that provided approval and the corresponding ethical approval code. 2.1. Study Subjects We searched our institution's electronic medical records and identified 523 treatment-naive patients at risk for HCC who underwent gadoxetate disodium-enhanced MRI between January 2017 and February 2022. We included patients who met the following criteria: (1) age >= 18 years; (2) high risk for HCC according to the LI-RADS v2018 (presence of cirrhosis or chronic hepatitis B infection regardless of the presence of cirrhosis); and (3) MRI findings of a focal hepatic solid nodule. We excluded 356 patients based on the following criteria: (1) inadequate final diagnosis such as unknown final diagnosis of malignancy as a result of immediate locoregional therapy or insufficient follow-up (<2 years) for benign lesions to determine size stability (n = 308); (2) poor quality of images for interpretation (n = 3); (3) only observations categorized as LR1, LR2, LR5, LR-TIV, or LR-M, according to the LI-RADS v2018 MFs and the algorithm (n = 45). Finally, 167 patients (132 males and 35 females; mean age, 62.8 +- 8.4 years) in whom gadoxetate disodium-enhanced MRI showed at least one untreated LR3 or LR4 observation were included in this study as a development group. For external validation, we collected 25 temporally separated patients who underwent gadoxetate disodium-enhanced MRI between March 2022 and October 2022. Excluding 5 patients due to nonavailable final diagnosis, 20 patients (17 males and 3 females; mean age, 64.4 +- 10.6 years) were enrolled as a test group. The LR3 and LR4 observations were categorized using only the MFs per the LI-RADS v2018 as follows: LR3, nonrim APHE, size 10-19 mm, and no additional MFs; LR4, nonrim APHE, size 10-19 mm, and enhancing "capsule" only as MFs; or nonrim APHE, size >= 20 mm, and no additional MFs. We did not consider threshold growth because we included only observations that first presented on gadoxetate disodium-enhanced MRI. 2.2. MRI Techniques The gadoxetate disodium-enhanced liver MRI examinations were conducted using a 3-T MRI scanner (MAGMETOM Vida, Siemens Healthcare; SIGNA Architect, GE, Erlangen, Germany). The imaging protocol included the following sequences: axial T2-weighted single-shot fast spin echo; axial T2-weighted fast spin echo; axial dual-gradient-recalled echo (GRE) T1-weighted sequence (in-phase and opposed-phase); and axial T1-weighted three-dimensional (3D) GRE with fat suppression (liver acquisition with volume acceleration--LAVA, or volumetric interpolated breath-hold examination--VIBE) obtained before and after the intravenous bolus injection of 0.025 mmol/kg gadoxetate disodium at a rate of 1.0 mL/s, followed by a subsequent 20 mL saline flush. Postcontrast axial 3D GRE images were obtained during the late hepatic arterial phase (AP; 5 s after peak aortic enhancement determined using 1 mL test bolus injection), portal venous phase (PVP; 50 s), transitional phase (TP; 3 min), and hepatobiliary phase (HBP; 20 min). Diffusion-weighted images were acquired using a maximum b-value of 800 s/mm2. Details of the MRI parameters are shown in Table A1 in Appendix A. 2.3. Image Analysis Image analyses were performed by two board-certified radiologists with >9 years of experience in hepatic imaging, who were blinded to any information about clinical history or final diagnosis. Any disagreement was resolved in consensus. Nodule size and the presence or absence of MFs (nonrim APHE, nonperipheral washout, or enhancing capsule) according to the LI-RADS v2018 were analyzed. The following AFs were also assessed based on the LI-RADS v 2018: (1) AFs favoring malignancy in general: mild-moderate T2 hyperintensity, corona enhancement, fat sparing in a solid mass, iron sparing in a solid mass, TP hypointensity, HBP hypointensity, and restricted diffusion; and (2) AFs favoring malignancy in particular: nonenhancing capsule, nodule-in-nodule, mosaic architecture, blood products in mass, and fat in mass more than that in the adjacent liver. Imaging features regarding interval change in tumor size (threshold growth, subthreshold growth, size stability >= 2 years, or size reduction) and discrete nodules observed on ultrasound were not assessed because only focal lesions initially detected on MRI were included, and prior imaging studies for the comparison were not provided. AFs favoring benignity were also not analyzed in this study because no observation showed AFs favoring benignity in preliminary imaging analysis. 2.4. Reference Standard The diagnosis of HCC was performed through histopathological confirmation or diagnosis of definitive HCC (LR5) on follow-up imaging (either contrast-enhanced computed tomography [CT] or MRI) within 12 months . Benignity was diagnosed based on histopathological results or shrinkage or stability of the solid mass as seen on follow-up serial imaging (contrast-enhanced CT or MRI) for a minimum of 24 months . The mean imaging follow-up period for benign observations was 28.1 +- 23.0 (range, 24.7-52.3) months. Histopathological diagnosis was performed after surgical resection (n = 11) or core-needle biopsy (n = 30). 2.5. Extracting Important Features and Constructing a Machine-Learning-Based Algorithm for Applying AFs Evaluating the influence of each factor is important for understanding the prediction process. In this study, we evaluated the feature importance using a random forest model. A random forest is an ensemble of decision trees. First, we sampled a dataset by allowing duplication from the training data, and many decision trees were generated using the sampled data. Second, various types of tree structures, which are called clusters of random forest trees, were generated. Important features are located at the top of each tree to predict the results, and the importance of each feature can be determined by statistically analyzing the number of trees. After determining the feature importance by random forest, we used the decision tree model for HCC prediction. A decision tree is one of the most famous machine learning algorithms. The advantage of a decision tree is that it is very intuitive and explainable for classification and regression; therefore, it is easy to understand why the results are predicted by the decision tree. In other words, unlike other machine learning algorithms such as KNN and SVM, a decision tree has the advantage of using both the results and a prediction process because of its explainability. In this study, we used scikit-learn (v1.1.1) in Python for decision tree training. The classification and regression tree (CART) method in scikit-learn is used to train the decision tree. The CART divides the data into two subsets depending on the characteristics that distinguish the data in the training set. The CART sets a threshold for one input feature, divides the data into two subsets according to the threshold, and sets the input factor and its threshold to minimize the impurity between the two divided subsets. The cost function of the classification CART is described by the following Equation (1):(1) Jk,tk=mleftmGleft+mrightmGright where is the kth feature of the input, tk is the threshold of the kth feature of the input, Gleft/right is the impurity of the left/right subset, mleft/right is the number of samples of the left/right subset, and m is the total number of samples. The CART builds its subtree using a recursive method of dividing the subset. The threshold is determined using two criteria: GINI and entropy. In this study, we used the GINI method to determine the threshold of the subset tree. Most ML algorithms cannot be reproduced due to the random characteristics of some hyperparameters. To solve the random characteristics of these ML algorithms, most ML libraries, including scikit learn, fix the random characteristics, enabling the generation of reproducible random variables. In other words, we used the randomness fixing technique of scikit-learn to perform fixing of both the randomnesses of the data splits and the hyperparameters. 2.6. Statistical Analysis All statistical analyses were performed on an observational basis. Continuous variables are presented as mean +- standard deviation (SD) or as median and interquartile range (IQR) and compared between HCC and non-malignant nodules using the Student's t-test or the nonparametric Mann-Whitney U test. Categorical variables or MFs and AFs are expressed as numbers and frequencies and compared using chi-squared test or Fisher's exact test, as appropriate. To identify significant AFs suggestive of HCCs rather than non-malignant nodules in initial LR3 or LR4 observations (LR3 or LR4 determined with only MFs, regardless of AFs), univariate and multivariate logistic regression analyses were performed. In the multivariate analysis, variables that showed a positive association with HCC (p < 0.05) in the univariate analysis were entered, and backward stepwise elimination was performed. The inter-reader agreement was evaluated using kappa statistics. The important AFs were identified using random forest analysis, and a decision tree algorithm was constructed for the application of AFs to improve diagnostic performance in the LR3 and LR4 categories. Sensitivity, specificity, accuracy, and area under the receiver operating characteristic (ROC) curve (AUC), positive predictive value, and negative predictive value were calculated to evaluate the diagnostic performance of our diagnostic systems. Statistical significance was set at p < 0.05. Statistical analyses were performed using SPSS version 25.0 (IBM Inc., Armonk, NY, USA) and MedCalc version 19.4.0 (MedCalc Software). The important AFs were identified, and a decision tree algorithm for applying AFs to the LR3 and LR4 categories was constructed using Python 3.8.13 module scikit-learn (v1.1.1) (Python Software Foundation, Wilmington, DE, USA). 3. Results 3.1. Baseline Characteristics of Patients and Observations The final study sample included 167 patients (mean age, 62.8 years; range, 33-84 years) with 245 observations (median size, 13 mm; IQR 10-17.8 mm) in the development group and 20 patients (mean age, 64.4 years; range, 37-81 years) with 30 observations (median size, 11 mm; IQR 6-23 mm) (Table 1) in the test group. Among 167 patients, hepatitis B was the most common cause of liver cirrhosis or chronic liver disease (n = 117, 77%). One hundred twelve (67.1%) patients had one lesion, 36 (23.7%) had two lesions, and 19 (12.5%) had three or more lesions. The median size of 245 observations was 13 mm (IQR, 10-17.8 mm). Among MFs, nonrim APHE was observed in 96 (39.2%) nodules, nonperipheral washout in 74 (30.2%) nodules, and enhancing capsule in 15 (6.1%) nodules. Overall, 137 HCC lesions and 108 benign lesions were identified. The test group included 20 patients. 3.2. Comparison of Imaging Features between HCCs and Non-Malignant Nodules and Important Features for Diagnosis HCC in LR3 and LR4 Observation Comparative analyses of the imaging features between HCC and non-malignant nodules are summarized in Table 2. The most common AFs recorded in HCC were HBP hypointensity (131, 95.6%), followed by TP hypointensity (113, 82.5%), restricted diffusion (88, 64.2%), and mild-moderate T2 hyperintensity (86, 62.8%). Fat sparing and iron sparing in the solid mass were not observed. Univariate analyses demonstrated that restricted diffusion, mild-moderate T2 hyperintensity, TP hypointensity, HBP hypointensity, nonenhancing capsule, and nodule-in-nodule appearance were significantly associated with HCC (all p < 0.05). In the multivariate analyses, restricted diffusion (odds ratio [OR], 12.4; 95% confidence interval [CI], 5.1-30.35; p < 0.001) and mild-moderate T2 hyperintensity (OR, 2.5; 95% CI, 1.1-5.3; p = 0.02) were independent significant features associated with HCC (Table 3). Random forest analysis showed restricted diffusion as the most important feature (feature importance ratio: 0.48), followed by mild-moderate T2 hyperintensity . Interobserver agreement of the AFs are presented in Table A2. We observed a range from 0.21 to 0.74 of kappa values for each AF. Among the AFs, hepatobiliary-phase hypointensity showed the highest kappa value. Restricted diffusion and mild-moderate T2 hyperintensity showed moderate agreement with kappa value of 0.55, in both. Nodule-in-nodule architecture showed a relatively high proportion of agreement (82.7%). However, it showed the lowest kappa value (0.21) due to its low prevalence. 3.3. Development of Decision Tree Algorithm for Application of AFs to LR3 and LR4 Observation Figure 2 shows the decision tree algorithm for the application of AFs to LR3 and LR4 observations for HCC diagnosis. Restricted diffusion was the first partitioning imaging feature of the decision tree algorithm. Further branching was performed using other features such as nodule-in-nodule, mild-moderate T2 hyperintensity, blood in mass, TP hypointensity, corona enhancement, HBP hypointensity, and fat in mass. This decision tree algorithm yielded an AUC of 0.84 (95% CI, 0.84-0.85); sensitivity of 92.0% (95% CI, 91.6-92.4); specificity of 71.1% (95% CI, 70.9-71.4); and accuracy of 84.5% (95% CI, 84.1-84.8) in the development group. In the test group, the decision tree algorithm showed good diagnostic performance: an AUC of 0.82 (95% CI, 0.76-0.88); sensitivity of 94.0% (95% CI, 89.3-98.2); specificity of 68.7% (95% CI, 58.0-79.4); and accuracy of 81.9% (95% CI, 75.8-87.9). 3.4. Comparison of Diagnostic Performance of Decision Tree Algorithm with Alternative Criteria of Applying Afs We also established alternative criteria for the application of Afs to LR3 and LR4 for HCC diagnosis. The diagnostic performance of these criteria for the application of Afs (according to the number of Afs and exclusive usage of significant Afs or their combination) is presented in Table 4. Figure A1 in Appendix A shows the diagnostic performance of the application criteria according to the number of Afs favoring malignancy at every cutoff point, that is, the number of Afs >= 1 to >=6. A cutoff value of >=3 showed the highest diagnostic performance, with an AUC of 0.75 (95% CI, 0.75-0.76); sensitivity of 77.6% (95% CI, 76.4-78.8); specificity of 72.9% (95% CI, 72.2-73.7); and accuracy of 75.5% (95% CI, 75.0-76.1). We also analyzed the application criteria via various combinations of independently significant AFs (restricted diffusion and mild-moderate T2 hyperintensity) identified from the multivariate and random forest analyses. Among those criteria, the criterion of "restricted diffusion only" yielded the highest AUC (0.78) compared with the criterion of "restricted diffusion or mild-moderate T2 hyperintensity" (AUC, 0.76; p = 0.025); "restricted diffusion and mild-moderate T2 hyperintensity" (AUC, 0.75; p = 0.01); and "mild-moderate T2 hyperintensity only" (AUC, 0.73; p < 0.001; Table A3 in Appendix A). Our decision tree approach had higher AUC, sensitivity, and accuracy than the other criteria ; however, it showed a significantly reduced specificity compared with the criterion of "restricted diffusion only" (Table 4). 4. Discussion The current study revealed two AFs favoring malignancy (restricted diffusion and mild-moderate T2 hyperintensity) as significant independent features for the diagnosis of HCC in LR3 and LR4 observations on gadoxetate disodium-enhanced MRI in both multivariate and random forest analyses. Furthermore, we presented a strategy for applying AFs in the diagnosis of HCC from the initial LR3 and LR4 observations, which are categorized using only the MFs of the LI-RADS v2018. We developed a decision tree algorithm to apply AFs to LR3 and LR4 observations. This approach was compared with other alternative approaches using the number of AFs or various combinations of significant AFs identified from multivariate and random forest analyses. Our decision tree approach showed the highest AUC, sensitivity, and accuracy compared with other criteria, albeit with somewhat compromised specificity. Our results showed that restricted diffusion and mild-moderate T2 hyperintensity were independent and important features for the diagnosis of HCC from the LR3 and LR4 observations in both multivariate and random forest analyses. In particular, restricted diffusion showed the highest odds ratio (12.4; 95% CI, 5.1-30.0) and importance ratio (0.48), which is consistent with the results of previous studies . Diffusion restriction is not specific to HCC but is more often used for detecting hepatic lesions and discriminating malignant lesions from benign lesions . It is important to understand hepatocarcinogenesis in the early diagnosis of HCC from a premalignant lesion such as a dysplastic nodule. Hepatocarcinogenesis is a multi-step process that starts from a regenerative nodule in cirrhosis or as a dysplastic nodule and progresses to advanced HCC . Given that one of the major histologic differences between dysplastic nodules and early HCC is the degree of cellular density, restricted diffusion reflecting the high cellularity of lesions might help in the better discrimination of HCC from non-malignant lesions . T2 hyperintensity is a typical imaging feature of HCC and helps differentiate hypovascular HCC from dysplastic nodules . We observed that mild-moderate T2 hyperintensity has an OR of 2.5 and an importance ratio of 0.21 and is the second most significant feature after restricted diffusion. According to previous studies, mild-moderate T2 hyperintensity has been proven to be a suggestive feature of progressed HCC rather than early HCC . When a focus of HCC develops within a dysplastic nodule, a mildly elevated signal may be observed on T2-weighted images, representing the focus of HCC within the hypointense dysplastic nodule, and has been described as a "nodule-in-nodule" appearance. This is consistent with our results showing that presentations of a nodule-in-nodule appearance were significantly more frequently encountered with HCCs than with non-malignant nodules, although their numbers were small. Thus, the entire change in T2 signal intensity of the observation may reflect the progressive biological characteristics of HCC. In our study, among AFs favoring malignancy, the most commonly encountered feature was HBP hypointensity. However, this feature was not significantly associated with HCC in the multivariate analysis. This might be because this feature appears to be frequent, even in non-malignant nodules, which is consistent with the results of previous studies . Because organic anion-transporting polypeptides that mediate hepatic uptake of gadoxetic acid may decrease in expression in the early stage of hepatocarcinogenesis, dysplastic nodules or regenerative nodules, and even hemangioma cysts, can present with HBP hypointensity . Therefore, applying this characteristic to the LR3 and LR4 categories may cause concerns regarding false positivity when diagnosing HCC. In this study, we found consistent results: HBP hypointensity was a significantly frequent finding among AFs in misclassification cases using our decision tree algorithm (Table A4). In the decision tree algorithm for the application of AFs in LR3 and LR4 observation, the results showed a good ability of the method to diagnose HCC, with an AUC of 0.84 (95% CI, 0.84-0.85); sensitivity of 92.0% (95% CI, 91.6-92.4); specificity of 71.1% (95% CI, 70.9-71.4); and accuracy of 84.5% (95% CI, 84.1-84.8). The decision tree algorithm consists of restricted diffusion, nodule-in-nodule, mild-moderate T2 hyperintensity, blood in mass, TP hypointensity, corona enhancement, HBP hypointensity, and fat in mass. Interestingly, in the decision tree algorithm, nodule-in-nodule, blood in mass, TP hypointensity, corona enhancement, HBP hypointensity, and fat in mass, which failed to demonstrate independent associations in the present study, were correlated with restricted diffusion and mild-moderate T2 hyperintensity. This may indicate that minor AFs still play important roles in the diagnosis of HCC among observations that already exhibit significant weighting features. We also evaluated an alternative application algorithm using a criterion based on the number of AFs and criterion utilizing independent features identified by multivariate analysis. These approaches have been addressed in previous studies . Kang et al. reported that criteria with the number of AFs >= 4 showed a sensitivity of 80.6% and a specificity of 70.0% , and Cannella et al. reported that criteria with the number of AFs >= 2 showed a sensitivity of 72.6% and a specificity of 91.5% . The present study also showed good diagnostic ability for HCC using criteria with the number AFs >= 3, with a sensitivity of 77.6% and a specificity of 72.9%. Direct comparison between results among the studies may be unnecessary because of the differences in the characteristics between study populations. Nevertheless, our results have strength in overcoming the overestimation of sensitivity because of the exclusion of the LR5 observation. In addition, this approach showed significantly lower sensitivity and specificity than those of the decision tree algorithm. Cannella et al., Lee et al., and Jeon et al. showed how to incorporate AFs identified as significant independent features in multivariate analysis to enhance diagnostic performance in the LR3 and LR4 categories in the LI-RADS diagnostic table . In the present study, we identified that the highest diagnostic performance for HCC was achieved using the exclusive application of restricted diffusion to the LR3 and LR4 categories, among other combinations of significant AFs. This approach had significantly higher specificity than our decision tree algorithm (91.3% vs. 71.1%, p < 0.001). Nevertheless, our decision tree algorithm showed significantly higher AUC, sensitivity, and accuracy in HCC diagnosis in LR3 and LR4 observations (AUC, 0.84 vs. 0.78, p < 0.001; sensitivity, 92.0% vs. 64.5%, p < 0.001; and accuracy, 84.5% vs. 76.4%, p = 0.032) than that of the criteria utilizing the exclusive application of restricted diffusion to the LR3 and LR4 category. Indeed, the LI-RADS is designed to promote the specific diagnosis of HCC . Other Western countries also adopt specific diagnostic algorithms to avoid false-positive diagnoses of HCC, because liver transplantation is the only potentially curative treatment in patients with advanced cirrhosis, who predominantly constitute individuals with a high risk of HCC in Western countries . Meanwhile, Asian countries prefer sensitive diagnosis of HCC to detect HCC in its early stages and to provide patients with HCC with local treatment, such as resection or ablation, as a curative treatment . Therefore, despite significantly reduced specificity, our decision tree algorithm for the application of AFs with significantly high sensitivity can be used more in Asian societies. This decision tree algorithm is a conceptually simple decision-making model and provides the diagnosis process of HCC in an easy-to-understand classification system. Thus, it may be useful in situations in which a decision must be made effectively and reliably. Although there were some limitations in the absence of AFs favoring benignity in our decision tree algorithm, it may be still useful in daily practice, as compared with other alternative approach. Our study had several limitations. First, there may have been an inevitable selection bias owing to the retrospective nature of this study. Among the initially eligible patients, approximately 308 patients were excluded from the study population due to a lack of a final diagnosis. Among these, the majority showed LR4 observation. They tended to be treated with locoregional treatment without pathological confirmation, especially when they exhibited a co-existing LR5 observation. Second, our study conducted LR3 and LR4 observations simultaneously. As LR3 and LR4 may express different distributions of AFs between HCC and non-malignant nodules, subgroup analysis of LR3 and LR4 showed more confident study results. Although subgroup analysis could not be performed in this study due to the lack of LR4 lesions, a larger study should be conducted in the future. Lastly, the majority of benign lesions in this study were not confirmed using biopsy but by follow-up imaging. To minimize misdiagnosis, we considered benignity based on long-term stability (>=24 months), whereas HCC was considered based on the presence of LR5 observation on follow-up imaging . 5. Conclusions In conclusion, among the LI-RADS v2018 AFs favoring malignancy, restricted diffusion and mild-moderate T2 hyperintensity showed a strong association for the diagnosis of HCC in LR3 and LR4 observations. Our decision tree algorithm for applying AFs to LR3 and LR4 observations provides significantly increased AUC, sensitivity, and accuracy but reduced specificity. These appear to be more appropriate for application under certain circumstances with an emphasis on early detection of HCC. Acknowledgments The authors thank the reviewers and editors from the CANCERS journal. Author Contributions Conceptualization, J.B.; methodology, S.P. and J.B.; software, S.P.; validation, J.B.; formal analysis, J.B.; investigation, S.P. and J.B.; resources S.P. and J.B.; data curation, S.P., S.M.H. and J.B.; writing--original draft preparation, S.P. and J.B.; writing--review and editing, S.P., S.M.H. and J.B.; visualization, S.P. and J.B.; supervision, J.B.; project administration, J.B.; funding acquisition, S.P. and J.B. All authors have read and agreed to the published version of the manuscript. Institutional Review Board Statement This study was approved and exempted from informed consent by the Institutional Review Board of Ewha Womans University Seoul Hospital (2022-09-071-001), date of approval 15 November 2022. Informed Consent Statement Informed consent was waived by the Institutional Review Board of Ewha Womans University Seoul Hospital due to the nature of the retrospective study design. Data Availability Statement The data presented in this study are available in this article. Conflicts of Interest The authors declare no conflict of interest. Appendix A cancers-15-01361-t0A1_Table A1 Table A1 Parameters of the MRI sequences. Sequence T1 LAVA/VIBE Dual-echo T1 GRE Navigator-triggered TSE T2 DWI + SIGNA Architect MAGNETOM Vida SIGNA Architect MAGNETOM Vida SIGNA Architect MAGNETOM Vida SIGNA Architect MAGNETOM Vida Repetition time (ms) 43.42 3.2 125 164 2100 1300 2600 2300 Echo time (ms) 1 1 1, OP; 3, IP 1, OP; 3, IP 96 83 67 60 Flip angle (deg) 10 11 60 57 90 120 90 90 Matrix 260 x 220 352 x 209 260 x 220 320 x 216 260 x 260 256 x 216 260 x 260 130 x 106 Field of view 360 x 360 400 x 338 360 x 360 400 x 338 360 x 360 400 x 338 360 x 360 400 x 326 Section thickness (mm) 4 3 5 5 5 5 5 5 No. of signal acquisitions 1 1 1 1 1 1 4 2 DWI--diffusion-weighted imaging, GRE--gradient-recalled echo, IP--in-phase, NA--not applicable, OP--out-of-phase, T1--T1-weighted, T2--T2-weighted, TSE--turbo-spin echo, VIBE--volumetric interpolated breath-hold examination, LAVA--liver acquisition with volume acceleration. + Diffusion-weighted imaging was performed using four b-values of 0, 50, 400, and 800 s/mm2. cancers-15-01361-t0A2_Table A2 Table A2 Inter-observer agreement for AFs in the 274 nodules. Favoring Malignancy in General, Not HCC in Particular Agreement Proportion Kappa Value Corona enhancement 98.1 (269) 0.66 Restricted diffusion 78.8 (216) 0.55 Mild-moderate T2 hyperintensity 78.8 (216) 0.55 ron sparing in solid mass 100 (274) 0.05 Fat sparing in solid mass 100 (274) 0.50 Transitional-phase hypointensity 85.4 (234) 0.47 Hepatobiliary-phase hypointensity 94.2 (258) 0.74 Favoring HCC in particular Nonenhancing capsule 96.2 (264) 0.65 Nodule-in-nodule architecture 82.7 (227) 0.21 Mosaic architecture 96.2 (264) 0.48 Fat in mass, more than adjacent liver 92.3 (253) 0.73 Blood products in mass 96.2 (264) 0.49 Data in parentheses are number of nodules. HCC--hepatocellular carcinoma. Figure A1 Receiver operating characteristic curve of the number of ancillary features favoring malignancy for the diagnosis of hepatocellular carcinoma and diagnostic performance of each criterion values. cancers-15-01361-t0A3_Table A3 Table A3 Diagnostic performance of criteria of applying AFs with various combinations of significant associations. AUC (95% CI) Sensitivity (95% CI) Specificity (95% CI) Accuracy (95% CI) PPV (95% CI) NPV 95% CI) (1) 0.78 (0.77, 0.78) 64.5% (63.6, 65.4) 91.3% (90.8, 91.7) 76.4% (75.7, 77.0) 90.3% (89.9, 90.7) 67.2% (66.1, 68.2) (2) 0.73 (0.72, 0.73) 63.1% (61.7, 64.5) 82.3% (81.4, 83.1) 71.6% (70.9, 72.3) 81.7% (80.8, 82.6) 64.0% (62.8, 65.1) (3) 0.76 (0.75, 0.76) 72.7% (71.8, 73.7) 78.3% (77.5, 79.0) 75.2% (74.8, 75.6) 80.8% (80.0, 81.5) 69.6% (68.5, 70.6) (4) 0.75 (0.74, 0.76) 54.9% (53.6, 56.1) 95.3% (94.9, 95.7) 72.8% (71.9, 73.6) 93.6% (93.1, 94.1) 62.7% (61.6, 63.8) Comparisons of each criterion, p-value AUC sensitivity specificity accuracy PPV NPV (2) (3) (4) (2) (3) (4) (2) (3) (4) (2) (3) (4) (2) (3) (4) (2) (3) (4) (1) <0.01 0.03 0.01 0.91 0.18 0.13 0.08 0.01 0.37 0.27 0.84 0.42 0.12 0.08 0.6 0.66 0.77 0.48 (2) <0.01 0.12 0.12 0.209 0.57 0.01 0.42 0.85 0.99 0.03 0.41 0.91 (3) 0.35 0.003 <0.01 0.62 0.02 0.27 (1) Restricted diffusion; (2) mild-moderate T2 hyperintensity; (3) restricted diffusion or mild-moderate T2 hyperintensity; (4) restricted diffusion and mild-moderate T2 hyperintensity; AUC--area under curve; PPV--positive predictive value; NPV--negative predictive value; CI--confidential interval; AF--ancillary feature; DWI--diffusion-weighted image. cancers-15-01361-t0A4_Table A4 Table A4 Comparison between true prediction and false prediction using decision tree algorithm in development group. True Prediction (n = 200) False Prediction (n = 45) p-Value Favoring malignancy in general Corona enhancement 3 (1.5) 1 (2.2) 0.56 Fat sparing in solid mass 0 (0.0) 0 (0.0) Restricted diffusion 89 (44.5) 8 (17.8) 0.01 Mild-moderate T2 hyperintensity 93 (46.5) 12 (26.7) 0.02 Iron sparing in solid mass 0 (0.0) 0 (0.0) Transitional phase hypointensity 154 (77.0) 32 (71.1) 0.4 Hepatobiliary phase hypointensity 180 (90.0) 45 (100.0) 0.03 Favoring HCC in particular Nonenhancing "capsule" 12 (6.0) 0 (0.0) 0.13 Nodule-in-nodule appearance 12 (6.0) 1 (2.2) 0.472 Mosaic architecture 5 (2.5) 0 (0.0) 0.59 Fat in mass, more than adjacent liver 28 (14.0) 8 (17.8) 0.52 Blood products in mass 5 (2.5) 0 (0.0) 0.59 Data are the number of patients with percentages in parentheses. Figure 1 Ranking of feature importance. Figure 2 Decision tree algorithm for applying ancillary features to LR3 and LR4 categories. Figure 3 Histologically proven hepatocellular carcinoma (HCC) in a 67-year-old man with hepati-tis-B-virus-related liver cirrhosis. Gadoxetate disodium-enhanced MRI (a-e, arrow) shows a 10-mm observation with nonrim arterial phase hyperenhancement (a) and lack of definitive nonperipheral washout or enhancing capsule on portal venous (b) and transitional phase image (c). Thus, it is categorized as LR3 according to major features only. The observation shows restricted diffusion (b = 800 s/mm2; (d)) and mild T2 hyperintensity (e). Our decision tree algorithm classifies this nodule as HCC. Figure 4 Histologically proven hepatocellular carcinoma (HCC) in a 35-year-old woman with chronic hepatitis B. Gadoxetate disodium-enhanced MRI (a-e, arrow) shows a 17-mm observation with a smaller inner nodule showing different imaging characteristics than the larger outer nodules on precontrast T1 image (a). This demonstrates the nodule-in-nodule. This observation does not show nonrim arterial phase hyperenhancement (b) in the nonperipheral washout on portal venous phase. (c) It shows enhancing capsule in the portal venous (c) and transitional phase (d). Thus, it is cate-gorized as LR4 according to major features only. The observation shows suspicious mild T2 hy-perintense foci within the nodule (arrowhead; (e)). Our decision tree algorithm classifies this nodule as HCC. cancers-15-01361-t001_Table 1 Table 1 Baseline characteristic of 245 observations in 167-patient development group and 30 observations in 20-patient test group. Characteristics Development Group Test Group Age * 62.8 +- 8.4 64.4 +- 10.6 Sex Male 132 (79.0) 17 (85.0) Female 35 (23.) 3 (15.0) Underlying disease Hepatitis B with/without cirrhosis 117 (77.0) 13 (65.0) Hepatitis C with cirrhosis 10 (6.6) 2 (10.0) Alcoholic liver disease with cirrhosis 30 (19.7) 4 (20.0) NASH with cirrhosis 3 (2.0) 0 (0.0) Cryptogenic cirrhosis 7 (4.6) 1 (5.0) Laboratory + AST (IU/L) 36 (27.0, 52.5) 46 (25, 96) ALT (IU/L) 28 (19.0, 41.8) 28 (6, 148) Total bilirubin (mg/dL) 0.8 (0.5, 1.2) 1.2 (0.4, 5.7) Prothrombin time (s) 12.7 (12.0, 13.6) 13.3 (12, 22) Platelet (x1000/mL) 123 (82.5, 177.5) 116 (37, 299) AFP (ng/mL) 7.1 (3.5, 26.7) 10.4 (1.8, 239) No. of nodules included in the analysis 1 112 (73.7) 13 (65.0) 2 36 (23.7) 4 (20.0) 3 16 (10.5) 3 (15.0) 4 2 (1.3) 5 1 (0.7) LiRADS category LR3 199 (81.2) 20 (66.7) LR4 46 (18.8) 10 (33.3) Major feature Size + 13 (10.0, 17.8) 11 (6, 23) APHE 96 (39.2) 20 (66.7) Non-peripheral washout 74 (30.2) 9 (30.0) Enhancing capsule 15 (6.1) 6 (20.0) Reference standard Pathologic diagnosis 41 (16.7) 11 (36.7) Imaging follow-up 204 (83.3) 19 (63.3) Final diagnosis HCC 137 (55.9) 16 (53.3) Non-malignant nodule 108 (44.1) 14 (46.7) Unless otherwise indicated, data are the number of patients with percentages in parentheses. * Data are mean +- standard deviation. + Data are median value, and data in parentheses are range. NASH--non-alcoholic steatohepatitis; AFP--alpha-fetoprotein; ALT--alanine aminotransferase; AST--aspartate aminotransferase; APHE--arterial phase hyperenhancement. cancers-15-01361-t002_Table 2 Table 2 Distribution of ancillary features favoring malignancy between HCC and non-malignant nodule. Development Group Test Group HCC (n = 137) Non-Malignant Nodules (n = 108) p-Value HCC (n = 16) Non-Malignant Nodules (n = 14) p-Value Favoring malignancy in general Corona enhancement 3 (2.2) 1 (0.9) 0.633 1 (6.3) 0 (0.0) 1.0 Fat sparing in solid mass 0 (0.0) 0 (0.0) NA 0 (0.0) 0 (0.0) NA Restricted diffusion 88 (64.2) 9 (8.3) <0.001 13 (81.3) 0 (0) <0.001 Mild-moderate T2 hyperintensity 86 (62.8) 19 (17.6) <0.001 14 (87.5) 1 (7.1) <0.001 Iron sparing in solid mass 0 (0.0) 0 (0.0) NA 0 (0.0) 0 (0.0) NA Transitional phase hypointensity 113 (82.5) 73 (67.6) 0.007 9 (56.3) 11 (78.6) 0.20 Hepatobiliary phase hypointensity 131 (95.6) 94 (87.0) 0.015 12 (75.0) 12 (85.7) 0.46 Favoring HCC in particular Nonenhancing "capsule" 11 (8.0) 1 (0.9) 0.011 0 (0.0) 0 (0.0) NA Nodule-in-nodule appearance 12 (8.8) 1 (0.9) 0.007 1 (6.3) 0 (0.0) 1.0 Mosaic architecture 5 (3.6) 0 (0.0) 0.069 2 (12.5) 0 (0.0) 0.53 Fat in mass, more than adjacent liver 25 (18.2) 11 (10.2) 0.077 1 (6.3) 3 (21.4) 0.50 Blood products in mass 5 (3.6) 0 (0.0) 0.069 1 (6.3) 0 (0.0) 1.0 Data are the number of patients with percentages in parentheses. cancers-15-01361-t003_Table 3 Table 3 Univariable and multivariable analysis of AFs associated with HCC. Univariable Analysis Multivariable Analysis OR (95% CI) p-Value OR (95% CI) p-Value Favoring malignancy in general Corona enhancement 2.4 (0.2, 23.4) 0.452 Fat sparing in solid mass NA Restricted diffusion 19.8 (9.2, 42.5) <0.001 12.4 (5.1, 30.3) <0.001 Mild-moderate T2 hyperintensity 7.9 (4.3, 14.5) <0.001 2.5 (1.1, 5.3) 0.022 Iron sparing in solid mass NA Transitional phase hypointensity 2.3 (1.2, 4.1) 0.008 0.9 (0.4, 2.1) 0.883 Hepatobiliary phase hypointensity 3.3 (1.2, 8.8) 0.020 5.5 (0.9, 32.1) 0.057 Favoring HCC in particular Nonenhancing "capsule" 9.3 (1.2, 73.5) 0.034 15.8 (0.8, 318.2) 0.072 Nodule-in-nodule appearance 10.3 (1.3, 80.3) 0.026 16.5 (0.9, 142.1) 0.051 Mosaic architecture 1,321,752,144.2 (0.0) 0.999 Fat in mass, more than adjacent liver 2.0 (0.9, 4.2) 0.081 Blood products in mass 1,321,752,144.2 (0.0) 0.999 OR--odds ratio; CI--confidence interval. cancers-15-01361-t004_Table 4 Table 4 Comparison of various approaches for applying AFs to LR3 and LR4 for diagnosis of HCC. AUC (95% CI) Sensitivity (9% CI) Specificity (95% CI) Accuracy (95% CI) PPV (95% CI) NPV (95% CI) I. Decision tree algorithm Development cohort 0.84 (0.84, 0.85) 92.0% (91.6, 92.4) 71.1% (70.9, 71.4) 84.5% (84.1, 84.8) 70.5% (70.2, 70.9) 92.2% (91.8, 92.7) Test cohort 0.82 (0.76-0.88), 94.0% (89.3-98.2) 68.7% (58.0-79.4) 81.9% (75.8-87.9) 70.7% (63.0-78.5) 92.3% (87.5-97.2) II. Number of AFs >= 3 0.75 (0.75, 0.76) 77.6% (76.4, 78.8) 72.9% (72.2, 73.7) 75.5% (75.0, 76.1) 78.3% (77.4, 79.1) 72.2% (70.9, 73.6) III. Restricted DWI 0.78 (0.77, 0.78) 64.5% (63.6, 65.4) 91.3% (90.8, 91.7) 76.4% (75.7, 77.0) 90.3% (89.9, 90.7) 67.2% (66.1, 68.2) Comparison of each approach for applying AFs I vs. II p < 0.001 p = 0.002 p = 0.886 p = 0.017 p = 0.216 p < 0.001 I vs. III p < 0.001 p < 0.001 p < 0.001 p = 0.032 p < 0.001 p < 0.001 II vs. III p = 0.011 p = 0.024 p < 0.001 p = 0.899 p = 0.025 p = 0.469 AUC--area under curve; PPV--positive predictive value; NPV--negative predictive value; CI--confidential interval; AF--ancillary feature; DWI--diffusion-weighted image. Disclaimer/Publisher's Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

PMC10000174

The concept of advocacy is of increasing importance to the veterinary profession internationally. However, there are concerns around the ambiguity and complexity of acting as an advocate in practice. This paper explores what 'animal advocacy' involves for veterinarians working in the domain of animal research, where they are responsible for advising on health and welfare. In focusing on the identity of veterinarians working in an arena of particular contestation, this paper provides empirical insights into how veterinarians themselves perform their role as an 'animal advocate'. Analysing interview data with 33 UK 'Named Veterinary Surgeons', this paper therefore examines what 'counts' as animal advocacy for veterinarians, considering the way their role as animal advocate is performed. Focusing on the themes of 'mitigating suffering', 'speaking for', and 'driving change' as three central ways in which veterinarians working in animal research facilities act as animal advocates, we draw out some of the complexities for veterinarians working in areas where animal care and harm coexist. Finally, we conclude by calling for further empirical exploration of animal advocacy in other veterinary domains and for more critical attention to the wider social systems which produce the need for such advocacy. veterinary profession advocacy responsibility laboratory science animal research Wellcome Trust205393/B/16/Z This work was funded in whole, or in part, by the Wellcome Trust (grant number 205393/B/16/Z). For the purpose of open access, the authors have applied a CC BY public copyright licence to any Author Accepted Manuscript version arising from this submission. pmc1. Introduction 1.1. Animal Advocacy and the Veterinary Profession Acting as an 'advocate' for animals is framed as a key principle for the modern veterinary profession. For example, in their 2016 animal welfare strategy report entitled 'Vets speaking up for animal welfare', the British Veterinary Association (BVA) highlight the importance of advocacy to the veterinary profession, claiming that 'Our opportunity to be advocates for animals may be the greatest of all and we have clear social, professional and legal responsibilities to do so' (p. 9). Also cited here is the World Organization for Animal Health's assertion that veterinarians should provide leadership on ethical issues and 'should be the leading advocates for the welfare of all animals' (ibid), illustrating the global proliferation of the language of 'advocacy'. Relatedly, in their 'recalibration' of the veterinary profession for the 2020s, De Paula Vieira and Anthony argue that 'society expects veterinarians to be animal advocates' (p. 7), adding that 'veterinarians will need to utilise their voice more' when called upon to provide societal leadership on moral and welfare issues involving animals (ibid, p. 2). Despite this shared language of veterinary 'advocacy', the concept of advocacy is complex and eludes a single definition, encompassing multiple, sometimes incongruent, values and necessitating different approaches in its practice. Capturing this ambiguity in their review of the theory and practice of advocacy in relation to social work, Forbat and Atkinson (p. 322) observe that 'At its simplest, advocacy means 'speaking up' for oneself or others. However, it is rarely that simple'. In the nursing profession, the nurse has long been theorised as a 'patient advocate', representing and intervening on behalf of those who are vulnerable . Indeed, Gadow (p. 81) put forward the influential concept of 'existential advocacy' as core to the nurse's role, with nursing described as a process of participating with the individual's unique experience of health and illness. For Gadow, existential advocacy is 'based upon the principle that freedom of self-determination is the most valuable human right' (ibid, p. 84). Such humanistic conceptualisations of advocacy rely on principles of self-determination and 'Promoting and protecting patients' rights to be involved in decision-making and informed consent' (p. 35). These ideas are not easily transferable to non-human animal advocacy, given communication barriers between humans and animals. A possible parallel is paediatrics. Here, Waterston (p. 155) summarises that 'Paediatricians advocate for children because they are vulnerable and not usually able to speak for themselves' and claims that such advocacy is importantly embedded in the principle of partnership with the family (p. 587). However, this differs with animal patients, as Ashall, et al. (p. 255) affirm that 'Whilst medical consent protects a patient's rights to make autonomous decisions concerning their own body, veterinary informed consent aims to protect an owner's right to make autonomous decisions concerning their legal property'. This imperfect comparison illustrates the complexity of applying a concept such as advocacy across medical and veterinary contexts. Indeed, existing veterinary literature confirms the challenges of advocacy in the veterinary context, including the question of balancing what the vet and the client (owner) may desire. For example, Morgan (p. 116) distinguishes between the 'animal advocate' and the 'client advocate' professional model, with the former prioritising the interests of the animal and the latter prioritising those of the client, though they acknowledge that such models 'do not have firm boundaries' in practice. Others have utilised ideas of partnership similar to that raised by Waterston. For example, Gray and Fordyce (p. 10) suggest that both the veterinarian and owner should act as advocates for the animal patient but add that this framework 'requires elevation of the status of the animal patient to more than just 'property' or legal 'object''. Yet, if it is the case that the principle of autonomy has been applied to owners rather than animal patients, Hiestand (p. 6) claims that 'this misstep has serious consequences for both the integrity of the profession and animal patients'. While the 'opportunity' for veterinarians to be 'advocates for animals' may be great as the BVA urges, there is evident complexity for the individual professional in performing this advocacy on the ground. In thinking about the current veterinary profession and the extent to which principles of advocacy inform the veterinary role, Main has claimed that despite evidence of public trust in the veterinary profession, the profession needs to work harder to actually fulfil societal expectations regarding their advocacy role. Main adds that although clinicians are justified in their daily ambition to promote animal interests, 'a minimal scratch below the surface reveals obvious tensions in this well-intentioned mantra within the profession'. Furthermore, there is no 'one' singular veterinary profession, and particular challenges exist in different contexts. For example, in companion animal medicine, Kipperman and German (p. 5) argue that failing to address problems such as obesity represents an abdication of their animal advocate role. Both Coghlan and Hernandez et al. promote a strong and active approach to patient advocacy in the veterinary profession. However, as the latter indicates, 'Veterinarians embedded within certain animal production industries may find it particularly challenging to separate their ethical obligations to animals from their professional responsibilities to the corporation within which they are employed' (p. 6). Another domain in which veterinarians are tasked with managing multiple responsibilities which may challenge their ethical obligations to animals is in animal research. In the UK, 'Named Veterinary Surgeons' (henceforth 'NVSs') advise on the welfare of animals kept at scientific establishments. As will be discussed, these veterinarians are involved in a contested practice in which animal care often coalesces with harm . Attending to how such veterinarians perform their role through the 'animal advocate' identity stands to offer important empirical insights into some of its key characteristics, complexities, and limits in practice. 1.2. Animal Research, the NVS, and Animal Advocacy In the UK, the presence of a veterinarian in all research facilities using animal models is mandated by law under the Animals (Scientific Procedures) Act 1986--'ASPA'--which governs UK scientific animal use . Being 'nominated by the establishment license holder and specified in the establishment license', the NVS is 'responsible for, monitors and provides advice on the health, welfare and treatment of animals, and should help the establishment license holder to fulfil his/her responsibilities' (p. 150). Working under the ASPA, the NVS is also required to uphold the principles of the 3Rs (Russell and Burch 1959). This requires that researchers must aim for 'the replacement of animals with alternative mechanisms, where possible; the reduction of the number of animals required for a given procedure through statistical or other improvements; and the refinement of experimental procedures to minimise suffering and improve animal welfare' (pp. 605-606). Crucially, in addition to these core requirements under the ASPA, the NVS is also governed by their professional accreditation and have 'professional responsibilities to the animals under their care, to other veterinary surgeons, to the public, and to the Royal College of Veterinary Surgeons (RCVS) (under the VSA (Veterinary Surgeons Act 1966))' (p. 72). This means that the NVS must 'actively navigate the boundary between these two pieces of legislation, exercising professional judgement to reconcile potentially conflicting tensions arising from multiple professional accountabilities within the laboratory' . Existing empirical literature on the role of the NVS is sparse, but has focused, for example, on their route into the role from clinical practice ; their relationship with the 3Rs ; geographical dimensions of their ethical boundary work and their role in promoting standardisation and regularisation . Historians have argued that animal welfare groups were the first to propose the role, imagining the veterinarian to act as 'the animal's friend' (pp. 117-118). Today, the NVS is claimed to be an 'advocate of the animal' (p. 26) and, indeed, acting as an advocate is described as the role's 'principal reward' (p. ii). However, existing literature also highlights some of the complexities of performing this advocacy role in practice. For example, Brouwer-Ince (p. ii) has neatly summarised some of these challenges, which involve: 'Challenges to your professional judgement, because your decisions may not be popular with some researchers; challenges to your scientific judgement where the broad scientific training of a veterinary surgeon may exceed the knowledge of a researcher in a narrow specialism; challenges to your personal ethics in making cost/benefit assessments if you feel that the scientific benefits are sometimes being overstated by researchers [...]; challenges to your ability to give truly impartial advice if that advice costs your employer time and money; challenges to your personal integrity when researchers recognise that your views might carry more weight than theirs; and challenges to your personal social life because involvement in animal research still carries a stigma in some quarters.' This list of challenges is evidence of the ethical complexity of the NVS role, with multiple factors complicating the veterinarian's responsibilities for the welfare of research animals. In short, the NVS must balance multiple care obligations: caring for research animals, with the welfare of each individual being interwoven with the cohort, caring for the specific and broader scientific goals, and caring for patients and publics invested in both the expected scientific outputs and the protection of animal welfare. Given these complexities, it is arguably unsurprising that some critics have voiced concern about the extent to which the veterinary profession should even be involved in the practice of animal research. Indeed, during the formalisation of the NVS role through A(SP)A (1986), there was some dissensus within the veterinary profession. As historians Kirk and Myelnikov (pp. 117-118) observe, although the establishment of a veterinarian in the animal research domain was viewed as a compromise between welfare and scientific interests, the prioritisation of the veterinarian's 'duty of care for the animal made experiments a difficult sell to many veterinarians'. This is part of a critical debate beyond animal research, which continues today, about whether and how veterinarians are complicit in profiting from the bodily labours of animals , their commodification in both life and afterlife , and whether or not the profession itself is compatible with ideas of animal advocacy . With this wider context in mind, it becomes increasingly urgent to understand more about how the identity of the veterinarian as an animal advocate is performed by those working within the complex practice of animal research, in which animal care coincides and converges with deliberate harm. In this paper, we therefore examine the performance of veterinary advocacy within animal research facilities through analysis of interviews with NVSs. Our analytical approach draws on the literature already discussed in both the human and veterinary fields. In particular, being inspired by Friese , who, following Despret, asks 'what counts' to animal technicians 'in the doing of their work', we are similarly keen to explore what 'counts' as animal advocacy for NVSs. More specifically, we anticipate that the insertion of a veterinarian into the animal research facility as an animal advocate creates the potential for dissensus, establishing 'the presence of two worlds in one', to briefly borrow terminology from the political philosophy of Ranciere (p. 43), in which the logics of veterinary advocacy within the laboratory to 'count' the animals in one way exists alongside the logics of science, which include the requirement to 'count' the animals in another. 2. Methods This study focusing on NVSs is part of a broader constellation of work under the Animal Research Nexus Programme (Animal Research Nexus Programme 2019; Davies et al. 2020). The aim of this specific study was to understand more about the role of veterinarians as key and underexplored actors in animal research facilities. Ethical approval for data collection was granted by the School of Veterinary Medicine and Science at the University of Nottingham (approval number 1800160608), and data collection took place in 2018. An interview guide was developed and discussed with an expert advisory panel of three NVSs and was trialled during two pilot interviews with NVSs. The interview format and question focus were subject to revision as the data collection progressed. NVS participants were contacted through snowball sampling initiated via personal networks and a call out during a specialist conference. Thirty-three interviews were carried out in person at a location chosen by the participant or over the telephone. Interviews were transcribed by a third-party under a confidentiality agreement. Transcripts were anonymised and decontextualised and each transcript was assigned a random but gender-specific pseudonym beginning with the letters M, N, O, or P. An initial inductive thematic analysis of the transcripts was originally undertaken by the second author, using the qualitative data analysis software NVivo 12. This aimed to create analytic themes that reflect meaning-based patterns across the interview dataset. The use of this inductive approach was particularly important as NVSs are an understudied group, and there was minimal prior research on this group to draw upon to inform the study and analysis. Through two coding cycles, this phase of analysis concentrated on drawing out participants' physical and conceptual actions using their own phrasing and language to prioritise their voices in driving the analysis and then worked to broaden and deepen these categories, focusing on the patterns underpinning them . This coding process was not linear, and in developing the second cycle of codes, the first cycle categories were sometimes modified in title or content as the analyst clarified the key themes. Following the initial phase of coding, a second phase of thematic analysis was undertaken by the first author. Working with the second author's coding structure in NVivo 12, the first author created subcodes within the thematic category of 'Advocate for the animals'. With animal advocacy already identified as an important theme in the dataset, this round of coding was guided by a broad questioning of what 'animal advocacy' means to Named Veterinary Surgeons. In doing so, the first author sought to examine how the advocate identity might be configured through particular orientations, enactments, and understandings of their role. These issues formed the basis of discussions between the authors. In drawing on this analysis, this paper therefore explores how the enactment of animal advocacy was characterised by Named Veterinary Surgeons, focusing on the three key aspects of mitigating suffering; speaking for; and driving change that were prominent throughout many of the interviews. Though overlapping in many ways, these themes are organised separately for clarity. This analysis aims to elucidate how veterinarians working in the ethically complex and contested arena of animal research perform 'advocacy' and draw out the tensions that may exist within this. Such tensions have the potential to complicate the dominant image of the veterinarian as 'the kindly, trained person who knows how to take care of [one's] companion animal' (p. 69). In other words, focusing on how veterinarians frame their advocacy for research animals within complex structures in which care and harm coalesce may offer important insights into the reality of the veterinary profession, which is 'considerably more murky' than the dominant image (p. 69). As will be returned to in the discussion, such analysis may reveal the limits of veterinary animal advocacy in the research context, how it might be expanded, better supported, and what can be expected of Named Veterinarians as advocates for research animals. 3. Results 3.1. Mitigating Suffering 3.1.1. Preventing and Limiting 'Unnecessary' Suffering A key way in which participants described enacting their role as an 'animal advocate' was through preventing and limiting suffering via the safeguarding of animal welfare. This may sound simple initially, but the analysis reveals how complex this can be in practice. As the following examples illustrate, this can mean drawing lines between 'necessary' and 'unnecessary' suffering : 'So, whatever is in the best interest of animals here. Like, say you have the animals occasionally suffer and you have to distinguish between the scientific benefits for study, and animals shouldn't suffer. And it depends on type of the study [...] you have to really be aware of these things and you have to stop everything, even despite millions of pounds invested and you have to say, 'Look...'' (Obadiah). 'Really just to prevent pain, suffering, distress and lasting harm or if you can't prevent it and obviously, if you've got justification and you're allowed to carry it out, that's what it's all about. But to mitigate it as much as possible and so I would say it entirely centres around how we control pain, suffering, distress and lasting harm [...] I mean the lasting harm is a bit difficult but the pain, suffering and distress' (Owen). As both participants indicate, some amount of animal suffering may be an accepted part of a study and so the NVS must work, as Owen states, 'to mitigate it as much as possible'. However, at certain points, the NVS may call for an end to an animal's use within a study, meaning the possible disruption of research aims, as Obadiah notes 'even despite millions of pounds invested and you have to say, 'Look...''. To give another example, the extract below from Mia's interview demonstrates their desire to ensure that the animals are 'looked after properly' and that research does not go 'too far' in compromising animal welfare. This is also linked, however, with apparent acceptance of the value of animal research: '[...] my role is there to protect them and to stand up for them, so that's how I, presumably, in my own mind, justify it to myself, that I am involved in research in that way. Partly because I see that there's a benefit in the long run but because I feel I'm there to stand up and make sure that those animals are looked after properly and appropriately and that research isn't allowed to go too far' (Mia). Through their focus on animal welfare, NVSs can therefore be seen to articulate the importance of preventing and limiting 'unnecessary' suffering to their performance of the animal advocate role. However, the analysis reveals that there are limits to this in practice and that, sometimes, the NVSs' animal advocacy may require 'ending' suffering. 3.1.2. Ending Suffering In some cases, preventing or minimising suffering is not deemed possible or sufficient and thus NVSs report the use of 'euthanasia' to end the suffering of particular animals. For instance, Oliver indicates that if measures to make animals more comfortable are not working then, as in general practice, the NVS will recommend euthanasia: '[...] it is the NVS job, to be the animal's advocate and if we feel that the animal's uncomfortable we will always recommend euthanasia but then that's the same as in general practice. I think where the problem is sometimes I think, I'll see an animal and I'll think well this might get better if I do this or do that. And two days later it's still not much better and you think well do we carry on? Because Home Office guidelines would almost certainly say no, you stop at this point' (Oliver). In animal research, such decision making reflects the guidance against using death as an endpoint for an animal's use in a study. Rather, researchers are encouraged to implement 'humane endpoints'. This involves defining 'clear, predictable and irreversible criteria which substitute for more severe experimental outcomes such as advanced pathology or death', and, when these are reached, the animal is humanely killed . However, decision-making around euthanasia or humane killing can involve more than assessments of individual animal welfare when deciding what counts as 'too much' suffering. In general veterinary practice, it may be complicated by the human-animal bond , the client's judgment of the animal's health , their wider interests, and their finances . In animal research, with the 'two worlds' of veterinary professionalism and scientific practice existing 'in one', decisions around killing an animal may also be complicated by the competing interests of researchers and other institutional actors, obligations towards the research aims and helping to ensure meaningful outputs from the experiences of animals, and the statistical value of each animal which interweaves their individual welfare. Indeed, as Michelle discusses, this can create areas of conflict: 'I think you can have areas of conflict where the researchers say, 'This is a very valuable animal and ...', it always happens, whenever anything, there's a problem with the animal, it always becomes the most valuable animal they have in their research and they want to continue using it to get the results they want. And then you can sometimes have conflict with them in that you're saying, 'No, I'm sorry, you have to finish, it has to go', particularly if they are a researcher who's got a very big grant and is basically going to run to the Vice Chancellor at the university and take the attitude, 'You're trying to stop my research'. So, you can get into situations like that and you basically, I think you have to stand up for the animal' (Michelle). In describing a situation in which the NVS's call to remove or discount an animal from a study may meet challenge from researchers, Michelle states that 'you have to stand up for the animal'. In this example, this can mean ending their life. This extract raises difficult questions, discussed in the wider literature, about the extent to which euthanasia should be understood as a 'gift' , or whether it should be identified as a harm . For example, Coghlan (pp. 360-361) contends that 'The denial that animals can be harmed by death is often connected with a quasi-technical notion of animal 'welfare' and 'Since veterinarians have power over animal life and death, this denial is significant'. In the animal research context, one could also question whether the relative absence of opportunities for rehoming research animals ( ) could be seen to reinforce decisions around humane killing. In other words, the wider context of animal research needs to be appreciated, within which the advocacy of NVSs is articulated. Overall, we have demonstrated that the NVS's work to prevent, limit, and end animal suffering is an important part of their advocacy. However, this is arguably premised on a professional acceptance of 'necessary' suffering. Critics have argued that such a conceptualisation of 'necessary' suffering is itself morally flawed. For instance, Fox (p. 30) has argued that 'if animals' lives have value independent of their interests to others, all of their suffering is morally unjustified'. Likewise, in challenging the 'necessity' of scientific animal use more broadly, Francione (p. 248) asserts that, although 'problematic in a number of respects', the 'use of nonhumans in biomedical research may involve a plausible claim of necessity [...] But such a claim, even if justified, cannot serve to provide a satisfactory moral basis for this use of animals'. Such contestation around the premise of 'necessary' suffering which is seen to inform how the welfare of research animals is conceived therefore highlights tensions within the identity of the NVS as an animal advocate within the worlds of scientific practice. 3.2. Speaking for 3.2.1. Speaking for Research Animals As well as constructing the performance of advocacy for research animals as intervening in suffering, the NVSs interviewed also emphasised the representational dimensions of their role, describing their work to 'speak for' the animals and act on behalf of their best interests. As Margaret discussed: 'I guess what I do is, it's really just to try and speak for them, be an advocate for the animals and safeguard them. Most researchers are not intending to be cruel to the animals, it's more sometimes they just don't understand them well enough to know the consequences of what will happen for the animal if they do something, or they don't see the signs which can be quite subtle, particularly if you're dealing with mice or sheep or rats or prey type animals, they can look at an animal and they won't see what I will see or the technician would see, that's not intentional, it's just they don't know. So, it's really being there for the animal and taking care of them' (Margaret). This 'speaking for' is here intended to safeguard animals from the harms of the research and the researchers. Margaret clarifies that the harms posed by the latter are not intentional but rather result from a lack of certain knowledge. Indeed, as she puts it, researchers 'can look at an animal and they won't see what I will see or the technician would see'. With their embodied expertise and the identification of welfare impacts and diagnosis of illness and disease this enables, Margaret's description portrays the NVS as thus able to count the animals as part of an 'intervention of the visible and sayable' (p. 45). This means representing the interests of research animals, speaking up for them on issues that may be missed by researchers who lack the same levels of knowledge of or familiarity with the presentation of animal suffering. Similarly framing their advocacy for research animals as juxtaposed to the interests of researchers, Owen describes how they are responsible for speaking up for the research animals 'against' the positions of the researchers. In this extract, the initial 'NACWO' refers to the Named Animal Care and Welfare Officer: 'The Home Office love to call ASPA "a caring Act" because there were two care people in there, the NACWO and the NVS and we are named care people, and I see my job as being a care person to speak up on behalf of the animals against the wicked scientists who are wanting to do all sorts of naughty things to them [...] it is what I feel, we are. So when I'm called in, in a dispute situation, the dispute is always between the NACWO and the scientist and it's always that the NACWO is saying, 'I think this animal ought to be put down' and the scientist saying, 'can't we hang on a bit longer?'' (Owen). Defining the NVS role as a 'care person', Owen here identifies their role and that of the NACWO as posing a challenge to the scientists, whose interests concern the animal's use as an experimental model. Perhaps half-joking in their description of the moral character and interests of scientists, portraying an oppositional relationship between an animal-loving veterinary surgeon and 'wicked scientists', Owen illustrates the conflict that can be expected as part of their role. Yet, later in the interview, Owen discusses how their advocacy can be enacted in conjunction with rather than against scientists if a working relationship has been built from the project's nascent stages: '[...] but if you've met them before their application goes in and you've talked to them and you've advised them and they've explained what they're trying to do and you've explained how they can best do it in your opinion, hopefully you're all on the same side and I think in the best units, that's what's happened, so there isn't much of a conflict' (Owen). Owen's claim here is that it is good practice in regard to animal welfare if the NVS is involved in a project from its inception. In this case, the NVS's' advocacy can take the form of sharing expertise, with the NVS advising how best the research aims can be met whilst in-keeping with animal welfare and meaning that all actors are 'on the same side'. Other participants described how an embracing of the 3Rs by scientists can have a significant impact in changing the NVS's role from one of opposition to one of collaboration. Relatedly, in representing and speaking up for the interests of research animals, some participants framed independence as a crucial part of the NVS role, as Nadir expresses: 'What I hope I achieve here is to be seen just as an honest broker, someone who can be relied upon to say what they think, and to not carry any political baggage. My job is to represent the interests of the animals as best I can in a complicated structure where the interests of the animals are one part of a larger programme that the institute or the company is trying to achieve' (Nadir). With the NVS's responsibility to speak for research animals often necessitating challenging the decisions and interests of others, their independence from the interests of researchers and the institution is significant for maintaining appropriate levels of impartiality. However, as Nadir implies, such independence relates not only to the research infrastructure, but also wider political agendas. In being seen as uninvested not only in the research outcomes and institutional goals but also in relation to other stakeholder agendas, Nadir discusses how they aim to present their role to others in the facility as an 'honest broker'. Interestingly, in Pielke's influential typology of the roles that scientists may play in political issues, the 'honest broker' is distinct from the role of 'issue advocate'. Indicating the key difference between the two, Pielke observes that the broker seeks to 'expand (or at least clarify) the scope of choice' whilst the advocate 'seeks to reduce the scope of available choice' (ibid, p. 18). In advocating on behalf of the research animals' interests, the NVS may be seen as carrying 'political baggage' of a kind. However, Nadir's excerpt suggests that, to perform their role effectively, it is important to be perceived as helping and not hindering researchers, providing impartial insights into how animals can be used and counted within welfare parameters instead of working to discount or limit their use altogether. 3.2.2. Speaking for Society The advocacy element of the NVS role may be framed, not only as representing the interests of research animals within a facility, but also representing wider society's interests in animal welfare. As Nicholas suggests: '[...] The other aspect which sometimes gets lost is that the NVS is the people's representative of the animals, they're there to be on the side of the animals and the role was created to put an independent person into the facility. And one of my friends, just coincidentally, was part of the campaigning around the '86 Act, he said one of the things we were really pleased about was that we got the vets in there because they wanted someone on the side of the animals in the facility, and I think that's probably the area that we need to make sure is still understood' (Nicholas). As well as caring for the research animals in their charge, Nicholas indicates that an important responsibility of the NVS is to act as 'the people's representative of the animals', representing the wider public interest in the protection of animal welfare. This perspective emphasises that the NVS is more than 'just' a representative for the animal, they also perform a social role mediating between science and society. This reflects the importance of public opinion in shaping the governance of animal research, a topic that has been itself explored in some depth . In this case, public concern for animal welfare and trust in the veterinarian to embody 'the side of the animals' is considered to have created a professional niche for veterinary surgeons in the establishment of the NVS role. Other participants invoked wider publics in their claims about their complex professional responsibilities: 'At the end if the animal is not fine, that research is not going to be valuable. So, I see that as a double responsibility. I've got the responsibility for the animal and the animal needs to be fine, so it should be done properly on the animal and everything. Because otherwise those days are not worthy, if those days are not worthy, I've wasted those animals. At the same time, what I want as a taxpayer or whoever will benefit one day, I've also wasted the money, time, effort. So, research at the end comes from money, that comes from people via tax, via charities, whatever you want that believes in that, and they want to make a change for the future' (Nathalie). Here, publics are figuratively present in the form of taxpayers who contribute to the funding of scientific research and as patients who are expected to ultimately benefit from the research. In this framing, the NVS is constructed as responsible for assisting the return on this financial and affective investment by ensuring that animal research is conducted to appropriate standards that facilitate the materialisation of promised and expected outputs. Publics are thus enrolled and counted here as one of the 'objects of care' (p. 60) for the veterinarian, alongside the animals and the scientific output. In summary, NVSs in our study articulate obligations for multiple objects of care, with their relationship to animal welfare being entangled with the project's scientific aims and also the wider interests of publics invested in both the research outputs and the protection of animals. Indeed, Ashall and Hobson-West (p. 292) argue that 'NVS responsibilities to a scientific establishment under A(SP)A should be viewed as additional to the multiple responsibilities which are faced by all veterinary surgeons, and which have previously been identified as a potential source of ethical conflict'. The plurality of the NVS's care obligations suggests that although their independence from the research itself is important, they must negotiate logics that count the animal as an experimental model, shaping the NVS's advocacy for research animals. 3.3. Driving Change 3.3.1. Changing from the 'Inside' Thus far, this analysis has highlighted the tensions involved in the NVS's performance of the animal advocate role. Given the limits of advocacy in the scientific context within which NVSs operate, it is perhaps understandable that some participants connected their role as an animal advocate to driving change in research practice. Indeed, prompted by the interviewer's discussion of the potential conflict in constructing the NVS as an animal advocate whilst their work can be seen as facilitating uses of animals that contravene their interests, Margaret argues that by being on the 'inside', the NVS role provides an opportunity to affect change: 'One of the reasons I sort of got involved in it because with research, it is a difficult thing but I think if you're going to actually make a difference and influence anything, you've got to be in amongst it, it's no good kind of standing outside and shouting at the walls, you've got to be there to see what happens and understand what's happened and try and influence it. And that's partly why I've got involved with it and partly why I've taken up some difficult conversations with some researchers and the Dean [...] I'm asking 'why?', but you've got to have that conversation because if you don't, you're failing really because you've got to point it out because otherwise nothing will change' (Margaret). The NVS here is framed as an agent of change within animal research facilities, able to initiate difficult conversations with scientists and organisational actors which would not be possible if they were 'standing outside'. This was contextualised differently by Nadir, who contrasted professional responsibilities towards animals with ethical responsibilities to advancing human medicine and preventing human illness and deaths. Nadir listed multiple relatives and members of their local community who had died of various diseases or lived with chronic conditions, and having justified animal research in this context echoed Margaret's sentiment: 'I have a foot in both camps. Although my professional responsibilities are to the animals I look after, I also have moral and ethical responsibilities to my species, particularly to my own family and friends and relatives, many of whom have struggled with the sorts of diseases which the use of animals here is trying to address. So there is that conflict going on all the time. [...] That's where animals come into the research picture. However reluctantly they appear on the horizon, they are there, and once you accept the fact that research is probably justified, then I'd rather be involved in it, doing it as well as I can, than standing on the outside griping' (Nadir). Nadir draws a clear distinction here between human and non-human animals, having a 'foot in both camps' that contextualises their ethical and professional responsibilities towards the two groups. However, following Clarke and Knights (p. 269), this assumption that animals are needed to address historic failures in human medicine is to reproduce narratives of 'anthropocentric masculinities' which are common in veterinary practice, whereby 'men seek to transform animals and nature into orderly, predictable and serviceable objects of human(istic) desire'. Interesting here, is the way in which, Nadir works through the tension they articulate between their ethical and professional responsibilities by being on the 'inside' of animal research. Such justifications of the NVS role and the possibilities they provide to advocate for research animals, rather than 'standing on the outside griping', raise broader questions around the lack of opportunities for both publics and professionals outside of organisational frameworks to participate in decision-making processes and influence change within animal research. 3.3.2. Changing Practice In discussing their desire to affect change, several participants referred to the implementation of the 3Rs as a key responsibility of the NVS. In working to implement the 3Rs, the NVS' animal advocacy is framed as necessitating an approach which goes beyond individual animal welfare and is also concerned with driving best practice. As Parker discussed: 'Yeah, because your main job as a vet, you are there for the animals, but the reason you are employed by the institute is not only to tick boxes, so you are there to provide, basically you have to justify your salary. So, you've got to try to be an [inaudible 64:49], a 3R Hercules. OK, so you've got to try to stimulate refinement all the time, in the husbandry, on the experiments, and then bring people to use more reduction, and the replacement is very difficult as an NVS because it's really, your mind is about the animals so replacement is very difficult' (Parker). Parker differentiates between the core work of a veterinarian who is 'there for the animals' and the work of the NVS who is 'employed by the institute [...] not only to tick boxes', describing the latter as going further than caring for animal health and welfare and also working to drive uptake of 3Rs approaches and techniques. In particular, Parker characterises their work around replacement as especially difficult, with their role embedded in the day-to-day animal use. Similarly, other participants highlighted the important yet tricky nature of the NVS's advocacy for the replacement of animal models. As Maeve describes in this interview exchange: 'I still see myself as an advocate for the animal. I think as a professional, well I'm maybe more outspoken than others but I'm not pro animal research, I never was but professionally, I can live with rules and if the product licence says this is what they're allowed to do, I'm fine with that. I think we should be much more involvement in replacement, which we aren't because...' Interviewer: NVSs should? Yes, we should understand it, we push it away from us because it's complex and we don't have time to look into this and also, I'm still seeing myself as an advocate. I give independent advice, this is what I do, I should be there to question, that doesn't mean I should make life difficult because I don't like somebody's notes, that is completely different from giving independent advice in a welfare situation but I will be the first one who, if somebody is hammering on about needs to do single housing, I'll be saying, "If you need to do single housing and you do surgeries, you need to single house earlier so that once stress is over and then do the surgery and then you do this", and a lot is to teach people to think about what they don't think about' (Maeve). In this excerpt, Maeve makes a distinction between 'giving independent advice in a welfare situation' and challenging decisions which may impact on animal welfare (e.g., single housing of animals). Such a differentiation suggests that for the NVS to be effective in their animal advocacy, they must recognise the appropriate moments when challenge is called for and appreciate the equal importance of advice and education. Indeed, in the context of paediatric medicine, Waterston (p. 156) states that 'Advocacy does not mean storming the White House or number 10 Downing Street; instead, it is using the wherewithal of professional expertise, credibility and experience to draw attention to issues and execute beneficial changes'. Yet, the specific challenges that surround the NVS's work in the area of replacement mean that the ways they can advocate is more easily directed at improving the ways in which animals are used (in terms of getting the best scientific output from the animals and safeguarding animal welfare) rather than preventing their use at all. Such barriers to advocating for replacement may hint at the pitfalls of becoming professionalised within systems one may aim to ultimately transform or disrupt. Hence, as some extracts included earlier in this section suggest, although being on the 'inside' may be said to enable veterinarians to question scientists on their animal use and hold challenging but important conversations aimed at stimulating better practice, the orientation of the NVS role towards animal use may impede their advocacy around the ways in which animals might be replaced. 4. Discussion In seeking to enrich understandings of what 'animal advocacy' means for veterinarians working in animal research, this paper has focused on three key ways in which Named Veterinary Surgeons enact their role as an 'animal advocate', that is, through mitigating suffering, speaking for, and driving change. The first thematic section analysed the link between the NVS's advocacy and their work to mitigate animal suffering and considered how the requirements of the research context may orient the advocacy of NVSs towards protecting animals from the impacts of a study, rather than cultivating lives 'worth living' per se. This section also highlighted how the mobilisation of distinctions between necessary and unnecessary involves some acceptance of degrees of animal suffering; in other words, NVSs bring together logics of veterinary professionalism and scientific practice. Finally, this section touched on how the work of NVSs to limit unnecessary suffering plays out within the confines of the research infrastructure when an animal's suffering cannot be controlled, with euthanasia or 'humane killing' discussed by several participants as an important part of how their advocacy is practiced. Given existing challenges around the rehoming of research animals , advocating for the humane killing of animals may often be one of the only options available to NVSs when advocating for the removal of an animal from a study. Scholars are increasingly recognising the way in which care, harm, and killing coalesce in animal research and in wider areas of veterinary practice. Indeed, Venkat (p. 1) considers that cruelty 'might be figured as an unavoidable aspect of the relation of dependency between animals and their human caretakers' and suggests that in and through veterinary medicine 'humans and their particular forms of cruelty are perhaps unavoidably implicated in the deaths of animals' (ibid, p. 14). This reminds us that the case of veterinary involvement in animal research is not the only example of how veterinary animal advocacy may be conflicted or contested. Our analysis thus might contribute to these wider debates by serving to highlight the ways in which veterinary animal advocacy is inherently complicated and open to contestation and involves a negotiation of multiple and often divergent interests within broader paradigms of anthropocentricism. The second thematic section, that of 'speaking for' research animals and wider society, illustrated how many NVSs framed their relationship with researchers as often being one of challenge, with the professional dynamics of their two different worlds associated with a common occurrence of conflict when they exist together. However, some participants described how when the NVS's involvement from the formation of a study and the prioritisation of animal welfare and the 3Rs is evident throughout, then the NVS's advocacy can more often take the form of advising rather than challenging researchers. Although the participation of the NVS in their institution's Animal Welfare and Ethical Review Body (AWERB) is mandatory in the UK, this analysis suggests the importance of relational dimensions of cooperation, beyond the formal institutional processes of ethical review. However, we also considered how presenting themselves as an impartial mediator may stir tensions with their identification as animal advocates, with their advocacy framed here as assisting with the scientific use of animals within acceptable welfare parameters, rather than advocating for their interests outside of this paradigm. This last point raises questions beyond the identity of veterinarians and encourages us to consider which versions of the animal are being counted or represented within animal advocacy. Here we find Weich and Grimm's analysis useful in arguing for more 'critical scrutiny of the normative social structure' in which an animal patient's interests emerge and an 'interrogation of animals' activities within that structure'. With understandings of research animals fluctuating between the naturalistic and the analytical , model and pet , individual and colony, wild and domestic, this provocation pushes us to consider what kinds of patients are produced through animal research for which the veterinarian is responsible. Future research might therefore focus more directly on how Named Veterinarians and other professionals who advocate for the welfare of research animals construct their animal patients, the meanings they make of their health and illness, what these involve, and where they begin and end. Further work could also explore where this differs internationally (as called for by Anderson and Hobson-West ). Future studies might therefore also question the extent to which animal advocacy in the research context is complicated by conceptualisations of the animal as both patient and scientific tool, and how their other roles and accordant interests may or may not be given opportunities to flourish in research facilities. The final theme explored the way in which NVSs discuss advocacy as partly about driving change in practice. That some participants described the NVS role as enabling them to advocate for research animals by permitting them entry inside the research infrastructure arguably draws attention to the current lack of opportunity for veterinary professionals outside of animal research to participate in driving good practice and inform policymaking on scientific animal use. This point relates to broader calls to open up this arena to more actors, including publics . The current paper also highlights how the construction of the role of the NVS as an 'independent insider' might generate both opportunities and limitations for veterinary animal advocacy for research animals. For example, this analysis has pointed to the ways in which a focus on the day-to-day use of animals may restrict the NVS's animal advocacy around the replacement of animals. Given the relationship between the NVS and wider publics discussed in the first analytical section, and evidence that publics are invested in full replacement of animals with alternative models , this may prove problematic in the longer term. 5. Conclusions This paper began by demonstrating the way in which the veterinary identity is closely associated with acting as an animal advocate. However, in doing so, we have also argued that definitions of advocacy are problematic and illustrated that transferring the concept from the human to the animal patient is not straightforward. We then introduced the role of the NVS and the ways in which assumptions about advocacy are built into this role. Whilst the role of the NVS is quite specific, we hope that this analysis will serve to enrich the definition of 'animal advocacy' in veterinary practice more broadly. Indeed, as Anderson and Hobson-West (p. 6) have previously argued, 'Focusing on the experiences of Named Veterinarians may therefore help us grapple with some of these fundamental questions about the future of the profession and the role of veterinary expertise in society'. This is arguably particularly urgent given the veterinary profession finds itself at 'somewhat of a crossroads' regarding the nature of its professionalism and the future of the profession in the UK as it addresses a number of concurrent crises . Returning to the BVA's proclamation that veterinarians' 'opportunity to be advocates for animals may be the greatest of all and we have clear social, professional and legal responsibilities to do so' (p. 9), this paper has illustrated the trickiness in defining veterinary advocacy for animals, demonstrating the tensions surrounding the veterinarian's advocacy for animals whilst working within arenas governed by both care and harm. This description not only applies to the animal research context, but arguably pertains to the majority of human-animal relations and the veterinarian's work as a, not independent, but thoroughly partial mediator of them . After all, veterinarians are not external to the cultures which radically shape their patients' experiences of health and illness. Given this broader context, we would encourage more practical support for veterinarians working in all fields, to scrutinise the specific structures and practices of power which produce animals as patients in the first place. This argument has implications for the social scientific study of veterinarians more broadly, signalling the need for further research which focuses, not just on the experiences or perspectives of veterinarians, or the interactions between veterinarian and client, but which critically examines the relationship between the veterinary profession and wider society. This in turn would demand further exploration of the kinds of veterinary patients that come into being through human-animal relations and allow consideration of the extent to which the animal as more-than-patient might be included in veterinary animal advocacy. Author Contributions Conceptualization, R.M. and A.A.; methodology, A.A., R.M. and P.H.-W.; software, A.A. and R.M.; validation, R.M., A.A. and P.H.-W.; formal analysis, R.M. and A.A.; investigation, R.M., A.A. and P.H.-W.; resources, R.M., A.A. and P.H.-W.; data curation, A.A. and R.M.; writing--original draft preparation, R.M. and A.A.; writing--review and editing, R.M., A.A. and P.H.-W.; visualization, R.M., A.A. and P.H.-W.; supervision, R.M., A.A. and P.H.-W.; project administration, P.H.-W.; funding acquisition, P.H.-W. All authors have read and agreed to the published version of the manuscript. Institutional Review Board Statement The study was conducted according to the guidelines of the Declaration of Helsinki, and approved by the Ethics Committee of the School of Veterinary Medicine and Science, University of Nottingham, United Kingdom (approval number: 1800160608; date of approval: 16 June 2016). Informed Consent Statement Informed consent was obtained from all subjects involved in the study. Data Availability Statement The dataset on which this paper draws has been anonymised and deposited in the UK Data Archive, subject to an embargo: Davies, G., Greenhough, B., Hobson-West, P, Kirk, R., and Roe, E. (2033) Animal Research in the UK, 2017-2023. UK Data Service. SN: 8942, DOI:10.5255/UKDA-SN-8942-1. Conflicts of Interest The authors declare no conflict of interest. Disclaimer/Publisher's Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

PMC10000175

Reproductive disorders caused by porcine reproductive and respiratory syndrome virus-1 are not yet fully characterized. We report QuPath-based digital image analysis to count inflammatory cells in 141 routinely, and 35 CD163 immunohistochemically stained endometrial slides of vaccinated or unvaccinated pregnant gilts inoculated with a high or low virulent PRRSV-1 strain. To illustrate the superior statistical feasibility of the numerical data determined by digital cell counting, we defined the association between the number of these cells and endometrial, placental, and fetal features. There was strong concordance between the two manual scorers. Distributions of total cell counts and endometrial and placental qPCR results differed significantly between examiner1's endometritis grades. Total counts' distribution differed significantly between groups, except for the two unvaccinated. Higher vasculitis scores were associated with higher endometritis scores, and higher total cell counts were expected with high vasculitis/endometritis scores. Cell number thresholds of endometritis grades were determined. A significant correlation between fetal weights and total counts was shown in unvaccinated groups, and a significant positive correlation was found between these counts and endometrial qPCR results. We revealed significant negative correlations between CD163+ counts and qPCR results of the unvaccinated group infected with the highly virulent strain. Digital image analysis was efficiently applied to assess endometrial inflammation objectively. CD163 digital image analysis endometrium inflammatory PRRSV QuPath reproductive Boehringer Ingelheim Vetmedica GmbH.RRF-2.3.1-21-2022-00001 National Recovery FundRRF-2.3.1-21 funding scheme The study was funded by Boehringer Ingelheim Vetmedica GmbH. Project no. RRF-2.3.1-21-2022-00001 has been implemented with the support provided by the Recovery and Resilience Facility (RRF), financed under the National Recovery Fund budget estimate, RRF-2.3.1-21 funding scheme. pmc1. Introduction Porcine reproductive and respiratory syndrome (PRRS) caused by PRRS virus (PRRSV) is one of the most economically devastating infectious diseases of pigs . PRRSV strains belong to the Nidovirales order and Arteriviridae family . These are small, enveloped viruses with a positive-sense single-stranded RNA genome belonging to two species: PRRSV-1 (Betaarterivirus suid 1, formerly European genotype 1) and PRRSV-2 (Betaarterivirus suid 2, formerly North American genotype 2). The haptoglobin scavenger receptor CD163 is an essential entry receptor for PRRSV. After the pH-dependent, receptor-mediated endocytosis, the viruses primarily replicate in differentiated alveolar macrophages of pigs (PAMs). Upon knocking out the whole CD163 encoding gene or specifically the receptor binding site (scavenger receptor cysteine-rich domain 5 or SRCR5), pigs and their PAMs become resistant to PRRSV infections . Sialoadhesin (Sn), a member of the siglec family of sialic acid-binding molecules, is also involved in the viral entry of PRRSV-1 and can be solely found on specific subsets of differentiated tissue macrophages . Mostly, the viruses cause respiratory disease in growing piglets and fattening pigs and reproductive failure in pregnant sows . The clinical appearance is very diverse; both asymptomatic infection and severe disease may develop, which is influenced, among others, by the virulence of the PRRSV strain and the immunity and genetics of the host . In 2006, a highly virulent strain emerged in the Far East characterized by severe symptoms and high mortality in all age groups, and strains of similar virulence have since appeared also in Europe . Initially, PRRSV-1 strains belonging to subtype 3, such as Lena and SU1-bel, were considered highly pathogenic; however, more recently, strains from subtypes 1 and 2 were found to cause fatal PRRSV-1 outbreaks. Thus, the "highly virulent" PRRSV-1 strains BOR59 (subtype 2), PR40/2014 (subtype 1), and AUT15-33 (subtype 1) have been reported from 2009 to 2015 in farms from Belarus, Italy, Austria, Germany , and most recently from Spain . While several respiratory models have been published so far, a lot fewer data are available regarding the pathology of reproductive disorders, especially for PRRSV-1 . According to reproductive challenge trials conducted in Canada, PRRSV-2 appears in the endometrium after systemic infection and replicates in CD163+ macrophages. It induces apoptosis in numerous cell types, including macrophages, other inflammatory cells, uterine epithelium, and fetal trophoblasts. Together with the inflammatory lesions, these changes may adversely affect the transfer of nutrients to the fetuses. The viral load of the maternal-fetal interface is a strong predictor of fetal viral load, and its low viral load, together with high fetal thymic viral load, is associated with an increased probability of fetal death . According to these findings, viral transmission from maternal to fetal tissues, fetal infection, viral replication in fetal tissues, and viral spread to adjacent fetuses may be cardinal for the development of reproductive disorders in PRRS. Uterine, fetal, and placental factors may also be responsible for disease development . Significantly higher viral loads were found in the endometrium, fetal thymus, and fetal serum in the case of meconium-stained fetuses than in other preservation categories in one study, suggesting that these are the most applicable for routine diagnostics . The duration of infection and fetal viral load are probably the leading factors for the prediction of fetal resistance . CD163+ and CD169+ macrophages at the maternal-fetal interface are significantly increased in pregnant gilts infected with PRRSV-2. The viral load of the fetal thymus has a positive correlation with the number of CD163+ macrophages in the endometrium, suggesting the important role of these cells in the pathogenesis of reproductive disorders caused by PRRSV-2 . Macrophages are able to activate certain immune cells, including natural killer (NK) cells; their interaction was already demonstrated in the human uterus. Increased numbers of Sn-positive macrophages and CD8-positive cells were observed in the maternal-fetal interface of sows infected with PRRSV-1. Based on these findings, Sn-positive macrophages can activate endometrial NK cells, which may contribute to the development of histopathological lesions . Anatomically, pigs have incomplete diffuse epitheliochorial placentation without invasion. The maternal and fetal tissues are clearly divided, and the circulation of the mother and the fetus is separated by six tissue layers; therefore, maternal antibodies cannot be transferred during gestation. The embryonic development mostly depends on endometrial gland secretions . Typical histopathological lesions caused by PRRSV infection in the uterus are myometritis and endometritis associated with vasculitis. In the endometrium, the inflammatory infiltrate is predominantly lymphoplasmacytic and histiocytic. If there are fetal lesions, they are varied and not characteristic . However, fetal and umbilical lesions were found to be strongly associated with fetal impairment . Digital pathology or virtual microscopy requires the digitization of histopathological slides using whole slide scanners, their storage, and the display and interpretation of the resulting whole slide images (WSI) on a monitor. It has several potential benefits in diagnostics, research, and medical/veterinary education . Software capable of digital image analysis (DIA) uses machine learning, meaning that they learn from data and make decisions about new inputs (cells and structures) without human assistance. In the case of supervised learning, the user verifies and modifies the data from which the program can learn . The analyzes performed with these types of software can be more objective and reproducible . The method is increasingly used for the accurate identification and quantification of certain biomarkers, especially in human oncology . The variety of colors and shades in routine staining procedures can confuse machine learning techniques and classifications; therefore, in the future, it may be necessary to standardize the entire histopathologic workflow for truly effective digital pathology analyses . However, advanced data-driven algorithms are able to learn a certain degree of image variability . QuPath is an open-access and user-friendly software with various image analysis applications in the field of pathology . It is able to classify the detected cells into different categories (classes) using machine learning algorithms in WSIs of hematoxylin and eosin (H&E) stained slides . The aims of our study were to digitally quantify the total inflammatory cell population and the CD163+ macrophages in the endometrial lamina propria of pregnant gilts experimentally infected with PRRSV-1 strains of different virulence. To demonstrate the superior statistical utility of this numerical data compared to manual scoring methods, we also determined the association between the number of these cells and endometrial, placental, and fetal features. The main objective was to determine the validity of this supervised machine learning method in comparison with a semiquantitative method described in a similar experimental setup. 2. Materials and Methods 2.1. Animal Ethics Approval The original animal study was reviewed and approved by the Institutional Ethics and Animal Welfare Committee (Vetmeduni Vienna) and the Austrian national authority according to SSSS 26ff. of Animal Experiments Act, Tierversuchsgesetz 2012--TVG 2012 (accession number: GZ 68.205/0142-WF/V/3b/2016). 2.2. Challenge Experiment The study material consisted of formalin-fixed and paraffin-embedded (FFPE) blocks of endometrial tissue obtained from an earlier challenge experiment, during which 24 pregnant gilts were infected with a low (720789, 99.76% sequence homology to the PRRSV field isolate IVI-1173 (Genbank Accession number KX622783.1)) or a highly virulent (AUT15-33) PRRSV-1 strain, which has been used for several experimental infection studies in the past whereas the low virulent strain also considered causing reproductive pathogenesis in the field, but so far without experimental confirmation . The experimental setup was published in detail by Kreutzmann et al. ; briefly, half of the animals were vaccinated with a modified live virus vaccine (Reprocyc PRRS(r) EU, Boehringer Ingelheim Vetmedica GmbH, Ingelheim am Rhein, Germany) twice before insemination, and then on the 53rd day of gestation according to the manufacturer's protocol. The gilts were randomly distributed into six groups: non-vaccinated and non-infected, vaccinated and non-infected, non-vaccinated and infected with the low virulent strain, vaccinated and infected with the low virulent strain, non-vaccinated and infected with the high virulent strain, and vaccinated and infected with the high virulent strain (4 gilts/group). The groups were named no_vacc/no_chall, vacc/no_chall, no_vacc/chall_L, vacc/chall_L, no_vacc/chall_H, and vacc/chall_H, respectively. The experimental design specifically sought to avoid cross-contamination and factors interfering with the evaluation of its outcomes. Four vaccinated and four non-vaccinated gilts were inoculated with 3 x 105 TCID50 of either AUT15-33 (H) or 720789 (L) in a total volume of 4 mL; 2 mL were administered intramuscularly, and 1 mL was administered into each nostril on the 84th day of gestation. The simplified experimental setup is shown in Figure 1. The negative control group was sham inoculated with Dulbecco's modified Eagle's medium. At 21 +- 2 dpi, the animals were humanely euthanized. Intravenous ketamine (Narketan(r) 100 mg/mL, Vetoquinol Osterreich GmbH, Vienna, Austria; 10 mg/kg body weight) and azaperone (Stresnil(r) 40 mg/mL, Elanco GmbH, Cuxhaven, Germany; 1.5 mg/kg body weight) were injected before the intracardiac injection of T61(r) (embutramide, mebezonium iodide, and tetracaine hydrochloride; Intervet GesmbH, Vienna, Austria; 1 mL/10 kg body weight). All fetuses were individually evaluated and dissected, according to Ladinig et al. . Endometrial samples collected during necropsy were used in our study. 2.3. Tissue Processing and Routine Histology After 24 h of fixation at room temperature in formaldehyde solution, samples were trimmed and dehydrated with a series of ethanol and xylene in an automatic tissue processor. The dehydrated tissue samples were embedded in paraffin blocks, and 4 mm thin sections were cut manually and mounted onto Superfrost+ adhesion slides (Thermo Fisher Scientific, Waltham, MA, USA). The unstained sections were deparaffinized and rehydrated in xylene and alcohol, respectively. Routine H&E staining was performed in an automatic staining instrument. 2.4. Section Selection Our aim was to determine the validity of digital pathology in similar experimental setups by objectively quantifying endometrial inflammatory cells. To illustrate the superior statistical applicability of the obtained numerical data, we also examined associations between digitally quantified total inflammatory cell numbers and manual scorings, CD163+ numbers, PRRSV genome equivalent (GE) values measured by qRT-PCR and the different groups defined by vaccination status and challenge strain virulence. We have chosen all H&E-stained endometrial slides with a manual endometritis score of at least 1 (n = 131): 50 slides were included from gilts that were non-vaccinated and challenged with the low virulent strain ("no_vacc/chall_L"), 45 slides from non-vaccinated gilts challenged with high virulent strain ("no_vacc/chall_H"), 21 slides originated from vaccinated gilts challenged with the high virulent strain ("vacc/chall_H") and 15 from vaccinated cases infected with the low virulent strain (belonged to the "vacc/chall_L" group). We used another 10 sections that scored 0 for both endometritis and vasculitis for the calculations performed with the manual scorings. The sections originated from non-infected and infected gilts with no inflammatory lesions in the uterine samples. The weight of the fetuses from cases with endometrial inflammation ranged from 273 g to 1481 g (mean = 768.2692, median = 793.5, standard deviation = 218.0419, n = 130). The manual scoring grades, grouping, weight of the fetuses, and log10 transformed qPCR results are presented in Supplementary Table S1. 2.5. CD163 Immunohistochemistry From the 131 FFPE blocks with inflammation, we randomly selected 35 for CD163 IHC. Rabbit polyclonal antibodies (ab87099, Abcam, Cambridge, MA, USA) were used as primary antibodies against the CD163 glycoprotein. Sections were deparaffinized in xylene and rehydrated in a graded ethanol series. The 15-min endogenous peroxide inhibition (97 mL distilled water + 3 mL H2O2) was followed by phosphate-buffered saline (PBS) rinsing. Antigen retrieval was performed in citrate buffer (pH 6.0) in a microwave (800 Watts for 5 min, then 180 Watts for 10 min) then the sections were flushed with PBS. We incubated the slides in 2% porcine albumin (Albumin from porcine serum, Sigma Aldrich) for 1 h in a moist chamber; then, without flushing, the primary antibody was added and incubated at a 1:500 dilution in a wet chamber for 30 min. The slides were rinsed in PBS before and after the 20 min incubation of the secondary antibody (Dako, EnVision Flex HRP, Agilent Technologies, Santa Clara, CA, USA). The staining was displayed with DAB Chromogen (Dako Envision Flex DAB + Chromogen 1 drop + 1 mL Envision Flex Substrate Buffer) for 3-4 min, and thereafter the sections were rinsed in PBS. The slides were counterstained with hematoxylin according to GILL II for 30 s; bluing was performed in PBS. After dehydration in ethanol and xylene, the slides were covered with coverslips. Negative tissue control samples were stained without adding the primary antibody, whereas the positive control was a fetal lymph node sample. 2.6. Manual Scoring Inflammation of the endometrium was assessed by two blinded examiners independently according to the method described by Novakovic et al. . Briefly, the severity of the endometrial inflammation (0-4) and vasculitis (0-3) was scored. A 1.1 mm and a 0.55 mm field of view were used for 200x and for 400x magnification, respectively. In the case of endometritis, the method determines the percentage of the area infiltrated with inflammatory cells and the distribution of the lesions at a 200x magnification. In the case of grade 0, inflammatory cells were rarely present; in grade 1, inflammatory cells were multifocally present in less than 10% of the slide; in grade 2, multifocal to coalescing inflammatory cell infiltration could be identified in 10-25% of the slide, in grade 3 a diffuse inflammatory cell infiltrate appeared in 25-50% of the slide, and in grade 4 inflammatory cells were diffusely present in more than 50% of the slide. In the case of vasculitis, three areas of 200x magnification were randomly selected, and three blood vessels were evaluated at a 400x magnification based on the inflammatory cell infiltration of their walls and/or their level of degeneration. In grade 1, inflammatory cells appeared within the blood vessel wall; in grade 2, besides inflammatory cells, the degeneration (vacuolation and splitting of smooth muscles) or necrosis of the blood vessel wall could be identified, and in grade 3, the above-mentioned lesions were present simultaneously. A single vasculitis severity score was obtained by averaging the scores of the above-mentioned nine vessels. 2.7. Section Scanning and Software Analysis The slides were digitized with a Pannoramic Midi slide scanner using a 20x objective (3D Histech, Budapest, Hungary). Whole slide images were further analyzed with QuPath (version 0.2.3.) . We applied the "estimate stain vectors" command in each slide to refine the hematoxylin and DAB stain estimates. We made annotations of 2.37 mm2 in the endometrial lamina propria of the H&E-stained slides, which is equivalent to 10 high power field (HPF) areas of conventional light microscopy . On contrary to the manual method, vasculitis was not scored separately; its inflammatory cells were quantified together with the selected propria area. The cell-poor, almost cell-free propria layer of healthy maternal endometrium helped the cell quantification even in the case of routine, H&E-stained slides. The "cell detection" command was selected, then the supervised machine learning method was applied using the "random trees classifier" method within the "train object classifier" command. Altogether 15 distinct classifiers were trained and used in the subsequent analyzes. Each one was trained on a 1 mm2 area of slides with distinct color shades and quality. This was necessary due to diverse section quality and color features. All measurements (41 morphological features) were computed for each cell and used as input for classification. Five classes were set to each: "immune cells", "stromal cells", "vessels", "glands", and "artifacts" . The use of the artifact class was necessary as the cell detection command also identified several non-cellular structures that interfered with the final numbering of the cells. For the slides labeled with CD163 IHC, the methodology was the same until cell detection. After that, we created six single measurement classifiers using "DAB channel filter" and "Cell: DAB OD std dev" as measurements. For each WSI, the different classifiers were evaluated and compared, and the most accurate was selected . The data were exported to separate Excel sheets (.xlsx) according to the identifier of the case. A detailed step-by-step description of the processes is provided in Supplementary File S2. 2.8. PRRSV qRT-PCR The qRT-PCR assay to detect and quantify the PRRSV genome was performed on fetal serum and thymus and on endometrial and placental tissues. PRRSV detecting qRT-PCR was carried out as described by Kreutzmann et al., 2022 . Briefly, tissue and organ sections (50 mg) were homogenized in 600 mL Qiazol (QIAGEN) in a TissueLyser II instrument (QIAGEN) for 3 min. After a quick spin, 300 mL of chloroform was added, and the mixture was thoroughly vortexed. Subsequently, the tubes were centrifuged at 13,000x g for 5 min. 200 mL of the aqueous phase was further processed using the cador Pathogen Kit in a QiaCubeHT instrument (QIAGEN) according to the manufacturer's protocol. ORF7-specific RT-qPCR was carried out using the Luna Onestep RT PCR Kit (New England Biolabs). The primer sequences were adapted from Egli et al. to fit the sequence of PRRSV-1 strains AUT15-33 and 720789 . As nucleic acid extraction control, porcine b-actin mRNA was quantified by qPCR in all samples according to Toussaint et al. . The absolute quantity of the genome equivalents (GE) was calculated from serially diluted SP6 transcripts of cloned AUT15-33 cDNA fragment 13261-3' end in a pGEM-T (Promega GmbH, Walldorf, Germany) plasmid (pLS69) . Quantitative RT-PCR was done in an Applied Biosystem 7300 instrument (Applied Biosystems, Thermo Fisher Scientific Inc., Waltham, MA, USA). 2.9. Statistical Analyzes For statistical evaluation, we used the R program (4.1.1) (R Foundation for Statistical Computing, Vienna, Austria). The concordance of the two examiners' scorings was compared using Fleiss' kappa. We compared the distributions of inflammatory cell counts (total and CD163+ cells) between the groups and examiner1's manual endometritis scoring grades, and the distributions of log10 transformed qPCR-results between the same grades by Kruskal-Wallis test with Holm correction (alpha = 0.05). This method was selected due to unsatisfactory normality conditions. The correlation between endometritis, vasculitis scores, and digitally quantified inflammatory cell numbers were examined by Kendall correlation. The concordance between the total cell count and examiner1's endometrial inflammation scoring was calculated by Fleiss' kappa. Thresholds between the five endometritis intervals (0-4) were calculated with 1000 simulations, considering a randomly selected 2/3 of the data and validating the results on the remaining 1/3. For demonstrative purposes, we used plots with different bandwidths, with the x-axis showing the cell number and the y-axis showing the proportion of examiner1's endometritis scores in the (x +- bandwidth) range of total cell counts. Pearson correlation coefficient was used to evaluate the correlation between inflammatory cell number and fetal weight, as well as between inflammatory cell count and GE PRRSV values measured by qRT-PCR. The data analyzed in this study are available in Supplementary Table S1. 3. Results 3.1. Comparison of the Manual Scorers In the case of vasculitis severity, an equal decision was made in 79.4% of all observations between the two examiners, the Fleiss' kappa was 0.68 (p < 0.0001). There was a greater agreement among the examiners in the case of endometritis, where the frequency of the same decision was 90.0%, and the Fleiss' kappa was 0.85 (p < 0.0001). The results indicate a strong concordance between the results given by the two examiners. 3.2. Section Scanning and Software Analysis Altogether 141 H&E-stained and 35 CD163 IHC-labeled histopathological slides were digitalized and analyzed. The inflammatory cell numbers of each slide with the associated data are shown in Supplementary Table S1. The total number of inflammatory cells calculated on 2.37 mm2 areas ranged from 92 to 5200 (mean = 1279.94, median = 798.0, standard deviation = 1205.64, n = 141). Similarly, the number of CD163+ macrophages ranged from 38 to 910 (mean = 177.83, median= 127, standard deviation= 156.39, n = 35). Numerical summaries of total inflammatory cell counts in examiner1's endometritis grades and within the infected groups are seen in Table 1 and Table 2. 3.3. Distributions of Cell Counts and qPCR-Results The distributions of total inflammatory cell counts differed significantly between each manual endometritis grade of examiner1 (p < 0.0001), although remarkable overlaps were found between the different levels . The difference was significant between the groups as well (p < 0.0001), except between the two non-vaccinated ones . The distribution of CD163+ cell counts showed no statistically significant difference between examiner1's manual endometritis grades or between the different groups. The distributions of endometrial and placental qPCR GE values differed significantly between examiner1's manual endometritis grades (p = 0.0001 and 0.039) . 3.4. Associations of Cell Counts and Manual Scores Kendall correlations show that regardless of the examiner, a higher vasculitis score is associated with a higher endometritis score, and a higher total inflammatory cell count is also expected with a high vasculitis or endometritis score (tau = 0.68-0.78, p < 0.0001). No statistically significant associations were found between the number of CD163+ cells and total inflammatory cell count or manual scores. 3.5. Cell Number Thresholds of Endometritis Grades According to simulations, the first threshold (grade 0-1) fell between 95.5 and 161 in 95% of cases, the second (1-2) between 817.5 and 1012, the third (2-3) between 2275 and 2752, and the fourth (3-4) between 3985 and 5038.5. Hit rates ranged from 66% to 87.2% in 95% of cases (Table 3). The proposed thresholds were 105.0 (0-1), 948.5 (1-2), 2398.0 (2-3), and 5038.5 (3-4), in which case there was 78.72% agreement with the manual scoring. A bandwidth of 300 was found to be optimal for the demonstration of total inflammatory cell count thresholds . 3.6. Correlations between Cell Counts and Fetal Weights/qPCR-Results A significant correlation between fetal weights and total inflammatory cell counts was only found in the unvaccinated groups . In group "no_vacc/chall_L", there was significant moderate positive (Pearson's correlation coefficient = 0.34, p = 0.0213, 95% CI: 0.05 and 0.58), whereas in the "no_vacc/chall_H" group, there was a significant moderate negative correlation (Pearson's correlation coefficient = -0.35, p= 0.0151, 95% CI: -0.57 and -0.07). A significant overall positive correlation was found between total inflammatory cell counts and endometrial PRRSV GE values (Pearson's correlation coefficient = 0.32, p = 0.0002, 95% CI: 0.16 and 0.47) . No statistically significant correlation was found between CD163+ cell counts and fetal weights; however, we identified statistically significant negative correlations between the number of CD163+ cells and PRRSV GE values of fetal serum, fetal thymus, endometrium and placenta in the "no_vacc/chall_H" group (Pearson's correlation coefficient = -0.56, -0.72, -0.61, -0.71, p= 0.0469, 0.0060, 0.0271, 0.0070, 95% CI: -0.85, -0.91, -0.87, -0.91 and -0.01, -0.27, -0.09, -0.25, respectively) . 4. Discussion The aim of our study was to apply digital image analysis and virtual microscopy on endometrial whole slide images to assess its applicability to replace semiquantitative, more subjective, and less reproducible scoring methods. Total inflammatory cell numbers and CD163+ cell numbers were both quantified and statistically compared to different variables measured during our experiment. Due to the case selection bias, we could not reveal real associations, yet our digital methodology may be useful for more robust future studies using similar variables and statistical analyses. Digital pathology is widely used in human medicine; however, recently, numerous data have been published about its applicability in veterinary pathology. Bertram et al. compared the diagnostic performance of the evaluation of WSIs and traditional histopathological slides in canine cutaneous tumors without using DIA. There was only a slight difference between the two methods; in the case of round-cell tumors, the digital version had some minor limitations. The mitotic count can vary enormously between areas of canine cutaneous mast cell tumors, and advanced machine learning (deep learning) systems can potentially detect the regions with the highest mitotic count more accurately and reproducibly, even if the current manual grading method is also highly reproducible among pathologists in most cases . Seung et al. analyzed the association between HER2 mRNA expressions of canine mammary tumors and their IHC stainings and quantified the in situ hybridization (ISH) results with open-source image analysis software. A significant correlation was found, and due to the similarities of spontaneous canine mammary tumors to human breast cancers, studies of this nature are also valuable to human research. Barrera-Zarate et al. used digital pathology for the examination of the porcine maternal-fetal interface. They evaluated the changes in angiogenesis and cell proliferation after PRRSV-2 infection using ImageJ software combined with vascular endothelial growth factor (VEGF) and Ki-67 immunofluorescence. According to the study, fetal resilience may be associated with greater uterine epithelial proliferation and fetal compromise with lower uterine submucosal angiogenesis. Intrauterine growth-restricted fetuses with inherently lower submucosal angiogenesis may be protected to a certain extent against PRRSV-2 infection. Since its release in 2017, QuPath has also been used for DIA in several studies evaluating animal tissues. QuPath could reliably identify gliotic expansion, axon loss, and morphological changes in degenerating optic nerves of brown Norway rats suggesting its usefulness in estimating therapeutic efficacy in preclinical treatment trials of glaucoma . Finney et al. developed an automated method to quantify reactive astrocytic responses in WSIs of rat brains using glial fibrillary acidic protein (GFAP) and 3,3'-diaminobenzidine (DAB) IHC. QuPath also has the potential to evaluate diffuse brain injury and any kind of disorder having extensive but slight effects on protein expression levels . Last year our research group successfully applied QuPath for the detection and quantification of RNAscope ISH signals of the PRRSV genome in porcine lung tissues infected with PRRSV-1, and a significant association was found between the proportion of the infected cells and the PRRSV GE numbers measured by qPCR in the same lung lobe . At first, we compared the different inflammation scores between the two examiners. A fair agreement was found between the manual scoring points at the applicability of this traditional, semiquantitative method despite its potential subjectivity. As the scoring of grades did not show a significant difference, for further statistical analysis, we used only examiner1's grades due to his expertise. Significant differences were found in the distribution of total inflammatory cell numbers between the manual endometritis grades of examiner1, proving that digital image analysis can be used to objectively characterize and even categorize endometrial inflammation comparable to manual scoring. It has to be stressed that there were notable overlaps and standard deviations between the grades, probably because the methods compared are substantially different, and there is no standardized scoring system for porcine endometrial inflammation. The distribution of total inflammatory cell counts also differed significantly between groups, except between the two non-vaccinated ones. Even though the aim of our study was not the comparison of lesion severity between the strains, unvaccinated animals may have similar inflammatory cell numbers in their endometrium regardless of the viral strain and fetal preservation. In the future, more robust studies are necessary to reveal the true associations between viral strains and inflammatory cell counts. Total cell number thresholds of different endometritis severity categories can be demonstrated by different bandwidth curves or determined by simulations. Our attempt to grade and determine absolute scales based on cell numbers is indicative only, but with a large number of sections, we would likely be able to develop a more objective digital scoring method that automatically assigns endometritis sections to the appropriate severity category. Manual grading of endometritis may potentially always have a higher error rate than DIAs due to the lack of a standardized, well-established scoring method. A significant correlation between fetal weights and total inflammatory cell numbers was found only in the non-vaccinated groups. Interestingly, the correlation coefficient showed a similar value, but it was positive for the "no_vacc/chall_L" group and negative for the "no_vacc/chall_H" group. Most of the slides used in this study came from these unvaccinated groups, so it is conceivable that we could have obtained similarly significant correlations in the vaccinated groups if we had had more samples with inflammation in them. Vaccination most likely had a protective effect on the endometrium as well. The positive correlation between total endometrial inflammatory cell counts and PRRSV qPCR GE values suggests that higher viral loads in the fetomaternal interface elicit a stronger inflammatory response. The latter was also suggested by a significantly different distribution of endometrial and placental PRRSV GE values between manual endometritis grades of examiner1. The statistically significant negative correlation between the number of CD163+ cells and PRRSV GE results of the fetal serum, thymus, endometrium, and placenta in the "no_vacc/chall_H" group is in contrast with the result of Novakovic et al. . In their publication, the PRRSV-2 viral load of the fetal thymus was positively related to CD163+ cell counts in the endometrium. As highlighted before, due to the case selection bias, we could not reveal real statistical associations. However, the role of these cells could be more complex, and CD163 IHC supplemented with cell quantification may not be sufficient to analyze the subtle mechanisms of reproductive disorders caused by PRRSV and the differences between the pathogenesis of PRRSV-1 and PRRSV-2 infection. Our study had some limitations. Mostly slides with lesions were selected, and these were not necessarily representative of the group. The number of slides obtained from the different groups was very uneven, as there were a lot more endometrial inflammatory lesions in the different groups. We had to work with a relatively small number of samples, especially in the case of higher manual grades and IHC slides. The random trees classifiers used to exclude cells of the proprial glands sometimes made errors by adding inflammatory cells to glandular cells in cases of high-grade inflammations. In these cases, manual corrections were applied. Classification of lymphocytes and plasma cells was the most dependable, granulocytes were occasionally evaluated as artifacts, and macrophages with diverse morphologies were sometimes classified as other cell types. Summarizing our data, it can be concluded that we have successfully applied digital image analysis in endometrial sections of pregnant gilts experimentally infected with PRRSV-1 for the quantification of inflammatory infiltration. The open-source QuPath software was shown to be an excellent platform for algorithm training and revision. It was capable of detecting and classifying different cells, but the manual correction was often required due to the varied quality and hues of slides. To our knowledge, this is the first study to investigate the use of DIA to assess PRRSV-induced inflammatory lesions in porcine endometrium. 5. Conclusions In conclusion, DIA can be an efficient method for tissue examination in similar research procedures in addition to or instead of semiquantitative scoring. With its methodology, we can gain new knowledge about the reproductive pathology of PRRSV-1. This was the first study that investigated PRRSV-induced inflammatory lesions in porcine endometrium using digital pathology. Acknowledgments Gyula Balka was supported by the Janos Bolyai Research Scholarship of the Hungarian Academy of Sciences. The authors would like to thank Renata Pop for performing the CD163 immunohistochemical procedure. Supplementary Materials The following supporting information can be downloaded at: Supplementary Table S1 and Supplementary File S2. Supplementary Table S1 includes the digitally quantified inflammatory cell numbers, the manual scores, groupings, fetal weights and PRRSV GE results. In Supplementary File S2 the detailed digital methodology and the computed morphological features can be found. Click here for additional data file. Author Contributions Conceptualization, A.L. and G.B.; methodology, D.G.H., Z.A.-T., A.L., M.P., T.R., C.K. and H.K.; software, D.G.H.; writing--original draft preparation, D.G.H.; writing--review and editing, G.B. and A.L.; visualization, D.G.H. and A.M.S.; supervision, G.B.; funding acquisition, A.L. and G.B. All authors have read and agreed to the published version of the manuscript. Institutional Review Board Statement The original animal study was reviewed and approved by the Institutional Ethics and Animal Welfare Committee (Vetmeduni Vienna) and the Austrian national authority according to SSSS 26ff. of Animal Experiments Act, Tierversuchsgesetz 2012--TVG 2012 (accession number: GZ 68.205/0142-WF/V/3b/2016). Informed Consent Statement Not applicable. Conflicts of Interest The authors declare no conflict of interest. Figure 1 The simplified formation and distribution of the experimental groups. Figure 2 Digital quantification of total inflammatory cells with QuPath software. To train the object classifiers, we manually set the detected cells into the corresponding classes in a 1 mm2 area. Figure 3 Digital quantification of total inflammatory cells with QuPath software. After the implementation of the cell detection command in a 2.37 mm2 area, one of the previously created object classifiers was loaded. If the classifying results were unsatisfactory, we trained a new one based on that image. Figure 4 Digital quantification of CD163+ macrophages with QuPath software. In the case of CD163 IHC, we applied the single measurement classification, which was considered the most reliable method for identifying positive macrophages. The DAB-labeled brown macrophages appear red after the classification. Figure 5 Distributions of total inflammatory cell counts. The boxplots of total inflammatory cell counts show the separation of examiner1's manual endometritis grades (a) and the markedly different distributions of total inflammatory cell counts between the vaccinated and unvaccinated groups (b). The circles above the bars are individual cases with substantially higher cell numbers. Figure 6 Violin plots depicting the distributional differences of log10 endometrial qPCR values between different endometritis grades as evaluated by examiner1. Figure 7 Violin plots depicting the distributional differences of log10 placental qPCR values between different endometritis grades as evaluated by examiner1. Figure 8 The proportion of examiner1's manual endometritis scores according to the total cases in the interval of inflammatory cell number +- 300. As the inflammatory cell count increases, the manual endometritis scores also increase, but there is considerable overlap between the different severity categories. Figure 9 Correlations between fetal weights and total inflammatory cell counts. Based on the width of confidence bands, a significant correlation between fetal weight and total inflammatory cell count only appears in the unvaccinated groups. Figure 10 Scatterplot indicating the association between the total inflammatory cell count and endometrial PRRSV GE values. Figure 11 Scatterplot on the association between CD163+ cell counts and log10 qPCR values of the fetal serum, fetal thymus, endometrium, and placenta samples. animals-13-00830-t001_Table 1 Table 1 Numerical summaries of total inflammatory cell count according to examiner1's manual endometritis grades. Grade Mean Standard Deviation Minimum Median Maximum Grade0 (n = 10) 166.6 65.5 92 178 285 Grade1 (n = 67) 521.9 249.57 97 490.0 1167 Grade2 (n = 37) 1477.57 658.52 631 1289.0 3869 Grade3 (n = 23) 3096.13 730.6 2273 2842.0 5167 Grade4 (n = 4) 4489.25 928.8 3128 4814.5 5200 animals-13-00830-t002_Table 2 Table 2 Numerical summaries of total inflammatory cell count according to treatment groups. Group Mean Standard Deviation Minimum Median Maximum no_vacc/chall_L (n = 45) 1669.49 1505.69 154 950 5200 no_vacc/chall_H (n = 50) 1577 978.3 337 1184 3775 vacc/chall_L (n = 21) 827.95 832.29 151 529 3813 vacc/chall_H (n = 15) 496.07 644.35 97 260 2632 animals-13-00830-t003_Table 3 Table 3 Determining the thresholds of different endometritis severity categories using the digitally quantified total inflammatory cell numbers. The first threshold value indicates the cell count above which we would score grades 1 and 0 below; therefore, the four threshold values would separate the four endometritis grades. Thresholds, p-values with extreme values, and confidence intervals based on 1000 simulations. The simulations were used to evaluate sensitivity. Two-thirds of the cases were randomly selected to determine the threshold values; then, the hit rate was tested on the remaining 1/3 of whether there were tendency differences. First Threshold Second Threshold Third Threshold Fourth Threshold Hit Ratio p-Value Minimum 95.5 776.5 2156 3404 0.5745 0.0001 Maximum 221 1156.5 2803.5 5038.5 0.9362 1 CI 2.5% 95.5 817.5 2275 3985 0.6596 0.0215 CI 97.5% 161 1012 2752 5038.5 0.8723 1 Disclaimer/Publisher's Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

PMC10000176

Characterising genomic variants is paramount in understanding the pathogenesis and heterogeneity of normal-karyotype acute myeloid leukaemia (AML-NK). In this study, clinically significant genomic biomarkers were ascertained using targeted DNA sequencing and RNA sequencing on eight AML-NK patients' samples collected at disease presentation and after complete remission. In silico and Sanger sequencing validations were performed to validate variants of interest, and they were followed by the performance of functional and pathway enrichment analyses for overrepresentation analysis of genes with somatic variants. Somatic variants involving 26 genes were identified and classified as follows: 18/42 (42.9%) as pathogenic, 4/42 (9.5%) as likely pathogenic, 4/42 (9.5%) as variants of unknown significance, 7/42 (16.7%) as likely benign and 9/42 (21.4%) as benign. Nine novel somatic variants were discovered, of which three were likely pathogenic, in the CEBPA gene with significant association with its upregulation. Transcription misregulation in cancer tops the affected pathways involving upstream genes (CEBPA and RUNX1) that were deregulated in most patients during disease presentation and were closely related to the most enriched molecular function gene ontology category, DNA-binding transcription activator activity RNA polymerase II-specific (GO:0001228). In summary, this study elucidated putative variants and their gene expression profiles along with functional and pathway enrichment in AML-NK patients. acute myeloid leukaemia next generation sequencing gene expression somatic variants functional enrichment Universiti Sains MalaysiaGIPS-PhD 311/PPSP/4404814 This research was funded by Universiti Sains Malaysia, grant number GIPS-PhD 311/PPSP/4404814 (23 April 2020). Universiti Sains Malaysia funded the APC. pmc1. Introduction Acute myeloid leukaemia (AML) is a clonal haematopoietic stem cell disorder characterised by genetic heterogeneity with diverse clinical outcomes in patients. For decades, cytogenetics has been crucial in diagnosing AML, as it provides a snapshot of genome-wide structural and copy number changes. Established recurrent structural variations such as t(8;21), t(15;17), inv(16) and del(5/7) are pivotal in the diagnosis and prognosis of AML, as these abnormalities partake in leukaemogenesis . However, almost half of all adult AML cases are diagnosed with a normal karyotype (NK) without structural aberrations identified during cytogenetic analysis. Varying frequencies of normal karyotypes were reported by different countries, and those frequencies ranged between 25% to 70% . Although the European Leukaemia Network (ELN) in 2017 and 2022 classified AML-NK as an intermediate prognosis, the clinical outcomes of the patients in this group are diverse and arduous to define . In recent years, the advent of novel and more sensitive platforms such as next-generation sequencing enabled the interrogation of the AML-NK genome to detect cryptic and subtle genomic aberrations that are otherwise undetectable with cytogenetics analysis. These genomic aberrations were indicated to be useful, and several findings were applied to the National Comprehensive Cancer Network (NCCN) guidelines . Some of the genomic aberrations that are included in the diagnosis and prognosis of AML-NK are FMS-like tyrosine kinase 3 internal tandem duplication (FLT3-ITD) mutations that confer poor prognoses if detected without nucleophosmin (NPM1) concomitant mutations. NPM1 mutations without FLT3-ITD mutations are associated with good prognoses, and CCAAT enhancer-binding protein alpha (CEBPA) mutations also indicate a good prognosis if biallelic mutation is present. This is also true of other myeloid gene-related mutations. Although some genomic aberrations have been established as diagnostic and prognostic markers, some genetic aberrations, such as Runt-related transcription factor 1 (RUNX1) and Tet methylcytosine dioxygenase 2 (TET2) , are still controversial, as their role in clinical studies is yet to be fully understood. On the other hand, gene expression profiling improves the understanding of the AMK-NK subgroup and further refines risk stratification and clinical outcomes. Several studies elucidated the distinct gene expression profiles between the AML-NK patients and refinement based on genomic aberrations such as the mutational status of prognostic genes (FLT3-ITD, CEBPA and NPM1) . The study of differentially expressed genes (DEG) profiles provides insights into the perturbed pathways associated with genomic markers in AML-NK. The discovery of activated and inactivated pathways enables prediction of a patient's response to specific targeted therapies. For example, the perturbed RAS signalling pathway inspired the development of RAS-targeted therapy in AML patients . Although considerable progress has been made in discovering genomic biomarkers in AML-NK, elucidating the genetic heterogeneity of this disorder, the backbone of induction chemotherapy with cytarabine (Ara-C) and anthracyclines has not changed in the last three decades. Monitoring patients' responses to chemotherapy was limited in AML-NK patients due to the paucity of suitable markers for minimal residual disease (MRD) assessment in the past. Nevertheless, now, NGS technologies enable deciphering the clonal heterogeneity of the AML-NK genome to identify specific genomic biomarkers suitable for MRD monitoring . The persistence of the genomic biomarkers of MRD at the time of complete remission (CR) serves as an independent prognosis for survival, as reported by Jongen-Laverencic et al. They analysed 482 AML patients using a targeted NGS panel at two time points: at presentation and CR after induction chemotherapy. In that study, the authors reported that about 50% of the mutations detected at presentation persisted after CR and that most of these mutations conferred increased relapse risk . Other studies suggested that NGS-based MRD monitoring has better sensitivity and specificity than conventional MRD monitoring techniques such as morphological review and flow cytometry immunophenotyping . To our knowledge, no comprehensive study on AML-NK integrating DNA and RNA sequencing with functional enrichment has been published. Therefore, we explored the genomic aberrations in the AML-NK genome using a targeted NGS panel and RNA sequencing for gene expression profiling to disclose DEGs. Next, we elucidated functional and pathway enrichment by performing overrepresentation analysis (ORA) in AML-NK patients affected by genomic aberrations and DEGs. Subsequently, we identified genomic biomarkers that could be useful in MRD monitoring for AML-NK patients. 2. Materials and Methods 2.1. Patient Samples and Ethics Statement A cohort of eight matched de novo AML-NK patient samples at two time points, presentation (DX1-8) and after remission attainment (RX1-8) following completion of consolidation chemotherapy, were included in this study. All patients were diagnosed based on the latest WHO criteria, and their responses to treatment were classified based on the International Working Group Criteria . Patients were included if their cytogenetic findings were normal at presentation using the standard G-banding karyotype analysis of 20 metaphase chromosomal spreads obtained from bone marrow aspirates. Multiplex RT-qPCR was performed using a Leukaemia Q-Fusion Screening Kit (QuanDx, San Jose, CA, USA) for simultaneous detection of 30 fusion genes to rule out chromosomal aberrations. All samples were screened for prognostic markers using qualitative PCR and high-resolution melting analysis to detect FLT3-ITD and NPM1 mutations. The remission statuses of patients at CR1 were assessed based on post-induction chemotherapy that included a combination of cytarabine and anthracycline (daunorubicin) and consolidation chemotherapy that included high-dose cytarabine (HIDAC), a combination of mitoxantrone and cytarabine (MIDAC) or a combination of fludarabine, high-dose cytarabine and granulocyte colony-stimulating factor (FLAG). Patients' remission at CR2 was assessed after salvage therapy using a combination of fludarabine, cytarabine, granulocyte-colony stimulating factor and idarubicin (FLAG-IDA). MRD monitoring was based on a morphological leukaemia-free state (bone marrow blast count of less than 5%) and either negativity of genetic markers (NPM1 and/or FLT3-ITD) or multiparametric flow cytometry for cases without genetic markers (absence of a leukaemia-associated immunophenotype) . These samples were retrospectively selected from the Clinical Haematology Referral Laboratory, Hospital Ampang. Ethical approvals were obtained from the Medical Research Ethics Committee of the Ministry of Health Malaysia (NMRR 17-1929-36614) and Universiti Sains Malaysia (USM/JEPeM/21010107). 2.2. Targeted DNA Sequencing Using Archer Dx Patients' gDNA was extracted from blood collected in K2-EDTA using a Qiamp DNA Blood Minit Kit (Qiagen, Hilden, Germany) according to the manufacturer's protocol. Genomic DNA concentration and purity were assessed by using a NanoDrop ND-1000 UV-VIS Spectrophotometer. Library preparation was performed using Archer HGC and VariantPlex Myeloid (Boulder, CO, USA) (Table SW1, Supplementary File S1). The following Illumina's adapter sequences were used for library preparation: read 1 (AGATCGGAAGAGCACACGTCTGAACTCCAGTCA) and read 2 (AGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT). Libraries were then quantified and normalised using qPCR (Kapa Biosystems, Wilmington, MA, USA). Sequencing was performed by Theragen Etex Inc. (Seoul, Korea) using Novaseq 6000, 150 bp paired-end analysis with a minimum 7M read per sample (presentation sample) and 30M (CR1/CR2 sample). Data analyses that included read quality cleaning, error correction, genome alignment and variant detection and annotation were performed on the ArcherDx platform (version 6.2) (Table S1, Supplementary File S2). The settings were optimised to include coding and non-coding sequences with default read filtering criteria, including a cut-off of allele frequency (AF) >= 0.027 and gnomAD AF <= 0.05. Raw sequencing data in Fastq format were used as the input file, and reads were mapped to the Hg 19 build of the human genome. The variant calling was conducted using three algorithms: Vision, Freebayes and LoFreq tools. A vision variant caller was used to call the somatic variants confined within the targeted mutation genes (Table SW1). The Freebayes variant caller utilised somatic variants, whereas Lofreq was used to detect somatic variants with AF frequencies. Variant annotation was done using the ArcherDx platform and verified using the Variant Effect Predictor tool (Ensembl) . The variant allele frequency (VAF) was used to deduce the origin of a variant: germline or somatic. A variant was considered to be of germline origin if the VAF was between 50% to 100%, and it was then excluded from the analysis to ensure only somatic variants were included in this study . All annotated variants were reviewed manually, and a series of filtering and prioritising was applied based on the following criteria: (1) variant consequences as defined by Sequence Ontology (SO) based on Ensembl's variation calculation filtering to include high-impact and selected moderate-impact variants (i.e., missense and protein-altering variants) , (2) exclusion of variants with minor allele frequencies (MAFs) of >= 1% in healthy populations' databases (1000 Genomes Project, ExAC, gnomAD, and ESP6500) and (3) exclusion of likely sequencing errors (variants with VAF < 5%) . Next, filtered variants were manually inspected using Integrated Genome Viewer (IGV) software (Broad Institute, Cambridge, MA, USA) and scrutinised against Varsome , dbSNP , Clinvar and COSMIC to assess previous reports on pathogenicity. Novel putative variants that were not reported were then evaluated based on computational variant effect prediction tools for coding regions (SIFT , Provean , Mutation Assessor , FATHMM , LRT ) and non-coding regions (CADD ). All variants were classified based on the American College of Medical Genetics (ACMG)'s recommendations and those of the Genomics and the Association for Molecular Pathology (AMP) and the American Society of Clinical Oncology . Variants were categorised into five main groups: (1) pathogenic, (2) likely pathogenic, (3) uncertain significance, (4) likely benign and (5) benign. The ArcherDx platform provided AMP guideline-specified somatic categories as Tier I to IV, with the first tier indicating strong clinical significance and the fourth tier deemed benign/likely benign . 2.3. Sanger Sequencing Sanger sequencing was performed on a recurrent ASXL1 c.1934dup G646WfsTer12 variant detected in 75% of the samples to verify if the variants with VAF < 5% were genuinely sequencing errors in this study (Supplementary S1: ASXL1 NM_015338.5:c.1934dup (VAF < 5%) validation, Supplementary File S1). The IDT Primer Quest (Integrated DNA Technologies, Inc. was used to design the primers for the variant of interest (ASXL1c.1934dup) and checked for specificity using the NCBI Primer-BLAST. Optimisation of the annealing temperature DNA input was performed, followed by mastermix preparation and PCR cycles. The PCR thermocycling protocols were as follows: 98 degC for 10 s for initial denaturation, 60 degC for 5 s and 72 degC for 15 s (30 cycles), followed by a final extension at 72 degC for 1 min. The PCR products were visualised using Agilent's D1000 Screentape with Agilent's 2200 TapeStation system. PCR product purification was performed using Geneall Exspin PCR SV (103-102) according to the manufacturer's instructions, and the samples were assessed for purity using the Nanodrop spectrophotometer. Samples with acceptable concentration and purity ratios were sent for Sanger sequencing. The Sanger sequencing data were reviewed using the Chromas software (version 2.6.6) (C. McCarthy; accessed on 20 January 2022), ). 2.4. RNA Sequencing Total RNA from the whole blood was isolated using QIAamp(r) RNA Blood Mini and processed according to the manufacturer's instructions. The concentration and purity of the extracted RNA was assessed using the Nanodrop ND-1000 UV-VIS Spectrophotometer. RNA integrity numbers (RIN) were determined using an Agilent RNA 6000 Nano Kit with the Agilent 2000 Bioanalyser. Only samples with RIN > 7 were selected for RNA sequencing. Messenger RNA (mRNA) library preparation was done using Agilent's SureSelect Strand-Specific RNA library preparation for Illumina Multiplex Sequencing. The steps for library preparation were as follows: (1) poly(A) RNA purification from total RNA; (2) first-strand cDNA synthesis and purification using AMPure XP beads; (3) second-strand cDNA synthesis, end repair and purification using AMPure XP beads; (4) dA-Tail of the cDNA 3' ends; (5) ligation of the adapters, followed by their purification using AMPure XP beads; (6) amplification and indexing of the adapter-ligated cDNA library and purification with AMPure XP beads; (7) quality assessment using DNA 1000 chip (Bioanalyser 2100) and ScreenTape (using Tapestation system). The library concentration was visualised using an electropherogram, which displayed a single peak with a size between 200-600 bp. All library preparations were sent to Theragen Etex Inc. (Seoul, Korea) for transcriptome sequencing, which was done using Illumina's NovaSeq 6000 (150 bp paired-end reads; 200 million reads per sample). Results were received in a raw format and processed in-house. The following Illumina adapter sequences were used in the library preparation: read 1 (AGATCGGAAGAGCACACGTCTGAACTCCAGTCA) and read 2 (AGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT). The quality of the sequencing raw data was inspected using FastQC (version 0.11.8). Low-quality reads (reads with Phred scores less than Q20), adapter and poly G sequences were removed using Fastp (Supplementary File S2, Table S2(a): RNA sequencing reads and alignments, Supplementary File S2, Table S2(b): RNA Sequencing FASTP summary). The cleaned reads were referenced against Ensembl's Human Reference Genome (version 38, Ensembl, Cambridge, United Kingdom) using HISAT2 (version 2.1.0). Aligned reads were quantified using featureCounts (version 1.6). Differential gene expression analyses were performed using DESeq2. Raw read and internal normalisation were performed across all samples, followed by customised sample-level and gene-level quality controls. Principal component analysis (PCA) was conducted to exclude potential outliers that could affect downstream analyses. Next, DEGs were filtered based on the following criteria: baseMean > 100, p-adjusted value (padj) < 0.01 and log2 fold change (lfc) > 1 and <-1. DEG profiles for genes with variants discovered in the ArcherDx targeted DNA sequencing panel were selected, and expression profiles were inspected during disease presentation and after attaining CR1/CR2. A heatmap to represent the DEG of the genes with variants was generated with hierarchical clustering based on a Euclidean distance metric. 2.5. Functional and Pathway Enrichment Analysis of Genes with Somatic Variants Using WEB-Based Gene Set Analysis Toolkit (WebGestalt) for Overrepresentation Analysis (ORA) WebGestalt was utilised to perform gene ORA on genes present with somatic variants that were deregulated in this cohort . ORA analysis was conducted to determine if the a priori gene set with variants in this study was present more than would be expected by chance . A total of 26 genes with somatic variants were included in the ORA analysis for pathway and gene ontology analysis using WebGestalt. Customised filtering parameters were applied for ORA analysis as follows: (1) a minimum of three genes for a category, (2) Benjamini-Hochberg multiple test adjustment was chosen and (3) categories were first ranked based on their FDR, and then the most significant categories were selected for significance-based filtering. The Kyoto Encyclopaedia of Genes and Genomes (KEGG) database was utilised to identify affected pathways based on a p-value < 0.05 and a false discovery rate of <=0.05. Based on the filtering criteria, seven out of ten pathways were inferred as significant, as summarised in Table S3, Supplementary File S2 . Next, gene ontology for biological processes (BP), cellular components (CC) and molecular functions (MF) was conducted using WebGestalt for the 26 genes present with somatic variants. Customised filtering parameters were applied for ORA analysis as follows: (1) a minimum of three genes for a category, (2) Benjamini-Hochberg multiple test adjustment was chosen and (3) categories were first ranked based on their FDR, and then the most significant categories were selected for significance-based filtering based on p-value <0.05 and a false discovery rate of <=0.05. A total of 10 BP categories were significantly enriched, as summarised in Table S4, Supplementary File S2. As for the MF, 1/10 and 3/10 for the CC categories met the filtering criteria (Tables S5 and S6, Supplementary File S2). 2.6. Statistical Analysis Descriptive statistics were generated using IBM SPSS Statistics 22 software (SPSS, Chicago, IL, USA). The gene mutations and gene expression profiles were assessed using the Fisher exact test, and a p-value of less than 0.05 was considered significant to scrutinise if there was concordance between the mutational and gene expression profiles of these genes. 3. Results 3.1. Demographic and Clinical Summary of Patients in This Study A total of eight de novo AML-NK patients whose DNA and RNA were collected at presentation and after CR1/CR2 were retrospectively included in this study. The median age of diagnosis was 40.5 years (range: 21-55 years). Six out of eight patients were female. All patients were negative for 30 fusion genes tested using a Leukaemia Q-Fusion Screening Kit (QuanDx, San Jose, CA, USA). The genotype for the FLT3-ITD and NPM1 mutations revealed that four patients' genotypes were FLT3-ITD+/NPM1+, two were NPM1+FLT3wt, and two were NPM1wt FLT3wt. Aberrant antigen expressions were seen in four cases (three CD2positive and one CD56positive). The remission status of patients at CR1 was assessed based on post-induction chemotherapy that included a combination of cytarabine and anthracycline (daunorubicin) (DA 3+7) and consolidation chemotherapy that included HIDAC, MIDAC or FLAG. The patient's remission at CR2 was assessed after salvage therapy using FLAG-IDA. MRD monitoring was based on either negativity for genetic markers (NPM1 and/or FLT3-ITD) or multiparametric flow cytometry for cases without genetic markers . Based on the ELN 2022 risk classification by genetics, 50% (4/8) of the patients had intermediate prognoses, and the others had good prognoses. Patients (DX1-7) were given the standard induction chemotherapy of daunorubicin and cytarabine (DA 3+7) followed by HIDAC/MIDAC/FLAG as the consolidation for patients who attained CR1. Patient DX8 was given salvage chemotherapy with FLAG-IDA, and they attained CR2. Four patients underwent stem cell transplantation, and all of the patients had an overall survival of more than five years in this study. The patients' demographic, clinical and laboratory details are listed in Table 1. 3.2. Variants Discovered in This Study An initial total of 208 variants (122 in the presentation and 86 in the CR1/CR2) were detected across the eight AML-NK samples. Rigorous filtering criteria were applied to exclude variants with MAF >= 1% and potential sequencing artefacts. Sanger sequencing was performed on the ASXL1 c.1934dup G646WfsTer12 variant recurrently seen in 12/16 samples to confirm if variants with VAF < 5% and/or that were recurrent in most samples were true findings. Sanger sequencing confirmed that this finding was a sequencing artefact. Hence for subsequent analyses, only cases with VAF > 5% were included to avoid the false positive inclusion of variants. After filtering, 131 recurrent (23/42) and non-recurrent (23/42) somatic variants involving 26 genes were identified, and they are summarised in Table S7, Supplementary File S2. Of these 42 variants, 21 missense, seven frameshifts, one splice donor variant, two in-frame insertions, and 11 untranslated regions (UTR)/intronic variants were affirmed. The oncogenic effects and role of the somatic variants were assessed in various databases (dbSNP, Clinvar and COSMIC) and published in the literature. Novel putative somatic variants were assessed using computational variant effect prediction tools. Based on ACMG and AMP classification, the pathogenicity of the somatic variants was determined as follows: 18/42 (42.9%) as pathogenic, 4/42 (9.5%) as likely pathogenic, 4/42 (9.5%) as variant of known significance (VUS), 7/42 (16.7%) as likely benign and 9/42 (21.4%) as benign. Overall, the types of somatic variants that were detected were SNVs (26/42,61.9%), insertions (11/42, 26.2%) and deletions (5/42, 11.9%). The pathogenic variants were mainly SNVs (12/22, 54.5%) and insertions (8/22, 36.4%), and the rest were deletions (2/22, 9.1%). A total of nine novel somatic variants were discovered in this study (three likely pathogenic, one VUS and four benign/likely benign (Table S7, Supplementary File S2). Three likely pathogenic novel somatic variants were detected in the CEBPA gene (DX5 and DX8) involving regions that are within previously reported pathogenic variants in the COSMIC database . 3.3. Size of FLT3 Internal Tandem Duplication (ITD) We detected FLT3-ITDs in four patients in this cohort (DX1, DX4, DX6 and DX7), as summarised in Table 1. The length of the ITDs ranged between 42-144 bp, with a median ITD length of 49.5 bp. The longest ITD was selected for patients with more than one ITD. 3.4. Evaluation of Somatic Variants Detected at Presentation and CR1/CR2 The samples in this cohort appeared to harbour between seven to thirteen somatic variants per sample; the minimum number of pathogenic somatic variants per sample was two, and the maximum was six. The mutational spectra of all somatic variants and their corresponding functional groups at disease presentation and after CR1/CR2 are depicted in Figure 1. Most recurrent somatic variants were detected belonging to cell signalling (FLT3), transcription (RUNX1 and CEBPA), methylation (DNMT3A) and nucleoplasmin (NPM1) functional groups. A total of 28 pathogenic/likely pathogenic variants (15 non-recurrent and 7 recurrent) were detected during disease presentation in all patients. Five somatic pathogenic/likely pathogenic variants were persistent in the CR1/CR2 samples (DNTM3A p.Arg659His, IDH2 p.Arg140Gln, LUC7L2 p.Glu253ArgfsTer34, NOTCH1 p.Ala1740Val and RUNX1 p.Leu56Ser genes). About 82% (23/28) of pathogenic/likely pathogenic variants seen during the presentation were lost after CR1/CR2. The VAF of pathogenic/likely pathogenic variants detected during the presentation and after CR1/CR2 in this study are depicted in Figure 2. A total of twelve clonal haematopoiesis driver mutations were seen in eight genes, according to the 5th edition of the World Health Organization Classification of Haematolymphoid Tumours. The myeloid and histiocytic/dendritic neoplasms that were detected in this study that contribute to the increased risk of developing AML, as reported in COSMIC and published in the literature, are listed in Table S8, Supplementary File S2 . Of the twelve CH driver mutations, the persistently detected mutations in CR1/CR2 samples were DNMT3A p.Arg659His in patient DX1, IDH2 p.Arg140Gln in patient DX3 and NOTCH1 p.Ala1740Val in patient DX5. The mutated genes were then classified into mutational classes (Class I, II and III) based on their putative effects that led to leukaemic transformation based on established leukaemogenesis models. Class I mutations confer proliferation and survival, such as activation of signal transduction pathways, whereas Class II gene aberrations affect differentiation and apoptosis, and Class III gene alterations promote epigenetic modifications. A total of 13/42 somatic pathogenic variants were classified into Class I (n = 1, gene: FLT3), Class II (n = 7, gene: CEBPA, NPM1, WT1) and Class III (n = 3, gene: DNMT3A, IDH2, TET2) as depicted in Figure 3. All of the patients had at least one mutation in one of these classes; five patients had mutations in Class II and Class III genes, one patient had mutations in all three gene classes and two patients had mutations in the Class II gene. Only two mutations in Class III, those in DNMT3A c.1976G > A and IDH2 c.419G > A that encode epigenetic modifiers, were retained, with VAFs of 41.68% and 44.83%, respectively, at disease presentation and 9.55% and 30.4%, respectively, after CR1. Somatic variants in recurrently mutated genes in AML (FLT3, NPM1, CEBPA and IDH2) were detected in all patients implicated as classic AML drivers and/or CH driver and hotspot mutations. Two VUS were recurrently detected in three patients (RUNX1 NM_001754.4:c.1270T > C) and two patients (ZRSR2 NM_005089.3:c.283G > A) that need further investigation to elucidate their roles in the leukaemogenesis of AML. Differential gene expressions for the 26 variants were determined from the RNA sequencing data to compare the expression profiles of patients during disease presentation and after attaining CR1/CR2 based on the normalisation and fold change information obtained from FeatureCounts (version 1.6) and DESeq2. Findings from the DEG profiles of patients during the presentation and after CR1/CR2 are summarised in Table 1 and depicted in a heatmap . The relationship between the presence of mutations and gene expression was investigated. We observed no significant relationship between mutations and gene expression in all genes except in the biallelic mutations in the CEBPA genes detected in patients DX5 and DX8. Although upregulation of the CEBPA gene was observed in other patients during the presentation, in cases with biallelic CEBPA gene mutations (DX5 and DX8), the fold changes were almost three times higher than in other cases with overexpression of the CEBPA gene. RUNX1 gene mutations were detected in patients DX1-2 and DX7-8, and overexpression was detected in patients DX1, DX3-4 and DX6-7, indicating no concordance between the mutation and gene expression profiles of this gene. Upregulation of the FLT3, WT1 and NPM1 genes was seen in all presentation samples, though a heterogeneous level of expression was observed across disease presentation and CR1/CR2. In terms of overall DEG expression (presentation samples versus CR1/CR2 samples), log2-fold changes of 5.07, 7.06 and 1.37 (Padj 0.01 for both) were observed in FLT3, WT1 and NPM1 genes, respectively. On the other hand, downregulation of genes was observed among the presentation samples compared to the CR1/CR2 samples in CBL, NOTCH1, PPM1D, RAD21, STAG2 and TET2. The normalised data for the presentation and CR1/CR2 samples, fold change and padj values generated from the DESeq2 are summarised in Tables S9 and S10, Supplementary File S2. 3.5. CEBPA Mutation and Gene Expression Profile Mutations in the CEBPA gene (known as transcription factor CCAAT/enhancer binding protein a) were detected in the frequently reported two prototypical classes of mutations: N-terminal mutations and C-terminal basic leucine zipper (bZIP) regions. Patients DX5 and DX8 had double (bi-allelic) mutations in both of these regions, as depicted in Figure 5. Further evaluation of the DX5 and DX8 patients' CEBPA-specific gene expression profiles revealed significant upregulations in both patients . Fisher's exact test statistically confirmed a significant association between the biallelic CEBPA mutation and its expression (p = 0.036). 3.6. Functional and Pathway Enrichment Analysis of 26 Genes with Somatic Variants Using WebGestalt ORA Analysis To elucidate the relevance of all of the genes present with somatic variants, over-representation analysis (ORA) using the WebGestalt tool for GO and KEGG pathway enrichment. Enriched KEGG pathways are summarised in Table S3, Supplementary File S2. Pathway analysis of the KEGG pathway is depicted in Figure 6 (transcriptional misregulation and pathways in cancer). Other significantly enriched pathways are appended in Supplementary Materials documents . Gene ontology depicting the genes' biological processes, cellular components and molecular functions are available in Figure SW5, Supplementary File S1. All significantly enriched GO terms in BP, MF and CC are summarised in Tables S4-S6, Supplementary File S2. All of the enriched GO terms in BP, MF and CC are also illustrated using volcano plots in Figures SW6-SW8, Supplementary File S1. 4. Discussion The genetic heterogeneity of AML could explain the varying responses of patients to the treatment regime, necessitating the need for tailored therapy based on the patient's genomic findings. However, the challenges lie in identifying the oncogenic effect of specific genetic aberrations and their role in the progression of leukaemogenesis. With the advent of NGS technology and bioinformatics tools, more sensitive assessments of genomic aberrations and MRD genomic markers are available. Recent evaluations of genomic MRD markers and their associations with patient outcomes and survival were explored and reported in multiple studies . This study compared the mutations detected in eight matched presentation and CR1/CR2 samples using a high-throughput 75-gene panel by Archer HGC VariantPlex Myeloid. We performed paired-end deep sequencing on the samples collected at disease presentation (a minimum of 7 million reads) and increased the sequencing depth to 10X (a minimum of 30 million reads) to ensure mutations with low VAFs and minor leukaemic cell clones were not missed in the CR1/CR2 samples. As suggested by Yan et al. (2021), Sanger sequencing was performed on the selected variant (ASXL1 c.1934dup G646WfsTer12) with VAF < 5% . Sanger sequencing disconfirmed this variant as a sequencing artefact; hence, for all of the subsequent analyses, only variants with VAF > 5% were included in this study. We disagree with Wang et al. (2020), as they have included all variants with VAF > 1%, suggesting that there are no standardised cut-offs . We affirm that the laboratory's internal validation can determine cut-offs for VAFs by performing Sanger sequencing for validating low VAFs in samples as performed in this study. We also added another facet by performing high throughput transcriptomic deep sequencing (200 million reads, PE, 150 bp) for the same cohort of patients during disease presentation and CR1/CR2. We then compared the gene mutations and DEG profiles of the genes to discover the impact of pathogenic mutations on gene expression. We found that cases with biallelic CEBPA mutations were significantly associated with its upregulation in two patients (DX5 and DX8). Nevertheless, no significant association was observed between mutation and expression profiles in other genes investigated in this cohort. Next, gene enrichment was assessed via ORA using the WebGestalt tool to investigate the pathways that are affected among KEGG pathways. As an outcome, five pathways were significantly enriched, affecting several hub genes: FLT3, AML1 (also known as RUNX1) and HRAS. This cohort consisted of AML-NK patients below 60 years of age with good prognoses (n = 4) and intermediate prognoses (n = 4) according to the ELN 2022 classification. All the patients were treated with the same treatment protocol during induction therapy (DA 3+7), including personalised HIDAC/MIDAC/FLAG for consolidation/salvage therapy, and they attained CR (n(CR1) = 7; n(CR2) = 1). Four patients had undergone SCT: n(allo-SCT = 3); n(auto-SCT = 1). All patients had an excellent OS of above five years with no indication of relapse (Table 1). The number of pathogenic somatic variants detected in the patients ranged between two and six per patient with no association with age, unlike previous reports by other studies that disclosed the number of mutations was lower in younger patients (18 to 39 years old) than in those above 40 years of age . This study's findings concur with another local study that performed whole-exome sequencing (WES) and reported fewer somatic variants in their AML-NK cohort (n = 6) . Although they performed WES sequencing with greater genome coverage, the number of somatic variants was relatively low, especially in the myeloid-related genes included in our targeted NGS panel. The number of somatic mutations detected in our cohort was lower than in other studies with similar targeted myeloid panel designs . In this study, the overall ratio of indels to SNVs was 1.58 at presentation and 2.4 at CR1/CR2, whereas the ratio of pathogenic somatic indels to SNVs was 1.15 at presentation and 0.25 at CR1/CR2, in contrast to previous publications . The higher overall ratios of indels to SNVs in our study are due to recurrent benign indels seen in FLT3, RAD21 and EZH2 genes, as illustrated in Table S1. Our findings did not concur with another WES study conducted on AML-NK in another local study that exhibited a lower ratio of indels to SNVs, with SNVs being almost twice as common as indels . The findings could be due to differences in experimental design, such as WES versus the 75-myeloid gene panel utilised in this study. The variants detected in this study are depicted in Figure 1 based on their types and functional groups. This study saw mutations in the signalling and kinase pathway-related genes that caused abnormal activation and proliferation of cellular signalling pathways, also known as Class 1 mutations, seen in 6/8 cases in this study, which agrees with other studies . Similar to other studies, we also identified mutations in the epigenetic modifiers that include regulation of DNA methylation and chromatin modification in 6/8 cases in this cohort, which are recognised as drivers in leukaemogenesis. Mutations in epigenetic modifiers result in clonal outgrowth but require subsequent mutations to initiate leukaemic transformation . Mutations in the myeloid transcription factors were seen in 3/8 cases, which concords with other studies that reported their presence in approximately 20% to 25% of AML cases . CEBPA is an essential transcription factor for haematopoietic lineage-specific myeloid differentiation. Studies have reported that CEBPA gene mutations were found in 10-15% of de novo AML cases, especially in AML-NK patients . Typically, CEBPA mutations cluster in two focal hotspots: N-terminal frameshifts insertions/deletions and/or C-terminal in-frame insertions/deletions. Mutations in the N-terminal give rise to the truncated p30 protein, which presides negatively to other full-length p42 proteins, whereas mutations in the C-terminal will obstruct the binding of CEBPA to DNA or dimerisation. In this cohort, we found biallelic CEBPA mutations in two cases (patients DX5 and DX8) at the hotspot regions of the N-terminal (within the first 357 bp of coding sequence) and the C-terminal bZIP region (between c.834-1074) which likely yielded the truncated p30 protein and led to the disruption of the binding of CEBPA to DNA or to dimerization . Upregulated CEBPA genes were observed distinctly in cases with biallelic CEBPA mutations, as discovered in other studies . AML with a CEBPA mutation is still retained as a unique entity in the 2022 WHO classification, and patients with biallelic CEBPA mutations often have good prognoses . CEBPA gene upregulation and the outcomes of AML patients were inconclusive in several studies . Although ELN 2022 suggested in-frame mutations affecting the bZIP region of CEBPA, irrespective of whether monoallelic or biallelic occurrence is the favourable prognosis, we could not ascertain whether biallelic mutations accompanied with the upregulation of the CEBPA gene is a favourable prognostic factor because the DX5 patient attained CR1 following induction and had OS > 5 years without SCT, whereas the DX8 patient only attained CR2 and had undergone allo-SCT in this study. The length of internal tandem duplication in the FLT3-ITD cases in this cohort was above the cut-offs (>30) used in several studies for prognosis based on the ITD's length . There are contradictory findings about the size of FLT3-ITD insertion length and the patient's clinical outcome. Several studies have shown poor OS with increasing ITD length, whereas increased ITD insertion conferred better OS in some studies . However, some studies did not agree with either of these findings and indicated that the ITD length has no significance in the prognosis or clinical application of AML . In this study, all patients had a good OS of above five years with concomitant NPM1 mutation; hence, their prognoses were categorised as intermediate based on the ELN 2022 classification. In addition, we did not observe any relationship between the presence of FLT3-ITD and the ITD's length with the expression of FLT3 genes, as FLT3 was overexpressed in all cases in this cohort. Moreover, the sample size was insufficient to infer the relationship between ITD insertion length and patient prognosis in this study. Of the five pathogenic/likely pathogenic somatic variants that were persistent in the CR1/CR2 samples, three variants (DNMT3A p.Arg659His, RUNX1 p.Leu56Ser in Patient DX1 and NOTCH1 p.Ala1740Val in Patient DX5) were reported as having a germline predisposition in AML . Two other recurrent pathogenic somatic variants that persisted after CR1/CR2, variants in IDH2 p.Arg140Gln (seen in Patients DX3 and DX6) and LUC7L2 p.Glu253ArgfsTer34 (seen in Patients DX2, DX3 and DX8), were reported as somatic in the Clinvar database and in other publications . We deduced that the pathogenic somatic variant detected in NPM1 p.Trp288CysfsTer12, which was recurrent in six cases (Patients DX1, DX2, DX3, DX4, DX6, DX7), is suitable for MRD monitoring for treatment response in this cohort where the VAF at presentation ranged between 25-29% and was not detectable at CR1/CR2. We also observed interesting findings in the DEG profiles of FLT3, WT1 and NPM1 that exhibited upregulation and those of CBL, NOTCH1, PPM1D, RAD21, STAG2 and TET2 that were downregulated at presentation versus the CR1/CR2 patients. Studies revealed the utility of FLT3, WT1 and NPM1 expression for MRD monitoring in AML, which supports the findings in this study . Functional enrichment analysis using ORA (WebGestalt) revealed that five pathways were significantly enriched (p-value < 0.01, FDR < 0.05), as illustrated in Table S3, Supplementary File S2. In addition, several hub genes were identified in several pathways, such as FLT3, RUNX1 and HRAS . Transcription misregulation in cancer tops the list of affected pathways, as some of the upstream genes in the transcriptional regulation, such as CEBPA and RUNX1, were deregulated in most cases, as shown in Figure 6. The overexpression of FLT3 impacted JAK-STAT signalling and cytokine-cytokine receptor interaction, as this gene is also located upstream of the pathway . Overexpression of RUNX1, which is a hub-gene crucial in haematopoietic differentiation, was observed in 62.5% (5/8) of presentation samples . Overexpression of CEBPA genes was observed in 88% (7/8 of the presentation samples) of the patients with concurrent biallelic mutations (Patients DX5 and DX8), supporting the notion that haematopoietic resistance is evident in the pathogenesis of AML-NK . The variant in the HRAS gene detected in this study (Sample DX7) was benign, occurred in the 5' UTR region, and did not affect gene expression in this study. However, two patients (DX1 and DX2) exhibited upregulation of this HRAS gene, as depicted in Figure 4. Upregulation of the HRAS gene could impact the deregulation of several pathways: PI3K-Akt signalling pathway, Jak-STAT signalling pathway, MAPK signalling pathway and calcium signalling pathway, as HRAS is a hub-gene in the RAS signalling pathway . The most significantly enriched pathway in this study was transcriptional misregulation in cancer (hsa05202) (p < 0.01, FDR = 0.001), which was closely related to the most enriched GO MF category, DNA-binding transcription activator activity related to RNA polymerase II-specific (GO:0001228) (p < 0.01, FDR = 0.4), with four overlapping genes that includes hub genes such as FLT3 and CEBPA, as listed in Tables S4 and S6, Supplementary File S2. The ancestral GO:0140110 (Transcription regulation activity) for GO:001228 ensures transcriptional regulators are modulating the gene expression at the right cells at the right time, which were disrupted in this study as the upstream genes (AML1/RUNX1 and CEBPA) at the hsa05202 were dysregulated, as depicted in the DEGs of genes during disease presentation and after CR1/CR2 in Figure 4. This explains the transcriptional dysregulation because the majority of the signalling pathways target transcription machinery, which provides insights into the mechanism of AML-NK leukaemogenesis . Significance of the Study Primarily, this study classified mutations discovered in AML-NK patients based on various classification guidelines (ACMG/AMP), databases (dbSNP, Clinvar and COSMIC) and variant effect prediction tools. Based on the ACMG/AMP recommendation, the variants were classified as benign, likely benign, VUS, likely pathogenic and pathogenic. The variants were then assessed based on mutational classes (Class I, II and III) and their functional classes to explicate their putative effects that enabled leukaemic transformation based on established leukaemogenesis models. Then, the variants detected during disease presentation and after CR1/CR2 were assessed to identify potential MRD biomarkers for treatment monitoring. Next, gene expression profiles for 26 genes with somatic variants were determined to assess the effect of these variants on their DEG profiles. Next, DEG profiles for 26 genes with somatic variants were assessed during disease presentation and after CR1/CR2. Finally, ORA analysis was conducted using the WebGestalt tool for GO and KEGG pathway enrichment analyses of the 26 genes with their variants in this study. To recapitulate, this study demonstrated the mutational profiles depicting AML's genomic landscape heterogeneity. Each patient had a unique list of somatic pathogenic variants that belonged to Class I/II/III mutations. In some cases, CH-related variants were identified that conferred an increased risk of developing AML. The deregulation of genes with mutations impacted the pathways in cancer and various signalling pathways, as these genes are either hub genes or were upstream of pathways, as described earlier. Based on ELN 2022 classifications, the two patients (DX5 and DX8) with biallelic CEBPA gene mutations affecting the bZIP regions were assigned to a favourable prognosis group (Table 1). We also verified the NPM1 p.Trp288CysfsTer12 mutation as a recurrent biomarker that conferred a favourable prognosis in our cohort. In addition, we also exemplified the usefulness of the DEG profiles of FLT3, WT1 and NPM1 for MRD assessment in AML-NK patients. 5. Conclusions This study elucidated the genomic mutations and gene expression profiles of myeloid genes using a 75-gene targeted DNA panel (Archer HGC VariantPlex Myeloid) during disease presentation and after CR1/CR2. Diverse mutations and expression profiles were observed in each patient during disease presentation, indicating that AML-NK is indeed a heterogeneous disorder requiring further risk-based stratification. This study discovered three novel pathogenic somatic variants at the reported prototypical regions, N-terminal and bZIP regions of the CEBPA gene that were significantly associated with its upregulation. Although the cohort size is relatively small, we identified potential biomarkers that indicate favourable prognoses and are suitable for MRD monitoring, including NPM1 p.Trp288CysfsTer12, which was recurrent in 75% (6/8) of the patients and is in line with the ELN 2022 recommendation. We also suggested DEG profiles for FLT3, WT1, and NPM1 for MRD as potential biomarkers for MRD assessment in AML-NK patients. This multifaceted study comprehensively integrated DNA and RNA sequencing findings with functional enrichment in AML-NK that has not been described elsewhere. Our next step is to expand this study into a larger cohort of AML-NK patients to formulate a hierarchical prognosis model with unique mutations and gene expression profiles. Acknowledgments The authors thank the following: the Director General of Health Malaysia for his permission to publish and the Department of Haematology, Hospital Ampang, for permission to access the patients' samples and data; the School of Medical and Health Sciences, Universiti Sains Malaysia for permission to conduct the research, for funding the research (GIPS-PhD 311/PPSP/4404814) and for publication permission. Special thanks to Syed Carlo Edmund, Hospital Ampang, for assistance in patient survival data retrieval and Wee Kai Li, Rose Ismet, Leong Wai Mun and Mohd Norhisam Hamid, Neoscience Malaysia, for technical assistance during experimental design. All of this was made possible because of the graciousness of Almighty God. Supplementary Materials The following supporting information can be downloaded at: Supplementary File S1, Table SW1: HGC VariantPlex(r) Myeloid panel, Supplementary File S1, Supplementary S1: ASXL1 NM_015338.5:c.1934dup (VAF < 5%) validation, Supplementary File S1, Figure SW1: Acute myeloid leukaemia pathway, Supplementary File S1, Figure SW2: Central carbon metabolism in cancer, Supplementary File S1, Figure SW3: Pathways in cancer, Supplementary File S1, Figure SW4: RAS signalling pathway, Supplementary File S1, Figure SW5: Gene Ontology (GO) summary for the 26 genes with somatic variants, Supplementary File S1, Figure SW6: Volcano plot depicting biological process GO for the 26 genes with somatic variants, Supplementary File S1, Figure SW7: Volcano plot depicting molecular function GO for the 26 genes with somatic variants, Supplementary File S1, Figure SW8: Volcano plot depicting cellular component GO for the 26 genes with somatic variants, Supplementary File S1, Figure SW9: Directed acyclic graph (DAG), Supplementary File S2, Table S1: Archer HGC VariantPlex Myeloid Sequencing Results, Supplementary File S2, Table S2(a): RNA sequencing reads and alignments, Supplementary File S2, Table S2(b): RNA Sequencing FASTP summary, Supplementary File S2, Table S3: Summary of enriched KEGG pathway, Supplementary File S2, Table S4: Summary of enriched GO for Biological Processes (BP), Supplementary File S2, Table S5: Summary of enriched GO for Molecular Function (MF), Supplementary File S2, Table S6: Summary of enriched KEGG for Cellular Component (CC), Supplementary File S2, Table S7: Summary of variants detected in this study during presentation (DX1-8) and after attainment of CR1/CR2 (RX1-8), Supplementary File S2, Table S8: CH variants detected in this cohort in patients during presentation (DX1-8) and after attainment of remission (RX1-8), Supplementary File S2, Table S9: Normalised count of samples at presentation (DX1-8) and after attainment of CR1/CR2 (RX1-8), Supplementary File S2, Table S10: Log2fold change information derived from DeSeq2 (patients at disease presentation versus after attainment of CR1/CR2). Click here for additional data file. Author Contributions A.A. and R.H. conceptualised the study. A.A. prepared the manuscript. A.A. collected patient data and performed the experiments. A.A. performed the statistical analysis and prepared all figures and tables. R.H., S.S., R.R. and J.S. oversaw the study and supervised. R.H., S.S., R.R., J.S., Y.Y.Y. and E.S.Z. reviewed and edited the manuscript. R.H., J.S., S.S. and R.R. critically reviewed the technical aspects of the study. All authors have read and agreed to the published version of the manuscript. Institutional Review Board Statement The study was conducted according to the guidelines of the Declaration of Helsinki and approved by the Medical Research Ethics Committee of the Ministry of Health of Malaysia (NMRR 17-1929-36614) on 29 December 2017 and Universiti Sains Malaysia (USM/JEPeM/21010107) on 15 June 2021. Informed Consent Statement Informed consent was obtained from all subjects in this study as approved by the Ministry of Health, Malaysia (NMRR 17-1929-36614). Data Availability Statement The datasets generated and/or analysed during the current study are available in the Sequence Read Archive, National Center for Biotechnology Information [SRA, NCBI] repository, Accession: PRJNA902950. Other data are available in Supplementary Files S1 and S2. Conflicts of Interest The authors declare no conflict of interest. Figure 1 The mutational spectra of all somatic variants (pathogenic/benign/VUS) and their corresponding functional groups at disease presentation and after CR1/CR2. The coloured boxes indicate the types of variants detected in the study SNV/insertion/deletion/ITD as depicted in the legend. P refers to samples collected at presentation, and CR refers to samples collected after remission (CR1/CR2). * Refers to the internal tandem duplication (ITD). Figure 2 Comparison between VAFs during disease presentation and after CR1/CR2. Each line in the graph corresponds to a somatic pathogenic variant detected during disease presentation and after CR1/CR2. Only pathogenic variants were included in each patient's VAF comparison during the presentation and after CR1/CR2. Figure 3 Representation of somatic variants during disease presentation and after attainment of CR1/CR2 based on the mutational classes. Figure 4 Heatmap of genes with variants during disease presentation and after attainment of CR1/CR2. Figure 5 A schematic representation of the CEPBA gene structure with two alternative start sites that give rise to different isoforms (p42 and p30). Patients DX5 and DX8 had biallelic frameshift mutations at the N-terminal region and in-frame insertion at the C-terminal bZIP mutations. Figure 6 Transcriptional misregulation pathway and pathways in cancer (reference: KEGG pathways) . This pathway illustrates the affected hub-gene RUNX1 and the upstream gene, CEBPA, in red boxes, which elucidates the differentiation resistance in AML-NK patients in this cohort in cases with deregulated CEBPA and RUNX1 genes. Upregulation of the RUNX1 and CEBPA genes could lead to impaired resistance to normal haematopoietic differentiation. cancers-15-01386-t001_Table 1 Table 1 Demographic, clinical and laboratory information of patients in this cohort. Patient Details DX1 DX2 DX3 DX4 DX5 DX6 DX7 DX8 Demographic data Age 31 29 54 55 36 49 45 21 Ethnicity Malay Malay Indian Malay Chinese Malay Indian Malay Gender Female Female Female Female Female Female Male Male No. of mutations 3 3 1 1 1 3 1 5 Karyotype G-banding 46 (X,X) 46 (X,X) 46 (X,X) 46 (X,X) 46 (X,X) 46 (X,X) 46 (X,Y) 46 (X,Y) Cell counts White blood cell count (109/L) 26.9 57.7 31.1 170 19.5 45.8 39.5 152.8 Haemoglobin (g/dL) 7.3 8.1 8.1 8.4 11.3 7.9 9.9 6.1 Platelet (109/L) 32 76 83 70 68 21 22 27 % Blast (PB) 20% 55% 89% 90% 40% 90% 95% 94% % Blast (BMA) 90% 80% 90% 92% 90% 96% 90% 95% Flow cytometry immunophenotyping Aberrant antigen expression CD2+ CD2+ - CD56+ CD2+ - - - Gene mutations Leukaemia Q-Fusion (30 fusion genes) Negative Negative Negative Negative Negative Negative Negative Negative FLT3-ITD Detected wt wt Detected wt Detected Detected wt NPM1 mutation Detected Detected Detected Detected wt Detected Detected wt Archer HGC VariantPlex Myeloid Panel Genes with pathogenic/likely pathogenic somatic variants/ITD CBL CBL IDH2 TET2 CEBPA FLT3 IDH1 CEBPA DNMT3A IDH1 NPM1 NPM1 IDH2 NPM1 CUX1 RUNX1 LUC7L2 FLT3-ITD U2AF2 FLT3-ITD GATA2 STAG2 NPM1 NPM1 IDH1 NPM1 FLT3-ITD LUC7L2 FLT3-ITD WT1 FLT3-ITD insertion length (Archer HGC) 42 - - 45 - 54 144 - Number of mutations per patients 4 3 1 1 1 3 1 5 DEG Upregulated genes EZH2 EZH2 CUX1 CUX1 EZH2 ANKRD26 CUX1 EZH2 FLT3 FLT3 FLT3 ETV6 CEBPA CUX1 FLT3 CEBPA HRAS HRAS GATA2 FLT3 CUX1 FLT3 GATA2 CUX1 IDH1 LUC7L2 LUC7L2 GATA2 DNMT3A GATA2 NPM1 DNMT3A NPM1 NPM1 NPM1 LUC7L2 FLT3 LUC7L2 RUNX1 ETV6 RUNX1 SMC1A RUNX1 NPM1 GATA2 NPM1 WT1 FLT3 U2AF2 WT1 RUNX1 IDH1 RUNX1 GATA2 WT1 NPM1 SMC1A IDH1 WT1 LUC7L2 NPM1 Downregulated genes ATRX ATRX CBL CBL CBL CBL CBL TET2 CBL CBL NOTCH1 NOTCH1 NOTCH1 NOTCH1 KDM6A STAG2 KDM6A KDM6A PPM1D PPM1D PPM1D PPM1D NOTCH1 RAD21 NOTCH1 NOTCH1 RAD21 RAD21 RAD21 RAD21 PPM1D PPM1D PPM1D PPM1D STAG2 STAG2 STAG2 STAG2 RAD21 NOTCH1 RAD21 RAD21 TET2 TET2 TET2 TET2 STAG2 KDM6A STAG2 STAG2 TET2 CBL TET2 TET2 ELN 2017 Classification Prognosis Intermediate Good Good Intermediate Good Intermediate Intermediate Good Treatment Protocol Induction DA 3+7 DA 3+7 DA 3+7 DA 3+7 DA 3+7 DA 3+7 DA 3+7 DA 3+7 Consolidation MIDAC/HIDAC HIDAC/HIDAC/HIDAC MIDAC/HIDAC/FLAG MIDAC/FLAG/HIDAC MIDAC/HIDAC/ARAC MIDAC/FLAG/FLAG MIDAC/HIDAC/ARAC FLAG-IDA/HIDAC/ARAC SCT Nil Nil Nil Allo-SCT after CR1 Nil Auto-SCT after CR1 Allo-SCT after CR1 Allo-SCT after CR2 Response to treatment Remission status CR1 CR1 CR1 CR1 CR1 CR1 CR1 CR2 OS >5 >5 >5 >5 >5 >5 >5 >5 Disclaimer/Publisher's Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). 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PMC10000177

It is well appreciated that the social determinants of health are intimately related with health outcomes. However, there is a paucity of literature that explores these themes comprehensively for the indigenous people within Micronesia. Certain Micronesia-specific factors, such as transitions from traditional diets, the consumption of betel nut, and exposure to radiation from the nuclear bomb testing in the Marshall Islands, have predisposed certain Micronesian populations to an increased risk of developing a variety of malignancies. Furthermore, severe weather events and rising sea levels attributed to climate change threaten to compromise cancer care resources and displace entire Micronesian populations. The consequences of these risks are expected to increase the strain on the already challenged, disjointed, and burdened healthcare infrastructure in Micronesia, likely leading to more expenses in off-island referrals. A general shortage of Pacific Islander physicians within the workforce reduces the number of patients that can be seen, as well as the quality of culturally competent care that is delivered. In this narrative review, we comprehensively underscore the health disparities and cancer inequities faced by the underserved communities within Micronesia. cancer disparities Micronesia Palau Nauru Kiribati Mariana Islands Guam Pacific Islands health disparities Oceania Stanford Cancer Institute, an NCI-designated Comprehensive Cancer CenterStanford Cancer Institute Women's Cancer Center Innovation AwardStanford Cancer Institute Fellowship AwardThis work was supported by the Stanford Cancer Institute, an NCI-designated Comprehensive Cancer Center. K.T. was funded by a Stanford Cancer Institute Women's Cancer Center Innovation Award. K.T. was funded by the Stanford Cancer Institute Fellowship Award. pmc1. Introduction 1.1. Methods/Review Approach Narrative reviews are a type of qualitative research that outline key results of scholarly work focusing on a topic of interest without focusing on the statistical significance of the findings . We conducted a keyword search-based literature review using PubMed Advanced Search on or before 1 December 2022, for studies with abstracts or titles containing terms such as "Micronesia", "Guam", "Mariana", "risk factors", "social determinants of health", "healthcare", "diet", "radiation", "cancer", and "oncology". We limited this review to research on human subjects only and included English language-based peer-reviewed articles (e.g., primary research, reviews, and editorials), online reports, electronic books, and press releases. Based on the available studies at the beginning of this search, we focused our review on studies performed in the US, Micronesia, Australia, and New Zealand. All publications identified for review were assessed for relevance by the team of authors. 1.2. An Introduction to Pacific Islanders and Micronesia The Pacific Ocean covers one-third of the planet's surface . Within the Pacific Ocean are the three ethnogeographic regions of the continent of Oceania from which Pacific Islanders share ancestry: Micronesia, Melanesia, and Polynesia . Together, these three regions comprise a collection of over 25,000 islands occupying over 300,000 square miles . Each island population has its own distinct language, culture, and history . Micronesia, more specifically, is comprised of over 2000 smaller islands and atolls dispersed across a region of the Pacific Ocean bordered by the Philippines to the West, Hawaii to the East, and Papua to the South . Micronesia is approximately 3000 miles across, comparable to the entire landmass of the US and the European continent . The island nations and territories in the Micronesian region include the Commonwealth of the Northern Mariana Islands (CNMI; a US commonwealth), Guam (a US territory within CNMI), the Republic of Belau (Palau), the Federated States of Micronesia (FSM; which consists of Yap, Pohnpei, Kosrae, and Chuuk), the Republic of the Marshall Islands (RMI), Nauru, and Kiribati . Notably, the FSM, which is an island nation, should be distinguished from the ethnogeographic region of Micronesia, which encompasses all of these island nations and territories. Moreover, many of these Micronesian island nations and territories represent political groupings consisting of multiple islands and not single island nations. The region of Micronesia encompasses nations and territories historically and politically tied to high-income countries (HICs) and low-middle income countries (LMICs). Independent islands, including Nauru and Kiribati, have ties to Australia and New Zealand, which represent LMICs. Five islands within the region of Micronesia have international relationships with the US and are referred to as US-Affiliated Pacific Islands (USAPI) and are naturally classified as having ties to HICs . The current state of Micronesian international relations is deeply rooted in military strategy with a colonization history of Spain, Germany, Japan, and the US since World War I . Following World War I, in 1947, the United Nations granted the US trusteeship over RMI, Micronesia, CNMI, and Palau, which allowed for subsequent US military occupation and weapons testing throughout the islands . Decades later in 1986, a joint resolution known as the Compact of Free Association Act (COFA) established a relationship between these Freely Associated States (RMI, FSM, and Palau) and the US to help these Pacific Islands advance their economic development and self-sufficiency . COFA granted the continued exclusive and strategic US military control over a large region of the Pacific; in return the USAPI were entitled to US security guarantees, disaster relief, health coverage, and economic assistance . Political and economic affiliations to HICs or LMICs continue to have significant implications for health care resources and, more broadly, the local economy. 1.3. Cancer Disparities in Micronesia Over the last half century, indigenous populations within USAPI have had dramatic shifts in health outcomes with some of the highest incidences in the world of preventable chronic diseases . Cancer remains the second most common cause of death in nearly all USAPI nations. The sociocultural determinants of health are intimately intertwined with the cancer disparities that Pacific Islanders face in Micronesia. The historical trauma from nuclear weapons testing has led to generations of radiation-related diseases impacting both physical and mental wellbeing. Cultural practices, such as betel nut chewing, also remain an ongoing problem particularly among the indigenous populations of Micronesia, which contributes to cancer disparities among patients with oral cavity cancer. Transitions from traditional dietary behaviors, exacerbated by sedentary lifestyles, have led to the highest rates of obesity in the world, which have known associations with developing other chronic conditions such as cancer. The incidence of cancer in Micronesia is particularly staggering with the indigenous population experiencing an unequal burden of the disease . The Pacific Regional Central Cancer Registry report cancer incidence rates (per 100,000 people) in USAPI from 2007-2018 . Lung and bronchus, breast, invasive cervical, tobacco-related oral cavity and pharynx, liver, and prostate cancers are among the most common cancers affecting the people of Micronesia (Table 1). The incidence of cervical cancer is as high as 65.8 in RMI and 40.7 in Pohnpei, 5-8-times higher than the rates in the US. Similarly, incidence rates of oral cavity and pharynx cancer are 4-times higher in Yap, and rates of liver cancer are 2-3-times higher in Palau and Yap, compared to rates in the US (Table 1). For cancers of the lung, we see some of the lowest 5-year survival rates ranging from 4% in Yap to 30% in Guam. Breast cancers, which typically have higher survival rates, are lower in places such as RMI and Pohnpei, with many of the cancers being detected at stage 3 or later (Table 1). The incidence of nasopharyngeal cancers in Micronesian men is up to 4-times higher compared to men in the US state of Hawaii . Given these cancer disparities, throughout this narrative review, we aim to underscore the multitude of factors that influence the significant cancer disparities observed across Micronesia. 2. The Social Determinants of Health in Micronesia 2.1. Social Determinants of Health The social determinants of health are essential social factors or conditions that directly or indirectly contribute to the development of disease among the general population including access to resources, power, prestige, and class . The Kaiser Family Foundation defines Social Determinants of Health as the "conditions in which people are born, grow, live, work, and age" . Cancer risk is irrevocably linked to the social conditions of diet, environmental exposure, substance use, education, physical activity, socioeconomic status, and the infrastructure of a country, affecting health outcomes, survivorship, and quality of life . Hence, Micronesian-specific social determinants of health are important to discuss, as they can alter the course of an individual's prognosis, may provide an explanation for disparate rates of certain cancers, and are an important source of knowledge to improve cancer interventions . While social determinants of health vary widely among and between island nations and territories across Micronesia, the purpose of this review is to highlight general themes within the literature that underscore the health-related disparities that may influence the observed cancer disparities throughout the region. Throughout this review, we will touch on aspects of Micronesian Cancer Disparities, as they pertain to the conceptual framework of the social determinants of health . 2.2. Diet and Nutrition Dietary factors have a significant impact on overall cancer incidence . For example, a large meta-analysis of 45 observational studies demonstrated that lower colorectal cancer risk was associated with higher intake of fiber, dietary calcium, and yogurt . Historically, Indigenous Pacific Islanders had incredibly diverse agroecosystems that were cultivated in sustainable practices and had the ability to support hundreds of thousands of individuals on some of the most geographically remote islands in the world . Pacific Island communities traditionally adopted variations on a common diet depending on the produce, fish, and fauna that were accessible. For example, within the FSM, bananas, taro, copra, and breadfruit are considered staple foods and are incorporated into local cuisine . This is due in part to the fact that the majority of accessible produce is optimally grown within the tropical Micronesian climate. For protein, Pacific Islanders traditionally had access to locally sourced poultry, fish, crustaceans, and mollusks; pork has often been reserved for special occasions. Tropical fruits, such as mango, guava, sour-sop, papaya, and pineapple, are also widely available between islands . As a result of colonization and globalization, modern-day supply chains connected to the Micronesian Islands have increased the import of more refined grains and processed foods from overseas such as white rice, high fructose corn syrup products, and red meats . Over the past century, many of these foreign imports have slowly replaced traditional Pacific Islander diets due to availability, affordability, and convenience . For example, due to the influence of Japanese imperialist occupation, rice has become an integral part of the modern FSM diet . One participatory mixed methods study including nearly 300 adult Pohnpeian women used a 7-day food frequency questionnaire to assess diet and found that 96% of participants reported eating rice frequently (3-7 days/week); only 75% consumed locally grown carbohydrates as a part of their normal diet . A shift from foods that are imported, with low nutritional value, back to indigenous, high fiber, healthier foods require culturally sensitive and innovative strategies to promote local and sustainable food production. The introduction of the Western diet with US influence has also drastically changed the food landscape and dietary behaviors among the people of Micronesia . Given the remoteness of the Pacific Islands, historically, many foods imported into the islands had to withstand long distances of travel, necessitating foods and goods that were highly processed. These canned and highly processed food products are widely consumed among Pacific Islanders and may have some level of carcinogenicity, with several studies demonstrating increased risk of stomach and colorectal cancers . In 2015, the International Agency for Research on Cancer Working Group made a formal recommendation that the consumption of processed meat be classified as a Group 1 carcinogen, labeling these products as "carcinogenic to humans" given associations with colorectal and gastric cancer . This inequity in food accessibility is exacerbated by a culture of misinformation, as the giant swamp taro, once widely cultivated and consumed by Micronesians, is in some cases shunned for being "starchy" and is instead being fed to pigs and other farm animals . Vegetable and fruit consumption is also disproportionally low throughout Micronesia. For example, over 80% of the Pohnpei population consume less than five servings of fruits and vegetables per day with an average of 1-2 servings per day, while in Majuro 40% and 23% of youth were found to not eat a single serving of "a green salad" or "fruit" at least once per week, respectively . Sociocultural factors also influence the diet of Micronesians in the Pacific Islands. Imported food are perceived as "higher quality," which gives these items a level of societal "prestige" . Conversely, in some Micronesian communities, the practice of farming is becoming culturally stigmatized. For example, in the Cook Islands, those who farm are called "Repo Taro" or "Dirty Taro" . This derogatory term is used to refer to men who perform manual labor (e.g., taro farming) and are stigmatized as men who "are nothing" and "have nothing", such as money, land, or other capital . Simultaneously, traditional knowledge and practices of food cultivation, preservation, and farming are being lost with each subsequent generation . As a result, local subsistence agriculture and fishing have all steadily decreased over time, leading to increased economic costs to families and to Pacific Island nations . Poverty and a growing reliance on government food subsidies has also contributed to the loss of farming and maintenance practices for many indigenous crops, such as taro, arrowroot, breadfruit, and other long storage crops . This is exacerbated by some islands lacking refrigerators, as low as only 10% of households in more geographically isolated islands, such as Pingelap, which creates greater dependence on canned foods . 2.3. Exercise and Obesity Physical activity, even if only leisurely, has demonstrated significant benefit to overall health and has been associated with a decreased risk for both cardiovascular and cancer mortality . A large meta-analysis demonstrated that obesity is associated with worse overall and cancer-specific mortality among patients with breast and colorectal cancer . Globally, the top 10 countries with the highest rates of obesity are all Pacific Island nations, including those within Micronesia . In many islands, including Pohnpei and Kosrae, over 80% of the adult population are obese . This observation applies outside of the geographical region of Micronesia, as a study conducted in Hawaii demonstrated that ethnically Pacific Islander children are nine-times more likely to be overweight or obese when compared to White children . However, in Micronesia today, physical activity and exercise rates are low among the general population, with most Pacific Islanders failing to vigorously exercise 3-times a week, moderately exercise 5-times a week, or achieve over 600 metabolic equivalents per week . For example, over 60% of residents of Chuuk reported low levels of daily physical activity with lifestyles centered around sedentary activities instead (e.g., "talking story" and weaving) . Weight management and exercise implementation programs are rare and largely non-established in Micronesia . Dietary factors, coupled with cultural beliefs, a lack of exercise, and a sedentary lifestyle, have led to an increase in non-communicable diseases, such as diabetes, hypertension, and obesity, as well as deficiencies in essential vitamins and nutrients . These comorbidities have been shown to be associated with an increased risk for several different types of cancer, as well as other adverse health outcomes . 2.4. The Economy and Infrastructure Socioeconomic status is fundamentally linked to health outcomes . In the US, patients with cancer who live in counties with lower income are associated with higher cancer-specific mortality . Within Micronesia, the majority of independent states are classified as low-income countries with GDPs far below the world average, with among the slowest GDP growth globally and heavy dependence on US aid . Poverty rates and resource access differ between communities. For example, in the CNMI, due to a decrease in tourism, garment industry closures, and the rising cost of fuel, over half of Micronesians live below the poverty line, and more than 1800 families make less than $5000 US dollars annually as recently as the 2010s, given a total population of approximately 50,000 . Whereas in Palau, 25% of people live below the US poverty line, and in the FSM, 15% of people live on less than $1.90 per day . Opportunities for work are predominantly in government or tourism industries, while growing overreliance on imported goods and services further stunts the growth of the economy . A shift away from the agricultural industry, due in part to the perceived undesirability of the career and wage differential compared to other sectors of the workforce, has further increased the economic challenges these islands face . These challenges have reportedly been associated with an alarming increase in the rates of drug use and reported suicides, especially among youths . It is well appreciated that developing countries tend to have worse cancer mortality due in part to the lack of healthcare access, limited infrastructure, and low availability of cancer screening . These disparities are apparent throughout Micronesia. For example, in Chuuk, water is primarily gathered from rain run-off, while cleaning facilities are rated as poor by members of the community . Some of the outer islands do not have consistent access to power, relying on kerosene. These are invaluable resources that are necessary to the infrastructure of a functioning healthcare system, let alone a specialized cancer center. Despite efforts to urbanize and improve infrastructure, most of the islands within the FSM do not have paved roads, making it difficult to travel to nearby community health centers . These structural factors put a significant strain on Pacific Islander patients and abrogate access to the most essential healthcare. Beyond the accessibility of these facilities, many of the island's clinics are limited in the scope of healthcare they can offer due to underequipped and aging facilities . Overall, the state of healthcare access throughout Micronesia is limited and impacts the overall health and wellbeing of the indigenous people who reside in these islands. 2.5. Educational Attainment among Micronesians In developed countries, such as the US, there is a well-known association between lower educational attainment and higher mortality . Since 1998, for patients with cancer, there has been an increasing gap in cancer-specific mortality between the least and most educated patients . Throughout Micronesia, educational and vocational attainment varies across islands. In Palau, 92% of the country is literate, and more than 75% of men and 82% of women have attended some college education . In comparison, a report in 2013 showed 43% of CNMI adults aged 25 and older have completed high school, while only 18% of FSM Micronesians attended college . Furthermore, most of the FSM have limited English proficiency. Barriers to educational attainment include a lack of effective policy framework, a developing educational and economic infrastructure, financial constraints, and the lack of a culturally appropriate "Western education" . Lower educational attainment is linked to worse health outcomes, limited economic mobility, as well as adverse risk behaviors (e.g., substance use, tobacco smoking) . This is not just limited to Micronesians living in Micronesia. In Hawaii, between 2020-2021, 59% of Micronesian students were unable to pass their high school mathematics classes . Given an individual's level of education is associated with health literacy and the ability to navigate the healthcare system and use of primary care services, improving educational equity among Micronesians may provide a steppingstone towards improved cancer outcomes and health equity in general . 2.6. Summary of Diet, Exercise, Economy, and Education in Micronesia In this section, we reviewed some of the major social determinants of health as they pertain to Micronesian patients in the setting of cancer. This section included diet and nutrition, exercise and obesity, economy and infrastructure, and educational attainment. Table 2 highlights critical areas of each of these factors along with possible solutions moving forward. 3. Exposure to Radiation and Substances 3.1. Radiation Exposure in Micronesia Historical nuclear weapons testing by the US government in the Marshall Islands from 1946-1958 has led to the deposition of radioactive materials in the air, water, and land . Many studies have demonstrated the associated increased risk of developing cancer with exposure to the radioiodine fallout following nuclear bomb testing in the Pacific, particularly cancers of thyroid origin . Although a mass evacuation was orchestrated by the US government, islanders from the CNMI were found to have radionuclides within their urine following atomic bomb testing in the Marshall Islands . Furthermore, despite the discontinuation of these tests several decades ago, radioactive particles remain in significant amounts and are still distributed by winds to populated areas . Some studies have suggested this phenomenon has contributed to the increased risk of acquiring thyroid and hematological cancers by 20% and 5%, respectively . The risk of radiation exposure varies by island, with those closest to the detonations or downwind of the detonations having more exposure than others . Residents of the Marshall Islands born in the 1950-1970s (the decades during or directly following radioactive testing) were also at increased risk given chronic exposure to greater concentrations of background radiation in-utero or as children/adults . There is also an individual risk of radiation exposure due to contaminated food and water. For example, some fruits on Rongelap remain contaminated with levels of radionuclide Cesium-137 that far exceed safe consumption standards . In addition to radiation exposure related to colonization and militarization, fish have been contaminated by polychlorinated biphenyl pollutants released by ships used for the nuclear test targeting . The impact of nuclear bomb testing is still an area of great research interest, due to the lack of research and data on these populations immediately following the nuclear bomb testing. Although radiation has declined over the years, there is still an inherent risk of radiation exposure associated with living within the Marshall Islands . For example, efforts to resettle the affected islands of Rongelap, Bikini atoll, and the various outer islands are currently unadvisable . The increased levels of background radiation may continue to pose future problems to the ecosystem and people living in adjacent areas . Years of trauma associated with radiation testing has led to generations of perceived radiation-related birth defects, cancers, and other chronic disease, which may explain in part why Pacific Islanders are particularly at risk of refusing potentially curative radiation therapy in the US . 3.2. Betel Nut and Substance Use Consumption of the carcinogenic areca palm seed, better known as the betel nut, is a growing epidemic directly associated with the development of aggressive oral cavity cancers across the region within Micronesia . The practice of areca nut chewing is also associated with an increased risk of metabolic disease, cardiovascular disease, and all-cause mortality . The areca nut can be consumed simply by itself or in conjunction with slaked lime, tobacco, and/or the betel leaf (this combination is referred to as betel-quid) . This sociocultural practice is commonly called "chewing betel nut." Over half of the adult population in Micronesia report betel nut chewing, compared to the world average of 20% . The 5-year areca nut chewing prevalence in Guam was 11% and increased among non-Chamorros, primarily other Micronesians, from 7% in 2011 to 13% in 2015. Nearly half of adult chewers in Guam preferred areca nut with betel leaf, slaked lime, and tobacco, while nearly half of youth chewers preferred areca nut only . The World Health Organization reports that betel nut chewing is an epidemic particularly among Micronesian youths who have the highest rates of betel nut chewing in the world among school-aged children . In Yap and Pohnpei, betel nut continues to be chewed by the majority of 7th and 8th graders, with 75% of Palauan high school students having tried betel nut at least once. The increased prevalence and adverse outcomes associated with oral cavity cancers and lesions can be attributed to high levels of betel nut consumption . In Yap, oral cavity cancer has an incidence of 22 per 100,000, which is two-fold greater than the US national average of 11 per 100,000 . In the CNMI, between 2005-2019, 79% of all head and neck cancers were oral cavity cancers, with those who used betel nut being diagnosed at younger ages (47 years versus 55 years) and with a decreased five-year survival rate of 50% . In addition to the betel nut, tobacco is regularly co-consumed, increasing the risk of oral cavity and other related cancers . Betel nut chewers co-consume tobacco at a rate of 90% and 80% in Pohnpei and Palau, respectively . Most adult smokers in Pohnpei started smoking before the age of eleven . Guam also has a high prevalence of current smokers at 25% . Adolescents are at risk for these behaviors and exposure to secondhand smoke. There are reportedly high rates of adolescents who observe regular smoking in public places or have parents who smoke . Likewise, although alcohol consumption varies between island populations, heavy drinking is another risk factor for cirrhosis, liver failure, and certain cancers . For example, on Nauru, the Cook Islands, and Chuuk, men who consume alcohol are more likely to be specifically "heavy drinkers" defined as consuming more than six drinks per day . This may be a possible explanation for the development of hepatocellular carcinoma seen in Micronesians . When compared to other US born Pacific Islanders, Micronesians born outside of the US are more likely to be diagnosed at an earlier age of hepatocellular carcinoma (52 years versus 60 years) and have more tumors (64% versus 32%), often associated with hepatitis B and liver cirrhosis . Overall, there are multiple highly prevalent addictive substances consumed within Micronesia that have direct implications for cancer incidence and clinical outcomes. 3.3. Summary of Micronesian Radiation Exposure and Substances In this section, we reviewed two types of exposures that have been associated with cancer specific disparities among the Micronesian population. We reviewed both radiation exposure related to nuclear weapons testing, betel nut chewing, and the consumption of other substances. Table 3 highlights critical areas of each of these factors along with possible solutions moving forward. 4. Cancer Care in Micronesia 4.1. Health Care Access and Cancer Surveillance, Diagnostics, and Treatment Compared to the integrated supply networks of continental landmasses that freely transfer goods and services from one region to another, the islands of Oceania are the most remote regions on the planet, which significantly limits their ability to transfer and access essential health care resources . For example, Pohnpei is 70 km (43 miles) from the closest populated island, with nothing but open ocean between . With over 600 islands across 18,000 miles in the FSM, just a few islands contribute to most of the country's population . Atolls are the most isolated landmasses, with often only a few families living on the entire island . Many of the islands rely heavily on outside support and foreign aid, but the considerable distance from major shipping ports, airports, and cities adds difficulty in collecting medical supplies. Not only are the physical equipment and supplies in shortage, but many islands throughout the Pacific face significant staffing shortages due to emigration of physicians who leave practices on remote Pacific Islands for opportunities in the continental US or elsewhere . These problems are more apparent in the outer islands of Micronesian nations, which depend on infrequent cargo shipments . Like many developing countries, the healthcare infrastructure of Micronesia is underdeveloped. Many of the major islands have clinics and healthcare services, but the quality, variety, and level of specialty care varies between islands . Furthermore, major hospitals are not always located in proximal islands, but rather are scattered throughout the Pacific. For example, the only major hospital in the entire US territory of the CNMI that provides primary care, dialysis services, disease screening tests, substance abuse treatment, mental health evaluations, and all public health services is located on the island of Saipan. In the RMI, the major hospitals are on the islands of Majuro and Ebenye, which serve twenty-four islands in the country. Not only are these hospitals scattered throughout the Pacific, but they are only accessible by boat or plane, which make emergency care for those living on atolls very difficult . Cancer surveillance in Micronesia under current healthcare infrastructure varies depending on proximity and access, both physical and economic, to major hospitals. Most USAPI nations and territories, with the exception of FSM, have access to CDC breast and cervical cancer early detection programs, prostate cancer screening, and colonoscopy capabilities. Specific tests, such as transrectal ultrasound, are not available in certain locations, including CNMI and Palau (Table 4) . In other cases, the island of Yap might have the equipment, such as a colonoscope, but at certain times of the year no specialist who is trained in performing the procedure, such as a board-certified gastroenterologist . Cancer diagnosis and treatment is limited by availability of equipment and personnel. Many islands have available general surgeons and obstetricians and gynecologists, but most had a shortage of or complete absence of oncologist or urologist (Table 5) . Guam was the only island with MRI and PET imaging capacity. Chemotherapy and radiation therapy was largely unavailable in most islands, except for Guam (Table 5) . All of these factors have the potential to delay cancer diagnosis and treatment, limiting the options for Pacific Islanders who are already at high risk for developing chronic diseases, such as cancer. Given specialty care is inaccessible across many USAPI, expensive off-island referrals and medical evacuations have become normalized . This poses a significant healthcare cost. For example, in ten years, Tuvalu in Polynesia spent 44% of its budget on outside medical referrals . The total cost of medical referrals is estimated to be $125 million as of 2017 . This is a major issue in healthcare delivery and equity, as the financial costs and geographical distance prevent many Pacific Islanders from receiving the care they need until it is too late . 4.2. Health Insurance Insurance status is known to be associated with access to medical care, specialty services, and severity of health outcomes . Many of the islands, including Chuuk and the Marshall Islands, have government plans for citizens to access regional health facilities within geographical proximity and even larger, more specialized Hawaii hospitals . However, up until recently, most COFA migrants were underinsured due to being ineligible for Medicaid (US-sponsored insurance program for low-income individuals and families) . Despite the Affordable Care Act of 2010 increasing health insurance coverage for most Americans in the 50 states, Micronesian migrants were one subgroup that were significantly disadvantaged by this policy and exempted from Hawaii coverage and access to specialty care, including cancer care . This resulted in more uninsured emergency medicine department visits in 2015, the year that COFA migrant coverage expired . Soon after from 2015-2018, mortality rates for COFA migrants concurrently increased by 21% and the overall healthcare utilization (net inpatient and emergency room visits) decreased . If care is not being utilized appropriately due to a lack of insurance, greater risks of developing later stage cancer and worse health outcomes are to be expected due to decreased screenings and delayed treatment . COFA healthcare coverage was reinstated in 2020, with the passage of the Consolidations Appropriations Act . Despite changes in policy, COFA migrants and other Pacific Islanders were disproportionately affected by the COVID-19 pandemic . Pacific Islanders in general, despite making up 4% of the population, made up 24% of the COVID cases in Hawaii , with people of Chuukese and Marshallese heritage comprising 24% and 22% of all Pacific Islander cases, respectively. Furthermore, mortality rates (319.6 per 100,000 people) far exceeded the mortality rates of other demographics . For those with specific cancers or undergoing systemic therapy, COVID-19 can be especially deadly due to the immunocompromised state of patients with cancer undergoing chemothearpy . This disparity highlights that the presence of health insurance, while necessary for good health outcomes, is not sufficient. More research measuring insurance coverage and barriers to obtaining healthcare among this underserved population is needed, although this poses a challenge since the US census does not directly disaggregate data on Micronesian ethnicity . 4.3. Perceptions of Cancer Care Cultural beliefs also contribute to barriers in accessing preventative cancer screening. In a study using the Health Information National Trends Survey data, 63% of all Pacific Islanders over 50 years of age in Hawaii had not underwent routine cancer screening as recommended by a physician . Further, Marshallese and Chuukese people in Hawaii are twice as likely to trust information from priests compared to information from doctors . In other cases, there is a strong desire to be screened and receive periodic check-ups, but there is a lack of access in general. In Yap, only 28% of all cervical cancers are caught at stage 1, significantly less than the 56% of invasive cancers diagnosed in the US . This may be due to low accessibility of urine and Pap screens within these islands, despite research showing that women in Yap express a preference to undergo recommended cancer screening . Results from a pilot community-based participatory randomized control study of women in the FSM indicate that 95% of women were comfortable with urine tests, and 82% of women were comfortable with a Pap smear for cancer screening . Overall, there is a discordance between people in Micronesia who want cancer screening and those who receive cancer screening. 4.4. Coalition Building in Micronesia A need for a comprehensive approach to cancer management in Micronesia has led to innovative solutions . In 1997, a regional, concerted approach was developed with a coalition of health leaders from USAPI to strengthen management of cancer care and prevention . Funding from the US National Cancer Institute and National Institutes of Health led to the formation of the Pacific Regional Comprehensive Cancer Control Program and partners (PRCP) in 2002. Since its inception, a number of partners were established that enabled the development of a comprehensive cancer control program aimed at improving key areas such as collaboration and sharing of resources between islands, cancer management in children, point-of-care testing and treatment, and surgical services . The PRCP regional central cancer registry was also formed, which allowed for systematic collection of cancer data in the region. Data collected since 2007 has already informed policy decisions and loco-regional health care practices . 4.5. Summary of Healthcare and Health Perceptions In this section, we reviewed themes within healthcare including access, insurance, and perceptions related to cancer within Micronesia. Table 6 highlights critical areas of each of these factors along with possible solutions moving forward. 5. Climate Change in the Pacific 5.1. The Impact of Climate Change on Cancer and General Health From the increase in environmental carcinogen exposures to disruptions to cancer care access, global warming and climate change directly impact the accessibility and quality of care for patients with cancer around the world . Pacific Islanders are among those disproportionately affected and are in direct significant danger from the impeding threat of climate change. Small, low-elevation islands in Micronesia are in jeopardy due to sea level rise, risk of tsunami, flooding, and other adverse weather events, which threaten the livelihoods of small communities that inhabit remote islands throughout the Pacific . On a global level, climate change has shown to negatively impact cancer treatments and clinical outcomes. In a study looking at over 1700 patients undergoing radiation therapy for non-operable non-small cell lung cancer during hurricane disasters, compared to propensity matched patients who underwent similar treatment without exposure to natural disasters, the group exposed to natural disasters had worse overall survival . These findings suggest the need for effective public health research and sustainable interventions that will monitor and shape the health of small island populations predicted to be at high risk for adverse health effects due to climate change . Compared to continental land masses, island topography tends to be close to sea level and thus low-lying islands, such as those in Kiribati and RMI, are susceptible to sea level rise, capable of rendering entire islands uninhabitable and impacting healthcare access . Food insecurity, malnutrition, fresh water accessibility, and displacement due to climate change and sea level rise are real threats that can have a disproportionate burden on patients from marginalized communities, including those with cancer in Micronesia . Severe weather events make it difficult to make medical appointments, face-to-face encounters, and routine cancer screenings. This was apparent in 2007, when an acute-onset sea level rise event occurred in two atoll islands of Micronesia, which resulted in extensive coastal erosion, shoreline inundation, and saltwater intrusion . The impact of the sea level rise went beyond the coastal shores, but also led to disastrous losses of crop productivity and limitations of freshwater resources. Of a total of 112 households surveyed, representing nearly 1000 residents in Micronesian islands, nearly all families had a partial loss, and one in three households reported complete loss of critical farmed produce, including taro or breadfruit . Even islands that have mountainous landscapes with high elevation are at risk for a rise in health problems over the coming decades, which will only increase the burden on an already challenged healthcare system . For example, climate change has been associated with a rise in infectious vector borne diseases, heat effects, and malnutrition, which impacts patients from Micronesia . The World Health Organization projects approximately 250,000 additional deaths per year by 2030 from malnutrition, diarrhea, and heat stress attributable to climate change . These more global health impacts can certainly impact the overall health trajectory of Micronesian patients with cancer. Thus, islands throughout Micronesia are vulnerable to multiple types of climate change and natural disasters capable of stripping islanders from already limited resources essential for health and wellbeing. Overall, there is a great unmet need to better understand how climate change may impact cancer care throughout Micronesia to build more climate resilient health systems. 5.2. Summary of the Impact of Climate Change in Micronesia In this section, we reviewed how climate change impacts health care and cancer care. We discussed how climate change, particularly with a focus on Micronesia, may impact cancer care. Table 7 highlights critical areas of each of these factors along with possible solutions moving forward. 6. Micronesia and Physician Workforce Representation 6.1. Review of Physician Workforce Diversity in Micronesia In general, there is a shortage of physicians in Micronesia, with many islands including FSM, the RMI, Kiribati, and Vanuatu have less than two physicians per 1000 people . This is due, in part, to multiple factors including the lack of medical training programs in Micronesia, requiring prospective physicians to go off-island for medical training. Many motivated Micronesians are unable to leave and face barriers to entry including prohibitive training costs, familial obligations, and a lack of social or familial support critical to success in medical training . Furthermore, there is a phenomena of "brain-drain," where highly-skilled physicians do not return to their home communities or are poorly retained, instead choosing to work in other areas, including New Zealand, Australia, and the US, resulting in a decreased density of doctors in certain areas . This data underscores the need for more pipeline programs throughout Micronesia, and Oceania more broadly, with the support of government funding to bolster the next generation of physicians with ties to these islands to promote high-quality, accessible, and longitudinal care for their communities . In the US, Pacific Islanders are among the least represented racial groups in medical schools and are significantly underrepresented throughout the physician pipeline . The number of Pacific Islander medical students, residents, and academic faculty in the US appear to be significantly declining in the past twenty years . Moreover, there is virtually no Pacific Islander representation within oncology in Micronesia, let alone throughout the US . Increasing the representation of Micronesian healthcare providers allows for cultural connection and understanding between Micronesian patients and their physicians, which may strengthen the physician-patient relationship and improve healthcare delivery . In the US, many Chuukese people have experienced a cultural disconnect with their physician, insensitive comments, or racial discrimination, which overall serve as barriers to health equity among Micronesians . Improving representation of Pacific Islanders in medicine should be prioritized to promote health equity for Pacific Islander patients through culturally competent care. 6.2. Summary of Micronesian Physician Workforce Representation In this section, we reviewed limitations to the Pacific Islander physician workforce representation in Micronesia and the importance of developing strong pipelines throughout Oceania to improve health equity for Micronesians. Table 8 highlights critical areas of each of these factors along with possible solutions moving forward. 7. Conclusions Micronesia is a vast network of islands with unique healthcare needs. To promote a healthy Micronesian community, a thorough understanding of the social, cultural, and economic determinants of health is important. Tremendous innovation and work have already been accomplished in building coalitions to promote positive public health outcomes. Further research and attention to Pacific Islanders in Micronesia, and Oceania more broadly, is warranted to reduce cancer disparities and promote health equity. Author Contributions Conceptualization, E.P. and K.T.; methodology, K.T. and N.V.D.; investigation, E.P., R.B., M.Y.G., N.V.D. and K.T.; resources, K.T.; data curation, E.P., R.B., M.Y.G., N.V.D. and K.T.; writing--original draft preparation, E.P. and R.B.; writing-- E.P., R.B., M.Y.G., N.V.D. and K.T.; supervision, K.T. and N.V.D.; All authors have read and agreed to the published version of the manuscript. Conflicts of Interest The authors declare no conflict of interest. Figure 1 The three ethnogeographic regions from which Pacific Islanders trace their ancestry throughout the continent of Oceania: Micronesia, Melanesia, and Polynesia. The shaded area in blue represents the region and collection of island nations within Micronesia. The collection of approximately 2000 islands throughout the Federated States of Micronesia, Guam, Kiribati, the Republic of Marshall Islands, Nauru, Northern Mariana Islands, and Palau. Map provided by CartoGIS Services, Scholastic Information Services, The Australian National University. Figure 2 Satellite images displaying the relative size of (a) the Micronesian region within the continent of Oceania, (b) the continent of Europe, and (c) the United States of America. The white bar approximates 3000 miles to scale for all images. Adapted Google maps satellite images used with permission. Imagery (c)2022 NASA, Terrametrics, Map Data (c)2022. Figure 3 Conceptual framework of the social determinants of health as it pertains to Micronesian cancer disparities. Some of the known social determinants of health include economic stability, physical environment, education, food, social context, and the healthcare system. cancers-15-01392-t001_Table 1 Table 1 Cancer incidence, 5-year survival, and % diagnosed late-stage based on the Pacific Regional Central Cancer Registry 2007-2018 data. Incidence rates are reported as incidence per 100,000 people, age-adjusted to the US standard population . Top Three Most Frequent Cancers in USAPI, 2007-2018 Guam Lung Breast Prostate Incidence 5-year Survival Diagnosed Late-Stage Incidence 5-year Survival Diagnosed Late-Stage Incidence 5-year Survival Diagnosed Late-Stage 52.6 30% 90% 82.1 91% 63% 84.6 88% 49% Commonwealth of the Northern Mariana Islands Breast Oropharynx Lung Incidence 5-year Survival Diagnosed Late-Stage Incidence 5-year Survival Diagnosed Late-Stage Incidence 5-year Survival Diagnosed Late-Stage 34.5 90% 73% 9.7 58% 76% 16.8 38% 86% Republic of Marshall Islands Cervical, Invasive Lung Breast Incidence 5-year Survival Diagnosed Late-Stage Incidence 5-year Survival Diagnosed Late-Stage Incidence 5-year Survival Diagnosed Late-Stage 65.8 57% 49% 31.4 8% 86% 23.3 72% 61% Republic of Palau Lung Liver Prostate Incidence 5-year Survival Diagnosed Late-Stage Incidence 5-year Survival Diagnosed Late-Stage Incidence 5-year Survival Diagnosed Late-Stage 30.3 10% 82% 23.8 7% 84% 49.9 68% 71% Federated States of Micronesia (FSM), combined Oropharynx Lung Cervical, Invasive Incidence 5-year Survival Diagnosed Late-Stage Incidence 5-year Survival Diagnosed Late-Stage Incidence 5-year Survival Diagnosed Late-Stage 15.8 49% 67% 19.9 6% 91% 22.7 46% 72% Chuuk, FSM Lung Liver Breast Incidence 5-year Survival Diagnosed Late-Stage Incidence 5-year Survival Diagnosed Late-Stage Incidence 5-year Survival Diagnosed Late-Stage 13.5 9% 91% 6.2 14% 100% 9.4 31% 94% Pohnpei, FSM Cervical, Invasive Oropharynx Breast Incidence 5-year Survival Diagnosed Late-Stage Incidence 5-year Survival Diagnosed Late-Stage Incidence 5-year Survival Diagnosed Late-Stage 40.7 54% 66% 25.2 47% 64% 40.6 52% 77% Yap, FSM Oropharynx Lung Liver Incidence 5-year Survival Diagnosed Late-Stage Incidence 5-year Survival Diagnosed Late-Stage Incidence 5-year Survival Diagnosed Late-Stage 53 50% 71% 32.2 4% 8% 20.5 0% 73% United States (cancer incidence per 100,000 people) Lung Liver Breast Oropharynx Cervical, Invasive Prostate 60.2 8.1 124.7 12.0 7.5 109.5 cancers-15-01392-t002_Table 2 Table 2 Risk factors and possible solutions for diet and nutrition, exercise and obesity, economy and infrastructure, and educational attainment related to Micronesians with respect to cancer. Key Concept Risk Factor Possible Solutions Diet and Nutrition Increased reliance on imported canned foods Traditional fibrous starches replaced with imported and refined grains Low fruit and vegetable diets Stigmatization of cultural farming Loss of traditional fishing and agricultural practices Create culturally informed interventions to promote sustainable local food production Government subsidized programs to promote and access traditional fishing and agricultural practices Exercise and Obesity High rates of child and adult obesity Sedentary lifestyle Lack of facilities and programs to promote routine exercise Provide incentives to engage in physical exercise programs Develop resources such as gyms and health centers Economy and Infrastructure Lack of economic diversity contributing to stunted economic growth Low household incomes Lack of transport infrastructure Aged and underfunded clinical facilities Diversify economy and workforce opportunities and stimulate economic growth Invest in national transportation system Modernize clinical facilities Educational Attainment Low educational attainment Limited English while living in the US Low health literacy Invest in and encourage educational equity for Micronesians through leadership cancers-15-01392-t003_Table 3 Table 3 Risk factors and possible solutions for radiation exposures and substances related to Micronesians with respect to cancer. Key Concept Risk Factor Possible Solution Radiation Exposure Exposure to radioactive air particles Radiation contaminated food supply Radiation-related trauma Expand publicly available research on radiation levels in Micronesia to inform public policy Create culturally competent interventions addressing radiation-exposure trauma Betel Nut and Substance Use Chewing betel nut with or without tobacco Adolescents chewing betel nut Smoking tobacco Adolescent smoking and secondhand smoke exposure Heavy alcohol consumption (>6 drinks/day) Develop clinical trials that focus on improving the cancer outcomes for patients with betel nut related oral cavity cancers Improve health literacy around betel nut and substance use in relation to cancer cancers-15-01392-t004_Table 4 Table 4 Summary table of cancer surveillance in the USAPI. Available resources are categorized by island. Data based on Pacific Regional Central Cancer Registry 2007-2018 report . Cancer Surveillance in USAPI CDC Breast and Cervical Early Detection Program Mammography Pap Smears Prostate Cancer Screening (PSA) CT On-Island Colonoscopy Transrectal Ultrasound Guam Available Available Available Available Available Available Available Commonwealth of the Northern Mariana Islands Available Available Available Available Available Available Unavailable Republic of Marshall Islands Available Available Available Available Unavailable Available Available Republic of Palau Available Available Available Available Available Available Unavailable Federated States of Micronesia (FSM) Chuuk, FSM Unavailable Unavailable Available Unavailable Unavailable Unavailable Unavailable Kosrae, FSM Unavailable Unavailable Available Unavailable Unavailable Unavailable Unavailable Pohnpei, FSM Unavailable Private Provider Available Available Private Provider Unavailable Unavailable Yap, FSM Unavailable Unavailable Available Available Unavailable Available Unavailable cancers-15-01392-t005_Table 5 Table 5 Summary table of available cancer diagnostic and treatment services in the USAPI. Data based on Pacific Regional Central Cancer Registry 2007-2018 report . Cancer Diagnosis and Treatment Pathologist Radiologist FNA MRI/ PET Scan Surgical Specialist Chemotherapy Radiation Therapy Off-Island Referral System Guam Available Available Available Available Gen. Surg., Urologist, OB-Gyn, Oncologist, Surg. Subspecialist Available Available Available Commonwealth of the Northern Mariana Islands Available Available Available Unavailable Gen. Surg., OB-Gyn, Oncologist, Surg. Subspecialist Available Unavailable Available Republic of Marshall Islands Unavailable Available Available Unavailable Gen. Surg., OB-Gyn, ENT Unavailable Unavailable Available Republic of Palau Unavailable Unavailable Available Unavailable Gen. Surg., OB-Gyn Unavailable Unavailable Available Federated States of Micronesia (FSM) Chuuk, FSM Unavailable Unavailable Unavailable Unavailable Gen. Surg., OB-Gyn, Surg. Subspecialist Unavailable Unavailable Available Korae, FSM Unavailable Unavailable Unavailable Unavailable Gen. Surg., OB-Gyn Unavailable Unavailable Available Pohnpei, FSM Unavailable Unavailable Available Unavailable Gen. Surg., OB-Gyn, Ortho Available Unavailable Available Yap, FSM Unavailable Unavailable Available Unavailable Gen. Surg., OB-Gyn Available Unavailable Available cancers-15-01392-t006_Table 6 Table 6 Risk factors and possible solutions for healthcare access, health insurance, and perceptions of cancer care related to Micronesians with respect to cancer. Abbreviation: COFA--Compact of Free Association. Key Concept Risk Factor Possible Solutions Healthcare Access Extremely remote, water-bound island locations with limited healthcare resources Inaccessible inter-island transportation for goods, patients, and healthcare workers Scarcity or complete lack of physician specialists Strengthen transportation access, particularly around healthcare systems Increase recruitment of and accessibility to physician specialists, particularly oncologists through telehealth technologies Health Insurance Underinsurance and lapses in insurance coverage with COFA Prior exemption of Micronesian migrants from Medicaid expansion Expand research on current Micronesian COFA migrant insurance coverage Perceptions of Cancer Care Low cancer screening rates Trust in physicians lower than spiritual leaders Lack of cancer screening access in populations that highly desire screening Improve trust in physicians with community engagement programs Increase access to cancer screening Strengthen the local physician workforce pipeline cancers-15-01392-t007_Table 7 Table 7 Risk factors and possible solutions for healthcare access, health insurance, and perceptions of cancer care related to Micronesians with respect to cancer. Key Concept Risk Factor Possible Solutions Climate Change Sea level rise impacting low-lying islands and atolls Wetland erosion with severe weather events threaten critical facilities Food source disruptions Potential increase in atmospheric carcinogen exposure Develop data systems to monitor changes in Micronesian climate correlated with health status Create effective health-protective interventions for high-risk islands Develop protocols for climate resilient healthcare systems within hospitals throughout Micronesia cancers-15-01392-t008_Table 8 Table 8 Risk factors and possible solutions for physician workforce diversity related to Micronesians with respect to cancer. Key Concept Risk Factor Possible Solutions Physician Workforce Diversity Underrepresentation of indigenous Pacific Islander physicians "Brain-drain" causing lapses in continuity of care Lack of specialty care, including oncology services, throughout Micronesia Improve Pacific Islander physician representation to maintain local talent with a vested interest in the care of Micronesian communities Create lasting pipeline programs for aspiring prospective physicians with strong Micronesian community ties Disclaimer/Publisher's Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

PMC10000178

Gradually, concern for the welfare of aquatic invertebrates produced on a commercial/industrial scale is crossing the boundaries of science and becoming a demand of other societal actors. The objective of this paper is to propose protocols for assessing the Penaeus vannamei welfare during the stages of reproduction, larval rearing, transport, and growing-out in earthen ponds and to discuss, based on a literature review, the processes and perspectives associated with the development and application of on-farm shrimp welfare protocols. Protocols were developed based on four of the five domains of animal welfare: nutrition, environment, health, and behaviour. The indicators related to the psychology domain were not considered a separate category, and the other proposed indicators indirectly assessed this domain. For each indicator, the corresponding reference values were defined based on literature and field experience, apart from the three possible scores related to animal experience on a continuum from positive (score 1) to very negative (score 3). It is very likely that non-invasive methods for measuring the farmed shrimp welfare, such as those proposed here, will become a standard tool for farms and laboratories and that it will become increasingly challenging to produce shrimp without considering their welfare throughout the production cycle. aquaculture grow-out pond larval rearing penaeid shrimp farming well-being FAI Farms LimitedFAI Farms Limited funded this work. pmc1. Introduction 1.1. Animal Welfare and Progress in Shrimp Farming Agricultural, aquacultural, and industrial production chains only develop (and grow) because they change. The shrimp farming production chain is considered one of the world's most controversial agri-food systems for animal protein production . The evolution of shrimp farming practices is currently taking place on several fronts. For example, there is a trend towards intensification of production systems for better utilisation of resources ; the pursuit of certifications and regulations to adapt shrimp production to the new demands of the market and society in general ; genetic improvement of animals ; improvement of shrimp feeding and nutrition ; and increased efforts towards hygienic-sanitary controls and biosecurity in shrimp farms , to name a few. This scenario of change, based on the scientific and technological development of the sector, in turn, helps to understand why the global production of a single species, the white-leg shrimp Penaeus vannamei, has increased by almost 53% in 5 years, from 3803.6 thousand tonnes to 5812.2 thousand tonnes between 2015 and 2020, so that this species already accounts for 51.7% of total shrimp production . It is estimated that more than 167 billion shrimps of this species are produced annually and that the total number of shrimps and prawns farmed annually reaches about 440 billion . However, it is human nature that despite all this progress, there is often a temptation to cling to traditional production methods and concepts that seemed to work satisfactorily in the past, even though they certainly no longer work in the same way or are no longer acceptable under present and future conditions. Perhaps this is a challenge to overcome regarding welfare in shrimp farming. The term "welfare" describes a potentially measurable quality of an animal's life at a particular point in time and is, therefore, a scientific concept . However, this concept is fundamentally dynamic and related to the adaptive abilities of the individual . In other words, the welfare of a particular animal is always a temporary state related to the animal's experiences and moves on a continuum from negative to positive . Thus, to improve animal welfare, it is necessary to ensure that the animal's experience is as positive as possible. Although society has recognised the importance of animal welfare in the commercial production of homeothermic vertebrates and even ectotherms , there is still a glaring lack of recognition of the welfare of higher invertebrates, as in the case of shrimps . The discussion on the need to apply the principles of animal welfare to shrimp farming includes two main aspects: those related to the current scientific evidence for sentience in these animals and the ethical/legal aspects associated with the issue . 1.2. Shrimp Sentience From the first perspective, the welfare of an organism is inseparable from the degree of suffering and the positive states that this individual experiences at a certain point in time . Sentience, thus, refers to the ability of an animal to consciously perceive what is happening to it and what surrounds it, consciously perceive through the senses, and consciously feel or subjectively experience . Crump et al. explained that sentience needs to be understood and addressed in a broad sense, including sensory experiences (visual, auditory, tactile, and olfactory) as well as feelings of warmth or cold, fatigue, hunger, thirst, boredom, excitement, fear, pain, pleasure, and joy. These authors also pointed out that this ability to feel must be distinguished from other related abilities, since a sentient being, for example, is not necessarily able to think about or understand the feelings of other animals. Finally, they argue that in answering questions about the ability of invertebrate animals to feel, we must rely (at least partly) on behavioural and cognitive markers associated with knowledge of their nervous systems. According to Birch et al. and Feinberg and Mallatt , invertebrates' brains differ even more radically from those of mammals and fish, the vertebrates most distant from humans. These authors explain that, although there are more than 500 million years of evolution between invertebrates and humans, it cannot be assumed that invertebrates lack sentience simply because their cerebral ganglia are organised differently from the brain of vertebrates . In the higher invertebrates (chordates, echinoderms, arthropods, and mollusks) there are a variety of complex interspecific neuronal structures that determine the degree of their sentience . According to Conte et al. , decapod crustaceans have sensory cells that enable the perception of noxious stimuli and show behavioural responses to these stimuli. However, the authors added that these responses could be nociceptive reflexes, that it cannot be ruled out that they occur without the involvement of sufficiently complex central processing thought to be necessary for the animals to feel pain, and that it has not yet been conclusively demonstrated that decapods possess sensory structures of sufficient complexity to feel pain. On the other hand, Langworthy et al. reported that some decapod crustaceans have cerebral ganglia as large and well-articulated as the brains of some fish. However, this does not seem to be the case with penaeid crustaceans. Crump et al. established eight criteria, four neural and four cognitive-behavioural, focusing on pain to assess the sentience of crustaceans. They found evidence that true crustaceans (Infraorder Brachyura) met five criteria, leading the authors to classify the result as solid evidence for sentience. The anomural crustaceans (suborder Anomura) and lobsters (suborder Astacidea) would meet three criteria, leading the authors to classify them as substantial evidence for sentience. In contrast, for other suborders, such as farmed shrimps (suborder Penaeidea), the evidence for sentience is less obvious, although the authors point to the glaring gaps in knowledge that still exist for these organisms. The lack of studies specifically aimed at assessing the sentience of decapod crustaceans should, therefore, not be confused with the absence of sentience in these organisms . As Birch suggested, "Where there are threats of serious, negative animal welfare outcomes, lack of full scientific certainty as to the sentience of the animals in question shall not be used as a reason for postponing cost-effective measures to prevent those outcomes". 1.3. Shrimp Welfare and Legislation Even if the question of the sentience of the Penaeidea has not yet been settled among researchers, it is a fact that it is no longer possible to treat shrimps (as well as any other animal) as simple "production machines"--they are not. Whether due to scientific findings or ethical reasons, several countries have already started to enact regulations for the species-appropriate and humane slaughter of crustaceans. In Europe, although the European Union only sets rules for the slaughter of vertebrates (and excludes reptiles and amphibians) , the EFSA (European Food Safety Authority) concluded that decapod crustaceans could feel pain and suffering . Countries such as Norway and Switzerland already include crustaceans in animal welfare legislation . France enacted a "service directive" more than 10 years ago to ensure the proper handling of crustaceans before sale and consumption . The Swiss government has established as a rule that only electrical stunning or "mechanical destruction" of the brain are recognised stunning methods, including for crustaceans, and requires lobsters and crayfish to be stunned before slaughter . The UK government amended its Animal Welfare (Sentience) Act 2022 to include decapod crustaceans , which are also protected by animal welfare legislation in New Zealand and Australia , a country that adopted the Australian Animal Welfare Strategy (AAWS) in 2004, but where the protection of crustaceans is limited to a few states. In Asia, where some of the world's largest shrimp producers are located, codes of conduct already include animal welfare as one of the pillars, along with food safety, animal health, and environmental integrity . There is, thus, a clear tendency for the world's largest shrimp import markets, particularly the North American and European markets, to require their suppliers to comply with norms and animal welfare standards during the production process of these organisms . Thus far, the most important economic power in the world, the United States, has taken an opposite position to that of the European Union in conflicts between trade and animal welfare, prioritising economic concerns . Nevertheless, due to scientific advances in recognising the sentience of decapod crustaceans or consumer demand--probably both--it will become increasingly challenging to produce shrimp without regard to animal welfare. This is due to consumer pressure, which affects all actors in the value chain, as well as issues such as sustainability certification, organic farming, and animal welfare, which have already been included in the social responsibility programmes of large companies . The challenge, then, is to define indicators that are more measurable and less subjective that encompass different aquaculture production systems and that focus on the welfare of farmed animals and not just the quality of the product because although these two factors are linked, they are not the same thing . However, since it is impossible to ask a shrimp directly how it is doing, it is assumed that the animal's welfare is directly related to satisfying its needs . That is how the animal relates biologically, behaviourally, and emotionally to its environment . Albalat et al. suggested using the five domains of animal welfare (i.e., nutrition, physical environment, health, behaviour, and psychological needs) as indicators of penaeid shrimp welfare, which in turn correspond to the five freedoms established by the Farm Animal Welfare Council . In this way, any measurements or observations made in a shrimp laboratory or on a shrimp farm that provide information about the extent to which the needs of the shrimp are being met can be considered potential indicators of their welfare. This paper aims to propose protocols consisting of the indicators, respective reference values and scores for assessing the welfare of P. vannamei in the phases of reproduction, larval rearing, transport, and growth in earthen ponds, and to discuss, based on a literature review, the processes and perspectives related to the development and application of shrimp welfare protocols. 2. Materials and Methods 2.1. The Welfare Domains and Indicators The indicators selected to assess the welfare of P. vannamei at the different stages of the production process (reproduction, larval rearing, transport, and grow-out) in earthen ponds were established following the same logic already used for farmed fish species such as Atlantic salmon , Nile tilapia , and grass carp . These indicators were grouped according to four of the five domains: (1) environmental, (2) sanitary, (3) nutritional, and (4) behavioural. The indicators related to psychological freedom were not considered a separate category, as the other proposed indicators assessed this freedom indirectly. 2.2. Identification and Selection of Documents for Bibliographic Review and Reference Levels The environmental, health, nutritional, and behavioural domains associated with P. vannamei and the indicators and their respective reference values during the reproductive, larval rearing, transport, and grow-out phases were identified based on a literature search using Google Scholar as the research platform. Books, technical and scientific articles, case studies, manuals, and handouts developed by international institutions, theses, and dissertations were sought. The search period covered 1976 to 2023. To determine the indicators and their respective reference values, texts and documents were evaluated with the following combination of previously defined terms in their title, abstract or keywords: vannamei AND shrimp AND "pond" -biofloc AND; the specific indicators are shown in Table 1. A database of preselected texts and documents was then set up. Subsequently, this material was analysed to evaluate the results and the methodology used by the authors to obtain them. The results were selected in situations likely to reflect biological and/or operational conditions in shrimp production. Based on the information available in the literature, three scores were assigned (1, 2, and 3). Score 1 can be interpreted as covering the ideal variation limits for the target species. Score 2 refers to variations within the limits that animals usually tolerate. Even if such variations cause or adversely affect the animals, these are sublethal. An exception to this criterion is the indicator of mortality, which is evaluated at an intensity that can be lethal as long as it is at a lower level than in nature and, at the same time, at an intermediate level when the rearing environment is taken into account. Score 3 refers to reference levels that affect the animals' physiological, health, and behavioural status to an unacceptable degree, so their welfare and survival are at risk. 2.3. Field Evaluation of the Preselected Indicators The applicability and operational feasibility, as well as the measurement technique of each of the preselected indicators, were assessed in the field. To this end, technical visits were conducted to monitor procedures and routine management in laboratories and farms producing marine shrimp in northeastern Brazil. More specifically, we visited one farm, and one larval rearing laboratory in the state of Rio Grande do Norte (municipality of Canguaretama), three farms (in the cities of Aracati and Jaguaruana), and two larval rearing laboratories (in Aracati and Parajuru), in the state of Ceara. The companies were selected after prior contacts and expressions of interest from people responsible for improving the welfare of farmed shrimp. The indicators that could not be collected on-site for technical or operational reasons were discarded. After this final check, the protocols were restructured in the format in which they are presented in Tables 2-14. 3. Results 3.1. Reproduction Stage The reproductive stage ranges from the selection of animals for the formation of breeding banks to their care (in tanks or earthen ponds), to mating and spawning. According to Ceballos-Vazques et al. the optimal reproductive potential of females of P. vannamei, based on the frequency of spawning and the quality of larvae, is reached at 12 months. As these authors point out, body weight appears to be of secondary importance at this age, although optimal breeding conditions are required for adequate growth and nutrition. This exact age is also recommended for males since the sperm quality of the animals is high from this period . Eleven environmental indicators were selected to assess the welfare of shrimp-farmed breeders. In addition to the parameters commonly used to determine water quality in a farm (temperature, pH, alkalinity, ammonia, nitrite, and salinity), photoperiod (when this variable is controlled), absence of predators (terrestrial or aquatic), and stocking density were considered (Table 2). The controlled presence of terrestrial predators means that the predators are physically present in the environment but do not have direct access to the shrimp. This is the case, for example, when protective fencing prevents birds from accessing ponds. In these cases, however, even indirect contact with the predator could be detrimental to the welfare of the shrimp, e.g., as a carrier of infectious diseases. For the aquatic predators indicator, we considered controlled presence of inter-specific inhabitants in case that the producer obtained knowledge of the existence of other species in the pond, for instance, in case of polyculture. Health indicators of P. vannamei breeding animals can be primarily measured by direct visual observation of the anatomical features of the animals. Luminescence, on the other hand, when observed in animals in dark environments, either in rearing facilities or in the laboratory, is indicative of the presence of bacteria of the genus Vibrio. Sexual maturity refers to the characteristics of animals that have already been selected for reproduction and spawning in the laboratory. Invasive procedures (especially removal of the eyestalk in females) are considered the most critical point in shrimp welfare at this stage of the production process and should be avoided. Mortality rates must be assessed cumulatively (from the beginning of this stage to the time of analysis or at the end of the reproductive process). Genetic selection means the application or non-application of properly standardised protocols used by the laboratory in the selection and husbandry of farmed animals (Table 3). Nutritional indicators of P. vannamei breeders include, in addition to a direct indicator (the filling of the digestive tract with food), some essential aspects of the feeding routine, such as the composition and type of feed offered, the proportion of crude protein in the breeders' artificial diet, the amount fed (as a percentage of shrimp biomass) and the feeding frequency (Table 4). Behavioural indicators refer to shrimp swimming, feeding behaviour during management, and outcomes related to the use of stunning methods when invasive procedures are performed on the animals (Table 5). 3.2. Larvae Rearing Phase According to Kitani , the eggs of P. vannamei are 0.26-0.29 mm in diameter. About 13 h after spawning, they hatch into a naupliar larva that feeds on its yolk and passes through six sub-stages (N1 to N6) before growing to about 0.46 mm in length, transforming into a zoea. At this stage, the larva feeds on phytoplankton and passes through three sub-stages (Z1 to Z3). When it reaches a size of about 2.1 mm in length, it transforms into a mysis larva. When shrimp in captivity reach this larval stage, they prefer to feed on rotifers, Artemia, or artificial food. The mysis measures up to 3.80 mm after passing through three sub-stages (M1 to M3) and swims upside down or holds its body in a vertical position with its head down. In the next stage, postlarva (PL1), the shrimp, about 4.2 mm long, holds its body horizontally and becomes a benthic organism. This stage continues until the animal completes its morphological development and becomes a juvenile. In a laboratory, larval rearing extends from nauplii to postlarvae when the animals are ready for marketing. The environmental Indicators adopted for the larval rearing stage here are essentially those already used for livestock rearing, except for the presence of predators, which are unlikely to be present in the ponds used for larval rearing. The reference values are adjusted accordingly for this life stage of the shrimp (Table 6). The health indicators for the larvae and postlarvae of P. vannamei are pretty specific and concern issues related to the laboratory itself (whether or not health certificates are issued for the animals sold), the conditions in the larval rearing tanks (presence of bioluminescence in the larvae or even in the water); questions directly related to the health status of the larvae at the time of assessment (uniformity of larval stages present in the tank, presence of poorly formed larvae, presence of epibionts, muscle necrosis or melanisation of the exoskeleton, presence of lipid droplets and staining of the hepatopancreas of the postlarvae); and finally, the observed cumulative mortality rate concerning the batch analysed (Table 7). Nutritional indicators of larval and postlarval welfare are analysed directly about shrimp feed intake (analysis of gastrointestinal tract filling) and also to diet (size of feed at each larval stage, composition of feed, and percentage of crude protein in artificial feed), as well as to the diet chosen and handling (analysis of gastrointestinal tract filling, frequency of artificial feed intake, and composition of feed), as shown in Table 8. The behavioural indicators of larvae and postlarvae were limited to the positive phototaxic behaviour of nauplii and zoeae and the swimming activity of the animals (Table 9). 3.3. Postlarvae Transport Although it is known that in some cases, transport of nauplii takes place (sold between laboratories doing reproduction and larval culture and others doing larval culture only), only transport of postlarvae (which takes place between laboratories and farms) was considered here. We have proposed 12 indicators for assessing the welfare of P. vannamei postlarvae, to be evaluated when the postlarvae arrive at the farm, including six environmental, two health, two nutritional, and two behavioural indicators (Table 10). 3.4. Grow-Out Stage The growth phase begins with the transfer of postlarvae to the nurseries (in the case of biphasic rearing) or the grow-out facilities (in the case of monophasic rearing) and ends with the selection of animals for breeding banks or slaughter for marketing and consumption. The indicators proposed here and their respective reference values have been established based on rearing in earthen ponds and do not necessarily apply to other rearing systems such as bioflocs or raceways. The environmental indicators are practically the same as those already described for the breeders, except for the photoperiod, because since the cultures are carried out in earthen ponds, the photoperiod is always the natural one. However, water transparency was included here, an indirect indicator of the number of planktonic organisms in the water (Table 11). It can be observed that the reference values for parameters such as temperature, pH, dissolved oxygen, salinity, and stocking density also differ between juveniles and breeders. The welfare degree of the young can be assessed by anatomical indicators that can be analysed without invasive methods (in Table 12, it is suggested that producers/caretakers can identify such parameters during the biometric survey or harvest). Mortality rates can be assessed through daily monitoring, removal, and counting of dead shrimp from the pond, assessment of feed intake, and accurate quantitative surveys after harvest. The nutritional indicators used to assess the welfare of the juvenile are essentially indirect, except for the visual assessment of the filling of the digestive tract (Table 13). The aim is to ensure adequate feeding conditions and, thus, good nutrition for the animals. Therefore, the reference values and corresponding scores for some of the indicators (size of feed, amount of initial feed, frequency of feeding in the ponds, percentage of crude protein in the feed, and apparent feed conversion ratio) were established based on four size classes in the production process: for shrimp below 0.9 g, for juveniles from 1 to 3.9 g, and in two size classes where they can already be marketed for consumption, from 4 to 8.9 g and from 9 to 15 g. The other three indicators are independent of the size of the animals. Two scenarios were considered for the distribution of the feed: the distribution of the feed over the pond surface and the use of feeding trays. The behavioural indicators (Table 14) are specific to the different stages of the production process in a shrimp farm. During routine management, it is often possible to observe the swimming behaviour of shrimp, especially at the surface and edges of the ponds or near the water inlet and outlet structures. During harvesting, it is possible to assess the behaviour of the animals depending on the harvesting method used (fixed nets, at the monk's exit, or trawls). The more restless the animals become, the more they jump (escape behaviour) and become stressed. The clinical reflexes detected during stunning are technical analyses that must be performed in detail because they are essential to assess the effectiveness of the slaughter, which is one of the most critical points for the welfare of the animals in the final grow-out stage. 4. Discussion 4.1. Indicators for Shrimp Welfare Sustainability of production, the environment, and animal welfare are critical issues in global food production and part of meaningful international sustainability discussions and policies, such as the United Nations Sustainable Development Goals . In this context, the welfare of farmed shrimp cannot be neglected, considering how representative they are of aquaculture and how many billions of animals are produced and marketed worldwide each year . The (few) studies on this subject still focus mainly on the reproduction and rearing/growth stages. As far as breeders are concerned, the principal reported risks are related to the removal of the eyestalk of females , which has several undesirable consequences, including physiological and biochemical imbalance, reproductive exhaustion, physical trauma, stress, increased energy requirements, weight loss, and increased animal mortality . The main risks to shrimp welfare during the grow-out phase are related to the occurrence of diseases, especially those of viral and bacterial origin and to the processes that eventually lead to the outbreak of these diseases, such as the problematic environmental factors related to loss of water quality or other stressful conditions to which shrimp are exposed during rearing . Another risk to shrimp welfare that has merited the attention of researchers relates to inadequate slaughter conditions, mainly due to the absence or inadequacy of prior stunning, since immersion of animals in ice, a widely used method in shrimp slaughter worldwide, is not a stunning procedure, unlike electric shock . In most publications, however, the term "shrimp welfare" now appears only sporadically and not infrequently in overly general contexts, generally referring to the most diverse aspects of the production process. However, for shrimp welfare to be put into practise by aquaculture producers and entrepreneurs or even included in international certification standards for companies and product marketing, concepts, and indicators that can effectively measure welfare need to be defined. Indicators of animal welfare can describe the extent to which nutritional, environmental, health, behavioural, and psychological needs are met or not met, and thus, provide information on the state of the quality of life of farm animals . Preferably, these indicators should be relevant, valid, standardised, easy to use, reliable, comparable, and appropriate for specific systems or routines, and it is also desirable that they are simple and inexpensive . We have attempted to incorporate such requirements into the indicators proposed here, following a method already proposed by our group in defining indicators of grass carp welfare . In both cases, we used protocols that were always scored with a maximum of three scores (1, 2, 3) for each indicator, and we tried to define the most significant possible number of non-invasive indicators of welfare. The scores range from a positive state (score 1) to a negative one (score 3). This makes the assessment less subjective and variable compared to welfare protocols that use a larger number of scores. We also advocate for indicators of P. vannamei welfare that can be effectively and accurately measured and scored in the field. Indeed, some of these indicators are already integrated into routine data collection and monitoring of the production process by laboratories and shrimp farms, mainly those historically applicable through good aquaculture practises (GAP) (see examples in ). We have tested and confirmed the practical feasibility of the proposed indicators in Brazilian shrimp farms and commercial laboratories and concluded that the protocols presented here do not affect the operations of these enterprises, although they may require operational adjustments. This does not mean animal welfare protocols must be mandated in aquaculture. However, if they are difficult to apply or require high investment, they are unlikely to be adopted voluntarily by the production sector, which, as Barreto et al. warned, would limit their relevance to a purely experimental context, which is not our aim. In this way, we would like to reinforce the concept that it is not incompatible in principle to adopt farming practises that promote the welfare of farmed shrimp while bringing technical, economic, and commercial benefits to aquaculture producers and entrepreneurs. On the other hand, we do not include physiological biomarkers that require invasive interventions among our indicators. Since there are no uniform definitions for the terms 'invasive' and 'non-invasive' when applied to animal-assisted methods , we assume here that "invasive" involves those procedures in which the integument of an animal is injured . That is, we do not propose here methods in which haemolymph is collected, as in the analysis of haematological , physiological , or biochemical biomarkers of shrimp. However, this does not mean that the animals cannot be manipulated at some point to obtain relevant information. Direct observations and measurements are always essential and provide information about the welfare of the animals, as they are not themselves the cause of excessive impairment of the welfare of these animals. In the case of shrimp, invasive interventions are likely to affect their welfare seriously, so we tried to minimise them in our protocols. We believe there is always a "maximum acceptable price" to be paid for scientific knowledge. As Valente questioned, is it ethically and morally correct to subject potentially sentient animals to painful or distressing tests to obtain information about their welfare? In our opinion, no. 4.2. The Establishment of Farmed Shrimp Welfare Protocols There have been several challenges in establishing indicators that reflect the welfare of P. vannamei. The first is related to the considerable gap that exists due to the lack of information on many aspects of the biology of this species, as pointed out by Saraiva et al. . Such a gap does not mean that assumptions about animal welfare are invalid, but without it, the debate about stress or shrimp welfare runs the risk of not getting beyond the emotional or subjective level . In other words, robust animal welfare protocols cannot be established without consistent scientific data. We have identified and used the most up-to-date and reliable information available in the technical and scientific literature to meet this challenge. However, we hope, recommend, and wish that the indicators proposed here, as well as the respective reference values for each of the three associated scores, will be regularly analysed and revised in the light of the knowledge that can be gained on the individual topics. The second challenge is related to the life stages of animals. We are trying to establish protocols that apply to all stages of the production cycle of farmed P. vannamei and, thus, to all stages of its life cycle, including the larval stages. However, if the understanding of the sentience of juvenile and adult shrimps is still full of doubts and gaps, what about their sentience during the larval stages? This question is not as evident as it may seem. It is already known that the shrimp's nervous system begins to structure itself immediately after hatching. Already at the beginning of the naupliar stage (N1), the neuropil is formed; the anterior part of the nervous system of P. vannamei begins to take structural shape in N3 when the ganglia in the anterior part of the head coalesce; and the complete structure of the central nervous system, including proto-, deutero-, and tritocerebrum, can be seen in N6 . Without going into the core of the debate as to whether the larval stages of P. vannamei are sentient or not--which is not our aim here--we have, therefore, decided to follow the precautionary principle proposed by Birc : "Where there is a threat of serious negative consequences for animal welfare, the absence of complete scientific certainty about the sentience of the animals concerned should not be used as a reason for postponing cost-effective measures to avoid these consequences". Furthermore, we believe that if we can propose methods and breeding procedures that better meet the biological needs of shrimp even at this life stage, and if we can help to ensure that producers benefit after the larval stage by including them in the GAP, why not? The third challenge is to recognize that the degree of tolerance or the desired level of a particular factor/indicator depends not only on the animal's life stage but also on the production system used and the environmental conditions in which they are kept . In this article, due to space constraints, it is neither possible nor necessary to discuss individually all the indicators proposed here for the welfare of P. vannamei at the different breeding stages, as both the indicators and the reference values are intended to be self-explanatory. However, it should be emphasised that the welfare indicators almost always interact directly and indirectly. In the following, we present two classic cases that illustrate this: the indicators "salinity" and "stocking density". Penaeus vannamei is known as one of the euryhaline shrimps . Their reproduction takes place in oceanic waters (with high salinity), while postlarvae are settled in coastal areas (estuaries and lagoons with variable salinity) , where they arrive after developing their physiological abilities to overcome the fluctuating nature of these environments . In these coastal regions, animals grow throughout the juvenile stage before returning to marine areas, where they reach sexual maturity and reproduce . It is known that the mechanisms of euryhalinity and osmoregulation change during the ontogenetic development of P. vannamei. The hyper-hypo osmoregulatory pattern exhibited by juveniles and adults appears to be established at the beginning of the first postlarval stage (PL1), with greater tolerance to salinity fluctuations observed in PL2, PL4, and PL22, suggesting that P. vannamei exhibits a progressive increase in the efficiency of the osmoregulatory mechanism after the last larval stage . However, it is known that salinity is not only an indicator that directly affects growth, survival , and, thus, the welfare of the species, but that in a husbandry system, it also directly affects changes in other parameters that determine water quality and thus shrimp welfare, such as solubility of oxygen . At the same time, salinity can also influence the oxygen consumption of the shrimp themselves and even their tolerance to low dissolved oxygen concentrations . The stocking density in the rearing phase is admittedly a critical decision in farmed shrimp production . The density, and thus, the sustainable biomass to be achieved, is closely related to the carrying capacity of the chosen system and varies considerably between different farming systems . In extreme cases, shrimp may arrive at a farm and initially be kept in nursery tanks with densities exceeding 1500 postlarvae/m2 and then exceed 400 shrimp/m2 during the final stages if the chosen husbandry system is Biofloc , or exceed 150 shrimp/m2 in earthen ponds . From an animal welfare perspective, it is recommended that the maximum stocking density in ponds during the rearing phase does not exceed 50 shrimp/m2 . Higher stocking densities tend to increase the stress to which shrimp are subjected, which from a technical point of view can compromise the results to be achieved by the operator (e.g., by reducing and compromising growth and survival rates, feed consumption, and final biomass achieved) and from the animals' point of view can severely damage their immune system affecting their welfare . The expression of a particular disease--another critical issue for the welfare of farmed shrimp--is almost always the result of a complex interplay of factors related not only to stocking density but also to intrinsic aspects of the cultivated species, the pathogen in particular, and the environment . This requires that disease control programmes take a multifactorial approach and that any innovation introduced should be seen as just one more element in this complex process . The ecological and biological variables are so dynamic and interactive that we believe it is impossible to think of standardised management techniques or control environmental variables in ponds and hatcheries for shrimp farming. On the other hand, it is perfectly possible, for example, to establish acceptable ranges of variation in environmental parameters; to ensure the supply of natural food to the animals; to offer probiotics as part of the shrimp diet; to stimulate the growth of beneficial microbial communities; to carry out regular tests on the health status of the farmed animals; to promote their stunning before slaughter. Our protocols aim to help measure the impact of all these practises on the welfare of farmed P. vannamei. 5. Conclusions The welfare of farmed shrimp has only recently attracted the interest of researchers, producers, policy makers, and other stakeholders in this valuable production chain . As we noted in our literature review, this paper is the first attempt to propose indicators that encompass all stages of the production process of a shrimp species and, in this case, an essential shrimp species for global aquaculture. However, it is foreseeable that technologies for monitoring the welfare of farmed shrimp and those specifically targeting GAP will increasingly converge from now on. This development will typically take place through so-called "precision aquaculture" , which will include technologies such as the use of biosensors, data loggers, and early warning systems ; computer vision for animal monitoring, sensor networks (wireless and long range), robotics, and decision support tools, such as algorithms, the Internet of Things, and decision support systems ; as well as the use of high-quality and sensitive cameras for automatic detection and analysis of shrimp behaviour even in turbid waters . Such technologies will, in turn, provide shrimp farmers with important information to manage feed supply with minimum waste and maximum feed efficiency; assess organic waste accumulation in ponds to optimise the water quality available to shrimp; improve management decisions related to animal health; and increasingly use animal behavioural signals as indicators of their welfare and the efficiency of management practices applied. Likely, these non-invasive methods of measuring shrimp welfare will soon become routine in farms and laboratories, and it will become increasingly challenging to produce shrimp without taking into account the welfare of the organisms that are farmed, slaughtered, and offered to consumers, whether due to scientific advances in decapod sensitivity, consumer market demand, or changes in international regulations governing shrimp production and marketing--or all of these factors combined. The best evidence that this is entirely plausible is that remote water quality monitoring technologies are already a reality in several places worldwide . Further evidence is that large companies are already incorporating issues such as sustainability certification, organic farming, and animal welfare into their social responsibility programmes . Although the possible technological revolution has the potential to facilitate the assessment of welfare indicators, it will not render the use of these indicators superfluous. On the contrary, as is already the case with remote water quality monitoring, there is a tendency to integrate it with this and other technological tools. However, until this happens, any shrimp farmer can already measure the welfare of P. vannamei during the different stages and breeding processes. Acknowledgments We thank all the aquaculturists involved for their trust and permission to carry out this work and the CNPq (Conselho Nacional de Desenvolvimento Cientifico e Tecnologico) for awarding a productivity and research grant to Antonio Ostrensky (Process 304451/2021-5). Author Contributions Conceptualization, A.S.P. and A.O.; methodology, A.S.P., C.P.d.S.T. and A.O.; validation, N.C. and U.d.A.T.d.S.; investigation, A.S.P., C.P.d.S.T., N.C. and A.O.; resources, A.S.P., C.P.d.S.T. and M.H.Q.; data curation, N.C. and A.O.; writing--original draft, A.S.P., N.C. and A.O.; writing--review and editing, A.S.P. and A.O.; supervision, M.H.Q. and A.O.; project administration, M.H.Q.; formal analysis A.S.P. and A.O. All authors have read and agreed to the published version of the manuscript. Institutional Review Board Statement The study was developed in accordance with the Directives of the Ethics Committee on Animal Use of the Agrarian Sciences Sector of the Federal University of Parana (CEUA-UFPR), Brazil. Informed Consent Statement Not applicable. Data Availability Statement Not applicable. Conflicts of Interest M.H.Q. was employed by FAI Farms. The remaining authors declare that the research was conducted without any commercial or financial relationships that could be construed as a potential conflict of interest. Figure 1 Domains and welfare indicators during the production process of white-leg shrimp, Penaeus vannamei. animals-13-00807-t001_Table 1 Table 1 Specific indicators used to obtain bibliographic data to define the reference values for the four domains of marine shrimp welfare studied. Domain Specific Indicators Environmental water quality; temperature; pH; transparency; dissolved oxygen; ammonia; nitrite; salinity; alkalinity; predators; competitors; photoperiod; stocking density Health health; diseases; eyes; exoskeleton; gills; hepatopancreas; antennae; rostrum; mortality Nutritional protein; feeding frequency; feed amount; food distribution Behavioural behaviour; respiratory frequency; swimming; feed intake; anaesthesia; handling; slaughter animals-13-00807-t002_Table 2 Table 2 Environmental indicators for Penaeus vannamei breeders. Indicators Score Reference Values References Temperature (degC) 1 24.5-32.5 2 15.6-24.4 or 32.6-35.4 3 <=15.5 or >=35.5 pH 1 7.5-8.5 2 5.0-7.4 or 8.6-9.0 3 <=4.9 or >=9.1 Photoperiod (Light: Dark) 1 Natural or 12L:12D-14L:10D 2 15L:9D-16L:8D 3 17L:7D or clearer; 11L:13D or darker Alkalinity (mg/L CaCO3) 1 100-140 2 51-99 or 141-199 3 <=50 or >=200 Dissolved oxygen (% saturation) 1 >=62 2 46-61 3 <=45 Non-ionised ammonia (mg/L NH3) 1 0.00-0.10 2 0.11-0.30 3 >=0.31 Nitrite (mg/L NO2-) 1 0-0.6 2 0.7-1.6 3 >=1.7 Salinity (psu) 1 28-35 2 10-27 or 36-40 3 <=9 or >=41 Stocking density (g/m2) 1 <=150 2 151-299 3 >=300 Terrestrial predators * 1 Absence 2 Controlled presence 3 Uncontrolled presence Aquatic predators and interspecific inhabitants ** 1 Absence 2 Controlled presence 3 Uncontrolled presence * Birds, mammals, and reptiles. ** Fish, other crustaceans (crabs and shrimps), molluscs (snails), amphibians (frogs), and reptiles (water snakes). animals-13-00807-t003_Table 3 Table 3 Health indicators for Penaeus vannamei breeders. Indicators Score Description or Reference Values References Antennae 1 Healthy appearance, no changes 2 Focal lesion, shortening or darkening 3 Absence, blueness, wrinkling, multifocal dark spots Rostrum 1 Healthy appearance, no changes 2 Mild injury, erosion or necrosis 3 Severe injury, erosion or necrosis, deformity, bending to one side, upwards or downwards Eyes 1 Healthy appearance, no changes 2 Unilateral lightening, injury, softening or swelling 3 Bilateral lightening, injury, softening or swelling, absence of one or both organs Gills 1 Healthy appearance, no changes 2 Focal lesion or darkening 3 Pale, yellowish, general redness, darkening, whitish spots, or erosion Hepatopancreas 1 Healthy appearance, no changes 2 Discrete volume reduction 3 Atrophy, stiffness, flaccidity, colour change (abnormal pallor or darkening, appearance of streaks of different shades), volume change, tearing, presence of worm-like structures, presence of fluid (oedema) Motor appendages (pereiopods/uropods/pleopods) 1 Healthy appearance, no changes 2 Focal absence or erosion 3 Severe or complete absence, colour change, necrosis (lightening), dark spots, rough and darkened edges Exoskeleton (cuticle) 1 Healthy appearance, no changes 2 Slight lesion or focal darkening, presence of debris 3 Tissue loss, necrosis, focal or generalised colour change, deformity, calcified round white patches, crusting Musculature 1 Healthy appearance, no changes 2 Focal necrosis (lightening) 3 Generalised necrosis, colour change (yellowing, redness, opacity, milky appearance), atrophy, swelling, stiffness (constant bending), zigzag shape Gastrointestinal tract 1 Healthy appearance, no changes 2 Presence of sand particles or debris 3 Atrophy, empty, pale, whitish bowel, whitish stools, faecal streaks Luminescence 1 No luminescence is observed in breeders in complete darkness 2 Not applicable 3 Luminescence is observed in breeders in absolute darkness Sexual maturation 1 Females: Enlarged ovaries, distinct olive-green colour (stage IV). Males: Shiny sperm ampullae 2 Not applicable 3 Females: Small, translucent ovaries (stage I); opaque and yellowish (stage II); bright yellow (stage III); spawned (stage V). Males: opaque or brownish (necrotic or melanised) sperm ampullae Invasive procedures (ablation of the eyestalk, extrusion of the spermatophore) * 1 No invasive intervention 2 Invasive procedure with effective anaesthesia and postoperative analgesia 3 Invasive procedure without effective anaesthesia and postoperative analgesia Mortality during breeding and spawning (%) ** 1 <=10 2 11-25 3 >=26 Genetic selection 1 Genetic selection and or improvement programme with inbreeding control 2 Genetic selection and or improvement programme without inbreeding control 3 No genetic improvement programme * There is not yet sufficient scientific evidence that the signs known as surgical staging signs cause adequate analgesia and unconsciousness in shrimp. ** More research is needed in order to understand how to decrease larval mortality in captive shrimp. animals-13-00807-t004_Table 4 Table 4 Nutritional indicators for Penaeus vannamei breeders. Indicators Score Reference Values References Filling of the digestive tract * 1 Full 2 Medium 3 Empty Frequency of feeding (times/day) 1 >=3 2 2 3 <=1 Composition/type of diet 1 Special artificial feed for breeders + fresh feed 2 Special artificial feed for breeders only 3 Fresh feed only Amount of food (% of biomass **) 1 >=4.0 2 2.4-3.9 3 <=2.3 Crude protein in the breeders' artificial diet (%) 1 >=35 2 29-34 3 <=28 * Observation after a minimum of 30 min and a maximum of 2 h after offering the food. ** Fresh + artificial feed. animals-13-00807-t005_Table 5 Table 5 Behavioural indicators for Penaeus vannamei breeders. Management Indicators Score Description References Routine management Swimming behaviour 1 Animals at the bottom that stop, walk or feed, or normally swim in the water column 2 Few animals with irregular swimming or with partial loss of balance at the tank bottom 3 Many animals with irregular swimming (off balance or in a spiral), crowding of shrimp in certain parts of the tank Feeding behaviour Reaction to offered food 1 Most animals react quickly by moving to the newly offered food Authors' suggestion 2 Only some of the animals react to the newly offered food 3 Most animals do not react to the newly offered food Invasive methods Anaesthesia--surgical phase * (loss of balance and response to external stimuli) 1 Induction and recovery in 3-5 min 2 Induction and or recovery in 6-9 min 3 Absence of anaesthetic procedures Induction and or recovery <= 2 min or >=10 min; death * There is not yet sufficient scientific evidence that the signs known as surgical staging signs cause adequate analgesia and unconsciousness in shrimp. animals-13-00807-t006_Table 6 Table 6 Environmental indicators for larvae and postlarvae of Penaeus vannamei. Indicators Score Reference Values References Temperature (degC) 1 26.5-32.4 2 19.5-26.4 or 32.5-35.4 3 <=19.4 or >=35.5 pH 1 7.5-8.5 2 5.0-7.4 or 8.6-9.0 3 <=4.9 or >=9.1 Photoperiod (Light:Dark) 1 18L:6D-24L:0D 2 17L:7D-12L:12D 3 11L:13D or darker Alkalinity (mg/L as CaC O3) 1 100-140 2 51-99 or 141-199 3 <=50 or >=200 Dissolved oxygen (% saturation) 1 >=64 2 49-63 3 <=48 Non-ionised ammonia (mg/L NH3) 1 0.00-0.01 2 0.02-0.06 3 >=0.07 Nitrite (mg/L NO2-) 1 0.0-0.1 2 0.2-0.7 3 >=0.8 Salinity (psu) 1 30-36 2 20-29 or 37-44 3 <=19 or >=45 Stocking density (Larvae or postlarvae/L) 1 <=250 (larvae) <=100 (postlarvae) 2 251-300 (larvae) 101-210 (postlarvae) 3 >=301 (larvae) >=211 (postlarvae) animals-13-00807-t007_Table 7 Table 7 Health indicators for larvae and postlarvae of Penaeus vannamei. Indicators Scores Reference Values or Description References Health certificate 1 SPF 1 + SPT 2 and or SPR 3 2 SPF 1 3 Does not have a health certificate Luminescence 1 No luminescence is observed in the larvae tank under absolute darkness 2 Not applicable 3 luminescence is observed in the larvae tank under absolute darkness Uniformity of larval stages in a tank * 1 >=75% of the sampled larvae are in the same larval stage 2 50-74% of the sampled larvae are in the same larval stage 3 <=49% of the sampled larvae are in the same larval stage Malformations (%) 1 Low (<=5% of the sampled larvae have deformities) 2 Moderate (6-10% of the sampled larvae have deformities) 3 Severe (>=11% of the sampled larvae have deformities) Staining of the hepatopancreas * 1 >=90% of larvae have darkened hepatopancreas 2 70-89% of larvae have darkened hepatopancreas 3 <=69% of larvae have darkened hepatopancreas Condition of the hepatopancreas * 1 >=90% of sampled larvae have abundant lipid vacuoles 2 70-89% of sampled larvae have abundant lipid vacuoles 3 <=69% of the sampled larvae have abundant lipid vacuoles Epibiont encrustation on the exoskeleton, appendages and gills * 1 <=5% of sampled larvae have fouling 2 6-10% of sampled larvae have encrustations 3 >=11% of sampled larvae suffer from fouling Necrosis * (%) 1 Absence of necrosis in larvae 2 <=15% of larvae show opacities of the muscles or limbs 3 >=16% of larvae show necrosis in muscles or limbs Melanisation * (%) 1 >=90% of larvae show up to 5% melanisation 2 70-89% of larvae show up to 5% melanisation 3 <=69% of larvae show up to 5% melanisation Mortality at the end of larval rearing ** 1 <=30 2 31-49 3 >=50 1 SPF: Specific pathogen-free postlarvae. Postlarvae must be negative for YHV, IHHNV, WSSV, TSV, IMNV, and NHP according to PCR analyses. 2 SPT: Specific pathogen tolerant postlarvae. 3 SPR: Specific pathogen-resistant postlarvae. * These analyses require a magnifying glass or microscope and should only be carried out if this is already part of the laboratory's quality control. ** More research is needed in order to understand how to decrease larval mortality in captive shrimp. animals-13-00807-t008_Table 8 Table 8 Nutritional indicators for larvae and postlarvae of Penaeus vannamei. Indicators Score Reference Values References 0.002-0.08 g 0.081-1.0 g 1.1-2.5 g Size of the food (mm) 1 Fed mash-700 701-1800 1801 mm -3.2 mm 2 >=701 <=701 <=1801 3 Not applicable >=1801 >=3.3 Crude protein in the artificial feed (%) 1 >=25 >=25 >=40 2 20-24 20-24 25-40 3 <=19 <=19 <=24 Analysis of PL gastrointestinal tract (% of sampled animals with complete tract) 1 Does not apply Does not apply >=90 2 70-89 3 <=69 Frequency of feeding--artificial feed (times/day) 1 >=6 2 3-5 3 <=2 Composition of the diet (type of food) 1 Specific artificial food for each larval stage + live food 2 Only specific artificial food for each larval stage 3 Only live food animals-13-00807-t009_Table 9 Table 9 Behavioural indicators for larvae and postlarvae of Penaeus vannamei. Indicators Score Description or Reference Values References Phototaxis of nauplii and zoea 1 >=95% of sampled larvae are attracted to the light from the water surface 2 70-94% of sampled larvae are attracted to the light from the water surface 3 <=69% of sampled larvae are attracted to the light from the water surface Swimming behaviour of larvae and postlarvae 1 >=95% of the sampled larvae are active 2 70-94% of the sampled larvae are active 3 <=69% of the sampled larvae are active animals-13-00807-t010_Table 10 Table 10 Indicator for the transport of postlarvae of Penaeus vannamei. Category Indicators Score Description or Reference Values References Environmental Temperature (degC) 1 22.5-25.4 2 19.5-22.4 or 25.5-29.5 3 <=19.4 or >=29.6 pH 1 6.5-8.0 2 5.0-6.4 or 8.1-8.5 3 <=4.9 or >=8.6 Salinity (psu) 1 Acclimatised to the salinity of the farm 2 Need to acclimatise for up to 5 ups 3 Need to acclimatise for more than 5 ups Dissolved oxygen (% saturation) 1 >=80 2 60-79 3 <=59 Transport density (postlarvae/L) * 1 <=750 2 751-1000 3 >=1001 Non-ionised ammonia (mg/L NH3) 1 0.00-0.01 2 0.02-0.06 3 >=0.07 Health Lesions, necrosis and/or malformations (rostrum, appendages and or gills) (%) 1 <=5 2 6-10 3 >=11 Mortality (%) 1 <=5 2 6-10 3 >=11 Nutritional Feeding (time interval) ** 1 <=3 h during transport 2 >=4 h during transport 3 No feeding during transport Type of food added 1 Artemia nauplii + industrial feed 2 Only industrial feed or artemia nauplii 3 No additional feed during transport Behavioural Swimming behaviour after transport (no anaesthesia) *** 1 >=95% of active postlarvae and normal swimming ability 2 70-94% of active postlarvae and normal swimming ability 3 <=69% of active postlarvae and normal swimming ability Swimming behaviour after transport (with anaesthesia) 1 Sedation (loss of balance with reaction to external stimuli) 2 Anaesthesia (complete loss of equilibrium with no response to external stimuli) 3 No induction or recovery, death * Stocking density applies to PL10 to PL20 in shipping bags or transport crates. ** Offering food during transport is done when using transport boxes. When transporting plastic bags, there is no possibility of offering food after the bags have been sealed. *** Abnormal swimming includes jumping at the surface, swimming at an angle, swimming in figure eights, swimming at the surface, and no swimming. animals-13-00807-t011_Table 11 Table 11 Environmental indicators for juvenile and adult Penaeus vannamei. It is recommended that postlarvae are previously acclimatised in ponds for 10 to 15 min for every degree of temperature difference and salinity unit (81; 149) and at least 60 min per different 0.5 pH unit (81; 88). Indicators Score Reference Values References Temperature (degC) 1 25.5-32.4 2 14.5-25.4 or 32.5-35.4 3 <=14.4 or >=35.5 pH 1 6.5-8.5 2 5.0-6.4 or 8.6-9.0 3 <=4.9 or >=9.1 Transparency (cm) 1 35-50 2 21-34 or 51-59 3 <=20 or >=60 Alkalinity (mg/L CaCO3) 1 100-140 2 51-99 or 141-199 3 <=50 or >=200 Non-ionised ammonia (mg/L de NH3) 1 0.00-0.10 2 0.11-0.30 3 >=0.31 Dissolved oxygen (% saturation) 1 >=65 2 49-64 3 <=48 Nitrite (mg/L NO2) 1 0.0-0.6 2 0.7-1.6 3 >=1.7 Salinity (psu) 1 10.0-40.9 2 0.6-9.9 or 41.0-59.0 3 <=0.5 or >=60.0 Stocking density (shrimp/m2) 1 <=40 2 41-60 3 >=61 Terrestrial predators * 1 Absence 2 Controlled presence 3 Uncontrolled presence Aquatic predators and interspecific inhabitants ** 1 Absence 2 Controlled presence 3 Uncontrolled presence * Birds, mammals, and reptiles. ** Fish, other crustaceans, molluscs, amphibians, and reptiles. animals-13-00807-t012_Table 12 Table 12 Health indicators for Penaeus vannamei juveniles. Indicators Score Description or Reference Values References Antennae 1 Healthy appearance, no changes 2 A focal lesion, shortening, or darkening 3 Absence, blueness, wrinkling, multifocal dark spots Rostrum 1 Healthy appearance, no changes 2 Mild injury, erosion, or necrosis 3 Severe injury, erosion or necrosis, deformity, bending to one side, upwards or downwards Eyes 1 Healthy appearance, no changes 2 Unilateral lightening, injury, softening or swelling 3 Bilateral lightening, injury, softening or swelling, absence of one or both organs Gills 1 Healthy appearance, no changes 2 Focal lesion or darkening 3 Pale, yellowish, general redness or darkening, whitish spots, erosion Hepatopancreas 1 Healthy appearance, no changes 2 Discrete volume reduction 3 Atrophy, stiffness, flaccidity, colour change (abnormal pallor or darkening, appearance of streaks of different shades), volume change, tearing, presence of worm-like structures, presence of fluid (oedema) Motor appendages (pereiopods/uropods /pleopods/telson) 1 Healthy appearance, no changes 2 Focal absence or erosions 3 Severe or complete absence, colour change, necrosis (lightening), dark spots, rough and darkened edges Exoskeleton (cuticle) 1 Healthy appearance, no changes 2 Slight lesion or focal darkening, presence of debris 3 Tissue loss, necrosis, focal or generalised colour change, deformity, calcified round white spots, encrustation Musculature 1 Healthy appearance, no changes 2 Focal necrosis (lightening) 3 Generalised necrosis, colour change (yellowing, redness, opacity, milky appearance), atrophy, swelling, stiffness (constant bending), zigzag shape Mortality (%) * 1 <=10 2 11-25 3 >=26 * More research is needed in order to understand how to decrease larval mortality in captive shrimp. animals-13-00807-t013_Table 13 Table 13 Nutritional indicators for Penaeus vannamei juveniles and adults. Indicators Score Weight (g) References <=0.9 1.0-3.9 4.0-8.9 9.0-15.0 Size of food (mm) 1 0.1-0.5 0.6-1.0 1.1-2.0 2.1-3.0 2 >=0.6 >=1.1 >=2.1 >=3.1 3 <0.1 <=0.5 <=1.0 <=2.0 Amount of initial food (% of biomass) 1 6.0-10.9 4.0-6.9 4.0-6.9 2.0-3.9 2 4.1-5.9 2.1-3.9 2.1-3.9 1.1-1.9 3 <=4.0 or >=11.0 <=2.0 or >=7.0 <=2.0 or >= 7.0 <=1.0 or >=4.0 Frequency of feeding in the ponds (times/day) 1 >=4 >=2 >=2 >=2 2 2-3 1 1 1 3 <=1 <1 <1 <1 Feed crude protein (%) 1 >=35 >=35 >=32 >=32 2 32-34 32-34 25-31 25-31 3 <=31 <=31 <=24 <=24 Apparent feed conversion rate (FCR) * 1 Does not apply Does not apply <=1.5 <=1.7 2 1.6-2.0 1.8-2.0 3 >=2.1 >=2.1 Feed distribution Distribution of feed over the surface (% of pond surface) OR 1 >=75 2 50-74 3 <=49 or no control of coverage area Use of trays (number of trays/ha) 1 >=20 2 15-19 3 <=14 Digestive tract filling index ** 1 Full 2 Medium 3 Empty * Calculated at the end of culture for shrimps with an average of 15 grammes. FCR = 100 x [ln (FW - IW)/T]: FW: final weight; IW: Initial weight; T: Time interval (days). ** Observation after a minimum of 30 min and a maximum of 2 h after offering the feed. animals-13-00807-t014_Table 14 Table 14 Behavioural indicators for Penaeus vannamei juveniles and adults. Management Indicators Score Reference Values References Routine management Swimming behaviour 1 No shrimp on the pond surface or irregular swimming 2 Few animals on the pond surface or irregular swimming 3 Reduced, irregular or "spiral" swimming, accumulation of shrimp at the edges of the pond or near the water inlet, many animals exposing their bodies at the water surface Partial or complete harvesting Escape behaviour (successive tail movements by flexion and extension of the abdomen) 1 Few jumping shrimps during harvest, with low frequency and intensity 2 Few jumping shrimps, but with high frequency and/or intensity during harvesting 3 Many jumping shrimps, high frequency and/or intensity during harvesting Stunning at slaughter * Clinical reflexes 1 Immediate loss of response to external stimuli; balance (with cephalothorax in horizontal and descending position); movement of pleopods and pereiopods; and movement of scaphognathites 2 Progressive loss of response to external stimuli; balance (with cephalothorax in horizontal and descending position); movement of pleopods and pereiopods; and movement of scaphognathites in <=30 s 3 Progressive loss of: Response to external stimuli; balance (with cephalothorax in horizontal and descending position); movement of pleopods and pereiopods; and movement of scaphognathites in >30 s * The use of ice or simple exposure to air is not considered a stunning method. Therefore, the treatment is given a score of 3. Disclaimer/Publisher's Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

PMC10000179

The study aimed to compare the dose distribution in permanent low-dose-rate brachytherapy (LDR-BT) and high-dose-rate brachytherapy (HDR-BT), specifically focusing on the impact of a spacer and prostate volume. The relative dose distribution of 102 LDR-BT patients (prescription dose 145 Gy) at different intervals was compared with the dose distribution of 105 HDR-BT patients (232 HDR-BT fractions with prescription doses of 9 Gy, n = 151, or 11.5 Gy, n = 81). A hydrogel spacer (10 mL) was only injected before HDR-BT. For the analysis of dose coverage outside the prostate, a 5 mm margin was added to the prostate volume (PV+). Prostate V100 and D90 of HDR-BT and LDR-BT at different intervals were comparable. HDR-BT was characterized by a considerably more homogenous dose distribution and lower doses to the urethra. The minimum dose in 90% of PV+ was higher for larger prostates. As a consequence of the hydrogel spacer in HDR-BT patients, the intraoperative dose at the rectum was considerably lower, especially in smaller prostates. However, prostate volume dose coverage was not improved. The dosimetric results well explain clinical differences between these techniques reported in the literature review, specifically comparable tumor control, higher acute urinary toxicity rates in LDR-BT in comparison to HDR-BT, decreased rectal toxicity after spacer placement, and improved tumor control after HDR-BT in larger prostate volumes. prostate cancer interstitial brachytherapy 125I 192Ir dosimetry spacer This research received no external funding. pmc1. Introduction Brachytherapy (BT) is associated with a high conformity--a high dose to the tumor region and a steep dose drop-off, with consequential optimum protection of organs at risk (OAR) . BT for prostate cancer was evaluated in many studies, confirming an excellent local control and acceptable side effects . It can be applied as a permanent interstitial BT (LDR, low dose rate) and a temporary interstitial BT (HDR, high dose rate) and can also be applied alternatively in many centers . BT is a standard treatment method for prostate cancer in national and international guidelines , with a considerably reduced integral dose in comparison to external beam radiotherapy (EBRT). Several studies compared the dosimetry between EBRT and BT . Our study included a large group of patients treated in clinical routine in the last years. A hydrogel spacer was only injected before HDR brachytherapy, as this treatment was usually applied as a boost to EBRT, thus resulting in a higher radiobiological dose and a larger risk for rectal toxicity. The aim was to analyze and compare the dose distribution in (dose to the macroscopic tumor) and outside (dose to potential microscopic spread beyond the capsule) the prostate between LDR brachytherapy at different intervals (intraoperative, day 1, and day 30 dose distribution) and HDR brachytherapy. The impact of a hydrogel spacer and different prostate volumes was evaluated to specify the additional advantage in respect of the dose to the organs at risk (predominantly dose to the rectum) and the dose to the prostate (possibly improved coverage with a larger distance to an organ at risk). The dose distribution outside the prostate, comparison at different LDR brachytherapy planning and post-planning intervals, evaluation of the advantage of applying a hydrogel spacer, and consideration of different prostate volumes have not been considered in previously published literature and thus improve our knowledge in understanding differences between different brachytherapy techniques. A literature review was performed to explain the clinical consequences that could result from the dosimetric findings in this study. 2. Materials and Methods The intraoperative dose distribution of 102 LDR-BT (125-I sources with a source activity of 17.8 MBq [0.48 mCi]) patients was compared with the dose distribution of 105 HDR-BT (192-Ir source) patients (232 HDR-BT fractions). The injection of 10 mL hydrogel spacer (SpaceOAR(r) System, Boston Scientific, Marlborough, MA, USA) was performed under transrectal ultrasound (TRUS) guidance after dissecting the space between the prostate and rectum with a saline/lidocaine solution under local anesthesia, as explained in detail in prior publications . A hydrogel spacer (10 mL) was only applied before HDR-BT, as HDR-BT was commonly applied as a boost to EBRT (resulting in a higher radiobiological dose), whereas LDR-BT was always performed as a monotherapy treatment in a single intervention. The target volume for all patients was the prostate volume and the base of seminal vesicles without additional margins (CTV, clinical target volume = PTV, planning target volume). The extent of seminal vesicles included was based on individual patient factors, in particular the localization of the tumor in magnetic resonance imaging (MRI) and the localization of positive probes. The prescription dose was 145 Gy for all LDR-BT patients, using 62 +- 10 (mean +- standard deviation) stranded sources and 21 +- 3 application needles. All patients have been treated with brachytherapy as monotherapy. The prescription dose was 9 Gy (151 fractions, treatment as a boost to EBRT, 1.8-2 Gy single fractions up to 50-50.4 Gy total dose) or 11.5 Gy (81 fractions, treatment as monotherapy in three fractions) for HDR-BT patients, using 14 +- 2 application needles. Treatment planning for LDR-BT was performed using VariSeed 9.0, and treatment planning for HDR-BT was performed using Vitesse 3.0 (Varian, Palo Alto, CA, USA). The comparison was based on dose levels relative to the respective prescription dose. For the analysis of the dose coverage outside the prostate, a 5 mm margin was added to prostate volume (PV+) as a surrogate of potential microscopic tumor extension and thus potential impact on tumor control. Prostate V100 and V150 denote the PV covered by 100% and 150% of the prescription dose, while prostate D90 and urethra D30 denote the maximum dose in 90% of the PV and 30% of the urethral volume and rectum D2 cm3 and rectum D0.1 cm3 the maximum dose to 2 cm3 and 0.1 cm3 of the rectal wall. Urethral volume corresponds to the volume of the transurethral catheter inside the prostate, and the rectum volume corresponds to the anterior rectal wall that could be defined in the transrectal ultrasound. For the comparison of different doses, all dose levels are reported as the percentage of the prescription dose, corresponding to the 100% dose level. Prostate D90 was required to receive at least 100% of the dose in treatment planning . Dose homogeneity indices were calculated to characterize and compare dose homogeneity: Dose Homogeneity Index DHI = (V100 - V150)/V100 (ratio of the target volume which receives a dose in the range of 1.0 to 1.5 times of the reference dose to the volume of the target that receives a dose greater than the reference dose); Overdose Volume Index ODI = V200/V100 (ratio of the target volume that receives a dose equal to or greater than 2.0 times of the reference dose to the volume of the target that receives a dose equal to or greater than the reference dose); Dose Nonuniformity Ratio DNR = V150/V100 (ratio of the target volume that receives a dose equal to or greater than 1.5 times of the reference dose to the volume of the target that receives a dose equal to or greater than the reference dose) . Patients after LDR-BT received a post-planning CT on day 1 (with a urethral catheter) and day 30 after implantation. Urethral contour on day 30 was based on estimation. Statistical analysis was performed using IBM SPSS Statistics 28.0 software (IBM, Armonk, NY, USA). An unpaired t-test was applied to compare volumes and dose levels of different techniques, and a paired t-test to compare volumes and dose levels in LDR-BT patients at different intervals. A chi-square test was applied to compare categorical variables. All p-values reported are two-sided; p < 0.05 is considered significant. 3. Results As demonstrated in Table 1, patients treated with LDR-BT as a monotherapy are low-risk or favorable intermediate-risk patients who tended to be younger than patients treated with HDR-BT. Many HDR-BT patients are patients with considerable risk factors and more aggressive tumors. The comparison of volume and dose values is shown in Table 2. HDR-BT patients had a slightly larger PV but considerably larger PV+. The only not significantly different value comparing both methods was prostate V100, whereas prostate D90 was slightly higher in LDR-BT (mean difference of 4%). LDR-BT was characterized by a considerably more inhomogeneous dose distribution, characterized by a prostate V150 twice as high compared to HDR-BT. Accordingly, the Dose Homogeneity Index is less than half in LDR-BT compared to HDR-BT. The Overdose Volume Index and Dose Nonuniformity Ratio are about twice as high in LDR-BT compared to HDR-BT. As a consequence of higher doses within the prostate, considerably higher doses to the urethra resulted (mean difference of 32%). The injection of a hydrogel spacer between the prostate and anterior rectal wall before HDR-BT resulted in considerably lower doses to the rectal wall. A mean rectum 2 cm3 reduction of about 30% and a mean reduction of the maximum dose (rectum 0.1 cm3) of about 70% could be achieved in comparison to LDR-BT patients. Comparing day 1 and day 30 post-planning CT, a significantly lower prostate V150 (55 +- 9% on day 1 and 61 +- 10% on day 30) and prostate D90 (98 +- 8% on day 1 and 102 +- 10% on day 30) resulted on day 1, respectively (p < 0.01 for all comparisons). Urethra D30 was also significantly lower on day 1 (114 +- 18% on day 1 and 125 +- 21% on day 30; p < 0.01). However, the dose to the rectum increased considerably (rectum 2 cm3 of 77 +- 18% on day 1 and 88 +- 21% on day 30; p < 0.01). Thus, though differences between LDR-BT and HDR-BT were still highly significant, differences respecting dose inhomogeneity and dose to the urethra decreased in post-planning CT evaluations. However, differences respecting the dose to the rectal wall increased. In Table 3, the impact of prostate volume on dose distribution is demonstrated (LDR-BT: 52 patients < 41 cm3; 50 patients >= 41 cm3; HDR-BT: 115 fractions < 41 cm3; 117 fractions >= 41 cm3). The dose to the prostate was found to be higher in smaller prostates for LDR-BT but not for HDR-BT. PV + 5 mm D90 was identical for smaller prostates comparing LDR-BT vs. HDR-BT. A difference resulted only in larger prostate volumes, with a considerably higher PV + 5 mm D90 in HDR-BT. PV + 5 mm D90 in HDR-BT was higher for larger prostates in comparison to smaller prostates. However, dose inhomogeneity and dose to the urethra were similar in smaller and larger prostates for both BT techniques; rectal doses tended to be higher in larger prostates (differences between LDR-BT and HDR-BT dose distribution independent of prostate volume). Dose indices were independent of the prostate volume. 4. Discussion 4.1. Dose Distribution An adequate radiotherapy dose is needed to achieve adequate local tumor control for prostate cancer . Brachytherapy, either as monotherapy or a boost to external beam radiotherapy, is a method of dose escalation for prostate cancer. Several studies, including randomized controlled trials, reported superior local tumor control and biochemical control compared to EBRT alone . Higher rectal toxicity rates in combined modality treatments are the rationale for using a hydrogel spacer in this patient group. In a systemic review including 14 clinical trials with 3534 patients who have been treated with HDR-BT as monotherapy, Viani et al. reported excellent results and effectiveness not only for local control after 5 years but also comparable toxicity rates to EBRT. The mean 5-year biochemical recurrence-free survival (bRFS) for low, intermediate, and high-risk patients were 98%, 94%, and 91%, respectively . In the present study, treatment planning was based on the same average relative prostate V100 (92%). The crucial difference between the techniques was a considerably higher prostate V150 in LDR-BT in comparison to HDR-BT, consequently a lower dose homogeneity and a higher overdose volume, also leading to a considerably higher dose to the urethra. The dose to the rectal wall can be substantially reduced after a hydrogel spacer injection. Technical or anatomical differences might result in dosimetric differences. Though different mean needle numbers and activities have been used in prior comparisons of HDR-BT vs. LDR-BT dose distributions , the results of our study are well supported. A former study compared the dose distribution in ultrasound-based planning for prostate cancer brachytherapy as monotherapy with single fraction HDR-BT (19 Gy prescription doses, 192-Ir, and a mean needle number of 18--in contrast to 14 needles in this study) in comparison to LDR-BT (145 Gy, I-125, a median seed activity of 0.56 mCi, and a mean source number of 47--in contrast to 0.48 mCi and 62 in this study). As in our study, relative prostate V150 in LDR-BT was about twice as high as prostate V150 in HDR-BT and urethra D30 was found >10% higher . Mean prostate V150 in our LDR-BT patients corresponded to the objective in a recently published consensus statement (<=65%) . In the publication by Solanki et al., the mean prostate V150 was found to be only about 50% higher in LDR-BT (mean 40%) in comparison to HDR-BT (mean 27%). However, the mean prostate V100 in LDR-BT was only 82% vs. 96% in HDR-BT, thus considerably lower in comparison to our study . Anatomical differences respecting prostate volume have been analyzed in our study in a large patient number. Studies comparing the dose to potential microscopic tumor spread and including postimplant dosimetry have not been published before. A hydrogel spacer has not been used in prior comparisons, and increasing the difference in the dose to the rectum between LDR-BT and HDR-BT has been found to be higher in LDR-BT even without a spacer . Nevertheless, a hydrogel spacer (HDR-BT) did not result in an improved PTV coverage that could be assumed with a larger distance between the prostate and anterior rectal wall, as reported in a recently published study using stereotactic body radiotherapy . A similar prostate coverage can be explained by the planning process within our center that is focusing on an adequate, but not increased, minimum dose to the prostate. However, a higher dose resulted in larger prostates with a margin of 5 mm, which is comparable to the PTV definition in EBRT that includes the prostate with a margin. In contrast to EBRT , potential mitigation of prostate or seminal vesicle motion during treatment in patients with a spacer is less relevant in brachytherapy, as needles and sources are implanted during continuous prostate visualization in TRUS. In spite of the dose distribution differences reported in our study, additional technical and radiobiological differences need to be considered. In contrast to HDR-BT, the dose in LDR-BT is delivered over a period of many weeks. With increasing and decreasing prostate edema, sources are slightly displaced and dose analyses on day 1 and day 30 differ from intraoperative dose planning . HDR-BT dose is delivered in a short period and is thus more consistent. However, fractionation concepts often differ between centers. A higher prostate V150 in LDR-BT might be associated with improved tumor control or increased urinary toxicity. Though the dose distribution is important to guide our treatments, the most important results for our clinical practice and guidelines are long-term clinical results. 4.2. Association of Dosimetric Results with Clinical Results Several clinical studies analyzing differences between LDR-BT vs. HDR-BT have been published. Burchardt et al. evaluated differences in a PSA bounce between LDR-BT and HDR-BT and found a similar rate (20-30%) but significantly earlier and shorter PSA bounces after HDR-BT in comparison to LDR-BT . Grills et al. evaluated the potential for differing toxicities between LDR-BT and HDR-BT (38 Gy delivered in four fractions) after a median follow up of 35 months . HDR-BT was associated with decreased rates of acute urinary frequency, urgency, and dysuria compared with LDR-BT. Chronic urinary frequency, urgency, and Grade 2 rectal toxicities were also decreased with HDR. The treatment methods maintained the same biochemical control. A phase 2 randomized pilot study compared PSA outcomes, quality of life, and late toxicity between LDR-BT and HDR-BT (single 19 Gy fraction). Quality of life, including IPSS (International Prostate Symptom Score) and EPIC 26 (Expanded Prostate Cancer Index Composite) urinary irritative scores were significantly more favorable for patients treated with HDR-BT over the first 36 months . This can well be the consequence of a lower relative dose to the urethra, as reported in this study. However, a significantly larger proportion of patients after LDR-BT reached PSA < 0.04 ng/mL. This can indicate a larger radio-biologically effective dose, possibly also a result of a considerably larger prostate V150. In a study comparing LDR-BT vs. HDR-BT boost for external beam radiotherapy, acute urinary toxicity (patient-reported using IPSS and EPIC scores, but also physician-assessed Grade 2 or greater genitourinary toxicity) has been found to be significantly lower (again resulting from a lower dose to the urethra) after HDR-BT boost, though the effect size diminished over time . We have found a significantly higher dose around the prostate (PV + 5 mm D90) in larger prostates--a dose that might cover microscopic tumors around the prostate. In a publication by Le et al. , the biochemical control 6 years after HDR-BT was found to be significantly higher for patients with prostate volumes >= 60 cm3 in comparison to volumes < 60 cm3 (91% vs. 78%; p < 0.01), though dose prescription to the prostate was found to be comparable. The findings in our analysis could explain these clinical results. Experience with the hydrogel spacer to reduce the dose to the rectal wall and thus reduce toxicity has also increased considerably in recent years, in particular for EBRT. A systemic review showed not only a significant reduction in the dose to the rectal wall--as demonstrated for our patients in this study--but also reduced the late gastrointestinal and genitourinary toxicity rates . Long-term studies showed significantly improved bowel quality of life for patients treated with a hydrogel spacer . Studies evaluating the advantage of a hydrogel spacer for prostate cancer BT in combination with EBRT or BT as monotherapy have also been published recently . A spacer can be advised especially for patients with a higher risk of rectal toxicity . 5. Conclusions In comparison to HDR-BT, LDR-BT was characterized by a more inhomogeneous dose inside the prostate, a higher dose to the urethra, and a lower dose around the prostate in larger prostate volumes. The dose to the rectal wall could be considerably reduced by applying a hydrogel spacer. However, the prostate volume dose coverage, as determined by prostate V100 and prostate D90, has not been improved with a larger distance between the prostate and anterior rectal wall. These results explain the clinical differences that have been found in other studies: comparable tumor control (with comparable prostate V100 and dose drop-off around the prostate), higher acute urinary toxicity rates (dose to the urethra), and lower PSA nadir (larger V150 volume) in LDR-BT; higher tumor control in larger prostates after HDR-BT (higher dose around the prostate); and lower rectal toxicity resulting from the application of a hydrogel spacer (considerably lower rectal dose). Author Contributions Conceptualization, H.H. (Hathal Haddad) and M.P.; methodology, H.H. (Hathal Haddad) and M.P.; validation, H.H. (Hathal Haddad) and M.P.; formal analysis, H.H. (Hathal Haddad) and M.P.; investigation, H.H. (Hathal Haddad), H.H. (Horst Hermani), H.H. (Herbert Hanitzsch), A.H. and M.P.; resources, M.P.; data curation, H.H. (Hathal Haddad); writing--original draft preparation, H.H. (Hathal Haddad) and M.P.; writing--review and editing, H.H. (Horst Hermani), H.H. (Herbert Hanitzsch), A.H. and M.P.; resources, M.P.; supervision, M.P.; project administration, M.P. All authors have read and agreed to the published version of the manuscript. Institutional Review Board Statement The study was conducted in accordance with the Declaration of Helsinki. Ethical review and approval were waived for this study due to local law (retrospective anonymous plan evaluation). Informed Consent Statement Informed consent was obtained from all subjects involved in the study for the treatment. Data Availability Statement The data presented in this study are available on request from the corresponding author. The data are not publicly available, as patient consent has not been given for the publication of individual details. Conflicts of Interest The authors declare no conflict of interest. Figure 1 LDR-BT plan and HDR-BT plan with prostate, urethra, rectum contours, 50%-, 100%- and 150%-isodoses (example). cancers-15-01396-t001_Table 1 Table 1 Patient characteristics. LDR-BT (n = 102) HDR-BT (n = 105) p-Value age/years 69 +- 9 72 +- 8 0.06 PSA/ng/ml <10 91% 61% <0.01 10-20 9% 25% >20 0% 14% Gleason score 6 54% 10% <0.01 7 46% 50% 8-10 0% 40% T stage 1c-2b 96% 65% <0.01 2c-3b 4% 35% cancers-15-01396-t002_Table 2 Table 2 Comparison of volume, dose values and homogeneity indices (p-value considering comparison of HDR-BT with the respective LDR-BT evaluation). HDR-BT (n = 232) LDR-BT Intraop. (n = 102) LDR-BT Day 1 (n = 102) LDR-BT Day 30 (n = 102) p-Value Intraop. p-Value Day 1 p-Value Day 30 prostate volume 44 +- 17 cm3 41 +- 12 cm3 40 +- 11 cm3 36 +- 10 cm3 0.02 <0.01 <0.01 prostate V100 92 +- 5% 92 +- 6% 88 +- 5% 90 +- 5% 0.63 <0.01 <0.01 prostate V150 33 +- 4% 65 +- 9% 55 +- 9% 61 +- 10% <0.01 <0.01 <0.01 prostate D90 103 +- 6% 107 +- 14% 98 +- 8% 102 +- 10% <0.01 <0.01 0.60 urethra D30 107 +- 5% 141 +- 20% 114 +- 18% 126 +- 20% <0.01 <0.01 <0.01 rectum D2 cm3 53 +- 9% 69 +- 14% 77 +- 18% 88 +- 20% <0.01 <0.01 <0.01 rectum D0.1 cm3 68 +- 11% 116 +- 32% 138 +- 45% 163 +- 50% <0.01 <0.01 <0.01 Dose Homogeneity Index 0.64 +- 0.05 0.30 +- 0.08 0.38 +- 0.09 0.33 +- 0.09 <0.01 <0.01 <0.01 Overdose Volume Index 0.15 +- 0.03 0.39 +- 0.08 0.30 +- 0.07 0.38 +- 0.09 <0.01 <0.01 <0.01 Dose Nonuniformity Ratio 0.36 +- 0.05 0.70 +- 0.08 0.62 +- 0.09 0.67 +- 0.09 <0.01 <0.01 <0.01 cancers-15-01396-t003_Table 3 Table 3 Comparison of intraoperative dose values and homogeneity indices depending on prostate volume (smaller or larger than median volume = 41 cm3). LDR-BT (n = 102) HDR-BT (n = 232) p-Value prostate V100 small 94 +- 5% 93 +- 4% 0.05 large 90 +- 7% 92 +- 6% 0.05 prostate V150 small 66 +- 9% 33 +- 4% <0.01 large 63 +- 7% 32 +- 5% <0.01 prostate D90 small 111 +- 14% 103 +- 6% <0.01 large 102 +- 13% 102 +- 5% 0.84 urethra D30 small 141 +- 20% 107 +- 6% <0.01 large 141 +- 20% 108 +- 4% <0.01 rectum D2 cm3 small 68 +- 14% 50 +- 9% <0.01 large 71 +- 15% 58 +- 7% <0.01 rectum D0.1 cm3 small 117 +- 36% 65 +- 11% <0.01 large 115 +- 28% 72 +- 8% <0.01 PV + 5 mm D90 small 69 +- 9% 68 +- 6% 0.83 large 65 +- 8% 73 +- 5% <0.01 Dose Homogeneity Index small 0.30 +- 0.08 0.64 +- 0.05 <0.01 large 0.30 +- 0.08 0.65 +- 0.06 <0.01 Overdose Volume Index small 0.38 +- 0.08 0.15 +- 0.03 <0.01 large 0.39 +- 0.08 0.14 +- 0.03 <0.01 Dose Nonuniformity Ratio small 0.70 +- 0.08 0.36 +- 0.05 <0.01 large 0.71 +- 0.08 0.35 +- 0.06 <0.01 Disclaimer/Publisher's Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

PMC10000180

Reduced-fat cured sausages were evaluated as a strategy to reduce boar taint in entire male pork products with high levels of androstenone and skatole, both lipophilic. Three fuet-type sausages (two replicates each) were developed: the control (C) (60% lean, 33.69% fat), and two reduced-fat (60% lean 21.19% fat) products; R1, 6% inulin, 0.5% b-glucan and R2, 3% inulin, 0.5% grape skin and 1% b-glucan. All of them were prepared from entire male pork with an androstenone concentration of 6.887 mg/g and 0.520 mg/g of skatole. Significant differences (p <= 0.001) in the moisture content were described between the fuet R1 and the C and R2, which obtained the highest percentage. Regarding the CIELAB, the C samples had the highest L* value, while the R2 sausages were the darkest. Boar taint was reduced in both R1 and R2, with a greater reduction in R2 (p <= 0.000). The addition of inulin and b-glucan in fuet R1 provided a similar technological and sensory profile to C. However, both strategies provided a reduction of sexual odour, which was higher when grape skins were included. In addition, R2 gave a characteristic sausage with more odour and flavour, dark colour and overall rating than C and R1. reduced-fat dietary fibres fuet sausage boar taint entire male pork MCI (Ministerio de Ciencias e Innovacion)RTA 2017-00039-02-02 AEI (Agencia Estatal de Investigacion)INIA (Instituto Nacional de Investigacion Agraria) of SpainAcknowledgement to MCI (Ministerio de Ciencias e Innovacion: RTA 2017-00039-02-02), AEI (Agencia Estatal de Investigacion) and INIA (Instituto Nacional de Investigacion Agraria) of Spain for their financial support. pmc1. Introduction The EU animal welfare recommendations to end the surgical castration of piglets without anaesthesia and/or analgesia have increased the production of entire male pigs, as it is seen as the most cost-effective alternative due to its better feed-to-gain ratio and healthiness (as this meat contains a higher percentage of lean meat) . However, the rearing of entire male pigs remains problematic, due to the possible occurrence of boar taint, so castration of the entire male pig has traditionally been carried out to control this undesirable odour . Boar taint is a disagreeable odour and flavour that arises mainly through the accumulation of androstenone (AND: 5a-androst-16-3one) and skatole (SKA: 3-methylindole) in fatty tissue. AND is a male sex pheromone with an odour similar to urine or sweat that is perceived by approximately 40-50% of the meat consumers, while SKA is a metabolite of tryptophan associated with faecal odour or naphthalene, and is recognised by 99% of meat consumers . Therefore, it is necessary to search for alternatives to reduce the boar taint in this meat, and to guarantee its commercialisation in both national and international markets. The use of spices, curing, fermentation, and smoking have been shown to reduce the boar taint of entire male pork . However, even with the combination of several of these techniques, it is complicated to obtain entire male pork products under high-quality standards if high levels of AND and SKA are found in the meat. Cured sausages made from this meat are darker, harder, dryer, and saltier, due to the lower fat content , and are traditionally produced with high percentages of fat (20-30%), which leads to the presence of high concentrations of the compounds responsible for sexual odour (AND and SKA), as they have a lipophilic character . As a consequence, effective technological strategies are needed to process such meat and obtain products that meet sensory quality standards, since fat from entire male pigs could be a handicap in the processing due to the main unsaturated profile. Although the production of low-fat meat products could contribute to reducing boar taint perception, it comes with some technological drawbacks, such as reduced performance, juiciness, and cured flavour, or increased toughness and therefore lower consumer acceptance . In recent years, some strategies have been studied to produce healthier meat products without affecting their technological and sensory properties. Replacing pork fat with vegetable fibres or functional ingredients is a common practice in the meat industry, as it has shown a positive effect on the texture of low-fat meat products . However, its effect on the boar taint perception in fuet sausages, a traditional Spanish meat product, is unknown. Among the fat substitutes utilised, inulin, a soluble fructooligosaccharide (FOS), is considered as a prebiotic agent due to its functional effects on the gastrointestinal microbiota , and is often used in numerous food formulations (dairy, meat, beverages, cereals, etc.) due to its lower caloric content . It also improves organoleptic properties, water retention capacity and emulsion stability, and provides a fat-like colour . b-glucan is a glucose polymer with prebiotic properties that improve the stability of emulsions by increasing the viscosity of formulations . The use of other plant fibres such as wheat by-products or fruit fibres , has also been shown to improve the technological properties of meat products. In general, these dietary fibres are mainly non-digestible polysaccharides that can contribute towards the improvement of the appearance and texture of meat products, as they slow down the drying rate, thus reducing moisture loss and providing more juiciness . However, there are no studies in the bibliography consulted on the use of vegetable fibres for fat reduction in dry-cured sausages made from entire male pork, as the use of these ingredients could result in the loss of the characteristic flavour derived from fat . Therefore, the objective of the present study was to assess if the fat reduction could diminish the boar taint in dry-cured sausages made from entire male pork with high levels of AND and SKA, and to study the technological and sensory quality by using inulin, b-glucan and grape fibre as fat substitutes in reduced-fat products. 2. Materials and Methods 2.1. Ingredients The experimental procedures used in this study complied with the EU guidelines for the care and handling of research animals . Thirty-one carcasses of entire male pigs with high boar taint levels were selected with human nose (trained panel) methodology , from three hundred animals in a slaughterhouse located in Catalonia (Spain). Subcutaneous fat from the dorsal neck region of these carcasses was analysed for androstenone (AND) with gas chromatography-mass spectrometry , while the presence of skatole (SKA) was analysed with high performance liquid chromatography . Once analysed, pig meat from the carcasses with the highest level of boar taint; 6.887 mg/g AND/0.520 mg/g SKA, was selected. Inulin (Guinama, Barcelona, Spain) and b-glucan (Guanjie Biotech, Shaanxi, China) were used in powder concentrate form (Inulin at 3% or 6% level (w/w)/b-glucan at 0.5% or 1%), to reach the desired final content in sausage production. The red grape extract was obtained from a local purveyor (Cimusa-Dallant, SA, Murcia, Spain) and incorporated into the formulation at a concentration of 0.5% (w/w), or not used (Table 1). The rest of the ingredients used during fuet manufacturing were salt (Aliada, Madrid, Spain), spice mix blend (Catalina Food Solutions SL, SCM-074-SCH, Murcia, Spain; including salt, potato starch, soy protein, spices, spice extract, dextrose, sugar, lactose, sodium triphosphate (E-451), sodium ascorbate (E-301), potassium nitrate (E-252), sodium nitrite (E-250), carmine (E-120)), and water. All the ingredients of the fuet composition were kept constant in each formulation and replicated. 2.2. Fuet Preparation Fuet sausages were produced in the pilot plant of the Faculty of Veterinary Medicine (University of Murcia). Lean pork ham and fat with a proximate composition of lean meat (1.25% fat; w/w) and fat (71.98% fat; w/w) from the selected carcasses of entire male pigs were used for the elaboration of three formulations of fuet-type sausages . A control formulation with a regular fat content (60% lean ham/25% fat) and two reduced fat productions, R1 and R2 (60% lean and 16% fat), including two different proportions of dietary fibre (inulin, b-glucan, and grape extract), which were added separately to these formulations, were created, as shown in Table 1. An overall control formulation without extracts was also prepared to obtain a total of three different formulations of fuet sausages, with the production of two replicates of each formulation on independent days, within one week of each other. Meat and backfat were processed under refrigerated conditions for removing excess fat and connective tissues. Pork meat and fat were separately ground twice (Mainca, Barcelona PM98, III, 380 V, 3Hp, 50 Hz) through two orifice plates (meat; 25 and 10 mm ph, fat; 10 and 3 mm ph) in a meat grinder, weighed (Tanita BSE-860), packed into bags (vacuum bags, 350*550, 140 my, La Bolsera Murciana, Murcia), vacuum sealed with a packaging machine (INEI, Barcelona, Model 500), and frozen at -18 degC until product formulation, within one month. Before fuet processing, frozen meat and backfat were thawed overnight in a refrigerator at 4 degC. The appropriate amount of ingredients for each formulation (lean meat, fat, inulin, b-glucan, grape skin, salt, commercial mix, and water), were placed into a bowl and manually mixed until a highly homogeneous mass was obtained (approximately 5 min). The mixed mass was then stored in the refrigerator (4 degC) for 1 h until the mixture was compact. The refrigerated mass (approximately 4 kg) was transferred into a hand stuffer (Garhe S.A., Vizcaya, Spain), and then stuffed into artificial collagen casings 45 mm in diameter (Edicas, Salamanca, Spain), previously soaked in water at 4 degC. The fuet sausages were manually tied into approximately 15 cm links, washed to remove materials on the outside of the casing, weighed for subsequent weight control, and hung in a drying chamber for 7 days at 4 degC-80% RH, and another 3 days at 22 degC-84% RH, until the curing process of the product was completed and an optimum point of moisture was reached (% moisture of fuet control: 43.6%, R1: 45.63% and R2: 45.71%; Aw control: 0.618, R1: 0.646, R2: 0.607). Finally, fuets were weighed, vacuum packed, and frozen at -18 degC in a freezing chamber until analysis, within three months, to prevent excessive dryness. 2.3. Colour Measurement Colour coordinates CIE L*a*b* were measured using a CR-400 Chroma Meter (Minolta Ltd., Milton Keynes, United Kingdom) calibrated against a standard white tile (8 mm diameter aperture, d/0 illumination system, illuminant D65, and a 2deg standard observer angle). L* (lightness), a* (redness), and b* (yellowness) were measured on the cutting surface from three randomly chosen spots of three slices from each fuet sausage . 2.4. Moisture and Fat Determination The determination of moisture content in the meat product samples was carried out following the process in , and the intramuscular fat content was established according to the process in , using petroleum ether (40-60 degC) as the solvent in a Soxhlet extraction equipment. Moisture and fat contents were measured in duplicate for each type of fuet. 2.5. Texture Profile Analysis (TPA) The texture profile analysis (TPA) of the sausages was carried out using a Texturometer QTS-25 (Brookfield CNS Engineering Labs. Inc., Harlow, Essex, UK) with TexturePro CT V1.8 software . The sausages were cut into pieces 2 cm in diameter x 2 cm in length to obtain cylindrical cores for TPA measurement, after conditioning at 23 degC. Core samples were compressed twice with a 10 mm diameter cylindrical probe (TA 10) provided with a 25 kg load cell, until they reached 50% of their original height. The force-time deformation curves were obtained with a crosshead speed of 2 mm/s and a trigger point of 5 g. TPA parameters; hardness (g), adhesiveness (mJ), chewiness (mJ), gumminess (g), cohesiveness, elasticity (mm), resilience (ability of the product to return to its initial position, J/m3), and extensibility (mm), were determined in triplicate. 2.6. Sensory Analyses Eight panellists were selected from a panel previously trained in the sensory evaluation of meat products from entire male pork , and trained in AND (from 0 to 7 mg/g) and SKA (from 0 to 1.5 mg/g) detection . Six theoretical-practical sessions lasting 1.5 h were held for specific training with reduced-fat products of entire and castrated (commercial product) male pigs. A quantitative descriptive analysis (QDA) test was performed using an unstructured 10-point scale (0 = not perceptible; 10 = maximum perception). The sensorial attributes for analysis were selected according to ; colour (colour intensity, brightness and homogeneous colour), odours (sausage odour, acid odour, boar taint and other odours), taste (acid, salty and bitter), flavours (sausage flavour, boar taint and other flavours), and texture (hardness, cohesiveness, chewiness and juiciness). Finally, an overall rating was included to assess the success of the strategies developed (0 = low rating; 10 = high rating). The panellists were also asked to provide their overall rating of the sausage samples. The sensory evaluation of the sausages was carried out according to the process in in a standardised room at the Food Technology Department (University of Murcia). The analyses were performed at the same hour of the day (at 12:00 h) for each session. A total of six samples per treatment (C, R1 and R2) and replicate (two batches) were tasted by each panellist. The samples were cut into 2 mm thick slices, and two slices coded with a random three-digit number per treatment were presented to each panellist, at a temperature of 23 degC. Sample presentation was balanced to account for order and carryover effects . The panellists were provided with mineral water and unsalted bread between sample tastings to rinse their palates and to avoid flavour retention in the mouth. 2.7. Statistics The data were analysed with the SPSS 24 statistical software package . The colour, texture and sensory parameters were analysed with a one-way analysis of variance (ANOVA), considering the effect of the different formulations (C, R1, and R2) as fixed sources of variation, and the two replicates, session and panellists as a random effect. The comparisons between means were performed using Tukey's test, and differences were considered statistically different when p < 0.05. 3. Results and Discussion 3.1. Composition and Instrumental Colour Table 2 shows the results of the composition and instrumental colour of the different fuet sausage treatments. As observed, there were significant differences (p <= 0.001) in the moisture content between the fuet produced with inulin plus b-glucan (R1), and the control and that produced with grape skin sausage samples (C and R2), which obtained the highest percentage in this parameter. The grape-skin addition could retain water, resulting in values similar to those of the control, in agreement with other authors, who reported how fibres, gums, or starches as fat substitutes can help slow down the drying rate during maturation, thus reducing moisture loss . Nutrition and health claims made in food regulation indicate that fat reduction must be at least 30% as compared to a similar product in order for the sausages to be considered as "reduced fat". Thus, although significant differences (p <= 0.000) were found between control and reduced-fat formulations, these fat reductions established were not sufficient for the R1 and R2 sausages to be considered "reduced fat" products. Regarding the CIELAB colour parameters, statistical differences (p < 0.001) were observed between the cured sausages, as the control samples had the highest L* coordinate value (48.4), while the R2 sausages were the darkest (41.8), as luminosity is directly related to the percentage of fat and water-retention capacity of meat products . Similar results were reported by on dry fermented sausages with a 16% inulin-gelled emulsion, showing how the addition of inulin provided meat products that were more similar to conventional ones, as compared with the use of other fat substitutes. The authors of also obtained similar values using inulin and b-glucan in meat emulsions. Furthermore, Ref. observed that the higher the concentration of inulin added, the higher the brightness values in this type of product. In the a* and b* colour coordinates, no significant differences were observed between R1 and the control sausages (p >= 0.05), but the comparison between these two and R2 (p = 0.008) showed that the latter obtained the lowest values for the a* coordinate. It has been observed that substitutes with a similar colour to fat, such as inulin, provide a similar colouring to conventionally cured sausages . Therefore, the sausages that were made with inulin powder tended to have a more yellowish colour and a less blue one (higher value of b*), giving them the characteristic colour of this type of product . On the other hand, a decrease in the reddish and yellowish colouring was obtained in the R2 fuet. Similar results were obtained by other authors, such as in , in meat emulsions where fat was partially replaced by grape seed oil at 0, 5, 10 and 15% and 2% bran fibre, or in cooked pork sausages made with 0.5, 1 and 2% grape marc powder (composed of stems, skins and pips) , and reduced in fat . The main molecules involved in the colour of grape dietary fibre are flavonoids; therefore, the observed a* and b* values could be related to the anthocyanins present in grape skins, as it should be noted that the colour of anthocyanins can vary from red to blue depending on the pH value . However, the antioxidant activity of phenolic compounds from grape skins could influence the colour of the meat, acting mainly on the heme group of myoglobin during the fermentation of these products, thereby helping to preserve the reddish colouring . 3.2. Instrumental Texture The results for the instrumental texture analysis are presented in Table 3, where statistically significant differences were observed between the three formulations (p <= 0.05) in all the parameters, except for hardness and cohesiveness (p > 0.05). It could be expected that fat reduction is accompanied by an increase in toughness and the instrumental chewiness of meat products, as the use of short-chain soluble fibres, such as inulin or wheat fibre (rich in b-glucan), forms a more compact emulsion than the protein matrix . Furthermore, showed that hardness increases with higher inulin concentration (7-7.5%). The results of the present study are in agreement with those from , who observed that hardness was not affected by the addition of 2% dietary fibre powder (inulin, FOS, cyclodextrin) as a fat substitute, since the higher moisture content and moisture/protein ratios could explain the differences in low-fat salami. However, other authors showed how the incorporation of konjac gel or inulin reduced hardness values in sausages . Therefore, the effects of dietary fibres on this parameter depend on both the amount of fibre and the type (powder or gel), as gels provide softer sausages. The impact of dietary fibre addition on chewiness indicated that the R2 formulation had a higher value (201.2) than the control (147.76) (p <= 0.002). The increase could probably be attributed to the addition of grape fibre and the higher levels of b-glucan in this formulation, as some dietary fibres have the ability to strengthen the connections between different matrix components . The control group reached the highest fat %, as fat promotes the stimulation of salivary receptors. Thus the higher the intramuscular fat content, the greater the juiciness and the lower the chewiness, as previously described . Similar results were obtained by , as the addition of inulin or FOS in reduced-fat cured sausages increased chewiness. Inulin promotes harder and chewier low-fat products, but in excess of 6%, it can lead to poorer texture characteristics. However, in the present study, an acceptable texture profile was described for both reduced-fat products. Furthermore, Ref. observed a lower chewiness with the addition of inulin in reduced-fat meat matrices (Spanish sausages), although other authors, such as , described an increase with the incorporation of gel agents such as cellulose-chitosan. For gumminess, R1 and R2 sausages obtained a higher value with respect to the control (p <= 0.004). Similar results were reported by other authors for gumminess in sausages and Spanish sausages reduced in fat with FOS or grape pomace, as gumminess and chewiness, being the secondary parameters of toughness, behave similarly . With the substitution of entire pork loin fat with inulin and b-glucan, no significant differences were observed for the parameters of adhesiveness, extensibility, cohesiveness, and elasticity, with respect to the control sample (p > 0.05). On the other hand, the addition of grape skin reduced the adhesiveness, had no effect on cohesiveness, and increased the extensibility and elasticity of the R2 sausages (p <= 0.05). For resilience (the ability of food to absorb energy when deformed, before reaching its yield strength, and to release it when the load is released), statistically significant differences were obtained between the three formulations (p <= 0.000), with the control sausage obtaining the highest value for this parameter, due to the higher fat content, followed by R1, while the lowest value was obtained by R2. Thus, the results revealed that the fibres employed varied widely in their effects on the parameters studied. In general, the effect observed was influenced both by the type of dietary fibre used, as well as its concentration and hydrophobicity, and the interactions established between the main components of the meat matrix (fat, proteins, and hydrocolloids) . Many studies have analysed the instrumental profile of dry-cured sausages, obtaining results that differed from each other, depending on the type and concentration of the fat substitute. In this sense, Ref. showed an increase in the adhesiveness of sausages with the use of inulin gel instead of powder, while observed how cohesion decreased as more fat was replaced by quinoa. On the other hand, Ref. analysed how binary mixtures of inulin and FOS increased cohesion to resemble the control product, as described in the present study. With respect to elasticity, Ref. found that sausages made with 4% starch as a fat substitute obtained higher values. However, elasticity was not affected with the addition of 3% inulin in salami . These parameters are important in the handling and slicing of sausages and can make it difficult to do . In addition, cured sausages made from entire male pork are tougher and drier due to the lower fat content in this meat, which causes higher processing losses . Therefore, the maintenance of adequate textural properties is desirable. The doses used in the R2 formulation resulted in less adhesive, and more elastic and chewy sausages, with a correct cohesiveness and hardness similar to the control sample. 3.3. Sensory Analyses The results of the sensory evaluation of the three fuet formulations are shown in Table 4. Statistically significant differences (p <= 0.05) were observed in all the attributes studied, except for homogeneity, acid odour/flavour, bitterness, cohesiveness, or juiciness (p > 0.05). The scores provided by the panellists for the appearance attributes (colour, brightness, and homogeneity) were in accordance with the results obtained in the instrumental analysis. The R1 formulation showed a similar behaviour to the control sample, as described in other studies on cured sausages reduced in fat with inulin . In contrast, R2 obtained a higher colour score and lower brightness (p <= 0.000) than the control and R1 samples, due to the colour of the anthocyanins present in the grape skin and the lower fat content of this formulation , in comparison with the control group. The R2 formulation obtained the highest score (6.8 and 7.0) for sausage odour and flavour, respectively, while R1 had a higher value (5.9 vs. 4.9) only for sausage flavour as compared to the control sample (p <= 0.000). The grape skin inclusion in the reduced-fat R2 fuet sausage decreased the boar-taint defect by masking unpleasant odour and flavour and highlighting the traditional organoleptic features of a dry-fermented sausage . The authors of Ref. described an intense fermented odour in the meat preparations with the addition of grape pomace. Furthermore, phenols and organic acids in the pomace can cause astringency, acidity, and bitterness. Our results showed slightly higher scores for the attributes of off-odour and flavour (0.2 and 0.3, respectively), and salty, in the R2 formulation (p <= 0.007), but no higher acidity than the control and R1 samples (p > 0.05). Boar-taint odour and flavour were reduced in both R1 (35.4% and 41.6%) and R2 (77.6 and 80.8%) formulations, with a greater reduction in R2 (p <= 0.000). Dry-fermented products require high percentages of fat that could imply high concentrations of boar taint, because the molecules involved are lipophilic . However, the dry-fermentation process can decrease boar taint, as fermentation results in changes in aroma , and the drying process leads to oxidation of the fat fraction . Moreover, as fuet sausages are consumed cold, the boar taint is more difficult to perceive . However, these processes are insufficient to eliminate this perception when very high levels of AND and SKA are present. Dry-cured products usually lose more than 30% of their weight during the curing process, thus increasing the absolute concentration of these compounds and their perception in the final product . Previous studies have shown how the use of conventional additives such as spices, smoking , and dilution of 10% entire male pork with uncontaminated meat , successfully masked the boar taint in dry-fermented sausages. It was therefore to be expected that the R2 formulation would obtain the lowest scores on these attributes, as the reduction in fat, together with the addition of grape skins, which intensifies the fermented odour/flavour typical of these products , helped to minimize their perception. Hence, it can be concluded that fine tuning of production processes (cooking and/or smoke condensates), and mixing with non-boar tainted meat (targeted dilution), can significantly reduce the perception of boar taint, thus potentially reducing rejection by consumers . In terms of textural attributes, the reduced-fat formulations obtained the highest scores for hardness and chewiness (p <= 0.05). As proven in the instrumental texture analysis, dietary fibres contribute to the strengthening of the bonds between the different components of the protein matrix . For cohesiveness and juiciness, no statistically significant differences were observed (p > 0.05), so that the fat-reduced formulations with inulin, b-glucan and grape skin had textural characteristics similar to the control sausages. Fat reduction could affect the juiciness of the products, but the addition of the vegetable fibre seemed to be sufficient to obtain similar values as the control sample. Similar results were obtained in previous works with reduced-fat Frankfurter sausages with the addition of inulin, b-glucan, and grape pomace, as inulin has a high capacity to bind water and form juicy and stable emulsions similar to control ones . In dry-fermented, reduced-fat sausages with inulin and FOS, no differences in sensory texture were observed either . Finally, the level of masking by the strategies (overall rating) was significantly higher for R2, followed by R1 and C (p <= 0.000). The R2 formulation provided improved odour and cured flavour, as previously described in fresh and Frankfurter reduced-fat sausages with grape skins added . 4. Conclusions The development of reduced-fat fuets (R1 and R2) under a high sensory and technological quality profile was carried out in the present study. In terms of boar-taint masking, both formulations (R1 and R2) reduced its sensory perception on the product, but R2 obtained the best masking properties by preserving the sausage odour and flavour of the traditional Spanish fuet sausages. Therefore, the incorporation of inulin, b-glucan and grape fibre, as fat substitutes in fuet production made with entire male pork with high levels of AND and SKA, could be considered as a useful technological masking strategy for boar-tainted pork. The formulation R1 (6% inulin and 0.5% b-glucan) showed a similar technological behaviour to the control sausages, while R2 (3% inulin, 1% b-glucan and 0.5% grape skin) showed the darkest product with the highest overall sensorial rating. Author Contributions Conceptualisation, D.A., M.E. and M.B.L.; data curation, I.P. and D.A.; formal analysis, I.P. and M.E.; funding acquisition, M.D.G.; investigation, M.D.G., M.B.L., I.P., M.E. and D.A.; methodology, D.A. and I.P.; project administration, M.D.G. and M.B.L.; resources, I.P., M.E. and D.A.; software, I.P.; supervision, M.B.L. and M.E.; validation, M.D.G. and M.B.L.; visualisation, D.A.; roles/writing--original draft, I.P.; writing--review and editing, M.D.G., M.B.L., I.P., M.E. and D.A. All authors have read and agreed to the published version of the manuscript. Institutional Review Board Statement Not applicable. Informed Consent Statement Not applicable. Data Availability Statement Data supporting reported results are available upon request. Conflicts of Interest The authors declare no conflict of interest. Figure 1 Fuet sausage and formulations. C: control with regular fat content; R1: reduced fat with inulin + b-glucan; R2: reduced fat with inulin + b-glucan + grape skin. animals-13-00912-t001_Table 1 Table 1 Formulations used in preparing the fuet sausages (%). Formulation (%fat) Lean Fat * Inulin Grape Skin b-Glucan Salt Mix Water C (25) 60 33.69 1 4 1.31 R1 (16) 60 21.19 6 0.5 1 4 7.31 R2 (16) 60 21.19 3 0.5 1 1 4 9.31 C: control fuet made with 25% fat. R1/R2: Low fat fuets made with 16% fat and different combinations of inulin, b-glucan and grape skin. * Final fat content in raw material used for fuet sausage production according to the proximal composition of meat and fat samples. animals-13-00912-t002_Table 2 Table 2 Composition and instrumental colour of fuet sausages (means +- SD). Parameters C R1 R2 p-Value Moisture (%) 21.4 +- 0.00 b 19.5 +- 1.43 a 21.3 +- 0.36 b 0.002 Fat (% MM) 35.2 +- 2.65 b 26.9 +- 1.93 a 26.2 +- 1.93 a 0.000 CIELAB colour L* 48.4 +- 1.83 c 45.1 +- 1.38 b 41.7 +- 1.17 a 0.000 a* 11.6 +- 1.78 b 11.7 +- 1.68 b 9.1 +- 1.22 a 0.004 b* 6.3 +- 0.53 b 5.9 +- 0.94 ab 5.2 +- 0.27 a 0.008 C: control regular fat content; R1: reduced fat with inulin + b-glucan; R2: reduced fat with inulin + b-glucan + grape skin. L*: luminosity, a*: red-green; b*: yellow-blue. a-c: Tukey's test p < 0.05. Fat (% MM): fat percentage in moisture matter. Sample size (n) 18 (6 samples per type of fuet). animals-13-00912-t003_Table 3 Table 3 Instrumental texture of fuet sausages (means +- SD). Parameters C R1 R2 p-Value Hardness (g) 9418.3 +- 464.24 10,069.0 +- 1146.32 10,206.0 +- 1007.07 0.212 Gumminess (g) 2907.2 +- 140.90 a 3348.7 +- 172.03 b 3341.6 +- 398.36 b 0.004 Chewiness (mJ) 147.8 +- 13.10 a 178.8 +- 24.99 ab 202.0 +- 36.04 b 0.002 Adhesiveness (mJ) 4.4 +- 0.43 b 3.9 +- 0.60 ab 3.6 +- 0.49 a 0.014 Resilience 1.5 +- 0.19 c 1.4 +- 0.06 b 1.2 +- 0.09 a 0.000 Extensibility 21.5 +- 5.47 a 18.0 +- 3.84 a 29.0 +- 5.22 b 0.001 Cohesiveness 0.3 +- 0.02 0.3 +- 0.02 0.3 +- 0.03 0.718 Elasticity (mm) 5.2 +- 0.29 a 5.4 +- 0.76 ab 5.9 +- 0.36 b 0.039 C: control regular fat content; R1: reduced fat with inulin + b-glucan; R2: reduced fat with inulin + b-glucan + grape skin. a-c: Tukey's test p < 0.05. Sample size (n) 18 (6 samples per type of fuet). animals-13-00912-t004_Table 4 Table 4 Sensory analyses of fuet sausages (means +- SD). Attributes C R1 R2 p-Value Colour 5.6 +- 0.80 a 6.4 +- 0.56 b 8.3 +- 0.72 c 0.000 Brightness 4.8 +- 0.70 b 4.9 +- 1.01 b 3.6 +- 0.88 a 0.000 Homogeneity 9.9 +- 0.43 9.9 +- 0.46 9.7 +- 0.63 0.390 Sausage odour 4.9 +- 0.69 a 5.7 +- 1.41 a 6.8 +- 1.82 b 0.000 Acid odour 0.9 +- 0.58 0.7 +- 0.76 0.8 +- 0.75 0.469 Off odour 0.0 +- 0.00 a 0.0 +- 0.00 a 0.2 +- 0.37 b 0.007 Boar taint odour 4.3 +- 1.32 c 2.8 +- 1.08 b 1.0 +- 0.53 a 0.000 Acid 1.1 +- 0.88 1.0 +- 0.98 1.1 +- 1.04 0.938 Salty 5.4 +- 0.46 a 5.6 +- 0.44 ab 5.9 +- 0.70 b 0.021 Bitter 0.2 +- 0.56 0.2 +- 0.57 0.2 +- 0.39 0.986 Sausage flavour 4.9 +- 0.92 a 5.9 +- 0.78 b 7.0 +- 1.23 c 0.000 Off flavour 0.0 +- 0.00 a 0.0 +- 0.00 a 0.3 +- 0.52 b 0.000 Boar taint flavour 5.3 +- 1.95 c 3.1 +- 1.38 b 1.0 +- 0.64 a 0.000 Hardness 5.7 +- 0.61 a 6.0 +- 0.66 ab 6.5 +- 1.15 b 0.016 Cohesiveness 5.9 +- 1.63 5.8 +- 0.59 6.1 +- 0.87 0.627 Chewiness 5.3 +- 0.50 a 5.9 +- 0.49 b 5.9 +- 1.16 b 0.016 Juiciness 5.0 +- 0.75 4.5 +- 0.93 4.6 +- 1.50 0.366 Overall rating 4.1 +- 1.57 a 5.8 +- 1.19 b 8.0 +- 1.58 c 0.000 C: control regular fat content; R1: reduced fat with inulin + b-glucan; R2: reduced fat with inulin + b-glucan + grape skin. a-c: Tukey's test p < 0.05. Scores from 0--not perceptible to 10--maximum perception, an unstructured 10-point scale. Sample size (n) 36 per panellist (12 samples per type of fuet). Disclaimer/Publisher's Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

PMC10000181

Locomotion in non-human primates, including walking, climbing, and brachiating among other types of movement (but not pacing), is a species-typical behavior that varies with age, social housing conditions, and environmental factors (e.g., season, food availability, physical housing conditions). Given that captive primates are typically observed to engage in lower levels of locomotor behaviors than their wild counterparts, increases in locomotion are generally considered to be indicative of improved welfare in captivity. However, increases in locomotion do not always occur with improvements in welfare, and sometimes occur under conditions of negative arousal. The use of time spent in locomotion as a welfare indicator in studies of well-being is relatively limited. We conducted focal animal observations on 120 captive chimpanzees across a series of studies and found higher percentages of time spent in locomotion (1) upon transfer to a new enclosure type, (2) in larger groups with wider within-group age ranges, and fewer males, and (3) with participation in an experimental medication choice paradigm. We also found that, among geriatric chimpanzees, those housed in nongeriatric groups exhibited more locomotion than those living in geriatric groups. Lastly, locomotion was significantly negatively correlated with several indicators of poor welfare and significantly positively correlated with behavioral diversity, one indicator of positive welfare. Overall, the increases in time spent in locomotion observed in these studies were part of an overall behavioral pattern indicative of enhanced welfare, suggesting that an increase in time spent in locomotion itself may be an indicator of enhanced welfare. As such, we suggest that levels of locomotion, which are typically assessed in most behavioral experiments, may be used more explicitly as indicators of welfare in chimpanzees. chimpanzee welfare locomotion positive welfare National Institutes of HealthU42-OD 011197 This research was funded by the National Institutes of Health, grant number NIH U42-OD 011197 and the University of Copenhagen. pmc1. Introduction There are various definitions of locomotion in studies of animal behavior, but all refer to the movement of the animal in relation to the environment . In chimpanzees, locomotion refers to movement from one location to another as a result of walking, climbing, jumping, or brachiating. It is a species-typical behavior that varies in frequency and duration among wild chimpanzees according to season and availability of food. Individuals travel between sites within their home range or territory to obtain food, access sleeping sites, and find areas that promote other activities necessary for their survival . Importantly, locomotion in captivity must be distinguished from pacing, which is used as an indicator of negative welfare. Pacing is distinct from locomotion in both form and function, as pacing occurs in a repetitive and stereotypical fashion and occurs with no apparent objective or goal . We want to make it clear that the behavior we refer to throughout this manuscript is locomotion, and not pacing. Locomotion accounts for approximately 12-15% of the daily time budget of wild chimpanzees , whereas locomotion accounts for 5-10% of observation time in studies of captive chimpanzees . Given that levels of locomotion are lower in captive chimpanzees than in their wild counterparts, and increases in species-typical behaviors in captive populations are typically equated with increases in well-being, increases in locomotion are generally considered to be related to positive welfare . Although locomotion is a behavior of interest in many studies, it is not often explicitly used as a measure of NHP welfare or well-being, at least not to the extent that other behavioral indicators of welfare are used (e.g., abnormal behaviors) . "Gold-standard" indicators of negative well-being include abnormal, stereotypic, and self-directed behavior (and sometimes rough-scratching) . However, there are very few "gold-standard" behavioral indicators of positive welfare in chimpanzees. Social play is often used to indicate positive welfare, but this behavior occurs at low frequencies (approximately 0.5-2% of daily time budget) in adult geriatric, and/or female chimpanzees (Neal Webb, unpublished data), making it a difficult behavior to use for welfare assessments of an aging chimpanzee population. Furthermore, social play may have a more complicated relationship with positive welfare than has been posited in the past, as adult chimpanzees may use social play to cope with social tension . Other studies have used the level, rate, or presence/absence of species-typical behaviors to make inferences about positive welfare states. For chimpanzees, this includes behaviors such as tool use, nesting, and grooming . In a comprehensive survey examining behavioral indices of welfare in facilities housing chimpanzees across the United States, Bloomsmith and colleagues asked about the presence or absence of four species-typical behaviors (i.e., tool use, nesting, grooming, and copulation) and seven abnormal behaviors (including self-directed activity) to provide a snapshot of the state of chimpanzee care and management . Although the authors described the ways that increased activity levels can reflect enhanced welfare, they did not include activity or locomotion in their survey. This illustrates the currently dominant approach to chimpanzee welfare assessment: the use of abnormal behaviors as indicators of negative welfare and species-typical behaviors as indicators of positive welfare. As mentioned above, locomotion likely falls into the category of species-typical behaviors, yet is not commonly used as an indicator of positive welfare . Previous studies have examined locomotion in relation to well-being through changes to the physical environment, such as increased enclosure size or complexity, with associated increased time spent in locomotion . Other studies have included locomotion as a component within a composite behavioral measure, making it difficult to determine the effects of experimental manipulations on locomotion itself. For example, captive chimpanzees showed decreased activity (including locomotion, exploration, and play) and agitation, as well as increased inactive/relaxed behavior, following the playback of different types of music . The authors suggested that this pattern of behavioral change (including decreased activity) meant that the music had a calming effect on the chimpanzees, thereby enhancing their well-being. However, it is unknown whether locomotion increased or decreased, as the authors did not examine the individual behaviors within the composite measure. Still, other studies found increased locomotion that seemed to be consistent with enhanced well-being but did not explicitly make that connection. For example, Jensvold and colleagues reported an increase in certain types of locomotion after chimpanzees were transferred to a larger, more complex enclosure . The authors suggested that the housing improvements that increased locomotion (indicative of increased species-typical behavior) are important elements in the quality of life of captive primates but did not explicitly link locomotion to welfare. Lastly, several studies have examined inactivity as a measure of welfare, finding decreased inactivity as a function of enrichment, transfers to new enclosures, social housing conditions, etc. . However, decreases in chimpanzee inactivity do not necessarily occur in conjunction with increases in locomotion . As such, inactivity levels cannot be used as a perfect proxy for levels of locomotion. Decreased inactivity suggests increased activity, but this increased activity may or may not include increased locomotive behavior. Further complicating the relationship between locomotion and welfare is the fact that increased locomotion has been found in negatively arousing contexts, or conjunction with negative welfare-related behaviors. For example, chimpanzees that were singly-housed long-term showed higher amounts of time spent in locomotion, aggression toward humans, tension-related behaviors, and scratching compared to socially-housed chimpanzees . The authors suggested that increased stress led to higher levels of locomotion, indicating decreased welfare. Furthermore, increased locomotion under conditions of negative arousal has been found in other NHP species. In rhesus monkeys (Macaca mulatta), locomotion increased following maternal separation, a stress-induction paradigm. This increased locomotion coincided with increased cortisol and lower social play, both of which are indicative of decreased well-being . Additionally, Erikson and colleagues found that locomotion was highest in rhesus macaques characterized as "high withdrawal", compared to those classified as low and moderate in withdrawal behavior . Furthermore, this increased locomotion coincided with increases in self-directed behaviors, and long-term separations were associated with decreased locomotion. In marmosets (Callithrix jacchus), several studies have shown that locomotion increases following the presentation of a predator in an experimental paradigm . Although these studies were performed in other NHP species, it is likely that similar responses would be seen in chimpanzees. Given the lack of a clear connection between locomotion and welfare in the existing literature, we aimed to further refine the utility of locomotion as an indicator of positive welfare in captive chimpanzees. Here, we first summarize findings from four previously published studies focused on changes to, or characteristics of, the physical and social environment that included locomotion as a dependent variable. We then present unpublished correlational data between locomotion and welfare-related behaviors. 2. Materials and Methods 2.1. Subjects A total of 120 chimpanzees housed at the National Center for Chimpanzee Care (NCCC), The University of Texas MD Anderson Cancer Center (UTMDACC), Michale E. Keeling Center for Comparative Medicine and Research (KCCMR) in Bastrop, Texas were included in this study. The Keeling Center has been continuously accredited by AAALAC-I for over 40 years. At the conclusion of data collection, chimpanzees were housed in 17 separate social groups ranging in size from three to 10 animals per group. There were 73 females and 47 males (M age = 31.39 years, age range = 15-56), 15 of whom were wild-born (unknown rearing history), 27 were nursery-reared, and 78 were mother-reared . Chimpanzees were housed in PrimadomesTM (KIVA, Buda, TX, USA) or corrals with indoor/outdoor access. Both PrimadomesTM and corrals included various physical environmental enrichment items, including, but not limited to, wooden climbing structures with platforms, telephone poles, culvert sections, tractor tires, various sizes of plastic balls, 55-gallon barrels, and fire hose rope/swings. Chimpanzees were also provided with daily foraging opportunities, enrichment devices, positive human interaction, and positive reinforcement training . 2.2. Procedure Behavioral observations served as the basis for the procedures of the four studies. One observer (SNW) familiar with each chimpanzee and trained in chimpanzee behavioral data collection, collected live observations using 15-min focal animal sampling (Altmann, 1974) on a laptop computer running Noldus Observer XT 10 (Noldus, Wageningen, The Netherlands,) between 0700 and 1600 from July 2016 to May 2018. An ethogram of behaviors was constructed from an existing chimpanzee ethogram with added definitions and behaviors taken from and the Chimpanzee Care Manual . This ethogram was used for each of the studies. Locomotion was operationally defined as time spent hanging, walking, brachiating, and/or climbing (see Table 1). Locomotion was only recorded if it was independent of a social context. For example, brachiating that occurred during a play bout was recorded as play, not locomotion, and walking that occurred during a foraging bout (i.e., manipulating the food item and bringing food to the mouth) was recorded as foraging, not locomotion. Overall, each chimpanzee was observed for a minimum of 22 observation sessions, although some studies included more observations per chimpanzee. Across the studies, a total of 3936 observations, or 984 h of behavioral data, were collected. The research conducted in this study complied with the approved protocols of the UTMDACC Institutional Animal Care and Use Committee and complied with the legal requirements of the United States and the American Society of Primatologists' Principles for the Ethical Treatment of Primates. 2.3. Study-Specific Methods 2.3.1. Locomotion and Changes in Space per Animal and Type of Housing (Previously Published) We examined changes in behavior as a function of increases or decreases in living space and changes in the type of housing. For full methodological details, please see . Briefly, 22 captive chimpanzees in three social groups were moved through three living space and enclosure scenarios. One group that was housed in a single PrimadomeTM (approximately 1000 ft2 total or 142 square feet per animal) was moved to a double PrimadomeTM (approximately 2000 ft2; 284 square feet per animal). A second group was moved from a single PrimadomeTM to a corral (approximately 4500 ft2 or 645 square feet per animal), and a third group was moved from a corral to a double PrimadomeTM. Behavioral data were collected as described above for four months prior to the transfer (pre-transfer period) and five months following the transfer (post-transfer period). We used bootstrapped paired samples t-tests to examine within-subject changes in behavior post-transfer . 2.3.2. Locomotion and Group Size and Group Composition (Previously Published) We examined changes in behavior as a function of differences in group size and group composition. The group size was dichotomized along previous NIH group size guidelines as either seven or more compared to six or fewer animals per group. Group composition included three categories: (1) average age of the group, defined as the mean age of all animals in the social group; (2) within-group age range, defined as the oldest animals' age minus the youngest animal's age within the group; and (3) percentage of males within the group, dichotomized as less than half males within the group compared to at least half males. Please see for full methodological details. Focal animal observations on all 120 chimpanzees were conducted as described in the General methods above. A bootstrapped MANCOVA with group average age as a covariate was used to examine differences in behavior as a function of these social environment characteristics. 2.3.3. Locomotion and Geriatric Group Status (Previously Published) We compared the behavior of elderly (n = 35) chimpanzees housed in geriatric (average age of the group >=35 years) and non-geriatric (<=34 years) social groups. Compared to non-geriatric groups, geriatric groups were characterized by smaller size, smaller within-group age ranges, fewer males within the group, a higher mean age of the group, and a larger number of chimpanzees that exhibited impairments in mobility. Please see for full methodological details. Focal animal observations on the 35 chimpanzees were conducted as described in the General methods above. A multivariate ANCOVA with impairment mobility scores as a covariate was used to examine behavioral differences between elderly chimpanzees housed in geriatric groups and those housed in non-geriatric groups. 2.3.4. Locomotion and Voluntary Participation in a Medication Choice Paradigm (Previously Published) As part of a study examining chimpanzee preferences for medications to treat arthritis, we examined changes in behavior as a function of voluntary participation in the medication choice protocol. For full methodological details, please see . Briefly, four arthritic chimpanzees that were receiving daily analgesic medication to treat the symptoms of their arthritis and their mobility impairments were chosen as participants for a medication choice paradigm. In the paradigm, chimpanzees were assigned to one of two groups that determined the order of medications (either ibuprofen or meloxicam) that they would receive in either blue or green Gatorade. The first phase was a baseline phase in which the chimpanzees received their normal analgesic medication (meloxicam in pill form). In the second, third, fourth, and fifth phases (following an ABBA design), two chimpanzees received ibuprofen in blue Gatorade (A), then meloxicam in green Gatorade (BB), then ibuprofen again in blue (A), respectively. The remaining two chimpanzees received ibuprofen in green and meloxicam in blue Gatorade. This procedure allowed the chimpanzee to form an association between the color of the Gatorade and the way that that color made them feel (based on which medication was included in that color). In the final phase of the study (the choice phase), each chimpanzee was allowed to choose the color of the Gatorade (and thus, which medication) they preferred each morning for one month. During each of these phases, behavioral data were collected as described above in the General Method section. Given the small sample size, we used nonparametric Friedman's rank tests with Wilcoxon posthoc tests for within-group comparisons across phases . 2.3.5. Correlational Data between Locomotion and Other Welfare-Based Behaviors (Unpublished Data) We used Pearson's bivariate correlation to explore the relationships between locomotion and other behaviors that are often used as indicators of welfare, including social play, social grooming, self-grooming, scratching, abnormal behavior, and inactivity (see Neal Webb et al., 2019 for full ethogram). Briefly, inactivity included "inactive rest" (i.e., immobile, generally relaxed) and "inactive alert" (awake and alert but not involved in any active behavior such as locomotion, foraging, etc.). Abnormal behavior was a composite measure that included regurgitation and reingestion, feces smearing, idiosyncratic body movement (e.g., rocking), idiosyncratic body manipulation (e.g., thumb-sucking), hair plucking, and any other abnormal behavior not listed (see for supplementary ethogram). We also examined the relationship between each chimpanzee's behavioral diversity score (the average number of different positive welfare-related behaviors exhibited by the subjects during an observation period) and their level of locomotion using Pearson's bivariate correlation. The calculations for behavioral diversity are described in detail in . Briefly, to calculate this score, the number of different, species-typical behaviors exhibited by the chimpanzee was counted, excluding negative welfare-related behaviors (i.e., abnormal behaviors, rough-scratching), which ensured that behavioral diversity would not be confounded by the possibility that the increase in diversity was due to the addition of an abnormal behavior to the repertoire. Therefore, increases in this measure signified an increase in the number of species-typical behaviors that comprised the observed repertoire of the subject . 2.4. Data Analyses First, the total duration of "out-of-view" for each observation for each chimpanzee was subtracted from the total observation time (900 s) to create total "within-view" durations (chimpanzees were in-view an average of 98% of each observation). Then, the total durations of each behavior for each chimpanzee were summed across all observations for that chimpanzee and divided by the total in-view duration time to create average durations of behavior. These averages were then converted into percentages ((average behavior duration in seconds/in-view duration in seconds) * 100). All analyses were performed in SPSS Statistics 22 (IBM Corporation, Chicago, IL, USA). The significance level for all analyses was set at p < 0.05. 3. Results 3.1. Locomotion and Changes in Space per Animal and Type of Housing (Previously Published) Locomotion was higher post-transfer across all transfer types, regardless of increases or decreases in space per animal or the type of change in housing (although not significantly so in the group that transferred from the corral to the double dome). As shown in Figure 1, chimpanzees exhibited up to a 5% increase in locomotion following transfer. Furthermore, these increases in locomotion were accompanied by significant decreases in rough scratching in the group that moved from the dome to the corral (p < 0.05) and by increases in behavioral diversity scores across all transfer types (p < 0.05) . 3.2. Locomotion and Group Size and Group Composition (Previously Published) There was a trend toward higher levels of locomotion in groups with a larger within-group age range . Furthermore, these changes coincided with lower levels of inactivity (p < 0.05). Locomotion was similar across group sizes if the group contained at least 50% males, but locomotion was approximately 4.5% higher in larger groups (>=7) that contained less than 50% males . 3.3. Locomotion and: Geriatric Group Status (Previously Published) Among elderly chimpanzees, those housed in geriatric groups (average age of group members >35 years) showed lower percentages of locomotion compared to those housed in non-geriatric groups , and this difference in behavior coincided with lower levels of rough scratching and submissive behaviors (p < 0.05). 3.4. Locomotion and Voluntary Participation in Medication Choice (Previously Published) Locomotion was significantly higher across all study phases (when subjects received their arthritis medication dissolved in Gatorade) compared to the baseline phase . There were also decreases in rough scratching during the choice phase compared to the other phases (p < 0.05) . 3.5. Correlational Data between Locomotion and Other Welfare-Based Behaviors (Unpublished Data) As shown in Table 2, locomotion showed weak to moderate, yet still statistically significant correlations with several behaviors, including negative relationships with rough scratching, gentle scratching , and inactivity , and a positive correlation with behavioral diversity scores . There was also a statistically significant negative relationship between abnormal behavior and locomotion . Upon further inspection of the data, two outliers seemed to be skewing the relationship (one chimpanzee that displayed rocking behavior and another that displayed thumb-sucking). Upon removal of these two outliers, the relationship between abnormal behavior and locomotion was no longer significant (p = 0.10). 4. Discussion This paper summarizes and extends findings related to chimpanzee locomotion in captivity, specifically in terms of (1) manipulations to certain physical and social housing conditions and (2) participation in experimental paradigms. Across these four studies, a pattern emerged that seems to show a meaningful relationship between locomotion and conditions related to enhanced welfare, including increased space per animal, changes in housing type, housing in more diverse social groups, and voluntary participation in a self-medication program. Furthermore, the increased locomotion observed in these studies in response to these positive environmental conditions coincided with changes in other "positive" welfare-related behaviors, including decreased inactivity levels and behavioral diversity, and decreased rough scratching and submissive behavior. In two of the studies summarized here, we used changes in time spent in locomotion across experimental conditions to assess changes in well-being. For example, the increased time spent in locomotion following changes in space per animal and type of housing suggested an increase in well-being post-transfer. Similarly, the increased locomotion in Phases 2-6 of the medication choice study indicated an improvement in welfare from the baseline condition. In the other two studies, individual chimpanzees served in only one condition/group and well-being was compared across groups. The higher level of locomotion in groups with larger within-group age ranges, in larger groups with less than half males, and among elderly chimpanzees housed in non-geriatric groups suggested higher welfare of those groups compared to the other groups included in each respective study. These are similar, but also slightly different, comparisons, and locomotion was sensitive enough to differ in both types of assessments. We also found a statistically significant positive correlation between locomotion and another behavioral indicator of positive well-being (behavioral diversity), a negative correlation with an indicator of stress (scratching), and a negative correlation with inactive behavior (a behavior typically associated with decreased welfare when levels are high). We also found a negative correlation between locomotion and abnormal behavior when including all individuals in the analysis. However, after removing two outliers from the analysis, this relationship was no longer significant. Given these outliers, and the fact that the level of abnormal behavior in our sample was very low (mean = 0.67, SEM = 0.19, median = 0.00), this particular result should be interpreted with caution (although it is interesting to note that the two outliers that were removed from the analysis showed low levels of locomotion, one in the bottom 10th percentile and the other in the 20th). We did not find relationships with social play or grooming behaviors, which are often associated with positive well-being. Regardless, these relationships with other behavioral welfare indicators provide some additional evidence supporting the use of locomotion as an indicator of positive well-being. There are several benefits to the use of locomotion as a welfare indicator. First, there are fewer widely accepted behavioral indicators of positive welfare in chimpanzees (e.g., social play) than there are of negative welfare (abnormal behavior, self-directed behavior, rough scratching), and there has been a push toward the validation and increased use of positive welfare indicators . Based on the current findings, along with some of the research summarized earlier, we suggest that locomotion can be profitably added to the small list of positive welfare-related behavioral indicators available for use in behavioral research studies. Second, locomotion seems to be more sensitive to environmental differences or experimental changes than other measures of well-being. For example, rough scratching, abnormal behavior, and social play often occur at such low rates within individuals (sometimes below 1%) that statistical analyses can be difficult to perform, and/or the differences in levels across conditions are so small that they are not necessarily meaningful . On the other hand, baseline levels of locomotion in captive chimpanzees range from 5 to 12% . Changes across conditions and/or differences across groups in time spent in locomotion in the studies reported here were between 2 and 5%, equating to a difference of between 14 and 36 min per day of additional walking, climbing, hanging, and/or brachiating. In an aging chimpanzee population that is experiencing health-related issues (e.g., obesity, arthritis, cardiovascular disease, etc.), an average of an extra 20 min per day of voluntary movement can be an impactful and meaningful enhancement to welfare. Although locomotion occurs at higher rates than some other behavioral welfare indicators, levels of locomotion would likely increase only to a certain point under captive conditions. Time spent in locomotion in captivity is, under current management practices, going to be lower than time spent in locomotion for wild chimpanzees . Wild chimpanzees must travel to search for and access required resources (e.g., food, sleeping sites, water, patrols, etc.) . On the other hand, captive chimpanzees are provided the majority of these resources . It is possible that, when NHPs (including chimpanzees) do not need to move to access resources, they simply do not. As such, we should not expect locomotion to increase to a level that is comparable to that of wild chimpanzees following attempted enhancements to welfare. It is important to note that the relationship between locomotion and welfare is nuanced. First, the meaning or implication of changes in locomotion is context-dependent. For example, increases in locomotion in response to increased space per animal may be considered indicative of positive welfare, whereas increased locomotion in response to a predator is indicative of tension or anxiety . As such, it is important to consider the context when making conclusions about the meaning of the overall level of, or change in, locomotion in response to a manipulation. Second, locomotion as a welfare indicator must be distinguished from other behaviors that may resemble the behaviors that comprise locomotion. For example, stereotypic or repetitive locomotion (i.e., pacing) is indicative of poor welfare . As such, locomotion must be precisely operationally defined so as not to include behaviors such as pacing or agitated locomotion (e.g., ). In the current studies, the operational definitions in the ethogram included only normal locomotive behaviors, including walking, climbing, brachiating, and hanging, which were not characterized by the repetitive, unfocussed movement that distinguishes pacing behavior. In fact, pacing was purposefully excluded from locomotive behaviors, as it was included under "idiosyncratic movement" as an abnormal behavior . Additionally, locomotion was further demarcated to exclude "agitated locomotion", which was defined as "brisk, or rapid walking that often occurs with increased vigilance toward event, animal, or object" . Therefore, these findings reflect normal locomotion and do not reflect pacing or other forms of locomotion that are typically related to negative welfare states. Lastly, it is worth noting that locomotion has been found to be unrelated to certain measures of physical well-being. Previous studies have found that rates of locomotory behavior were not significantly correlated with scores on the Mobility Scoring System (MSS) or Fluency Scoring System , both of which reflect ease of movement. As such, it may be that, although chimpanzees' ease of movement may decrease (for example, with age, injury, or illness), their rates of locomotory behavior remain stable . 5. Conclusions We would like to note that we are not specifically advocating for particular changes to characteristics of the physical or social environment, or certain health or behavioral study practices (although the data across the studies outlined here do suggest some potential for welfare enhancements in these areas). Our purpose was to summarize and link findings regarding locomotion across the several published studies from our facility to highlight the pattern that emerged. We suggest that locomotion while considering contextual factors, may be a sensitive, and perhaps underutilized, measure of well-being in studies examining captive chimpanzee welfare. Overall, locomotion is easily observable, is of appreciable duration (allowing for robust statistical analyses), and is typically relatively easy to record, all of which make it a useful measure. As such, we are advocating for empirical evaluations that include time spent in locomotion as an indicator of well-being. Furthermore, since it is likely that previous behavioral studies included measures of normal locomotion in their ethograms (as these studies did), retrospective analyses of locomotion from such studies would also be useful. Acknowledgments We would like to thank the care staff at the Center for Chimpanzee Care for their unwavering care of the chimpanzees. Author Contributions Conceptualization, S.N.W.; methodology, S.N.W. and S.S.; formal analysis, S.N.W.; investigation, S.N.W. and S.S.; data curation, S.N.W.; writing--original draft preparation, S.N.W.; writing--review and editing, S.N.W. and S.S.; supervision, S.S. All authors have read and agreed to the published version of the manuscript. Institutional Review Board Statement The animal study protocol was approved by the Institutional Review Board of UTMDACC. Informed Consent Statement Not applicable. Data Availability Statement Data used in the four published studies as well as the correlational data included in this manuscript are available upon reasonable request. Please contact the corresponding author. Conflicts of Interest The authors declare no conflict of interest. Figure 1 Mean percentage of time spent locomoting across the three transfer types. Error bars represent the standard error of the mean. Locomotion was trending higher post-transfer in the dome (~142 square feet per animal) to corral (~645 square feet per animal, p <= 0.08) and significantly higher in the dome to double dome (~284 square feet per animal, p <= 0.05) transfers, but not significantly so in the corral to double dome transfer (p > 0.10). Figure 2 Mean percentage of time spent locomoting as a function of within-group age range. Error bars represent the standard error of the mean. Locomotion was trending higher in groups with a larger within-group age range (p = 0.067). Figure 3 Mean percentage of time spent locomoting as a function of group size and percentage of males in the group. Error bars represent the standard error of the mean. Within smaller groups (six or fewer chimpanzees), locomotion was significantly higher when at least half the chimpanzees in the group were males (p <= 0.05). Within larger groups (seven or more chimpanzees), the mean percent time spent locomoting was significantly higher in groups that comprised less than half male chimpanzees (p <= 0.05). Lastly, the mean time spent locomoting was highest in larger groups that contained less than half male chimpanzees (p <= 0.05). Figure 4 Mean percentage of time spent locomoting as a function of housing in a geriatric (group average age greater than or equal to 35 years) compared to non-geriatric (group average ages less than 35 years) groups. Error bars represent the standard error of the mean. Elderly chimpanzees housed in non-geriatric groups spent more time locomoting than those housed in geriatric groups (p <= 0.05). Figure 5 Mean percentage of time spent locomoting as a function of the experimental phase in the medication choice study. Error bars represent the standard error of the mean. The percentage of time spent locomoting was higher during the experimental phases of the paradigm, including the choice phase (during which chimpanzees received a choice between meloxicam and ibuprofen) compared to the baseline phase (prior to the start of the study) (p <= 0.05). Figure 6 Negative relationship between the mean percentage of time spent locomoting and the percentage of time spent rough scratching (dark circles and solid trend line) and gentle scratching (light squares and dotted trendline). Chimpanzees that exhibited higher levels of scratching showed lower levels of locomotion. Figure 7 Negative relationship between the mean percentage of time spent locomoting and time spent inactive. Chimpanzees that spent more time inactive exhibited lower levels of locomotion (p <= 0.05). Figure 8 Positive relationship between behavioral diversity scores and mean percentage of time spent locomoting. Chimpanzees that spent more time locomoting had higher behavioral diversity scores (i.e., exhibited a higher number of different positive welfare behaviors) (p <= 0.05). Figure 9 Relationship between mean time spent engaging in abnormal behaviors and mean percentage of time spent locomoting. Once the two outliers (circled) were removed, the relationship was no longer significant (p = 0.10). animals-13-00803-t001_Table 1 Table 1 Ethogram of locomotive behaviors. Ethogram of Locomotive Behaviors Hang All of the animal's weight is supported by wire of walls (i.e., animal is grasping wire with hands and feet), or the animal is hanging beneath the climbing bars. Walk Moving through space at a calm, steady pace on horizontal surface (may be on ground, plank, or platform). If walking occurs with play face, then it is considered play. Includes walking with food. Includes bipedal walking as a means of travel from one point to another. Climb Individual is ascending from one point to another in normal location (e.g., onto a platform, from top of cage, to side of cage, etc.). If this occurs with play face, then it is considered play. Includes climbing with food. Brachiate Animal uses arms to swing from one location to another. If this occurs with play face, then it is considered play. Includes brachiating with food. animals-13-00803-t002_Table 2 Table 2 Relationships between locomotion and welfare-related behaviors. Behavior r p-Value Social Play 0.01 0.88 Social Groom -0.10 0.30 Groom self -0.05 0.60 Rough Scratch ** -0.21 0.02 Gentle Scratch ** -0.25 0.01 Abnormal Behavior ** -0.20 0.03 Abnormal Behavior (excluding 2 outliers, n = 118) -0.15 0.10 Inactive ** -0.42 0.00 Behavioral Diversity Score ** 0.18 0.05 Note: n = 120 for all analyses unless otherwise noted. r = Pearson's correlation coefficient. ** Statistically significant. Disclaimer/Publisher's Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

PMC10000182

Probiotics as novel antibiotics' substitutes are verified to provide barriers for hindering the colonization of enteric bacterial pathogens with nutritional benefits. For enhancement of the probiotics' effectiveness, their integration within nanomaterials is a paramount tool to support the progress of new compounds with functional features. Therefore, we addressed the impact of effective delivery of probiotics (Bacillus amyloliquefaciens) loaded nanoparticles (BNPs) on performance and Campylobacter jejuni (C. jejuni) shedding and colonization in poultry. Two hundred Ross broiler chickens were divided into four groups fed various BNP levels: BNPs I, BNPs II, BNPs III, and BNPs-free diets for 35 days. Nanoparticles delivery of probiotics within broiler diets improved growth performance as reflected by higher body weight gain and superior feed conversion ratio, especially in BNPs BNPs III-fed groups. In parallel, the mRNA expression levels of digestive enzymes encoding genes (AMY2a, PNLIP, CELA1, and CCK) achieved their peaks in BNPs III-fed group (1.69, 1.49, 1.33, and 1.29-fold change, respectively) versus the control one. Notably, with increasing the levels of BNPs, the abundance of beneficial microbiota, such as Bifidobacterium and Lactobacillus species, was favored over harmful ones, including Clostridium species and Enterobacteriaceae. Birds fed higher levels of BNPs displayed significant improvement in the expression of barrier functions-linked genes including DEFB1, FABP-2, and MUC-2 alongside substantial reduction in cecal colonization and fecal shedding of C. jejuni. From the aforementioned positive effects of BNPs, we concluded their potential roles as growth promoters and effective preventive aids for C. jejuni infection in poultry. probiotic-loaded nanoparticle performance C. jejuni colonization barrier function poultry Deputyship for Research and Innovation, Ministry of Education in Saudi ArabiaINST007 This research was funded by the Deputyship for Research and Innovation, Ministry of Education in Saudi Arabia through Project number (INST007). pmc1. Introduction Feed additives as growth promoters have a robust impact on the production cost of broilers for covering alterations in profits due to instability in feed costs . The extensive use of antibiotics as feed additives for enhancing poultry growth performance and treatment of bacterial infections leads to the emergence of multidrug resistant (MDR) bacterial strains . Up until now, research is in progress exploring feed additives that can substitute antibiotics as prophylactics and growth promoters in poultry production. Probiotics became prospective antibiotic alternative additives in broilers owing to their impact on growth performance and the well-being of broilers compared to antibiotics . The main favorable effects of probiotics correlate largely with improved feed bioavailability and digestibility, boosting the immune system, saving health, and providing superior carcass composition . They have immunomodulatory roles in enhancing immunity against microbes and preventing exaggerated inflammatory responses, and they serve as biological barriers to protect the epithelial cells from being breached by pathogenic bacteria and to maintain epithelial integrity . As well, probiotic bacteria are able to impede the growth of pathogenic microflora in the gastrointestinal tract of birds owing to their roles in nutrient depletion, blockage of pathogens' target receptors on epithelial cells, and the creation of natural antibacterial products known as bacteriocins . In addition, the early institution of probiotics in the gut can provide a barrier against foodborne pathogen colonization . Bacillus-based probiotics have unique properties comprising growth promotion, immunomodulation, competitive exclusion , and production of a variety of extracellular substances and antimicrobial peptides against a wide range of pathogens . Bacillus amyloliquefaciens (B. amyloliquefaciens) is a strong Bacillus species that produces numerous extracellular enzymes such as cellulase, metalloproteases, proteases, and a-amylases that augment digestibility and nutrient absorption in addition to overall gut immune functions . Moreover, B. amyloliquefaciens produces bacteriocins with a consequence of gut resistance to infection, and it reduces noxious gases emission in chickens . Additionally, B. amyloliquefaciens CECT 5940 possesses a wide range of antimicrobial activity that can ultimately improve broilers' health . Disruption in the elegant interaction between gut microbiota, intestinal epithelium barrier, and host immunity plays a crucial role in the development of acute bacterial enteritis including campylobacteriosis , which is caused by Campylobacter jejuni (C. jejuni) and is considered one of the most serious foodborne pathogens causing zoonotic bacterial gastroenteritis in humans. Poultry are a major reservoir for C. jejuni as they provide it with an optimal temperature (42 degC) in their gastrointestinal tract, which is required for its colonization and proliferation . Campylobacter jejuni inhabits avian cecum with approximately 106-108 colony forming unit (CFU)/g without clinical illness . Pondering this situation, exploring ongoing promising strategies to reduce C. jejuni colonization in poultry and therefore in humans is needed. Administration of beneficial live probiotics can rescue the intestinal microbiota ecosystem balance, inhibit exaggerated immune responses against innocuous antigens, and hinder pathogenic bacterial colonization by competitive exclusion . Recently, B. amyloliquefaciens CECT 5940 could probably improve growth rate, nutrient intake, gut function, and negative impacts of necrotic enteritis challenge . However, its usage as an alternative to antibiotics is still limited because a harsh gut environment of gastric acids and bile salts may kill it and decrease its bioavailability with a consequent demand for high doses and long-term use of probiotics to exert their curative functions . Therefore, to overcome these limitations and use the advantages of available advancement in technology, recent studies have investigated different formulation methods for effective oral delivery of probiotics to protect them from harsh gut environments and sustain their therapeutic effects . Nanoparticle formulations provide a new gateway for achieving a safe delivery system for the ingredients used in feed by increasing their potency and concentration at their target sites . Moreover, using natural biodegradable biopolymers for encapsulation of these ingredients is widely applied . Based on this technology, co-encapsulation of probiotics strains for their engineering to improve their therapeutic efficiency has been reported . However, the effect of using B. amyloliquefaciens-loaded nanoparticles (BNPs) as alternatives to antibiotics for growth promotion and competing against campylobacteriosis needs to be defined. Therefore, we investigated for the first time in this study the effect of feeding poultry with BNPs on growth performance, barrier function, and immunity of broiler chickens in addition to its efficiency on cecal colonization of C. jejuni and its shedding in poultry excreta. 2. Materials and Methods 2.1. Ethical Statement All experimental procedures, bird rearing, and management were conducted upon approval from animal resources at the Faculty of Veterinary Medicine, Zagazig University, Egypt according to the rules of the Institutional Animal Care and Use Committee (ZU-IACUC/2/F/337/2022 approval number). 2.2. Bacillus amyloliquefaciens-Loaded Nanoparticles (BNPs) The strain of B. amyloliquefaciens probiotic CECT 5940 obtained from Evonik Nutrition and Care GmbH was propagated in Luria-Bertani (Oxoid, Hampshire, UK) broth and incubated at 37 degC. Then, B. amyloliquefaciens was stored in the bacterial cryopreservation fluid at -80 degC for further experiments. The BNPs were prepared through incorporation of the bacteria into chitosan (0.4%, w/v) (Sigma-Aldrich, St. Louis, MO, USA) nanoparticles as previously described . The prepared BNPs were stored by freezing at -20 degC in a cryoprotectant agent and were then dried and condensed for 18 h at -40 degC using LyoBeta 25TM freeze-dryer (Telstar, Terrassa, Spain). Finally, the dried cells were kept at 4 degC to be protected from light. The nano size and shape of the formulated BNPs were confirmed using transmission electron microscopy and Fourier-transform infrared spectroscopy characterization . 2.3. Birds and Experimental Design Considerations Two hundred Ross 308 male broiler chickens (42.21 g average initial weight) at 1 day old were provided from a commercial hatchery (Dakahlia Poultry Company, Dakahlia, Egypt). The birds were housed in divided floor pens for separation between groups at the Animal Care Unit at the Faculty of Veterinary Medicine, Zagazig University. The birds were divided randomly into 4 groups; each group had 5 replicates (10 birds each). The first group defined as control was fed the conventional diet and the three other groups were offered the conventional diet supplemented with B. amyloliquefaciens-loaded nanoparticles (BNPs) with three different doses [BNPs I (2.5 x 105 CFU/g of feed), BNPs II (5 x 105 CFU/g of feed) and BNPs III (7.5 x 105 CFU/g of feed)]. Both control and tested groups were fed the conventional or BNPs-containing diets for 35 days during the experimental period. All chicks in control and tested groups were co-housed at the same rearing conditions: temperature (33 +- 1 degC), which was gradually decreased every week until it reached 24 +- 2 degC at the end of the experimental period and humidity (around 60%) was constant during the whole experimental period. The diet was formulated according to nutrition specification of a Ross broiler handbook as presented in Table 1. Chemical analyses of all feed ingredients were performed using the standard method as endorsed by the Association of Official Analytical Chemists, AOAC77 . All animals were provided with ad libitum access to water and food during the whole experimental period. 2.4. Growth Performance and Mortality Indicators Individual birds were weighed and feed residues were determined to calculate feed intake for evaluation of different growth parameters of broiler chickens within the growth phases (1-35 days) including body weight gain for each phase [final body weight (g/bird) - initial body weight (g/bird)] and feed conversion ratio (FCR) [feed intake (g/bird)/weight gain (g/bird)] . Moreover, mortality rate was recorded during the period from 1 to 35 days. 2.5. Sampling Procedure At the end of the experimental period, the birds (5 per replicate) were weighed and euthanized by cervical dislocation. The intestinal contents and cecal and fecal samples were then aseptically placed in sterile Eppendorf tubes and kept at -80 degC for further quantification of intestinal microbial populations and evaluation of C. jejuni colonization and shedding via real-time (quantitative) polymerase chain reaction (qPCR) strategies. Moreover, pancreatic and jejunal samples (around 1 cm each) were excised, flushed three times with phosphate buffered saline, and subjected later to genes expression analysis utilizing reverse transcription-quantitative polymerase chain reaction (RT-qPCR) assays. 2.6. Reverse Transcription Quantitative Polymerase Chain Reaction (RT-qPCR) The expression of genes associated with digestive enzymes [alpha 2A amylase (AMY2A), pancreatic lipase (PNLIP), cholecystokinin (CCK), and chymotrypsin-like elastase family member 1 (CELA1)], barrier functions and antimicrobial defense [beta-defensin 1 (DEFB1), fatty acid-binding protein-2 (FABP-2) and mucin-2 (MUC-2)], and proinflammatory cytokines [interleukin-1beta (IL-1b) and tumor necrosis factor-alpha (TNF-a)] was done. Total RNA was extracted via RNeasy Mini kits (Qiagen, Cat. No. 74104) following the kits' instructions. RNA purity and concentration were determined using NanoDrop ND-8000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). The cDNA was then synthesized using RevertAidTM H Minus kits (Fermentas Life Science, Pittsburgh, PA, USA) according to manufacturer's instructions. The RT-qPCR assays were done using SsoAdvancedTM Universal SYBR Green(r) Supermix (Bio-Rad, 1725274) according to the manufacturer's instructions and analyzed using StratageneTM MX3005P qPCR thermocycler (Agilent Technologies, Santa Clara, CA, USA). The sequences of primers of target genes are illustrated in Table 2. Target genes expression was normalized using glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as a housekeeping gene. Relative fold changes in the target genes' expression were determined using 2-DDCT method . 2.7. Campylobacter jejuni Challenge Model A pandrug-resistant (PDR) and multi-virulent field C. jejuni strain was used in this experimental trial. It was previously recovered from cloacal swabs of recently slaughtered broiler chickens based on a previous research by one of the co-authors . The strain was inoculated into Bolton broth (Oxoid, UK) for 48 h at 42 degC in microaerophilic conditions (10% CO2, 85% N2 and 5% O2) using an anaerobic jar (Sigma-Aldrich) with CampyGen sachets (Oxoid). The inoculated broth was then streaked onto modified charcoal cefoperazone deoxycholate agar (Oxoid, UK) plates that were incubated under the previous microaerophilic conditions. The strain was then evinced to be resistant to 24 antimicrobials (amoxicillin, ampicillin, sulbactam-ampicillin, amoxicillin-clavulanic acid, cephalothin, cefoxitin, cefoperazone, cefepime, imipenem, aztreonam, nalidixic acid, ciprofloxacin, trimethoprim-sulfamethoxazole, doxycycline, erythromycin, azithromycin, clarithromycin, tobramycin, gentamicin, amikacin, linezolid, chloramphenicol, colistin, and clindamycin) of 10 different antimicrobial categories being identified as PDR. The stain was also proved to harbor three substantial virulence factors, which have vital roles in its pathogenesis (wlaN, virB11, and flaA) as previously detailed) . The challenge inoculum was adjusted to provide a viable concentration of 108 CFU/mL . At 30 days of age, the birds in all groups including control were orally infected through the crop gavage with 108 CFU/mL of C. jejuni (1 mL per bird). The experimental infection was then affirmed following the appearance of clinical signs via re-isolation and identification of the infecting C. jejuni strain in addition to re-investigating its antimicrobial resistance pattern and virulence genes profiling. 2.8. Real-Time Quantitative Polymerase Chain Reaction (qPCR) for Evaluating C. jejuni Shedding and Colonization Quantitative PCR assays were carried out to quantify the microbial populations including Bifidobacterium, Enterobacteriaceae, Lactobacillus, and Clostridium species in the avian intestinal contents at the end of the experimental period and to determine C. jejuni colonization in chickens' cecal samples and its shedding in their fecal samples 3 and 7 days post-infection (33 and 35 d of age). Genome DNA was extracted with QIAamp DNA fast DNA stool kit (Qiagen, Hilden, Germany) adopting the guidance of the manufacturer. Purity and concentration of extracted DNA were determined using NanoDrop (Thermo Fisher Scientific). The sequences of the primers used for quantification of intestinal microbial populations and C. jejuni are illustrated in Table 2. The number of copies of DNA was determined, in triplicate, using Stratagene MX3005P(r) RT-PCR instrument considering the created standard calibration curves prepared from serially diluted pure bacterial cultures and then the bacterial quantities were expressed as log10 of the CFU/g of the sample. 2.9. Statistical Analysis The results were evaluated via one-way ANOVA test using SPSS Inc. program version 20 (IBM Corp., Armonk, New York, NY, USA). Differences among the results were expressed as the standard error of the mean (SEM) and variations between means were assessed at a probability level of 5% using Tukey's test. All graphs were made using GraphPad Prism program Version 8 (San Diego, CA, USA). 3. Results 3.1. Efficacy of B. amyloliquefaciens-Loaded Nanoparticles on Growth Performance and Mortality Percentage of Broiler Chickens The effect of adding BNPs in different doses to broilers' diets within the whole experimental period (35 days) is shown in Table 1. Our data revealed that broiler chickens fed BNPs with higher doses (BNPs II and BNPs III) for 35 days exhibited significant (p < 0.05) increases in their body weight gain (2499 and 2569 g/bird, respectively) compared to those fed BNPs I (2430 g/bird) and control diets free of BNPs (2297 g/bird). While there was a reduction in food intake in the groups of birds fed BNPs compared to those in the control group, their efficiency for food utilization was improved (p < 0.05) specifically with higher BNPs doses as indicated by lower FCR in these groups compared to that in the control one. Notably, inclusion of BNPs with higher doses significantly (p < 0.05) decreased mortality rates compared with the challenged group (Table 3). 3.2. Efficacy of B. amyloliquefaciens-Loaded Nanoparticles on Expression of Digestive Genes Controlling Digestion The analysis of genes expression associated with digestive enzymes including AMY2a, PNLIP, CELA1, and CCK is illustrated in Figure 2. The results indicated that the expression levels of AMY2a and CCK genes attended their peaks (p < 0.05) following supplementation of BNPs III. A concentration-dependent manner upregulation of PNLIP gene (p < 0.05) was detected following BNPs' dilatory inclusion. Significant (p < 0.05) upregulation of relative expression of CELA1 gene was noticed in broilers fed diets supplemented with higher levels of BNPs compared to control ones fed diets free of BNPs. 3.3. Efficacy of B. amyloliquefaciens-Loaded Nanoparticles on Broiler Chickens' Intestinal Microbial Populations The abundance of intestinal microbial populations following BNPs supplementation is illustrated in Figure 3. As expected, there were increases in the abundance of Lactobacillus and Bifidobacterium and reductions in Clostridium and Enterobacteriaceae as indicated by qPCR in groups of birds fed BNPs compared to control ones. Although the differences among the three groups of birds fed different doses of BNPs did not reach statistical significance in reduction of Enterobacteriaceae and Clostridium species abundance unlike the control group, PNPIII showed the highest tendency in their reduction. Conversely, a high tendency in increasing Bifidobacterium populations was noticed in BNPs-fed groups compared to the control one. Likewise, BNPs III-fed birds had significant (p < 0.05) highest counts for both Lactobacillus and Bifidobacterium species. 3.4. Efficacy of B. amyloliquefaciens-Loaded Nanoparticles on Intestinal Barrier and Cytokines-Associated Genes Analysis of the relative mRNA expression of genes related to intestinal barrier and antimicrobial defense including DEFB1, FABP-2 and MUC-2, and proinflammatory cytokines comprising IL-b and TNF-a is depicted in Figure 4. We observed an overwhelming increase in the expression of MUC-2 gene after supplementing BNPs in broiler chickens' diets in a dose-reliant way. In addition, a proportional (p < 0.05) increase in the expression of FABP-2 gene was demonstrated in broiler chickens with increasing the dose of dietary BNPs when compared with the control group. The expression of DEFB1 gene achieved its highest peak in BNPs III-fed birds. Notably, prominent downregulation (p < 0.05) was noticed regarding IL-1b and TNF-a genes, especially after feeding higher BNPs levels when compared to the control group. 3.5. Efficacy of B. amyloliquefaciens-Loaded Nanoparticles on Shedding and Colonization of C. jejuni Investigating C. jejuni shedding and colonization in fecal and cecal samples 3 and 7 days post-infection using qPCR assay is shown in Figure 5. After 3 and 7 days of C. jejuni infection, there was a tendency of fecal and cecal C. jejuni counts toward reduction within groups of birds fed BNPs with different doses (BNPs I, BNPs II, and BNPs III) compared to those in the control group fed diets free of BNPs. Notably, a lower trend of C. jejuni counts was detected in fecal samples than cecal ones 3 and 7 days post-infection. At both intervals, inclusion of BNPs III in birds' diets remarkably (p < 0.05) reduced C. jejuni populations considering both sample types unlike the control group. Seven days post-infection, C. jejuni counts were significantly (p < 0.05) reduced in fecal and cecal samples of birds fed BNPs III compared to those in the control group (2.93 and 4.22 CFU/g vs. 6.79 and 8.16 CFU/g), respectively. 4. Discussion Higher productivity in the poultry industry has been complemented by the balance between nutrition, intestinal health, and animal welfare. Additionally, there are many impacts threatening this productivity such as the emergence of a large assortment of pathogens and bacterial resistance . The concept of using in-feed live probiotics strains in the poultry industry not only exerts a beneficial effect on overall growth performance parameters , but also signifies their use in reducing and eliminating the colonization of pathogenic bacteria, such as C. jejuni . Nevertheless, gut colonization and effectiveness of supplemented probiotics depend on various aspects involving the specificity of the strains relative to the host, digestive enzymes, bile acids, availability, nutritional status of the host, intestinal pH, stress, and form of substrate (prebiotics) . Thus, offering probiotic living cells with a physical barrier for escaping from unfavorable environmental conditions is a crucial consequence, which currently has obtained substantial interest . In this study, encapsulation of B. amyloliquefaciens in chitosan nanoparticles had beneficial effects on broiler chicken growth performance and reduced the colonization and shedding of C. jejuni. C. jejuni represents the most prevalent species accountable for 84% of cases diagnosed recently in Europe . Poultry is the main carrier of the bacteria and is responsible for the most outbreaks of campylobacteriosis through consumption of contaminated poultry meat . The bacteria can colonize and persist in the chicken gut during their lifetime without causing diseases . The emergence of MDR, extensively drug-resistant, and PDR strains , especially C. jejuni, which is resistant to the main drugs used in the treatment of severe post-acute sequelae of campylobacteriosis (fluoroquinolone and macrolides) evoked a warning sign about hazardous uses of antibiotics for enhancing poultry growth performance and treatment of bacterial infections . The use of probiotics such as Lactobacillus, Bacillus, and Bifidobacterium has been shown to be a promising alternative solution for reduction of pathogen burden in the avian gut and consequently breaks the transmission cycle to humans through the food chain. However, the effects of inclusion of BNPs in poultry diets to decrease the colonization and shedding of C. jejuni need to be defined. In this study, we have shown that supplementation of B. amyloliquefaciens coated within chitosan nanoparticles, especially at high doses in broilers' diets for 35 days, promoted their growth performance and increased their body weight gain. Moreover, feed utilization efficiency of broilers was improved as indicated by significant (p < 0.05) reduction in FCR and increase in BWG, especially in BNPs III-fed group. These results are consistent with previous data regarding the improvement in growth performance of broilers fed B. licheniformis spores supplement either alone or combined with mannan oligosaccharide feed additives . Correspondingly, probiotics can modify the intestinal ecosystem by supplying digestion enzymes, reducing pH, and increasing the activity of enzymes in the gastrointestinal tract . Similarly, reported a significant improvement in FCR and growth performance of broilers fed dried brewer grain (10%) subjected to probiotics fermentation. Different formulations used for probiotics delivery would affect their absorption; therefore, in this study, chitosan nanoencapsulated probiotics improved their resistance against gastric and bile acids, which consequently increased their bioavailability, absorption, and sustained effectiveness during the overall period of experiments (35 days). The growth performance of birds was strongly correlated with the digestibility and utilization efficiency of feed . Moreover, it has been known that dietary composition of broilers' rations could modulate the expression of digestive enzymes and nutrient transport-related genes . Therefore, our study revealed that a group of birds fed BNPs III for 35 days exhibited significant increase in relative mRNA expression of pancreatic digestive enzyme-linked genes, such as AMY2A, PNLIP, CELA1, and CCK. Aligned with our data, stated that feeding microbially fermented dried brewer grains not only improved growth performance and carcass dressing, but also stimulated expression of pancreatic digestive enzymes encoding genes as AMY2A, PNLIP, CELA1, and CCK. Microbiota and their metabolites prompt gut enteroendocrine cells to secrete gut hormones, which consequently influence metabolism . Our results indicated the ability of BNPs to stimulate gut hormones, such as CCK, which plays important roles in gastric motility, appetite, bile acids, and pancreatic enzymes release . Although less is known about how probiotics regulate CCK secretion, a recent research showed that feeding broilers for 42 days on microbially fermented olive pomace via two stages of solid fermentation using Bacillus subtilis followed by Lactobacillus casei upregulated the genes encoding pancreatic enzymes: CCK, AMY2A, PNLIP, and CELA1 . Furthermore, dietary inclusion of microbially fermented soybean meal enhanced pancreatic enzymes' activities in broilers . Moreover, dietary supplementation of B. amyloliquefaciens had significant efficacy in enhancing chymotrypsin, amylase, and lipase activities compared to groups fed antibiotics alone . Similarly, it has been reported that B. amyloliquefaciens (2 x 105 CFU/g diet) pretreatment either alone or combined with mannan oligosaccharides improved metabolic activity of intestinal cell energy-related genes through upregulation of mRNA genes expression of enzymes implicated in protein digestion. The mechanism by which probiotics regulate digestive enzymes secretion needs to be deciphered. However, this may be attributed to the role of probiotics in restoring intestinal normal architecture and achievement of larger healthy surface area for nutrient assimilation . Moreover, the role of probiotics in increasing the relative mRNA expression of pancreatic digestive enzymes could be related to their vital role in the activity of enteroendocrine cells to express the pancreatic enzymes encoding genes. Additionally, increasing the expression of genes encoding pancreatic lipase associated enzymes (PNLIP and CCK) could be secondary to the production of probiotics' metabolites including short chain fatty acids . Our data indicated increases in the abundance of Bifidobacterium and Lactobacillus species, which was accompanied by reduction in Enterobacteriaceae and Clostridium species populations. In parallel, probiotics not only exert their beneficial effects on host resistance through improving its immunity, epithelial function, and competitive exclusion of pathogens burden within the avian gut, but also through their effects on rescuing the normal microbial populations of the intestine via favoring the growth of beneficial commensals and inhibiting that of the opportunistic ones . Moreover, Bao et al. . reported an increase in the abundance of Lactobacillus species combined with reduction in Enterobacteriaceae and Clostridiales counts after inclusion of B. amyloliquefaciens in poultry diet compared to poultry fed a basal diet . The increase of Lactobacillus and Bifidobacterium within the cecal contents of broilers may be attributed to the ability of BNPs to produce extracellular enzymes as phytase, amylase, xylanase, and b-glucanase, which could improve food digestibility and utilization . In addition, accumulation of short chain fatty acids produced within microbial metabolic processes confers a suitable pH that would support the growth of beneficial bacteria and prevent that of harmful ones . Both Lactobacillus and Bifidobacterium provide substrates for augmenting the growth of butyrate-producing bacteria, which stimulates epithelial regeneration and improves host energy metabolism . Improvement of epithelial barrier functions as indicated by upregulation of MUC2 and IgA genes expression was observed following administration of Bacillus coagulans to chickens . Consistent with this finding, we found that improvement of growth performance of birds was correlated with upregulation of epithelial barrier functions-related genes including MUC2, DEFB1, and FABP2, specifically, in birds supplemented with BNPs III in their diet for 35 days. Avian defensin represents the innate antimicrobial peptide with a wide range of spectrum against bacteria, fungi, and protozoa . Moreover, its ability to kill bacteria is through alteration in cell membrane permeability and immunomodulatory functions involving chemoattraction for leukocytes to the injury site and elimination of bacterial lipopolysaccharides and lipoteichoic acids triggering proinflammatory response . The upregulation of avian defensin following BNPs supplementation suggests its role as an antimicrobial peptide against MDR bacteria as proved previously . Mucus is the first physical barrier protecting epithelial cells from microbial translocation and inflammatory response . Likewise, intestinal barrier is strengthened by a glycosylated mucin-rich layer produced by goblet cells . Supporting this view, probiotics have been proven to strengthen the integrity of the intestinal barrier by elevating the number of goblet cells that support the mucus layer . A previous study described that many Bacillus species were evidenced to upregulate the expression of intestinal mucin as demonstrated in our study. FABP-2 gene expression has been indicated as a biomarker for intestinal barrier in broilers' gut and its higher expression signifies its superior role in intestinal function . Similarly, our data showed upregulation of FABP-2 relative mRNA expression, which suggested that BNPs enhanced epithelial integrity and functions. In the present study, downregulation of intestinal gene expression levels of proinflammatory cytokines (TNF-a and IL-1b) were observed in broilers fed higher levels of BNPs. This observation indicated their suppressive role on proinflammatory cytokines, which in turn counteracted the inflammation . Several studies evaluated the in vitro and in vivo anti-campylobacter activities of different probiotics strains . However, the effect of dietary inclusion of probiotics-loaded nanocarriers on the improvement of birds' resistance against C. jejuni and their interaction with host epithelial cells need to be explicitly defined. Our data further demonstrated that the aforementioned beneficial effects of BNPs, especially with higher doses, led to significant reduction in cecal colonization and fecal shedding of C. jejuni. These data suggested the ability of BNPs to competitively exclude C. jejuni from broilers' intestine and prevent its colonization. From what has been declared above, our data explained several possible mechanisms by which BNPs could mediate the exclusion and inhibition of C. jejuni colonization and translocation within the avian gut. These mechanisms are mediated firstly through the improvement of nutrient digestibility and assimilation and secondly via their effects on improving intestinal morphology and functions through upregulation of FABP2 and MUC2 genes, which increased mucus secretion and prevented bacterial adhesion. Lastly, they exert antimicrobial impacts either through secretion of antimicrobial peptides, such as b-defensin, that breaks down the bacterial cell wall and consequently activates the innate immune response to control inflammatory process and prevent bacterial translocation. 5. Conclusions Our data suggested that encapsulation of B. amyloliquefaciens as a probiotic bacterium by chitosan nanoparticles enhanced its bioavailability and maintained its beneficial effects in the broiler chicken's gut. The augmented growth promoter properties of BNPs were detected in our study and supported by higher expression of digestive enzymes-related genes. Friendly gut bacterial species including Bifidobacterium and Lactobacillus species outnumbered the unfriendly ones following inclusion of dietary BNPs. Supplementation of BNPs could also enhanced gut barrier functions and consequently decrease C. jejuni colonization with a superior outcome of minimizing its fecal shedding. Acknowledgments The authors extend their appreciation to the Deputyship for Research and Innovation, Ministry of Education in Saudi Arabia, for funding this research work (Project number INST007). Author Contributions Conceptualization, H.I., D.I., S.E.S., A.W., R.M.E.-T. and M.I.A.E.-H.; methodology, D.I., A.W., R.M.E.-T., W.R.E.-G., B.A.A., B.A.-H.Y.A., S.M.A.-R. and M.I.A.E.-H.; software, D.I., A.W., R.M.E.-T., W.R.E.-G., B.A.A., S.E.S. and M.I.A.E.-H.; validation, H.I., D.I., R.M.E.-T., W.R.E.-G., B.A.A. and M.I.A.E.-H.; formal analysis, D.I. and M.I.A.E.-H.; investigation, H.I., D.I., A.W., R.M.E.-T., W.R.E.-G., S.E.S., B.A.A., B.A.-H.Y.A., S.M.A.-R. and M.I.A.E.-H.; resources, D.I., A.W., R.M.E.-T., W.R.E.-G., B.A.A., S.M.A.-R. and M.I.A.E.-H.; data curation, H.I., D.I., A.W., R.M.E.-T., W.R.E.-G., B.A.A., S.E.S., B.A.-H.Y.A., S.M.A.-R. and M.I.A.E.-H.; writing--original draft preparation, H.I., S.E.S., D.I., A.W. and R.M.E.-T.; writing--review and editing, D.I. and M.I.A.E.-H.; visualization, H.I., D.I., A.W., R.M.E.-T., W.R.E.-G., B.A.A., B.A.-H.Y.A., S.M.A.-R. and M.I.A.E.-H.; supervision, H.I., D.I., A.W., B.A.-H.Y.A., S.M.A.-R. and M.I.A.E.-H.; project administration, H.I., D.I., A.W., R.M.E.-T., W.R.E.-G., B.A.A., B.A.-H.Y.A., S.M.A.-R. and M.I.A.E.-H.; funding acquisition, D.I., A.W., R.M.E.-T., W.R.E.-G., S.E.S., B.A.A. and M.I.A.E.-H. All authors have read and agreed to the published version of the manuscript. Institutional Review Board Statement All experimental procedures, bird rearing, and management were conducted upon approval from animal resources at the Faculty of Veterinary Medicine, Zagazig University, Egypt according to the rules of the Institutional Animal Care and Use Committee (ZU-IACUC/2/F/337/2022 approval number). Informed Consent Statement Not applicable. Data Availability Statement Data presented in this research are offered upon request from the corresponding author. Conflicts of Interest The authors declare no conflict of interest. Figure 1 Characterization of Bacillus amyloliquefaciens-encapsulated nanoparticles using transmission electron microscopy (a) and Fourier-transform infrared spectroscopy (b). Figure 2 Effect of feeding Ross broilers (n = 5/group) different levels of Bacillus amyloliquefaciens-encapsulated nanoparticles (BNPs I, II, and III) on the expression of digestive enzymes-related genes compared to birds fed basal diet. Graphs represented the relative mRNA expression of alpha 2A amylase (AMY2A, (a)), chymotrypsin-like elastase family member 1 (CELA1, (b)), pancreatic lipase (PNLIP, (c)), and cholecystokinin (CCK, (d)). The data represented the relative mRNA expression normalized to GAPDH and the significance is p < 0.05. a-d Mean values within columns showing numerous letters differ statistically. Figure 3 Effect of feeding Ross broilers (n = 5/group) different levels of Bacillus amyloliquefaciens-encapsulated nanoparticles (BNPs I, II, and III) on the intestinal microbial populations compared to birds fed basal diet. Graphs represented the counts of Clostridium (a), Lactobacillus (b), Bifidobacterium (c), and Enterobacteriaceae (d) expressed as log10 of the CFU/g of the sample and the significance is p < 0.05. a-d Mean values within columns showing numerous letters differ statistically. Figure 4 Effect of feeding Ross broilers (n = 5/group) different levels of Bacillus amyloliquefaciens-encapsulated nanoparticles (BNPs I, II, and III) on the relative mRNA expression of mucin-2 (MUC-2, (a)), fatty acid-binding protein-2 (FABP-2, (b)), beta-defensin 1 (DEFB-1, (c)), interleukin-1beta (IL-1b, (d)), and tumor necrosis factor-alpha (TNF-a, (e)). The data represented the relative mRNA expression normalized to GAPDH and the significance is p < 0.05. a-d Mean values within columns showing numerous letters differ statistically. Figure 5 Effect of feeding Ross broilers (n = 5/group) different levels of Bacillus amyloliquefaciens-encapsulated nanoparticles (BNPs I, II, and III) on colonization and shedding of Campylobacter jejuni (C. jejuni) in cecal (a) and fecal (b) samples at 3 and 7 days post-infection compared to birds fed basal diet and the significance is p < 0.05. a-d Mean values within columns showing numerous letters differ statistically. animals-13-00775-t001_Table 1 Table 1 The ingredients of basal diets and nutrient composition. Item Starter (Days 1-10) Grower (Days 11-20) Finisher (Days 21-35) Ingredient (%) Soybean meal (48) 34.4 30.8 25.8 Ground yellow corn 59 61.5 65.5 Soybean oil 1.8 3 4 Calcium carbonate 1.2 1.2 1.2 Dicalcium phosphate 1.5 1.5 1.5 Common salt 0.3 0.3 0.3 Choline chloride 0.2 0.2 0.2 Anti-mycotoxin 0.1 0.1 0.1 L-Lysine HCL (Lysin, 78%) 0.35 0.3 0.3 DL-Methionine (Methionine, 99%) 0.25 0.2 0.2 Premix * 0.3 0.3 0.3 Chemical composition Lysine (%) 1.5 1.3 1.2 Methionine (%) 0.6 0.5 0.5 Available phosphorus (%) 0.5 0.5 0.5 Calcium (%) 1.2 1.2 1.2 Ether extract (%) 4.3 5.6 6.6 Crude fiber (%) 2.6 2.6 2.5 Crude protein (%) 23 21.5 19.5 Metabolizable energy (kcal/kg) 3106 3103 3200 * Premix provided per kilogram of diet: Se (selenate), 0.3 mg; I (iodide), 1.3 mg; Mn (sulphate and oxide), 100 mg; Fe (sulphate), 30 mg; Cu (sulphate), 14 mg; Zn (sulphate and oxide), 120 mg; thiamine, 4 mg; pyridoxine, 6 mg; pantothenate, 14 mg; niacin, 50 mg; biotin, 200 mg; cyanocobalamin, 15 mg; riboflavin, 7 mg; cholecalciferol, 6000 IU; retinol, 10.000 IU; folate, 3 mg; and tocopherol acetate, 70 mg. animals-13-00775-t002_Table 2 Table 2 Target genes and primers used for qPCR assays. Specificity/Target Gene Primer Sequence (5'-3') Accession No./ Reference Digestive enzymes AMY2A F: CGGAGTGGATGTTAACGACTGG R: ATGTTCGCAGACCCAGTCATTG NM_001001473.2 CELA1 F-AGCGTAAGGAAATGGGGTGG R-GTGGAGACCCCATGCAAGTC XM_015300368.1 CCK F: AGGTTCCACTGGGAGGTTCT R: CGCCTGCTGTTCTTTAGGAG XM_015281332.1 PNLIP F: GCATCTGGGAAG|GAACTAGGG R: TGAACCACAAGCATAGCCCA NM_001277382.1 Intestinal microbiota/16S rRNA Lactobacillus species F: CACCGCTACACATGGAG R: AGCAGTAGGGAATCTTCCA Bifidobacterium species F-GCGTCCGCTGTGGGC R-CTTCTCCGGCATGGTGTTG Enterobacteriaceae F: CATTGACGTTACCCGCAGAAGAAGC R: CTCTACGAGACTCAAGCTTGC Clostridium species F: AAAGGAAGATTAATACCGCATAA R: ATCTTGCGACCGTACTCCCC Barrier functions and antimicrobial defense MUC-2 F-AAACAACGGCCATGTTTCAT R-GTGTGACACTGGTGTGCTGA NM_001318434 FABP-2 F: AGGCTCTTGGAACCTGGAAG R: CTTGGCTTCAACTCCTTCGT NM_001007923 DEFB1 F: AGCCTGTCTGCCTGGAGTAG R: GATGAGGAGAGGCTTCATGG XM017337690.1 Proinflammatory cytokines IL-1b F: GCTCTACATGTCGTGTGTGATGAG R: TGTCGATGTCCCGCATGA NM_204524 TNF-a F: CGTTTGGGAGTGGGCTTTAA R: GCTGATGGCAGAGGCAGAA NM_204267.1 Campylobacter jejuni mapA F: CTATTTTATTTTTGAGTGCTTGTG R: GCTTTATTTGCCATTTGTTTTATTA Internal control GAPDH F: GGTGGTGCTAAGCGTGTTA R: CCCTCCACAATGCCAA X01578.1 AMY2A: alpha 2A amylase, CELA1: chymotrypsin-like elastase family member 1, CCK: cholecystokinin, PNLIP: pancreatic lipase, MUC-2: mucin-2, FABP-2: fatty acid-binding protein-2, DEFB1: beta-defensin 1, IL-1b: interleukin-1beta, TNF-a: tumor necrosis factor-alpha, GAPDH: glyceraldehyde 3-phosphate dehydrogenase. animals-13-00775-t003_Table 3 Table 3 Effect of feeding male Ross broilers a diet formulated with three different levels of Bacillus amyloliquefaciens-encapsulated nanoparticles (BNPs I, II, III) on growth performance parameters. Parameter Control BNPs I BNPs II BNPs III p Value SEM Initial body weight (g/bird) 42.00 41.40 42.60 42.60 0.99 0.71 Overall growth Final body weight, g/bird 2339 c 2471 b 2542 a 2612 a <0.001 21.54 Body weight gain, g/bird 2297 c 2430 b 2499 a 2569 a <0.001 18.63 Feed intake, g/bird 4163 a 3925 b 3740 c 3800 bc <0.001 29.78 Feed conversion ratio 1.82 a 1.61 b 1.50 bc 1.48 c <0.001 0.05 Mortality % (1-35 d) 3 a 2 ab 1 b 1 b 0.025 0.37 Control: group of birds fed basal diet; BNPs I: group of birds fed diet containing Bacillus amyloliquefaciens-encapsulated nanoparticles at the level of 2.5 x 105 CFU/gm; BNPs II: group of birds fed diet containing Bacillus amyloliquefaciens-encapsulated nanoparticles at the level of 5 x 105 CFU/gm; BNPs III: group of birds fed diet containing Bacillus amyloliquefaciens-encapsulated nanoparticles at the level of 7.5 x 105 CFU/gm; SEM: standard error of the mean. Values with various letters in the same row differ significantly (p < 0.05). 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PMC10000183

The African tropical forests host an inestimable number of resources, including food, medicine, vegetal and animal species. Among them, chimpanzees are threatened with extinction by human activities affecting their habitats, such as forest product harvesting, and/or more directly, snaring and trafficking. We aimed to better understand the spatial distribution of these illegal activities, and the reasons for setting snares and consuming wild meat in an agricultural landscape (subsistence farming and cash crops) densely populated near a protected area (Sebitoli, Northern part of Kibale National Park, Uganda). To carry out this study, we combined GPS records of illegal activities collected with group counts (in total, n = 339 tea workers, 678 villagers, and 1885 children) and individual interviews (n = 74 tea workers, 42 villagers, and 35 children). A quarter of illegal activities collected (n = 1661) targeted animal resources and about 60% were recorded in specific areas (southwest and northeast) of the Sebitoli chimpanzee home range. Wild meat consumption, which is illegal in Uganda, is a relatively common practice among participants (17.1% to 54.1% of respondents depending on actor types and census methods). However, consumers declared that they eat wild meat unfrequently (0.6 to 2.8 times per year). Being a young man coming from districts contiguous to Kibale National Park particularly raises the odds of consuming wild meat. Such an analysis contributes to the understanding of wild meat hunting among traditional rural and agricultural societies from East Africa. biodiversity management wild meat chimpanzee social science illegal harvesting spatial analysis Uganda wildlife conservation Fondation Prince Albert II de Monaco and the Great Ape Conservation ProjectThis research was funded by Fondation Prince Albert II de Monaco and the Great Ape Conservation Project. pmc1. Introduction Besides high human, developmental, and financial investments in wildlife conservation worldwide and access limitation to protected areas (PAs) , illegal wild meat hunting (here, defined as poaching) continues to threaten terrestrial mammals' extinction . Wild meat (a wild animal killed for consumption, which is different from bushmeat killed for trade ) serves as a vital resource in many rural, lower-income regions of sub-Saharan Africa, and near tropical forests it is often a primary source of protein for rural populations . Its consumption tends to prevail in areas with greater biodiversity indices, which also frequently experience higher poverty and food insecurity . Nevertheless, the dependence of households on wild meat is particularly lacking documentation in East Africa . Recent increases worldwide in illegal wildlife harvesting are less related to local use and traditions in provenance countries than to an increase in wealth and a decrease in resources in countries importing harvested species or body parts (sent from Africa to Asia; ). Therefore, wild meat hunting and poaching (hunting becomes poaching when the practice does not follow the law/rules) can respond to different motivations such as harvesting wild meat for consumption or trade (locally and abroad ). To regulate both practices, most countries require hunters to have hunting permits and refer to quotas. Following international guidelines, countries also regulate subsistence hunting through laws that restrict hunting certain species (protected or non-protected), during certain time periods (months, day/night, seasons, etc.), and using specific weapons/tools (riffles, snares, traps, dogs, etc.). However, in practice, it is not this simple: reforming and adapting the regulatory framework of local communities hunting for food is necessary , conservation militarization is questionable , and development projects aiming to reduce wild meat hunting and/or poaching and maintain food security need a large, adaptable, and clear framework . More research on the prevalence of wild meat consumption and its drivers has been conducted in West Africa and Central Africa than in East Africa. Additionally, across Africa, cultural, sociopsychological, and sociodemographical factors driving wild meat consumers' behaviors remain understudied . Some studies show that wildlife illegal harvesting is determined by non-exhaustive ecological, social, and economic drivers such as the frequency of game species, poverty, countries/areas of provenance, ethnic groups, cultural values/beliefs, revenge from crop-raiding animals, lack of access to alternative incomes, heath issues, the distance to local markets, and/or the frequency of patrols . Species abundance and wild meat affordability are often cited as main predictors of harvest levels , variables such as taste or health issues are often considered secondary factors for wild meat food choices , and remoteness or landscape characteristics have been rarely investigated . Indeed, the driving factors of wild meat consumption are complex and variable . In the end, motives to consume legal or illegal wild meat are distinct, mainly relying on case-by-case studies , and are more likely to result from a combination of factors, with some being more prevalent than others depending on each case. Consequently, different responses should target different factors , which is our aim here. In East Africa, and specifically, in Uganda, wild meat hunting is forbidden within parks and their surrounding areas . The continued existence of a global market for trade in natural resources acquired through illegal means, as well as a lack of data and research for decision and policy makers to implement successful wildlife management and regulation, has been noticed . Between 2005 and 2009, over 71% of households interviewed on their incomes (cash or subsistence) that were located around two protected areas and one unprotected area reported having participated in wild meat hunting . Such practices were related to different reasons in different areas . Yet, conservation strategies used over the last two to five decades in/around a Ugandan National Park seem to have been effective for protecting the park and animals living within . Here, we aim to add a new wild meat consumption case in East Africa to the literature, and identify the drivers of wild meat consumption around a PA in order to design better local alternatives and interventions to reduce pressure on wildlife in a context where both human density and animal species diversity are high. We focus on the Sebitoli area, located in the extreme north of Kibale National Park (hereafter, Kibale NP), southwestern Uganda. Besides a combination of international and national policies trying to conserve different wildlife forms , the illegal activity index is high in the area . Chimpanzees (Pan troglodytes schweinfurthii; CITES and IUCN) are endangered. In the Sebitoli community they are indirectly victims of illegal snare injuries . Indeed, snares are likely directed at small game meat (duikers, bush pigs, etc.) not at primates. However, it threatens primate conservation and a non-negligible part of tourism income . Additionally, over the course of the study a chimpanzee was found dead and smoked in a surrounding household (august 2016). Therefore, to estimate the threat animals face in the Sebitoli area, we aimed to obtain a spatial understanding of illegal activities targeting wildlife (objective 1), to identify the targeted species and evaluate the frequency of wild meat consumption (objective 2), and to discuss, in general, drivers of domestic and wild meat consumption in the area (objective 3) with six-year-old data. This study will contribute to the PA's management plan, help identify and compare wild meat consumption drivers with those identified in other studies, and add to the understanding of this practice in Uganda in particular, and in East Africa in general. 2. Materials and Methods 2.1. Study Area and Inhabitants Kibale NP (795 km2) was established in 1993 and is currently under Uganda Wildlife Authority (UWA) management. It covers three districts: Kyenjojo, Kabarole, and Kamwenge. Local communities can access the park by request to the UWA through resource use agreements . The area of Sebitoli (25 km2) is densely populated with humans and wildlife . Landscapes are anthropogenic, combining tea, plantain, and eucalyptus plantations; small-scale food gardens; a busy tarmac road ; and a high human population density that tripled between 1959 and 1990 and is currently as high as 293 inhabitants/km2 in subcounties including Kibale NP . As wild animals from the park (especially elephants, baboons, and chimpanzees) raid crops in local communities' gardens (maize, cassava, bananas, etc.), human-wildlife conflicts around Kibale NP are recurrent . Batooro and Bakiga are the main tribes in the area , and Batooro are particularly represented in the north of the park . Batooro are rare meat eaters and their feeding taboos regarding meat consumption can favor the conservation of some wild species, including primates . Additionally, there is not a large wild meat market around Kibale NP: most demands are minimal because most illegally harvested meat (bush pigs and small antelopes) are consumed locally and are not supplied to large external markets . Outside of the park, inhabitants practice subsistence farming, and some families work in the large tea plantations . Migrant workers from other districts (not contiguous to Kibale NP) or other countries such as Rwanda can comprise up to 40-60% of the tea workforce, depending on the size of the tea concessions . Tea plantations cover a particularly large surface outside of the park and play a buffering role between the park's edge and palatable crops (maize, potatoes, millet, etc.). However, few studies have focused on tea workers' way of life . 2.2. Snare Removal and Illegal Activity Patrols in the Sebitoli Area The Sebitoli area benefits from the presence of UWA rangers patrolling the park to monitor illegal activities and interacting with the local communities neighboring the park about wildlife conservation. It also has benefitted from the presence of the Sebitoli Chimpanzee Project (SCP) since 2008. In addition to UWA patrols, research teams studying chimpanzees in Kibale NP also run snare removal projects (Kanyawara Snare Removal Project; Ngogo Chimpanzee Project; and SCP). At SCP, three local community members were recruited for this task beginning on May 29th, 2015 (study period: 29 May 2015-30 November 2016, 271 patrol days). Using transects and their knowledge of the park, they patrolled the Sebitoli chimpanzee home range 5/7 days for 6-8 h/day using GPS to record illegal activity locations where the targeting of natural resources occurred (snaring, tree cutting, charcoal burning, etc.) and datasheets to record illegal activity characteristics (date, hour, GPS location, type and oldness of activity, amount of evidence, etc.). They disactivated and confiscated any evidence (snares, spears, cables, etc.) that they brought back to the Sebitoli research site, where they were stored. This spatial information was later mapped by SB. 2.3. Individual Interview and Group Count We aimed to compare the legal and illegal animal protein access of tea workers and villagers since both populations live and work within a close distance of the chimpanzee home range and seem to experience sociodemographic, economic, and cultural differences . Children were also included in our sampling, as they represent an important proportion of the local populations and are sensitive to environmental degradation, such as species loss . Group counts and individual interviews were carried by the first author (SB) and a local translator. The two methods were designed to be complementary to estimate wild meat consumption in the area. In group counts, votes (via boxes and hands) were opportunistically set up before other SCP activities and described wild meat consumption in a quantitative way from a large sample of respondents (no sociodemographic characteristics identified outside of the actor type--villager, tea worker, and children). In semi-structured interviews, wild meat consumption was qualified with more details and time from a smaller sample of respondents (including sociodemographic characteristics and the discourse of participants). 2.3.1. Individual Interviews Interview Process Tea workers, villagers, and children had the context of the survey verbally explained to them (e.g., a postdoctoral research study on domestic and wild meat consumption, and relationships with wildlife), the anonymity of their name and the possibility to withdraw from the survey at any time were guaranteed, and both SB and interviewees signed a consent form after each participant agreed to participate in the survey. A 30 min semi-structured interview was then administered by SB and a translator . Questions focused on: (1) domestic animal protein consumption (frequency, buying location, and transport means); (2) wild meat consumption and illegal activities knowledge (frequency of consuming wild meat, and if respondents did not eat bushmeat they were asked the frequency they come across with it, number of poachers known, targeted species, and reasons to eat wild meat); (3) relationship with wildlife (chimpanzee knowledge and crop-raiding levels); and (4) sociodemographic characteristics. Most questions were closed, but questions related to wild meat consumption were more open and allowed for free speech. In this case, responses were later classified by SB into the main categories. Tea Workers A tea company granted us access to tea worker camps and allowed us to carry out interviews with their employees during working hours between May and July 2016. SB came to an agreement with 8 tea estate managers on when to carry out interviews at tea camps located around the Sebitoli chimpanzee home range (average distance of 309 m, range: 151-576 m from Kibale NP). In the morning, interviewers came to a tea camp, presented the survey to tea workers, and interviewed workers who volunteered one after the other in an isolated place near the tea fields. A total of 74 workers were interviewed (4 to 11 workers interviewed per camp). The number of tea workers working at each camp varied between 62-178 workers (median: 143). Some workers reside in villages, whereas some reside at the tea camps for free, with a proportion varying between estates (18.6-88.7%; median: 49.6%) (Rwenzori Commodities Ltd., Fort Portal, Uganda, unpublished data). At the tea camps, workers benefit from individual bedrooms where they can occasionally host their family and share common cooking fireplaces and sanitary installations with other workers staying at the camp. Additionally, the tea company provides free lunches to tea workers. Villagers and Children Four village chiefs (LC1) and two head teachers (primary schools) located around the Sebitoli chimpanzee home range granted us permission to interview local inhabitants and children, choosing days when they would be available between October and November 2016. The same voluntary selection process was applied as for the tea workers. A total of 42 adults (10-12 per village) living around Kibale NP (average distance of 300 m, range: 2-572 m from the park's edge) were interviewed at their household in four villages during their daily activities. Additionally, 35 children (15-20 per school) living at the park's edge were also interviewed in two schools (average distance of 2000 m, range: 1349-2668 m from Kibale NP) during class hours. 2.3.2. Group Counts We took advantage of a chimpanzee awareness presentation on chimpanzee biology and behavior conducted by SCP that is a regular program of the project to ask questions about wild meat consumption. The program was conducted in 11 tea worker camps, 20 villages, and 14 schools located around the Sebitoli chimpanzee home range between June and December 2016. More than 3900 persons attended presentations, mainly in schools (n = 2722 persons, median: 181 pers/school, range: 22-598; 1 nursery school, 8 primary schools, 1 high school, and 4 institutes/vocational schools), villages (n = 739 persons, median: 49 pers/village, range: 15-99) and tea worker camps (n = 456 persons, median: 41 pers/camp, range: 25-64). No specific sampling method was used to select respondents. After setting up an appropriate time with tea managers, LC1s, and head teachers, the aim of the presentation was explained to the people who came, and it was explained that on a voluntary and anonymous basis, the SCP team would like to ask them two questions about wild meat consumption. Informed consent was verbally given by adults and children, and only people who volunteered to participate contributed. A total of 2902 persons answered: "Do you eat bushmeat?", and if yes, "Do you eat bushmeat more than once a month?". Two different methods to collect responses were used for the different age groups. The ballot-box method was used with adults (villagers and tea workers): two boxes were set out of sight and participants dropped a paper (one paper yes, and one paper no) in each of these boxes to answer questions . This method has the advantage of reducing the social desirability bias and the disadvantage of showing a trend without the possibility to link individual behavior and explanatory variables . With children in schools, hand votes were used to respond to the same two questions. This method has the advantage of reducing children's confusion that can occur with two sets of boxes, but has a social desirability bias due to other pupils' presence (teachers were asked to leave the classroom) during votes. 2.4. Analyses Data acquisition types, methods, sample sizes, research questions, and analyses are presented in Figure 1 and Figure A4 to help synthesize the key information. 2.4.1. Geospatial Analyses Illegal activity locations were recorded by the SCP snare removal team during their patrol. SB took the locations of each tea camp, village, and school and georeferenced them all using GPS coordinates (GPS Garmin 64s; ArcGIS 10.2; geodesic system-WGS 84; cartographic projection-UTM 36 N). To facilitate spatial analysis, the Sebitoli chimpanzee home range was divided in four areas of relative equivalent size (northwest (NW) 7 km2, northeast (NE) 6 km2, southwest (SW) 8 km2, and southeast (SE) 6 km2). Euclidean distances of illegal activities to the edge and to the road were assessed to evaluate which border of the forest was more at risk. The Euclidean distance was also calculated between participants' residences and the sellers of domestic animal protein by using the centroids of villages/trading centers/camps to evaluate their accessibility. 2.4.2. Statistical Analyses We used bi-/multivariate analyses and regression models to estimate the relationships between variables depending on key research questions . With the GPS sample (n = 1661), the relationships between types of illegal activities, Euclidean distances to the road and the edge, and the sides of the park were assessed though a multiple logistic regression. In the group count sample (n = 2902), we used a chi2 test to estimate the relationship between actor type and wild meat consumption . In the individual interview sample (n = 151), five distinct analyses were conducted: (i) A chi2 test was used to estimate the relationship between actor type and wild meat consumption . (ii) To assess which variables were associated with wild meat consumption, we carried out a multiple generalized linear regression model. Given the distribution of the response variable (frequency of consuming wild meat, i.e., a count variable) and the hierarchical structure of the data (151 individuals nested into 28 residency locations), we estimated the following random intercept multilevel (i.e., mixed) Poisson model:yij~Poisson mij for i=1,...,151andj=1,...,28lnmij=b0+b1xi1++bkxik+(eij+m0j) where i denotes the individuals, j is the residency locations, b is the parameters estimated by the maximum likelihood (Laplace approximation), xik is the value of the kth covariate for individual i, and eij and uoj are the two residual components at both the individual and location residency levels. Adding location residency as a random effect allowed us to account for between-location heterogeneity while controlling for within-location spatial dependence. Multicollinearity among regressors was previously verified through VIF values. Covariates with a VIF > 2 were removed, leading to the exclusion of the side of the park and ethnicity variables. The final model includes the five following regressors: sex, age, actor type (children, tea workers, or villagers), provenance (contiguous/non-contiguous to Kibale NP, Kabale, and Rwanda) and number of people in the household. (iii) We used the Wilcoxon test to assess the relationship between wild meat consumption and salary , as well as for Euclidean distance to market and actor type . (iv) The relationship between the percent of salary respondents spent on food (response variable) and actor type was estimated through a beta regression. Beta regression is a class of model used when the response variable is beta-distributed, which is commonly true when it is between 0 and 1 for such proportions. As other generalized linear models, beta regression relates the mean response to the regressors through a link function. (v) To assess whether the Euclidean distance to the market was equivalent in function of the side of the park respondents reside in, we used a Kruskal-Wallis test. In all the GLM presented in this study, coefficients were exponentiated (i.e., leading to odds ratios (OR)) for an easier interpretation of elasticities. R software with betareg and lme4 packages was used to perform statistical analyses. 3. Results 3.1. Illegal Activity Spatial Distribution Between May 2015 and November 2016, the SCP georeferenced a total of 1661 illegal activities (9 different types, targeting animal and vegetal species) inside (n = 1436) and outside (n = 255) the park in the Sebitoli area . Overall, illegal activities targeted more vegetal (74.7%) than animal resources (24.7% including 24.6% of snares), and were found closer to the park's edge (mean: 165.2 m) than the road (mean: 1529.2 m) . Animal resources were more likely to be found in the NE (concentration of 1/3 of snares collected in the area during the study period). On that side, the tea company had eight tea camps/factories at the border of the forest. The logistic regression confirmed that animal resources have fewer chances to be found in the northwestern (OR = 0.28, 95% CI = [0.19-0.41], p < 0.001) and southern (OR = 0.33, 95% CI = [0.23-0.49], p < 0.001) sides of the park compared to the NE side and the area close to the road . 3.2. Wild Meat Consumption in Group Counts Wild meat was said to be eaten by 27.3% of overall participants according to the group counts (n = 2902, Table 1). It was reported to be more consumed among villagers (47.9%) than tea workers (31%) or children (19.3%), and this difference was significant (kh2 = 209.08, df = 2, p < 0.001). Additionally, the proportion of children eating wild meat more regularly throughout the year was more important (38.9%) compared to villagers (31.2%) or tea workers (26%). 3.3. Wild Meat Consumption in Individual Interviews 3.3.1. Descriptive Statistics According to individual interviews, meat consumption is generally low in the Sebitoli area. About 60% of respondents do not eat domestic animal protein , eat it less than once a month, or eat it once a month when it is accessible . Additionally, even if about 40% of respondents in individual interviews declare to eat wild meat, it happens rarely: 2.8 times/year for tea workers, 1.3 times/year for villagers, and 0.6 times/year for children (Table 2). Individual interview results differ from group counts. Here, more tea workers declare to eat wild meat (54.1%) compared to villagers (35.7%) and children (17.1%), and this difference is significant (kh2 = 13.975, df = 2, p < 0.001). From the interviews, the main reasons to eat wild meat were that its cheaper in price compared to domestic meat (43.7%), unknown motives (20.5%), culture/tradition (19.9%), taste (9.9%), and other motives (medicine, distance to access domestic meat, and revenge against crop-raiding animals-6%). Participants in interviews mainly consumed bushbuck (24.5%; Tragelaphus scriptus), red duiker (15.9%; Cephalophus harveyi), bush pig (12.6%; Potamochoerus porcus), and edible rat (6.6%; unknown species). Primates were cited by 5.3% of respondents (seven out of eight were children). Additionally, 26.5% of respondents did not know which species were consumed. 3.3.2. Results of the Mixed Poisson Model of Wild Meat Consumption Frequency The mixed Poisson model (location residencies as a random variable) exhibited four significant variables (Table 3). Among them, the strongest association was sex. On average, males consume 3.08 (95% CI = [1.83-5.19]) times more wild meat than females, whereas all other variables held constant. Respondents coming from the Kabale district consume 0.50 (95% CI = 0.29-0.85) less than those coming from districts contiguous to Kibale NP. Regarding continuous variables, age was negatively associated with wild meat consumption (IRR = 0.96, 95% CI = [0.94-0.99]; meaning that a one-year increase in age was associated with 0.04% less meat consumption), whereas the number of people sustained in the household had a positive effect (IRR = 1.13, 95% CI = [1.03-1.23]). The regression's interclass correlation coefficient (ICC = 0.43) indicates a strong effect of residency locations in the model, but no clear patterns appear between tea camps and village residencies. In summary, being a young man coming from districts contiguous to Kibale NP particularly raises the odds of consuming wild meat when all other covariates are kept constant and controlled for within-residency location dependence. 3.3.3. Complementary Factors from Individual Interviews (Adults Only) This section offers complementary data analyses from variables that were not available for the entire individual interview sample. Indeed, for some questions (salary and market location for domestic meat) only adults were able to answer. Yet, this information is useful when discussing our results. Revenues and Percentage Spent on Food The difference between adult tea workers' (n = 74) and villagers' (n = 42) revenues were not significant . Villagers tend to spend more money on food than tea workers (97.6% vs. 13.5% had a self-sufficient garden and most tea workers lived in camps without their family where they were offered free lunches) while their main occupation was subsistence agriculture (61.9%), but this relationship was not significant according to the beta regression model . Geographical Distance to Domestic Meat Three market centers, Fort Portal, Kaswa, and Ntoroko, with distances of 16 km, 4.4 km, and 2.5 km from the forest edge, respectively, attracted domestic meat buyers . However, to access domestic animal protein, adult respondents travelled a longer or shorter distance. Significant differences existed in the distance to access domestic meat yearly (Kruskal-Wallis chi-squared test= 26.434, df = 3, p < 0.001), and this difference was stronger between the NE and SW (respective mean: 25.6 km and 32.8 km, pairwise comparison p < 0.001) than the NE and SE (respective mean: 25.6 km and 20 km, p < 0.01), and the SE and SW (p < 0.05). No significant difference existed between the NW (mean: 24.5 km) and other areas of Sebitoli. Additionally, a significant difference existed between tea workers' and adult villagers' Euclidean distance to access/buy domestic meat (Wilcoxon test W = 1191.5, p < 0.05; villager mean: 21.5 km; tea worker mean: 29.2 km). 4. Discussion Over an 18-month study period, 24.6% of overall censused illegal activities consisted of targeting animals (snares), putting them at risk to be trapped, especially in the northeastern and southwestern sides and at the forest edges. Results estimating wild meat consumption among the three groups differed between group count and individual interview methods. Children were always consuming wild meat the least, but different results regarding adult and tea worker consumption occurred depending on the methods used. Results from the individual interview method may be more reliable as more time and attention were taken in collecting information. 4.1. Spatial-, Social-, and Species-Related Factors Affecting Wild Meat Consumption In the Sebitoli area, the park is probably accessed from its edges (rather than the road) to extract resources. This is common in such a conservation context , especially for forest products . Surprisingly, the setting of snares occurred close to the forest edge, where chances of being caught are likely higher. More snares were collected in the NE where crop-raiding impacts are of a medium level relative to other locations around the park , but they were also collected in the SW of the Sebitoli area where group count participants mostly declared to eat wild meat, distances are longer to access domestic meat, and crop-raiding impacts are high . This edge effect supports the belief that large PAs provide a good option to prevent species losses . The stability of illegal activity hotspots (e.g., location in previous years) may be a good predictor for illegal activities in PAs , which can be helpful to establish de-snaring strategies . Our interview methods highlight that between 17.1% and 54.1% of our samples declared to eat wild meat in the Sebitoli area, but more on an occasional (a few times a year) than regular basis. These proportions are lower than the 71% respondents from the three Ugandan sites , and are close, on average, to the 31% of households wanting to access wild meat around Kibale NP . Regarding our results, we reviewed common but non-exhaustive motives found in previous research that should be considered if trying to increase the sustainability of wild meat consumption at a study site:Price: In interviews, wild meat appearing to be cheaper than domestic meat (or free if the harvester) is a common pattern among poor African households that can explain its consumption frequency compared to other meats . As in other studies, our results highlight that respondents' frequency of eating wild meat increases with the number of people in the household , as eating wild meat can decrease spending on food , and it is an important factor in poverty reduction in rural areas . Taste: Species preferred as wild meat, such as bushbuck, red duiker and wild pig, are known to be hunted for wild meat in Kibale . None of them are threatened with extinction . Wild meat is believed to be "sweeter" than livestock meat and several people believe that it contains fewer chemicals than other meat around the park . Across Africa, wild meat is generally preferred to domestic meat, with arguments of higher quality and better taste . However, sometimes, the wild meat sold is misrepresented as another species of wild meat . Ungulates constitute more than the majority of all hunted animals in West, Central, and East Africa , and among wild meat, antelopes are often cited as a preferred species . Compared to other meats, ungulates have a superior quantity of meat with less fat , and a greater amount of edible protein per unit of live weight than domestic animals (Ledger, 1967, cited in .) Bush pigs are also advantageous animal protein sources, representing an important quantity of meat with a low level of total fat . Remoteness of domestic meat: The northeastern part of the Sebitoli area experiences more snares targeting wildlife and it is also where the distance to access domestic meat is one of the longest. Most studies and field programs on wild meat consumption focus on individual preferences and/or the role of sociodemographic variables in such behavior. Remoteness and, more generally, landscape characteristics, which can limit access to marketed domestic animal protein, are less frequently used to account for wild meat consumption despite it being a relevant factor linked to dependence on hunting for subsistence . 4.2. Increasing Human Livelihoods to Promote Wildlife Conservation In the Kabarole district (connected to Kibale NP), animal protein contributes a small percentage of the total protein intake, as families seem to strive to eat and buy fish or red meat once a week and chicken (the most expensive animal protein) is eaten more rarely . The Batooro and Bakiga are mainly subsistence-level agriculturalists , and have many taboos regarding meat; it is considered "pure" when it is coming from ruminants (or, generally, animals eating plants), but "impure" and inconsumable when coming from carnivore and omnivore animals . Relative to other foods, meat and fish have a high "diet impact ratio'' (e.g., a high environmental impact per calorie of food supplied ) and the amount of meat consumed through the world is not predicted to decrease . Therefore, increasing domestic animal production is not necessarily the most suitable perspective for biodiversity conservation . In Uganda, increasing the supply of domestic animal protein and reducing its price locally could be a viable policy option to reduce wild meat quantity demand and hunting pressure on Kibale NP's wildlife, as it was also suggested in other locations . As for tea workers specifically, implementing lunches provided by the tea company with meat (for example, once a week) may be an incentive for wild meat consumption as well. Therefore, changes in diet would have to be followed up carefully as some studies suggest that a substitution away from wild meat can mean a significant increase in fish consumption , which could also cause biodiversity conservation issues. New ways to involve and sustain local communities' needs while contributing to wildlife conservation are developing. For example, mini/micro-livestock (the rearing of small wild mammals ), eating insects , or sun-dried meat in times of food shortage can enhance the animal product supply in sub-Saharan Africa. Regarding the taste issues mentioned earlier, meat should be produced locally to be positively perceived , and it could be sold in the Sebitoli area through community markets for conservation . Other means, such as reducing the impact of crop raiding with the use of wildlife deterrents to avoid risks of reprisals from agriculturalists , incentivizing poachers , and developing programs about wildlife conservation and/or spiritual associated knowledge may be effective at strengthening and combining the positive support and sustainable attitudes toward wildlife. 5. Conclusions A combination of factors leads to illegal hunting and consumption of wildlife in the Sebitoli area. The driving factors are spatial (proximity to park's edges) and social (sex, age, provenance, and household size). The wild meat hunting crisis is a fundamentally distressing problem to address because it is intimately tied to human development challenges such as food insecurity, emergent disease risks, and land-use changes . Therefore, efforts to promote biodiversity conservation need to be integrated, conjoint, multilevel, interdisciplinary, progressive, and sustained for the long term. Acknowledgments We are grateful to the Uganda Wildlife Authority, Uganda National Council for Science and Technology, Rwenzori commodities Ltd. And the Mukwano Group, local councils, and local communities for granting us authorization to conduct research and for facilitating the survey. We thank SCP for hosting our research, and thank our informant R. Asiimwe and SCP field assistants for their work in Sebitoli, particularly C. Aliknyera, D. Birungi, P. Kusemererwa, P. Musinguzi, R. Nyahuma, and C. Twesige. We are grateful to our colleagues from the Museum national d'Histoire naturelle and McGill University, and to reviewers for their worthy comments on our work. Author Contributions Conceptualization, S.B. and S.K.; methodology, S.B. and S.K.; software, S.B. and T.F.; validation, S.B., S.K., T.F., W.K., E.A. and R.N.; formal analysis, S.B. and T.F.; investigation, S.B.; resources, S.B., S.K., W.K., E.A. and R.N.; data curation, S.B. and S.K.; writing--original draft preparation, S.B. and S.K.; writing--review and editing, S.B., T.F., S.K., W.K., E.A. and R.N.; visualization, S.B. and S.K.; supervision, S.K. and E.A.; project administration, S.K.; funding acquisition, S.K. All authors have read and agreed to the published version of the manuscript. Institutional Review Board Statement The study was conducted in accordance with the Declaration of Helsinki, and approved by the Ethics Committees through a Memorandum of Understanding (MoU) for research and conservation in Kibale National Park between the Museum national d'Histoire naturelle, Uganda Wildlife Authority, and Makerere University (named SJ445-12, and signed in 2012 for 10 years). Institutions involved in the MoU were the only ones in charge of evaluating the ethical aspects of the research developed within this frame (for both non-human-related study protocols). Informed Consent Statement Informed consent was obtained from all subjects involved in group counts (orally) and individual interviews (written format) after it was explained that the study was voluntary and anonymous in English and Rutooro. It was not obtained from LC1 and tea camp managers as they did not participate in the study outside of facilitating contacts. See the consent form in Appendix A. Data Availability Statement The data presented here are sensitive (related to illegal practices). They are only available on request and under certain conditions to respect the authors' commitment to participants. Conflicts of Interest The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results. Appendix A Figure A1 Interview consent form used around Sebitoli area, Kibale National Park, Uganda. Interview respondent and interviewer each had a copy. Figure A2 Interview form used around Sebitoli area, Kibale National Park, Uganda. Figure A3 Ballot boxes used for group counts in one tea camp to answer the questions: "Do you eat bushmeat?", and if yes, "Do you eat it more than once a month?" around Sebitoli area, Kibale National Park, Uganda. Figure A4 Variables used for illegal activity and wild meat consumption statistical modeling of key questions, Sebitoli area, Kibale National Park, Uganda. Figure A5 Number of illegal activities collected by SCP (29/05/2015-30/11/2016) per section of Sebitoli area, Kibale National Park, Uganda. Figure A6 Multiple logistic regression between type of illegal activities, Euclidian distances to the road and the edge, and sides of Sebitoli area, Kibale National Park. Figure A7 Frequency of domestic animal protein consumption (A); access time to domestic animal protein among consumers who eat, buy, and raise domestic animals (B); access mean to domestic animal protein among consumers who eat and buy domestic animals (C); Euclidian distance (kilometer) to domestic animal protein among consumers who eat and buy domestic animals (D) around Sebitoli area, Kibale National Park, Uganda. Figure A8 Residency locations of domestic animal meat buyers and locations where they acquire all types of meat combined in Sebitoli area, Kibale National Park, Uganda (May-December 2016). Figure A9 Residency locations of domestic animal meat buyers and locations where they acquire each type of meat in Sebitoli area, Kibale National Park, Uganda (May-December 2016). Figure 1 Data acquisition types, collection methods, and sample sizes used in determining spatial distribution of illegal activities and wild meat consumption analyses in Sebitoli area, Kibale National Park, Uganda. Figure 2 Illegal activities identified by SCP snare removal team (29 May 2015-30 November 2016), Sebitoli area, Kibale National Park, Uganda. animals-13-00771-t001_Table 1 Table 1 Wild meat consumption among group count participants, Sebitoli area, Kibale National Park, Uganda. Consume Wild Meat More than Once a Month (12 Times a Year) Total Yes No Total Yes No Group counts n n n n n n % % % % % % Villagers 678 325 353 247 77 170 23.3 47.9 52.1 32.2 31.2 68.8 Tea workers 339 105 234 150 39 111 11.7 31 69 19.6 26 74 Children 1885 363 1522 370 144 226 65 19.3 80.7 48.2 38.9 61.1 Total 2902 793 2109 767 260 507 100 27.3 72.7 100 33.9 66.1 animals-13-00771-t002_Table 2 Table 2 Interview respondents' description, Sebitoli area, Kibale National Park, Uganda. Variables Modalities Interviews of Tea Camps (n = 74) Interviews of Villagers (n = 42) Interviews of Children (n = 35) Wild meat consumption (percent) Yes 54.1 35.7 17.1 No 45.9 64.3 82.9 Frequency of consuming wild meat (count) Mean (SD) Range 2.8 (12.3) 1.3 (3.9) 0.6 (2.1) 0-104 0-24 0-12 Sex (percent) Woman 21.6 47.6 54.3 Man 78.4 52.4 45.7 Age (year) Mean (SD) 29.4 (6.7) 37.4 (14.7) 11.7 (2) Range 18-50 18-78 7-16 Provenance (percent) District contiguous to Kibale NP 39.2 71.4 88.5 Kabale district 32.4 16.7 2.9 District non-contiguous to Kibale NP 8.1 7.1 8.6 Rwanda 20.3 4.8 0 People sustained (Number) Mean (SD) Range 4.4 (2.2) 6.1 (3.1) 6.2 (2.3) 1-15 1-14 2-11 animals-13-00771-t003_Table 3 Table 3 Mixed Poisson model of wild meat consumption frequency in Sebitoli area, Kibale National Park, Uganda. Fixed Effects Regressors Incidence Rate Ratios 95% CI p-Value (Intercept) 0.44 0.15-1.29 0.133 Actor Type (Tea worker) 1.61 0.90-2.88 0.17 Actor Type (Child) 0.44 0.16-1.16 0.096 Sex (Male) 3.08 1.83-5.19 <0.001 Provenance (Kabale) 0.50 0.29-0.85 0.011 Provenance (Noncontiguous) 0.43 0.18-1.05 0.065 Provenance (Rwanda) 0.81 0.44-1.50 0.56 Age 0.96 0.94-0.99 0.002 Number of people in household 1.13 1.03-1.23 0.009 Random effects s2 0.97 t00 village 0.74 ICC 0.43 N villages 28 Observations 151 Conditional R2 0.560 Disclaimer/Publisher's Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

PMC10000184

Persons with disabilities, who own service dogs, develop strong relationships with them. Since the COVID-19 pandemic decreased the possibility of social contact and modified human relationships, we hypothesized that the COVID-19 lockdown would influence people with disabilities--service dog relationships. An online survey was conducted during the first COVID-19 lockdown in France and included information (e.g., MONASH score) both in the general context prior to and during the COVID-19 lockdown. Seventy owners participated. Compared to the general context, scores for the Perceived Emotional Closeness and Perceived Costs subscales were significantly higher during the COVID-19 lockdown, while scores for the Dog-Owner Interaction subscale were significantly lower during the COVID-19 lockdown. Our study confirmed that service dogs, like other pets, were a source of emotional support for their owners during the COVID-19 lockdown. However, people with disabilities found their relationship with their service dog costlier (e.g., my dog makes too much mess). Our study highlights that, in extreme situations, characteristics of a human-animal relationship can be exacerbated in both positive and negative ways. COVID-19 lockdown interspecific relationship service dog social isolation Adrienne & Pierre Sommer FoundationThis research was funded by Adrienne & Pierre Sommer Foundation. pmc1. Introduction According to Hinde , "a relationship between two individuals involves a series of interactions [...] over time, such that each interaction may be affected by preceding ones". Relationships are thus not a fixed entity but are dynamic over time since various emotional processes are involved--such as feelings, hopes and distress--which may influence individual expectations of the relationship. Furthermore, this bond is built between two individuals, and the cultural, temporal and social contexts in which a relationship is embedded must be considered since these factors may influence the relationship . For example, stress or other challenges in families (e.g., unemployment) can result in a reduction in satisfaction in a relationship . Although relationships and interactions were historically conceptualized in intraspecific contexts, these concepts can be applied at the interspecific level . Thus, like the relationships and interactions between humans, external factors can influence human-animal relationships and interactions. For example, inappropriate human management of horses (e.g., food, spatial and social restriction) may compromise the horse's welfare and consequently alter the human-horse relationship . Additionally, just as peers and family represent an essential part of the human environment, pets are often partners in our immediate environment . Families, including animals, could thus be viewed as a group with ongoing and constantly changing interactions among members . As a consequence, similar to human intraspecific relationships , interspecific relationships with pets may be impacted by major contextual changes. One could argue that the worldwide health context associated with COVID-19 is a major event with multiple consequences on our interspecific relationships. In less than three months, at the end of January 2020, a public health emergency was declared by the World Health Organization, and 6 weeks later, it was upgraded to pandemic status, i.e., the COVID-19 pandemic . Consequently, unprecedented restrictions on movements, travel, work, social contacts and leisure activities were set globally to prevent the spread of the virus. For example, in France, the first lockdown was enforced for 2 months from 17 March 2020 (with an additional month with strict movement restrictions). Additionally, the COVID-19 pandemic clearly caused stress and anxiety, affected mood, and sometimes, in extreme situations, increased the risk of developing post-traumatic stress disorders and suicides, especially due to social isolation (e.g., ). Different studies demonstrated that, in this specific context, pets and especially dogs alleviated the negative impacts of the COVID-19 lockdown in the general population. For example, in the USA, using an online survey during the first month of COVID-19 (April 2020), Bussolari et al. investigated the thoughts, experiences and concerns of adults living with pet dogs. Having a pet dog clearly reduces the feeling of isolation and loneliness, as well as sustains both the mental and physical health of their owners. Similar benefits were observed in British pet dog owners between April and June 2020 and Spanish owners in March 2020 . Furthermore, pet cats were valuable and comforting human companions during lockdowns, as were other animals , but to a lesser extent . As expected, the COVID-19 pandemic, including lockdown periods, influenced the owners' interspecific relationship with their pets. For example, using a questionnaire based on the Monash Dog-Owner Relationship Scale or MDORS to evaluate the social support obtained from pet dogs, Bowen et al. showed that, during the lockdown, pet dog-owner relationships differed from that in their normal daily life context. Owners engaged in more shared activities with their dogs, hugged them more often, turned to them more as a source of companionship and comfort, and confided more in them. However, using a revised version of MDORS, Howell et al. reported that although dogs can be social substitutes for humans in some crisis situations involving social isolation, such as COVID-19 lockdowns, their influence was not as great as expected, since humans could keep in touch with one another via telecommunications . Consulting over 400 diaries, Holland et al. found specific changes in pet dog-owner relationships during the lockdown, e.g., pet dogs enjoying increased human company or more time for their training. However, as explained by Bowen et al. and Christley et al. , pets can also experience the negative consequences of lockdown since their quality of life "is highly influenced by the characteristics of their physical and social environment, as well as the behaviour and lifestyle of their owners , all of which would be substantially changed during an official lockdown" . Consequently, pet dogs displayed more behavioral problems (e.g., object destruction, barking all day) during lockdown periods, probably resulting from changes in their daily routines. For example, around 80.0% of UK dog owners reported that their dog's routine had changed compared to pre-lockdown . Most Spanish dog owners reported that their dog's behaviors "got worse", with more attention-seeking behavior (41.6%), annoying or excessive vocalizations (24.7%) or excitable behavior (20.8%) . In another British survey, pet dog owners reported, in their diaries, new undesirable behaviors during the lockdown period, such as attention-seeking or destructive behaviors . One could argue that these changes are subject to variation according to the populations surveyed and the type of questions asked. For example, only 3.2% of English-speaking respondents (e.g., USA, UK, Canada, Australia) mentioned a strained relationship with their pet dog . In addition, results could be different if the animals at home are not pets but trained animals such as service dogs. Service dogs have been specifically trained to cope with particular situations and to perform a specific physical or functional task(s) to aid - and adult-owners with various forms of disabilities . First used to help adults with mobility challenges, service dogs are now also trained to help individuals with a wide range of developmental disorders, disabilities or chronic health conditions such as diabetes, post-traumatic stress disorder (PTSD), epilepsy and autism spectrum disorder (ASD) . This training may lead to particular relationships between the owners and their service dogs. For example, using the MDORS, two recent studies indicate that the dog-owner bond--and hence attachment between dog and owner--was significantly higher with seizure-alert dogs than with non-alert dogs . Service dogs also provide companionship, e.g., for people with ASD . These bonds also lead to numerous benefits for the recipients, as reviewed by Lindsay and Thiyagarajah : quality of life, reduced stress and anxiety, enhanced self-confidence, social interaction and physical health. Considering the stress, anxiety and loneliness generated by the COVID-19 pandemic, especially during the first lockdown, in various countries around the world , recipients of service dogs may have also been affected by such difficulties and/or be impacted at a higher (or at least different) level than the general population, as observed in other at-risk populations . Surprisingly, even though Ratschen et al. mentioned that their sample involved 57 service dogs (e.g., guide dogs), no analyses focusing on human-service dog relationships have been done. To our knowledge, no other study mentioned service dog-recipient relationships during the COVID-19 pandemic, especially during the first lockdown period. There is no doubt that pets, especially pet dogs, provided social support to their owners during COVID-19 lockdowns and that human-pet relationships could change during this period. Thus, we hypothesized that a similar phenomenon would also be observed for humans with disabilities and service dog dyads. Based on the previous literature, we hypothesized that relationships between people with disabilities and their service dogs would become stronger during COVID-19 lockdowns than before. It has been established that recipients have strong relationships with their service dogs in their daily life , and COVID-19 lockdowns could change these dyad interactions--and hence, relationships. We investigated the relationship between owners with disabilities and their service dogs in a French population, using an auto-evaluated MDORS questionnaire and three additional sub-scales, as well as measures of other components of daily life prior to and during lockdown (e.g., location, working or not, living alone or not). 2. Materials and Methods 2.1. Context of the Study In France, the first cases of COVID-19 were diagnosed at the end of 2019, and a pandemic expansion at the beginning of 2020. The French government established a lockdown of the whole population from 17 March with strict restrictions on time spent outdoors (e.g., 1 h per day for walking within an authorized radius of 1 km from residence). Each family was confined to their own home, without outside visitors, except in case of an emergency. Confinement was progressively reduced with authorization to travel freely within French borders at the beginning of June 2020. We solicited participants and collected data between 15 May and 30 June 2020 survey's link is not available anymore). 2.2. Subjects 2.2.1. Ethics The survey complied with the French law on digital information and was approved by the University of Rennes 1. Participants were fully informed about the aim of the study before the survey completion and informed about the hypothesis of the study after the survey completion. As the study survey was entirely anonymous, informed signed consent was not required from participants. Participants could withdraw from the online survey at any time. 2.2.2. Human Participants Using the Handi'Chiens association's database (containing contacts of people with disabilities and service dogs), all listed recipients and their families were invited to participate in the survey. The survey was available online on LimeSurvey (i.e., an online surveying tool). We used the contact e-mail addresses in the database to email invitations to 729 potential respondents, providing detailed information about the aim of the survey, as well as the digital link to the online questionnaire. A total of 380 individuals opened the email, and 127 answered the survey (final rate of 17.4%). Among the 127 respondents, 70 completed the survey completely (58 by recipients and 12 by caretakers). A flow chart of the survey process is presented in Figure 1. 2.3. Survey Design The online survey could be completed either by the recipients themselves or with the help of their caretakers (e.g., a recipient's parent for children with ASD). Data were collected through close-ended questions (e.g., yes/no, male/female), open-ended questions (i.e., comment on the answer when "other" was selected) and a 5-point Likert scale. The survey was performed only at one time during Spring of 2020, and we collected information during the COVID-19 lockdown period and for the general context (i.e., prior to COVID-19 lockdown). "In general context" was explained at the beginning of the survey as corresponding to the condition before and outside COVID-19 lockdown, as the daily life routines. The online survey included five categories of questions: Recipients' relationship with their service dog (n = 37 questions). This category is composed of two subparts. For the first subpart, we used the Monash Dog-Owner Relationship Scale (MDORS), a validated questionnaire aimed at evaluating human-dog relationships. The MDORS includes 28 questions. The item "How often do you take your dog in the car?" was removed since most recipients did not have a car or drive a car. Thus, our survey was comprised of 27 questions, contributing to three different subscales: (1) Dog-Owner Interaction; (2) Perceived Emotional Closeness; and (3) Perceived Costs. The Dog-Owner Interaction sub-scale corresponds to activities related to the care of the dog and close activities (e.g., kiss, pet, groom the dog). As mentioned previously , "such activities indicate the amount of time spent together in a relationship as well as the opportunity for shared emotional experiences and reciprocal interactions, which are known to be important elements in the formation of affectional bonds". The Perceived Emotional Closeness reflects social support, attachment and companionship, an important component of human-dog bond. The Perceived Costs explore the potential perceived costs associated with a dog by the owner, independent of the perceived benefits , such as money, mess and difficulties in looking after the dog. The answers to subscales 1 and 2 were rated from 1 to 5 (never/totally disagree to always/strongly agree), and the answers to subscale 3 were rated from 1 to 5 (never/totally disagree to always/strongly agree). The MDORS was back-translated to French, as previously done . For the present study, each item of the MDORS was adapted to two versions of the questions: a) relationship with the service dog during the COVID-19 lockdown; and b) relationship with the service dog in a general context prior to the COVID-19 lockdown. For example, for item 1 of Perceived Emotional Closeness subscale, people answered the sentence: during general context, my dog helps me get through tough times; then, during this COVID-19 context, my dog helps me get through tough times. Next, people answered item 2 and so on. In the second subpart, additional elements previously pointed out in the literature relative to service dogs were collected, such as the dog's walks (i.e., frequency and duration) and possible changes in behaviors during the COVID-19 lockdown (i.e., boredom, excitement, objects' destruction, vocal noises, attention seeking, other). Other animals at recipients' home (n = 1 question). Information was gathered about other dog(s) and/or other species at recipient's home. COVID-19 contagion context for the recipients (n = 6 questions). Participants were asked if and how COVID-19 had impacted them (i.e., recipients), their family and/or their friends. They were asked (1) if they knew anybody personally who had contracted COVID-19, if these people had been (2) hospitalized or (3) died, (4) whether they had been positively-tested for COVID-19, (5) felt COVID-19 symptoms, and lastly (6) to describe their general change in stress level since the beginning of the COVID-19 pandemic (4-point Likert-scale from more stress to no stress). Since these questions could be emotionally difficult, participants could skip them. Recipients' daily life (n = 7 questions). We collected information about the dog recipients' work or institution, their accommodation generally, and about the impact of COVID-19 on these elements. We asked (1) what kind of activity the recipient had done (e.g., work, school, unemployment), (2) if and how it changed during lockdown (e.g., teleworking), (3) their type of housing during lockdown: lived in their regular residence or had to move, the (4) type of residence: flat, house or other, (5) its location: urban, semi-urban or in a rural area, (6) presence--or not--of an outside area (e.g., garden, balcony), and (7) if they lived alone or not. General information (n = 10 questions). Questions from this category yielded general information concerning the recipients (e.g., sex, age) and their service dog (e.g., age, service type, breed). Since France was divided into green zones (i.e., where COVID-19 occurred to a lesser extent, with fewer people at hospitals and fewer deaths due to COVID-19) and red zones (i.e., where COVID-19 was more prevalent, with more people in hospitals and more deaths due to COVID-19) during lockdown, the participants' regional location was recorded since it may have affected recipients' perceived stress. 2.4. Data Collection and Analyses Only fully completed questionnaires were considered for analysis (n = 70). All results were analyzed using Statistica 13 software accessed on 1 July 2022) statistical tests. Since the data did not fit a normal distribution, non-parametric statistics were used . The significance threshold was set at p = 0.05. Previously, some authors using the MDORS analyzed total scores for all items while others analyzed either mean scores only or both ; we chose the last option in this study for 2 reasons: there was no defined procedure, plus these two ways of analyses have previously been shown to complement each other . MDORS scores were calculated for the whole scale and its three subscales (i.e., Dog-Owner Interaction, Perceived Emotional Closeness, and Perceived Costs). First, a total score was calculated by adding the scores of all items (for the whole MDORS and each of its subscales). Then, mean scores (i.e., the total divided by the number of items) were calculated for the three subscales and the total MDORS. These scores on the MDORS were calculated for answers concerning the COVID-19 lockdown period and for answers concerning the period outside COVID-19. The MDORS scores (i.e., total scores and scores for each subscale) and specific items of the three subscales were compared between COVID-19 lockdown and the period outside COVID-19 using Wilcoxon matched pairs test. Spearman correlation tests were used to analyze the relationships between subscales during and prior to COVID-19 lockdown. Spearman correlation tests were used to compare MDORS scores on subscales between contexts (i.e., scores during COVID-19 lockdown minus scores for the general context) to evaluate links between variations on subscales. Finally, using Kruskal-Wallis, Mann-Whitney U and Pearson correlation tests, we compared scores (i.e., total and for each subscale) for the MDORS subscales and other parts of the questionnaire (i.e., other animals at home, COVID-19 context, recipient daily life, general information). 3. Results 3.1. Information about the Participants and Service Dogs Details about the recipients and their service dogs are presented in Table 1. Overall, 70 recipients answered the survey (n = 47 , 67.1%; n = 23 , 32.9%; mean age +- SD = 37.4 +- 15.3 years old, range 8 to 72; 10 under 18 years old). The survey was mostly completed by the recipients themselves (n = 58), while 12 completed it together with their caretaker (n = 11 mother, n = 1 father). The highest level of education (for the recipient or for a parent if the recipient was under 18 years old) was a high school diploma (54.2%). The employment status of the recipient, or the highest one for parents if the recipient was under 18 years old, was mainly unemployed (54.2%). Thus, by the time of the survey completion, only a few recipients were employed (22.9%) or went to school or a specialized institution (15.7%). These activities suddenly stopped or changed drastically during lockdown for almost all the participants; only 1 of the 16 employed recipients continued to go to work, while 10 were working online, and all schools/institutions were closed. During the COVID-19 lockdown, most recipients did not live alone (71.4%) and were confined in their regular housing (92.9%). Their housing conditions were mainly in a house (64.3%), with an outside area (i.e., at least a balcony: 93.2%) with a quasi-equal distribution between urban, semi-urban and rural areas (35.7%, 28.6%, and 35.7%, respectively). Most lived in an area with low COVID-19 contagion rates (i.e., green area: 65.7%). The 70 service dogs involved in the survey were well balanced according to sex ratio (n = 33, 47.1% ; n = 37, 52.9% ), breed (n = 35, 50.0% Labradors; n = 34, 48.6% Golden retrievers) and well distributed according to age (mean age +- SD = 5.72 +- 2.48 years old, range 2 to 12). They were mainly service dogs for individuals with physical disabilities (n = 62, 88.6%), while others were service dogs for ASD children (n = 7, 10.0%) and one was a seizure alert service dog (n = 1, 1.4%). Such an unbalanced sample did not allow comparison between types of recipients. All had lived with the recipient for several years (mean +- SD = 42.0 +- 30.7 months). Most recipients (58.6%) owned one or more other animals (i.e., pet dog, cat). Approximately half of the service dogs were reported by their recipient to have displayed behavioral changes during lockdown (48.7%; Table 1). The most frequently reported changes in these 34 service dog behaviors were human attention seeking (64.7%), boredom (41.2%), vocal emissions (32.3%), excitement (14.7%) and destruction of objects (5.9%) occasionally. Some of the 18 recipients (25.7%) who reported other behavioral changes during lockdown mentioned, for example, a decrease in obedience (in general or specific contexts such as running away; n = 3), an increase in time spent sleeping (n = 1) and that the service dog became cuddlier and closer (n = 4). None mentioned aggressiveness. Interestingly, behavioral changes were not associated with changes in living place (only one recipient who was not living in her regular housing during lockdown reported that her service dog manifested behavioral changes). Similarly, they were not associated with housing conditions during the COVID-19 lockdown (house versus flat: X2 = 2.02, p = 0.154; urban, semi-urban versus rural: all p > 0.05; presence versus absence of an outside area: X2 = 0.2, p = 0.653; housing alone versus not: X2 = 0.02, p = 0.879). 3.2. Impact of the COVID-19 Context on Relationships between Recipients and Their Service Dogs For the sake of clarity, only significant results are discussed. The COVID-19 lockdown modified the relationships between recipients and their service dogs. Scores for the three MDORS subscales differed significantly between the COVID-19 lockdown and the general context prior to COVID-19 lockdown (all p < 0.05; Table 2). The Perceived Emotional Closeness subscale scores were significantly higher during the COVID-19 lockdown than for the general context (total score, Z = 1.99, p = 0.047; mean score, Z = 2.07, p = 0.038). Interestingly, the Perceived Costs subscale scores were also significantly higher during the COVID-19 lockdown than for the general context (total score, Z = 2.17, p = 0.030; mean score, Z = 2.10, p = 0.036). Conversely, the Dog-Owner Interaction subscale scores were significantly lower during the COVID-19 lockdown than for the general context (total score, Z = 3.30, p < 0.001; mean score, Z = 3.30, p < 0.001). No significant differences were reported between COVID-19 lockdown (Sum +- SD = 90.46 +- 8.64, Mean +- SD = 3.35 +- 0.32) and general context (Sum +- SD = 90.46 +- 8.7, Mean +- SD = 3.35 +- 0.32) for total scores on the MDORS (Wilcoxon tests, Z = 0.461, p = 0.645 and Z = 0.409, p = 0.683, respectively). Comparisons of scores during and outside the COVID-19 lockdown for each item of the MDORS revealed significant differences between contexts for specific items of the three subscales (Table 2). The Perceived Emotional Closeness subscale yielded higher recipient scores during the COVID-19 lockdown than for the general context concerning the trauma the service dog's death might cause them (Z = 2.02, p = 0.043) and the tendency to tell things to their service dog they would not tell anyone else (Z = 2.82, p = 0.005). Scores for two items from the Perceived Costs subscale were higher: the difficulty to look after the service dog (Z = 2.76, p = 0.006) and the fact that the service dog made too much mess (Z = 2.92, p = 0.004) was higher during the COVID-19 context than for the general context. Multiple significant differences were observed for items on the Dog-Owner Interaction subscale: recipients gave significantly more hugs (Z = 3.18, p = 0.002) and kisses (Z = 2.52, p = 0.012) to their service dog during the COVID-19 lockdown than in the general context; they played less with the service dog (Z = 3.24, p = 0.001), bought it fewer presents (Z = 3.76, p < 0.001) and took it less with them to visit people (Z = 5.11, p < 0.001) during the COVID-19 lockdown than in the general context. Correlation analysis revealed similar significant relationships between MDORS scores on subscales for the general context and the COVID-19 lockdown. MDORS scores on the Perceived Emotional Closeness subscale were positively correlated with scores on the Dog-Owner Interaction subscale (General context: r = 0.577, p < 0.001; COVID-19 lockdown: r = 0.503, p < 0.001) and negatively correlated with scores on the Perceived Costs subscale (General context: r= -0.465, p < 0.001; COVID-19 lockdown: r = -0.390, p < 0.001). Although the correlation was not significant, scores on the Dog-Owner Interaction and the Perceived Costs subscales tended to correlate negatively for the General context only (r = -0.224, p = 0.06). Analyses of differences in MDORS scores on subscales between contexts (i.e., scores for the COVID-19 lockdown minus scores for the general context) revealed the presence of a significant positive correlation between score differences on the Perceived Emotional closeness subscale and the Dog-Owner Interaction subscale (r = 0.325, p = 0.005). Thus, an increase in the score on the Perceived Emotional Closeness subscale during the COVID-19 lockdown compared to the general context was also associated with an increase in the score on the Dog-Owner Interaction subscale. No other significant correlations on score differences on MDORS subscales were observed. 3.3. Factors Affecting the Impact of the COVID-19 Lockdown on Recipient-Service Dog Relationships Significant differences were found when comparing MDORS scores on subscales (i.e., score during the COVID-19 lockdown minus the score for the general context prior to the COVID-19 lockdown) according to recipients' daily routine and other general information. First, comparisons revealed that recipients' scores increased more on the Perceived Costs subscale when they indicated that their service dog manifested: (1) changes in their behavior during the COVID-19 lockdown (Yes Mean +- SD = 1.68 +- 3.16, No Mean +- SD = -0.33 +- 1.79, U = 329, p < 0.001), (2) frequent boredom (Yes = 2.43 +- 2.47, No = 0.20 +- 2.62, U = 177, p = 0.001), (3) increased excitement (Yes = 4.40 +- 2.51, No = 0.35 +- 2.53, U = 27, p < 0.001), (4) more frequent vocal manifestations (Yes = 3.64 +- 3.47, No = 0.09 +- 2.18, U = 103, p < 0.001). The same was true when the participants said they felt more stressed during the COVID-19 lockdown (Not/Less Stressed = -0.54 +- 0.53, Same = 1.60 +- 0.68, More Stressed = -1.10 +- 0.47; H = 11.67, p = 0.009; Not/Less versus Same, p = 0.009; Not/Less versus More, p = 0.075) and when their housing did not include a garden (Yes = 0.11 +- 2.83, No = 1.54 +- 2.54, U = 401, p = 0.03). Finally, the scores on the Perceived Costs subscale of participants who indicated that they had shorter walks during the COVID-19 lockdown increased (Shorter = -1.80 +- 0.49, Same = -0.67 +- 0.73, Longer = -0.50 +- 0.89; H = 11.04, p = 0.004; Same versus Shorter, p = 0.01). Scores on the Dog-Interaction subscale of participants decreased more between the general context and the COVID-19 lockdown when they reported that (1) they decreased the frequency of walks with their dog during the COVID-19 lockdown (No walks = -3.63 +- 1.01, Less = -2.35 +- 0.60, Same = -0.33 +- 0.55, More = 0.17 +- 0.83; H = 14.07, p = 0.003; Less versus Same and More, both p < 0.05), (2) they did not remain in their regular housing during this period (Yes = -1.0 +- 2.69, No = -5.0 +- 5.52, U = 78.5, p = 0.05), and (3) their housing did not include an outdoor space (Yes = -0.90 +- 2.84, No = -3.89 +- 4.20, U = 153, p = 0.03). In addition, a significant negative correlation was found with the dogs' age: younger dogs were associated with a greater increase in the score on the Dog-Interaction subscale (COVID-19 lockdown, r = -0.240, p = 0.045). Finally, we found only one significant difference between the Perceived Emotional Closeness data. Scores of participants who indicated that they had more than one dog (i.e., in addition to their service dog) increased between the general context and the COVID-19 lockdown (Yes = 2.30 +- 0.75, No = 0.37 +- 0.30, U = 156, p = 0.011). However, having other types of animals did not seem to have any significant effects. 4. Discussion Previous studies showed that pets and service dogs provide social support for people and help them cope with difficult situations in their daily life and in a crisis such as the COVID-19 pandemic . Using the MDORS scale, the present study confirms that service dogs are a source of support for their recipients and demonstrates how the COVID-19 lockdown modified relationships between recipients and their service dogs. While recipients of service dogs perceived more emotional closeness (Perceived Emotional Closeness subscale), they also reported an increase in costs (Perceived Costs subscale) and a decrease in interactions (Dog-Owner Interaction subscale) with their service dogs during the COVID-19 lockdown compared to the general context prior to the COVID-19 lockdown. Interestingly, some factors modulated the effects of the COVID-19 lockdown on the recipient-service dog relationships, such as the service dog's behavioral changes, the recipient's stress level, and the decrease in the time taken for walks, as well as the dyad's living environment. Our results revealed that the COVID-19 lockdown enhanced Perceived Emotional Closeness between recipients and their service dogs. A similar effect was observed during the COVID-19 lockdown in Spanish populations and their pets . Modulation of emotional closeness included notably the trauma the recipients pictured experiencing if their service dog died and the tendency to tell it things they would not tell anyone else. As previously reported, service dogs clearly appear as a confidant and a source of support that helped the owners to cope with the challenging COVID-19 lockdown . Interestingly, recipients that owned more than one dog (i.e., other than their service dog)--but not other animals--felt emotionally closer to their service dog. Such cumulative effects should be explored further. Our study revealed that the COVID-19 lockdown involved a decrease in dog-owner interactions. Specifically, recipients of service dogs played less with their dogs, bought them fewer presents and took them less often to visit people during the COVID-19 lockdown. If the changes related to the latter two activities are probably a direct consequence of the lockdown conditions (i.e., shops closed, strict restriction of time spent outdoors), the decrease in time spent playing is surprising. One could argue that lockdown conditions offered more time to play with pets, as reported in a British survey and the Dogs Trust's COVID-19 Report . However, the participants in the present study were individuals with several disabilities that may compromise their ability to play inside their home (e.g., difficulty playing in a small indoor space with a service dog if the recipient is in a wheelchair). This assumption would explain why the interaction score of recipients without an outdoor space decreased more. Conversely, service dogs may not be used to playing inside their home, where they are mostly in their working role helping their recipient. Lastly, some service dogs seemed to display boredom due to a lack of stimulation that could impact interactions with humans . However, one aspect of the Dog-Owner Interaction that improved during the COVID-19 lockdown was that the recipients hugged and kissed their service dogs more often. This corroborates the fact that, although interactions were affected by the COVID-19 lockdown, attachment to their service dog did not seem to be impaired per se. Taken together, we may remain circumspect about this decrease in Dog-Owner Interactions as the main changes concerned two items directly affected by lockdown conditions. One may argue that a revised version of the MDORS should have been used, as by Bowen et al. , who excluded three items in their study (i.e., How often do you take your dog to visit people? How often do you take your dog out in the car? How traumatic do you think it will be for you when your dog dies?). However, we considered using the whole tool would yield more information about human-dog relationships. The most surprising and troubling change relates to the increase in Perceived Costs during the COVID-19 lockdown. Specifically, the main issue faced by the recipients was the difficulty of looking after their service dog and the fact that their service dog made too much mess. This latter information was reinforced by the fact that recipients felt more costs in taking care of their service dogs when they displayed behavioral changes, especially boredom, excitement and frequent vocal manifestations. These behavioral changes were not specific to our study since other surveys, including pet dogs, reported similar modifications that could be costly for people with disabilities. We propose two non-exclusive hypotheses to explain why such difficulties increased when the recipients felt more stressed. On the one hand, being more stressed may have increased the recipients' sensitivity to their service dog's behavioral problems (i.e., already present before the COVID-19 lockdown) and hence led them to perceive these later as challenging. On the other hand, the recipients' stress may have consequences on their service dog that elicited the development of behavioral problems (e.g., not present before the COVID-19 lockdown). Interestingly, previous surveys focusing on pet dog owners reported that the Perceived Costs items tended to remain the same or be reduced during lockdown . We suspect these adults have fewer or no needs (or difficulties) than the recipients in our study. Thus, Bowen et al.'s explanation that the support gained by pet owners from their pets increased when the owner's quality of life was more impaired does not seem to apply to everyone. Our subjects who, in addition to their disabilities, reported more costs were those who felt more stressed during the COVID-19 lockdown, had no garden and took shorter walks. All these traits are elements that could lead to a reduction in their quality of life during the COVID-19 lockdown. Since people with disabilities are already in vulnerable situations (e.g., altered physical, health and mental conditions), the costs in terms of social isolation (e.g., no outing, no shopping, no visit to museums) and their anxiety would be exacerbated. Moreover, reports have shown that owning pets can intensify depressive symptoms in depressive individuals due to too many responsibilities . Finally, we cannot exclude the fact that differences between ours and other studies could be linked to the time between the beginning of the lockdown and the timing of the survey (i.e., 3 weeks versus 10 weeks in our study). The changes in costs perceived could have taken longer to emerge than other dimensions of the human-dog relationship measured by MDORS. Indeed, as Hinde stated: "the progress of a relationship is often traced through periods of marked change or crisis, but it is essential to remember that change may also be gradual, occurring in the course of everyday life". Longitudinal studies in crisis situations would be useful to further understand human-dog relationship dynamics. The service dogs in our sample were reported to have displayed behavioral changes during the COVID-19 lockdown, as reported for pet dogs . Our study found that approximately half of the service dogs displayed behavioral changes, thus agreeing with Bowen et al.'s report but not other studies . These discrepancies could be explained by methodological differences (e.g., standardized questionnaire, diary data or directed content analysis). Similar to Bowen et al. and the Dogs Trust's COVID-19 Report , more human attention-seeking was the most reported behavioral change, followed by vocal emissions (e.g., barking, whining). Boredom levels also commonly changed . Holland et al. reported that owners perceived a state of depression in their pet dogs as well. These authors also reported that owners explained this change in their pet dog by the reduced variety of activities, insufficient exercise and stimulation associated with the COVID-19 lockdown since we found a reduction of walking time. All the other previously mentioned studies also observed other behavioral changes. Thus, as lockdown clearly impacted human well-being (e.g., , this might also have been the case for both pet and service dogs. As Bowen et al. mentioned, such behaviors need to be taken into consideration as they "could easily lead to other problems if the lockdown continued or these changes were mishandled by owners". The generalizability of our findings is limited by several factors. First, our sample size (n = 70) was smaller compared to other studies on similar topics (e.g., 5926 participants ; 4105 participants ; 1297 participants ). Nevertheless, our sample focused on service dog recipients, not on pet dog owners. Additionally, contrary to previous investigations based on convenience samples (i.e., mainly animal lovers), we used a list of recipients of service dogs, and this limited the eventual recruitment bias. Another limitation concerns our survey design. Due to the sudden COVID-19 lockdown, we could not apply a strict longitudinal design with a before and during lockdown data collection. We could only collect one set of data points from questionnaires asking respondents to provide answers for two specific periods (i.e., prior to and during lockdown), i.e., thus including a form of retrospective survey. However, since the information reported referred to a period that had occurred not long prior to the data collection, the quality of the report could be considered excellent . Other limitations should be addressed since we had a limited ability to examine the relationships between the variables. In the present study, we did not perform direct observations of the relationships. Thus there is a lack of objective measures of the dogs' behavior and welfare (self-reported by the handler). We also need to be cautious about the generalizability of these results since the sample was from one specific service dog provider. We only provided a broad explanation of what was intended by the "general context" situation. Repeating this information at each item of the MDORS could have better aided the recipients in their responses throughout the survey. Our sample was mainly composed of individuals with physical disabilities, and these results may be more specific to this population and not to all recipients of service dogs. Further studies should compare people with different psychological and/or physical disabilities. 5. Conclusions To conclude, our study confirms the fact that service dogs constituted a source of emotional support for their owners during the COVID-19 lockdown, as previously shown with other pets. Service dogs fulfil the role of a pet as well as their professional role (e.g., perform a specific physical or functional task(s) to aid their owners). However, contrary to previous observations of pet dog owners, it appears that the COVID-19 lockdown elicited a costlier relationship for service dog recipients. Our study highlights the fact that, in extreme situations, characteristics of human-animal relationships can be exacerbated, both positively and negatively. These results are of importance for organizations delivering service dogs. For example, using the MDORS at key moments of the service dogs' life would give organizations an objective tool to measure and follow the quality of their relationship with their recipient and evaluate the support the organizations might have to provide. For example, an increase in the Perceived Costs subscale could be a warning sign. In a crisis, they must be sufficiently aware of taking into account the recipients' difficulties with their service dogs and their daily living issues since both factors could have negative impacts on the service dogs and their relationship with their owners. Some warning factors (e.g., living alone, having no outdoor space), as we highlighted here, are indicators of the need for closer monitoring and better assistance. As suggested by Bowen et al. , this type of research should be reproduced at the international level to better understand what happens between animals and owners in social deprivation contexts, and especially how human-animal relationships are affected by this context. Since relationships are dynamic over time and are influenced by factors that are extrinsic and intrinsic to each individual , it would provide a unique opportunity to better understand the dynamic nature of human-animals relationships. Acknowledgments We are grateful to the service dog owners and their families for participating, the Adrienne & Pierre Sommer Foundation for their support, and Jean-Thibault Daniel from Handi'Chiens for sending survey emails. We thank Ann Cloarec and Neville Pillay for their assistance with the English. Author Contributions Conceptualization, M.G.; methodology, M.G. and N.D.; validation, M.G. and N.D.; formal analysis, N.D.; investigation, M.G. and N.D.; resources, F.A., M.G. and N.D.; writing--original draft preparation, M.G. and N.D.; writing--review and editing, F.A., C.R., M.G. and N.D.; visualization, N.D.; supervision, M.G.; project administration, M.G.; funding acquisition, M.G. and F.A. All authors have read and agreed to the published version of the manuscript. Institutional Review Board Statement The study was conducted in accordance with the Declaration of Helsinki and approved by the Institutional Review Board of Rennes 1 University (no protocol code, date of approval: 19 May 2020). Informed Consent Statement Participants were fully informed about the aim and hypothesis of the study. As the study survey was entirely anonymous, informed signed consent was not required from participants. Participants could withdraw from the online survey at any time. Data Availability Statement The data presented in this study are available on request from the corresponding author. Conflicts of Interest The authors declare no conflict of interest. Figure 1 Flow chart showing the survey process and criteria used to include survey's answers. animals-13-00914-t001_Table 1 Table 1 Characteristics of our sample. Information about Recipients Information about Service Dogs Gender 47 23 Mean age in years (range) 5.7 +- 2.5 (2-12) Mean age in years (range) 37.4 +- 15.3 (8-72) Mean time since attribution to recipient (months) 42.0 +- 30.7 Under 18 yo/Over 18 yo 10/60 Sex 33 37 Activity (and change during lockdown) Breed Employed 16 (15) Labradors 35 School/specialized institution 11 (11) Golden retrievers 34 Highest level of education (for recipient or for parents if recipient was under 18 yo) No information 1 High school diploma or less 38 Dog type Bachelor's degree 15 For individuals with physical disabilities 62 Master's degree 11 For ASD children 7 Above Master's degree 6 For epileptic people 1 Employment status (for recipient or parents if recipient was under 18 yo) Changes in behavior during lockdown Tradesman, company head 1 All behaviors (Yes/No) 34/36 Manager and intellectual worker 10 Boredom 14/56 Employee 17 Excitement 5/65 Student 2 Destruction of objects 2/68 Retired 2 Vocal noises 11/59 Unemployed 38 Attention seeking 22/48 Condition of housing during lockdown Other 18/52 Alone or not 20/50 Regular housing or not 65/5 Flat, house or other 25/45/0 Urban, semi-urban or rural area 25/20/25 Presence of outside area or not 61/9 Regional location (green/red zones) 46/24 yo--years old. animals-13-00914-t002_Table 2 Table 2 Scores for items on each subscale of the MDORS during the COVID-19 lockdown and in a general context (mean +- SD). Significant differences between contexts (i.e., the COVID-19 lockdown vs. General context prior to COVID-19 lockdown) are in bold--level of significance: p < 0.05 (Friedman tests). Number of item corresponds to the order of items in the MDORS questionnaire developed by Dwyer et al. . Perceived Emotional Closeness Subscale Perceived Costs Subscale Dog-Owner Interaction Subscale General Context COVID-19 Context Wilcoxon Test General Context COVID-19 Context Wilcoxon Test General Context COVID-19 Context Wilcoxon Test Item 1 4.53 +- 0.76 4.53 +- 0.83 Z = 0.03, p > 0.05 1.37 +- 0.82 1.34 +- 0.80 Z = 0.43, p > 0.05 4.13 +- 1.30 4.26 +- 1.28 Z = 2.52, p = 0.012 Item 2 4.56 +- 0.79 4.59 +- 0.75 Z = 0.46, p > 0.05 1.64 +- 1.18 1.63 +- 1.23 Z = 0.68, p > 0.05 3.93 +- 0.79 4.23 +- 0.78 Z = 3.24, p = 0.001 Item 3 4.84 +- 0.58 4.87 +- 0.56 Z = 0.80, p > 0.05 1.37 +- 1.01 1.44 +- 0.99 Z = 0.20, p > 0.05 4.29 +- 0.93 2.87 +- 1.58 Z = 5.11, p < 0.001 Item 4 4.69 +- 0.75 4.70 +- 0.69 Z = 0.28, p > 0.05 1.87 +- 1.15 2.20 +- 1.37 Z = 2.76, p = 0.006 3.29 +- 1.02 2.76 +- 1.26 Z = 3.76, p < 0.001 Item 5 4.61 +- 0.75 4.77 +- 0.66 Z = 1.70, p > 0.05 1.64 +- 1.19 1.63 +- 1.07 Z = 0.96, p > 0.05 3.70 +- 0.92 3.60 +- 1.12 Z = 1.07, p > 0.05 Item 6 4.46 +- 0.76 4.50 +- 0.74 Z = 0.60, p > 0.05 1.56 +- 0.96 1.64 +- 1.05 Z = 0.38, p > 0.05 3.47 +- 1.02 3.56 +- 1.04 Z = 1.30, p > 0.05 Item 7 4.26 +- 1.09 4.27 +- 1.13 Z = 0.84, p > 0.05 1.33 +- 0.83 1.37 +- 0.97 Z = 0.53, p > 0.05 4.50 +- 0.74 4.70 +- 0.62 Z = 3.18, p = 0.002 Item 8 4.57 +- 0.79 4.64 +- 0.82 Z = 1.27, p > 0.05 1.73 +- 1.15 1.69 +- 1.10 Z = 0.74, p > 0.05 4.61 +- 0.62 4.66 +- 0.59 Z = 1.10, p > 0.05 Item 9 4.66 +- 0.70 4.74 +- 0.65 Z = 2.02, p = 0.043 1.44 +- 0.93 1.66 +- 1.10 Z = 2.92, p = 0.004 - - - Item 10 3.41 +- 1.25 3.61 +- 1.30 Z = 2.82, p = 0.005 - - - - - - Total score 44.59 +- 5.21 45.23 +- 4.9 Z = 1.99, p = 0.047 13.96 +- 5.88 14.6 +- 6.05 Z = 2.17, p = 0.030 31.91 +- 4.47 30.63 +- 4.54 Z = 3.30, p < 0.001 Mean score 4.46 +- 0.52 4.52 +- 0.49 Z = 2.07, p = 0.038 1.55 +- 0.65 1.62 +- 0.67 Z = 2.10, p = 0.036 3.99 +- 0.56 3.83 +- 0.57 Z = 3.30, p < 0.001 Perceived Emotional Closeness subscale: Item 1: my dog helps me get through tough times; Item 2: my dog is there whenever I need to be comforted; Item 3: if everyone else left me, my dog would still be there for me; Item 4: I would like to have my dog near me all the time; Item 5: my dog provides me with constant companionship; Item 6: my dog is constantly attentive to me; Item 7: My dog gives me a reason to get up in the morning; Item 8: I wish my dog and I never had to be apart; Item 9: How traumatic do you think it will be for you when your dog dies? Item 10: How often do you tell your dog things you don't tell anyone else? Perceived Costs subscale: Item 1: How often do you feel that looking after your dog is a chore? Item2: How often does your dog stop you doing things you want to? Item 3: How often do you feel that having a dog is more trouble than it is worth? Item 4: How hard is to look after your dog? Item 5: There are major aspects of owning a dog I don't like; Item 6: It is annoying that I sometimes have to change my plans because of my dog; Item 7: It bothers me that my dog stops me doing things I enjoyed doing before I owned it; Item 8: My dog costs too much money; Item 9: My dog makes too much mess; Dog-Owner Interaction subscale: Item 1: How often do you kiss your dog? Item 2: How often do you play games with your dog? Item 3: How often do you take your dog to visit people? Item 4: How often do you buy your dog presents? Item 5: How often do you give your dog food treats? Item 6: How often do you groom your dog? Item 7: How often do you hug your dog? Item 8: How often do you have your dog with you while relaxing, i.e., watching TV? Disclaimer/Publisher's Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

PMC10000185

The present study aimed to determine the content of health-promoting compounds, and fatty acids, with particular emphasis on the content of cis9trans11 C18:2 (CLA) acid, selected minerals, folates in organic and commercial goat's milk and fermented goat's milk drinks. The analyzed milk and yoghurts had various contents of particular groups of fatty acids, CLA, minerals, and folates. Raw organic goat's milk had a significantly (p < 0.05) higher content of CLA (3.26 mg/g fat) compared to commercial milk (2.88 mg/g fat and 2.54 mg/g fat). Among the analyzed fermented goat's milk drinks, the highest CLA content (4.39 mg/g fat) was determined in commercial natural yoghurts, while the lowest one was in organic natural yoghurts (3.28 mg/g fat). The highest levels of calcium (1322.9-2324.4 mg/g), phosphorus (8148.1-11,309.9 mg/g), and copper (0.072-0.104 mg/g) were found in all commercial products and those of manganese (0.067-0.209 mg/g) in organic products. The contents of the other assayed elements (magnesium, sodium, potassium, iron, and zinc) did not depend on the production method, but only on the product type, i.e., the degree of goat's milk processing. The highest folate content in the analyzed milks was found in the organic sample (3.16 mg/100 g). Organic Greek yoghurts had a several times higher content of folates, reaching 9.18 mg/100 g, compared to the other analyzed fermented products. short-chain branched and odd-chain fatty acids conjugated fatty acid (CLA) folates mineral composition goat's milk goat's milk products This research received no external funding. pmc1. Introduction Milk and dairy products contain many bioactive compounds valuable for human health and occurring in an easily absorbable form, such as fatty acids, micronutrients (especially calcium, magnesium, potassium, zinc, and phosphorus), and folates-B vitamins . However, cow's milk and dairy products may not be suitable for all consumers, for instance, children with protein diathesis, allergic patients, convalescents, the elderly, and people suffering from anemia, osteoporosis, and malabsorption syndrome. Goat's milk is recommended for these groups of the population as an alternative . It has a high biological value and exhibits nutritional properties. It is also characterized by a smaller diameter of fat globules than the milk of cows and contains more short-chain and polyunsaturated fatty acids . Furthermore, it is digested faster than cow's milk and hence can be recommended to people allergic to the latter . Milk fat is a complex mixture of over 400 different fatty acids (FAs) with four to twenty-six carbon atoms, making it the most complex natural fat . Fatty acids of milk fat are acids with different degrees of saturation (saturated, monounsaturated, and polyunsaturated). Unsaturated acids are mainly cis acids and trans isomers of these acids are found in smaller amounts. In milk fat, the fatty acids are mostly straight-chain and have an even number of carbon atoms, but fatty acids with an odd number of carbon atoms and branched-chain fatty acids are present as well. Studies conducted by many authors have indicated that the fatty acids present in milk fat may affect human health in different ways . The main isomer of conjugated dienes in milk fat, acid cis9trans11 C18:2 (CLA), has, among others, anti-carcinogenic, anti-atherosclerotic, antioxidant, and anti-inflammatory properties . Trans-vaccenic acid, which is the major trans C18:1 isomer in milk fat, shows anti-carcinogenic and anti-atherogenic effects . In turn, the main MUFA of milk fat, oleic acid (cis9 C18:1), enhances the activity of low-density lipoprotein receptors, while decreasing the cholesterol concentration in the serum . Short-chain fatty acids (SCFAs) also exhibit various bioactivities, such as anti-inflammatory and immunoregulatory effects, as well as preventive and therapeutic effects on several diseases, whereas branched-chain fatty acids (BCFAs) have been shown to elicit anti-carcinogenic effects . In turn, n-3 PUFAs play a meaningful role in heart disease prevention and immune response improvement. Linolenic acid (C18:3) exhibits anti-carcinogenic and anti-atherogenic properties , while (n-6) PUFAs improve insulin sensitivity and thus, reduce the incidence of type 2 diabetes . Mammalian milk is also a significant source of minerals in the human diet. It contains, for example, calcium, phosphorus, sodium, potassium, chlorine, iodine, magnesium, and small amounts of zinc, iron, and manganese . In the European diet, milk is regarded as the best source of calcium, and its high bioavailability is closely correlated with a higher content of casein, lactose, and vitamin D. The absorption of calcium from milk reaches 80%, while the availability of this element from other products, for example, vegetables or cereals, is limited due to the presence of fiber, phytic and phenolic compounds, and oxalic acid . Calcium deficiency in food increases the risk of developing such diseases as arterial hypertension, colorectal cancer, ischemic heart disease, osteoporosis, and kidney stones . For consumers with malabsorption syndrome, it can be extremely beneficial to consume goat's milk because the retention of ingredients from this product is better than from cow's milk. In addition to calcium, this also applies to iron, which results from a greater share of nucleotides than in cow's milk, which contributes to better absorption of this element in the intestines . Iron plays an important role in supplying oxygen to human organs and muscles, and its deficiency can lead to anemia , which, in 2019, was diagnosed in almost 30% of women across the world aged 15-49 and in about 40% of the children aged 6-59 months (in Africa the rate was 60%) . The alkaline pH of goat's milk is associated primarily with the content of calcium, potassium, and sodium, and its salty aftertaste is due to the high content of chlorine. Compared to cow's milk, goat's milk has a relatively high level of magnesium, which prevents stress, increases the body's immunity, and is a cofactor in many enzymatic reactions . The contents of micro and macroelements in goat's milk and products make it vary greatly and depend on many factors, for example, species, breed, or breeding conditions of animals, including, especially, their feeding, or lactation phase . Folates occur naturally in foods, both those of plant and animal origin, as reduced folic acid derivatives. These essential dietary components belong to the family of water-soluble B vitamins. The name folic acid refers only to the synthetic form of the vitamin . Folates are indispensable in the reactions of one-carbon groups, especially for the synthesis of DNA, RNA, and proteins. They are donors of methyl groups used in the remethylation of homocysteine to methionine . Unfortunately, their deficiency is common worldwide . Prolonged too low folate consumption promotes, among others, atherosclerotic processes, increasing the risk of development of cardiovascular (ischemic heart disease, stroke, thromboembolism) and neurodegenerative (Alzheimer's, Parkinson's) diseases. Adequate folate intake and status have also been associated with a reduced incidence of certain types of cancer (colon cancer, breast, cervix, lungs, pancreas), depressive mental disorders, and macrocytic anemia . Young women of childbearing age are particularly vulnerable to deficiency of this vitamin. The results of epidemiological surveys indicate that an insufficient folate status in future mothers increases the probability of congenital heart defects, urinary tract abnormalities, cleft palate, limb defects, and neural tube defects (NTD) in newborns . However, deficiencies can be effectively prevented by providing a folic acid dose of 400 mg per day . In addition to the use of dietary supplements and foods enriched with synthetic folic acid, it is recommended, above all, to regularly consume products naturally rich in these vitamins. An example of products popular among consumers, which represent a natural source of bioactive substances, including folates, are milk and milk products . In Europe, cow's milk is most often used in dairy production. However, the popularity of goat's milk and goat's milk products among consumers is being observed to grow. These products are especially appreciated by pioneers of taste in regional and organic food who look for new and unique flavor sensations. The commercial offer of goat's milk products increases year by year. Local breeders and sellers appear on the market, offering cheeses, cottage cheese, yoghurts, and kefirs made of goat's milk, often of organic origin . Despite growing interest, the knowledge of the content of many valuable compounds in goat's milk is incomplete. Therefore, the present study aimed to determine the contents of health-promoting compounds, including fatty acids, with particular emphasis on the content of cis9trans11 C18:2 (CLA) acid, selected minerals, and folates in organic and commercial goat's milk and fermented goat's milk drinks. 2. Materials and Methods 2.1. Materials The experimental materials were organic goat's milk, ecological fermented goat's milk drinks, commercial goat's milk, and commercial fermented goat's milk drinks available on the Polish market (Table 1). The analyzed products were purchased in March 2021. 2.2. Methods 2.2.1. Analysis of Fatty Acids (FAs) The fat from the milk and yoghurts was extracted with the Folch method . The IDF method was used to convert fatty acids into the corresponding fatty acid methyl esters (FAME) (ISO 15884:2002) . The methyl esters obtained were then analyzed by gas chromatography (GC). Chromatographic separation was performed using a Hewlett-Packard 6890 gas chromatograph (Munster, Germany) with a flame-ionization detector (FID) and a capillary column with a length of 100 m and an internal diameter of 0.25 mm. The liquid phase was CP Sil 88 (Chrompack, Middelburg, The Netherlands) and the film thickness was 0.20 mm., The analyses were carried out under the following temperature conditions: column temperature 60 degC; 5 degC/min increase to 180 degC; injector temperature 225 degC; and detector temperature 250 degC. The sample injection volume was 0.4 mL (split mode--50:1). Helium was used as a carrier gas at a flow rate of 1.5 mL/min. Fatty acids were identified based on a comparison of their retention times, with the retention times of fatty acid methyl esters of reference milk fat (BCR Reference Materials, CRM 164 symbol)(Aldrich, Taufkirchen, Germany), and literature data . The cis9trans11 CLA isomer was identified using a mixture of CLA methyl esters (Sigma-Aldrich, St. Louis, MO, USA). Other positional trans isomers were identified using the standards of methyl esters of these isomers (Sigma-Aldrich, St. Louis, MO, USA, and Supelco, Bellefonte, PA, USA). The contents of fatty acids were expressed in mg/g fat according to the applicable standard (methyl ester of C21:0 acid, Sigma-Aldrich, St. Louis, MO, USA). 2.2.2. Analysis of Minerals The mineral content of milk and fermented dairy products was established by wet mineralization in a mixture of nitric acid and perchloric acid (3:1, v/v). The samples were mineralized in a block heating digester (DK 20, VELP Scientifica, Usmate, Italy) for 4-5 h by gradually increasing the temperature from 120 to 200 degC. The mineralized samples were transferred to 25-mL volumetric flasks, and the flasks were filled up with deionized water. Reagent samples were prepared simultaneously. The contents of copper (Cu--324.8 nm), manganese (Mn--279.5 nm), iron (Fe--248.3 nm), zinc (Zn--213.9 nm), magnesium (Mg--285.2), and calcium (Ca--422.7 nm) were determined with Flame Atomic Absorption Spectrometry (FAAS), using an iCE 3000 Series atomic absorption spectrometer (Thermo Scientific, Madison, WI, USA) equipped with a GLITE data station, deuterium lamp background correction, and various cathodic lamps. In determining the Ca content, an aqueous solution of lanthanum chloride was added to obtain an La+3 concentration of 0.5% to eliminate the effects of P. The contents of sodium (Na--589 nm) and potassium (K--766.5 nm) were determined with the Atomic Emission Spectrometry (AES) in an acetylene-air flame, using the same spectrometer, but working in emission mode. Phosphorus (P--610 nm) content was determined using the colorimetric method with ammonium molybdate (VI), sodium sulfate (IV), and hydroquinone. A VIS 6000 spectrophotometer (Thermo Scientific, Madison, WI, USA) was used for the absorbance measurements. 2.2.3. Analysis of Folates Folates: 5-methyltetrahydrofolate (5-CH3-H4folate) and tetrahydrofolate (H4folate) were obtained from Sigma Aldrich (St. Louis, MO, USA) and prepared according to Konings . The concentration of the standards was calculated using the molar absorption coefficients given by Blakley . Protease (Sigma Aldrich, St. Louis, MO, USA) was dissolved in 0.1 M phosphate buffer, pH 7.0, with 1% (w/v) sodium ascorbate and 0.1% (v/v) 2-mercaptoethanol (in the amount of 4 mg/mL). This was done just before the analysis to avoid contamination with bacteria that can synthesize folate during incubation. Some g-Glutamyl hydrolase, from rat blood plasma, was purchased from Europa Bioproducts Ltd. (Cambridge, UK) and prepared according to Patring et al. . Samples were prepared in triplicate under dim light. A 10 g sample (exact to 0.001 g) was weighed into 30-mL centrifuge flasks (30-mL PPCO Oak Ridge PPCO Nalgene centrifuge tube; Rochester, NY, USA). Then, 20 mL of an extraction buffer (0.1 M phosphate buffer, pH 7.0, with 1% (w/v) sodium ascorbate and 0.1% (v/v) 2-mercaptoethanol) were added. The mixture was shaken (2500 rpm/10 s Vortex 4 basic IKA Vortex 4 basic; Staufen, Germany) and heated in a water bath at 100 degC for 15 min with occasional shaking. Then, the samples were cooled in an ice bath to 20 degC, and 0.25 mL of g-glutamyl hydrolase and 1 mL of protease were added. The samples were incubated at 37 degC for 4 h. Then, to inactivate the enzymes, the samples were heated for 5 min at 100 degC and cooled in an ice bath. Afterwards, they were centrifuged for 20 min at 12,000 rpm/4 degC (MPW-350R; Warsaw, Poland). After centrifugation, the supernatant was poured into 50-mL dark glass measuring flasks. An amount of 10 mL of the extraction buffer was added to the sediment remaining in the tubes, and the mixture was shaken and centrifuged again. The resulting supernatant was poured into the same measuring flasks. The volume of the flasks was supplemented with the extraction buffer and the whole sample was filtered through paper filters into 25-mL bottles. Sample purification was carried out using Solid Phase Extraction (SPE) on Strong Anion Exchange (SAX) Bakerbond SPE JT cartridges (3 mL x 500 mg Solid Phase Extraction Column and PP (polypropylene), Quaternary Amine (N+) Anion Exchange; Philipsburg, MT, USA), as described by Jastrebova et al. . Folate separation was carried out using the HPLC system (Shimadzu Nexera-i LC-2040 C plus; Shimadzu Co.; Kyoto, Japan) and the C18 LC column (150 x 4.6 mm, 3 mm, Luna 100A; Phenomenex; Torrance, CA, USA) as described previously . Identification and calculation of folate content were performed based on a standard with a known folate content (fluorescence detection, 290 nm excitation, and 360 nm emission wavelengths). 2.2.4. Statistical Analysis The data were analyzed using one-way analysis of variance (ANOVA) to identify significant differences in the contents of fatty acids, minerals, and folates between organic and commercial goat's milk and fermented goat's milk drinks. Statistically different results were assessed using the Duncan's test at a significance level of p < 0.05. The Statistica software package version 13.3 (StatSoft, Krakow, Poland, 2016) was used . 3. Results and Discussion 3.1. Fatty Acid Composition Fat extracted from the analyzed goat's milk and fermented goat's milk drinks had various contents of fatty acids (Table 2). The major fatty acids identified in the tested samples were saturated fatty acids (SFAs). Their highest content was determined in fat from organic natural yoghurts (604.77 mg/g fat). Fat from the other analyzed yoghurts had a significantly (p < 0.05) lower content of SFAs. The highest content of branched-chain fatty acids (BCFAs) was found in fat from kefirs (15.54 mg/g fat), and that of odd-chain fatty acids (OCFAs) in fat from organic probiotic yoghurts (17.23 mg/g fat). The content of monounsaturated fatty acids (MUFAs) was the highest in fat from the raw milk (236.43 mg/g fat), while the lowest in fat extracted from UHT milk (160.23 and 159.34 mg/g fat, respectively). The highest content of PUFAs (29.82 mg/g fat) was determined in fat from natural yoghurts (producer 2). Significantly (p < 0.05) lower contents of PUFAs were determined in the other analyzed products. The present study indicates that fat extracted from commercial natural yoghurts (producer 2) had a significantly (p < 0.05) higher content of n-3 (4.69 mg/g fat) and n-6 PUFAs (16.80 mg/g fat) compared to the other tested products (Table 2). The lowest content of n-3 PUFAs was found in fat from commercial natural yoghurts (producer 1) (1.76 mg/g fat) and that of n-6 PUFAs in fat from organic probiotic yoghurts (10.42 mg/g fat). Determination of the contents of n-6 and n-3 fatty acids is particularly important from a nutritional point of view. An adequate supply of these acids in the diet is indicated to reduce the risk of the development of many diseases . These acids cannot be synthesized in the body and must be derived from the diet. Although their everyday content is important, even more important is their ratio. Excessive amounts of n-6 PUFAs and a high n-6/n-3 ratio in the diet contribute to the pathogenesis of many diseases. An increased level of n-3 PUFAs and a lower n-6/n-3 ratio have a suppressive effect . In our study, the n-6/n-3 ratio determined in organic goat's milk was 5.29, and in commercial milk--5.72 (milk, producer 2) and 4.76 (milk, producer 1) (Table 2). In the tested fermented products, the n-6/n-3 ratio was the lowest in fat from organic Greek and organic probiotic yoghurts (3.00 and 3.16, respectively). The highest ratio (8.37) was determined in fat from commercial natural yoghurts (producer 1) (Table 2). According to Cossignani et al. , the n-6/n-3 ratio in goat's milk was 5.8. In a previous study, Paszczyk et al. showed that the ratio of these acids in goat's milk was 6.98, and in goat yoghurts--6.04. Dairy products with a lower n-6/n-3 ratio are characterized by a better composition of fatty acids supporting the proper functioning of the body. The acid, cis9trans11 C18:2 (CLA), which is the major CLA isomer in food, is a very important acid from a nutritional point of view. In milk and dairy products, it accounts for over 80-90% of the total CLA content . The data presented in Table 2 indicate that the analyzed products had various contents of this acid. Among the goat's milk samples, a higher CLA content was determined in fat extracted from raw organic milk (3.26 mg/g fat). Both commercial UHT kinds of milk had a significantly (p < 0.05) lower average CLA content, i.e., 2.88 and 2.54 mg/g fat, respectively. Cossignani et al. reported a higher cis9trans 11 C18:2 acid content in goat's milk of 3.9 mg/g fat. Differences in the quality and composition of ruminant milk are due to many factors, such as animal breed, age, lactation stadium, and health condition as well as the production system, diet, etc. . Previous investigations have indicated that the composition of fatty acids, including the content of CLA, in fermented milk products may differ from the milk they were made from. The content of CLA in dairy products may be influenced by industrial technological treatments and the additives used . According to research by other authors, the content of CLA in fermented dairy products is influenced by the strains of lactic acid bacteria used in the production process. Studies have also shown that selected bacterial strains are capable of synthesizing CLA during fermentation, but this process can be influenced by many factors, such as the number of cells, appropriate substrate concentration, and incubation conditions. In the analyzed fermented goat's milk drinks, the highest CLA content (4.39 mg/g fat) was determined in fat from commercial natural yoghurts (producer 2) (Table 2). In the tested organic products, Greek yoghurt produced with the vaccine containing strains of Streptococcus thermophilus and Lactobacillus delbrueckii subsp. Bulgaricus, and probiotic yoghurts produced with strains of Streptococcus thermophilus, Lactobacillus bulgaricus, Bifidobacterium bifidum, Lactobacillus acidophilus, and Lactobacillus casei, also had a high content of this acid (4.19 and 4.07 mg/g fat, respectively). The lowest CLA content, 3.28 mg/g fat, was determined in fat from organic natural yoghurt (Table 2) produced with the same strains as Greek yoghurt, which confirms the importance of the quality of the yoghurt vaccines used and the fermentation conditions applied for the synthesis of bioactive ingredients, including CLA. The most important trans fatty acids (TFAs) in the human diet are monounsaturated fatty acids with 18 carbon atoms. The study showed that the average total content of trans C18:1 isomers in the fat extracted from the analyzed products varied (Table 2). The highest content of trans C18:1 was found in fat extracted from commercial natural yoghurts (17.19 and 16.61 mg/g fat), whereas the lowest was in fat from organic natural yoghurts (10.89 mg/g fat) and UHT milk (producer 2) (10.88 mg/g fat). Considering the group of trans C18:1, all samples contained the highest amounts of trans10 + trans11 isomers. The major TFA in milk fat is vaccenic acid (trans11 C18:1, VA). Its content in milk fat largely depends on the way animals are fed . In ruminant milk from conventionally fed cows, this acid constitutes about 40-50% of all trans C18:1. Contents of other trans C18:1 isomers, such as trans9 and trans10, are much lower (by 5% and 10% on average, respectively) . Milk fat also contains trans C18:2 isomers; however, their content is lower compared to the content of trans C18:1 isomers. The conducted research showed that the average content of trans C18:2 isomers in the fat extracted from the analyzed products was diversified, with the highest value noted for Greek yoghurt fat (4.97 mg/g fat), and significantly (p < 0.05) lower than for the other analyzed products (Table 2). 3.2. Mineral Composition The elements that were present in the tested samples in the largest amounts were potassium, calcium, and phosphorus, with their contents noted in the range of: 1117.4-1965.3 mg/g, 1125.5-2324.4 mg/g and 1130.5-11,309.9 mg/g, respectively, regardless of product type (Table 3). The high content of these elements in goat's milk and its products was also demonstrated by Mohammed et al. , who determined them in goat yoghurts at the level of 1873.3 mg/g (potassium), 1256.7 mg/g (calcium) and 923.3 mg/g (phosphorus). Bezerril et al. considered these compounds to be the most important minerals found in goat's milk products, emphasizing the role of calcium and phosphorus in the formation of the casein structure. They showed that one portion of yoghurt (100 g) would provide almost 78% of the recommended daily intake of phosphorus, 67% of calcium, and approx. 34% of potassium. These authors also claimed that goat yoghurts were a good source of sodium, which is consistent with our study results. Sodium content in all analyzed samples was in the range of 307.0-522.8 mg/g, and the next element in terms of content was magnesium (126.3-205.4 mg/g). Similar contents of these elements in goat's milk have been reported by many authors who point out the high bioavailability of magnesium from goat's milk products when compared to those obtained from cow's milk. The minerals that were present in the tested products, in the smallest amounts, were: zinc (2.556-4.663 mg/g), copper (0.024-0.104 mg/g), manganese (0.029-0.209 mg/g), and iron (0.208-0.300 mg/g). A similar content of these elements in goat's milk was also indicated by Zamberlin et al. , who determined zinc at the level of 2.420 mg/g, copper at 0.110 mg/g, manganese at 0.055 mg/g, and iron in the range from 0.360 to 0.750 mg/g, depending on milk origin and goat breed. Analyzing the effect of milk processing on the content of the tested minerals, it should be stated that the levels of potassium, calcium, phosphorus, and magnesium were related to the degree of milk treatment. They were present in greater amounts in fermented beverages compared to milk, but this relationship was observed only in the case of commercial products (Table 3). Bergillos-Meca et al. reported the similarity of quantitative changes of these components in raw and fermented goat's milk and they showed statistically significant correlations between the levels of calcium, phosphorus, magnesium, and zinc, proving their similar susceptibility to changes during milk processing. The higher content of these elements in yoghurts obtained from goat's milk in laboratory conditions was also demonstrated in our previous study . We assayed potassium at 1797.0 mg/g in raw goat's milk, and at 2486.2 mg/g in yoghurt obtained using a thermostatic method. These changes ranged from 1536.6 to 2178.5 mg/g in the case of calcium, from 1168.8 to 1566.2 in the case of phosphorus, and from 104.3 to 150.3 mg/g in the case of magnesium. The levels of sodium, iron, and zinc were not related to the degree of goat's milk processing neither in organic nor industrial production, while manganese turned out to be the element whose content was related to the degree of milk processing. This component was present in the highest amount in organic goat's milk (0.209 mg/g), and in products obtained from it were lower by two or even three times. The content of manganese depended significantly on the f treatment method because its level was almost 10 times higher in organic milk than in commercial milk. There is little information in the available literature on the changes in manganese levels that occur during the processing of goat's milk or milk from other mammals. Certain information on this subject was provided by Al Sidawi et al. , who analyzed 195 samples of cow's milk and 25 samples of cheese from different regions of Georgia and showed that the level of this element in the analyzed milk differed 4-fold, and in cheese 3-fold, depending on the origin of the samples. Some changes in the level of manganese occurring during milk processing were also indicated by Quintana et al. , but the differences they identified were primarily related to the type of milk, not the degree of process advancement. Comparing the content of mineral compounds in all organic and commercial products, regardless of their type, it should be stated that the processing method had a significant impact on the levels of calcium, phosphorus, and copper. A significantly higher level of these elements was found in all products from commercial production, and the most noticeable differences were observed in the case of phosphorus whose level was 6-10 times higher than in organic products. Such a high content of phosphorus in industrial products may be related to the level of this element in commercial feed obtained from raw materials cultivated in areas fertilized with phosphorus. This element is very important in the nutrition of mammals, because it is, in terms of content, the second highest component of the skeleton after calcium, and its deficiency causes growth inhibition, reduced productivity, and deterioration of animal fertility. Cereal grain and protein supplements, often used in industrial animal nutrition, are also good sources of phosphorus . Similarly, high differences in both types of production were observed in the case of manganese, significantly higher levels of which were detected in products of organic origin, compared to commercial products (definite differences were up to 10 times). The greatest impact on the observed relationships may have been the way the goats were fed, especially the share of some cereal products or legumes in feed mixtures, which are a particularly good source of this element . 3.3. Folate Content The available literature data mainly concern the content of folates in cow's milk and cow's milk products . Table 4 presents folate contents in the tested goat's milk and fermented goat's milk drinks. In all tested products, the main folate form was methyl form (5-CH3-H4folate). Small amounts of H4folate were also determined in most of the analyzed samples. Previous studies on cow's milk and beverages also indicated that 5-CH3-H4folate was the major or even the only identified form of folate . Folate content in the tested material differed significantly (p < 0.05) among products. Among the milk samples, the highest folate content was determined in raw organic goat's milk (3.16 mg/100 g). Both commercially sterilized milks, UHT 1 and UHT 2 had significantly (p < 0.05) lower folate contents, i.e., 1.87 and 2.24 mg/100 g respectively. In previous studies on cow's milk , transport and storage conditions as well as high-temperature treatment, solar radiation, and acidity below neutral were indicated as possible factors causing folate losses during milk processing. Folate content in all tested milk was higher than that presented in Food Composition Tables for goat's milk, 1 mg/100 g, but lower than that set for cow's milk with 3.2% fat content, 5 mg/100 g . Although according to other authors , the content of folates in cow's milk is not high (4-10 mg/100 g) compared to other rich sources of this vitamin, such as green leafy vegetables, yeast, grains, or animal liver, due to the high frequency of consumption of dairy products, they can be a significant source of this vitamin in an everyday diet . Available research studies indicate that milk and dairy products can contribute up to 15% of daily folate intake, especially in the young generation and in countries with high consumption of these products, like Sweden and the Netherlands . A much higher folate content of 9.18 mg/100 g, compared to other tested fermented products, was found in organic Greek yoghurt, with a simple composition of yoghurt vaccine including Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus (Table 4). The lowest folate content, 1.60 mg/100 g, was determined in probiotic yoghurt. In the study of Gujska et al. , the folate content in fresh one-day stored yoghurts from cow's milk did not exceed 3.5 mg/100 g, and a significant (p < 0.05) folate loss was observed within 34 days of cold storage. In the authors' previous study on cow's milk yoghurts fermented with different yoghurt vaccines, higher folate levels of 4.5-10.5 mg/100 g were determined in fresh products. However, even those higher folate amounts in commercial yoghurts, when eaten in a normal daily portion, cannot meet more than 10-20% of the daily recommended intake of this vitamin. In the work on fermented milk beverages, additional attention was also paid to the possibility of folate consumption by lactic acid bacteria (LAB) during storage . On the other hand, various studies have indicated that the selection of appropriate bacterial cultures can promote folate synthesis during fermentation . However, this promotion depends on many factors, such as the applied starter culture (the strain, the combination of LAB), cultivation conditions, the incubation time as well as the composition of the culture medium, and the presence of folate precursors . In the study of Laino et al. , among different combinations, a strain of Lactobacillus delbruecki subsp. bulgaricus and Streptococcus thermophilus yielded the best results in milk in terms of folate content, which is consistent with the results obtained in our study for organic Greek yoghurt. Similarly, previous studies on the folate content of various strains of Saccharomyces cerevisiae have shown that the selection of appropriate starter cultures is very important when improving the folate content in foods produced with the use of yeast. In the tested organic kefir, despite a very diverse mixture of starter cultures declared by the manufacturer, the determined folate content of 0.99 mg/100 g was the lowest among all analyzed products. 4. Conclusions The research provides basic knowledge on the content of selected health-promoting components of goat's milk and fermented goat's milk products. Until recently, it has been believed that fat content in products is a source of energy accumulated in the fatty tissue required to build cell membranes. Today, fat's role as a component demonstrating health-promoting properties in the human body is gaining emphasis. The study results show that goat's milk and fermented products are a good source of components valuable from a nutritional point of view, such as fatty acids, minerals, and folates. The amount of these nutrients depended on milk processing and production methods, which resulted in significant differences in the content of health-promoting compounds determined in goat's milk and fermented milk drinks, as well as in organic and commercial products. The potential for obtaining increased levels of such compounds as folates and CLA was observed for organic Greek yoghurt, as well as for selected minerals, depending on the production method. However, further studies are needed to identify the impact of the quality of the raw material and the selection of starter cultures on the content of different bioactive compounds in the final product. Author Contributions Conceptualization, B.P.; methodology, B.P., M.C.-K. and E.T. validation B.P., M.C.-K. and E.T.; data analysis, B.P., M.C.-K., J.K. and E.T. writing--original draft preparation, B.P., M.C.-K. and J.K.; writing--review and editing, B.P., M.C.-K. and J.K.; visualization, B.P., M.C.-K. and J.K. All authors have read and agreed to the published version of the manuscript. Institutional Review Board Statement Not applicable. Informed Consent Statement Not applicable. Data Availability Statement Data is contained within the article. Conflicts of Interest The authors declare no conflict of interest. animals-13-00907-t001_Table 1 Table 1 Test material description. Products Characteristics of Starter Culture (According to the Information Provided by the Producer) Organic products Raw milk Natural yoghurt Streptococcus thermophilus, Lactobacillus bulgaricus Probiotic yoghurt Streptococcus thermophilus, Lactobacillus bulgaricus, Bifidobacterium bifidum, Lactobacillus acidophilus, Lactobacillus casei Greek yoghurt Streptococcus thermophilus, Lactobacillus delbrueckii subsp. bulgaricus Kefir Streptococcus thermophilus, Lactococcus lactis subsp. lactis, Lactococcus lactis subsp. cremoris, Lactococcus lactis subsp. Lactis Biovar diacetylactis, Leuconostoc mesenteries subsp. cremoris, Debarymyces hansenii, Kluyveromyces marxianus subsp. marxianus Commercial products UHT milk (producer 1) UHT milk (producer 2) Natural yoghurt (producer 1) Lactobacillus delbruecki sub. bulgaricus, Streptococcus thermophilus Natural yoghurt (producer 2) Cultures of lactic acid bacteria animals-13-00907-t002_Table 2 Table 2 Content of fatty acid groups (mg/g fat) in fat from goat's milk and fermented goat's milk drinks (Mean +- SD). Products SSFA 1 SSCFA 2 SBCFA 3 SOCFA 4 SMUFA 5 SPUFA 6 n-3 n-6 n-6/n-3 trans C18:1 trans C18:2 cis9trans11 C18:2(CLA) Organic products Raw milk (n = 4) 528.40 d,e +- 17.38 105.41 e +- 2.37 12.74 c +- 0.46 14.11 b,c,d +- 0.57 236.43 a +- 7.01 21.18 d +- 0.51 2.16 g +- 0.10 11.43 c,d +- 0.35 5.29 c +- 0.10 14.66 b +- 0.39 4.32 b,c +- 0.10 3.26 d +- 0.06 Natural yoghurt (n = 4) 604.77 a +- 23.76 130.71 a +- 4.97 14.96 a,b +- 0.69 16.44 a,b +- 0.65 231.13 a,b +- 9.79 21.23 d +- 0.83 2.27 g +- 0.16 11.82 c +- 0.48 5.22 c +- 0.22 10.89 e +- 0.62 3.93 c,d +- 0.18 3.28 d +- 0.10 Probiotic yoghurt (n = 4) 584.70 a,b +- 4.58 129.23 a +- 1.77 14.54 a,b +- 0.22 17.23 a +- 0.14 180.79 d,e +- 3.60 22.08 d +- 0.34 3.30 c +- 0.04 10.42 e +- 0.19 3.16 g +- 0.03 14.79 b +- 0.26 4.29 b,c +- 0.09 4.07 b +- 0.08 Greek yoghurt (n = 4) 526.84 d,e +- 18.93 114.11 c +- 2.77 14.15 a,b,c +- 0.56 14.83 b,c +- 0.58 196.35 c +- 7.84 24.22 b,c +- 1.39 3.76 b +- 0.11 11.29 c,d +- 0.60 3.00 g +- 0.08 13.93 c +- 0.74 4.97 a +- 0.55 4.19 b +- 0.14 Kefir (n = 4) 587.52 a,b +- 27.67 122.56 b +- 4.97 15.54 a +- 0.81 16.25 a,b +- 0.98 220.82 b +- 10.24 20.96 d +- 0.85 2.66 e +- 0.15 10.86 d,e +- 0.52 4.09 e +- 0.08 12.65 d +- 0.43 3.67 d +- 0.05 3.77 c +- 0.19 Commercial products UHT milk (producer 1) (n = 4) 512.76 e +- 6.80 107.65 d +- 1.55 11.49 d,e +- 0.17 12.91 c,d +- 0.18 160.23 f +- 2.05 25.04 b +- 0.31 3.06 d +- 0.04 14.57 b +- 0.18 4.76 d +- 0.05 14.96 b +- 0.26 4.53 b +- 0.11 2.88 e +- 0.05 UHT milk (producer 2) (n = 4) 565.88 b,c +- 17.42 112.80 c,d +- 2.87 11.13 e +- 0.37 12.38 d +- 0.43 159.34 f +- 5.88 22.09 d +- 0.86 2.46 f +- 0.08 14.00 b +- 0.47 5.72 b +- 0.01 10.88 e +- 0.51 3.09 e +- 0.20 2.54 f +- 0.11 Natural yoghurt (producer 1) (n = 4) 547.98 c,d +- 9.14 127.22 a,b +- 3.20 13.83 b,c +- 2.43 12.23 d +- 2.65 190.98 c,d +- 4.10 23.80 c +- 0.42 1.76 h +- 0.06 14.68 b +- 0.28 8.37 a +- 0.29 17.19 a +- 0.25 3.54 d +- 0.30 3.82 c +- 0.08 Natural yoghurt (producer 2) (n = 4) 500.89 e +- 18.25 114.83 c +- 4.65 11.88 d,e +- 0.72 12.19 d +- 3.34 178.63 e +- 11.42 29.82 a +- 1.14 4.69 a +- 0.12 16.80 a +- 0.90 3.58 f +- 0.23 16.61 a +- 0.55 3.95 c,d +- 0.37 4.39 a +- 0.26 n--number of samples; Mean--mean value; SD--standard deviation; a,b,c,d,e,f,g,h--different letters within the column indicate a statistically significant difference (p < 0.05), 1 S SFA--sum of all saturated fatty acids; 2 S SCFA--sum of short-chain fatty acids (C4:0-C10:0); 3 S BCFA--sum of branched-chain fatty acids; 4 S OCFA--sum of odd-chain fatty acids; 5 S MUFA--sum of monounsaturated fatty acids; 6 S PUFA--sum of polyunsaturated fatty acids. animals-13-00907-t003_Table 3 Table 3 Minerals content in goat's milk and fermented goat's milk drinks (mg/g) (Mean +- SD). Products Mg Ca Na K P Cu Mn Fe Zn Organic products Raw milk (n = 4) 156.4 d +- 2.702 1125.5 h +- 5.725 347.1 e +- 6.881 1745.1 b,c,d +- 169.420 1373.7 e +- 3.731 0.040 e +- 0.004 0.209 a +- 0.005 0.286 c,d +- 0.004 4.398 b +- 0.185 Natural yoghurt (n = 4) 166.9 c +- 6.046 1165.4 g +- 23.985 365.3 d +- 7.781 1690.7 c,d +- 247.383 1365.1 e +- 22.682 0.040 e +- 0.002 0.118 b +- 0.003 0.267 e +- 0.011 4.239 c +- 0.095 Probiotic yoghurt (n = 4) 162.2 c +- 3.026 1237.6 e +- 28.320 319.6 f +- 5.278 1965.3 a +- 24.285 1143.9 f +- 39.792 0.024 f +- 0.003 0.067 e +- 0.004 0.330 a +- 0.018 3.681 d,e +- 0.067 Greek yoghurt (n = 4) 162.6 c +- 1.800 1195.2 f +- 14.710 365.6 d +- 7.998 1791.1 a,b,c +- 67.580 1331.1 e +- 26.655 0.052 d +- 0.004 0.091 d +- 0.004 0.275 d,e +- 0.014 3.863 d +- 0.088 Kefir (n = 4) 155.5 d +- 0.875 1193.9 f +- 16.575 341.8 e +- 2.113 1582.6 d +- 62.113 1130.5 f +- 18.438 0.028 f +- 0.002 0.097 c +- 0.002 0.289 c,d +- 0.011 3.678 e +- 0.065 Commercial products UHT milk (producer 1) (n = 4) 126.3 f +- 2.695 1322.9 d +- 4.343 522.8 a +- 1.749 1243.0 d,e +- 13.461 9382.9 c +- 71.219 0.104 a +- 0.001 0.029 g +- 0.0001 0.303 c +- 0.003 3.149 f +- 0.017 UHT milk (producer 2) (n = 4) 139.1 e +- 1.059 1554.6 c +- 9.720 307.0 g +- 1.802 1117.4 e +- 4.726 8148.1 d +- 22.030 0.072 c +- 0.002 0.030 g +- 0.0004 0.208 f +- 0.004 2.556 g +- 0.008 Natural yoghurt (producer 1) (n = 4) 205.4 a +- 2.869 1645.8 b +- 9.114 425.4 c +- 13.404 1931.1 a,b +- 24.932 10,591.2 b +- 232.535 0.104 a +- 0.005 0.044 f +- 0.002 0.278 d,e +- 0.001 3.729 d,e +- 0.068 Natural yoghurt (producer 2) (n = 4) 172.9 b +- 0.816 2324.4 a +- 21.891 454.2 b +- 11.079 1777.6 a,b,c +- 2.788 11,309.9 a +- 276.317 0.097 b +- 0.002 0.042 f +- 0.002 0.310 b +- 0.009 4.663 a +- 0.019 n--number of samples; Mean--mean value; SD--standard deviation; a,b,c,d,e,f,g--different letters within the column indicate a statistically significant difference (p < 0.05). animals-13-00907-t004_Table 4 Table 4 Folate content in goat's milk and fermented goat's milk drinks (Mean +- SD). Products Folates (mg/100 g) Organic products Raw milk 3.16 1,b +- 0.03 Natural yoghurt 2.36 c,d +- 0.09 Probiotic yoghurt 1.60 f +- 0.06 Greek yoghurt 9.18 a +- 0.42 Kefir 0.99 g +- 0.06 Commercial products UHT milk (producer 1) 1.87 e +- 0.01 UHT milk (producer 2) 2.24 d +- 0.05 Natural yoghurt (producer 1) 2.56 c +- 0.04 Natural yoghurt (producer 2) 3.32 b +- 0.18 1 Folate content is expressed as means with standard deviations from triplicates per fresh weight unit. Folates is the sum of 5-CH3-H4folate and H4folate expressed as folic acid content using a molar absorption coefficient given by Blakely ; a,b,c,d,e,f,g--different letters within the column indicate a statistically significant difference at p < 0.05 in the Duncan test. Disclaimer/Publisher's Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). 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PMC10000186

Animal diseases are a serious threat to animal husbandry production and diet health, and effective prevention and control measures need to be explored. This study investigates the factors influencing the adoption of biosecurity prevention and the control behaviours of hog farmers towards African swine fever and provides appropriate recommendations. Using research data from Sichuan, Hubei, Jiangsu, Tianjin, Liaoning, Jilin, and Hebei, we employed a binary logistic model to empirically analyse these factors. Regarding individual farmer characteristics, male farmers emphasised biosecurity prevention and control in farms, with higher education actively influencing the adoption of prevention and control measures. Farmers who received technical training were actively inclined to adopt such behaviours. Furthermore, the longer the duration of farming, the more probable the farmers were to neglect biosecurity prevention and control. However, the bigger and more specialised the farm, the more inclined they were to adopt prevention and control behaviours. With respect to disease prevention and control awareness, the more risk-averse the farmers were, the more they actively adopted epidemic prevention behaviours. As the awareness of epidemic risk increased, the farmers tended to adopt active epidemic prevention behaviours by reporting suspected outbreaks. The following policy recommendations were made: learning about epidemic prevention and improving professional skills; large-scale farming, specialised farming; and timely dissemination of information to raise risk awareness. hog farms animal diseases prevention and control behaviours African swine fever influencing factors National Natural Science Foundation of China72033009 Agricultural Science and Technology Innovation Program10-IAED-01-2022 ASTIP-IAED-2022-01 National Social Science Youth Fund Project22CGL025 the special fund of basic scientific research business of central public welfare research institutes1610052022028 161005202106 This research was funded by the key project of the National Natural Science Foundation of China (72033009), the Agricultural Science and Technology Innovation Program (10-IAED-01-2022, ASTIP-IAED-2022-01), the National Social Science Youth Fund Project (22CGL025), and the special fund of basic scientific research business of central public welfare research institutes (1610052022028, 161005202106). pmc1. Introduction The high incidence of and difficulty in controlling major animal diseases has led to a focus on animal health and its relationship with biosecurity . The Food and Agriculture Organization (FAO) considers biosecurity to be directly related to agricultural sustainability, food safety, and environmental protection . The application of biosecurity is the most important measure for avoiding the spread of disease . Disease prevention through biosecurity measures is considered an important factor in improving animal production . In August 2018, China experienced an outbreak of African swine fever (ASF), which spread expeditiously across several provinces, resulting in a steep decline in hog production and dramatic market fluctuations . The outbreak significantly impacted the hog industry, mainly in terms of markets and hog farming entities. The infection led to abnormal fluctuations in the prices of meat, especially pork, disrupting market development and resulting in considerable disparities in regional pork prices . Most farm owners lacked the experience and expertise to prevent and control the virus. Therefore, the measures taken were inadequate, culminating in low-level biosecurity in farming environments. Consequently, the epidemic brought huge economic losses to hog-farming entities . Differences in production size, biosecurity standards, production inputs, and sales practices between hog farms can affect the potential risk of disease transmission and lead to a wide variation in the ability to take preventive measures between farms . The current biosecurity situation in China is not promising; the state of some farms is alarming in the wake of the African swine fever, limiting their productivity, and causing heavy economic losses. Therefore, it is necessary to understand the current situation of biosecurity in Chinese hog farms and investigate the factors that influence the prevention and control of epidemics, to improve the level of biosecurity. Existing studies on the epidemic prevention behaviours of farmers have mainly focused on specific aspects. The epidemic prevention behaviour of farmers is the first step in animal epidemic prevention and control. Farmers usually perform four kinds of epidemic prevention behaviours, which include, the improvement of feeding conditions, the use of vaccines and veterinary drugs, the reporting of animal epidemics, and the treatment of sick and dead livestock . Strengthening biosecurity management measures is the most important means to prevent and control African swine fever in China's pig farms. The construction of a biosecurity system requires attention to the safety of inputs, optimisation of the feeding environment, management of personnel and vehicles, disinfection, and waste disposal . Zhou et al. examined the disease prevention and control behaviours of farmers and found that small-scale hog farms focused mainly on disinfection, feed and water management, disease prevention and control, and incoming and outgoing vehicle management. Meanwhile, retail farmers emphasised the regulation of the internal and external environment of hog farms, personnel management, and the use of vaccines and drugs. In a study on biocontainment measures in avian influenza prevention and control, Huang et al. focused on preventing rodents and birds, as well as measures to manage incoming vehicles and disinfection of personnel. It has also been suggested that four factors: vaccines, veterinary drugs, disinfection and cleaning, and quarantine, play a crucial role in the prevention and control of hog diseases . The immunisation file, also known as the animal household register, is also an important link in the animal epidemic prevention chain. It records in detail the ages of the animals, entry bar, immunisation date, immunisation identification number, vaccine type, injection, etc . The establishment of immunisation files by farmers is vital to ensure the implementation of compulsory immunisation programs, and the conducting of risk assessments for the epidemic control of animal diseases to accurately grasp the immunisation status and monitor animals, both overall and individually, to avoid missed and late immunisations and to ensure the overall quality of the immunisation . Moreover, ensuring drinking water quality is an important component of livestock rearing and management . Water quality can be evaluated by testing the farm's drinking water source to check whether the bacterial count exceeds the limit . Since all types of vehicles are in direct contact with the outside world, vehicle biosecurity and management are also important aspects of hog farm biosecurity controls . Scholars have long examined the factors influencing disease prevention and control in farms. He Zhongwei et al. studied the influencing factors of prevention and control behaviours in poultry farmers, taking the individual characteristics, breeding characteristics, epidemic awareness, external environment cognition, and policy implementation as explanatory variables, with which to study the impact of whether farmers adopt epidemic prevention behaviour. Li Yanling et al. analysed the factors affecting epidemic prevention behaviours from four aspects: farmer characteristics, production and operation characteristics, cognitive characteristics, and environmental characteristics. Studying the relationship between epidemic prevention behaviours and the characteristics of poultry farmers, Li Jie et al. used further four prevention behaviours: regular disinfection, whether to believe the official warning, whether to clean the chicken cage, and whether to adhere to the "all-in and all-out" system. Song et al. conducted an empirical analysis of hog farms in the Hubei province and concluded that factors such as farmer literacy, whether they had participated in the training, their knowledge of epidemics, the scale of farming, and whether they had joined a cooperative organisation significantly influenced whether the farmers adopted the disease prevention and control measures. Through a study in the Xinjiang Autonomous Region, Chen et al. . analysed disease prevention and the control behaviours of cattle and sheep breeding cooperatives and found that the education level of the breeders, whether they had received training, their annual income, and the provision of timely assistance were the main influencing factors. Luo et al. . studied the disease prevention and control behaviours in beef cattle and meat sheep farms in the Qinghai province and determined that the education level, whether they had received disease prevention and control training, if the farm maintained records of disease prevention and control, and previous certifications were the main influencing factors determining the adoption of the disease prevention and control behaviours by the farmers. Through literature reviews, it can be summarised that there are four main categories of factors that influence the disease prevention and control behaviours of farmers: the characteristics of the farmer, including their gender, age, and education level; the production and operation characteristics, such as the proportion of the farming income in the total household income; the cognitive characteristics of the farmer, such as their cognitive level, the willingness for disease prevention and control and safety awareness; and environmental characteristics, including socio-economic environment and government policies, and what social services are provided . While many scholars have adequately researched the disease prevention and control behaviours of farmers alongside any influencing factors, there remain some shortcomings. Most of this relevant research merely focuses on a specific region or province and the representativeness of the samples and the universality of the pattern are, thus, inadequate. For this reason, we used the logistic model to empirically analyse the main factors affecting the relevant animal disease prevention and control behaviours, from the perspective of the farmers. Moreover, we analysed the microscopic research data on hog farmers in Sichuan, Hubei, Jiangsu, Tianjin, Liaoning, Jilin and Hebei, and proposed more targeted countermeasures to improve the prevention and control capacity of the farmers, by providing policy recommendations to improve China's animal disease prevention and control at the grassroots level. 2. Materials and Methods 2.1. Data Sources The data used in this study were obtained from a survey of hog farming and disease prevention and control conducted by the research team in the third and fourth quarters of 2020. The survey area includes seven provinces (municipalities directly under the Central Government) in Sichuan, Hubei, Jiangsu, Tianjin, Liaoning, Jilin, and Hebei. The selection of the provinces and regions for the research mainly used the following approach: each province was ranked from the highest to lowest by the number of slaughtered fattened hogs in 2018, the top 18 provinces whose total production reached 90.78% of the national total were categorised into six grades, with each grade representing a different output level, and one province was selected in each grade as a representative of the corresponding production level. Moreover, considering the geographical variability of hog farming, we selected the provinces to research according to six major geographical sub-regions of China. The north-western region, where a very low number of pigs are produced, was not included in the 18 provinces. The remaining five regions were considered in an integrated/comprehensive manner. When sampling representative provinces in different grades, we selected provinces in different geographical subdivisions, as far as possible, to indicate the variability of the geographical areas used for farming. The specific selections are listed in Table 1. In addition, some of the provinces selected included those with African swine fever outbreaks, to consider the research content in terms of disease prevention and control. The samples were selected by adopting the principle of stratified stochastic sampling. To gain a comprehensive picture of the occurrence and non-occurrence of the African swine fever outbreak, we selected one infected and one non-infected county per province. Based on the above, we selected the specific locations for research, as shown in Table 2. The survey, which mainly targeted hog farmers, included a combination of field interviews and questionnaires. A total of 267 questionnaires were returned, and 262 valid samples were obtained, excluding those with missing or abnormal data. The items in the questionnaire mainly included basic characteristics of farming entities (age, gender, education level, and years of farming, etc.), the production and operation information of the farm (scale of farming, type of operation, and mode of farming, etc.) alongside the biosecurity prevention and control measures undertaken at the farm, including quarantine, disinfection, as well as vehicle and personnel management. 2.2. Descriptive Analysis of the Survey Sample The survey sample was selected in line with the basic characteristics of hog farming entities, and primarily included males, who accounted for 81.30% of the total sample. Individuals aged between 45-54 accounted for half of the overall group, and those aged 45 or above accounted for 73.66%, in line with the characteristic that the farming entities mainly consisted of middle-aged and elderly male groups. The majority had 6-15 years of education, and their education levels were dominated by junior secondary and senior secondary school, which accounted for 90.08%. The number of years of farming was evenly distributed, with the highest percentage (26.72%) having been engaged in hog farming for 6-10 years. Meanwhile, 79.39% of the farmers had received technical training, whereas approximately 20% had not. The percentage of entities whose farming income accounted for over 50% of their total household income was 92.75%. The proportion of farmers who adopted the farming mode of self-breeding and self-rearing was 69.47%, followed by commercial hog fattening at 26.34%. Additionally, 82.44% of the farms had not participated in industrial organisations, whereas the remaining 17.56% of farms had joined industrial organisations. The basic characteristics of the survey sample distributions are listed in Table 3. The detailed data can be found in the Supplementary Materials File (S1_survey data of pig farms). Epidemic prevention and control in the farm. According to the code of construction of ecological scale pig farms, the distance of the farm site from the main traffic routes and residential areas should meet certain requirements. The farm boundary should be no less than 300-metres from the main traffic routes, 300-metres from residential areas and other livestock farms, and 1000-metres from areas such as slaughterhouses, livestock and poultry processing plants, livestock and poultry trading markets, waste disposal sites and scenic tourist areas. With respect to the location of farms, the proportion of hog farms located over 300-metres from other farms was 68.70%, those located over 300-metres from residential areas were 69.47%, while 17.94% of the farms were located less than 300-metres from main traffic routes. With the above three indices taken together, the location of more than 60% of the survey sample farms was in accordance with construction regulations. The proportion of farms with an "all-in, all-out" management of feeder hogs is a mere 33.59%, indicating that most farmers buy and sell feeder hogs in separate batches. Furthermore, 90.08% of the farms have separate pens for sows, feeder hogs, and piglets, while 9.92% of farms had not conducted separate zoning management; and 76.72% of the farms had dedicated means of transport. With respect to immunisation records, 87.79% of the farms maintained them. Meanwhile, 83.59% of the farms had separate clean and dirty paths, but 40% of the farms did not conduct effective control of birds, as shown in Table 4. See a Supplementary Materials File (S1_survey data of pig farms) for complete information. 2.3. Variable Selection By reviewing the literature, we found that the disease prevention and control behaviour of hog farmers is mainly influenced by the characteristics of farmers, variables such as farm size and degree of specialisation, and risk awareness of farmers. The variables selected for this study included the following components. The characteristics of the farmers mainly included gender of the farmer, educational level, and whether the farmer had received training in hog farming techniques. The farm characteristics mainly included the number of years of farming, the scale of farming, and the degree of specialisation. The farmers' risk awareness included their investment risk appetite, whether the hog farms can effectively block the introduction of African swine fever, whether they will report any suspected outbreaks in hog farms, and whether they will report any suspected outbreaks in neighbouring hog farms. The farmers' disease prevention and control behaviours included, whether the farm has immunisation records, whether water sources were tested, and whether the farm has a dedicated means of transport. The descriptive statistical analysis of the variables is detailed in Table 5. The observed original data of the variables can be found in a Supplementary Materials File (S1_survey data of pig farms). 2.4. Model Construction This study constructed a logistic model to test the factors influencing the adoption of preventive and control behaviours by farmers, which was estimated using the maximum likelihood estimation method. The dependent variables are divided into two categories: y=1 if the hog farmers adopt disease prevention and control behaviours, while y= 0 if not. X=(x1,x2,,xk)T denotes the independent variable. The relationship between the probability P(y=1|X) of occurrence of the event and the independent variable was studied, with the logistic regression equation as follows:(1) P(y=1|X)=F(b0+b1x1++bkxk)=eb0+b1x1++bkxk/(1+eb0+b1x1++bkxk)+e 3. Results In this study, Stata software was used to regress a binary logistic model for each of the three factors influencing hog epidemic prevention behaviours. Prior to model regression, variance inflation factor tests were conducted separately to verify the existence of multicollinearity problems. The results showed that the highest value of the inflation factor of the variables did not exceed 2, implying that there was no multicollinearity problem. The estimation results show that the three models fit well and the LR statistical significance levels are all highly significant at the 1% significance level. As shown in Table 6, in terms of individual characteristics, the gender of the farmer has a significant effect, a 10% significance level, on the adoption of disease prevention and control behaviours, whereas having a dedicated means of transport in place, and its coefficient is significantly negative, indicating that male hog farmers are more inclined to adopt dedicated means of transport. This is related to the fact that male farmers, as the main breadwinners of the family, pay greater attention to taking precautionary measures to ensure that household assets are protected from epidemics. At the 1% level of significance, the number of years of education has a positive effect on the adoption of disease prevention and control behaviours by hog farmers. The higher the educational level, the more active a farmer is in adopting the behaviour of testing the water source. This indicates that people with higher educational levels have greater knowledge and access to information about epidemics and take biological prevention and control at the farm more seriously. Technical training had a positive effect on the building of immunisation records and the disease prevention and control behaviours of dedicated means of transport, at significance levels of 10% and 1%, respectively. This indicates that the farmers' participation in technical training helped to augment their knowledge of disease prevention and control, improved their relevant awareness, and promoted the relevant behaviours required to prevent epidemics. With respect to farming characteristics, the number of years of farming has a significantly negative effect on the adoption of disease prevention and control behaviours, with a 1% significance level. This indicates that the more years of farming they possess, the less of a priority the farmers attached to disease prevention and control. When they have adequate farming years, they tend to accumulate extensive farming experience, which, nevertheless, will prompt them to rely on their experience in animal disease prevention and control; thus, they are found to lack in science, which results in gaps in their disease prevention and control behaviours. The size of the farm had a significantly positive effect on the adoption of immunisation records and water testing behaviours at the 5% and 1% levels. This indicates that as the scale of farming increases, farmers pay additional attention to the adoption of disease prevention and control measures to avoid huge economic losses from the onset of diseases. At the 5% significance level, the degree of specialisation contributes positively to both the building of immunisation records and the adoption of dedicated means of transport, suggesting that the higher the proportion of total household income from farming, the greater the focus on the biosecurity situations in the farms, with more human, material, and financial resources invested in farming, which is conducive to the adoption of biosecurity prevention and control behaviours. With respect to disease prevention and control awareness, the farmers' appetite for risk had a significant negative effect on building immunisation records and water testing at the 10% and 1% levels, respectively. This suggests that the more risk-averse the farmers were, the more active they were in adopting disease prevention and control behaviours. Conversely, the more risk-seeking the behaviour, the more passive they were in adopting disease prevention and control behaviours. This can be attributed to both types of farmers adopting different attitudes towards risks: active and passive. At the 10% significance level, the more the farmers believed that the farm can effectively intercept the introduction of African swine fever, the more likely they were to adopt active disease prevention and control behaviours and test water sources. In contrast, farmers were more likely to build immunisation records when reporting suspected outbreaks at their farms. At the 1% significant level, farmers were more inclined to have their water sources tested and their means of transport dedicated to the farm when reporting suspected outbreaks found in neighbouring hog farms. These data show that the farmers' active awareness of disease prevention and control can also generate active disease prevention behaviours and exhibit a positive effect. 4. Discussion Biological vectors and trade are the main methods for African swine fever transmission. Biological vectors generally refer to organisms that infect and carry the African swine fever virus (ASFV). For example, ticks, wild suids, and domestic pigs. Susceptible pigs have direct or indirect contact with these infection sources, which then transmit the African swine fever. In trade, infected pork products, swill, alongside equipment and vehicles are the media of ASFV . Epidemiological studies revealed three main routes of ASFV transmission: 46% was spread by unsterilised vehicles and workers, 34% by swill-feeding pigs, and 19% by trans-regional transportation of pigs and products . Unsterilised vehicles and people are the main vectors in the spread of African swine fever, and strict control of vehicles and people entering farms is particularly necessary. The questionnaire survey revealed that most farms have strict control over people and vehicles, whereby foreign vehicles and people are not allowed to enter at will, and farms have special transport as well as vehicle decontamination areas, although this does not cover all farms, and a small number of farms are still loosely managed. In terms of feed and water, only 2.63% of farmers feed their pigs with slop, which is excellent. However, for drinking water, more than half of the farmers have not tested their water sources, which greatly increases the risk of contamination in the farms and poses a threat to the health of the hogs. Regarding the prevention and control measures of ASF, there is no commercialised vaccine at present, and the focus on prevention and control of ASF is "prevention". The only control measures are to find infected pigs as soon as possible, to either cull or treat them harmlessly, or block and isolate them . Chai Weihua et al. discussed the measures to strengthen pig breeding management and disease prevention and control based on the three elements of infectious disease control: controlling the source of infection, cutting off the transmission routes, and protecting susceptible populations. Starting with "prevention", represents cutting off all the paths through which the virus may spread. It is strictly forbidden for people outside the pig farm to enter, and the staff should be disinfected and protected when entering and leaving. Disinfect the materials and vehicles entering the pig farm, and when introducing breeding pigs from outside, it is necessary to set up an isolation and observation period before they become gregarious and attempt to adopt a population management mode of "all-in and all-out" to improve the biosecurity barrier. An Dong et al. concluded that ASF should be prevented and controlled by isolation measures, standardised disinfection procedures for farms, training of farmers on biosecurity knowledge, and international border epidemic prevention. The biosecurity measures taken on the surveyed farms focus on cutting off the means of transmission. Biological control is carried out to prevent birds, rodents, and insects from entering the farm, and to disinfect and control the people and vehicles. In terms of pig management, 90.08% of the farms are zoned, with sows, feeders, and piglets kept separately. However, only 33.59% of the farms adopt "all-in, all-out" management practices for their pigs, which undoubtedly increases the risk for the farms not currently adopting such management practices, as those newly introduced pigs may carry viruses into the farm, increasing the risk of infection for pigs throughout the farm. In terms of the farmers' characteristics, men are more aware of epidemic prevention and actively take epidemic prevention actions . As the head of the family, men bear the main business responsibilities, thus, taking active epidemic prevention actions to avoid economic losses. The more educated the farmers are, the more inclined they are to take epidemic prevention measures . The higher the educational background, the more knowledge you will receive, and the higher your ability to acquire information and learn new knowledge. When an epidemic occurs, you will take active defensive measures. Farmers with more years of farming possess rich farming experience, yet this also means that they will follow the old rules. Compared with farmers with fewer farming years, they pay attention to different disease prevention and control measures . The larger the size of the farm when faced with an epidemic, the greater the losses suffered will be, having the intrinsic motivation to adopt epidemic prevention and control behaviours as well as the financial ability to support them . The relationship between risk perception and the adoption of conservation behaviours by farmers has been examined by scholars. The results of Valeeva's study revealed the direct importance of the intrinsic risk characteristics of farmers for their adoption of risk management strategies . Self-protective (risk-averse) behaviours, in general, directly contribute to the farmers' decisions to exhibit more specific farm-protective behaviours. This is the exact opposite of the derived empirical results. Conversely, the results of the study indicated that farmers with a higher perception of animal disease risk tend to adopt bird-proofing measures, and those who prefer risk tend to disinfect their vehicles. This is consistent with the results of this paper. The reason for these different results is the different meanings of the measurement indicators. In the questionnaire, "high risk, high return", "medium risk, medium return" and "low risk, low return" were used as options to measure the farmers' risk preferences. The empirical results show that farmers with a 'high risk, high return' bias are more likely to adopt epidemic prevention behaviours. The outbreak of ASF in China led directly to a shortage of pig stocks and a sharp rise in pork prices . On the one hand, restocking brings huge economic benefits to farmers, but at the same time, they are exposed to the risk of further contracting the ASFV. These farmers, who prefer "high risk, high reward", are driven to replenish their farms by the huge economic gains, but at the same time take active measures to protect themselves against the risk of the ASFV. 5. Conclusions and Recommendations 5.1. Conclusions Most of the farmers in the survey area are middle-aged or older males, mostly above 45 years of age, who have a primary school and junior secondary school education and have received technical training. Most of the farms can implement zoning management, and dedicated means of transport, and have built immunisation records. Nevertheless, there are still approximately 30% of the farms whose layout does not meet the ecological codes of construction, and most of them have not adopted an "all-in, all-out" management style. With respect to individual farmers' characteristics, gender, education, and technical training had a significant impact on the adoption of biosecurity measures. Specifically, male farmers paid significant attention to biosecurity control in their farms. The more educated the farmers, the more active they were in adopting biosecurity measures; and farmers who had received technical training were more inclined to adopt biosecurity behaviour than those who had not. With respect to farming characteristics, the number of years of farming, the scale of farming, and the degree of specialisation had a significant effect on the adoption of disease prevention and control behaviours in the farms. Specifically, the more years of farming, the more likely the farm was to neglect biosecurity control. However, the larger the farm, the greater importance it placed on adopting disease control measures; and the more specialised the farm is, the more inclined it was to adopt disease prevention and control behaviours. With respect to risk awareness, the more risk-averse a farmer was, the more active they were in adopting disease prevention and control behaviours. Conversely, the more risk-seeking a farmer was, the more passive they were in adopting disease prevention and control behaviours. When farmers have an active awareness of disease prevention and control, they will adopt active disease prevention and control behaviours while reporting suspected epidemics found in neighbouring farms. 5.2. Policy Recommendations 5.2.1. Learning about Epidemic Prevention and Improving Professional Skills Farmers' knowledge reserves of farming techniques and animal epidemic prevention, their mastery of epidemic prevention measures, and their own scientific literacy, all have a direct impact on the epidemic prevention behaviours adopted by farmers. Therefore, it is important to provide farmers with training in epidemic prevention and control from multiple angles, subjects, and in a variety of ways. Knowledge of epidemic prevention and control should be promoted through the internet, APP, TV, and veterinary medicine dealers. Subjects such as animal husbandry and veterinary departments, scientific research institutes, breeding associations, and government departments should increase technical support for farmers and organise regular epidemic prevention training for farmers to improve their understanding of the principles of various epidemic prevention and control measures and their effectiveness, and to master the know-how of using various prevention and control measures to build the first barrier for epidemic prevention. 5.2.2. Large-Scale Farming, Specialised Farming Promoting large-scale farming models requires strengthening the promotion and guidance of large-scale farming. Regularly organised visits to large-scale farming demonstration farms for retail and small-scale farmers to raise their awareness of the importance of developing large-scale farming and carrying out biocontainment measures. Improve the degree of specialisation in farming. Promote semi-modern and fully enclosed management. Farms should strictly control the entry and exit of personnel and staff should strictly implement the bathing and disinfection entry system to avoid the risk of virus infection. Environmental disinfection is carried out regularly and quantitatively in pig houses. Establish a sound farm biosecurity prevention and control system to provide a healthier growing environment for pig breeding. 5.2.3. Timely Dissemination of Information, Raising Risk Awareness Animal husbandry and veterinary departments should release epidemic information in a timely manner. By using new media technology to build a smart platform for animal epidemic prevention and control, timely epidemic information can be released, making it possible for farmers to learn about epidemic information through their mobile phones. Making farmers aware of the serious dangers of epidemics and raising their risk awareness will help increase their willingness to implement epidemic prevention and control measures. The government should vigorously publicise relevant laws and regulations such as the Animal Epidemic Prevention Law and the Code of Practice for the Disposal of Major Animal Diseases to raise the awareness of farming subjects to understand the law and their responsibilities and to know the rules and regulations. Acknowledgments This research work was carried out by the members of the research group on animal disease prevention and control. The members of the research group would like to thank all participating farmers for their cooperation. Supplementary Materials The following supporting information can be downloaded at: Excel data file: S1_survey data of pig farms.xlsx. Click here for additional data file. Author Contributions Conceptualisation, J.W. and X.H.; methodology, J.W.; software, J.W.; validation, J.W. and X.H.; formal analysis, J.W.; investigation, J.W.; resources, J.W.; data curation, J.W.; writing--original draft preparation, J.W.; writing--review and editing, J.W.; visualisation, J.W.; supervision, X.H.; project administration, X.H.; funding acquisition, X.H. All authors have read and agreed to the published version of the manuscript. Institutional Review Board Statement Not applicable. Informed Consent Statement Informed consent was obtained from all subjects involved in the study. Data Availability Statement The data presented in this study are available in the article and supplementary materials. Conflicts of Interest The authors declare no conflict of interest. animals-13-00787-t001_Table 1 Table 1 Number of Slaughtered Fattened Hogs by Province in 2018. Grade Province Number of Slaughtered Fattened Hogs (1000 Head) Region 1 Sichuan 66,383 Southwest China 2 Henan 64,024 South Central China 3 Hunan 59,937 South Central China 4 Shandong 50,823 Eastern China 5 Hubei 43,635 South Central China 6 Yunnan 38,505 Southwest China 7 Guangdong 37,574 South Central China 8 Hebei 37,096 Northern China 9 Guangxi 34,658 South Central China 10 Jiangxi 31,240 Eastern China 11 Anhui 28,374 Eastern China 12 Jiangsu 26,809 Eastern China 13 Liaoning 24,958 Northeast China 14 Heilongjiang 19,644 Northeast China 15 Guizhou 18,699 Southwest China 16 Chongqing 17,582 Southwest China 17 Jilin 15,704 Northeast China 18 Fujian 14,213 Eastern China animals-13-00787-t002_Table 2 Table 2 Regional distribution of the survey sample. Region Province (Municipality Directly under the Central Government) Infected Areas Non-Infected Areas Sample Size Proportion Northern China Hebei Province Baoding (Anguo) Luannan County, Ningjin County 29 11.07% Tianjin Ninghe District / 33 12.60% Northeast China Jilin Province Tonghua (Meihekou, Liuhe) Changchun (Nong'an County, Dehui City) 35 13.36% Liaoning Jinzhou (Linghai) / 31 11.83% South Central China Hubei Province Huanggang (Tuanfeng) Huanggang (Xishui) 42 16.03% Eastern China Jiangsu Province Suqian (Muyang) Yancheng (Funing) 50 19.08% Southwest China Sichuan Chengdu (Jintang) Bazhong (Siyang) 42 16.03% Total 262 100% animals-13-00787-t003_Table 3 Table 3 Basic characteristics of the survey sample distribution. Variables Classification Sample Size Proportion (%) Variables Classification Sample Size Proportion (%) Gender Male 213 81.30 Number of years of education 0-5 years 13 4.96 Female 49 18.70 6-10 years 161 61.45 Age Under 35 years old 20 7.63 11-15 years 75 28.63 35-44 years old 49 18.70 16-20 years 13 4.96 45-54 years 128 48.85 Technical training Yes 208 79.39 55-64 years 58 22.14 No 54 20.61 Over 64 years old 7 2.67 Proportion of farming income Less than 25% 6 2.29 Years of farming 1-5 years 60 22.90 25%-49% 13 4.96 6-10 years 70 26.72 50%-74% 46 17.56 11-15 years 57 21.76 75% and above 197 75.19 16-20 years 46 17.56 Mode of farming Self-breeding and self-rearing 182 69.47 20 years or more 29 11.07 Level of operation scale Less than 100 heads 60 22.90 Commercial hog fattening 69 26.34 100-499 head 81 30.92 Piglet rearing 11 4.20 500-999 head 35 13.36 Joining an industrial organisation or not Yes 46 17.56 1000-4999 heads 76 29.01 Over 5000 heads 10 3.82 No 216 82.44 animals-13-00787-t004_Table 4 Table 4 Prevention and control of internal and external biosecurity in pig farms. Variables Classification Sample Size Proportion (%) External biosecurity Site selection Distance from other farms (0, 0.3) km 82 31.30 (0.3, 3) km 113 43.13 (3, 10) km 53 20.23 (10, 100) km 14 5.34 Distance from residential areas (0, 0.3) km 80 30.53 (0.3, 3) km 156 59.54 (3, 10) km 22 8.40 (10, 35) km 4 1.53 Distance from main traffic routes (0, 0.3) km 47 17.94 (0.3, 3) km 149 56.87 (3, 10) km 53 20.23 (10, 20) km 13 4.96 Existence of natural barriers around the farm (e.g., rivers, mountains, farmland) Yes 193 73.66 No 69 26.34 Biological control Whether birds can be effectively controlled Yes 157 59.92 No 105 40.08 Whether rodents can be effectively controlled Yes 176 66.17 No 86 33.83 Whether insects can be effectively controlled Yes 163 61.28 No 99 38.72 Personnel and vehicle management Can external personnel and vehicles freely enter the farm? Yes 23 8.65 No 239 91.35 Are there vehicles entering the decontamination site? Yes 219 82.33 No 43 17.67 Whether the farm has a dedicated means of transport Yes 201 76.72 No 61 23.28 Internal biosecurity Cleaning and disinfection Whether to disinfect the feed? Yes 207 77.82 No 55 22.18 Is there any decontamination operation in the workers' approach area? Yes 245 92.11 No 17 7.89 Feed and drinking water Whether feed uses kitchen waste (swill) Yes 7 2.63 No 255 97.37 Whether the water source has been tested Yes 123 46.95 No 139 53.05 Pig herd management "All-in, all-out" management of feeder hogs Yes 88 33.59 No 174 66.41 Are sows, feeder hogs, and hoglets managed separately on the farm? Yes 236 90.08 No 26 9.92 Infrastructure allocation Is there a transit site in the inner and outer sites? Yes 186 69.92 No 76 30.08 Separation of clean and dirty paths within the farm Yes 219 83.59 No 43 16.41 Immunisation records Built 230 87.79 Not built 32 12.21 animals-13-00787-t005_Table 5 Table 5 Variable definitions and descriptions. Variable Name Meaning of Variables Mean Standard Deviation Explained variable Whether the farm has immunisation records Yes = 1, No = 0 0.88 0.33 Whether the water source is tested Yes = 1, No = 0 0.47 0.50 Whether the farm has dedicated means of transport Yes = 1, No = 0 0.77 0.42 Explanatory variables Farmer characteristics Gender Male = 0, Female = 1 0.19 0.39 Years of education Actual number of years of education the farmer has received 9.63 3.08 Training in hog farming techniques Received = 1, not received = 0 0.79 0.41 Farming characteristics Years of farming Actual number of years the farmer has engaged in farming 12.31 7.43 Farming scale Less than 100 head = 1, 100-499 head = 2, 500-999 head = 3, 1000-4999 head = 4, more than 5000 head = 5 2.60 1.23 Degree of specialisation Proportion of farming income in total household income 84.26 21.82 Awareness of disease prevention Risk appetite High risk, high return = 1, medium risk, medium return = 2, low risk, low return = 3 2.42 0.72 Whether the farm is effective in blocking the introduction of African swine fever Yes = 1, No = 0 0.34 0.47 Whether the farm will report suspected outbreaks Yes = 1, No = 0 0.66 0.47 Whether the farm will report suspected outbreaks in neighbouring farms Yes = 1, No = 0 0.60 0.49 animals-13-00787-t006_Table 6 Table 6 Results of empirical analysis of factors influencing farmers' disease prevention and control behaviours. Explanatory Variables Immunisation Records Water Source Testing Whether the Farm Has Dedicated Means of Transport Coefficient Standard Deviation Coefficient Standard Deviation Coefficient Standard Deviation Gender -0.70 0.46 0.06 0.42 -0.76 * 0.40 Number of years of education 0.14 0.09 0.17 *** 0.06 0.09 0.06 Technical training 0.87 * 0.48 0.67 0.41 0.99 *** 0.39 Years of farming -0.04 0.03 -0.06 *** 0.02 -0.03 0.02 Farming scale 0.54 ** 0.23 0.41 *** 0.14 0.07 0.15 Degree of specialisation 0.02 ** 0.01 -0.003 0.01 0.02 ** 0.01 Risk appetite -0.81 * 0.43 -0.63 *** 0.23 -0.34 0.26 Whether the farm is effective in blocking the introduction of African swine fever -0.86 0.52 0.60* 0.34 -0.20 0.38 Whether the farm will report suspected outbreaks 0.91 * 0.54 -0.08 0.41 0.02 0.41 Whether the farm will report suspected outbreaks in neighbouring farms -0.19 0.54 1.36 *** 0.40 1.19 *** 0.41 Constants -0.19 1.83 -1.82 1.21 -1.09 1.30 Note: *, **, and *** denote significance levels of 10%, 5%, and 1%, respectively. 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PMC10000187

Urbanization negatively affects biodiversity worldwide. Consequently, alternative urban development styles are required for an eco-friendlier urbanization process. Thus, two development styles have been suggested: land-sharing (buildings mixed with dispersed green space) and land-sparing (buildings interspersed with large green patches). We assessed differences in species diversity and composition of bird assemblages between both development styles in two Argentinian cities: Santa Fe and Buenos Aires. We surveyed birds in land-sharing and land-sparing areas during the breeding and non-breeding seasons. As a control, we also surveyed birds in areas dominated by impervious surfaces. At a local scale, we also measured the environmental noise and pedestrian traffic. At a landscape scale, we measured the percent vegetation cover surrounding development styles and their distance to the main river. In Buenos Aires, species richness was higher in land-sparing than in land-sharing. However, the Shannon diversity and Simpson diversity were higher in land-sharing. In Santa Fe, both urban development styles supported similar species richness and diversity. Species composition varied between land-sharing and land-sparing in both cities during the breeding season. The pedestrian traffic was negatively associated with species diversity. Therefore, both development styles and strategies to reduce pedestrian traffic should be taken into account to enhance different components of species diversity and composition within the urban matrix. avian Latin America species composition taxonomic diversity urban planning Agencia Nacional de Promocion de la Investigacion, el Desarrollo Tecnologico y la InnovacionPICT 2018-03871 This research was funded by the Agencia Nacional de Promocion de la Investigacion, el Desarrollo Tecnologico y la Innovacion, PICT 2018-03871. pmc1. Introduction Urbanization is a complex socioeconomic process that has grown globally in recent decades . The world urban population increased by about 1.973 million inhabitants and the global urban surface area grew by about 80% between 1985 and 2015 . This accelerated urban development is leading to a strong transformation of the landscape and natural ecosystems . Numerous studies at a global scale have indicated that urbanization impacts biological communities causing changes in their ecological interactions, the behavior of individuals, and their biodiversity . Intensive urban development leads to the loss of species, mainly native species . Due to the negative impacts of urbanization on natural ecosystems, it is necessary to seek an alternative urban landscape planning that allows for biodiversity conservation . A multidisciplinary debate was recently raised between two models of urban development: land-sharing vs. land-sparing . The land-sharing model refers to the expansion of low-intensity urban areas that contain relatively small green spaces in a dispersed manner . In contrast, the land-sparing model promotes urban densification in some sectors to the extent that relatively large green spaces are established . Several studies have discussed the relevance of each model in different cities and taxonomic groups such as vegetation, beetles, mammals, and birds . Particularly for birds, the most current understanding of the response of bird assemblages to land-sparing and land-sharing development styles in urban environments is spatially biased towards a few cities or regions and with contrasting results. Additionally, urban landscapes are spatially and temporally heterogeneous, and consequently, the response of biological communities to land-sharing/land-sparing models may depend on factors such as the geographical, biological, and social context . The extent to which the patterns are generalizable remains unclear. Therefore, this highlights the importance of conducting studies in different cities to regionally differentiate the suitability of each urban development style for bird diversity . In urban areas, birds are one of the most frequently studied taxa, since they rapidly respond to anthropogenic changes, are easy to survey, and can function as surrogates of diversity for other taxa . Birds also provide ecosystem services such as pollination, seed dispersal, and pest control, and bird species richness may have a positive impact on human well-being . Although in recent years the study of urban birds has grown rapidly in highly biodiverse regions of Latin America such as Brazil, Argentina, and Mexico , gaps in the knowledge of the effect of urbanization on bird assemblages still remain . Additionally, as in many works from temperate zones (i.e., North America, Europe, and Australia) , studies from Latin America have reported a negative effect of urbanization and a positive influence of green space size on bird diversity . Since this region is one of the most diverse in avian species, but at the same time it is experiencing an unplanned lower-density growth in many cites , knowledge about the effect of urban development styles on urban bird assemblages is imperative as a base to guide future eco-friendlier urbanization. The effect of urbanization on bird diversity constitutes a process that varies according to different spatial scales . At the local scale, an increase in bird diversity is usually associated with greater plant diversity and stratification, the presence of natural or artificial waterbodies, and low levels of human disturbance such as pedestrian traffic and noise . At the landscape scale, greater species richness has been associated with greater proximity to waterbodies or green spaces in different cities . However, the relation between bird diversity and land-sharing/land-sparing models has not been analyzed considering the distance to other waterbodies or green areas. Moreover, the relationship between bird diversity and urbanization may change between seasons . In general, birds have a more restricted home range during the breeding season since they are associated to a nesting territory, whereas during the non-breeding season, birds have a more flexible home range because they are mainly influenced by the abundance and distribution of food . Therefore, the relationship between bird diversity and land-sharing/land-sparing models needs to be analyzed in different seasons . In this study, our aims were (1) to compare bird communities in land-sharing and land-sparing landscapes; (2) to analyze the role of human disturbance represented by pedestrian traffic and level of noise on bird communities; (3) to analyze the role of the amount of green cover and the distance to the main watercourses of urban landscapes on bird communities; and (4) to compare the relation between bird communities and different landscapes during breeding and non-breeding seasons. We expected higher bird diversity in land-sharing during the breeding season due to the presence and higher abundance of species that can nest on manmade structures and use the surrounding natural resources. In addition, we expected higher bird diversity in landscapes surrounded by more green cover and next to watercourses. Finally, we expected differences in bird diversity and species composition related to environmental variables only during the breeding season. 2. Materials and Methods 2.1. Study Area and Preliminary Classification of Urban Areas We assessed the taxonomic diversity and composition of bird assemblages in two cities from central Argentina, Buenos Aires (34deg35'59'' S 58deg22'55'' W, 3,075,646 inhabitants, 25 masl) and Santa Fe (31deg38'00'' S 60deg42'00'' W, 401,544 inhabitants, 25 masl) . Buenos Aires is located in an ecotone between the Pampean and the Paranaense phytogeographic regions . The Pampean region was originally dominated by grasslands, whereas the Paranaense region is composed of deltaic forests. Santa Fe is located in the ecotone between the wooded Espinal and the Paranaense phytogeographic regions. However, the surroundings of both cities are heavily impacted by human activities, being dominated by crops and exotic tree plantations. In each city, we selected 200 m x 300 m sampling units depending on the availability of landscapes with land-sharing or land-sparing (14 in Santa Fe city and 22 in Buenos Aires city) . Half of them presented a land-sharing urban development and the other half, a land-sparing urban development . To recommend a land-sharing or land-sparing urban development style for future urban planning, we need to provide evidence that at least one of them supports a different bird assemblage from highly urbanized areas. Thus, we considered 200 m x 300 m rectangles where pavement and buildings predominated (>50% impervious cover) and hereafter referred to them as "control" (7 in Santa Fe city and 11 in Buenos Aires city). Sampling units were separated by a minimum distance of 200 m to secure data independence. Sampling units were initially assigned to either one or another urban development style by visual inspection of satellite images available on Google Earth. Land-sharing units consisted of fragmented green areas interspersed by buildings, while land-sparing units corresponded with the majority (>50%) of their green surfaces aggregated into a single patch . All green spaces presented a certain level of management or human intervention, such as lawn mowing, irrigation, or pruning. Due to a positive relationship between the size of green areas and animal biodiversity (including birds) that has been extensively reported in urban landscapes , every land-sharing square in a given city was paired with another land-sparing square of the same city holding a similar overall green area. This procedure allowed us to test for the effect of urban landscape organization, avoiding bias associated with the size of green areas. 2.2. Classification of Urban Areas In order to confirm the initial assignment of each sampling unit to one of three urban development styles, we followed Ibanez-Alamo et al. . Using the satellite images from Google Earth and ImageJ package , we divided each 200 m x 300 m rectangle into 96 cells (25 m x 25 m) and estimated the percentage of vegetated and non-vegetated surface for each cell . Then, we used this information to calculate the following variables for each rectangle: (Single_patch), percentage of high vegetation cover cells (those with more than 50% green area) in a single patch (contiguous cells); (N_patches), number of green patches (a green patch was defined as having at least one high vegetation cover cell); (Per_built_cells), percentage of built cells of all vegetated cells; (Per_veg_cells) percentage of fully vegetated cells of all vegetated cells; (N_veg_cells), number of cells with vegetated surfaces; and (Per_veg_cover), percentage of vegetation cover in the sampling unit. Variables Single_patch and N_patches provide information on the land-sharing/sparing urban development style at the 200 m x 300 m square level with high values of variable Single_patch associated with land-sparing urban areas (i.e., vegetation in a single patch), while high values of variable N_patches are associated with land-sharing urban areas (i.e., vegetation distributed into many patches). Variables Per_built_cells and Per_veg_cells provide information on the within-cell land-sharing or land-sparing urban development, respectively, and variables N_veg_cells and Per_veg_cover estimate the overall amount of vegetation in the square . Since the width of greenways is often positively associated to the flow of individuals of many urban bird species , we used the Rook contiguity criteria to determine contiguous cells. With the previous six variables, we ran a principal component analysis (PCA) using the FactoMineR package version 2.7 from R . The first two PCA axes retained more than 80% of data variation for both cities . For both cities, the first axis contrasted control, land-sparing, and land-sharing rectangles based on variables: Single_patch, Per_veg_cover, Per_built_cells, and Per_veg_cells. The second axes contrasted land-sharing from land-sparing and control rectangles based on N_patches and N_veg_cells . Thus, we confirmed the suitability of our initial classification . 2.3. Bird Survey Data on bird species were collected using fixed-radius point counts carried out during the 2020 non-breeding season (May-August) and breeding season (October-December). Standardized point count surveys have been recommended to provide data resulting in indices of abundance that are comparable across years, habitats, and studies . In urban environments, point counts are as effective as other widely used techniques in determining patterns of relative abundance . Two professional ornithologists from Santa Fe and Buenos Aires with more than 10 years of bird-survey experience in their respective regions carried out all the surveys in each city. Within each 200 m x 300 m rectangle, 2 point counts were settled with a minimum of 100 m distance between them and from the border of the rectangle to avoid counting the same individual twice . At each point count, we recorded all birds seen and heard for 5 min within a 30 m radius. We considered all individuals perching, nesting, or feeding within the point counts for further analyses. Point counts were carried out during the morning (up to 4 h after local sunrise), only in working days to avoid excessive variation in the circulation of vehicles and people and with similar weather conditions (without rain and heavy winds). To capture potential temporal changes in bird assemblages within season, we carried out three surveys separated by a month in each season. 2.4. Environmental Variables Environmental variables were established at two different scales. At a landscape scale, we analyzed the distance to the nearest river and the vegetation coverage surrounding the sampling units. Landscape variables were estimated using a global land cover map with 10 m pixel resolution . In order to measure the distance to the nearest river, we calculated the Euclidean distance between the centroid of each sampling unit and the nearest river using the raster package version 3.6-11 (Hijmans, 2022). In order to calculate the vegetation coverage, we performed the following steps. First, the original 23 land use types were reclassified into 2 broad categories: vegetated and non-vegetated areas. Second, we made buffers of 500 m width from sampling units using the rgeos package version 0.6-1 . Finally, we calculated the vegetation coverage using the landscapemetrics package version 0 . At the sampling unit scale, we measured the pedestrian traffic and the environmental noise. To measure the pedestrian traffic, we recorded the number of people passing through the point count surface during bird surveys. Environmental noise was measured using the cellphone application "Sound Meter" as per de Camargo Barbosa et al. . The mean decibels per 30 s immediately before and after bird counts were estimated. Due to the fact that the sound meter was not calibrated, decibels measures should be taken only for relative comparisons between urban development styles. 2.5. Data Analysis 2.5.1. Taxonomic Diversity per Urban Development Style For each season, we measured taxonomic bird diversity using Hill numbers, which are the effective numbers of equally abundant species . Hill numbers differ by a parameter q that reflects their respective sensitivity to the relative frequency of a species. We used the hillR package version 0.5.1 to calculate Hill numbers with q = 0, q = 1, and q = 2, which can be interpreted as bird species richness (BSR), Shannon-Wiener (H), and Simpson's (S) index of diversity, respectively . Bird species richness was the total number of species, whereas Shannon-Wiener and Simpson diversities reflected the number of common and dominant species, respectively . To ensure our survey effort was comparable between urban development styles, we calculated rarefaction curves for each urban development style and city in relation to sample completeness using 999 bootstraps with the iNEXT package version 3.0.0 in R . Sample completeness is the proportion of the total individuals that belong to the species detected in the sampling unit . Significant differences between curves were established when 95% confidence intervals did not overlap . 2.5.2. Taxonomic Diversity per Sampling Unit BSR was calculated as the maximum number of recorded bird species at each sampling unit considering the three surveys within each season, whereas Hill numbers for Shannon-Wiener and Simpson diversities were calculated with the maximum individuals for each species recorded during the three visits. Environmental variables included urban development styles, cities, pedestrian traffic, noise, and landscape variables. The interactions between urban development styles and city and between urban development styles and landscape vegetation cover also were explored. Generalized linear models (GLMs) were performed to analyze association patterns between bird diversity (Hill numbers) and environmental variables. Models were obtained by backward elimination of non-significant variables (p > 0.05) from the full model using the anova function. Final models were compared with null models using a likelihood ratio test (LRT test) (p < 0.05). Differences of means between types of urban development styles were explored with Tukey tests using the function glht of the multcomp package version 1.4.20 . Multicollinearity among predictor variables was explored using the vif function of the car package version 3.1.1 . As gvif values were lower than 5, all variables were retained for further analyses. The pseudo-rsquare of final models were obtained using piecewiseSEM package version 2.1.2 . The final models were plotted with the visreg package version 2.7.0 . For species richness (q = 0) (count data), we assumed a Poisson distribution of errors and we checked for sub-dispersion. For the Shannon-Wiener and Simpson index (q = 1 and q = 2, respectively) (continuous data), we assumed a Gaussian distribution of errors, and homoscedasticity and normality were checked. All diagnostic analyses were carried out with the DHARMa package version 0.4.6 . 2.5.3. Taxonomic Composition To estimate the variation in species composition explained by environmental variables in each season, we used a distance-based redundancy analysis (dbRDA) using the vegan package version 2.6.2 . db-RDA is an ordination method which arranges data objects in a space defined by the linear combinations of explanatory (environmental) variables and, at the same time, quantifies the variation in species composition explained by the environmental variables . dbRDA is a reliable test for analyzing species-environment relations, especially with linear environmental gradients . The variation of species composition in db-RDA has to be expressed on the basis of a non-Euclidean distance response matrix. We examined a Bray-Curtis dissimilarity matrix along with urban development styles and environmental variables. Environmental variables included urban development style, cities, pedestrian traffic, noise, and landscape variables. Models were obtained by backward variable selection and comparisons with null models using a likelihood ratio test (LRT test) (p < 0.05). A db-RDA with the variable "Urban development style" as significant makes it possible to determine that species composition is different in at least one of the urban styles. However, our main objective was to determine the variation in bird species composition between land-sharing and land-sparing. Thus, if db-RDA showed significant differences between development styles, we would perform a new db-RDA considering only land-sharing and land-sparing (hereafter, "db-RDA2"). 3. Results In Buenos Aires city, we recorded a total of 6001 individuals of 48 species (Table 1). The most abundant species were Columba livia and Zenaida auriculata. In Santa Fe city, we recorded a total of 4391 individuals of 63 species (Table 1). The most abundant species were Zenaida auriculata and Passer domesticus. 3.1. Taxonomic Diversity per Urban Development Style During both seasons, species diversity in control was lower than in land-sharing and land-sparing for all Hill numbers . In Santa Fe city, species diversity did not differ between land-sparing and land-sharing during both seasons . Although we observed a higher species richness and Shannon diversity in land-sparing than in land-sharing during the breeding season, we did not find significant differences . In Buenos Aires city, species diversity did not differ between land-sparing and land-sharing during the non-breeding season . During the breeding season, species richness was higher in land-sparing than in land-sharing, whereas Simpson diversity was higher in land-sharing than in land-sparing . Shannon diversity did not differ between these two landscapes during both seasons . 3.2. Taxonomic Diversity per Sampling Unit Taxonomic diversity responded differently to environmental predictors at both landscape and local scales between seasons. During the non-breeding season, species richness was related to urban development styles, pedestrian rate, and the percentage of surrounding vegetation coverage (LRT = 79.69, df = 6, p < 0.001, pseudo-R2 = 0.77). Species richness was lower in control than in land-sparing and land-sharing, whereas we did not find significant differences between land-sparing and land-sharing (Tukey test, p > 0.05) . Pedestrian traffic was negatively associated with species richness . The relationships between species richness and surrounding vegetation coverage varied between cities . In Buenos Aires, species richness related negatively to vegetation coverage, whereas in Santa Fe, there was no clear relationship between variables . During the breeding season, we also found lower species richness in control landscapes and negative relationships with pedestrian rate . During the non-breeding season, Shannon diversity was related to urban development styles, pedestrian rate, and the percentage of surrounding vegetation coverage . Shannon diversity was lower in control than in land-sparing and land-sharing . We did not find significant differences between land-sparing and land-sharing (Tukey test, p > 0.05). Pedestrian traffic was negatively associated with Shannon diversity . The association between the surrounding vegetation coverage and Shannon diversity varied between cities, being negative in Buenos Aires and positive in Santa Fe . During the breeding season, Shannon diversity was related to urban development styles and the percentage of vegetation coverage . Shannon diversity was higher in land-sharing than in land-sparing and control . The association between Shannon diversity and surrounding vegetation coverage varied between urban development styles and cities . Control and land-sharing had a negative relationship, whereas land-sparing had a positive relationship . On the other hand, the relationship between Shannon diversity and vegetation cover was negative in Buenos Aires and positive in Santa Fe . During the non-breeding season, Simpson diversity was related to urban development styles and pedestrian rate . Simpson diversity was lower in control than in land-sparing and land-sharing . We did not find significant differences between land-sparing and land-sharing (Tukey test, p > 0.05). Pedestrian traffic was negatively associated to Simpson diversity . During the breeding season, Simpson diversity was related to urban development styles and the percentage of surrounding vegetation coverage . Simpson diversity was higher in land-sharing than in land-sparing and control . The association between Simpson diversity and surrounding vegetation coverage was negative in Buenos Aires city and positive in Santa Fe city . 3.3. Taxonomic Composition The results of the db-RDA showed that over 40% of the variation in species composition was associated to urban development style, city, and surrounding vegetation coverage during the non-breeding and breeding seasons . In ordinations for both the non-breeding and breeding seasons, the first axis showed differences in species composition between cities. The second axis was correlated with surrounding coverage of vegetation and urban development styles. The abundance of Columba livia and Passer domesticus tended to be higher in control from Buenos Aires and Santa Fe, respectively, and areas with the lowest landscape vegetation coverage . By contrast, the abundance of Pitangus sulphuratus, Furnarius rufus, Myiopsitta monachus, Turdus rufiventris, and Patagioenas picazuro tended to be higher in land-sharing and land-sparing areas and in areas with the highest surrounding vegetation coverage . However, species composition changed between land-sparing and land-sharing landscapes during the breeding season. During the non-breeding season, db-RDA2 only showed significant associations between bird composition and surrounding vegetation coverage and cities , but no bird composition differences between land-sparing and land-sharing development styles. During the breeding season, species composition was related to urban development style, cities, and surrounding vegetation coverage . The abundance of Zenaida auriculata, Myiopsitta monachus, Columba livia, Passer domesticus, and Turdus rufiventris was higher in the land-sparing landscape, whereas Patagioenas picazuro, Progne chalybea, Furnarius rufus, Molothrus bonariensis, Pitangus sulphuratus, Troglodytes aedon, Zonotrichia capensis, and Agelaioides badius were more abundant in the land-sharing landscape . The abundance of Zenaida auriculata and Passer domesticus was higher in Santa Fe city, whereas the abundance of Columba livia, Turdus rufiventris, and Patagioenas picazuro was higher in Buenos Aires city . Finally, Columba livia and Zenaida auriculata dominated sites with the lowest surrounding vegetation coverage . 4. Discussion Spatial configuration of green cover can affect bird assemblages. Land-sparing and land-sharing development styles supported bird assemblages with different species diversity and composition during the breeding season in the cities of Santa Fe and Buenos Aires. In Buenos Aires, land-sparing favored species richness while land-sharing enhanced the Shannon diversity and the Simpson diversity during the breeding season. In Santa Fe, both urban development styles supported similar species richness and diversity. Thus, our results support that both urban development styles can influence the diversity and composition of birds in Argentinian urban environments. On the other hand, differences in the response of bird assemblages between cities suggest that local knowledge about the effect of urbanization on bird assemblages is required for planning conservation strategies. In addition, pedestrian traffic and the amount of green cover surrounding sites affected bird communities. Our study showed that the relationship between urban development style and species richness varied according to cities and seasons. In Buenos Aires, land-sparing had higher species richness than land-sharing during the breeding season. Land-sparing may favor the presence of specialist bird species that require contiguous extensions of green cover . Simpson diversity, which represents the number of dominant species, was higher in land-sharing. Land-sharing has more edge habitats due to the impervious surface interspersed with a wide range of small public and private urban green spaces such as small parks, gardens, and wooded streets. This habitat structure may favor a greater number of dominant species than in land-sparing. In contrast, in Santa Fe city, species diversity had similar values between urban development styles. The lack of association between bird assemblages and the spatial configuration of vegetation was also reported in a previous work performed in another Latin-American city . Our results suggest that the underlying ecological processes that shape bird assemblages in both cities may be different. Further studies are required to provide more conclusive insights about the ecological processes that take place in both cities. Additionally, comparisons with previous studies are difficult because of the differences of methodological approaches, the scale of the study, and the attribute of the bird assemblage analyzed . In this sense, we followed the methodological approach applied in Ibanez-Alamo et al. and the response of bird assemblages in European cities is different from that of Argentinian cities. Therefore, it is recommended to extend the same methodological approach to different cities to analyze which patterns can be generalizable. Distinct urban development styles supported bird assemblages with different species composition. In land-sparing, there was a greater abundance of ground feeder species with a high propensity to form flocks for feeding and breeding such as Zenaida auriculata and Columba livia . These species may require contiguous extensions of lawn for feeding. Turdus rufiventris, which feeds on worms and insects on the ground, also was more abundant in land-sparing. In Argentinian cities, high abundances of these species were often reported in large urban parks, which resemble land-sparing development style . On the other hand, land-sharing can provide a mixture of artificial and natural resources. The highest abundances of species such as Furnarius rufus, Troglodytes aedon, and Progne chalybea may be associated with their ability to nest on artificial structures and feed on the surrounding vegetation or air . Other species more common in land-sharing, such as Pitangus sulphuratus, Molothrus bonariensis, Agelaioides badius, and Zonotrichia capensis, were often reported in residential areas along urbanization gradients and can use artificial structures for nesting (L. M. Leveau pers. obs.). Moreover, additional mechanisms, such as inter-specific interactions, have been associated with changes in species occurrences and abundances between land-sparing and land-sharing development styles in European cities . Studies aiming to analyze the ecological processes that shape species composition in urban environments are strongly encouraged due to gaps of knowledge in urban areas of Latin America . The relationship between bird communities and urban development styles varied according to seasons. We found differences in species diversity and composition between land-sparing and land-sharing development styles only during the breeding season. Most of the species recorded in both cities can exploit resources present in urban environments . As the home range of most of the bird species tends to be larger during the non-breeding season, the availability of resources and not their spatial arrangement in urban environments may influence the presence and abundance of species. However, the spatial arrangement of resources in urban environments may become relevant when species reduce their home range during the breeding season . For instance, birds may prefer to nest on sites where they can find resources without getting far from their offspring . However, in European cities, differences in the bird assemblage supported by land-sparing and land-sharing development styles were found during the non-breeding season . These patterns highlight the importance of conducting studies during the breeding and non-breeding seasons since the response of bird assemblages to urban development styles may be heterogeneous not only spatially, i.e., between urban development styles, but also temporally . Human disturbance is another factor that can influence bird diversity in urban development styles. It can be described in terms of pedestrian traffic, human density, and speed of approaching humans to birds . We found a negative relationship between pedestrian traffic and bird diversity . Pedestrian rate can reduce species occupation and persistence and affect the feeding activity of birds . According to our results, urban planners should take into account strategies for pedestrian traffic calming to enhance species diversity and composition of bird assemblages in any urban development style . However, additional mechanisms should be taken into account in further studies. During the survey, we recorded people that occurred within the point count, but we did not record their activity. It has been shown that patterns of human activity, such as bird feeding, can shape the presence and abundance of birds in urban green spaces . Therefore, future studies should shed light on this topic that is still poorly understood. On the other hand, no association pattern was found between species diversity and environmental noise. This result was unexpected since it has been shown that vehicle traffic, which is one of the most frequently cited sources of environmental noise, affected attributes of bird assemblages in both cities . However, environmental noise is often associated to changes in urban land use, i.e., urban cores (administrative and commercial areas) usually present higher levels of noise than suburban and peri-urban areas . In our study, changes in bird diversity associated to changes in environmental noise may not be significant since paired samples of urban development styles were located across the urban matrix. In this sense, bird diversity in control was always lower than land-sparing and land-sharing development styles, even if rectangles were located in the urban core or in suburban/peri-urban areas. It is often reported that bird diversity is negatively associated to distance to the surrounding natural habitats . Natural habitats can act as source habitats, and consequently, the flow of individuals from different species towards the urban landscape can take place. Riparian corridors of the La Plata basin represent highly dynamic and heterogeneous habitats with high levels of biodiversity as a result of an ecotone of species assemblages from tropical and temperate regions . In this sense, Santa Fe and Buenos Aires city are surrounded by these riparian environments, and consequently, we hypothesized a negative distance effect. The lack of association between the distance to the main watercourses and bird diversity may be related to the following ecological processes. First, bird assemblages within the urban matrix of both cities present a subset of native species than in surrounding riparian corridors . It has been shown that urban systems can filter bird species according to their ecological traits . In both cities, bird assemblages were composed of species that were often considered as urban exploiters and urban adapters, i.e., species that are able to inhabit and exploit urban habitats . In addition, the success of colonizing urban systems may also be associated with the abundance of species in surrounding non-urban areas . Since knowledge about ecological processes that shape bird assemblages can help to define conservation strategies within cities (e.g., Leveau ), future studies aiming to understand the effect of surrounding non-urban habitats on urban bird assemblages are required. Bird diversity was influenced by the coverage of vegetation at both the local and landscape level. It has been widely documented that the coverage of vegetation is positively associated to bird diversity since it implies more resources to species . Our results agree with these previous findings. However, in Buenos Aires, we found a negative association between the coverage of vegetation and bird diversity at the landscape level. Although these results were unexpected, this negative association may be caused by the low variability in the coverage of vegetation at the landscape scale between sample sites. Most of the landscapes surrounding our sites (>90%) presented a vegetation coverage lower than 40%. Buenos Aires is a compact city where impervious surfaces predominate over vegetation across the urban matrix . In this context, urban green spaces with the highest vegetation coverage within the city may support the highest levels of bird diversity because of a greater availability of resources than surrounding areas . On the other hand, not only the coverage of vegetation is a determinant of resources for birds in urban environments, but also the vegetation diversity and composition , which were not assessed in our study. The abundance of Zenaida auriculata, Furnarius rufus, Myiopsitta monachus, Turdus rufiventris, and Patagioenas picazuro was higher in land-sparing/land-sharing urban development styles than in controls and in areas with higher percentage of vegetation coverage at the landscape scale. These species can inhabit cities such as Buenos Aires and Santa Fe, where urban green spaces are characterized by a mixture of non-vegetated infrastructure and vegetation mainly composed of non-native and ornamental species . By contrast, the abundance of Columba livia and Passer domesticus increased in control sites with low vegetation coverage at the landscape scale. These species were often considered as urban exploiters in cities worldwide because of generalist habits which allow them to feed on food discarded by humans and nest in buildings . Land-sparing and land-sharing strategies should be taken into account in future urban planning to enhance biodiversity within the urban matrix in both cities. However, we emphasize three important potential extensions of our work. Firstly, we have considered taxonomic diversity and composition of bird assemblages. Follow-up work needs to examine other attributes of bird assemblages such as functional and phylogenetic diversity and abundance of native and exotic species to reduce gaps of information in the association between urban development style and bird assemblages . Secondly, it is necessary to analyze the potential synergistic and interactive role that the two urban development styles can play. For example, the inclusion of two urban development styles may have positive ecological impacts by increasing connectivity within the urban matrix. Habitats more typical of a land-sharing development style such as small urban parks and wooded streets were often reported as urban corridors, favoring connectivity between large urban parks and increasing bird diversity . Thirdly, enhancement of biodiversity in urban ecosystems can be quite important as some evidence suggests that personal exposure to natural features in everyday life is a major determinant of sensitivity to environmental issues . Consequently, the third worthwhile extension would examine how the diversity and composition of bird assemblages in each urban development style influence human well-being . Our conceptualization and spatial representation of land-sparing and land-sharing development styles corresponded to urban habitats with structural differences, i.e., differences in spatial configuration of vegetation cover. However, the association pattern between the urban development style and bird assemblages may vary according to land use. For example, the configuration of vegetation cover can be analyzed in different land uses, such as commercial and industrial areas. Previous works have shown differences in the diversity and composition of bird assemblages in urban areas related to differences in land use and/or land cover . This is an important extension of research since planning strategies generally reflect decisions based on both land use and land cover. 5. Conclusions We found that the response of bird assemblages to urban development styles varied according to the season and the city. Differences in species diversity between land-sparing and land-sharing were restricted to Buenos Aires city during the breeding season. In contrast, differences in species composition were found in both cities during the breeding season. Due to the fact that both development styles benefit different species, the design of equal proportions of land-sharing and land-sparing landscapes will enhance the total bird diversity in the cities of Santa Fe and Buenos Aires. However, differences in these association patterns with other studies question the extent to which these patterns can be generalizable, and consequently, further studies are required to shed light on the local ecological processes that shape bird assemblages in cities. Additionally, strategies for calming pedestrian traffic and increasing vegetation coverage should be taken into account in future urban planning in order to enhance bird diversity within the urban matrix. Acknowledgments The comments made by three anonymous reviewers greatly improved a first draft of the manuscript. The English writing was revised by Paloma Garcia Orza. Supplementary Materials The following supporting information can be downloaded at: Figure S1: Image processing for the classification of sampling units as an urban development style; Figure S2: Ordination of sampling units from the city of Santa Fe and Buenos Aires obtained from a Principal Component Analysis; Figure S3: Sample completeness curves for each urban development style in the city of Santa Fe and Buenos Aires; Figure S4: Diagnostics of fitted generalized linear models for all response variables. Click here for additional data file. Author Contributions Conceptualization, L.M.L. and M.A.C.; methodology, L.M.L. and M.A.C.; software, M.A.C.; validation, M.A.C.; formal analysis, M.A.C.; investigation, M.A.C., L.M.L. and I.N.G.; resources, M.A.C., L.M.L. and I.N.G.; data curation, M.A.C.; writing--original draft preparation, M.A.C.; writing--review and editing, M.A.C., L.M.L. and I.N.G.; visualization, L.M.L. and M.A.C.; supervision, L.M.L.; project administration, L.M.L.; funding acquisition, L.M.L. All authors have read and agreed to the published version of the manuscript. Institutional Review Board Statement Not applicable. Informed Consent Statement Not applicable. Data Availability Statement The datasets generated and/or analyzed during the current study are available upon request to the corresponding author. Conflicts of Interest The authors have no relevant financial or non-financial interests to disclose. Figure 1 Location of control (red circles), land-sharing (green), and land-sparing (blue) landscapes in Santa Fe (A) and Buenos Aires (B) city. Examples of sample units used in this study: (C(a)) land-sharing and (C(b)) land-sparing landscapes. The yellow line indicates the limits of the sample unit (300 x 200 m), whereas red points indicate the location of point counts. Figure 2 Rarefaction curves of Hill numbers (species richness, q = 0; Shannon diversity, q = 1; and Simpson diversity, q = 2) in relation to sample coverage for control, land-sharing (lsh), and land-sparing (lsp) landscapes during the non-breeding (above) and breeding seasons (below) in Santa Fe and Buenos Aires city (A-D, respectively), Argentina. Shaded bands indicate 95% confidence intervals. Figure 3 Representation of the best generalized linear model for species richness (q = 0) in Buenos Aires and Santa Fe (Argentina). Species richness in relation to urban development style (A), pedestrian rate (B), and the interaction between the percentage of surrounding vegetation coverage and city (C) during the non-breeding season. Species richness in relation to urban development style (D) and pedestrian rate (E) during the breeding season. Abbreviations: lsh--land-sharing; lsp--land-sparing. Grey dots refer to sampling units (200 m x 300 m rectangles). Blue lines indicate the parameter estimates with 95% confidence intervals represented by the grey area. Figure 4 Representation of the best generalized linear model for Shannon diversity (q = 1) in Buenos Aires and Santa Fe (Argentina). Shannon diversity in relation to urban development style (A), pedestrian rate (B), and the interaction between the percentage of surrounding vegetation coverage and city (C) during the non-breeding season. Shannon diversity in relation to urban development style (D), the interaction between the urban development style and the percentage of surrounding vegetation coverage (E), and the interaction between the surrounding vegetation coverage and city (F) during the breeding season. Abbreviations: lsh--land-sharing; lsp--land-sparing. Grey dots refer to sampling units (200 m x 300 m rectangles). Grey dots refer to sampling units (200 m x 300 m rectangles). Blue lines indicate the parameter estimates with 95% confidence intervals represented by the grey area. Figure 5 Representation of the best generalized linear model for Simpson diversity (q = 2) in Buenos Aires and Santa Fe (Argentina). Simpson diversity in relation to urban development style (A) and pedestrian rate (B) during the non-breeding season. Simpson diversity in relation to urban development style (C) and the interaction between the percentage of surrounding vegetation coverage and city (D). Abbreviations: lsh--land-sharing; lsp--land-sparing. Blue lines indicate the parameter estimates with 95% confidence intervals represented by the grey area. Figure 6 Distance-based redundancy analysis triplots showing the relationship between urban development styles (UDS) (points), species (code), and selected environmental variables (blue arrows) in Santa Fe and Buenos Aires city during the non-breeding (A,B) and breeding seasons (C,D). The right column shows analysis without control sites (B,D). The lines represent the direction (orientation with respect to the axis) and strength (length of the line) of the correlations between environmental variables and variation in species composition. Abbreviations: CITYstafe--Santa Fe city; CITYbsas--Buenos Aires city; VEGCOV--percentage vegetation cover surrounding sites; UDSc--control sites; UDSlsh--land-sharing sites; UDSlsp--land-sparing sites; coli--Columba livia; pado--Passer domesticus; zeau--Zenaida auriculata; mobo--Molothrus bonariensis; pisu--Pitangus sulphuratus; furu--Furnarius rufus; papi--Patagioenas picazuro; pama--Patagioenas maculosa; mymo--Myiopsitta monachus; brch--Brotogeris chiriri; psle--Psittacara leucophthalmus. animals-13-00894-t001_Table 1 Table 1 List of species observed in control, land-sharing, and land-sparing landscapes during breeding (BS) and non-breeding (NBS) seasons in Santa Fe and Buenos Aires, Argentina. The sum of the highest number of individuals recorded during three visits in each urban development style and season is shown for each species. Nomenclature follows the South American Classification Committee of the American Ornithologists' Union (Remsen et al., 2022). Scientific Name Santa Fe City Buenos Aires City Control Land-Sharing Land-Sparing Control Land-Sharing Land-Sparing NBS BS NBS BS NBS BS NBS BS NBS BS NBS BS Columba livia 12 5 27 32 4 8 50 92 7 12 31 32 Patagioenas maculosa 2 0 3 4 10 5 0 0 0 0 2 0 Patagioenas picazuro 0 0 3 2 3 2 3 5 12 10 32 12 Leptotila verreauxi 0 0 0 0 0 1 0 0 0 0 0 0 Zenaida auriculata 17 24 20 26 26 46 7 12 11 25 17 49 Columbina picui 0 1 1 1 16 2 3 2 0 0 0 1 Guira guira 8 0 3 8 5 8 0 0 0 0 0 0 Tapera naevia 0 0 0 0 0 1 0 0 0 0 0 0 Chlorostilbon lucidus 0 3 0 2 0 1 0 1 0 3 0 1 Hylocharis chrysura 0 0 1 0 1 1 1 1 2 2 1 1 Vanellus chilensis 0 0 2 3 3 2 0 0 0 0 0 0 Cathartes aura 0 0 0 0 0 1 0 0 0 0 0 0 Rostrhamus sociabilis 0 0 0 0 1 0 0 0 0 0 0 0 Rupornis magnirostris 0 0 1 1 1 0 0 0 0 0 0 0 Parabuteo unicinctus 0 0 0 0 0 0 0 0 2 3 0 1 Athene cunicularia 0 0 2 1 0 0 0 0 0 0 0 0 Picumnus cirratus 0 0 0 1 0 0 0 0 0 0 0 0 Melanerpes cactorum 0 0 2 1 2 0 0 0 0 0 0 0 Dryobates mixtus 0 0 0 1 0 1 0 0 1 0 0 0 Colaptes melanochloros 0 0 0 2 3 1 0 0 1 2 2 1 Colaptes campestris 0 0 1 1 0 1 0 0 0 0 0 0 Caracara plancus 2 0 1 0 2 0 0 0 0 2 2 2 Falco sparverius 0 0 1 2 0 1 0 0 0 0 0 0 Myiopsitta monachus 4 2 15 7 11 17 2 0 24 7 22 12 Brotogeris chiriri 0 0 0 0 0 0 0 0 2 4 21 6 Amazona aestiva 0 0 0 0 0 0 0 0 0 2 3 2 Pyrrhura frontalis 0 0 0 0 0 0 6 1 4 14 12 5 Pyrrhura molinae 0 0 0 0 0 0 0 0 0 0 0 2 Aratinga nenday 0 0 0 0 0 0 0 0 0 5 5 4 Psittacara leucophthalmus 0 0 0 0 0 0 0 2 0 11 0 8 Taraba major 0 0 0 0 1 0 0 0 0 0 0 0 Lepidocolaptes angustirostris 0 0 2 2 1 1 0 0 2 1 1 1 Furnarius rufus 5 5 6 5 12 8 2 2 5 9 5 4 Phacellodomus ruber 0 0 0 0 2 2 0 0 0 0 0 0 Pseudoseisura lophotes 0 0 3 1 3 2 0 0 0 0 0 0 Schoeniophylax phryganophilus 0 0 2 0 2 1 0 0 0 0 0 0 Camptostoma obsoletum 0 0 1 1 1 1 0 0 0 0 0 0 Serpophaga subcristata 0 0 0 0 0 0 0 0 1 2 1 1 Serpophaga griseicapilla 0 0 1 0 0 0 0 0 0 0 0 0 Pitangus sulphuratus 3 5 4 6 4 9 3 3 4 5 3 3 Machetornis rixosa 1 1 2 2 1 3 0 0 1 3 1 4 Myiodynastes maculatus 0 0 0 0 0 0 0 0 0 0 0 1 Tyrannus melancholicus 0 0 0 1 0 2 0 0 0 1 0 0 Tyrannus savana 0 0 0 2 0 3 0 0 0 1 0 1 Sublegatus modestus 0 0 0 1 0 0 0 0 0 0 0 0 Cyclarhis gujanensis 0 1 1 1 1 1 0 0 0 0 0 0 Progne tapera 0 0 0 5 0 4 0 0 0 1 0 1 Progne chalybea 0 4 0 6 0 7 0 5 0 6 0 4 Tachycineta leucorrhoa 0 1 3 2 0 4 0 1 5 5 0 3 Troglodytes aedon 1 3 3 3 2 2 2 2 3 4 2 4 Polioptila dumicola 0 0 3 2 2 2 0 0 0 0 0 1 Turdus rufiventris 1 0 2 2 2 2 3 4 11 7 12 8 Turdus amaurochalinus 0 0 5 1 2 1 0 0 0 1 0 1 Mimus saturninus 4 2 5 2 3 0 0 1 4 2 2 3 Mimus triurus 0 0 1 0 0 0 0 0 0 0 0 1 Sturnus vulgaris 0 0 4 2 0 2 0 1 0 3 7 16 Passer domesticus 13 18 15 9 11 12 6 5 2 5 12 13 Spinus magellanicus 0 1 0 0 2 2 0 0 0 3 2 1 Zonotrichia capensis 2 2 3 2 2 3 1 1 1 2 1 1 Icterus pyrrhopterus 0 0 2 0 0 0 0 0 0 1 0 1 Molothrus rufoaxillaris 0 0 0 0 1 1 0 0 1 2 0 3 Molothrus bonariensis 5 7 5 5 21 15 1 0 1 4 12 1 Agelaioides badius 0 0 4 4 4 0 0 0 7 3 7 4 Geothlypis aequinoctialis 0 0 0 0 0 1 0 0 0 0 0 0 Setophaga pitiayumi 0 0 0 1 0 1 0 0 1 1 1 1 Piranga flava 0 0 0 0 0 1 0 0 1 2 0 0 Sicalis flaveola 0 1 1 3 3 2 0 0 2 1 0 2 Sicalis luteola 0 0 0 0 0 2 0 0 0 0 0 0 Saltator coerulescens 0 0 0 0 0 1 0 0 0 0 0 0 Paroaria coronata 0 2 0 4 2 3 0 0 0 0 0 1 Paroaria capitata 0 0 0 1 0 1 0 0 0 0 0 0 Thraupis sayaca 0 1 1 2 2 2 2 2 1 2 0 2 animals-13-00894-t002_Table 2 Table 2 Final generalized linear models between bird diversity and environmental variables during (a) non-breeding and (b) breeding seasons: indicates interaction between variables. Response Variable Predictor Estimate Standard Error z Test/ t-test p (a) Non-breeding season Species richness Intercept 3.23 0.23 13.6 <0.001 Landscape_control -0.69 0.12 -5.66 <0.001 Landscape_land-sharing -0.16 0.1 -1.63 0.1 Pedestrians -0.01 0.004 -3.77 <0.001 Vegetation -0.01 0.01 -2.26 0.02 City_SantaFe -0.24 0.2 -1.17 0.24 Vegetation:City_SantaFe 0.01 0.01 2.23 0.026 Shannon diversity Intercept 12.39 1.55 7.97 <0.001 Landscape_control -4.29 0.79 -5.4 <0.001 Landscape_land-sharing -0.6 0.75 -0.81 0.42 Pedestrians -0.09 0.03 -3.55 <0.001 Vegetation -0.07 0.04 -1.87 0.07 City_SantaFe -2.19 1.3 -1.68 0.1 Vegetation:City_SantaFe 0.11 0.04 2.66 0.01 Simpson diversity Intercept 8.32 0.59 14.07 <0.001 Landscape_control -3.03 0.62 -4.93 <0.001 Landscape_land-sharing -0.29 0.63 -0.47 0.64 Pedestrians -0.06 0.02 -4.17 <0.001 (b) Breeding season Species richness Intercept 3.17 0.07 44.97 <0.001 Landscape_control -0.75 0.09 -7.92 <0.001 Landscape_land-sharing -5 0.07 -0.66 0.51 Pedestrians -0.01 0.003 -3.24 0.001 Shannon diversity Intercept 8.63 1.36 6.36 <0.001 Landscape_control -2.21 1.52 -1.45 0.15 Landscape_land-sharing 5.25 1.87 2.8 0.007 Vegetation 0.04 0.05 0.9 0.37 City_SantaFe -2.02 1.36 -1.48 0.14 Landscape_control:Vegetation -0.13 0.06 -2.07 0.04 Landscape_land-sharing:Vegetation -0.09 0.06 -1.6 0.11 Vegetation:City_SantaFe 0.13 0.05 2.67 0.01 Simpson diversity Intercept 6.84 1.02 6.69 <0.001 Landscape_control -2.71 0.79 -3.42 0.001 Landscape_land-sharing 2.82 0.71 3.99 <0.001 Vegetation -0.002 0.03 -0.05 0.96 City_SantaFe -2.35 1.11 -2.12 0.04 Vegetation:City_SantaFe 0.1 0.04 2.67 0.01 Disclaimer/Publisher's Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). 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PMC10000188

Wildlife can harbour Shiga toxin-producing Escherichia coli (STEC). In the present study, STEC in faecal samples from red deer (n = 106) and roe deer (n = 95) were characterised. All isolates were non-O157 strains. In red deer, STEC were detected in 17.9% (n = 19) of the isolates, and the eae/stx2b virulence profile was detected in two isolates (10.5%). One STEC strain harboured stx1a (5.3%) and eighteen STEC strains harboured stx2 (94.7%). The most prevalent stx2 subtypes were stx2b (n = 12; 66.7%), stx2a (n = 3; 16.7%), and stx2g (n = 2; 11.1%). One isolate could not be subtyped (NS) with the applied primers (5.6%). The most widely identified serotypes were O146:H28 (n = 4; 21%), O146:HNM (n = 2; 10.5%), O103:H7 (n = 1; 5.3%), O103:H21 (n = 1; 5.3%), and O45:HNM (n = 1; 5.3%). In roe deer, STEC were detected in 16.8% (n = 16) of the isolates, and the eae/stx2b virulence profile was detected in one isolate (6.3%). Two STEC strains harboured stx1a (12.5%), one strain harboured stx1NS/stx2b (6.3%), and thirteen strains harboured stx2 (81.3%). The most common subtypes were stx2b (n = 8; 61.5%), stx2g (n = 2; 15.4%), non-typeable subtypes (NS) (n = 2; 15.4%), and stx2a (n = 1; 7.7%). Serotype O146:H28 (n = 5; 31.3%) was identified. The study demonstrated that the zoonotic potential of STEC strains isolated from wildlife faeces should be monitored in the context of the 'One Health' approach which links human health with animal and environmental health. wildlife foodborne disease STEC 'One Health' infectious disease National Science Centre of Poland2020/04/X/NZ7/00896 Minister of Education and Science, "Regional Initiative of Excellence"010/RID/2018/19 This research was funded by the National Science Centre of Poland (grant No. 2020/04/X/NZ7/00896), and the project was financially supported by the Minister of Education and Science under the program entitled "Regional Initiative of Excellence" for the years 2019-2023, project No. 010/RID/2018/19, amount of funding PLN 12 000 000. pmc1. Introduction Wildlife is an important source of infectious diseases transmitted to humans. It has the potential to harbour foodborne pathogens such as Shiga toxin-producing Escherichia coli (STEC), Campylobacter spp., Yersinia enetrocolitica, or Salmonella spp. . Human activities have led to the loss of wildlife habitats and have increased the interactions between wildlife, humans, domestic animals, and livestock. The above has increased the prevalence of zoonotic diseases, and the 'One Health' holistic approach was introduced to sustainably regulate and optimise the health of humans, animals, and the ecosystem by meeting the need for clean and nutritious food, water, and air. 'One Health' is an interdisciplinary approach involving multiple disciplines, sectors, and communities who work together to develop sustainable natural policies and address the threats to human and ecosystem health. STEC should be addressed under 'One Health' research because these pathogens are ubiquitous in various ecosystem niches . In recent years, the European population of wild ruminants, including red deer (Cervus elaphus) and roe deer (Capreolus capreolus), has been increasing in density and distribution , and information about potential foodborne pathogens carried by these animals is available . However, little is known about the prevalence of STEC, their pathogenic potential, and the virulence profiles detected in different wildlife species. According to the European Food Safety Authority (EFSA), the European Centre for Disease Prevention and Control (ECDC), and the One Health 2020 Zoonoses Report, STEC infections were the fourth most common zoonoses after campylobacteriosis, salmonellosis, and yersiniosis . Currently, the categorisation of STEC pathogenicity is based on serotype; STEC belonging to O-group, namely O26, O103, O111, O145, and O157, are termed top-five and used to be the most frequently detected O-group STEC among patients with the haemolytic uraemic syndrome (HUS) . Public health authorities focus mostly on O157 STEC infections due to their pathogenicity, whereas non-O157 STEC serogroups such as O26, O103, O111, O121, and O145 cause twice as many infections in humans . According to the EFSA Authority Panel on Biological Hazards (EFSA BIOHAZ), the Food and Agriculture Organization of the United Nations, and the World Health Organization (FAO/WHO), the pathogenic capacity of STEC strains is determined mainly by the number of virulence factors such as Shiga toxin 1 (stx1), Shiga toxin 2 (stx2), E. coli attaching and effacing (eae), or transcriptional activator of aggregative adherence fimbria I (aggR) genes . STEC strains harbouring the eae gene are referred to as attaching-effacing Shiga toxin-producing E. coli (AE-STEC). There are at least three variants of the stx1 gene (stx1a, stx1c, and stx1d) and seven variants of the stx2 gene (stx2a, stx2b, stx2c, stx2d, stx2e, stx2f, and stx2g) ; however, new stx2 variants are being discovered all the time (stx2k, stx2i, stx2h, stx2d1, and stx2d2) . These variants have been linked to differences in clinical outcomes and toxicity. stx variants are also associated with disease severity. Some stx2 subtypes, such as stx2a, stx2c, and stx2d, are frequently linked to a higher risk of HUS, whereas other subtypes, such as stx2e, stx2b, stx2f, and stx2g, are linked to less severe illnesses . STEC strains are transmitted to humans mainly through the consumption of contaminated food products of animal origin , but also through vectors (ticks, fleas, mosquitoes, and rodents), scratches or bites, soil and water, direct contact with farmers, abattoir workers, veterinary practitioners, and animal faeces . Wild ruminants are a reservoir of STEC . Asymptomatic animals can harbour and shed STEC, including through faeces, thus contaminating the environment, water, and agriculture. The aim of the present study was to investigate the prevalence, virulence profiles, and pathogenic potential of STEC in faecal samples from red deer and roe deer living in their natural habitats. 2. Materials and Methods 2.1. Study Area, Sample Collection, and Initial Processing A total of 201 faecal samples were collected from wild animals including 106 red deer and 95 roe deer between May 2020 and May 2021 in north-eastern Poland (Warmian-Masurian and Podlaskie Voivodeships). Fresh faecal samples were collected in designated feeding locations in the forest. 'Point 0' was the observation of faeces upon arrival at the feeding site. From then on, freshly deposited faeces were collected every 3-4 h, always within 24 h of being passed by the target species at each location. Both hunters and foresters have specialised knowledge and are able to correctly identify both faeces and animal species. The samples were kept in a field cooler and stored at 4 degC until processing (within 24 h). In the laboratory, faecal samples of one g each were ground, combined with 10 mL of buffered peptone water (BPW) (BTL, Lodz, Poland) in aseptic conditions, and incubated overnight at 37 degC, with an aeration rate of 180 rpm. The resulting culture was used to extract DNA and isolate STEC. An amount of 500 mL of each enrichment culture was stored at -80 degC in 30% sterile glycerol. 2.2. Extraction of DNA and STEC screening by PCR DNA was extracted from 1 mL of each BPW (BTL, Lodz, Poland) enrichment culture using a Genomic Mini kit (A&A Biotechnology, Gdynia, Poland) according to the manufacturer's instructions. All samples were tested for stx1, stx2, and eae using the procedure recommended by the European Union Reference Laboratory for E. coli (EU-RL VTEC_Method 01 for E. coli) and aggR genes described previously . An amount of 50 mL of BPW (BTL, Lodz, Poland) enriched cultures of the samples with amplified stx1, stx2, eae or aggR genes were plated on CHROMagar STEC (1381)--a selective medium for the isolation of STEC (GrasoBiotech, Starogard Gdanski, Poland), and incubated at 37 degC for approximately 24 h. Mauve colonies were isolated, and the presence of stx1, stx2, eae, or aggR was confirmed by conventional PCR. After confirmation, each STEC isolate was stored at -80 degC in 30% sterile glycerol. 2.3. Shiga Toxin Subtyping and Molecular Serotyping The presence of stx1 and stx2 subtypes in the STEC isolates was determined according to the methods described by Scheutz et al. and the EU-RL procedure for E. coli (EU-RL VTEC_Method 06) . STEC serogroups associated mainly with human infections (O antigen-encoding genes wzx, wbgN, wzy, and rfb specific to thirteen serogroups: O26, O45, O55, O91, O103, O104, O11, O113, O121, O128, O145, O146, and O157) were identified according to the EU-RL procedure for E. coli (EU-RL VTEC_Method 03) . H antigens encoding fliC (specific to flagellar genes) related to H7, H8, H11, H21, and H28 were identified based on the protocols proposed by Durso et al., Gannon et al., and Mora et al. . 2.4. Statistical Analysis The Clopper-Pearson 'exact' method based on beta distribution at a significance level of a = 0.05 was used to evaluate the prevalence of STEC strains in red deer and roe deer populations. Statistical comparisons were conducted using EpiTools--free epidemiological calculators . 3. Results Fresh faecal samples were collected from 106 red deer. In the red deer population, STEC were detected in 19 isolates (17.92%, 95% CI = 11.15-26.57). All isolates were non-O157 strains, and two isolates belonged to the O103 serogroup (one of the top five serogroups) (10.53%, 95% CI = 1.30-33.14). All isolates were aggR-negative. Two isolates were eae-positive (10.53%, 95% CI = 1.30-33.14) with the eae/stx2b virulence profile, and one of them belonged to the O146 serogroup. The remaining isolates contained the stx1 gene or the stx2 gene (89.47%, 95% CI = 66.86-98.70). One STEC harboured stx1a (5.26%, 95% CI = 0.13-26.03) and belonged to the O103:H7 serotype. Sixteen isolates harboured only stx2 (84.21%, 95% CI = 60.42-96.62). stx2b was the most prevalent subtype that was detected in 10 isolates (62.50%, 95% CI = 35.43-84.80). Three isolates harboured stx2a (18.75%, 95% CI = 4.05-45.65), one isolate harboured stx2g (6.25%, 95% CI = 0.16-30.23), and one isolate could not be subtyped with the applied primers (6.25%, 95% CI = 0.16-30.23). O146 was the most prevalent O-group that was detected in six isolates (31.58%, 95% CI = 12.58-56.55); two isolates belonged to the O103 serogroup (10.53%, 95% CI = 1.30-33.14) and one isolate belonged to the O45 serogroup (5.26%, 95% CI = 0.13-26.03). The pathotypes, stx subtypes, and serogroups of STEC isolates collected from the red deer population are characterised in Table 1. Fresh faecal samples were collected from 95 roe deer. In roe deer faeces, STEC were detected in 16 isolates (16.84%, 95% CI = 9.94-25.90). All isolates were non-O157 strains, and none belonged to the O-group (top five serogroups). All isolates were aggR-negative. One isolate was eae-positive with the eae/stx2b virulence profile (6.25%, 95% CI = 0.16-30.23). Two STEC isolates harboured stx1a (12.50%; 95% CI = 1.55-38.35), but O-antigen groups could not be identified with the applied primers. One isolate harboured stx1NS/stx2b (6.25%, 95% CI = 00.16-30.23). Twelve isolates harboured only the stx2 gene (75.00%, 95% CI = 47.62-92.73). stx2b was the most prevalent subtype that was detected in seven isolates (58.33%, 95% CI = 27.67-84.83), one isolate harboured stx2a (8.33%, 95% CI = 00.21-38.48), two isolates harboured stx2g (16.67%, 95% CI = 2.09-48.41), and two isolates could not be subtyped with the applied primers (16.67%, 95% CI = 2.09-48.41). The most prevalent O-group in the roe deer population was O146, which was detected in five isolates (31.25%, 95% CI = 11.02-58.66). The pathotypes, stx subtypes, and serogroups of STEC isolates collected from the roe deer population are characterised in Table 2. 4. Discussion In the present study, the prevalence and pathogenic potential of STEC strains isolated from the faeces of 106 red deer and 95 roe deer were examined. Wildlife faeces were analysed in this study because they directly enter the environment, fields, and water. Agricultural products such as vegetables may become contaminated with STEC pathogens through direct or indirect contact with wildlife. STEC strains were isolated from 17.92% of faecal samples collected from red deer and 16.84% of faecal samples collected from roe deer. All STEC strains were serotyped as non-O157 and were aggR-negative. Serotypes O146:H28, O146:HNM, O103:H7, O103:H21, and O45:HNM, and the eae/stx2b, stx1a, stx1NS/stx2b, stx2a, stx2b, and stx2g virulence profiles were identified. According to the EFSA, ECDC, and the European Union One Health 2020 Zoonoses Report, STEC serogroups (based on the O antigen) O26, O157, O103, O145, O146, O91, O80, and O128 were most commonly identified in humans, whereas stx2a and stx2b virulence profiles were reported in patients with HUS, bloody diarrhoea, and hospitalised patients. Virulence profile stx1a was also identified in patients with bloody diarrhoea and patients requiring hospitalisation . The study demonstrated that red deer and roe deer are potential carriers of non-O-157 STEC isolates that may be pathogenic to humans. This is an important consideration because the red deer population has been increasing steadily in Europe, and wildlife have direct and indirect contact with humans, domestic animals, livestock, water bodies, and the environment . The results also confirmed that wildlife, including red deer and roe deer, could play a role as reservoirs and shedders of STEC in the environment . The prevalence and pathogenic potential of STEC strains isolated from rectal swabs from red deer and roe deer were examined in our previous study. Four O157:H7 (4.1%) strains were isolated from red deer and one O157:H7 (0.75%) strain was isolated from roe deer. STEC strains were identified in 21.65% and 24.63% of rectal swabs from red deer and roe deer, respectively . The most dangerous human STEC virulence profile, stx2a was identified in one STEC strain from roe deer and one strain from red deer . The STEC strains isolated in this study appear to have a lower pathogenic potential than those isolated from rectal swabs in our previous experiment. However, pathogenic potential exists. The environmental spread of STEC should be monitored and controlled, especially since rare stx human subtypes, such as stx2g, have been detected in human clinical samples in Denmark and Germany , and the stx2g virulence profile was associated with the outbreak of HUS in France . The results of studies conducted in different European countries indicate that wildlife such as red deer and roe deer could carry mainly STEC belonging to serogroups other than the top five serogroups (O26, O103, O111, O145, and O157). In Italy, the prevalence of STEC in free-ranging red deer was 19.9%, O146:H28 was the most frequently detected serotype (32.3%), and all isolated strains were non-O157:H7 . In Spain, the prevalence of STEC was 24.7% in red deer and 5% in roe deer, O146 was the most frequently detected serogroup, and all strains were non-O157 . However, in another Spanish study, the prevalence of STEC in faecal samples collected from red deer was 35%, four isolates (4.3%) belonged to O157:H7, and the remaining ones were non-O157 (95.7%) . In Portugal, the prevalence of STEC was 9.5% in red deer and 25% in roe deer; all STEC strains were non-O157, and serogroup O146 was the second (after O27) most frequently identified O-group . In Germany, none of the STEC isolated from faecal samples collected from roe deer belonged to the top-five serogroups . In the current study, two strains isolated from faecal samples collected from red deer belonged to group O103 (one of the top five serogroups) with the virulence profiles stx1a and stx2g. These results corroborate the findings of Spanish and Italian authors who found that STEC strains isolated from red deer were mainly eae-negative (89.47%), and stx2 was the most common stx type (84.21%) . Nevertheless, there is a need for more accurate and complete data about the pathogenic potential of STEC isolated from wildlife, in particular red deer and roe deer populations. In this study, STEC strains isolated from roe deer were mainly eae-negative (93.75%), and stx2 was the most common stx type (75%). Further research is needed to determine why STEC strains isolated from faecal samples collected from red deer and roe deer seem to have lower pathogenic potential than STEC strains isolated from rectal swabs taken from red deer and roe deer in our previous study . More information is needed to accurately characterise STEC strains present in the environment. There is no doubt that wildlife can carry STEC that are dangerous to human life and health. Due to the loss of natural wildlife habitats and rapid population growth, wild animals are increasingly observed in fields, near farms, and in other areas that are used by people and other animals. Humans, livestock, and wildlife belong to the same ecosystem, and the health of each should be equally important. Emergent zoonotic bacterial diseases should be identified and controlled under the 'One Health' approach, which integrates the efforts of physicians, veterinarians, epidemiologists, microbiologists, public health workers, and hunters. To prevent infections, the food chain should be controlled at all levels, from agricultural production, processing, and food preparation to production facilities and domestic kitchens. Hygiene education is indispensable because environmental strains should be monitored to minimise the risk of infection and outbreaks of foodborne diseases. 5. Conclusions The majority of STEC strains isolated from faecal samples collected from red deer and roe deer did not belong to the top five serogroups. O146 was the most prevalent O-group. All STEC isolates were aggR-negative, and most of them were eae-negative. stx2 was the most common stx gene type, and stx2b was the most common subtype of the stx2 gene. stx2a, stx2b, and stx2g virulence profiles were identified, and these profiles have been reported in patients with HUS and bloody diarrhoea. Wild ruminants such as red deer and roe deer are reservoirs of potentially pathogenic STEC. These animals can shed STEC in faeces and contaminate agricultural production systems, water, and the environment. According to the 'One Health' concept, naturally occurring STEC strains should be monitored in the environment, agriculture, and water to evaluate the risks to public health. New information about serogroups and virulence profiles is needed to expand our knowledge about the epidemiology and circulation of STEC in the environment. Knowledge and communication are necessary for prevention and control strategies. Author Contributions Conceptualization, A.S.-T.; methodology, A.S.-T. and F.C.; software, A.S.-T.; validation, A.S.-T., F.C.; P.S. and W.S.; formal analysis, A.S.-T.; investigation, A.S.-T.; writing--original draft preparation, A.S.-T.; writing--review and editing, A.S.-T., W.S., F.C. and P.S.; visualization, A.S.-T.; supervision, A.S.-T.; funding acquisition, A.S.-T. All authors have read and agreed to the published version of the manuscript. Institutional Review Board Statement Not applicable. Informed Consent Statement Not applicable. Data Availability Statement Not applicable. Conflicts of Interest The authors declare no conflict of interest. animals-13-00901-t001_Table 1 Table 1 Serotype, stx subtype, and the number of isolates harbouring virulence genes in STEC strains isolated from red deer (Cervus elaphus) faeces. Pathotypes Total stx Subtype Serogroups STEC stx1 1 stx1a (1) O103:H7 (1) STEC stx2 16 stx2a (3) stx2b (10) stx2g (2) stxNS1 (1) ONT2:HNM 3 (2), O45:HNM 3(1) O146:H28 (4), ONT2:HNM 3 (3), ONT 2:H7 (1) ONT 2:H21 (1), O146:HNM 3 (1), ONT 2:H28 (1), O103:H21 (1) ONT 2:H28 (1) AE-STEC eae/stx2 2 stx 2b ONT 2:HNM 3 (1) O146: HNM 3 (1) 1 NS, not subtyped with the use of the primers for Shiga-toxin encoding genes (stx). 2 ONT, O antigen non-typeable. 3 HNM, H antigen non-motile. animals-13-00901-t002_Table 2 Table 2 Serotype, stx subtype, and the number of isolates harbouring virulence genes in STEC strains isolated from roe deer (Capreolus capreolus). Pathotypes Total stx Subtype Serogroups STEC stx1 2 stx1a (2) ONT 2:HNM 3 (1) ONT 2:H21 (1) STEC stx1 + stx2 1 stx 1NS1 /stx 2b ONT 2:HNM 3 STEC stx2 12 stx2a (1) stx2b (7) stx2g (2) stxNS1 (2) ONT 2:HNM 3 O146:H28 (5), ONT 2:H21 (2) ONT 2:H28 (1) ONT 2:HNM 3 AE-STEC eae/stx2 1 stx 2b ONT 2:HNM 3 1 NS, not subtyped with the use of the primers for Shiga-toxin encoding genes (stx). 2 ONT, O antigen non-typeable. 3 HNM, H antigen non-motile. Disclaimer/Publisher's Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

PMC10000189

Omega-3 polyunsaturated fatty acids (n-3 PUFAs) have special physiological functions in both brain and retinal tissues that are related to the modulation of inflammatory processes and direct effects on neuronal membrane fluidity, impacting mental and visual health. Among them, the long-chain (LC) n-3 PUFAs, as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), are of special importance. Scarce data are available about the fatty acid (FA) composition of the ruminant brain in response to dietary intervention. However, we decided to examine the brain and retina FA composition of lambs supplemented with an EPA-rich microalga feed for 21 days, as it is known that despite the extensive biohydrogenation of dietary PUFAs in the rumen, ruminants can selectively accumulate some n-3 LC-PUFAs in their brain and retinal tissues. Twenty-eight male lambs were fed a control diet, or the same diet further supplemented with Nannochloropsis sp. microalga. Their brains and retina were collected for FA characterization. Overall, the brain FA profile remained unchanged, with little alteration in omega-3 docosapentaenoic acid (DPA) enhancement in both the hippocampus and prefrontal cortex. Retinal tissues were particularly responsive to the dietary intervention, with a 4.5-fold enhancement of EPA in the freeze-dried-fed lambs compared with the control lambs. We conclude that retinal tissues are sensitive to short-term n-3 PUFA supplementation in lambs. fatty acid hippocampus prefrontal cortex dimethyl acetal Fundacao para a Ciencia e Tecnologia (FCT)SFRH/BD/136609/2018 PTDC/CAL-ZOO/29654/2017 DL 57/2016/CP1438/CT0008 CIISAUIDB/00276/2020 AL4AnimalSLA/P/0059/2020 This research was funded by Fundacao para a Ciencia e Tecnologia (FCT) through the PhD grant SFRH/BD/136609/2018 awarded to ACM Vitor, the research contract to S.P.A. (DL 57/2016/CP1438/CT0008) and the research projects PTDC/CAL-ZOO/29654/2017 and UIDB/00276/2020 (CIISA) and LA/P/0059/2020 (AL4AnimalS). pmc1. Introduction Fatty acids (FAs) are the most abundant organic compounds in the brain. More than 90% of polyunsaturated FAs (PUFAs) in the mammalian brain are composed of long-chain PUFAs (i.e., >=20 C chain, LC-PUFA) such as arachidonic acid (AA, 20:4n-6) and docosahexaenoic acid (DHA, 22:6n-3) . Most of these PUFAs are esterified in brain membrane phospholipids (PLs), and they influence brain functions by altering the biophysical properties of cell membranes . Omega-3 PUFAs (n-3 PUFAs) have a special importance since they play a role in a variety of physiological functions related to neurogenesis, neurotransmission, and neuroinflammation, contributing to the development, functioning, and ageing of the brain. Furthermore, in humans, n-3 LC-PUFA dietary deficiencies are associated with an increased risk of developing various psychiatric disorders and neurodegenerative diseases . Among these FAs, eicosapentaenoic acid (EPA, 20:5n-3) and DHA have been linked to the maintenance of mental health, mediated by the modulation of inflammatory processes and direct effects on neuronal membrane fluidity and receptor function . N-3 docosapentaenoic acid (DPA, 22:5n-3) also plays an important role, as it is the second most abundant n-3 LC-PUFA in the brain after DHA. It is suggested to be specifically beneficial for elderly neuroprotection and early-life brain development . In addition to the brain, retinal tissue also is rich in lipids, which comprise approximately 20% of the retina's dry weight. Retinal membrane PLs have the highest level of LC-PUFAs of any tissue in humans (approximately 33%) . The retina is a tissue with a naturally high content of n-3, particularly DHA, which plays an essential role in optimizing the fluidity of photoreceptor membranes, retinal integrity and visual function . DHA also has a protective role in the retina, participating in the anti-inflammatory activity, anti-angiogenesis, anti-apoptosis and providing protection from neurotoxicity . The brain lipids of ruminant species, especially cattle, have been well characterized and appear to be very similar to those of the human brain in terms of both content and composition . Although they are less well-characterized, the lipids in the brains of sheep also do not seem to differ markedly from the ones found in cattle . In ruminants, brain characterization has been traditionally focused on the types of lipids and less so on a deeper characterization of FAs. Moreover, lipid analyses have been performed primarily in brain homogenates or gross anatomical structures within the brain , not in specific functional regions or tracts of the ruminant brain. Bovine and ovine retinal fatty acid compositions are similar: they are composed of appreciable amounts of palmitic acid (16:0), stearic acid (18:0), DHA and AA . Thus, despite the extensive biohydrogenation of dietary PUFAs in the rumen, ruminants can selectively accumulate n-3 LC-PUFA in their brain and retinal tissues, contrasting with the low deposition of these FAs in adipose tissue and muscle . Liver stores of n-3 LC-FA were reported to be the primary source of these FAs for the brain tissues of rats , even during periods of low dietary intake of these FAs . This makes it possible to maintain adequate levels of DHA and AA in the brains of AA-deprived animals . In a very recent publication, cattle supplemented with calcium salts of fish oil with 11% EPA and 8% DHA had, across most brain regions, greater EPA concentrations when compared to palm-oil-supplemented animals . The dietary supplementation of microalgae has been shown to enhance n-3 LC-PUFAs along lambs' gastrointestinal tracts . Moreover, Vitor et al. showed that the drying method applied to the microalgae strongly influenced the powder architecture and cell wall integrity, consequently affecting the degree of EPA protection against rumen microbes. Therefore, we hypothesise that lambs' brains and/or retinal tissues would be sensitive to the differences in n-3 LC-PUFA absorption due to the changes in rumen biohydrogenation associated with the processing of microalgae biomass. Thus, we collected brain and retina samples from six-month-old lambs used in a previous experiment and conducted a detailed FA composition of those tissues. 2. Materials and Methods 2.1. Animal Handling and Diets The current lamb trial was conducted in compliance with the ARRIVE and international guidelines. The trial was conducted in certified facilities and was approved by an ethical and animal well-being commission, as fully detailed in Vitor et al. . Twenty-eight sixty-day-old Merino Branco ram lambs with an average body weight of 21.8 +- 4.4 kg were housed in INIAV facilities in Santarem, Portugal. The animals were randomly allocated to individual pens (1.52 m2) with ad libitum access to clean water. The lambs were sorted into four experimental groups with seven replicates per group. The experimental diets included a control diet (C diet), consisting of pellets containing dehydrated lucerne, barley and soybean meal and no added sources of EPA, and three diets supplemented with the microalga Nannochloropsis sp., designed to provide approximately 3 g of EPA per kg of diet dry matter (DM). The average content of EPA (mg EPA/g product) in each microalgal format was 235 in the Nannochloropsis oil, 22.7 in the spray-dried Nannochloropsis oceanica and 30.8 in the lyophilized Nannochloropsis oceanica. Nannochloropsis sp.-containing diets were composed of the C diet plus 123 g/kg of spray-dried Nannochloropsis oceanica biomass (SD diet); 92 g/kg freeze-dried Nannochloropsis oceanica biomass (FD diet); and 12 g/kg of Nannochloropsis sp. free-oil (O diet) (Table 1). The trial had a 3 week duration limitation due to the high cost of the spray-dried Nannochloropsis oceanica biomass and the difficulty of obtaining enough freeze-dried biomass with lab-scale equipment. 2.2. Slaughter and Sample Collection After the end of the third week, the animals were slaughtered using a captive bolt. This was followed by exsanguination. The brain tissue was removed whole. It retained its shape and landmarks in spite of the captive bolt damage. The brain was cut on a sagittal plane and divided into two hemispheres, which were then frozen at -80 degC. After thawing, the different parts were individualized from the right hemisphere , stored in individual bags, frozen at -80 degC and lyophilized. Grey and white matter were collected from two different points in the brain and were considered samples from non-function-specific brain parts, representing only samples from the two histologic and physiological brain areas. The prefrontal cortex (cerebral cortex covering the front part of the frontal lobe), and hippocampus (located in the medial part of the temporal lobe and, on a mid-sagittal section of the brain, posterior to the amygdala extending posteriorly to the splenium of the corpus callosum) were selected as function-specific brain parts. Immediately after slaughter, the right eyeball of each lamb was removed with a spatula, stored in a bag and frozen at -80 degC. The eyeballs were thawed, and the retina and tapetum lucidum (RTL) were individualised . The liver was removed from the carcass, and a portion of the left lobe was stored in a bag and frozen at -80 degC. All brain parts, RTL and a portion of the liver were lyophilised prior to fatty acid extraction. 2.3. Fatty Acid Methyl Esters (FAMEs) and Dimethyl Acetals (DMAs) Analysis Fatty acid methyl esters (FAMEs) and dimethyl acetals (DMAs) of the brain, RTL tissues and liver samples were prepared by acid-catalysed transesterification in methanol . In plasmalogens, the sn-1 position of the glycerol contains a vinyl-ether that releases a DMA in the presence of acid methanol solution. Briefly, approximately 100 mg of lyophilised and ground sample was weighted to reaction tubes. Toluene and 1 mL of internal standard (methyl nonadecanoate -1 mg/mL) were then added, and the samples were placed in an ultrasound bath for ten minutes. A solution of 1.25 M HCl in methanol (3 mL) was added and left to react overnight at 50 degC in a water bath. Fatty acid methyl esters and DMAs were analysed by gas chromatography with flame ionization detection (GC-FID), using a Shimadzu GC 2010-Plus (Shimadzu, Kyoto, Japan) equipped with an SP-2560 (100 m x 0.25 mm, 0.20 mm film thickness, Supelco, Bellefonte, PA, USA) capillary column. The injector and detector temperatures were maintained at 220 degC and 250 degC, respectively. The carrier gas was helium at a constant flow of 1 mL/min. The GC oven temperature began at 50 degC for 1 min, then increased to 150 degC at 50 degC/min, held for 20 min, increased to 190 degC at 1 degC/min, and finally increased to 220 degC at 2 degC/min and held for 40 min. The identification of FAMEs was achieved by a comparison of the fatty acid retention times with those of commercial standards (FAME mix, 37 components from Supelco Inc., Bellefont, PA, USA) and with published chromatograms . Additional confirmation of FAMEs and DMAs were achieved by electron impact mass spectrometry using a Shimadzu GC-MS QP2010 Plus (Shimadzu, Kyoto, Japan) equipped with a SP-2560 (100 m x 0.25 mm, 0.20 mm film thickness, Supelco, Bellefonte, PA, USA) capillary column and similar GC conditions. 2.4. Statistical Analysis FAME and DMA data were analysed as a completely randomised experimental design using the MIXED procedure of SAS 9.4 (SAS Institute Inc., Cary, NC, USA). Diet was used as a fixed factor, and the animal was used as the experimental unit. Feed intake data were analysed as a completely randomised block design, in which an individual lamb was used as the experimental unit and the model included the treatment and initial live weight block as the fixed factors. The least square means and standard error of the mean (SEM) were reported, and the main effects and their interactions were considered significant at p < 0.05. The TFA + DMA content is presented in mg/g DM, and the FA individual composition is presented in % of TFA + DMA (g FA/100 g TFA + DMA). The sparse partial least squares discriminant analysis (sPLSDA) was performed using MetaboAnalyst 5.0 software, using the centred log ratio transformed FA data as input. 3. Results 3.1. Fatty Acid Intake The feed intake averaged 1.19 +- 0.13 kg (mean +- standard error of the mean) of DM/day during the experiment. It did not differ among treatments (p > 0.05) . A brief report of individual and total FA intake (mg/day) is presented in Table 2. Apart from 16:0, the intake of all the FAs analysed differed between the control and Nannochloropsis-diet-supplemented lambs (p < 0.05). The FA 14:0, 20:4n-6 and 20:5n-3 were only present in the Nannochloropsis-supplemented diets; therefore, their feed intake was zero in the control-fed lambs. The total FA intake differed between control and Nannochloropsis-supplemented lambs (p < 0.05), being higher in the latter. 3.2. Brain Fatty Acid and Dimethyl Acetal Profile The total FA and DMA (TFA + DMA) content (mg/g DM) and composition (g/100 TFA + DMA) of grey and white matter are presented in Table 3; those of the hippocampus and prefrontal cortex are presented in Table 4. Regarding the grey and white matter, the amount of TFA + DMA did not differ among treatments, averaging 191 and 227 mg/g DM, respectively. Additionally, in both the hippocampus and prefrontal cortex, TFA + DMA content did not differ among treatments, averaging 199 and 208 mg/g DM, respectively. No major effects of microalgal supplementation (p > 0.05) were observed for any of the FAs and DMAs in both the grey and white matter. Significant differences in the FA composition were observed in the hippocampus and prefrontal cortex (Table 4). In the hippocampus, 20:3n-6 was significantly (p < 0.05) lower in the C and SD diets and higher in the O and FD diets. In the prefrontal cortex, this FA was higher in all Nannochloropsis-supplemented lambs compared to C-fed lambs. Regarding 22:5n-6: it was significantly higher in the hippocampus in the C-fed lambs when compared to the Nannochloropsis-supplemented lambs. In the prefrontal cortex, this FA tended (p = 0.056) to follow a similar pattern to what was found in the hippocampus. DPA had its lowest values (p < 0.05) in the hippocampi and prefrontal cortices of C lambs, and its highest values were found in the tissues of lambs fed with the FD supplement. DPA was lower (p < 0.05) in these tissues in O-fed lambs than in SD-fed lambs. Supplement treatments did not affect (p > 0.05) the EPA or DHA concentrations in the hippocampus or prefrontal cortex. The sum of EPA + DHA averaged 10% in the hippocampus and 11% in the prefrontal cortex. The total PUFA and n-3 PUFA contents averaged 22% and 12% in both the hippocampus and prefrontal cortex, respectively. The content of TFA + DMA was not affected (p > 0.05) by dietary treatment. 3.3. Retina and Tapetum Lucidum (RTL) Similar to what was observed in the brain parts, the TFA + DMA content in the RTL tissues did not differ among treatments, averaging 53 mg/g DM (Table 5). However, more treatment effects occurred for RTL tissues than were observed in brain tissues. In RTL tissues, the c16-18:1 value was greater (p < 0.05) in SD lambs than in all other treatments. Regarding 20:3n-9, a higher content was found in both the SD-fed lambs when compared to the remaining groups. The content of 20:3n-6 was lower in C-fed lambs when compared to the remaining treatments, and 22:4n-6 was higher in both SD-fed lambs when compared to the remaining treatments. Both EPA and DPA were higher in the microalgae-biomass-fed lambs, although there were no differences between the FD-fed lambs. When compared to the C treatment, biomass-fed lambs had 4.6 times more EPA and twice more DPA in their RTL tissues. The DHA content in RTL tissues did not differ among treatments (p > 0.05). In the partial sums evaluated, the AA/EPA ratio differed between treatments, being higher in the C-fed lambs when compared to the remaining treatments (p < 0.001). The sum of the EPA + DHA averaged 7% TFA. N-3 PUFA averaged 10% +- 1.0, corresponding to approximately 45% of the total PUFAs. Similar to what was verified in the brain, none of the individual DMAs nor the total DMA content differed between treatments in the RTL tissues. Overall, the total DMA content was much lower than that found in the brain, averaging 4%. The fold change in the EPA, DPA and DHA content between FD-fed lambs and C-fed lambs was compared between the brain, RTL and liver (Table S1) and previously analysed subcutaneous adipose tissue (SC AT) and longissimus lumborum muscle samples . The EPA fold change was higher in the SC AT and similar between the liver and RTL. 3.4. sPLSDA Analysis Figure 3 illustrates a total of five sparse partial least squares-discriminant analysis (sPLS-DA) plots, corresponding to four plots that belong to all the brain parts evaluated (Panels A-D) and one plot belonging to RTL tissues (Panel E). It is possible to observe that there is no clear individualization of lambs belonging to the same diet in accordance with their brain FA and DMA compositions (%TFA) in all brain parts. However, in the RTL tissues , it is possible to clearly separate C-fed lambs from Nannochloropsis-supplemented lambs based on their retinal FA composition. 4. Discussion 4.1. Brain FA Composition The classic research on ruminant brain lipids has been based on evaluating the lipid classes and the FAs within the lipid classes of whole brain homogenates . In the present study, we present the FA and DMA profile (expressed as % of TFA + DMA) of the brain and RTL tissues of lambs fed Nannochloropsis sp. lipids. The most abundant FAs found in the ovine brain were 18:0 (18%), 16:0 (17%), c9-18:1 (17%), DHA (9%) and AA (5% of TFA + DMA). A similar pattern was also observed in the bovine brain regions , with the same five FAs also being the most abundant: 16:0 (18%), 18:0 (20%), c9-18:1 (24%), AA (6%) and DHA (9% of total FA). It was shown that linoleic acid (LA), often the major PUFA in muscle, and a-linolenic acid (ALA) were only present at very low levels. In fact, the longissimus lumborum muscles of the same animals presented LA and ALA proportions of circa 9 and 1% , contrasting with the proportions of 0.5% and 0.1% observed in the brain, respectively. Despite being the major dietary PUFA, LA can hardly be considered functional in the brain because of its low concentration (<0.5% of TFA). This low concentration is probably due to LA being extensively converted to AA, which plays a role in neurodevelopment . Moreover, the majority (59%) of LA entering the brain is rapidly b-oxidized . We hypothesised that lambs' brains and/or retinal tissues would be sensitive to the differences in n-3 LC-PUFA absorption due to the changes in rumen biohydrogenation associated with processing the microalgae biomass. Namely, FD Nannochloropsis oceanica-supplemented diets appeared to produce a higher n-3 LC-PUFA enhancement in the lambs' brains when compared to O and SD because freeze-drying better protects the integrity of the microalgal cell wall, reducing the access of ruminal microbiota to the n-3 LC-PUFA inside the cell. In ruminants, almost 90% of dietary lipids reach the duodenum as non-esterified saturated FAs , and PUFAs are selectively converted into phospholipid forms in the enterocyte . The transport and uptake of EPA and DHA within brain and retina involves their esterification into a lysophospahtidylcholine and a specific transporter (Mfsd2a) . Thus we anticipated that the uptake of EPA into the brain and retina would be efficient and responsive to the intestinal absorption of EPA. However, despite the dietary supplementation of EPA, EPA proportions in the brain were low (0.6%) and did not differ among treatments in any of the brain parts. This contrasts with the response to EPA deposition observed in the longissimus lumborum muscle of the same animals, in which EPA rose from 0.8% in the C treatment to 1.7% in the Nannochloropsis-supplemented treatments . The response in the liver was even more pronounced, with EPA rising from 0.9% up to 4% in the Nannochloropsis-supplemented treatments (Table S1). Thus, in general, the ovine brain was not responsive to dietary EPA supplementation. This contrasts with the results reported by Rule et al. , in which a similar intake of EPA (2.3 g/day) resulted in an EPA enhancement across various brain parts. However, the supplementation period in our study lasted 1/10 of the one in Rule et al. . As the uptake of EPA and DHA into the brain is similar , the lack of EPA enhancement in the lambs' brains in response to the treatments might be explained by the faster b-oxidation of EPA compared to DHA and/or by the extensive elongation to DPA and subsequent desaturation to DHA. Nevertheless, we also did not observe an increase in DHA. The long half-life of DHA in brain tissues can explain the slow turnover of these fatty acids, therefore explaining the lack of their enhancement in the brain . Moreover, there seems to be evidence that brain DHA and AA levels can be maintained by the liver stores once there is evidence that liver (but not brain) DHA synthesis is upregulated when the dietary content of n-3 PUFA is reduced . Similar responses in brain FAs to microalgae supplementation were observed for the hippocampus and prefrontal cortex, probably reflecting the extensive hippocampal-prefrontal interactions involved in various cognitive and behavioural functions in animals . Higher amounts of n-3 DPA and dihomo-g-linolenic acid (DGLA, 20:3n-6) were found in the hippocampi and prefrontal cortices of microalgae-fed lambs. DGLA is an intermediate of the elongation and desaturation of LA, being converted into AA through the activity of the D-5 desaturase enzyme . The increase in DGLA in the brains of lambs supplemented with microalgae was not obvious, as Nannochloropsis does not contain relevant amounts of LA and DGLA. DPA, a product of the elongation of EPA, was increased despite the lack of response in EPA and DHA. As it has been proposed that DPA constitutes a storage depot for EPA and DHA , its enhancement seems desirable. Although DPA was approximately 10 times lower than DHA in the brain, it was more responsive to the dietary supply of n-3 PUFA. The same pattern was also observed in the brains of lambs suckling from ewes fed with linseed and in the hippocampus of bovines fed fish oil . Most of the beneficial effects of marine oils (mainly fish oils) have been attributed to DHA and EPA . However, DPA, which is the intermediate between EPA and DHA in the n-3 LC-PUFA biosynthetic pathway, also presents beneficial biological effects. It reduces platelet aggregation, improves the lipid plasmatic profile, neural health and endothelial cell migration, and assists in the resolution of chronic inflammation . Plasmalogens are a subclass of glycerophospholipids that comprise part of biological membranes, including the plasma membrane and the membranes of intracellular organelles, affecting their biophysical properties. They are quantitively important in membranes of neuronal tissues, including the brain and the retina, and are associated with neurological and psychiatric disorders or are involved in the regulation of retinal vascular development, respectively . In the DMA, the backbone at the sn-2 position is mainly bonded to PUFAs such as DHA and AA, suggesting its protective role against lipoxidation . The DMA content of ruminant brains is not often reported . In our study, the high abundance of plasmalogens in the brain can be perceived through the high DMA content (10% of TFA + DMA). The average content of DMA was higher in the brain when compared to the retina (3.5% of TFA + DMA). 4.2. RTL Tissue FA Composition The retina is a thin, highly organised neural tissue lining the posterior aspect of the eye. It is responsible for initiating vision by transducing light into neural signals . The visual streak area of the retina is a narrow horizontal band. It runs parallel to the ventral edge of the tapetum . Therefore, due to anatomical proximity and for practical reasons, we collected both tissues simultaneously. The tapetum lucidum is a biologic reflector system that is commonly present in the eyes of vertebrates. It enhances visual sensitivity at low light levels by providing light-sensitive retinal cells with a second opportunity for photon-photoreceptor stimulation. Ovine tapetum lucidum belongs to the choroidal fibrous type, and the reflective material is made of collagen that constitutes 65% of the dry weight of the tapetum . When comparing the results with the literature, it is important to consider that the specialized retina lipids in the joint RTL samples will be diluted by the fibrous tapetum tissue. Studies with rodents demonstrated that the FA composition of the retina is influenced by diet and, for a given species, the retinal FA composition of each phospholipid class is comparable to that of the brain grey matter . The retina of sheep and cattle have a similar FA composition , and the main FA of bovine retina (% dry weight) are 16:0 (25%), 18:0 (17%), 18:1 (17%) and DHA (23%) . In the present study, 16:0 averaged 20%, 18:0 averaged 18%, c9-18:1 averaged 26% and DHA averaged 6.4%. AA averaged 6%, in line with it being the most abundant omega-6 PUFA in the retina . Contrary to what was observed in the brain, EPA supplementation increased the EPA content in the RTL tissues. In the C lambs, EPA averaged 0.18% TFA + DMA, which was in line with human EPA retinal content . The EPA content significantly increased in O-fed lambs (0.59% TFA + DMA) and particularly in FD-fed lambs (0.82% TFA + DMA; + 4.6 times the EPA content in the C-fed lambs). Contrary to the results achieved in the brain, our results showed that the RTL tissues of lambs are very responsive to EPA supplementation. The same magnitude of response was only comparable to what we found in the liver . The 4.6-fold increase was achieved despite the short duration of EPA supplementation. Consistent with a better responsiveness of RTL tissues to the experimental diets, control lambs were clearly separated from the lambs consuming Nannochloropsis-supplemented diets in the sPLSDA analysis. This shows, once again, that the RTL tissues seem to have been much more sensitive to dietary intervention. The high responsiveness of the retina is evident in rodent studies, in which EPA contents of 6 to 35 times greater have been reported following EPA supplementation . As in the brain, no alterations in DHA content were observed between different treatments. The content of DHA in RTL tissues averaged 6.4% of the total TFA + DMA. This is considerably lower that what was reported in previous studies for ruminants in which the DHA content averaged approximately 20-30% . 5. Conclusions After a short-term trail of EPA supplementation in lambs, achieved through feeding using three different diets containing Nannochloropsis sp. microalga, it was possible to conclude that the brain content of EPA was not responsive to dietary supplementation. However, the EPA content in the retina was highly responsive in lambs supplemented with Nannochloropsis, especially lambs consuming SD and FD diets. Although we could not confirm an advantage in freeze-drying over spray-drying Nannochloropsis oceanica with respect to the efficiency of EPA enhancement in the lambs' retinal tissues, we can confirm their advantage over the free oil. Overall, our results suggest that RTL is a good target to evaluate the differences in n-3 LC-PUFA absorption due to the changes in rumen biohydrogenation associated with dietary interventions. Acknowledgments The authors acknowledge the support from Fundacao para a Ciencia e Tecnologia (FCT) through the PhD grant SFRH/BD/136609/2018 awarded to ACM Vitor, the research contract to S.P.A. (DL 57/2016/CP1438/CT0008) and the research projects PTDC/CAL-ZOO/29654/2017, UIDB/00276/2020 and UIDB/05183/2020. The authors acknowledge the collaboration of Instituto Superior de Agronomia, University of Lisbon, for helping with the microalgae processing and helping with the management of the animals. The authors also acknowledge the INIAV staff for the support in animal management and slaughter. Supplementary Materials The following supporting information can be downloaded at: Table S1: Summary table for total fatty acid (TFA) and dimethyl acetal (DMA) (mg/g DM) content and composition (%TFA + DMA) of the liver tissues of lambs. Click here for additional data file. Author Contributions S.P.A. and R.J.B.B., were responsible for conceptualization and funding acquisition; A.C.M.V., R.J.B.B. and S.P.A., implemented the experiment; A.C.M.V. conducted the animal experiment; A.C.M.V. and S.P.A. conducted laboratory analysis; J.J.C. monitored and assisted histopathological analyses; A.C.M.V., R.J.B.B. and S.P.A. performed data analysis; A.C.M.V., R.J.B.B. and S.P.A. interpreted the results; A.C.M.V. drafted the manuscript; and J.J.C., S.P.A. and R.J.B.B. edited and revised the original draft. All authors have read and agreed to the published version of the manuscript. Institutional Review Board Statement The experimentation involving live animals was conducted under strict compliance with international guidelines (Directive 2010/63/EU) regulating the use of production animals in animal experimentation. Experimentation was conducted at the INIAV-Santarem facilities. The INIAV-Santarem facilities are certified by the competent veterinary authority (DGAV) to conduct animal experimentation (Ref: 04211000/000/2013). The experimental animal procedures were approved by the Ethical and Animal Well-Being Commission (CEBEA) of the Faculty of Veterinary Medicine, University of Lisbon, Portugal (Protocol FMV/CEBEA 007/2016). Animal management, handling, transport, and sacrifice were conducted replicating approved standard commercial practices regarding animal welfare except that animals were individually housed. The study was carried out in compliance with the ARRIVE guidelines. Informed Consent Statement Not applicable. Data Availability Statement The data presented in this study are available on request from the corresponding author. Conflicts of Interest The authors declare no conflict of interest. Figure 1 Lamb right brain hemisphere. The four collected parts are highlighted in the figure: prefrontal cortex and hippocampus are filled in blue and yellow, respectively; grey matter is identified with the yellow triangles and white matter with the blue circles. Figure 2 Dissection of the right eyeball of one lamb. From left to right: (A) right eyeball; (B) removal of eye structures (cornea, iris and lens) and tapetum lucidum evidenced; and (C) cutting of material used for FA analysis including tapetum lucidum and retina tissues. Figure 3 sPLSDA analysis including the FA + DMA profile. The different plots correspond to the different brain parts (A-D) and RTL tissues (E). (A) prefrontal cortex, (B) hippocampus, (C) grey matter, (D) white matter, and (E): RTL tissues. Diets are represented in different colours: red--C, control diet with no EPA sources; purple--O, diet with Nannochloropsis sp. oil; blue--SD, diet with spray-dried Nannochloropsis oceanica biomass; and green--FD, diet with freeze-dried Nannochloropsis oceanica biomass. Figure 4 EPA, DPA and DHA fold change in lambs' tissues. The fold change was calculated between the mean value determined in the tissues of freeze-dried Nannochloropsis oceanica-fed lambs versus the mean value for control-fed lambs. SC AT: subcutaneous adipose tissue; RTL: retina and tapetum lucidum. The reference value for the brain corresponds to the mean of all brain parts. animals-13-00828-t001_Table 1 Table 1 Total fatty acid content (g/kg dry matter) and fatty acids (FAs) profile (% of total fatty acids) of the experimental diets. Item Diets 2 C O SD FD Total FA 1 13.7 20.6 19.5 20.2 FA profile 14:0 n.d. 1.26 2.40 2.28 16:0 25.5 23 23.9 26.2 c9-16:1 0.51 5.15 10.2 8.22 17:0 0.66 0.34 n.d. n.d. 18:0 4.09 2.96 2.51 3.32 c9-18:1 18.6 14.5 11.7 13.7 c11-18:1 0.73 0.93 0.72 0.69 18:2n-6 41 31.5 28.2 29.1 18:3n-3 8.91 6.80 5.02 6.44 20:4n-6 n.d 2.58 3.69 3.17 20:5n-3 n.d. 10.40 11.3 6.88 22:0 n.d. 0.58 0.36 n.d. 1 FA--fatty acids. 2 C--control diet with no EPA sources; O--diet with Nannochloropsis sp. oil; SD--diet with spray-dried Nannochloropsis oceanica biomass; FD--diet with freeze-dried Nannochloropsis oceanica biomas; n.d.--not detected. In the FA notation (x:n-), 'x' represents the number of C atoms, ':' the number of double bonds and 'n-' the location, in its carbon chain, of the double bond which is closest to the methyl end of the molecule. c stands for cis. Adapted from . animals-13-00828-t002_Table 2 Table 2 Daily fatty acid (FA) intake (m/day) during the trial. FA 1 Diets 2 SEM 3 p-Value C vs. Nannochloropsis DIETS 4 C O SD FD 14:0 - 320 590 540 0.030 <0.001 16:0 4110 5780 5710 6200 0.466 0.217 c9-16:1 60 1300 2490 1970 0.119 <0.001 18:0 660 740 600 790 0.061 0.011 c9-18:1 3010 3620 2800 3260 0.277 <0.001 c11-18:1 120 230 110 160 0.014 <0.001 18:2n-6 6640 7900 6710 6910 0.607 <0.001 18:3n-3 1440 1700 1190 1530 0.129 <0.001 20:4n-6 - 670 890 750 0.058 <0.001 20:5n-3 - 2600 2710 1630 0.178 <0.001 Total 16,100 25,100 24,000 23,700 1.90 0.033 1 FA--fatty acid; 2 C-- control diet with no EPA sources; O--diet with Nannochloropsis sp. oil; SD--diet with spray-dried Nannochloropsis oceanica biomass; FD--diet with freeze-dried Nannochloropsis oceanica biomass; n.d.--not detected. 3 Standard error of the mean. 4 C vs. Nannochloropsis diets, compares C with O, SD and FD together. In the FA notation (x:n-), 'x' represents the number of C atoms, ':' the number of double bonds and 'n-' the location, in its carbon chain, of the double bond which is closest to the methyl end of the molecule. c stands for cis. animals-13-00828-t003_Table 3 Table 3 Total fatty acid (TFA) and dimethyl acetal (DMA) content (mg/g DM) and composition (% TFA + DMA) of the grey matter and white matter of lambs. FA and DMA 1 Grey Matter White Matter Diets 2 SEM 3 p-Value Diets 2 SEM 3 p-Value C O SD FD C O SD FD TFA + DMA 189 192 190 193 5.2 0.970 235 227 225 222 4.1 0.163 14:0 0.57 0.55 0.58 0.58 0.025 0.746 0.52 0.52 0.55 0.53 0.018 0.778 15:0 0.11 0.12 0.11 0.13 0.008 0.392 0.09 0.09 0.09 0.11 0.007 0.177 16:0 19 19 19 19 0.4 0.942 13 14 14 15 0.4 0.261 c7-16:1 0.42 0.42 0.40 0.42 0.021 0.934 0.35 0.37 0.35 0.36 0.016 0.722 c9-16:1 0.42 0.43 0.45 0.45 0.024 0.810 0.26 0.29 0.27 0.29 0.013 0.354 17:0 0.29 0.29 0.27 0.31 0.014 0.445 0.27 0.31 0.28 0.31 0.015 0.181 c9-17:1 0.11 0.11 0.10 0.11 0.010 0.940 0.11 0.17 0.14 0.15 0.019 0.223 18:0 21 21 20 21 0.5 0.964 15 16 16 16 0.4 0.176 c9-18:1 15 15 15 14 0.5 0.847 22 22 22 21 0.5 0.216 c11-18:1 3.4 3.3 3.3 3.3 0.07 0.433 3.0 3.0 2.9 3.0 0.05 0.671 18:2n-6 0.48 0.48 0.50 0.52 0.030 0.673 0.29 0.43 0.39 0.42 0.042 0.111 19:1 0.06 0.07 0.07 0.07 0.006 0.836 0.10 0.10 0.10 0.10 0.008 0.892 20:0 0.32 0.31 0.31 0.32 0.017 0.870 0.69 a 0.64 ab 0.62 b 0.62 b 0.019 0.045 18:3n-3 0.04 0.03 0.03 0.04 0.008 0.843 0.13 a 0.08 b 0.13 a 0.09 b 0.013 0.026 c11-20:1 0.86 0.83 0.88 0.80 0.080 0.902 2.2 2.0 2.1 1.9 0.11 0.372 20:1 0.27 0.27 0.27 0.26 0.021 0.941 0.62 0.62 0.59 0.56 0.035 0.542 21:0 0.06 0.06 0.07 0.07 0.005 0.428 0.09 0.10 0.11 0.10 0.012 0.868 20:2 0.10 0.12 0.11 0.12 0.014 0.761 0.26 0.25 0.24 0.23 0.023 0.803 20:2n-6 0.10 0.11 0.11 0.10 0.019 0.959 0.15 0.14 0.14 0.14 0.022 0.638 20:3n-9 0.54 0.54 0.59 0.49 0.068 0.764 0.69 0.69 0.69 0.65 0.054 0.943 22:0 0.71 0.66 0.64 0.66 0.069 0.915 1.90 1.84 1.68 1.69 0.078 0.140 20:3n-6 0.33 0.39 0.36 0.39 0.020 0.094 0.35 0.42 0.38 0.45 0.030 0.111 22:1 0.33 0.32 0.31 0.30 0.040 0.970 0.80 0.10 0.40 0.37 0.170 0.068 20:3n-3 0.13 0.13 0.12 0.11 0.020 0.941 0.57 0.78 0.62 0.60 0.083 0.334 22:1/20:3n-3 0.28 0.20 0.30 0.27 0.064 0.727 0.81 0.88 0.98 0.70 0.067 0.154 20:4n-6 6.3 5.8 5.7 6.2 0.24 0.312 3.8 4.0 3.8 4.2 0.16 0.176 23:0 0.21 0.20 0.21 0.20 0.028 0.992 0.50 0.39 0.39 0.37 0.050 0.262 22:2n-6 0.09 0.09 0.09 0.09 0.018 0.985 0.52 0.48 0.49 0.60 0.119 0.863 20:5n-3 0.26 0.29 0.32 0.32 0.040 0.655 0.71 0.60 0.69 0.69 0.053 0.502 24:0 0.66 0.63 0.61 0.63 0.139 0.997 2.3 2.3 2.2 2.0 0.11 0.115 c15-24:1 1.2 1.3 1.4 1.3 0.20 0.951 4.4 3.9 4.2 3.7 0.25 0.251 24:1 0.69 0.20 0.23 0.22 0.200 0.284 0.70 0.65 0.65 0.63 0.038 0.576 22:4n-6 2.5 2.5 2.6 2.9 0.268 0.864 2.8 2.4 2.5 2.7 0.14 0.194 21:5 0.11 0.03 0.03 0.03 0.035 0.334 0.14 0.17 0.12 0.10 0.030 0.448 22:5n-6 0.62 0.41 0.38 0.38 0.066 0.060 0.34 0.25 0.25 0.25 0.037 0.251 26:0 0.15 0.02 0.03 0.04 0.053 0.304 0.08 0.07 0.08 0.08 0.012 0.892 26:1 0.08 0.10 0.13 0.09 0.049 0.878 0.26 0.34 0.42 0.23 0.036 0.069 22:5n-3 3.0 0.76 1.05 1.21 1.111 0.504 0.51 0.32 0.57 0.65 0.150 0.454 22:6n-3 11 14 14 13 1.07 0.248 4.7 5.2 5.2 5.9 0.40 0.212 DMA DMA 16:0 2.2 2.2 2.3 2.2 0.10 0.911 3.7 3.6 3.7 3.5 0.09 0.644 DMA 17:0 0.19 0.16 0.16 0.19 0.013 0.320 0.23 0.24 0.23 0.26 0.012 0.319 DMA 18:0 3.9 4.7 4.7 4.7 0.35 0.275 4.5 4.6 4.6 4.7 0.09 0.543 DMA c9-18:1 0.92 1.01 1.11 1.01 0.128 0.784 2.8 2.5 2.3 2.5 0.24 0.458 DMA c11-18:1 0.95 0.96 1.00 0.95 0.071 0.941 2.0 1.9 2.0 1.9 0.075 0.645 Partial sums C18 4 39 39 39 39 0.3 0.526 40 41 41 40 0.5 0.207 C18:1 5 20 0.27 0.27 0.27 9.97 0.418 0.32 0.34 0.33 0.28 0.027 0.355 DMA 15 9.0 9.2 9.1 2.82 0.341 13 13 13 13 0.2 0.596 SFA 6 38 42 42 42 1.6 0.270 35 36 35 36 0.6 0.207 MUFA 7 22 22 22 22 0.8 0.901 35 34 34 32 0.8 0.156 cis-MUFA 22 21 21 21 0.9 0.455 32 32 32 30 0.7 0.223 PUFA 8 24 26 26 26 0.9 0.519 16 16 16 18 0.6 0.159 n-3 PUFA 14 15 15 15 0.5 0.121 6.7 7.0 7.3 8.0 0.41 0.162 n-6 PUFA 12 9.8 9.8 10 0.55 0.101 8.1 8.0 7.8 8.7 0.33 0.320 EPA + DHA 18 14 14 14 2.2 0.414 5.4 5.8 5.9 6.6 0.41 0.232 AA/EPA ratio 21 23 19 21 3.07 0.815 5.7 6.9 5.7 6.2 0.56 0.375 Means within a row with different letters are significantly different (p < 0.05). 1 FA and DMA--fatty acids and dimethyl acetals; 2 C--control diet with no EPA sources; O--diet with Nannochloropsis sp. oil; SD--diet with spray-dried Nannochloropsis oceanica biomass; FD--diet with freeze-dried Nannochloropsis oceanica biomass. 3 Standard error of the mean; the value presented corresponds to a pooled sample standard error of the mean. 4 Sum of C18 FA. 5 Sum of C18:1 FA. 6 Sum of saturated FA. 7 Sum of monounsaturated FA. 8 Sum of polyunsaturated FA. In the FA notation (x:n-), 'x' represents the number of C atoms, ':' the number of double bonds and 'n-' the location, in its carbon chain, of the double bond which is closest to the methyl end of the molecule. c stands for cis. animals-13-00828-t004_Table 4 Table 4 Total fatty acid (TFA) and dimethyl acetal (DMA) (mg/g DM) content and composition (% TFA + DMA) of the hippocampus and prefrontal cortex of lambs. FA and DMA 1 Hippocampus Prefrontal Cortex Diets 2 SEM 3 p-Value Diets 2 SEM 3 p-Value C O SD FD C O SD FD TFA + DMA 199 196 204 195 4.4 0.569 206 206 211 210 3.0 0.533 14:0 0.52 0.51 0.53 0.48 0.018 0.243 0.59 0.59 0.61 0.63 0.024 0.643 15:0 0.11 0.10 0.09 0.10 0.005 0.185 0.11 0.11 0.10 0.12 0.008 0.493 16:0 17 17 16 16 0.4 0.458 18 17 17 18 0.3 0.923 c7-16:1 0.43 0.43 0.40 0.38 0.017 0.101 0.43 0.42 0.40 0.41 0.018 0.649 c9-16:1 0.35 0.39 0.38 0.34 0.017 0.174 0.42 0.42 0.41 0.43 0.019 0.863 17:0 0.29 0.28 0.27 0.29 0.009 0.428 0.27 0.29 0.26 0.31 0.019 0.408 c9-17:1 0.11 0.12 0.12 0.12 0.009 0.937 0.13 0.13 0.13 0.15 0.010 0.263 18:0 18 19 18 18 0.4 0.642 19 19 19 19 0.4 0.951 c9-18:1 17 17 18 17 0.5 0.756 17 17 17 17 0.5 0.905 c11-18:1 3.4 3.3 3.2 3.3 0.05 0.325 3.4 3.3 3.2 3.2 0.05 0.107 18:2n-6 0.41 0.45 0.45 0.44 0.027 0.644 0.40 0.42 0.42 0.45 0.035 0.790 19:1 0.08 0.08 0.08 0.09 0.008 0.578 0.08 0.08 0.07 0.08 0.005 0.125 20:0 0.50 0.48 0.49 0.50 0.029 0.968 0.45 0.48 0.44 0.45 0.027 0.705 18:3n-3 0.09 0.08 0.09 0.06 0.013 0.481 0.07 0.08 0.06 0.06 0.008 0.443 c11-20:1 1.4 1.3 1.6 1.6 0.14 0.567 1.2 1.2 1.2 1.2 0.07 0.912 20:1 0.38 0.37 0.40 0.39 0.026 0.822 0.38 0.39 0.38 0.37 0.021 0.878 21:0 0.07 0.07 0.08 0.07 0.007 0.904 0.10 0.09 0.08 0.07 0.013 0.660 20:2 0.19 0.17 0.19 0.17 0.012 0.472 0.19 0.20 0.19 0.18 0.018 0.846 20:2n-6 0.12 0.12 0.12 0.15 0.013 0.297 0.08 0.11 0.10 0.11 0.007 0.075 20:3n-9 0.83 0.84 0.77 0.70 0.051 0.221 0.73 0.74 0.69 0.64 0.066 0.673 22:0 1.0 1.1 1.2 1.2 0.12 0.815 1.1 1.2 1.1 1.2 0.07 0.759 20:3n-6 0.37 b 0.44 a 0.42 ab 0.46 a 0.023 0.049 0.29 b 0.38 a 0.35 a 0.37 a 0.018 0.008 22:1 0.50 0.47 0.58 0.59 0.054 0.348 0.46 0.53 0.48 0.48 0.032 0.536 20:3n-3 0.24 0.21 0.25 0.24 0.022 0.588 0.26 0.29 0.25 0.27 0.022 0.572 22:1/20:3n-3 0.59 0.49 0.55 0.57 0.145 0.960 0.43 0.30 0.46 0.46 0.037 0.108 20:4n-6 5.4 5.4 4.9 5.1 0.26 0.476 5.3 5.0 4.9 5.1 0.17 0.346 23:0 0.38 0.33 0.36 0.37 0.030 0.641 0.24 0.25 0.24 0.25 0.022 0.980 22:2n-6 0.18 0.13 0.19 0.19 0.035 0.600 0.18 0.20 0.21 0.20 0.038 0.958 20:5n-3 0.56 0.59 0.63 0.62 0.035 0.580 0.55 0.65 0.63 0.65 0.061 0.596 24:0 1.4 1.4 1.5 1.5 0.14 0.971 1.1 1.3 1.2 1.2 0.10 0.805 c15-24:1 2.6 2.1 2.7 2.8 0.28 0.266 2.2 2.3 2.3 2.3 0.17 0.983 24:1 0.39 0.31 0.38 0.40 0.034 0.260 0.43 0.42 0.40 0.44 0.030 0.823 22:4n-6 3.2 2.8 2.8 2.9 0.16 0.235 2.8 2.5 2.6 2.6 0.13 0.329 21:5 0.09 0.08 0.09 0.10 0.014 0.861 0.06 0.07 0.07 0.06 0.008 0.413 22:5n-6 0.50 a 0.34 b 0.31 b 0.31 b 0.052 0.047 0.52 a 0.38 ab 0.34 b 0.33 b 0.052 0.056 26:0 0.05 0.04 0.05 0.05 0.005 0.286 0.05 0.05 0.05 0.05 0.005 0.675 26:1 0.26 0.18 0.23 0.25 0.067 0.846 0.52 0.35 0.60 0.57 0.078 0.256 22:5n-3 0.83 c 1.14 b 1.24 ab 1.36 a 0.066 <0.001 0.54 c 0.82 b 0.97 ab 1.06 a 0.069 <0.001 22:6n-3 9.0 9.6 8.9 9.0 0.38 0.573 10 10 10 10 0.53 0.956 DMA DMA 16:0 2.7 2.7 2.8 2.7 0.09 0.717 2.7 2.9 2.8 2.8 0.12 0.910 DMA 17:0 0.20 0.20 0.20 0.22 0.013 0.716 0.19 0.19 0.19 0.21 0.013 0.373 DMA 18:0 4.6 4.7 4.7 4.8 0.08 0.612 4.5 4.7 4.7 4.6 0.09 0.519 DMA c9-18:1 1.4 1.5 1.8 1.8 0.17 0.268 1.5 1.6 1.6 1.6 0.11 0.986 DMA c11-18:1 1.4 1.4 1.5 1.4 0.05 0.301 1.3 1.4 1.4 1.4 0.08 0.919 Partial sums C18 4 40 40 40 39 0.3 0.303 40 40 40 40 0.3 0.813 C18:1 5 0.37 0.32 0.33 0.33 0.018 0.265 0.29 0.28 0.26 0.27 0.019 0.807 DMA 10.3 10 11 11 0.3 0.320 10 11 11 11 0.3 0.830 SFA 6 39 40 39 39 0.6 0.311 40 40 40 40 0.5 0.934 MUFA 7 27 26 28 27 0.9 0.617 27 27 27 27 0.7 0.979 cis-MUFA 25 25 26 25 0.8 0.671 25 25 25 25 0.6 0.972 PUFA 8 22 22 21 22 0.7 0.744 22 22 22 22 0.6 0.952 n-3 PUFA 11 12 11 11 0.3 0.340 12 12 12 12 0.5 0.790 n-6 PUFA 10 9.7 9.2 9.6 0.41 0.362 9.6 9.0 8.9 9.2 0.29 0.309 EPA + DHA 9.6 10 9.5 9.6 0.39 0.600 10 11.0 11 11 0.5 0.960 AA/EPA ratio 9.8 9.2 8.1 8.4 0.72 0.366 10 8.4 8.2 8.3 1.13 0.476 Means within a row with different letters are significantly different (p < 0.05). 1 FA and DMA--fatty acids and dimethyl acetals; 2 C--control diet with no EPA sources; O--diet with Nannochloropsis sp. oil; SD--diet with spray-dried Nannochloropsis oceanica biomass; FD--diet with freeze-dried Nannochloropsis oceanica biomass. 3 Standard error of the mean; the value presented corresponds to a pooled sample standard error of the mean. 4 Sum of C18 FA. 5 Sum of C18:1 FA. 6 Sum of saturated FA. 7 Sum of monounsaturated FA. 8 Sum of polyunsaturated FA. In the FA notation (x:n-), 'x' represents the number of C atoms, ':' the number of double bonds and 'n-' the location, in its carbon chain, of the double bond which is closest to the methyl end of the molecule. c stands for cis. animals-13-00828-t005_Table 5 Table 5 Total fatty acid (TFA) and dimethyl acetal (DMA) (mg/g DM) content and composition (%TFA + DMA) of the RTL tissues of lambs. FA and DMA 1 Diet 2 SEM 3 p-Value C O SD FD TFA + DMA 54 61 43 53 8.9 0.591 10:0 0.05 0.06 0.08 0.06 0.014 0.376 12:0 0.26 0.24 0.23 0.25 0.036 0.955 14:0 2.2 1.3 1.6 1.9 0.25 0.069 i-15:0 0.08 0.05 0.05 0.02 0.014 0.063 a-15:0 0.18 0.06 0.07 0.05 0.066 0.433 c9-14:1 0.06 0.08 0.09 0.07 0.017 0.518 15:0 0.27 0.27 0.28 0.31 0.026 0.766 i-16:0 0.09 0.07 0.10 0.06 0.018 0.341 16:0 21 21 20 21 0.58 0.608 i-17:0 0.15 0.17 0.17 0.15 0.027 0.881 c7-16:1 0.29 0.30 0.25 0.29 0.034 0.717 c9-16:1 0.77 0.77 0.66 0.83 0.118 0.752 a-17:0 0.18 0.14 0.09 0.13 0.041 0.488 17:0 0.67 0.70 0.60 0.74 0.038 0.084 c9-17:1 0.31 0.32 0.27 0.27 0.033 0.543 18:0 18 18 19 18 0.5 0.591 t6/t7/t8-18:1 0.08 0.11 0.11 0.09 0.019 0.664 t9-18:1 0.07 0.12 0.11 0.11 0.018 0.198 t10-18:1 0.49 0.53 0.28 0.35 0.096 0.219 t11-18:1 0.66 0.87 0.88 0.87 0.114 0.455 t12-18:1 0.20 0.22 3.56 0.23 1.784 0.437 c9-18:1 29 28 21 27 2.4 0.121 c11-18:1 1.8 1.8 2.0 1.9 0.10 0.415 c12-18:1 0.09 0.11 0.15 0.10 0.023 0.241 c13-18:1 0.06 0.06 0.04 0.05 0.014 0.728 t16/c14-18:1 0.09 0.13 0.10 0.12 0.025 0.671 c15-18:1 0.04 0.04 0.05 0.03 0.014 0.799 tc/ct-18:2/cyclo-17 0.11 0.18 0.16 0.22 0.043 0.370 c9,t12/c9,t15/t8,c13-18:2 0.09 0.10 0.07 0.11 0.021 0.626 c16-18:1 0.03 b 0.03 b 0.08 a 0.04 b 0.012 0.021 t9,c12-18:2 0.04 0.06 0.07 0.05 0.013 0.385 t11,c15/t10,c15-18:2 0.06 0.16 0.08 0.14 0.033 0.094 18:2n-6 0.31 3.75 4.25 3.98 0.299 0.317 20:0 0.16 0.17 0.20 0.17 0.022 0.675 18:3n-3 0.63 0.67 0.65 0.66 0.038 0.873 c9,t11-CLA 0.26 0.37 0.30 0.38 0.057 0.339 20:2n-6 0.15 0.16 0.19 0.17 0.020 0.509 20:3n-9 0.29 a 0.20 bc 0.24 ab 0.14 c 0.032 0.014 22:0 0.05 0.07 0.07 0.06 0.012 0.414 20:3n-6 0.38 b 0.59 a 0.70 ab 0.58 a 0.056 0.004 20:3n-3 0.18 0.22 0.22 0.20 0.029 0.646 20:4n-6 6.4 5.4 6.5 5.6 0.54 0.391 20:5n-3 0.18 c 0.59 b 0.83 a 0.81 a 0.057 <0.001 22:4n-6 0.93 a 0.62 b 0.73 ab 0.58 b 0.071 0.008 22:5n-3 1.2 c 2.0 b 2.5 a 2.4 ab 0.17 <0.001 22:6n-3 5.7 6.8 6.9 6.2 0.90 0.752 DMA DMA 16:0 1.2 1.2 1.5 1.2 0.14 0.195 DMA 18:0 1.8 1.9 2.1 1.7 0.18 0.552 DMA 18:1 0.41 0.37 0.43 0.34 0.041 0.360 Partial sums C18 4 37 37 34 36 1.6 0.485 DMA 3.4 3.4 4.0 3.3 0.3 0.351 SFA 5 42 41 41 42 0.40 0.417 MUFA 6 34 33 30 32 1.7 0.330 cis-MUFA 32 31 25 30 2.4 0.141 PUFA 7 20 22 24 22 1.6 0.266 n3-PUFA 7.9 10 11 10 1.0 0.130 n6-PUFA 11 11 12 11 0.9 0.472 EPA + DHA 5.9 7.4 7.7 7.0 0.91 0.491 AA/EPA ratio 37 a 9.1 b 8.0 b 6.9 b 2.33 <0.001 Means within a row with different letters are significantly different (p < 0.05). 1 FA and DMA--fatty acids and dimethyl acetals; 2 C--control diet with no EPA sources; O--diet with Nannochloropsis sp. oil; SD--diet with spray-dried Nannochloropsis oceanica biomass; FD--diet with freeze-dried Nannochloropsis oceanica biomass. 3 Standard error of the mean; the value presented corresponds to a pooled sample standard error of the mean. 4 Sum of C18 FA. 5 Sum of saturated FA. 6 Sum of monounsaturated FA. 7 Sum of polyunsaturated FA. In the FA notation (x:n-), 'x' represents the number of C atoms, ':' the number of double bonds and 'n-' the location, in its carbon chain, of the double bond which is closest to the methyl end of the molecule. c stands for cis and t stands for trans. i stands for iso and a stands for anteiso. Disclaimer/Publisher's Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

PMC10000190

The most documented fibrates are gemfibrozil, clofibrate and bezafibrate, while for statins, the majority of the published literature focuses on atorvastatin and simvastatin. The present work reviews previously published research concerning the effects of these hypocholesterolaemic pharmaceuticals on fish, with a particular focus on commercially important species, commonly produced by the European aquaculture industry, specifically in recirculated aquaculture systems (RAS). Overall, results suggest that both acute and chronic exposures to lipid-lowering compounds may have adverse effects on fish, disrupting their capacity to excrete exogenous substances, as well as both lipid metabolism and homeostasis, causing severe ontogenetic and endocrinological abnormalities, leading to hampered reproductive success (e.g., gametogenesis, fecundity), and skeletal or muscular malformations, having serious repercussions on fish health and welfare. Nonetheless, the available literature focusing on the effects of statins or fibrates on commonly farmed fish is still limited, and further research is required to understand the implications of this matter on aquaculture production, global food security and, ultimately, human health. lipid-lowering agents fibrates statins fish gemfibrozil atorvastatin European Commission956481 Plan Nacional de InvestigacionPID2020-113221RB-I00 Ramon y Cajal contractRYC2019-026841-I Agencia Estatal de InvestigacionPID2020-117557RB-C21 This research was funded by the European Commission's Horizon 2020 research and innovation programme under the Marie Sklodowska-Curie (MSCA) grant agreement No 956481 through the RASOPTA Ph.D. fellowship. M.T. was supported through "Plan Nacional de Investigacion" with reference PID2020-113221RB-I00 and a Ramon y Cajal contract with reference RYC2019-026841-I. L.T. was supported by the "Agencia Estatal de Investigacion" with a "Plan Nacional de Investigacion" with reference PID2020-117557RB-C21. pmc1. Introduction The daily use of man-made products such as pharmaceuticals, cleaning agents and plastics is in constant and accelerating growth , and many of these contaminants have been detected in a variety of environments . This uncontrolled anthropogenic activity has triggered several potential risks for aquatic environments since several contaminants have been quantified in considerable amounts both in surface and ground waters, as well as within organisms . Conventional wastewater treatment processes are still unable to efficiently remove all contaminants from influents, and a substantial share of these pollutants, known as contaminants of emerging concern (CECs), remain in the effluent due to their high persistence and low degradation rates . The input of many compounds classified as personal care products (PPCP), flame-retardants, endocrine-disrupting chemicals, polyfluoroalkyl substances and pharmaceuticals from wastewater treatment plants are not monitored in water, potentially subjecting different organisms to chronic exposures to various chemicals . Improvements in analytical techniques allow for the detection of an increasing number of these compounds in a variety of bodies of water (e.g., stagnant and running water; surface and ground waters; salt, fresh and brackish water; and wastewater treatment plant influents, effluents and sludge) , as well as in food items . Pharmaceuticals play a major role in life quality and welfare, but the fast increase in their use has led to significant pressure on natural ecosystems . The misuse of pharmaceuticals, combined with their persistence in the environment, and their proven ability to bioaccumulate on both organisms and sediments , make these compounds, and their respective bioactive metabolites, potential chemical stressors to aquatic and cultured fish, and, consequently, a possible threat to humans. Common aquaculture practices face a number of challenges (e.g., disease outbreaks, feed components shortages, droughts, unexpected flow of contaminants and xenobiotics) and present multiple threats to natural environments, such as eutrophication, habitat destruction and organisms escaping from aquaculture facilities into the wild . To overcome the effects of those stressors on farmed fish, over the past few decades, recirculated aquaculture systems (RAS) have caught the attention of seafood farmers all over the world and have been increasingly developed at an international scale . RAS are based on the concept of reusing or recirculating, over 90% of the production water, depending on the specific design, therefore minimising water exchange with the surrounding environments compared to traditional aquaculture practices . The recirculated water is repeatedly treated with biological, mechanical, and often UV filters in order to maintain appropriate water quality . It is now widely accepted that the implementation of RAS provides a wide range of benefits and that this technology may be increasingly adopted to raise all fish species. Firstly, the considerable reduction in water use allows for RAS to be installed in a variety of environments, including areas where water is scarce . Second, being a virtually closed system, RAS enable the farmers to hold full control of water parameters that could naturally fluctuate in a body of water, such as oxygen levels, the concentration of nitrogenous compounds, temperature, and, eventually, the presence of pathogens . In addition, RAS are usually installed indoors, therefore being sheltered from external factors such as climatic events and the potential predation of cultured organisms from wild animals . Last, but not least, RAS significantly reduce the impact of aquaculture farms on neighbouring natural ecosystems. Indeed, by isolating the facility from the surrounding environments, the probabilities of eutrophication, escapees with potentially deleterious effects on wild populations, pathogen transfer or contamination outflow is minimised . The low rates of water exchange, which directly translate to a considerable reduction in water usage, prevent surrounding rivers or groundwater to be heavily polluted or depleted as a consequence of this activity , and minimise the risk of pathogens entering the systems, translating to a decreased use of antibiotics . These factors, in conjunction with the versatility of RAS regarding the species and life stages suitable for culture in such systems , as well as the different possible stocking and production densities , make RAS an appropriate approach to deal with the growing food demand while minimising the environmental impact of the food production industry . Nonetheless, the array of technicalities incurred by RAS entails farmers developing further skills and expertise and must therefore undertake specific training in order to properly manage the entire system . Furthermore, the different components of RAS, including power supply and sensory systems, are particularly expensive, and consequently, it has been observed that these constraints may restrict the development of these aquaculture practices to areas with greater financial power, such as European countries, North America or some Asian countries . Therefore, further technological advances are required to make RAS truly cost-effective, and to enable its expansion and implementation all over the world . In Europe, RAS farms are principally used to culture aquatic invertebrates (e.g., molluscs and crustaceans), and although the vast majority of farmed fish are salmonids (i.e., Salmo trutta, Oncorhynchus mykiss), other fish such as Anguilla anguilla (European eel) are also widely produced . As pharmaceuticals are now recognised as ubiquitous in aquatic systems , and with treatment plants proving unable to remove these from influents , it can be assumed that these contaminants are also present in RAS and will affect cultured fish, and consequently aquaculture production. Here we will focus on the effects of lipid regulators, a group of pharmaceuticals engineered to treat dyslipidaemias in humans, acting as lipid-lowering agents, and as primary prevention methods for cardiovascular diseases . Lipid regulators are usually manufactured from a variety of compounds such as atorvastatin, bezafibrate, clofibrate, ezetimibe, fenofibrate, fluvastatin, gemfibrozil, lovastatin, mevastatin, pravastatin, rosuvastatin or simvastatin. The first drug of its kind introduced to the market was clofibrate, followed by bezafibrate, fenofibrate and gemfibrozil . Due to the rising need for anti-cholesterol treatments, with earlier-stage treatments and higher dosages , lipid regulators are widely prescribed all around the world, and consequently, comprise some of the most reported pharmaceuticals in drinking and wastewater . The prescription and consumption of lipid regulators multiplied between 2000 and 2017 in countries members of the OECD, with the United Kingdom, Denmark and Belgium displaying the highest consumption rates . Lipid regulators can be sub-classified into statins and fibrates , and, even if both are ubiquitous in water bodies, fibrates are often found at higher concentrations . The present work aims to review the currently available literature on the presence of lipid-regulating agents (i.e., fibrates and statins) in the aquatic environment, and their effect on fish, including those commonly farmed in European RAS. 1.1. Fibrates Fibrates decrease levels of fatty acids, triglycerides and low-density lipoproteins, mainly by stimulating the peroxisomal, and partly the mitochondrial, b-oxidation pathways, lowering blood cholesterol levels . On the other hand, fibrates act as agonists of the peroxisome proliferator-activated receptors alpha (PPAR-a), expressed in the liver, heart and skeletal muscle . In the literature, the most commonly investigated fibrates are gemfibrozil, bezafibrate and clofibric acid, with the latter being the most commonly detected in drinking water and food . The most common lipid regulators in water bodies, namely, atorvastatin, bezafibrate, clofibrate, gemfibrozil and simvastatin, have been described to have similar effects in aquatic organisms, particularly vertebrates, as they do in humans, including the induction of changes in lipid levels . Hence, marine and freshwater organisms are vulnerable to the potentially deleterious effects that the presence of such contaminants might incur, making this an issue of major ecological concern. It is known that exposure to lipid regulators generally present in water (e.g., wastewater treatment effluents and drinking water) can have a range of consequences on fish, affecting physiology, metabolism, reproduction, behaviour and immunity . For instance, previous studies have shown that exposure to gemfibrozil may lead to decreased levels of cholesterol in plasma , and trigger the organism's antioxidant response, as well as tamper with a fish's capacity to efficiently swim in counter-current . Moreover, gemfibrozil, like many other compounds, can bioconcentrate in different tissues in fish , potentially having deleterious effects on human health through the ingestion of contaminated food. Gemfibrozil, is resistant to photodegradation, has a long half-life in water and is not completely removed by wastewater treatment plants (WWTP) . In Spain, for instance, removal rates of this pollutant from WWTP ranged between 10-75% . Its persistence in water allows us to find concentrations up to 4760 ng/L in effluents , and up to 758 ng/L in coastal waters . Moreover, values ranging between 6.69 and 10.34 ng/L were recorded in Portuguese surface waters and up to 0.8 and 1.7 ng/L in Italian tap and surface waters, respectively . In comparison, in Spain, values of up to 70.27 and 77.8 ng/L were detected in different sites of the Llobregat river . Bezafibrate was detected in wastewater treatment facilities effluents in Spain at concentrations ranging between 2 and 132 ng/L, and in estuarine environments at concentrations ranging between 2 and 67 ng/L . In comparison, Portuguese surface waters were contaminated with values of bezafibrate ranging from 11.86 to 15.52 ng/L . Moreover, bezafibrate, gemfibrozil and clofibric acid, an active metabolite of clofibrate, have been detected in groundwater in Barcelona, being found in concentrations of maximum 25.8, 751 and 7.57 ng/L, respectively . Nonetheless, the most predominantly detected fibrate in the aquatic environment is clofibric acid . 1.2. Statins Statins lower cholesterol levels through the suppression of its biosynthesis as a result of competitive inhibition of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoA reductase), which is responsible for cholesterol biosynthesis in the liver . Statins currently available on the market include atorvastatin, cerivastatin, fluvastatin, lovastatin, pitavastatin, pravastatin, rosuvastatin and simvastatin . However, data on the toxicity of statins on different organisms are very limited and are generally restricted to atorvastatin and simvastatin, the two most common statins . Few studies have focused on the environmental concentrations of statins despite the evident increase in their consumption. Statins appear to have more stable removal efficiencies compared to fibrates, but their presence has already been reported in both untreated and treated sewage water samples at values ranging from 4 to 117 ng/L and 1 to 59 ng/L, respectively . Atorvastatin, for example, was found in low amounts, although its metabolites, namely p-hydroxy atorvastatin, and o-hydroxy atorvastatin, were found in greater amounts in wastewater. A similar trend occurs with simvastatin, where this statin was not directly detected in surface water and sediments unlike its specific metabolite (i.e., simvastatin hydroxy carboxylic acid) and was found up to 108 ng/L in Norwegian cities . The inefficiency of wastewater treatment plants when it comes to the removal of these pharmaceuticals is translated by an inevitable exposure of both wild and cultured aquatic animals to these contaminants of emerging concern, even in closed systems, such as recirculated aquaculture systems (RAS), where they might accumulate in water. In both wild and laboratory-reared fishes, the consequences of exposure to fibrates and statins have been studied to varying extents, depending on compound and species, indicating the potentially deleterious effects of these contaminants on the overall health and welfare of fish . Here, we aim to compile existing knowledge on the effects of lipid-regulating agents, their environmental distribution, as well as their effects on fish, since these pharmaceuticals are likely to influence their lipid metabolism and nutritional quality, with a special interest in organisms that are highly relevant to aquaculture, and, therefore, directly linked to global food security and human health. We will also focus specifically on the most documented drugs, namely gemfibrozil, clofibric acid (a metabolite of clofibrate) and bezafibrate, representing the fibrates, and atorvastatin and simvastatin representing the statins. 2. Methodology Articles published from 2011 onwards were selected in July-October 2022, using "lipid regulators", "lipid-lowering agent", gemfibrozil", "clofibrate", "clofibric acid", "bezafibrate", "fibrates", atorvastatin", "simvastatin" and "statins" as keywords. Moreover, the combinations of the aforementioned keywords, in conjunction with "effects in fish", "aquaculture" and both the common and scientific names of the main species of interest for the European aquaculture industry (i.e., Salmo salar, Oncorhynchus mykiss, Anguilla anguilla and Clarias gariepinus) were also used. Due to the scarcity of recent studies focusing on the environmental presence and distribution of these pharmaceuticals in European countries, as well as their effects on the previously specified species of interest to the present work, articles related to these matters were selected, even if the publication was dated prior to 2011. 3. Effects of Lipids Regulators on Fish An increasing number of studies focus on the potentially deleterious effects that the presence of lipid-regulating agents may have on non-target organisms. These suggest that lipid regulators can act similarly in mammals (e.g., humans) and other vertebrates, such as fish, when exposed to these compounds through contaminated waters. This factor, combined with the high use of these drugs, their incorrect discard and their low removal rates, imply a major environmental problem. 3.1. Fibrates 3.1.1. Gemfibrozil A considerable quantity of the published literature has focused on the effects of gemfibrozil in aquatic organisms as it is now an issue of high concern (Table 1). In humans, gemfibrozil is capable of increasing serum aminotransferase, and, in isolated cases, of causing liver damage. The reduction in lipids is associated with the peroxisome proliferator-activated receptor (PPARa), involved in the gene expression of enzymes for the oxidation of fatty acids . In fish, as in other animals, an increase in levels of lipoprotein lipase can occur, subsequently increasing triacyl glyceride-rich lipoproteins . Danio rerio (zebrafish) exposed to gemfibrozil through diet for 30 days displayed decreased plasma levels of triglycerides and overall cholesterol levels . These results corroborate the idea that gemfibrozil can operate in similar ways in fish as in humans, notably generating changes in lipid homeostasis and altering the availability of energy resources. Nonetheless, a study with fathead minnow (Pimephales promelas) showed an inconsistent effect of chronic exposure to gemfibrozil on lipid metabolism, steroidogenesis and reproduction, and no effect when employing environmentally relevant concentrations . In gilthead seabream (Sparus aurata), gemfibrozil was found to affect swimming behaviour , with the ability to swim in counter-current for extended periods of time decreased by up to 65%. These behavioural effects were further associated with a significant increase in both catalase (CAT) and glutathione reductase (GR) activities in the gills of fish exposed to concentrations of the pharmaceuticals . Furthermore, exposure to gemfibrozil appeared to have genotoxic effects on this species, causing nuclear anomalies . In addition, exposure to gemfibrozil did not change gill total antioxidant capacity (TAC) but increased total oxidative status (TOS) and altered mitochondrial RNA of certain genes such as glutathione peroxidase 1 (GPx) and interleukin 1b in both gills and head kidney of the same species . The transcription of key genes involved in lipid homeostasis was also affected by exposure to gemfibrozil, being characterized as a stress-inducing agent. Furthermore, exposure of S. aurata to 15,000 ng/L of gemfibrozil induced the activation of the inflammatory response in the liver . Altogether, these studies suggest that, in S. aurata, this compound alters biochemical and gene functions involved in the immune and oxidative stress responses, having serious implications for the health and welfare of this species. The bioaccumulation of gemfibrozil in both muscle and adipose tissue of juvenile rainbow trout (Oncorhynchus mykiss) following an 8-day waterborne exposure to 3000 ng/L of gemfibrozil has been previously demonstrated using space-resolved solid-phase microextraction (SR-SPME) . Prindiville et al. investigated the effects of exposure to gemfibrozil on the lipoprotein metabolism of O. mykiss. The authors injected female individuals with 100 mg/kg of gemfibrozil every 3 days over a 15-day period and reported a significant decrease in the concentration of plasma lipids, as well as in lipoprotein concentrations. Such results might translate into a hindered ability to reproduce, to efficiently undergo homeoviscous adaption to changes in water temperature, as well as tampered locomotor capacities, impeding long-distance, constant swimming behaviours . Nonetheless, this is mostly speculative work and required additional research to be carried out to confirm these hypotheses. Furthermore, it was reported that exposure to pharmaceutical doses of gemfibrozil significantly reduced the concentration of n-3 fatty acids, which is likely to have direct repercussions on the nutritional quality of the fish . Gagne et al. further corroborated the presence of gemfibrozil in municipal wastewater treatment plants effluent in Canada, once again emphasizing the importance of studying the effects of emergent contaminants on the health of both wild and cultured fishes. However, the authors classified this specific drug as of little-to-no concern to fish, as they observed minimal toxicity of this pharmaceutical in rainbow trout hepatocytes. This is in accordance with previously published findings, also indicating that gemfibrozil had no significant effect on the hepatocytes of O. mykiss . On the other hand, Donohue et al. described a significant increase in bioindicators for oxidative stress on hepatocytes, caused by clofibric acid, the metabolite of clofibrate. Similarly, Triebskorn et al. reported a moderate reaction of the liver of rainbow trout to exposure to clofibric acid, including dilation of blood vessels, and no significant reaction in the kidney of the exposed fish. However, results indicated a more severe response from the gills of O. mykiss to this pollutant, as the authors described epithelial lifting, increased cell production (hyperplasia) and growth (hypertrophy) of mucus cells. Nonetheless, these reactions are non-specific, as they tend to occur when the gills enter into contact with waterborne contaminants and are often regarded as a measure to preserve ion balance . In addition, through an experiment focusing on the exposure of primary hepatocytes of rainbow trout to clofibric acid alone and combined with female sex hormone, it was demonstrated that this pharmaceutical is not a significant PPAR activator in hepatocytes in this species. In addition, neither lipid metabolism nor estrogenic activity resulted altered from this exposure . The available information on the impact that exposure to lipid-lowering compounds has on the European eel (Anguilla anguilla) is extremely limited. According to Lyssimachou et al. , gemfibrozil is not a significant PPARa activator in this species. Nonetheless, injections of gemfibrozil significantly decreased the activity of certain pathways directly linked to cytochrome P450, which are responsible for the metabolism of xenobiotics, as well as the oxidation of fatty acids. This could indicate a tampered ability of the fish to efficiently extract energy from their food source, and to develop and function efficiently , having potentially serious implications for aquaculture production. In zebrafish, chronic exposure to low concentrations of gemfibrozil can have transgenerational effects . In adults, reproductive ability is reportedly altered, accompanied by histological changes in ovaries and kidneys. Furthermore, adult zebrafish chronically exposed to 500 and 10,000 ng/L of this pharmaceutical displayed a decrease in reproductive output, with atretic oocytes and altered kidney histology . On the other hand, unexposed offspring of parents exposed to this contaminant display sex-dependent alterations in courtship activity and swimming behaviour, and abnormal sperm morphology, significantly affecting their fecundity and breeding success , although this seems limited to the first generation of offspring only . Similarly, in a long-term exposure (i.e., 155 days) experiment with Japanese Medaka (Oryzias latipes), a significant decrease in fecundity was observed, tied with significant decreases in both testosterone levels in female fish and hatchability of F1 fish. Moreover, the expression of genes related to oestrogen receptors and vitellogenin was increased in both gonads and livers . This suggests that, in some species, gemfibrozil can act as an endocrine disruptor, affecting reproduction and ontogeny. In male individuals of the same species, short-term exposure to gemfibrozil lowered levels of 17b-estradiol, 11-ketotestosterone and plasma cholesterol . The authors argued that the endocrine disruption observed as a consequence of the challenge might be directly linked to the hypocholesterolaemic nature of the medicine , altering the reproductive success of individuals, and having varying effects depending on the exposure duration. The inconsistency in the published results is likely linked to the dependence of the gemfibrozil's mechanisms of toxicity to exposure duration, administration method, species and life stage, as well as organ analysed. However, it appears as if exposure to gemfibrozil may have strong effects on reproduction (e.g., fecundity, gametogenesis), excretion of exogenous substances (i.e., detoxification process), lipid metabolism and the overall homeostasis. In addition, the presence of this pollutant in aquatic systems is a potential factor affecting larval development, leading to ontogenetic and behavioural anomalies in both adults and larval stages, and, to some extent, in the subsequent generations. 3.1.2. Clofibrate and Clofibric Acid Clofibric acid, the main metabolite of clofibrate, is a PPAR-a agonist that increases beta-oxidation and decreases triglyceride secretion in humans, but it is known to affect aquatic organisms . Even though some of the previous literature investigated the consequences of exposure to clofibrate itself, a major part of the studies hereby considered focused on the effects of clofibric acid (Table 1). In grass carp (Ctenopharyngodon idellal), it was observed that clofibrate decreased triacylglycerol, plasma cholesterol and overall lipid concentrations. Similarly, it increased hepatic enzymes involved in the synthesis of fatty acids, as well as lipoprotein lipase expression. Furthermore, a decrease in lipogenic enzymes, previously increased through feeding with a high carbohydrate diet, was observed . This suggests that clofibrate, like gemfibrozil, can act as a hypolipidemic agent and alter the lipid metabolism of some fishes. In zebrafish, exposure to clofibrate through ingestion leads to a modulation of genes such as fatty acid-binding proteins (fabp) and acyl-CoA oxidase 1 (acox1), a marker of PPAR-a activation in many tissues. Nonetheless, this process proved to be tissue dependent. Clofibrate is potentially unable to cross the blood-brain barrier of zebrafish, hence the lack of significant alterations in the transcription of acox1 in the brain . In zebrafish larvae, the exposed group displayed shorter body lengths compared to the control group, in addition to changes in morphological characteristics and lethargic behaviour. When fed with this compound, an upregulation of peroxisomes in liver and heart mitochondria in was observed , suggesting that waterborne exposure to clofibrate affects both the metabolism of adults and larvae, and may cause strong alterations in the development of embryos, leading to severe deformities and behavioural changes. On the other hand, it has been reported that clofibric acid can cause behavioural and metabolic alterations in adults and larvae of D. rerio both after acute and chronic exposures but does not act as an endocrine disruptor. This drug can influence the enzymatic activities involved in counteracting oxidative stress, like superoxidase dismutase (SOD), CAT and GPx, although the level of alterations depends on the pollutant's concentration. In addition, it has been found that this compound may cause alterations in biotransformation enzymes, as well as in lipid peroxidation levels . Zebrafish embryos have a high potential for metabolizing xenobiotics, involving a variety of enzymatic activities, and generating derivate products such as clofibric acid . It has been previously stated that a lifelong exposure of zebrafish to clofibric acid significantly affected the reproduction, spermatogenesis, growth and gene expression of fabp, in a transgenerational manner . It was determined that the effects are dependent on the individual's sex, and on the organ considered, and can express inter-generational variations, influencing fecundity, mortality and the occurrence of skeletal deformities. With the purpose of investigating key enzymatic activities with involvement in reproduction, an in vitro study in common carp (Cyprinus carpio) showed that clofibrate had an inhibitory effect upon CYP17, related to a synthesis of active androgens on gonads . In an experiment challenging C. carpio the results showed alterations in haematological parameters (decreased red blood cells count and increased white blood cell counts), biochemical endpoints (increased levels of glucose and proteins), ion regulation (decreased plasmatic sodium, increased Na+/K+ ATPase in gills) and on enzymatic responses (i.e., Lactate dehydrogenase, LDH; glutamic-oxaloacetic transaminase, GOT; and glutamate pyruvate transaminase, GPT) . In a different study from the same research group, Cirrhinus mrigala was exposed to the same concentrations resulting in alterations in enzymatic parameters, such as GOT and GPT after short-term exposure, and changes in the ion homeostasis of gills (Na+/K+ ATPase activity) after both long-term exposures to this clofibric acid . On the same species, clofibric acid influenced biochemical parameters, causing an increase in plasmatic glucose and Na+, and K+, as well as a decrease in plasma protein and short-term exposures. On the other hand, long-term exposure to this contaminant lead to increased levels of plasmatic Na+ and oscillations in the levels of plasma glucose, protein and K+ . Through a similar experiment, the authors also reported changes in thyroid hormones in the plasma of C. mrigala . The published literature focusing on the effects of these lipid-regulating agents on S. salar is extremely scarce. Rorvik et al. , briefly described a significant decrease in lipid content in Atlantic salmon following ingestion of a feed complemented with clofibrate when compared to a control group. Overall, these studies suggest, to a certain extent, inter-species variation regarding the effects of clofibric acid. However, the consequences of either chronic or acute exposure to this pharmaceutical often include strong alterations in lipid metabolism and homeostasis, ontogenetic disturbances, and effects on behaviour, haematological parameters and the detoxification process. Furthermore, and similarly to gemfibrozil, clofibric acid has been described as being, to a certain extent, an endocrine disruptor, and as having effects on fish displaying inter-generational and sex-dependent differences. Finally, it appears evident that rising environmental concentrations of this pollutant may have serious implications for the health and welfare of wild fish populations. 3.1.3. Bezafibrate Despite being one of the first lipid regulators to be introduced in the pharmaceutical market, there are not many studies investigating the effects of bezafibrate on aquatic organisms when compared to other fibrates, such as gemfibrozil and clofibrate, or its metabolite, clofibric acid. To the best of the authors' knowledge, the only article available that investigates the effects of this compound reported that exposure to bezafibrate through diet can act like an endocrine disruptor in male zebrafish. This drug can lower plasma cholesterol, decrease 11-ketotestosterone (11-KT), and induce changes at the histological and molecular level in the testis. In addition, the expression of a variety of genes was affected, being either down-regulated depending on exposure time (Table 1). Therefore, bezafibrate affects the gonadal steroidogenesis and spermatogenesis of this organism, consequently having strong implications on the reproduction of the species and affecting it at the population level . 3.1.4. Overall Effects of Fibrates in Fishes It is clear from the literature reviewed that the effects of fibrates in fishes depend strongly upon the still unresolved influence of species-specific physiologies and lifecycles, developmental stage, dosage differences across studies and the amount of time exposed to environmentally relevant concentrations of fibrates. To date, gemfibrozil seems to be the focus of most studies, but overall, the main deleterious effects of fibrates on fish physiology seem to be restricted primarily to alterations in cell and tissue lipid metabolism. This, in turn, may affect the timing and functionality of the reproductive axis and vitellogenesis and may result in abnormalities during the larval development, affecting normal swimming, and perhaps foraging, performance. From the data reviewed, it is not clear if these effects impair the immune responses in adults, but the few reports that indicate alterations in the expression of genes related to inflammation processes suggest that fibrates do not damage long-term immune reactivity. The antioxidant response associated with exposure to fibrates seems, in this sense, mostly related to aerobic metabolic outbursts as a byproduct of lipid oxidative alterations rather than to energy-consuming inflammatory responses. However, this may not be the case in cultured fishes, even in more advanced RAS systems, fed with lipid-enriched formulations, prone to elicit oxidative stress and inflammatory responses . It seems safe to assume that a short-life antioxidant response may dysregulate the metabolic budget during development but not the lifelong (adaptive) performance of adults. However, it is particularly worrying that some of the effects on lipid metabolism can be transgenerational as mentioned in zebrafish studies . Although chronic exposures are hard to reproduce in non-model, free-living species, in fishes, the long-term impact of stressors (including pharmaceuticals, endocrine disruptors and persistent changes in physical variables such as temperature), at the population level seems to rely, partly, on the transgenerational persistence of stress-related disruptive effects in the thyroidal-, estrogenic/ growth-related axis all of them highly sensitive to alterations in the metabolism of lipids. Moreover, sex plasticity in fishes depends on the concerted influences of lipid-, temperature-, stress-responsive gene pathways regulating gonadal maturation and fate . Pharmaceutical-related alterations in the availability and amount of lipid substrates may result in an impairment of fertility output and biases sex ratios. Of special concern are the changes of Na+/K+ ATPase transporters and activity resulting from exposure to fibrates in Cyprinus carpio and Cirrhinus mrigala . In salmonids, these changes have still not been described, but if present, they may interfere with the osmoregulatory adjustments (smoltification) required for the migratory phase of diadromous fishes, in which branchial mitochondrial-rich ionocytes and the management of hepatic lipid stores determine the onset of smoltification , a complex process still understudied. In Atlantic salmon, prior to sea migration, lipid metabolism becomes less plastic to changes in lipid diets as demonstrated by the variations in the expression of genes linked to lipid uptake in the gut and hepatic lipogenesis and lipid transport . These shifts in the metabolism of lipids are life-stage dependent, and constrict the growth rate that, in turn, acts as a trigger to undergo, or not, smoltification. In this sense, it would be worth studying the effects that exposure to fibrates during phases of major osmoregulatory and morphological changes may have on the obligate or facultative development of smoltification in migrant salmonids, a process that requires behavioural changes and modifications of swimming performance that may be influenced by the presence of gemfibrozil in the brain circuitry . 3.2. Statins Statins inhibit an enzyme called 3-hydroxy-3-methylglutaryl CoA reductase (HMGCR) that converts 3-hydroxy-3-methylglutaryl-CoA (HMG CoA) into the cholesterol precursor mevalonate. Thus, statins decrease cholesterol synthesis as they compete with HMG CoA for the active site of the enzyme, altering its conformation and inhibiting its function . Although waterborne statins are expected to be bioavailable to fish due to their high input rate and persistence, there are indications that they will not bioaccumulate in organisms . In addition to uptake via passive diffusion through membranes, statins may be taken up and excreted by membrane transporters. The activity and expression of membrane transporters in the gills of aquatic organisms may therefore influence their bioavailability by affecting their influx and/or efflux rates . Therefore, the effects of this contaminant on an aquatic organism may vary greatly between species, or life stages within the same species (Table 2), but might be less severe than those observed for fibrates. 3.2.1. Atorvastatin The prescription rate of statins continues to increase, mainly with the high sale of atorvastatin, often under the brand name "Lipitor" , representing a pioneering treatment for hypercholesterolemia but increasing the concentration of this pollutant in aquatic systems. Exposure to atorvastatin in rainbow trout was reported to alter genes involved in membrane transport, oxidative stress response, apoptosis and metabolism in gills at low concentrations. However, no effect was observed on genes involved in cholesterol biosynthesis or peroxisomal proliferation . In contrast, an experiment focusing on primary rainbow trout hepatocytes showed that this environmental pollutant may alter lipid synthesis and reduce cholesterol biosynthesis . In zebrafish, exposure to this emergent contaminant was directly linked to alterations in cholesterol metabolism, lipid regulation, and steroid production. Specifically, an observed reduction in cholesterol was associated with decreased levels of cortisol and sex steroids. Moreover, a reduction in triglyceride was associated with changes in mRNA levels involved with lipid regulation . Additionally, exposure to this compound resulted in signs of skeletal muscle breakdown, indicating rhabdomyolysis, and strongly tampering with the welfare of male fish. Interestingly, this effect on skeletal muscle seemed to be gender-specific, as different response between male and female was observed . Besides the effects observed on adult individuals, a strong, dose-dependent, myotoxic effect of this drug was reported in zebrafish larvae . In addition, exposure to atorvastatin significantly reduced the response to tactile stimuli, and caused a decrease in enzyme activity, like citrate synthase (CS) and cytochrome oxidase (COX). This suggests involvement in metabolism in larvae, thus, affecting the early development of the fish, and, ultimately, success in adults. . The effects of atorvastatin seem somewhat similar to those described for fibrates, with important implications for an individual's development, and lipid, as well as overall metabolism, also acting as an endocrine disruptor. 3.2.2. Simvastatin As with the other lipid-regulating agents mentioned before, exposure to simvastatin has been reported to have a variety of consequences in fish. For instance, exposure of fewer than 7 days was able to cause significant alterations in the antioxidant system of mosquitofish (Gambusia affinis) and related enzymatic activity, such as nuclear factor erythroid 2-related factor 2 (Nrf2) and mitogen-activated protein kinases (MAPK) . Furthermore, the presence of this contaminant in aquatic environments is reportedly responsible for strong histological changes in hepatic cells of the same species . Similarly, histological changes were reported to occur in Mugilogobius abei following short-term exposure to this statin. Furthermore, increased expression of genes related to xenobiotic resistance and detoxification (pregnane X receptor, PXR), including cytochromes P450 (i.e., CYP1A, CYP3A), has been shown to be somewhat induced by short-term exposure to this pollutant, concomitantly with alterations in enzymatic activity . In zebrafish, it has been reported that chronic exposure to simvastatin at environmentally relevant concentrations, may have, in certain parameters, both dose-dependent, and sex-dependent consequences . Indeed, following a 90-day waterborne exposure to this contaminant, the authors observed significant differences in the expression of genes responsible for energy metabolism (e.g., fatty acids synthesis and metabolism, glucose metabolism) in the brain . Further analyses performed during the same experiment indicated significant alterations in the mevalonate pathway (e.g., directly involved in the synthesis of cholesterol), as well as a sex-dependent variation in body weight and body length at the highest exposure concentrations. However, a significant increase in reproductive success (fertilisation %) was observed at 200 ng/L, although this was followed by an increase in developmental anomalies. In addition, chronic exposure to simvastatin resulted in significant changes in cholesterol and triglyceride levels, although this response was not dose dependent. Moreover, the authors described a significant decrease in the heartbeat rate of embryos following parental exposure to 8 ng/L of this statin . This agrees with what was previously described by Campos et al. , who also reported significantly slower heartbeat in zebrafish larvae as a consequence of exposure to simvastatin. Additionally, the authors reported decreased movement and significant structural alterations (e.g., changes in septum shape and size of somites, essential for locomotion in fish). The authors further speculate that exposure to this lipid-regulator may directly affect DNA replication, apoptosis, and muscle function . Zebrafish embryos exposed to high concentrations of simvastatin experienced important mortality, and sever morphological anomalies, compared to low concentrations, which induced barely any mortality, and no morphological alterations . In addition, it has been observed that exposure to this compound caused strong developmental issues, leading to abnormalities in the septum, somites and myofibrils and most likely tampering with the individual's ability to swim, further corroborating previous findings . On the other hand, Cunha et al. described no significant effects of simvastatin exposure on cholesterol levels of D. rerio larvae, although the experimental methods differed with regard to the developmental stages challenged, and the concentrations tested, potentially indicating dose-dependent differences in the effects of simvastatin. Furthermore, the authors reported that exposure to this lipid-lowering agent may cause either down-regulation of nuclear receptors (i.e., PPAR; PXR; constitutive androstane receptor, CAR; retinoid X receptors, RXR), as well as in aryl hydrocarbon receptor (AhR) in zebrafish, depending on the dose of simvastatin, and the exposure duration . In that regard, the authors had previously reported that waterborne exposure to simvastatin could cause severe developmental abnormalities in zebrafish embryos, particularly tail deformities . Moreover, the authors detected significant upregulation of ethoxyresorufin-O-deethylase (EROD), glutathione S-transferases (GST) and CAT as well as different ATP-binding cassette (ABC) transporters and cytochrome P450, indicating the induction of the cell detoxification process, and the related antioxidant procedure. Furthermore, the authors demonstrated the potential for simvastatin to interact with other chemical environmental pollutants, possibly having synergistic effects . In contrast, Rebelo et al. observed a significant decrease in SOD, GST and GPx, as well as thiobarbituric acid reactive substances (TBARS), in zebrafish embryos and juveniles following both acute and chronic exposures to simvastatin, indicating a reduction in antioxidant defences. In addition, significant behavioural changes were detected throughout these experiments, but no alterations in gonad development or sex determination were noted. 3.2.3. Overall Effects of Statins in Fishes The caveats mentioned above for fibrates are equally relevant for the effects of statins in the physiology and behaviour of fishes, with an amendment: statins are even less studied and in fewer species. Due to the pleiotropic nature of the effects of statins , some unrelated to their ability to reduce cholesterol levels, it is hard to say if the intensity and scope of statins' effects in fish are comparable to those described for fibrates beyond changes in lipid metabolism, abnormal development in larvae and oxidative stress. Either way, even conflicting results seem to indicate no major effects on long-term inflammatory outcomes. However, unexpected systemic alterations related to interferences with metabolic and mitochondrial oxidative pathways should not be ruled out. In mammals, statins have been linked to dose-dependent alterations in angiogenesis , and in this sense it would be worth further analysing their putative myotoxic effects during cardiovascular development in fishes , widening the focus beyond the classical zebrafish ecotoxicology resources to cover non-model species. 4. Concluding Remarks The manufacturing and use of lipid-regulating agents are constantly growing, and many of these compounds (e.g., statins and fibrates) are ubiquitous in aquatic systems. However, limited information is available on the effects of lipid-regulating agents on the most common species of fish in European RAS, with most research having focused on O. mykiss. Nonetheless, the published literature offers a worrying insight into the implications of having aquaculture fishes exposed to such hypocholesterolaemic agents. Indeed, it has been reported that fish might be continuously exposed to these contaminants, and that, although not all these compounds are particularly likely to bioaccumulate, prolonged contact may potentially lead to modified swimming, feeding and social behaviour in these organisms. Furthermore, it has been suggested that lipid-lowering agents may tamper with fish's ability to reproduce and develop properly, causing deformities if exposed during early life stages, and therefore, greatly affecting the welfare of farmed animals. In addition, preliminary findings indicate that the nutritional quality of cultured fish may be significantly decreased by these contaminants of emerging concern, which may have serious implications for food security, as fish represents a considerable proportion of protein sources worldwide. The low-level activation of inflammatory responses in fishes exposed to fibrates is intriguing and deserves more attention, considering the mutual influence between lipid and immune tissues . Some regulators of key metabolic pathways, such as the mTOR anabolic signalling pathway, participate not only in the hepatic balance between lipolysis and lipogenesis, but also regulate the onset of inflammatory responses . These pathways, together with the lipid tissue-dependent regulation of local immune responses in fishes enforce the crosstalk between visceral adipose masses and immune cell trafficking and antigen presentation in fishes, similar to what has been observed in mammals. More chronic studies are needed to ascertain the effects of lipid-regulating agents at the population level in free-living fishes, including those that, such as salmonids, have experienced several rounds of genome duplication, gained a duplicated set of genes related to lipid regulation and management, and, thus, may be more resilient to pharmaceutical-derived metabolic alterations . Seasonal trends in lipid allocation for vitellogenesis and gonadal maturation, coupled with transgenerational alterations due to chronic exposure to environmental stressors have been described in three-spined sticklebacks (Gasterosteus aculeatus) in an attempt to dissect the complexity of reproductive allocation of resources during the breeding season . These, and other species with context-dependent lifecycles deserve more studies concerning the synergistic or antagonistic effects of lipid regulators, acting alone or coupled with other stressors, on normal and maladaptive physiology of fishes. In this sense, the activation of the hypothalamic-pituitary-interrenal (HPI) stress axis in fishes responds not only to environmental cues but also to the presence of pharmaceuticals, xenobiotics and other contaminants that may bias the effect of HPI byproducts on the sex plasticity pathways , compromising the maintenance of healthy populations. All these factors suggest that, if the environmental concentrations of statins and fibrates continue to rise, the effects on both wild and cultured organisms will intensify. Therefore, it appears that both the health and welfare of fishes in aquaculture systems will be at risk. Nonetheless, the effects of lipid-regulating agents seem to be somewhat dependent on a variety of factors (e.g., dose, exposure time, sex, and life stage). Thus, although the available literature allows, to a certain degree, for extrapolation of results on farmed species, additional research, with a particular focus on commercially important species (e.g., cultured in RAS), should be carried out to fully understand the implications for aquaculture production and, ultimately, global food security. Author Contributions Conceptualization, M.B., J.L. and M.T.; methodology, M.B. and J.L.; validation, L.T. and M.T.; investigation, M.B. and J.L.; writing--original draft preparation, M.B. and J.L.; writing--review and editing, all authors; visualization, J.C.B. and C.G.; supervision, M.T. and L.T.; project administration, M.T. and L.T.; funding acquisition, M.T., L.T., M.B. and J.L. declare equal contributions to all aspects of the present work. All authors have read and agreed to the published version of the manuscript. Institutional Review Board Statement Not applicable. Informed Consent Statement Not applicable. Data Availability Statement Data sharing not applicable. No new data were created or analyzed in this study. Conflicts of Interest The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results. Figure 1 Putative effects of fibrates and statins in fish. Scheme of the effects of acute or chronic exposure to lipid regulators (see text for details) on major organs and tissues of fish. animals-13-00792-t001_Table 1 Table 1 The published literature on fibrates (gemfibrozil, clofibrate, clofibric acid and bezafibrate) and their main effects on fish. Species Drug Exposure Period Concentration Exposure Route Sample Type Main Effects Reference (2011-2022) Oncorhynchus mykiss GEM 15 d 100 mg/kg Injection (IP) Plasma, liver, adipose tissue, red muscle and white muscle | lipoprotein concentration | n-3 fatty acids Pimephales mykiss GEM 2, 8 and 21 d 1, 5, 15, 600, 1500 mg/L Waterborne Blood, liver, gonads, brain and pituitary | plasma cholesterol at 600 mg/L in both sexes | plasma Chol at 1500 mg/L in males | ppara after 8 d at 600 mg/L in males | lpl after 2 d at 600 mg/L in males | ldlr, fasn, apoeb, apoa1 in males | fasn, apoeb in females | apoa1 at 2 d in females | apoa1 at 21 d in females Danio rerio GEM 6 w 0.5 and 10 mg/L Waterborne Plasma, gonads and whole body | embryo viability | survival in exposed embryos from non-exposed parents | yolk-sac oedema | skeletal deformities | histopathological score | regressive changes in kidney tubule D. rerio GEM 6 w 0.5 and 10 mg/L Waterborne Plasma, gonads and whole body | embryo production | embryo mortality after direct exposure | histopathological score | regressive changes in kidney tubule | regressive changes in liver | irregular oocytes in females Anguilla anguilla GEM 24 and 96 h 0.1, 1, 10, 2, 20, 200 mg/g BW Injection (IP) Blood and liver | body weight | EROD activity (CYP1A-like) | BFCOD activity (CYP3A-like) | CYP2K-like activity | Hepatic AOX and CAT D. rerio GEM 6 w 0, 10 g/L Waterborne Embryos | breeding success | courtship time | courtship frequency | sperm size | sperm speed Solea senegalensis GEM 2 d 1 mg/kg BW Injection (IP) Blood and liver |CYP450-related activities | UDPGT | hepatic CAT, GPx and GST. D. rerio GEM 30 d 16 mg/g BW Diet Liver and brain | Chol | TG, and E | T in females | PPARa in females | PPARU | liver SREBP-2, CYP3A65, atrogin1 Sparus aurata GEM 96 h 0, 1.5, 15, 150, 1500 and 15,000 mg/L Waterborne Liver and blood | APOA1, LPL | IL-1b, TNF-a, and CASP3 genes at 15 mg/L. | cortisol D. rerio GEM 120 and 144 h 0, 1.5, 3 and 6 mg Waterborne Embryos | embryo and larvae survival | yolk-sac deformities and oedema | locomotion S. aurata GEM 96 h 1.5, 15, 150, 1500 and 15,000 mg/L Waterborne Blood | DNA damage |cortisol at 1.5 mg L-1 D. rerio GEM 42 d/67 d 0 and 10 mg/L Waterborne Blood, gonads, and whole bodies | reproductive output | whole body, plasma and testicular 11-KT D. rerio GEM 6 w 0 or 10 mg/L Cell culture Gonads | sexual differentiation in F1 offspring depending on which parent was exposed S. aurata GEM 96 h 0, 1.5, 15, 150, 1500 and 15,000 mg/L Waterborne Gills and head kidney | TOS, GPx1, and IL1b in gills and head kidney S. aurata GEM 96 h 1.5, 15, 150, 1500 and 15,000 mg/L Waterborne Liver, gills, brain and muscle | locomotion capacity. | CAT, GR, GPx | TBARS | LPO | activity of enzymes of antioxidant defence activity (15-15,000 mg L-1). D. rerio GEM 6 w 10 mg/L Water Sperm, whole fish, blood and organs | embryo production | 11-KT | courtship duration, | sperm size | sperm speed effects in unexposed F1, but not subsequent generations Oryzias latipes GEM 155 d (embryos) and 1 d (adults) 0, 0.04, 0.4, 3.7 and 40 mg/L Waterborne Blood, liver and gonads | T at long-term exposure | Hatching rate in F1 | ER and VTG | E2 and 11-KT at short-term exposure | Chol in males Cyprinus carpio GEM, CLO and CA - 1 mM In vitro Gonads GEM and CLO: | CYP11b CA: | CYP11b CLO: | CYP17 D. rerio CLO 4 w 0, 0.25, 0.50, 0.75 and 1.00% of the diet Diet Liver, intestine, muscle, brain and heart | FABP1, FABP7, FABP10, FABP11 | acox1 | peroxisomes (liver) and mitochondria (heart) Ctenopharyngodon idellal CLO 4 w 0, 1.25 g/kg Diet Liver, muscle and mesenteric fat | TG, cholesterol and total lipid concentration | hepatic ACO and LPL | in FAS and ACC C. carpio CA 96 h 1, 10 and 100 mg/L Waterborne Blood | RBC, plasma Na+, K+ and GOT | WBC, glucose, protein, LDH enzyme and gill Na+/K+ ATPase Cirrhinus mrigala CA 96 h and 35 d 1, 10 and 100 mg/L Waterborne Blood | plasma GOT, GPT and gill Na+/K+ ATPase at short-term exposure | gill Na+/K+ ATPase at long-term exposure C. mrigala CA 96 h and 35 d 1, 10 and 100 mg/L Waterborne Blood | plasmatic glucose, Na+ and K+ level at short-term exposure | plasma protein and Cl levels | plasma Na+ and Cl levels at long-term exposure C. mrigala CA 96 h and 35 d 1, 10 and 100 mg/L Waterborne Blood | TSH | T4 level at 1 and 100 g L-l at 96 h | T4 at long-term exposure | T3 D. rerio CA 5 to 140 d 1 and 10 mg/g Diet Muscle, gonads and liver |, growth of F1 generation, TG in muscle and fecundity at high doses | embryo's abnormalities | PPARa, PPARb and ACO transcript of F1 | weight of F2 generation (control diet) compared to F1 at low dose C. carpio CA 4 and 10 d 20 mg/L and 4 mg/L Waterborne Blood | PPAR |Acox1 | CYP4 and lpl, apoa1 | CYP27A | CYP2K, CYP3A, GSTP, MDR1, MRP2 D. rerio CA 3, 6, 24, 48, 72 and 96 h 50 mg/L Waterborne Embryos embryos present high metabolic potential to CA, with 18 transformation products detected D. rerio CA 120 h and 60 d 10.35, 20.7, 41.4, 82.8, and 165.6 mg/L Waterborne Eggs, embryos and adults | SOD, GPx and LPO at high doses. | CAT and GST at low dose | CAT and GST at high dose |LPO at low dose | total distance travelled and swimming time D. rerio BEZ 48 h, 7 and 21 d 1.7, 33 and 70 mg/g Diet Blood and gonads | Chol | 11-KT after 21 d | in PPARb and PPARU after 48 h | PPARU PPARb, StAR and CYP17A1 at 70 mg/g after 21 days. | CYP19A1a | cystic spermatocytes Abbreviations: 11-ketotestosterone (11-KT); Acetyl coenzyme A carboxylase (ACC); Acyl-CoA oxidase activity (ACO, Acox); Apolipoprotein AI (APOA1); Bezafibrate (BEZ); Chloride (Cl); Clofibrate (CLO); Clofibric acid (CA); Days (d); Estrogen receptor (ER); Gemfibrozil (GEM); Glutamate oxaloacetate transaminase (GOT); Glutathione peroxidase 1 (GPx1); Glutathione S-transferase P (GSTP); Hours (h); Interleukin 1b (IL1b); Intraperitoneal Injection (IP); Lactate dehydrogenase (LDH); Lipoprotein lipase (LPL); Potassium (K+); Red blood cell (RBC); Sodium (Na+); Testosterone (T); Thyroid stimulating hormone (TSH); Thyroxine (T4); Triiodothyronine (T3); Vitellogenin (VTG); Weeks (w); White blood cell (WBC); Peroxisomal proliferation-associated receptor (PPAR); Peroxisomal proliferation-associated receptor a (PPARa); Peroxisomal proliferation-associated receptor U (PPARU); Peroxisomal proliferation-associated receptor b (PPARb) Glutamate oxaloacetate transaminase (GOT); Glutathione peroxidase (GPx); Farnesyl diphosphate synthase (FDPS); Superoxide dismutase (SOD); Sulphotransferase (sult2b); Glucose transporters (glut1b); Catalase (CAT); UDP-glucuronosyl transferase (UDPGT); Tumor necrosis factor a (TNFa); Steroidogenic Acute Regulatory Protein (StAR); Total oxidant status (TOS); Lactate dehydrogenase (LDH); Glutamate-pyruvate transaminase (GPT); Alternative oxidase (AOX); Cytochrome P450 (CYP450); Cytochrome P450, family 11, subfamily B (CYP11B); Cytochrome P450, family 17, subfamily A, polypeptide 1 (CYP17A1); Cytochrome P450, 1A (CYP1A); Cytochrome P450, 3A (CYP3A); Cytochrome P450, 2K (CYP2K); Cytochrome P450, family 3, subfamily A, polypeptide 65 (CYP3A65); Cytochrome P450, family 4 (CYP4); Cytochrome P450, family 27, subfamily A (CYP27A); Cytochrome P450, family 19, subfamily A, polypeptide 1a (CYP19A1a); 17b-estradiol (E2); Estradiol (E); Triglycerides (TG); sterol regulatory element-binding protein 2 (SREBP2); Caspase 3 (CASP3); Glutathione reductase (GR); Multidrug Resistance Mutation 1 (MDR1); Multidrug Resistance-Associated Protein 2 (MRP2); and Cholesterol (Chol). animals-13-00792-t002_Table 2 Table 2 The published literature on statins (atorvastatin and simvastatin) and their main effects on fish. Species Drug Exposure Period Concentration Exposure Route Samples Type Main Effects Reference (2012-2022) Oncorhynchus mykiss ATV 7 d 200 ng/L and 10 mg/L Waterborne Gills and liver | SOD, MT, BAX, P-gp, MRP1 and SULT2b in gill at low concentration Danio rerio ATV 30 d 16 mg/g BW Diet Liver and brain | cortisol, Chol, T, E, and atrogin1 | TG and CYP3A65 in males | TG in females | PPARa in females | PPARU, SREBP1, SREBP2, HMGCR1 and HMGCR2 D. rerio ATV 120 h 0.045, 0.5, 1 mg/L Waterborne Larvae | mortality | spontaneous movement and response to physical stimuli | COX, CS and LDH | atrogen-1 at 0.045 mg/L and 1 mg/L | atrogen-1 at 0.5 mg/L | MURF | pgc-la at 0.045 mg/L | pgc-la at 0.5 mg/L and 1 mg/L O. mykiss ATV 3 or 6 h 45 ng mL-1 and 80 nM In vitro Hepatocytes | HMGCR1, LDLR, PPARa, PPARU, and SREBP1 (lipid metabolism) | CYP3A27 (xenobiotic metabolism) | Chol D. rerio SIM 18 or 13 h 0.3, 3 and 6 nM, 0.375, 0.5 and 0.75 mM Waterborne Embryos | mortality | movement and heart rate uneven distribution of septa | myofibril size | somite size D. rerio SIM 18 or 13 h 0.3, 3 and 6 nM, 0.375, 0.5 and 0.75 mM Waterborne Embryos | mortality | chol | yolk extension, tail length, somite length, septa angle | swimming capacity | pericardial oedema | heartbeat | total cell number D. rerio SIM 80 h 5 and 50 mg/L Waterborne Embryos | mortality, tail abnormalities, developmental delays, pericardium oedema | ABC proteins | EROD and GST | Cu/ZnSOD and CAT | accumulation | ABCB4, ABCC1, ABCC2, ABCG2A, CYP3A65, CYP1A1 and CAT at 50mg/l | GST, SOD and CAT at 5 mg/l D. rerio SIM 2 and 80 h 5 or 50 mg/L Waterborne Embryos | mortality | PPARa, PPARb, PPARU, PXR, AhR, RAR-a, RAR-b, RAG-a after 2 h exposure | rxraa, rxrab, rxrbb, rxrga, rxrgb after 2 h exposure | RAR-ab, and AhR. After 80 h exposure | PXR and rxrgb after 80 h exposure D. rerio SIM 90 d 8, 40, 200 and 1000 ng/L Waterborne Liver | body weight | fertilization rate | abnormalities (short tail, pericardium oedema, yolk-sac and eye deformities) | heartbeat after parental exposure | Chol and TG | HMGCRA | CYP51 in males Gambusia affinis SIM 24, 72 and 168 h 0.5, 5, 50 and 500 mg/L Waterborne Liver | PXR, CYP3A, P-gp, MRP2, UGT | PXR and MRP2 at high doses | ERND (72 h) | ERND (24) | hepatocyte abnormalities at 5 mg/L1 (168 h) Mugilogobius abei SIM 72 h 0.5, 5, 50 and 500 mg/L Waterborne Liver | P-gp, CYP1A, CYP3A, a-GST and PXR genes | miR-148a at 50 mg/L | miR-34c at 0.5mg/L (24 h) | miR-34b and miR-34c at 0.5mg/L, 5mg/L, and 500mg/L (72 h) | miR-34a at 500mg/L | miR-27b at low doses | miR-27b at high doses | miR-27a | ERND, T-SOD, CAT, GPX, MDA | GSH | hepatic vacuole diameter D. rerio SIM 90 d 8, 40, 200 and 1000 ng/L Waterborne Brain | glut1b and COX4I1 in females | glut1b and COX4I1 in males | GAPDH | ACADM and COX5aa in females | ACADM in males G. affinis SIM 3 or 7 d 0.5, 5, 50 and 500 mg/L Waterborne Liver | Nrf2 | SOD2, CAT and GST | GSTA (168 h) | GCLC and GSTA | NQO1 | GSH | GSH at 5 mg/L (24 h) | MDA | MDA (24 h) | ERK | JNK at low doses | JNK at high doses | histological changes in hepatic tissue (endoplasmic reticulum and mitochondria anomalies) D. rerio SIM 120 h and 60 d 92.45, 184.9, 369.8, 739.6 and 1479.2 ng/L Waterborne Embryos | and | locomotion (erratic vs. purposeful swimming, distance travelled and time) depending on dose and day/night phase | Cu-ZnSOD | GPx, GST, TBARS Abbreviations: Atorvastatin (ATV); Simvastatin (SIM); Superoxide Dismutase (SOD); Metallothionein (MT), bcl-2 associated X protein (BAX), P-glycoprotein (P-gp), multidrug resistance protein 1 (MRP1), Sulfotransferase Family 2B (SULT2b); cholesterol (Chol), testosterone (T); estrogen (E); triglycerides (TG); cytochrome P450, family 3, subfamily A, polypeptide 65 (CYP3A65); cytochrome P450, family 3, subfamily A (CYP3A); cytochrome P450, family 3, subfamily A, polypeptide 27 (CYP3A27); cytochrome P450, family 1, subfamily A, polypeptide 1 (CYP1A); cytochrome P450, family 1, subfamily A, polypeptide 1 (CYP1A1); cytochrome P450, family 51 (CYP51); Peroxisome proliferator-activated receptor alpha (PPARa); Peroxisome proliferator-activated receptor Beta (PPARb) peroxisome proliferator-activated receptor gamma (PPARU); sterol regulatory element-binding protein 1 (SREBP1); sterol regulatory element-binding protein 2 (SREBP2); 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase 1 (HMGCR1); 3-hydroxy-3-methyl-glutaryl-coenzyme A (HMGCRA); 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase 2 (HMGCR2); cytochrome oxidase (COX); citrate synthase (CS); lactate dehydrogenase (LDH); muscle RING-finer (MURF); peroxisome proliferator-activated receptor gamma coactivator 1a (pgc-1a); low-density lipoprotein receptor (LDLR); ATP-binding cassette transporter (ABC); ATP-binding cassette, subfamily B, member 4 (ABCB4); ATP-binding cassette, subfamily C, member 1 (ABCC1); ATP-binding cassette, subfamily C, member 2 (ABCC2); ATP-binding cassette, subfamily G, member 2A (ABCG2A); ethoxyresorufin-O-deethylase (EROD); glutathione S-transferases (GST); Copper (Cu); Zinc (Zn); catalase (CAT); pregnane X receptor (PXR); aryl hydrocarbon receptor (AhR); retinoic acid receptor alpha (RAR-a); retinoic acid receptor Beta (RAR-b); retinoic acid receptor alpha b (RAR-ab); recombination activating gene alpha (RAG-a); retinoid x receptor alpha a (rxraa); retinoid x receptor alpha b (rxrab); retinoid x receptor beta b (rxrbb); retinoid x receptor gamma a (rxrga); retinoid x receptor gamma b (rxrgb); multidrug resistance-associated protein 2 (MRP2); uridine 5'-diphospho-glucuronosyltransferase (UGT); Erythromycin-N-Demethylase (ERND); microRNA 148a (miR-148a); microRNA 148a (miR-148a); microRNA 34a (miR-34a); microRNA 34c (miR-34c); microRNA 34b (miR-34b); microRNA 27a (miR-27a); microRNA 27b (miR-27b); malondialdehyde (MDA); glutathione (GSH); Glutathione peroxidase (GPx); glucose transporter 1b (glut1b); cytochrome c oxidase subunits 4I1 (COX4I1); cytochrome c oxidase subunits 5aa (COX5aa); glyceraldehyde 3-phosphate dehydrogenase (GAPDH); medium-chain acyl-CoA dehydrogenase (ACADM); NF-E2-related factor 2 protein (Nrf2); superoxide dismutase 2 (SOD2); Alpha-glutathione S-transferase (GSTA); glutamate cysteine ligase catalytic subunit (GCLC); NAD(P)H quinone dehydrogenase 1 (NQO1); extracellular regulated protein kinase (ERK); c-Jun N-terminal kinase (JNK); and Thiobarbituric acid reactive substances (TBARS). 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PMC10000191

This study aims to investigate the effect of neem leaf supplementation on the feed intake, digestibility, performance, fermentation characteristics, and ruminal microbes in goats. We included 24 Anglo-Nubian Thai native male goats with a body weight of 20 +- 2.0 kg, using 2 x 2 factorial in a completely randomized design for the following four treatments: (1) control, (2) control + 15% PEG in the concentrate, (3) 6% NL in concentrate, and (4) 6% NL + 15% PEG in concentrate. The results show that supplementation with 6% NL + 15% PEG in the concentrate had a higher (p < 0.05) feed intake gDM/d, % BW, g/kgBW0.75, nutrient intake, nutrient digestion, weight change, and ADG than did the goats that were fed with 0% NL + 0% PEG, 0% NL + 15% PEG, and 6% NL + 0% PEG in concentrate, respectively. The feeding with 6% NL + 15% PEG had a higher (p < 0.05) level of propionic acid at 2 and 4 h post feeding compared to the other treatments. Supplementation with 6% NL + 15% PEG in the concentrate had the lowest (p < 0.05) methanogen, protozoa, blood urea nitrogen, ammonia nitrogen, acetic acid, and butyric acid, as well as a lower ratio of acetic acid to propionic acid at 2 and 4 h post feeding than the other treatments. However, supplementation with 6% NL + 15% PEG in concentrate had the highest values of Butyrivibrio fibrisolvens and Streptococcus gallolyticus at 2 and 4 h post feeding compared to the other treatments (p < 0.05). Collectively, this study indicates that neem leaf supplements can increase growth performance and propionic acid and can modulate the abundance of Butyrivibrio fibrisolvens and Streptococcus gallolyticus. Thus, neem leaf could potentially be a good supplement for goat feed. neem leaf polyethylene glycol digestibility growth performance rumen fermentation ruminal microbial population Suranaree University of Technology scholarship for External Grants and Scholarships for Graduate StudentsSUT-OROG scholarship Thailand Science Research and Innovation (TSRI)National Research and Innovation FundFF3-303-65-36-17 This research was funded by (i) Suranaree University of Technology scholarship for External Grants and Scholarships for Graduate Students (SUT-OROG scholarship), (ii) Thailand Science Research and Innovation (TSRI), (iii) National Research and Innovation Fund (No. FF3-303-65-36-17). pmc1. Introduction Animal nutritionists are interested in improving ruminant feed efficiency by modifying rumen fermentation. Feed additives that influence rumen fermentation improve feed utilization . Goat production in Thailand is increasing due to the rising demand for goat products as a result of human population growth . Goats are small ruminants adapted well to the environment. They are resistant to dry weather and heat. Neem (Azadirachta indica) is a tropical tree that grows in Thailand and India. The leaf extract is used as an antibacterial and bacteriostatic compound. It promotes digestibility and metabolism of proteins in ruminants. Neem leaves have been found to be a rich sources of secondary plant compounds, including alkaloids, flavonoids, polyphenolic components, and condensed tannins (CT). Moreover, they might have antimicrobial properties, particularly toward protozoal and methanogen populations. Neem leaf may prevent protein breakdown in the rumen and increase amino acid absorption in the small intestine. Animal feed containing protein-tannin compounds can increase protein production by microorganisms in goats. Tannins can decrease the bacterial population in the rumen, resulting in a decrease in the protozoa population, which could be caused by a decrease in cell membrane permeability. The effect of tannin supplementation on the methanogen population to help minimize methane permeability has been researched . Tannins have a major impact in that they decrease protein-producing bacteria, including Butyrivibrio fibrisolvens, Ruminobacter amylophilus, and Streptococcus bovis . Tannins directly inhibit methanogenic activity in the rumen by absorbing the microorganisms in the cell wall, but they also contribute to the decrease in cellulolytic bacteria in complex cellulose tannins. Fibrolytic microorganisms have a surface adhesion defect that reduces the availability of hydrogen, thereby reducing methanogens . Polyethylene glycol (PEG) and polyethylene supplementation glycol are polymers capable of adhering to tannins and reducing the aggregation of protein-tannin complexes. Animals can more rapidly absorb tannins and use their ability to allow the proteins to pass into the small intestine . However, the tannin and CT in neem leaves have not yet been studied for use in goats. As a result, this study was performed in order to test the following hypothesis: neem leaf feed supplements can be used in goat diets as a medicinal herb feed additive with no negative effects on dry matter, nutritional component intake, or apparent digestibility. This research also examines whether combining neem leaf with polyethylene glycol can improve feed intake, digestibility, performance, fermentation characteristics, and ruminal microbes in goats. 2. Materials and Methods 2.1. Nutrition Management All experimental procedures used in this study were approved by the Animal Ethics Committee of Suranaree University of Technology (SUT 4/2558). All the animal experiments were performed at the Suranaree University of Technology (SUT) goat and sheep research farm, Nakhon Ratchasima, Thailand. In all, 24 Anglo-Nubian male goats with a body weight (BW) of 20 +- 2.0 kg were assigned in a 2 x 2 factorial in CRD. This research had treatment 1, control; treatment 2, control + 15% PEG in the concentrate; treatment 3, 6% NL in concentrate; and treatment 4, 6%NL + 15% PEG in the concentrate. We used pangola hay for roughage. In this experiment, we investigated whether PEG can increase CP digestibility and improve palatability. Table 1 and Table 2 display the nutrient composition of neem leaf and the chemical composition of the experimental diets. During the 60-day experiment, all goats were kept in individual feeding pens. Mineral blocks and clean water were readily available to all animals. During the 60-day experiment, the goats were fed 1.5% BW DM/day of pangola (Digitaria eriantha) hay and 16% crude protein in a 60:40 ratio. Those goats were provided feed that was supplemented with their specific treatment. Goats were fed at approximately 8:00 and 17:00 in the morning and afternoon, and there was a 60-day growth performance measurement period and a 7-day digestibility measurement period. The diets were formulated according to . 2.2. Feed Analyses Throughout the experimental period, the quantity of nutrition and the quantity of refused feed were recorded on a daily basis. On the final 7 days of the experiment, feeds of given and refused feeds were collected each day. The feed (500 g) was dried for 72 h in a vacuum oven at 65 degC and ground in a Wiley mill (Retsch SM 100 mill; Retsch Gmbh, Haan, Germany) with a screen 1 mm in diameter. Chemical and nutritional tests were performed on the dried samples . To obtain the crude protein (CP) values, 6.25 was used as the conversion factor. Condensed tannins were determined by HPLC . 2.3. Feces Sampling and Analyses The total collection method was used to measure and sample daily feces during the last 7 days of each period when the animals were in metabolism cages to investigate feed digestion and nitrogen metabolism. Around 5% of the total fresh weight of fecal samples was collected and divided into two portions. The first portion was used to calculate daily dry matter (DM), while the second was stored in the refrigerator and pooled for the animals at the end of each period for chemical analysis. The method described in was used to calculate the acid detergent fiber (ADF) and the neutral detergent fiber (NDF). 2.4. Urine Sampling Procedures Urine was collected in the final 7 days at the end of the period when the animals were in metabolism cages. To evaluate N use, urinary samples were obtained at roughly 100 mL of total urine volume, kept in a refrigerator, and pooled at the end of each session. Total N was determined using AOAC procedures . 2.5. Apparent Digestibility The apparent nutritional digestibility (%) was calculated using the acid-insoluble ash (AIA) technique as follows: apparent nutritional digestibility (%) = 100 - ((100 x % AIA in diet x % AIA in fecal)/(% AIA in fecal x % AIA in diet)) . All the fecal samples were oven dried at 65 degC for 72 h crushed, passed through a 1 mm filter, and stored at 4 degC until analysis. 2.6. Rumen Fluid Sampling The final day of the experiment, ruminal fluid was collected using a stomach tube connected to a vacuum pump at 0, 2, and 4 h after the animals were fed. In each case, experienced collectors collected the fluid and discarded any fluid that had saliva so as to prevent interference with pH value assessment. About 30 mL of rumen fluid was obtained at the same time as the blood sampling. Four layers of cheesecloth were used to filter the rumen fluid samples. A total of 30 mL of rumen fluid was mixed with 5 mL of sulfuric acid (H2SO4). The rumen fluid solution was centrifuged at 16,000x g for 15 min, and the NH3-N content was determined using a Kjeltech Auto 1030 analyzer (Tecator, Hoganiis, Sweden). Total volatile fatty acid (VFA), acetate, propionate, and butyrate concentrations were determined using a Thermo Fisher Scientific Inc. (Waltham, MA, USA) analyzer. 2.7. Blood Sampling A blood sample (about 5 mL) was taken from the jugular vein of each goat at 0 h, 2 h, and 4 h after feeding at 8:00 am on the final feeding week. The sample was placed in a tube (without EDTA), allowed to coagulate for 20 min at room temperature, and centrifuged for 10 min at 1107x g to separate the serum (Table Top Centrifuge PLC-02, Enfield, CT, USA). The collected serum was kept at -20 degC until it was analyzed (within 1 day) for blood urea nitrogen (BUN). 2.8. Rumen Microbial Procedures Total DNA was collected from rumen fluid using QIAmp PowerFecal DNA kit (QIAGEN GmbH, QIAGEN Strasse 1, 0724 Hilden, Germany). A gel extraction kit (QIAGEN GmbH, QIAGEN Strasse 1, 0724 Hilden, Germany; Cat. No. 28704) was used. The purity of the extracted DNA was measured using a NanoVue spectrophotometer to measure the absorbance ratio of 260/280 (GE Healthcare Bio-Sciences, Pittsburgh, PA, USA). The samples were tested using a Roche LightCycler(r) 480 real-time PCR method for quantitative real-time polymerase chain reaction (PCR) amplification (Roche Diagnostics GmbH, Penzberg, Germany). There were five DNA target prime sequences: total bacteria, methanogen, protozoa, Butyrivibrio fibrisolvens, and Streptococcus gallolyticus. Real-time PCR amplifications were conducted on 10 mL reaction volume of 5 mL of 2x Roche 04707516001 LightCycler(r) 480 SYBR Green I Master (Roche Diagnostics GmbH, Mannheim, Germany), 2 mL of 10x diluted DNA, and 1 mL of forward and reverse primers. Next, the plates (LightCycler(r) 480 Multiwell Plate 96, white; Roche Diagnostics GmbH) were centrifuged at 4 degC at 1500 rpm for 3 min (Universal 320, Hettich Zentrifugen, Tuttlingen, Germany). The cycling conditions were as follows: the sample was kept at 95 degC for 10 min for pre-incubation, and then subjected to 40 cycles in 30s at the same temperature for amplification; the temperature was increased from 55 degC to 57.5 degC and maintained for 1 min (annealing temperature optimized based on the primer; Table 3); then, the temperature was reduced to 40 degC for cooling. For each gene, amplifications were carried out in triplicate. 2.9. Statistical Analysis All the equations were evaluated as a 2 x 2 factorial in a completely randomized design (CRD). The data collected from the experiment were analyzed for variance (ANOVA), and we compared the variations between the treatments using the Statistical Analysis System 9.1.3 (SAS Inst. Inc., Cary, NC, USA) method of Duncan's New Multiple Range Test (DMRT). According to the general model, Uijk = m + ai + bj + (ab)ij + e, where Yijk denotes the observation, m represents the overall mean, ai represents the results from the tannin that consists of i levels, bj represents the results from polyethylene glycol that consists of j levels, (ab)ij represents the result of the sum of the tannin factor at i and the polyethylene glycol factor at j, and e is the residual error value at k of the tannin factor at i and the polyethylene glycol factor where j is normally distributed independently and has a mean of 0 and a variance of s 2. There are random cage for goats, and a fixed influence is dietary treatment. Significant differences were considered at p < 0.05. 3. Results 3.1. Feed Intake Table 4 presents the effect of neem leaf supplementation on feed intake. There were significant differences in feed intake (gDM/d, % BW, and g/kgBW0.75) (p < 0.05) for group fed 6% NL + 15% PEG, which was the highest among all groups. This group also showed higher intake for OMI, CPI, and EE (p < 0.05). In this experiment, we found that neem leaf modulates feed intake without any negative influence on the animal. 3.2. Digestibility Table 5 displays the data on nutrient digestion. There were significant differences (p < 0.05). Supplementation with 6% NL + 15% PEG increased the nutrient digestion of DDM, DOM, DCP, and DEE and reduced the nutrient digestion of DNDF and DADF. 3.3. Nitrogen Utilization There were significant differences (p < 0.05). Supplementation with 6% NL + 15% PEG led to the highest N intake, N absorption, % N absorption, N retention, and % N retention, as shown in Table 6. Additionally, supplementation with neem leaves had no effect on N in feces and N in urine (p > 0.05). 3.4. Performance In terms of animal growth performance (Table 7), supplementation with 6% NL + 15% PEG led to the highest final weight, weight change, and average daily gain (g/d) compared with other treatments. 3.5. Rumen Fermentation Table 8 shows the effect of neem leaf in blood urea nitrogen and ammonia nitrogen in the four treatments (p < 0.05). Supplementation with 6% NL + 15% PEG reduced blood urea nitrogen and ammonia nitrogen at 2 and 4 h. Among goats fed neem leaf, there was no effect on pH at 0 h and 2 h; however, there was an effect on pH at 4 h. Supplementation with 6% NL + 15% PEG affected the level of propionic acid at 2 and 4 h (p < 0.05), as shown in Table 9. In addition, supplementation with 6% NL + 15% PEG in the concentrate did not reduce acetic acid, butyric acid, or the ratio of acetic acid to propionic acid at 2 and 4 h after the animal was fed. 3.6. Microbial Population in Rumen Table 10 shows the effect of neem leaf supplementation on rumen microbial populations. Supplementation with 6% NL + 15% PEG in the concentrate increased Butyrivibrio fibrisolvens and Streptococcus gallolyticus at 2 and 4 h post feeding (p < 0.05). The methanogen and protozoa values were reduced at 2 and 4 h after feeding (p < 0.05). However, the supplementation had no effect on total bacteria at 0 and 2 h, and reduced the total bacteria at 4 h after feeding. 4. Discussion 4.1. Feed and Nutrient Intake The tannin-rich neem leaf has been shown to enhance the flow of rumen undegraded protein, in addition to providing nutritional benefits , because the leaves of Leucaena spp. plants, cassava, and Siamese neem are among the local feed sources used for livestock, including ruminant development in tropical countries, due to their high nitrogen content . In this study, growing goats fed 6% NL + 15% PEG showed increased feed and nutrient intake. In , the authors have reported that diet containing 2-4% of tannins reduces digestion in the rumen to allow microbial proteins to pass through the small intestine and increase the absorption of essential amino acids. A study on passing of feed particles , which also corresponds to the study in , reported that rumen was not affected by tannin-yucca extracts at 8 g/d. However, this should not be used at a proportion of more than 9% in the feed, as it decreases the digestion of feed and also reduces feed intake, which can lead to death . In the current study, PEG in diet increased the intake of all nutrients. Dry matter, OM, CP, EE, and ADF intakes were optimized at 15% PEG supplementation. This finding is similar to , which found that PEG consumption enhanced tanniniferous foliage intake and that PEG in the diet degrades tannin-fiber complexes, allowing them to be digested by microbial enzymes. The intake of dry matter by ruminant animals varies according to their size, the type of feed, the level of proteins, and fiber the animal receives, as well as the type and condition of the animal's body and the management of the feeding process. 4.2. Digestibility The results of our study once again indicated that supplementation with 6% NL + 15% PEG resulted in the highest digestibility of protein, consistent with the findings of . Polyethylene glycol supplementation had no negative effects on the apparent digestibility of nutrients in the present study. The effect of neem leaf on the digestion, modifying the ruminal microbes and development of tannin-protein complexes, has been well established in ruminant diets . The addition of neem leaf in ruminant formulas for chewing was found to create a compound of tannin and digestive saliva. The use of condensed tannin from Lotus corniculatus in the late stage of milking feeding was found to increase milk production and milk proteins , increase the microbial proteins released from the rumen , and increase wool production in sheep . Ref. reported that PEG 4000 supplementation did not affect NDF and ADF digestibility in Pedi goats fed Acacia nilotica leaf meal. According to , PEG supplementation significantly improved the apparent digestibility of nutrients. It can be used to feed animals, and tannins can prevent digestion in the rumen and increase the protein microbes passing through the intestines. This in turn can increase the absorption of essential amino acids because condensed tannins in forage protein can replace proteins that are not digested in the rumen from other sources. However, they should not be added at a proportion of more than 9% in dry-matter feed, as this will reduce the feed intake and may lead to the animal's death . 4.3. Performance Studies have shown that supplementation with 6% NL + 15% PEG resulted in the highest weight change and ADG. The optimal amount of concentrated tannins for animal feed use is 2-4% in the concentrate, which can prevent digestion in the rumen, increase the amount of microorganisms and proteins that pass through the small intestine, and also increase the absorption of essential amino acids. They can also decrease the incidence of both . However, large doses may harm animals, in particular their reproductive and physiological systems. For example, changes in the dietary rates result in changes in the rate of feeding, digestion, growth rate, and movement of the rumen, and these can affect microbe function. Some studies have indicated that tannins exceeding 9% should not be added to the feed, as this can contribute to the death of the animal due to reduced assimilation via rumen digestion and decreased levels of nitrogen in the body . In this study, supplementation with 6% NL + 15% PEG in the concentrate led to the highest performance in goats, even at high condensed tannin concentrations. In addition, the recorded amounts had no adverse effects on the animals and were able to increase the efficiency of the goats because the polyethylene glycol in the concentrate combined with the tannins and decreased toxic tannins. Polyethylene glycol reduces the effectiveness of tannins. The addition of polyethylene glycol (PEG) to tannin-rich diets is another attractive alternative for enhancing the feeding value of such diets. Because PEG has a stronger affinity for tannins than proteins, it is thought to break tannin-protein structures. This product has been used to counteract the effects of tannins . 4.4. Rumen Fermentation Parameters Supplementation with 6% NL + 15% PEG in the concentrate had no effect on nutrient digestion or nitrogen excretion. Neem leaf has been shown to prevent digestion in the rumen and increase the amounts of nitrogen that accumulate and circulate in the body. Although condensed tannins can decrease the feed intake of animals, the optimal amount does not affect feed intake. The levels of condensate in the tannins influence the nitrogen levels in the bodies of dairy cows . The amount in the form of dry matter should not be more than 5% of the feed, which will provide benefits because of reduced digestion in the rumen. However, if more than 5% is used in the feed, the digestion in the rumen will increase, and the levels of nitrogen in the body will decrease. Supplementation with 6% NL + 15% PEG in the concentrate reduced the blood urea concentration. The blood urea nitrogen level indicates the mechanism of protein change in ruminant animals, related to the ammonia nitrogen in the rumen liquid. However, the values of blood urea nitrogen differ depending on, for example, the amount of protein received by the animal for digestion, the level of energy, and the oxidation of proteins in the body to produce energy during fasting, including amino acids that are not used in protein synthesis, which are converted into blood urea nitrogen. The amount of protein that animals obtain from feed may be influenced by high blood urea nitrogen levels . Supplementation with 6% NL + 15% PEG resulting in the highest pH value (6.52) revealed that there was no impact due to the use of neem as a source of polyethylene glycol condensate. The optimal pH value in the rumen is between 6.5 and 7.0, which is ideal for the growth of microorganisms in the rumen . The pH value of the rumen was within the appropriate range and suitable for microbial growth in the rumen. Moreover, tannins have an acidic influence, but the protein-tannin compounds were found to reduce rumen pH. In animal feed containing polyethylene glycol tannins, polyethylene glycol tends to decrease the potency of tannins, which means that this experiment used condensed tannins at amounts higher than the acceptable standard. In this research, the neem leaf did not harm the animals because we used polyethylene glycol with condensed neem tannin, a polyethylene combination that helps minimize the toxicity of condensed tannins. PEG improves the consumption of tannin-containing feeds by acting as a tannin binding agent without changing the genetic pool of tannin-containing plants. Supplementation with 6% NL + 15% PEG led to the lowest NH3-N levels at 2 h (11.33 mg/dL) in the rumen, and it is a nitrogen source essential for growth in the rumen in this experiment. This is because the effect of CTs on digestion by modifying the population of ruminal microbes and tannin-protein complexes has been well established in terms of reducing ruminal crude protein degradation in ruminants, leading to reduced ruminal NH3-N concentration . In , the optimum NH3-N levels were found to be in the range of 9.7-21.4 mg/dL. The study in stated that 17.6 mg/dL was the acceptable amount. Increased digestibility of dry matter, protein, and bacterial communities in the rumen results from this stage. The predetermined range of PEG with tannin over protein saves the protein for rumen fermentation, resulting in higher NH3-N levels in rumen fluid. The amount of ammonia nitrogen, which is related to the level of nitrogen in the bloodstream, also increases in the rumen, leading to increased blood urea nitrogen. Supplementation with 6% NL + 15% PEG resulted in high values of propionic acid at 2 and 4 h and a decrease in total VFA, but with values within the normal range for goats. Because of its high molecular weight, neem leaf has a strong effect on total VFAs and acetic acid production, unlike low molecular weight substances . Since tannins can bind to enzymes, especially in cellulose, the decrease in total VFAs caused by condensed tannin supplementation may be due to a reduction in microbial activity . The ratio of acetic acid to propionic acid, both before and after the use of neem leaf, was found to have no adverse effect on goat disease in this experiment. Furthermore, the condensed tannin-affected decrease in acetic acid and the increase in propionic acid proportions indicate that nutrients were partitioned more into microbial protein synthesis . Higher VFA production in 15% PEG is caused by particular tannin binding with PEG, which leads to enhanced fermentation. 4.5. Microbial Population in Rumen Supplementation with 6% NL + 15% PEG is reported to have antibacterial properties, helping to reduce gastrointestinal tract fermentation. In this study, the use of neem leaf in the concentrate led to the highest amount of tannins, which were found to increase Butyrivibrio fibrisolvens and Streptococcus gallolyticus at 2 and 4 h. It was also discovered that Streptococcus gallolyticus can assist animal health by helping improve goat growth and reducing the occurrence of mastitis. Neem leaf supplementation can reduce the quantity of Streptococcus gallolyticus, , increase the use of nutrients, and prevent mastitis. The study in found that Streptococcus gallolyticus, which improves the digestibility of nitrogen in sheep, may be reduced due to supplementation with tannins. The rumen bacterium Butyrivibrio fibrisolvens has been identified as undertaking biohydrogenation of fatty acids and forming conjugated linoleic acid (CLA), which is an intermediate isomer of C18:2, in the process. In this study, supplementation with 6% NL + 15% PEG reduced methane production because neem leaf contains tannins, compounds with good anti-protozoa properties. Adding condensed tannins with a higher molecular weight has been found to reduce acetic acid formation and CH4 production . Tannins enter through the cell membrane and attack the structure of the protozoa cell membrane because the cell membrane covers the entire inner portion consisting of fat and protein layers , which improves rumen fermentation and may increase high-protein microbes. In this experiment, supplementation with 6% NL + 15% PEG increased Butyrivibrio fibrisolvens at 2 and 4 h, which increased the amount of protein-producing microorganisms. Nevertheless, depending on the tannin and microbial levels in the rumen, roughage is one of the factors that affects the rumen microbe population. In , it was reported that feed concentrate with a high tannin content and tannins that bind to proteins resulted in complex structures that may affect the microbes that digest fiber and reduce the digestibility of proteins in the rumen. 5. Conclusions In our study, supplementation with 6% NL + 15% PEG in the concentrate increased the feed intake, nutrient intake, nutrient digestion, nitrogen utilization, and growth performance of goats. Moreover, supplementation with 6% NL + 15% PEG in the concentrate had the highest propionic acid, Butyrivibrio fibrisolvens, and Streptococcus gallolyticus, as well as decreased protozoa and methanogens. Further studies are warranted to investigate the effect of neem leaf on meat products and antioxidant activity in meat. Acknowledgments The Suranaree University of Technology farm, Siriwan Phetsombat, as well as the Center for Scientific and Technological Equipment and Section of Goat and Sheep SUT farm, are all thanked for their support in using the farm's research facilities and managing animals. Nittaya Taethaisong gratefully recognizes the Suranaree University of Technology scholarship for External Grants and Scholarships for Graduate Students (SUT-OROG scholarship) as a source of funding. Author Contributions Planning and design of the study, N.T. and P.P.; conducting and sampling, N.T.; sample analysis, N.T.; statistical analysis, N.T.; manuscript drafting, N.T.; manuscript editing and finalizing, N.T., S.P., W.K., N.O.-u., S.T. and P.P. All authors have read and agreed to the published version of the manuscript. Institutional Review Board Statement The Animal Ethics Committee of Suranaree University of Technology issued a statement approving the experimental protocol (approval number SUT 4/2558). The research was carried out in accordance with regulations on animal experimentation and the Guidelines for the Use of Animals in Research as recommended by the National Research Council of Thailand (10-11/3/2559; U1-02632-2559). Informed Consent Statement Not applicable. Data Availability Statement Not applicable. Conflicts of Interest The authors declare no conflict of interest. animals-13-00890-t001_Table 1 Table 1 Chemical composition of neem leaf. Ingredient Neem Leaf Chemical Composition (% DM) Dry matter 36.00 Crude protein 18.83 Ash 7.78 Ether extract 1.52 Non-fibrous carbohydrate 30.15 Neutral detergent fiber 41.72 Acid detergent fiber 31.52 % Condensed tannin 10.66 animals-13-00890-t002_Table 2 Table 2 Diets' nutrition and composition. Diet Items 0% NL + 0% PEG 0% NL + 15% PEG 6% NL + 0% PEG 6% NL + 15% PEG SEM p-Value Soybean meal 17.10 15.57 15.00 12.20 Rice bran 30.00 24.33 25.00 22.01 Cassava chip 22.00 25.00 25.40 22.61 Corn 29.80 19.00 27.50 21.08 Sodium chloride 0.40 0.40 0.40 0.40 Pure sulfur 0.20 0.20 0.20 0.20 Minerals and vitamins 0.50 0.50 0.50 0.50 Condensed tannin 0.00 0.00 6.00 6.00 Polyethylene glycol 0.00 15.00 0.00 15.00 Chemical composition (% DM) Dry matter 74.05 74.43 74.07 74.05 0.05 0.01 Ash 6.16 5.99 6.77 6.43 0.09 0.01 Crude protein 16.30 16.45 16.55 16.65 0.04 0.01 Ether extract 1.34 2.01 2.13 2.25 0.11 0.01 Non-fibrous carbohydrate 23.73 24.7 16.56 19.51 0.99 0.01 Neutral detergent fiber 52.47 50.85 57.99 55.16 0.82 0.01 Acid detergent fiber 24.23 23.65 32.21 31.40 1.19 0.01 TDN, % 88.70 88.64 87.20 87.15 0.23 0.01 Metabolizable energy, Mcal/kg DM 3.21 3.20 3.15 3.15 0.01 <0.01 NL = neem leaf, PEG = polyethylene glycol. animals-13-00890-t003_Table 3 Table 3 Primer sequences used in this analysis for real-time PCR amplification. Items Forward/ Reverse Temperature (degC) Product Size (bp) Primer Sequence (5'-3') Total bacteria F 55 130 CGGCAACGAGCGCAACCC R CCATTGTAGCACGTGTGTAGCC Methanogen F 58 140 TTCGGTGGATCDCARAGRGC R GBARGTCGWAWCCGTAGAATC Protozoa F 55 223 CTTGCCCCTCYAATCGTWCT R GCTTTCGWTGGTAGTGTATT Butyrivibrio fibrisolvens F 58 64 ACACACCGCCCGTCACA R TCCTTACGGTTGGGTCACAGA Streptococcus gallolyticus F 58 419 GAAAAGTACTCAACCAAATA R AGTAACGGTACTTAAATTGTTTA animals-13-00890-t004_Table 4 Table 4 Effect of neem leaf supplementation on feed intake in goats. 0% NL 6% NL p-Value TRT 0% PEG 15% PEG 0% PEG 15% PEG SEM NL PEG NL x PEG Feed intake gDM/d 456.84 d 469.91 c 476.96 b 495.87 a 0.57 0.44 0.44 <0.01 % BW 2.11 c 2.11 c 3.95 b 4.07 a 0.20 0.44 0.46 0.01 g/kgBW0.75 41.94 d 44.46 c 66.53 b 75.41 a 2.95 0.44 0.45 0.02 Nutrient intake g DM/d OMI 429.43 d 441.72 c 443.57 b 466.12 a 0.39 0.0015 0.02 <0.01 CPI 73.09 d 75.19 c 81.08 b 84.30 a 0.25 0.01 0.01 0.04 EEI 4.56 d 9.40 c 9.54 b 9.92 a 0.16 0.01 0.01 0.01 NDFI 237.56 d 239.65 c 276.64 a 272.23 b 0.93 0.01 0.01 0.01 ADFI 109.64 d 112.78 c 152.63 b 153.72 a 0.58 0.01 0.01 0.01 NL, neem leaf; PEG, propylene ethylene glycol; SEM, standard error of the mean; g DM/d, daily intake of dry matter; a, b, c, and d, in the same row, indicate statistically significant difference p < 0.05. %BW, ((g DM intake x 100)/ (body weight (kg) x 1000)); g/kgBW0.75, g DM intake/ (kg body weight)0.75. animals-13-00890-t005_Table 5 Table 5 Effect of neem leaf supplementation on nutrient digestion in goats. 0% NL 6% NL p-Value TRT 0% PEG 15% PEG 0% PEG 15% PEG SEM NL PEG NL x PEG Apparent Digestibility, % of intake DDM 72.80 d 76.83 c 79.85 b 82.03 a 0.76 0.44 0.46 0.08 DOM 75.97 b 76.68 b 81.45 a 82.39 a 0.63 0.44 0.09 0.81 DCP 40.81 33.03 45.49 60.70 0.32 0.47 0.66 0.42 DEE 79.99 c 82.18 b 85.95 a 85.55 a 0.58 0.45 0.12 0.03 DNDF 31.89 a 33.32 c 35.04 b 32.58 d 0.51 0.0076 0.46 0.08 DADF 21.78 c 23.16 b 26.34 a 21.04 c 0.47 0.0224 0.0006 0.01 NL, neem leaf; PEG, propylene ethylene glycol; SEM, standard error of the mean; DDM, digestibility of dry matter; DOM, digestibility of organic matter; DCP, protein digestibility; DEE, fat digestibility; DEE, digestibility fatty digestibility; DNDF, the digestibility of the crude fiber that cannot be dissolved in neutral solution; DADF, the digestibility of the fiber that cannot be digested in an acidic solution; a, b, c, and d, in the same row, indicate statistically significant difference p < 0.05. animals-13-00890-t006_Table 6 Table 6 Effect of neem leaf supplementation on nitrogen utilization in goats. 0% NL 6% NL p-Value TRT 0% PEG 15% PEG 0% PEG 15% PEG SEM NL PEG NL x PEG N intake (g/d) 36.39 c 26.64 d 37.59 b 45.72 a 1.43 0.44 0.23 <0.01 N feces (g/d) 21.54 17.84 20.49 17.97 0.37 0.30 0.45 0.17 N urine (g/d) 5.47 5.38 5.78 5.38 0.06 0.20 0.04 0.17 N absorption (g/d) 14.85 c 8.80 d 17.1 b 27.75 a 0.85 0.66 0.0014 0.28 N absorption (%) 40.81 c 33.03 d 45.49 b 60.70 a 1.02 0.66 0.001 0.28 N retention (g/d) 9.38 c 3.42 d 11.32 b 22.37 a 0.63 0.44 0.04 0.02 N retention (%) 25.78c 12.84 d 30.11 b 48.93 a 0.48 0.0004 0.02 0.07 NL, neem leaf; PEG, polyethylene glycol; SEM, standard error of the mean; a b c, d, in the same row there are significant differences in Statistics (p < 0.05). animals-13-00890-t007_Table 7 Table 7 Effect of neem leaf supplementation on performance. 0% NL 6% NL p-Value TRT 0% PEG 15% PEG 0% PEG 15% PEG SEM NL PEG NL x PEG Initial weight, kg 20.33 20.67 20.83 21.00 0.15 0.19 0.43 0.79 Final weight, kg 24.00 24.18 26.00 26.54 0.27 0.03 0.68 0.20 Weigh change, kg 3.67 c 3.51 d 5.17 b 5.54 a 0.19 0.06 0.44 0.31 ADG, g/d 40.78 c 39.00 d 57.44 b 61.56 a 3.42 0.44 0.12 0.46 NL, neem leaf; PEG = level of polyethylene glycol; SEM, standard error of the mean; a, b, c, and d, in the same row there are significant differences in Statistics (p < 0.05). animals-13-00890-t008_Table 8 Table 8 Effect of neem leaf supplementation on pH, ammonia nitrogen, and blood urea nitrogen. 0% NL 6% NL p-Value TRT 0% PEG 15% PEG 0% PEG 15% PEG SEM NL PEG NL x PEG BUN Mg% 0 h 23.03 21.23 17.66 20.09 0.56 0.0022 0.73 0.03 2 h 17.82 a 14.81 b 14.89 b 12.97 c 0.45 0.0002 0.47 0.29 4 h 18.20 a 17.82 b 15.88 c 14.44 d 0.40 0.46 0.06 0.25 Mean 19.79 a 17.90 b 16.11 c 15.82 c 0.26 0.01 0.007 0.04 Ruminal pH 0 h 6.76 6.67 6.74 6.90 0.04 0.27 0.66 0.17 2 h 6.58 6.53 6.69 6.68 0.03 0.06 0.59 0.71 4 h 6.43 b 6.24 d 6.32 c 6.52 a 0.02 0.44 0.58 <0.01 Mean 6.59 ab 6.48 c 6.58 cb 6.70 a 0.03 0.01 0.94 <0.01 Ruminal NH3-N mg/dL 0 h 16.92 14.24 14.79 14.6 0.43 0.23 0.09 0.13 2 h 15.08 a 13.49 c 14.23 b 11.33 d 0.31 0.0003 0.45 0.06 4 h 12.10 13.91 12.29 10.09 0.41 0.01 0.77 <0.01 Mean 14.59 a 13.80 a 13.77 a 12.00 b 0.33 0.01 0.01 0.30 NL, neem leaf; PEG, polyethylene glycol; BUN, concentration of urea in the blood; NH3-N, ammonia nitrogen; SEM, standard error of the mean; a, b, c, d, in the same row there are significant differences in Statistics (p < 0.05). animals-13-00890-t009_Table 9 Table 9 Effect of neem leaf supplementation on volatile fatty acids in goats. 0% NL 6% NL p-Value TRT 0% PEG 15% PEG 0% PEG 15% PEG SEM NL PEG NL x PEG Acetic acid (% Molar) 0 h 62.22 61.39 60.49 60.80 5.40 0.44 0.65 0.67 2 h 53.14 c 56.66 a 54.06 b 53.48 c 0.65 0.45 0.45 <0.01 4 h 53.69 50.56 52.51 51.14 0.32 0.04 0.47 0.71 Mean 50.20 45.85 46.19 45.12 1.30 0.22 0.16 0.37 Propionic acid (% Molar) 0 h 22.21 26.41 26.19 28.07 1.48 0.59 0.31 0.85 2 h 25.08 d 26.06 c 29.99 b 32.13 a 0.47 0.45 0.06 <0.01 4 h 22.96 d 27.51 b 26.51 c 28.51 a 0.48 0.46 0.46 <0.01 Mean 21.45 c 22.38 bc 23.56 ab 24.70 a 0.34 0.0003 0.05 0.82 Butyric acid (% Molar) 0 h 15.57 12.20 13.33 11.13 3.41 0.56 0.43 0.78 2 h 21.78 a 12.28 d 15.96 b 14.39 c 0.80 0.45 0.45 <0.01 4 h 23.36 a 21.92 b 20.92 c 20.35 c 0.25 0.01 0.06 0.95 Mean 18.25 a 14.22 b 14.03 b 12.82 b 0.80 0.03 0.03 0.22 Ratio of acetic acid to propionic acid 0 h 2.80 2.32 2.31 2.17 0.23 0.40 0.84 0.99 2 h 2.12 a 2.17 b 1.80 a 1.66 b 0.01 0.62 0.44 0.12 4 h 2.34 a 2.84 b 1.98 c 1.79 d 0.03 0.44 0.44 0.63 Mean 2.27 a 2.18 ab 2.10 ab 2.01 b 0.05 0.05 0.25 0.99 Total VFA (mmol/L) 0 h 83.57 76.98 75.04 74.32 3.96 0.94 0.22 0.66 2 h 96.64 a 79.98 c 82.73 b 82.82 b 1.45 0.44 0.45 <0.01 4 h 87.56 c 89.01 b 91.71 a 89.62 b 0.45 0.01 0.04 0.72 Mean 82.02 b 80.62 b 83.49 ab 86.82 a 1.01 0.02 0.51 0.11 NL, neem leaf; PEG, polyethylene glycol; VFA, volatile fatty acids; SEM, standard error of the mean; a, b, c, and d, in the same row there are significant differences in Statistics (p < 0.05). animals-13-00890-t010_Table 10 Table 10 Effect of neem leaf supplementation on rumen microbial populations in goats. 0% NL 6% NL p-Value TRT 0% PEG 15% PEG 0% PEG 15% PEG SEM NL PEG NL x PEG Total bacteria (lg10 copies/mL) 0 h 8.66 7.09 8.87 8.97 0.90 0.61 0.71 0.67 2 h 10.57 10.64 10.53 10.70 0.03 0.83 0.03 0.34 4 h 10.35 d 10.74 a 10.53 c 10.63 b 0.04 0.67 0.01 0.04 Mean 9.86 9.49 9.98 10.10 0.46 0.59 0.85 0.70 Methanogen (lg10 copies/mL) 0 h 7.71 7.45 7.64 7.36 0.06 0.51 0.03 0.90 2 h 7.40 b 7.50 a 7.58 a 7.34 c 0.02 0.81 0.02 <0.01 4 h 7.42 a 7.30 b 7.20 c 7.16 d 0.02 0.44 0.47 0.03 Mean 7.51 a 7.42 a 7.47 a 7.29 b 0.03 0.05 0.003 0.23 Protozoa (lg10 copies/mL) 0 h 4.85 c 6.30 a 5.47 b 3.11 d 1.76 0.19 0.63 0.05 2 h 6.58 a 5.43 b 6.65 a 4.66 c 0.17 0.46 0.44 <0.01 4 h 7.35 a 5.35 b 4.39 c 2.34 d 4.90 0.44 0.44 0.80 Mean 9.32 a 5.72 b 5.40 b 3.59 b 0.84 0.02 0.03 0.45 Butyrivibrio fibrisolvens (lg10 copies/mL) 0 h 8.17 8.45 8.37 8.74 0.26 0.37 0.21 0.87 2 h 7.95 c 8.13 b 8.25 b 9.27 a 0.03 0.44 0.44 <0.01 4 h 7.99 c 8.07 b 8.23 b 9.25 a 0.02 0.44 0.44 <0.01 Mean 8.04 b 8.22 ab 8.30 ab 8.41 a 0.06 0.02 0.10 0.67 Streptococcus gallolyticus (lg10 copies/mL) 0 h 8.66 7.09 8.87 8.97 0.90 0.61 0.71 0.67 2 h 10.56 b 10.56 b 10.58 b 11.64 a 0.01 0.44 0.44 <0.01 4 h 10.55 b 10.36 b 10.53 b 11.61 a 0.01 0.44 0.44 <0.01 Mean 9.86 9.45 10.00 10.02 0.47 0.60 0.77 0.74 NL, neem leaf; PEG, propylene glycol; SEM, standard error of the mean; a, b, c, and d, in the same row there are significant differences Statistics (p < 0.05). 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PMC10000192

This study was conducted to provide alternative high-quality feed and to reduce methane production using a mixture of the minimum effective levels of Euglena gracilis, EG, and Asparagopsis taxiformis, AT. This study was performed as a 24 h in vitro batch culture. Chemical analysis demonstrated that EG is a highly nutritive material with 26.1% protein and 17.7% fat. The results showed that the supplementation of AT as a feed additive at 1 and 2.5% of the diet reduced methane production by 21 and 80%, respectively, while the inclusion of EG in the diet at 10 and 25% through partially replacing the concentrate mixture reduced methane production by 4 and 11%, respectively, with no adverse effects on fermentation parameters. The mixtures of AT 1% with both EG 10% and EG 25% had a greater reductive potential than the individual supplementation of these algae in decreasing methane yield by 29.9% and 40.0%, respectively, without adverse impacts on ruminal fermentation characteristics. These results revealed that the new feed formulation had a synergistic effect in reducing methane emissions. Thus, this approach could provide a new strategy for a sustainable animal production industry. Asparagopsis taxiformis digestibility euglena feed additives global warming nutritive value rumen fermentation This research received no external funding. pmc1. Introduction Modern livestock systems are challenged by greenhouse gas (GHG) emissions, land degradation, and feed costs . The livestock industry, particularly the ruminant production sector, plays an important role in food security because it is capable of converting indigestible plant mass to meat and milk, which are sources of high-quality protein and essential nutrients for human well-being . However, this industry accounts for approximately 14-18% of global anthropogenic GHG emissions, of which enteric methane (CH4) production is the most significant source because its global warming potential is 28 times greater than that of carbon dioxide (CO2) on a 100-year timescale . Moreover, livestock occupies two-thirds of the world's agricultural land, either for grazing or crop cultivation . In addition, the production of conventional feed protein concentrates such as soybean is a very expensive process and may be viewed as being in direct competition with human food security because they are considered staple food crops for humans . On the basis of the aforementioned challenges, in addition to the rise in the global population and increased demand for animal-derived products, finding natural alternatives and high-quality feeds is of great importance for a sustainable animal production industry. These novel feeds may replace the expensive conventional sources in animal diets and help reduce the environmental impact of the animal husbandry sector. Algae are one of the suitable options that have that potential. Algae are classified into two categories: microalgae, which are microscopic, small unicellular organisms that are commonly referred to as phytoplankton, and macroalgae, which are multicellular and can be of very large size; these are commonly known as seaweeds. Microalgae supplementation is one of the prospective solutions as a promising alternative source of nutrition for cattle. Microalgae are photosynthetic microorganisms that consume CO2 via light energy to produce a variety of proteins, carbohydrates, and lipids . The nutritional profiles of microalgae species are quite variable; however, the majority are characterized by protein (25-40%), fat (10-30%), and carbohydrate (5-30%) contents that are comparable, if not superior, to traditional feed ingredients like soybean meal, corn, and wheat . Recent studies have shown that the inclusion of microalgae can improve the growth performance of animals for both meat and milk production . One example is Euglena gracilis (EG), a freshwater microalga that is characterized by its high protein and fat content . A recent study conducted with Holstein calves has shown that the supplementation of EG b-glucans to the milk replacer improved their growth performance and ameliorated diarrheal bouts . Studies on the effect of EG as a feed, considering its impact on ruminal fermentation and CH4 production, are scarce. In an in vitro study, the inclusion of up to 10% of EG to replace concentrates in the ruminant diet reduced CH4 production by up to 9% owing to the effect of fatty acids, without negative impacts on the fermentation profile . The EG is a commercially available product from Euglena Co., Ltd., Japan. This company has its own facilities at Ishigaki Island, Japan, for the large-scale cultivation and production of EG . Further research and innovation could improve the utilization of EG as a sustainable alternative feed with greater CH4-reduction potential. Recently, macroalgal seaweeds have emerged as a potential dietary strategy to reduce CH4 emissions from ruminants . The most effective candidate is the red seaweed, Asparagopsis taxiformis (AT). With an inclusion rate of 1-3%, AT can reduce CH4 production by more than 80% because of its main active component, bromoform . Bromoform has been shown to react with vitamin B12, which hinders the cobamide-dependent methyltransferase reaction, a terminal step in CH4 formation . Despite its strong reduction potential, AT also has high concentrations of heavy metals and minerals, such as iodine, which can be excreted in milk, leading to undesirably high levels in animal products, which can have adverse health impacts on humans . Notably, there are human safety concerns regarding the main active component of AT (bromoform) present in milk . Additionally, one study showed that the supplementation of sheep with AT led to pathological changes in the ruminal mucosa . However, the inclusion of low doses of AT in feed formulations can reduce these risks in both animals and humans, but this also diminishes the mitigation potential to reduce CH4 production . Another challenge is that the amount of AT required to be supplemented in ruminant feed (50-100 g/day for cattle) is tremendous. There is currently a growing interest in investing in that area through many startup companies such as CH4 GlobalTM, Inc. in Australia (CH4 Australia PTY Limited) and New Zealand (CH4 Aotearoa Limited)to ensure constant large-scale production. The company announced the first commercial supply of its proprietary AT-based product for ruminant animals in June 2022. However, the mass production needed to feed ruminants on a larger scale is currently infeasible . Therefore, considering the advantages of EG and AT, the main purpose of the present study was to formulate a new mixture composed of the minimum effective levels of EG and AT to reduce CH4 emissions without adverse impacts on ruminal fermentation. The selected dosages for AT and EG in the current study were based on previously conducted pre-trials. In this experiment, the minimum effective levels of EG (as a feed) and AT (as a feed additive) were used in different combinations to study the impacts on CH4 production and ruminal fermentation characteristics. We hypothesized that the new feed formulations (EG as a feed + AT as a feed additive) would efficiently substitute part of the concentrate mixture in the diet with EG and reduce CH4 production in an additive or synergistic manner without negatively affecting ruminal fermentation. 2. Materials and Methods This study was approved by the Animal Care and Ethics Committee of the Obihiro University of Agriculture and Veterinary Medicine, Japan (approval number 21-41). The animals involved in these experiments were kept and cared for by the Field Science Center. Animal management and sampling procedures were performed according to the established guidelines at the Obihiro University of Agriculture and Veterinary Medicine. The collection of seaweeds and experimental research complied with institutional, national, and international guidelines and legislation. The in vitro experiments were conducted during the period from 8 July to 18 September 2021. This study followed the standard procedures and is reported in accordance with ARRIVE guidelines. 2.1. Experimental Feeds Kleingrass (Panicum coloratum) hay and a commercial concentrate mixture (the composition of the ingredients is presented in Table 1) were used as the basal diet. They were ground using the Retsch SM-2000 cutting mill (Retsch, Germany) to pass through a 1 mm sieve. The proximate analyses of the grass hay and concentrate mixture are presented in Table 1. The tested material of EG in fine powder form (10-150 mm particle size) was obtained from Euglena Co., Ltd., Japan. More details about EG growth, mass cultivation, and processing were described previously . The tested material of EG in this study was characterized by high protein (26.1%), fat (17.7%), and carbohydrates (51.2%) (Table 1). Approximately 35% of the carbohydrate content in our material was paramylon. The tested material had all the essential amino acids (Table 2). The EG was rich in saturated fatty acids (59.7%), whereas the unsaturated fatty acid content was 34.3% (Table 3). The biomass of AT was harvested during the gametophyte phase at Kochi Bay, Kochi City, Kochi Prefecture, Japan on 25 May 2021 by Dr. Takuma Mezaki. The harvested biomass was stored on ice and shipped to the Obihiro University of Agriculture and Veterinary Medicine. Once the AT was received, it was stored at -20 degC until further processing. The biomass was kept at 4 degC overnight to thaw, then it was dried in an oven dryer at 45 degC for 24 h and then ground by a cutting mill (Retsch SM-2000, Haan, Germany) to pass through a 1-mm sieve on 05 July 2021. The ground material was packaged in aluminum laminated film zipper bags (Lamizip(r), AL-16, Seisan Nipponsha, Ltd., Tokyo, Japan) and kept at -20 degC until use. The OM content of the AT was 41.7% (Table 1), while the bromoform concentration in the AT was 953.8 mg/g DM. 2.2. Donor Animals and Rumen Fluid Collection Approximately 1.3 L of rumen fluid was collected 3 h after morning feeding from two ruminally cannulated non-lactating Holstein cows that were approximately 8 years old with an average body weight of 894 kg. The cows were fed at the maintenance level on a diet of orchard grass (Dactylis glomerata) hay (organic matter (OM), 980 g/kg; crude protein (CP), 132 g/kg; neutral detergent fiber (NDF), 701 g/kg; acid detergent fiber (ADF), 354 g/kg; acid detergent lignin (ADL), 40 g/kg; dry matter (DM) basis) with free access to clean drinking water and mineral blocks (Koen(r) E250 TZ, Nippon Zenyaku Kogyo Co., Fukushima, Japan). The collected rumen fluid was strained through four layers of surgical gauze, placed into a thermos flask that had been pre-warmed to 39 degC, and then immediately transferred to the laboratory within 15 min. 2.3. Experimental Design This study was conducted as an in vitro batch culture according to the procedure described by Menke and Steingass . This experimental design was composed of nine treatments: control (50% hay:50% concentrate), two inclusion levels of AT as a feed additive (1 and 2.5% of the basal diet), two inclusion levels of EG as a feed in the basal diet (10 and 25%) partially replacing the concentrate mixture, and four different formulations as a combination of EG (as a feed) and AT (as a feed additive) as follows: EG 10% + AT 1%, EG 10% + AT 2.5%, EG 25%+ AT 1%, and EG 25% + AT 2.5%. Each treatment had four replicates, and this experimental design was repeated in three separate runs in different weeks. Two bottles were used as blanks in each run. 2.4. In Vitro Incubation and Sample Collection Approximately 500 mg of substrate (basal diet) was added to pre-weighed nylon bags with a pore size of 53 +- 10 mm (BG1020, Sanshin Industrial Co., Ltd., Kanagawa, Japan), which were heat-sealed and placed in 120 mL glass bottles. Since the AT was applied as a feed additive, it was added directly to the bottles, while the EG was evaluated as a feed, so it was added to the nylon bags. Under continuous CO2 flushing, 40 mL of fresh buffer solution (pH 6.8) prepared according to McDougall and 20 mL of the collected rumen fluid were added to each fermentation bottle. The bottles were then flushed with CO2 before being sealed with butyl rubber stoppers and aluminum caps (Maruemu Co., Ltd., Osaka, Japan). All the bottles were incubated for 24 h at 39 degC. At the end of the incubation, the total gas production was measured using a calibrated syringe, and an aliquot of the headspace gas was collected from each bottle and stored in a vacutainer tube (BD Vacutainer, Becton Drive, NJ, USA) at room temperature until CH4 and CO2 were determined by means of gas chromatography (GC-8A, Shimadzu Corp., Kyoto, Japan), as described previously by Ahmed et al. . Then, the pH was determined, and 1 mL of the culture medium was collected in Eppendorf tubes (Eppendorf AG, Hamburg, Germany) and centrifuged at 16,000x g at 4 degC for 5 min. The supernatant was used to estimate the volatile fatty acids (VFA) by means of high-performance liquid chromatography (HPLC, Shimadzu Corp., Kyoto, Japan) and ammonia nitrogen (NH3-N) by using a microplate reader (SH-1000 Lab, Corona Electric Co., Ltd., Hitachinaka, Japan), as processed and reported previously . The nylon bags were rinsed with tap water until the effluent became clear, after which they were dried at 60 degC for 48 h and weighed to determine the in vitro dry matter digestibility (IVDMD) . The IVDMD was calculated as the DM that disappeared from the initial DM weight input into the bag. 2.5. Chemical Analysis The chemical compositions of grass hay, concentrate, EG, and AT were determined according to AOAC standard procedures . The DM content was determined by drying the samples in an air-forced oven at 135 degC for 2 h (method 930.15). The OM was measured by placing the samples in a muffle furnace at 500 degC for 3 h (method 942.05). The ether extract (EE) was determined according to method 920.39 while nitrogen was measured according to the method of Kjeldahl (method 984.13) using an electrical heating digester (DK 20, VELP Scientifica, Usmate (MB), Italy) and an automatic distillation apparatus (UDK 129 VELP Scientifica, Usmate (MB), Italy). The CP was then estimated as nitrogen x 6.25. The NDF, ADF, and ADL were estimated and expressed as inclusive residual ash using an ANKOM200 fiber analyzer (Ankom Technology Methods 6, 5, and 8, respectively; ANKOM Technology Corp., Macedon, NY, USA) . The NDF was measured using sodium sulfite without the heat-stable a-amylase. The non-fiber carbohydrate was estimated according to the following formula: 100 - (NDF %+ CP %+ EE % + Ash %). The chemical compositions of the experimental treatments are presented in Table 4. 2.6. Bromoform Concentration in Asparagopsis The analysis of bromoform was conducted using a gas chromatograph mass spectrometer (GCMS-QP2010Plus, Shimadzu Corp., Kyoto, Japan) combined with a TurboMatrix HS40 headspace sampler (PerkinElmer, Waltham, MA, USA) and equipped with an RTX-624 capillary column (60 m x 0.32 mm i.d.; film thickness 1.8 mm; Restek, Bellefonte, PA, USA). The column temperature was programmed to be maintained at 35 degC for 5 min, increased to 230 degC at a rate of 10 degC/min, and held at 230 degC for 5 min. The detector temperature was set at 200 degC. Pure helium was used as the carrier gas. The internal standard was a fluorobenzene stock solution (Kanto Chemical Co., Inc., Chuo-ku, Tokyo, Japan). This analysis was performed at a private institute (Zukosha Co., Ltd., Obihiro, Japan). 2.7. Fatty Acid and Amino Acid Composition of Euglena The fatty acid and amino acid profiles of EG were analyzed by the Japan Food Research Laboratories, Japan. The fatty acid composition was determined using gas chromatography (7890 B, Agilent Technologies, Inc. Wilmington, DE, USA) equipped with a flame ionization detector. Each fatty acid was calculated on the basis of the standard retention time and was reported as a percentage of the total fatty acid content. The amino acid composition, except for tryptophan, histidine, phenylalanine, and leucine, was determined using an automated amino acid analyzer (JLC-500/V, JEOL Ltd., Tokyo, Japan; column, LCR-6 with 4 mm x 120 mm ID, JEOL, Co., Ltd., Tokyo, Japan). Tryptophan was analyzed using HPLC (LC-40D, Shimadzu, Co., Ltd., Kyoto, Japan). The column was CAPCELL PAK C18 AQ (4.6 mm ID x 250 mm, Osaka Soda Co., Ltd., Osaka, Japan) with fluorescence detection (RF-20Axs, Shimadzu, Co., Ltd., Kyoto, Japan). Histidine, phenylalanine, and leucine were analyzed using HPLC (LA8080, Hitachi High-Tech Corporation, Tokyo, Japan) on a column packed with Hitachi custom ion exchange resin (4.6 mm ID x 60 mm, Hitachi High-Tech Corporation, Tokyo, Japan). Each amino acid was reported as a percentage of the total amino acid composition. Further information on the analytical specifications for both fatty acids and amino acids was previously provided by Ahmed et al. . 2.8. Statistical Analysis The data were screened for normality using the PROC UNIVARIATE of SAS statistical software version 9.4 (SAS Institute Inc., Cary, NC, USA). Then, data were analyzed using PROC MIXED with models including the treatments as a fixed effect, whereas the three experimental runs were considered random effects. Orthogonal polynomial contrasts were conducted to test the linear and quadratic responses to increasing levels of EG and AT. Comparisons of AT versus AT + EG (all dosages), EG versus AT + EG (all dosages), and AT 1% versus AT 1% + its combination with EG at 10 and 25% were conducted as well. The values are shown as means with pooled standard errors of the means. Differences in the means among the experimental groups were estimated using Tukey's test. Differences were considered statistically significant at p < 0.05. 3. Results The yield (mL/g) of CH4/DM significantly decreased by 21.3 and 80.1% after supplementation with 1 and 2.5% AT, respectively, compared to the control group (p < 0.01, Table 5). The inclusion of 10 and 25% EG in the diet decreased the CH4 yield by 4.4 and 11%, respectively; these findings were not significant (p > 0.05, Table 5) when compared with the control group. The combination of 1% AT and 10% EG in the diet led to a significant CH4 reduction potential of up to 29.9% compared to the control (p < 0.01, Table 5). A more reductive potential of up to 40.1% was observed with the combination of 1% AT and 25% EG (p < 0.01). In contrast, the combination of 2.5% AT with neither EG (10%) nor EG (25%) was effective in achieving a more significant reduction than individual supplementation of 2.5% AT (75.9 and 81.7% versus 80.2%, respectively, p = 0.09, Table 5). The fermentation characteristics in Table 6 show that the combination of 1% AT with 10% EG or 25% EG had no adverse effect on ruminal fermentation parameters or IVDMD. These combinations shifted the fermentation profile towards more propionate and less acetate (p < 0.01). The combination of 2.5% AT and 25% EG led to a decrease in the production of total VFA (p < 0.01). The concentration of NH3-N was higher (p < 0.05) with the inclusion of 25% EG, either individually or in combination with 1 and 2.5% AT, compared to the basal diet in the control group. 4. Discussion To the best of our knowledge, this is the first study to evaluate the combination of EG and AT as a novel sustainable feed for ruminants. It is important to mention that in vitro studies are a valuable tool for the study of ruminal CH4 production; however, any approach needs to be investigated with animals as well. The chemical composition analysis of EG performed in this study confirmed the previous findings of Aemiro et al. , who reported that EG is a rich source of amino and fatty acids. Aemiro et al. reported that EG has an essential amino acid profile very similar to that of soybean. The EG was also characterized by very low NDF content because it does not have a carbohydrate-based cell wall such as that found in terrestrial plants . It is noteworthy to mention that the carbohydrate storage in microalgae is different from that in vascular plants (e.g., grasses or herbs), and additionally, there is huge variation between different microalgae genera; it may even vary from species to species within the genera . The carbohydrate storage in EG is known as paramylon (classified as dietary fiber), an insoluble, highly crystalline, fibrillary b-(1,3) linked glucan . Paramylon crystallinity is comparable to that of cellulose . Therefore, the rumen microbiome might need some time to adapt to this carbohydrate mixture as a nutrient source. The ash content of the AT in the current study is relatively high (greater than 50%), which is the same as that which was observed previously . According to the previous studies where the nutritional value was evaluated, it was reported that the main elements that formed the major basis of ash were sodium and potassium . The bromoform concentration of the tested AT in the current study (953 mg/g DM) was much lower than that reported in other studies where the AT was collected from the Santa Catalina island, 35 km off the coast of southern California, United States (dried at 55 degC for 72 h, bromoform 2305 mg/g DM ), and Humpy island, Keppel Bay, Queensland, Australia (freeze-dried, bromoform 6550 mg/g DM , and 7800 mg/g DM ). The lower concentrations in our study can be attributed to the processing methods used. Vucko et al. found that drying the AT biomass at 45 degC led to a substantial reduction in bromoform concentration. This could explain the lower concentration of bromoform in our study, because the AT was dried at 45 degC for 24 h. Additionally, the concentration of bioactive components in seaweeds can vary across seasons and geographical locations , which might explain why the bromoform in the present study was lower than that in California, United States, where the authors dried AT 55 degC for 72 h . The current study showed that AT had a dose dependent potency in reducing CH4 production, as previously reported . This effectiveness has been linked to the halogenated bioactive compounds present in AT, such as bromoform, dibromochloromethane, bromochloroacetic acid, dibromoacetic acid, and dichloromethane . Bromoform is the most abundant compound in AT and has been shown to inhibit enzymatic activity by binding to vitamin B12 that chemically resembles coenzyme F430, a cofactor required by methyl-coenzyme M reductase, which catalyzes the last step of CH4 synthesis . It is well established that the addition of dietary fat, regardless of its source, can reduce CH4 production by approximately 2.2-5.6% . The potency of EG in reducing CH4 in this study could be attributed to its high fat content. One possible theory is that fat reduces the fermentation of organic matter, thus decreasing the available hydrogen for methanogens . However, the unsaturated fatty acid content in EG may have a direct role in reducing CH4 production through bio-hydrogenation as an alternative hydrogen sink . In addition, the saturated fatty acids, such as myristic and palmitic, which are abundant in EG, could have played a significant role in the reduction potential by increasing the cell membrane permeability of microorganisms associated with methanogenesis . Accordingly, the combination of different modes of action represented by EG and AT led to a synergistic effect in inhibiting methanogenesis, which was obvious with the mixture of EG 25% in the basal diet (as a feed) + AT 1% of the basal diet (as a feed additive), which achieved up to 40% CH4-reduction compared with their potential as individual supplementations: 11% for EG 25%, and 21.3% for AT 1%. Similarly, the combination EG 10% + AT 1% had a slight synergistic effect, leading to a 29.9% reduction in CH4 production. The anaerobic fermentation of feed by rumen microorganisms results in the production of VFAs, which are the major energy sources for ruminant animals . Any dietary intervention that can lead to the inhibition of methanogenesis at the expense of decreased VFA production is undesirable. The effect of AT supplementation on total VFA production varies in the literature, with some authors reporting a decrease , while others report no effect . The main reason for this discrepancy is attributed to the dosages used and the bromoform concentration . In the current study, AT supplementation at an effective dosage of 2.5% (0.112 mg bromoform/g DM feed incubated) with 80% CH4-reduction potential had no significant adverse effect on total VFA production, which might be related to the concentration of bromoform being lower than that reported in other studies where bromoform was 6.6 mg/g and 7.8 mg/g . Adding AT favored the fermentation pathway toward propionogenesis, which has been considered as a major hydrogen sink alternative to methanogenesis. This finding was also observed in other studies that evaluated the impact of AT on the fermentation profile . The production of total VFA was not adversely affected by EG up to 25% inclusion. Although there was a CH4-reduction with EG inclusion, the concentration of propionate did not increase. This may indicate that the propionogenic pathway is not the reason for the inhibition of CH4. Therefore, decreasing the fermentation rate by inhibiting cellulolytic bacteria, resulting in less hydrogen and CO2 being available for methanogenesis, might be the explanation for the inhibition of CH4 in this study. This theory is further supported by the lower production CO2 with increasing EG levels. The IVDMD was not affected by the effective level of AT, as the fermentation rate was not inhibited. The increased IVDMD with EG inclusion might be due to the lower fiber content and higher content of digestible nutrients, as reported previously . However, this is contradictory to the production of total VFA in the current study, which is the same as that observed in a previous in vitro trial conducted by Aemiro et al. . Another possible explanation for the increased IVDMD could be the loss of some fine EG particles from the nylon bag during the incubation and washing procedures. Therefore, to confirm this theory, further experiments must be conducted with bags with smaller pore sizes to ensure that there is no loss of EG from the bag. It is noteworthy that digestibility-associated parameters for EG as a feed should be interpreted with caution; therefore, we only considered yield (mL/g) of CH4/DM in our comparisons. The higher concentration of NH3-N with EG 25% might be attributed to the high CP content of EG, which may have stimulated the activity of proteolytic rumen microbes. This nitrogen source can be utilized as a precursor for amino acids and for the synthesis of microbial proteins . 5. Conclusions The current study demonstrated the high nutritive value of EG as an alternative high-quality feed to replace concentrate up to a 25% inclusion level in a ruminant diet without adverse effects on ruminal fermentation characteristics. This strategy was accompanied by a reduction in the CH4 yield of up to 11%. The AT harvested from Japan, when supplemented to a ruminant diet as a feed additive at 1 and 2.5% of the basal diet, led to a CH4-reduction of up to 21 and 80%, respectively. As a novel intervention, the combination of 25% EG as a feed and 1% AT as a feed additive had a synergistic effect, reducing CH4 production by up to 40%. This intervention has double-sided benefits, providing high-quality alternative feed and reducing CH4 with less AT. This study should be the first step toward further in vivo trials to ensure the efficacy of the mixture. Acknowledgments The authors would like to acknowledge Takuma Mezaki, Director of the Kuroshio Biological Research Foundation, Japan, for providing the AT used in this research. Additionally, many thanks are given to Kazutaka Umetsu, Masaaki Hanada, and Naoki Fukuma at Obihiro University for facilitating the use of their resources. We would also like to thank Belgutei Batbekh, a PhD student in Nishida's lab, for his assistance during this experiment. Author Contributions E.A.: Project administration, Conceptualization, Methodology, Investigation, Formal analysis, Visualization, Writing--Original Draft. T.N.: Supervision, Validation, Resources, Writing--Review and Editing. K.S.: Resources, Writing--Review and Editing. All authors have read and agreed to the published version of the manuscript. Institutional Review Board Statement The experimental procedures of this study were approved by animal care and ethics committee at the Obihiro University of Agriculture and Veterinary Medicine, Japan (approval number, 21-41). Informed Consent Statement Not applicable. Data Availability Statement The datasets supporting reported results in this study are available on reasonable request from the corresponding author. Conflicts of Interest K.S. is an employee of Euglena Co., Ltd. The authors declare no conflict of interest. animals-13-00796-t001_Table 1 Table 1 Chemical composition (% in dry matter) used for 24 h in vitro incubation. % Kleingrass Hay Concentrate Mixture Euglena gracilis Asparagopsis taxiformis Organic matter 89.19 93.73 95.56 41.74 Crude ash 10.81 6.27 4.44 58.26 Crude protein 14.58 18.83 26.10 13.28 Ether extract 3.68 4.32 17.65 2.31 Neutral detergent fiber 62.52 31.48 0.60 21.99 Acid detergent fiber 33.82 11.69 0.50 9.16 Acid detergent lignin 6.22 2.54 0.00 4.59 Non-fiber carbohydrates 8.41 39.1 51.21 4.16 Ingredients of the concentrate mixture (%) Corn 41.0 Wheat 5.0 Soybean meal 14.0 Rapeseed meal 11.0 Corn gluten 17.0 Dried distiller's grains with solubles 7.0 Wheat bran 1.0 Molasses 1.5 Calcium carbonate 1.5 Vitamin and mineral complex 1.0 animals-13-00796-t002_Table 2 Table 2 Amino acids profile (%) of Euglena gracilis. Amino Acid % In Amino Acid Profile Arginine 6.99 Lysine 7.28 Histidine 2.74 Phenylalanine 4.78 Tyrosine 4.20 Leucine 8.69 Isoleucine 4.20 Methionine 2.29 Valine 6.69 Alanine 7.44 Glycine 5.20 Proline 6.32 Glutamic acid 11.98 Serine 4.24 Threonine 4.99 Asparagine 8.48 Tryptophan 1.87 Cysteine 1.62 animals-13-00796-t003_Table 3 Table 3 Fatty acids profile (%) of Euglena gracilis. Fatty Acid % In Fatty Acid Profile 10:0 0.5 12:0 7.4 13:0 8.0 14:0 41.0 14:1 0.4 15:0 2.5 16:0 8.8 16:1 1.7 16:3 0.9 17:0 0.5 17:1 0.8 18:0 1.5 18:1 3.9 18:2n - 6 1.7 18:3n - 3 1.1 20:1 0.2 20:2n - 6 1.6 20:3n - 6 3.1 20:3n - 3 0.2 20:4n - 6 2.9 20:4n - 3 1.0 20:5n - 3 0.6 22:4n - 6 2.6 22:5n - 6 0.9 22:5n - 3 0.2 unknown 6.0 animals-13-00796-t004_Table 4 Table 4 Chemical composition (% in dry matter) of the experimental treatments with the combination of Euglena and Asparagopsis. % Control Euglena gracilis Asparagopsis taxiformis Euglena gracilis (EG) + Asparagopsis taxiformis (AT) 0% 10% 25% 1% 2.5% EG 10% + AT 1% EG 10% + AT 2.5% EG 25% + AT 1% EG 25% + AT 2.5% Dry matter 89.24 90.03 91.23 89.24 89.26 90.03 90.04 91.22 91.20 Organic matter 91.46 91.64 91.92 90.97 90.25 91.15 90.43 91.42 90.69 Crude ash 8.54 8.36 8.08 9.03 9.75 8.85 9.57 8.58 9.31 Crude protein 16.71 17.43 18.52 16.67 16.62 17.39 17.33 18.47 18.39 Ether extract 4.00 5.33 7.33 3.98 3.96 5.30 5.26 7.28 7.21 Neutral detergent fiber 47.00 43.91 39.28 46.75 46.39 43.69 43.38 39.11 38.86 Acid detergent fiber 22.76 21.64 19.96 22.62 22.42 21.51 21.33 19.85 19.69 Acid detergent lignin 4.38 4.13 3.75 4.38 4.39 4.13 4.14 3.75 3.77 Non-fiber carbohydrates 23.76 24.97 26.78 23.56 23.28 24.76 24.46 26.56 26.23 animals-13-00796-t005_Table 5 Table 5 Effect of Euglena, Asparagopsis, and their combination on gas production profile from 24 h in vitro incubation (n = 12). Treatments 1 Polynomial Contrast Contrasts between Treatments Parameter Control AT 1% AT 2.5% EG 10% EG 25% EG 10% + AT 1% EG 10% + AT 2.5% EG 25% + AT 1% EG 25% + AT 2.5% SEM AT EG AT x AT + EG EG x AT + EG AT 1% x AT 1% + EG (10% and 25%) Total Gas/DM 2 (mL/g) 123.99 ab 125.32 a 117.88 abc 118.91 abc 109.24 cd 114.36 bc 109.21 cd 102.60 de 93.15 e 1.46 Ns L <0.001 <0.001 <0.001 Total gas/DMD 3 (mL/g) 217.02 a 208.32 ab 201.82 bc 187.44 cd 157.41 e 188.78 cd 175.13 d 148.20 ef 139.57 f 2.83 L L <0.001 0.003 <0.001 CH4 (%) 7.32 a 5.69 b 1.46 c 7.29 a 7.38 a 5.55 b 1.93 c 5.27 b 1.70 c 0.24 L Q Ns 0.89 <0.001 0.33 CO2 (%) 92.68 c 94.32 b 98.54 a 92.71 c 92.62 c 94.45 b 98.07 a 94.73 b 98.30 a 0.24 L Q Ns 0.89 <0.001 0.33 CH4/DM (mL/g) 9.11 a 7.17 bc 1.81 d 8.71 a 8.11 ab 6.39 c 2.20 d 5.46 c 1.66 d 0.31 L Ns 0.089 <0.001 0.01 CH4/DMD (mL/g) 15.92 a 11.86 c 2.97 e 13.71 b 11.71 bc 10.51 c 3.45 e 7.88 d 2.51 e 0.49 L L 0.004 <0.001 <0.001 CO2/DM (mL/g) 114.88 ab 118.15 a 116.07 ab 110.21 abc 101.13 cd 107.97 b 107.00 bc 97.14 d 91.49 d 1.29 Ns L <0.001 0.02 <0.001 CO2/DMD (mL/g) 201.10 a 196.46 a 198.85 a 173.73 b 145.71 c 178.26 b 171.68 b 140.32 cd 137.06 d 2.60 Ns L <0.001 0.22 <0.001 1 EG: Euglena gracilis; AT: Asparagopsis taxiformis. 2 DM: Dry matter. 3 DMD: Dry matter degraded. SEM: Standard error of the mean. a-f Values with different superscripts in the same row are significant different among different groups by Tukey test (p < 0.05). L: Linear (p < 0.05); Q: Quadratic (p < 0.05); Ns: Not significant (p > 0.05), by orthogonal polynomial contrasts. animals-13-00796-t006_Table 6 Table 6 Effect of Euglena, Asparagopsis, and their combination on ruminal fermentation characteristics from 24 h in vitro incubation (n = 12). Treatments 1 Polynomial Contrast Contrasts between Treatments Parameter Control AT 1% AT 2.5% EG 10% EG 25% EG 10% + AT 1% EG 10% + AT 2.5% EG 25% + AT 1% EG 25% + AT 2.5% SEM AT EG AT x AT + EG EG x AT + EG AT 1% x AT 1% + EG (10% and 25%) pH 6.58 ab 6.56 bc 6.53 c 6.58 ab 6.60 a 6.58 ab 6.56 bc 6.58 ab 6.57 ab 0.01 Ns Ns 0.20 0.35 0.57 IVDMD 2 (%) 57.05 de 60.20 cde 58.28 de 63.54 bc 70.27 a 60.51 cde 62.39 bcd 69.23 a 67.02 ab 0.60 Ns L <0.001 0.03 0.001 Acetate (mmol/L) 257.13 a 255.80 a 236.84 b 258.46 a 255.00 a 252.30 a 237.40 b 248.12 a 228.37 b 4.78 L Ns 0.38 0.002 0.40 Propionate (mmol/L) 59.83 b 65.85 ac 68.75 a 59.54 b 55.92 d 63.31 bc 66.94 a 60.19 b 60.64 b 0.80 L L 0.002 <0.001 0.04 Butyrate (mmol/L) 23.59 bc 24.73 ac 25.70 a 24.29 ab 23.31 b 24.52 ab 25.46 a 23.24 b 23.63 bc 0.29 Ns Ns 0.18 0.55 0.38 Total VFA 3 (mmol/L) 340.56 ab 346.38 a 331.30 b 342.29 ab 334.23 ab 340.13 ab 329.79 b 331.55 b 312.64 c 5.61 Ns Ns 0.16 0.15 0.27 TVFA / DMD 4 (mol/g) 1.34 a 1.29 a 1.27 ab 1.20 bc 1.03 d 1.24 b 1.16 c 1.05 d 1.02 d 0.02 Ns L <0.001 0.88 0.002 Acetate (mol/100 mol) 75.27 b 73.57 d 71.13 f 75.23 bc 76.03 a 73.91 cd 71.68 f 74.57 c 72.79 e 0.26 L L Q <0.001 <0.001 0.01 Propionate (mol/100 mol) 17.71 e 19.18 cd 20.99 a 17.59 e 16.91 f 18.78 d 20.50 a 18.33 d 19.59 c 0.18 L L <0.001 <0.001 0.001 Butyrate (mol/100 mol) 7.03 d 7.25 bcd 7.87 a 7.17 bcd 7.06 bcd 7.32 bc 7.82 a 7.10 cd 7.62 a 0.09 L Ns 0.36 <0.001 0.78 A/P 5 ratio 4.27 b 3.86 d 3.42 e 4.31 b 4.52 a 3.96 d 3.52 e 4.10 c 3.76 d 0.05 L L <0.001 <0.001 0.001 NH3-N 6 (mg/dL) 8.73 c 8.16 c 8.54 c 9.58 bc 11.52 ab 8.50 c 8.48 c 11.87 a 10.83 ab 0.50 Ns Ns 0.18 0.59 0.22 1 EG: Euglena gracilis; AT: Asparagopsis taxiformis. 2 IVDMD: In vitro dry matter digestibility. 3 VFA: Volatile fatty acids. 4 DMD: Dry matter degraded. 5 A/P: Acetate/Propionate. 6 NH3-N: ammonia-nitrogen. SEM: Standard error of the mean. a-f Values with different superscripts in the same row are significantly different among different groups by Tukey test (p < 0.05). L: Linear (p < 0.05); Q: Quadratic (p < 0.05); Ns: Not significant (p > 0.05) by orthogonal polynomial contrasts. Disclaimer/Publisher's Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

PMC10000193

Proximal sesamoid bone (PSB) fracture is the leading cause of fatal musculoskeletal injury in Thoroughbred racehorses in Hong Kong and the US. Efforts are underway to investigate diagnostic modalities that could help identify racehorses at increased risk of fracture; however, features associated with PSB fracture risk are still poorly understood. The objectives of this study were to (1) investigate third metacarpal (MC3) and PSB density and mineral content using dual-energy X-ray absorptiometry (DXA), computed tomography (CT), Raman spectroscopy, and ash fraction measurements, and (2) investigate PSB quality and metacarpophalangeal joint (MCPJ) pathology using Raman spectroscopy and CT. Forelimbs were collected from 29 Thoroughbred racehorse cadavers (n = 14 PSB fracture, n = 15 control) for DXA and CT imaging, and PSBs were sectioned for Raman spectroscopy and ash fraction measurements. Bone mineral density (BMD) was greater in MC3 condyles and PSBs of horses with more high-speed furlongs. MCPJ pathology, including palmar osteochondral disease (POD), MC3 condylar sclerosis, and MC3 subchondral lysis were greater in horses with more high-speed furlongs. There were no differences in BMD or Raman parameters between fracture and control groups; however, Raman spectroscopy and ash fraction measurements revealed regional differences in PSB BMD and tissue composition. Many parameters, including MC3 and PSB bone mineral density, were strongly correlated with total high-speed furlongs. ash fraction computed tomography dual-energy X-ray absorptiometry Raman spectroscopy bone mineral density Harry M. Zweig Memorial FundEquine ResearchNational Science FoundationNSF CMMI 1452852 This research was funded by the Harry M. Zweig Memorial Fund for Equine Research (HLR) and the National Science Foundation NSF CMMI 1452852 (ED). pmc1. Introduction The metacarpophalangeal joint (MCPJ) is the most common site for fracture in Thoroughbred racehorses , and proximal sesamoid bone (PSB) fracture is the leading cause of fatal musculoskeletal injury in Hong Kong and the US . Prior studies have evaluated exercise history and PSB morphology using radiography and computed tomography (CT) to identify risk factors and imaging features associated with PSB fracture. Bone volume fraction , bone mineral density , sclerosis , and focal osteopenic lesions have been investigated as potential imaging features associated with catastrophic PSB fracture . Here, we investigated multiple modalities for assessing metacarpophalangeal joint pathology and bone mineralization in Thoroughbred racehorses sustaining catastrophic PSB fracture and controls, including dual-energy X-ray absorptiometry (DXA), computed tomography (CT), Raman spectroscopy, and ash fraction measurements. In humans, bone mineral density (BMD) is the most important quantitative predictor of fracture risk . UK racehorses sustaining lateral condylar fracture of the third metacarpal bone (MC3) had increased MC3 BMD adjacent to the fracture site as compared to the non-fractured condyle or non-fracture racehorse control MC3 . Conversely, in New Zealand racehorses, MC3 BMD did not differ between horses with condylar fractures and non-fracture controls but was greater in horses with more exercise . In New York racehorses, microCT-derived PSB bone volume fraction (BVF) was greater in horses sustaining catastrophic PSB fracture as compared to controls . Similarly, voxel-based morphometry revealed increased PSB BMD in Hong Kong racehorses with catastrophic PSB fracture as compared to controls . In California racehorses, a focal subchondral region of osteopenia and decreased tissue mineral density was identified in the abaxial, subchondral region of medial PSBs from racehorses that sustained catastrophic PSB fracture which was hypothesized to predispose to PSB fracture . Imaging modalities capable of quantifying bone mineral content in vivo include dual energy X-ray absorptiometry (DXA) and quantitative computed tomography (CT). DXA is the most common osteoporosis screening method in women , and DXA has demonstrated comparable accuracy to ash fraction measurements for quantifying bone mineral density of the mid-MC3 in an equine cadaver study . Likewise, a portable DXA device was used to measure BMD in the live, standing horse, suggesting that DXA could be adapted to equine practice for in vivo detection of BMD changes . Raman spectroscopy is being investigated for clinical use to predict age-related fragility fractures in humans and has been used to evaluate the equine MC3 in cadaveric tissues as well as in vivo . Radiography has been used to evaluate equine MCPJ pathology, including changes associated with palmar osteochondral disease (POD) and fetlock fractures . Prior studies have identified gross , radiographic , and histopathologic evidence of osteoarthritic changes associated with PSB fracture, including PSB discoloration; PSB radiolucency and subchondral bone osteopenia, osteophytosis, and enlarged vascular channels; and PSB articular cartilage fibrillation and chondrone formation. Interestingly, a cadaveric magnetic resonance imaging (MRI) study revealed that horses sustaining PSB fracture were more likely to have evidence of orthopedic disease in the contralateral limb compared to non-fracture controls, including subchondral bone densification, disruption of the subchondral bone plate or articular cartilage of the palmar distal MC3 condyle, and intraarticular osteochondral fragmentation . The objectives of this study were to investigate PSB density and mineral content using multiple modalities, including dual-energy X-ray absorptiometry (DXA), computed tomography (CT), Raman spectroscopy, and ash fraction measurements; and to investigate bone quality and MCPJ pathology in Thoroughbred racehorses with and without catastrophic PSB fracture. We hypothesized that horses sustaining PSB fracture would have greater bone mineral content and inferior bone quality as compared to controls and that cumulative high-speed furlong exercise would be predictive of bone volume fraction and bone mineral density measurements. 2. Materials and Methods 2.1. Study Population Forelimbs from Thoroughbred (TB) racehorses that died or were euthanized on New York racetracks were ethically obtained and chronologically enrolled in the study from 2019 to 2020. The study population consisted of 29 animals, including cases that sustained unilateral PSB fracture (n = 14, Supplemental File S1: Table S1A) and controls that died or were euthanized due to reasons unrelated to MCPJ fracture (n = 15, Supplemental File S1: Table S1B). Horses with bilateral fractures and horses with concurrent fracture of the distal MC3 or proximal P1 were excluded from the study. The fracture group consisted of 14 horses, including 8 females, 3 castrated males, and 3 intact males which ranged in age from 2 to 6 years with a median age of 3 years. The median age at death was 3 years and ranged from 2 to 6 years. The fracture group included horses that sustained a fracture of one or both PSBs in one forelimb, while bones in the contralateral limb remained intact (n = 14, 8 left forelimb fractures, 6 right forelimb fracture). The control group consisted of 15 horses, including 5 females, 7 castrated males, and 3 intact males which ranged in age from 2 to 6 years, with a median age of 4 years. The median age at death was 4 years and ranged from 2 to 6. The control group contained horses that were euthanized or died due to disease or injury unrelated to the fetlock joint including: cardiovascular collapse (n = 4), laminitis (n = 4), head trauma (n = 2), colic (n = 2), sudden death (n = 1), carpal fracture (n = 1), humeral fracture (n = 1). For the epidemiologic portion of the study, the mean percentage of exercise-related fatalities for 2-year-old horses during the 6-year period leading up to the 2020 Saratoga race meet was compared with that for the 2020 Saratoga race meet. 2.2. Training and Racing Exercise History For each fracture case and control, training and racing exercise history was curated using information provided by the New York State Gaming Commission (NYSGC). Data collected included total high-speed furlong workouts, defined as any session in which an animal runs one furlong in 14 s or faster (~14.3 m/s or faster). Total furlongs is a race training factor that can be altered in future training methods and has previously been used as a measure of exercise history for evaluating catastrophic PSB fracture . Additionally, from the exercise history provided, career duration (weeks), total weeks of rest, total weeks of work, total career furlongs, total number of races, number of breaks greater than 8 weeks of no work, and career work to rest ratio were calculated. The exercise-related fatality rate at Saratoga racetrack during the 2020 race meet was sorted by age and compared to the mean exercise-related fatality rate sorted by age from the 2014-2019 Saratoga race meets, using the New York State Gaming Commission Equine Breakdown, Death, Injury and Incident Database. 2.3. Imaging Acquisition and Processing All limbs were transected at the level of the mid-radius for imaging and stored at 4 degC for <24 h while awaiting imaging and dissection. 2.3.1. Dual-Energy X-ray Absorptiometry All limbs were analyzed using a Discovery A/216 DXA scanner (Hologic Canada ULC, Mississauga, ON, Canada). Images were acquired within a 42 cm sagittal plane that included the distal MC3, both PSBs, and the proximal first phalanx (P1). Images were analyzed using onboard software. Measurements included the area and radiographic bone density (g/cm2) of the distal MC3 condyle, the palmarodistal MC3 condyle, and the PSBs. 2.3.2. Computed Tomography Acquisition Images of all forelimbs (total n = 58: fracture n = 14, fracture contralateral n = 14, control n = 15, and control contralateral n = 15) were acquired with the limb's long axis perpendicular to the scan axis. The image acquisition was centered on the PSBs and extended approximately 30 cm proximally (distal 1/3 of MC3) and distally (proximal 1/3-1/2 of the proximal phalanx, P1) from the metacarpophalangeal joint using 16-slice helical computed tomography (Toshiba/Canon Aquilion Large-Bore, Toshiba/Canon America Medical Systems, Tustin, CA, USA). Images were acquired using 135 kVp with a scan field of view 240 mm and a standard reconstruction kernel. Images were reconstructed using 0.5 mm slice thickness at a 0.3 mm slice interval, reconstruction field of view 240 mm, pixel dimensions ranging from 0.296 to 0.468 mm, and 512 x 512 display matrix. All images were reconstructed in a bone filter, displayed with window width 4500, window center 1100, and variable tube current (SURE ExposureTM). 2.3.3. Computed Tomography Manual Analysis Images were exported and analysed using HorosTM (Nimble Co. LLC d/b/a Purview, Annapolis, MD, USA). The proximal first phalanx (P1), PSBs, and distal MC3 were assessed for signs of pathological changes, including osteophytes, osteochondral fractures or fragmentation, enthesiophytes, subchondral lysis, and subchondral cyst-like lesions. Distal MC3 measurements included areas of the medial and lateral condyles, depth, and area of sclerosis of the condyles, radiographic bone density as measured in Hounsfield Units (HU) of the complete condyle and of the sclerotic region, and standard deviation of the radiographic bone density of the distal condyle and sclerotic region in a sagittal plane centered on each condyle. All intact PSB bones were measured for maximum height, width, and depth. Area, radiographic bone density, and standard deviation of radiographic bone density were measured for all intact PSBs in the following planes: apical, mid-body, basilar, axial, mid-sagittal, abaxial, sub-condylar, medullar, and flexor (Supplemental File S2). Fractured PSBs were not analyzed. Proximal P1 measurements included the depth of subchondral bone sclerosis measured in the frontal plane at the level of the sagittal grove and the medial and lateral thirds of the bone. 2.3.4. Computed Tomography Radiomics Analysis DICOM images were exported to 3D Slicer (v4.11) (Irvine, CA, USA) for subsequent segmentation and analysis ("3D Slicer", n.d.). For each intact forelimb, four regions of interest (ROIs) were drawn on each forelimb scan: the visible MC3 within the image volume, the visible portion of proximal P1, and the medial and lateral PSBs. Because many CT scans did not include the entire MC3 or P1, two additional regions of interest were created for the MC3 and P1 bones that extended from the subchondral surface of the bone to approximately 5 cm either proximally or distally to the joint. Finally, a 15 mm spherical volume entirely contained within the PSBs was drawn, resulting in a total of eight ROIs analyzed. These ROIs are hereafter referred to as PSBs, MC3-Full, MC3-Partial, P1-Full, P1-Partial, and PSBs-15 mm. Bone mineral density (BMD), bone volume fraction (BVF), and total mineral density (TMD) were extracted from the CT data with scripts written in Matlab, using Otsu's method for thresholding image data . The conversion of CT Hounsfield Units (HU) to K2HPO4 density was performed by sampling the voxel intensities over a 1 cm diameter by 2 cm long cylinder within the 2 cm diameter phantom plugs, and an average HU to K2HPO4 density (mg*cm-3) calibration curve was applied to the CT data. Tabularized data of features were exported and analyzed in JMP. 2.4. Dissection and Gross Examination Following imaging, distal limbs from both fracture and control groups were dissected by a board-certified pathologist (SPM). Pathology descriptions included macroscopic observations of joint disease, including articular cartilage damage to the MC3, PSBs, and proximal P1 (none, mild, moderate, or severe) and the presence of osteochondral fragmentation. For fracture limbs, cartilage damage that was believed to be the result of trauma from the acute sesamoid fracture fragment was excluded to describe the health of the joint prior to fracture. Medial and lateral metacarpal condyles were assigned a grade for palmar osteochondral disease (POD), based on the degree of discoloration of the subchondral bone and disruption of overlying articular cartilage. Grades were assigned as follows: grade 0, no evidence of POD; grade 1, discoloration or subchondral bone without disruption of overlying articular cartilage; grade 2, discoloration of subchondral bone plus mild to moderate disruption of overlying articular cartilage; and grade 3, discoloration of subchondral bone plus collapse of overlying articular cartilage. As previously reported, the mean POD grade for each horse was calculated and reported as a categorical outcome ranging from 0 to 3 . Following necropsy, PSBs were analyzed for fracture configuration and abaxial subchondral bone lesions characterized by discoloration or an articular cartilage or subchondral defect as recently described by Shaffer et al. . The distal MC3, PSBs, and P1 were stored in saline-soaked gauze at -20 degC until further processing. 2.5. PSB Sectioning and Processing The medial sesamoid of the contralateral limb of the fracture group (n = 8) and the medial sesamoid of a randomly assigned limb in the control group (n = 8) were selected for Raman spectroscopy. Samples were cut into five parasagittal sections using a low-speed saw (Buehler IsoMet, Buehler, Lake Bluff, IL, USA) and stored at -20 degC until further processing. A 2 to 4-mm-thick section just axial to the mid-sagittal section was selected for Raman spectroscopy to maximize the surface area. These sections were adhered to a metal sample holder using mounting wax (Allied High Tech Products Inc., Rancho Dominguez, CA, USA) and polished on an automated/semi-automated polishing system (MultiPrepTM, Allied High Tech Products, Inc., Rancho Dominguez, CA, USA), using decreasing particle sizes of diamond wafer paper (30-micron to 3-micron, Allied High Tech Products, Inc., Rancho Dominguez, CA, USA), followed by 0.3-micron alumina slurry (Allied High Tech Products, Inc., Rancho Dominguez, CA, USA). Samples were sonicated in isopropyl alcohol to remove abrasive particles, removed from the sample holder, wrapped in lens paper, and stored at -20 degC in saline-soaked gauze until the time of characterization with Raman spectroscopy. 2.6. Raman Spectroscopy Raman spectra were acquired from the polished surfaces of mid-sagittal sections of the PSB. A confocal Raman microscope (WITec Alpha300R, WITech Instruments Corp., Ulm, Germany) with a 785 nm diode laser; 20x, 0.5 N.A. objective (Zeiss, Jena, Germany) and software that enables imaging guided by surface topography (TrueSurface Microscopy, WITech Instruments Corp., Ulm, Germany) was used to collect the spectra. The spot size was ~1900 nm. Nine rectangular regions in a 3 x 3 grid were identified on the sample surface and grouped for subsequent analysis both in the proximal-distal direction, to form the subchondral, medullary and flexor regions and in the dorsal-palmar direction to form the apical, midbody and basilar regions . Three spectra were collected in each region in a line along the proximal-distal direction with a 1000 mm spacing between each collection point. Thus, a total of 27 spectra were collected from each sample. For each spectrum, the total acquisition time was 30 s. Background fluorescence was subtracted from the Raman spectra using commercial software (WITec Project FIVE, WITech Instruments Corp., Ulm, Germany). All spectra were normalized to the n1PO4 peak (960 cm-1). Raman spectra were analyzed using custom code (Matlab, Mathworks, Natick, MA, USA) developed to calculate Raman compositional parameters (Supplemental File S3: Table S1) from integrated peak areas that characterize the bone mineral and matrix (Supplemental File S3: Table S2). The positions of several features that characterize the collagen matrix were determined through second derivative spectroscopy, including glycosaminoglycans (GAGs, derived from CH3 deformation [1365-1390 cm-1]) , extracellular matrix (derived from methylene side chains [CH2] deformation (1450 cm-1) , the mature trivalent enzymatic crosslink Pyridinoline (PYD, 1660 cm-1) , the immature divalent enzymatic crosslink dehydrodihydroxylysinonorleucine (de-DHLNL, 1690 cm-1) , and the AGE pentosidine (PEN, 1495 cm-1) . After initial peak positions were determined from the second derivative spectra, Gaussian functions were fit to the Raman spectra using spectroscopy software (GRAMS/AI, Thermo Galactic, Waltham, MA, USA) . A non-linear least squares method was used to find the final peak position of each peak with a +-5 cm-1 window . 2.7. Ash Fraction Three to four mm sagittal sections of the medial PSB from each case (n = 29, n = 14 fracture, and n = 15 control) were further sectioned into nine sub-regions: apical flexor, apical medullary, apical subchondral, mid-body flexor, mid-body medullary, mid-body subchondral, basilar flexor, basilar medullary, and basilar subchondral using a low-speed saw (IsoMet, Buehler, Lake Bluff, IL, USA). Each sub-region was individually placed into an Eppendorf microcentrifuge tube and stored at -70 degC prior to ash fraction measurements. Initial weights for each of the nine PSB sections were recorded. New microcentrifuge tubes were obtained for each bone section and filled with acetone such that the volume was approximately 10x the volume of the bone. Bone sections were individually submerged, and microcentrifuge tubes were sealed with laboratory film (Parafilm). The tubes were placed within a fume hood in end-over-end rockers for 72 h, and the acetone in the microcentrifuge tubes was replaced every 24 h. Bone sections were subsequently removed from acetone and placed in individual porcelain crucibles (High-Form Porcelain Crucibles, Fisherbrand, Leicestershire, UK) using thumb forceps and dried in an oven (Horizontal Air Flow 1370 FM, VWR Scientific Products, Radnor, PA, USA) at 60 degC for 23 h, followed by measurement of dry weight (scale description and manufacturer here). The sections were ashed in a muffle furnace (Type 1300 Furnace, Barnstead Thermolyne Corp., Dubuque, TX, USA) at 600 degC for a period of approximately 18 h. After cooling in a desiccator (Pyrex, Corning, NY, USA) for approximately one hour, a final ash weight was recorded. Ash fraction measurements were calculated by dividing weight by ash weight. Percent mineral by weight was calculated by dividing ash weight by dry weight and multiplying by 100. 2.8. Statistical Analysis To compare exercise variables between fracture and control groups, a paired t-test was performed for career duration, weeks of rest, weeks of work, total furlongs, total number of races, number of training breaks of >=8 weeks, and the ratio of work weeks to rest weeks. To determine associations between age and exercise variables, Pearson's correlation coefficients were calculated for age and these same exercise variables. To validate the repeatability of the measurements manually acquired from CT images by a boarded radiologist (IRP) and an equine veterinarian (KJN), an intra-class correlation coefficient (ICC) analysis was performed. All nominal and ordinal data were assessed for normality. Nominal data were analyzed for differences between fracture and control groups using Fisher's Exact tests, and ordinal data were analyzed using ChiSquare likelihood ratios. Continuous data obtained from DXA, CT, and ash fraction measurements were compared using linear mixed-effects models. The fixed effects consisted of group (fracture vs. control), sex, and high-speed furlongs (HSF). The random effects included horse, limb (left vs. right), and, when appropriate, laterality (medial vs. lateral). Radiomic features for the medial and lateral PSB were examined together when exploring differences in the case and control image sets, as previously described . Linear mixed-effects models were also used to compare Raman parameters across the fracture and control groups and across the anatomic regions (Supplemental File S4). Comparisons across anatomic regions were performed using three models: a model that included all nine anatomic regions and two additional, simplified models that included three anatomic regions grouped in the dorsal-palmar direction (creating subchondral/medullary/flexor regions) or in the proximal-distal direction (creating apical/midbody/basilar regions) . Fixed effects included group (fracture vs. control), sex, total high-speed furlong workouts (HSF), region and the interaction between region and group; random effects included the sample to account for multiple spectra collected within each animal. A Tukey posthoc test was used for multiple comparisons of the estimated marginal means of the regions. The significance level was set to p = 0.05. Raman analyses were performed using R studio (R Studio, PBC, Boston, MA, USA). Finally, a multivariate analysis with pairwise correlations was performed to compare between modalities and reported using Pearson's correlation coefficients. This included a comparison between DXA radiographic bone density of the sesamoids in the lateral projection, CT-derived Hounsfield Units of the mid-sagittal PSB, radiomics derived bone mineral density of the mid-sagittal PSB, and ash fraction. To maintain independence of observations and assess correlations across modalities, mean values for combined left and right forelimbs were calculated as appropriate. All analyses were performed in JMP Pro 16 (Cary, NC, USA) unless stated otherwise. 3. Results 3.1. Study Population Within the fracture group, 13 of 14 horses (92.9%) sustained fractures to both the medial and lateral proximal sesamoid bones, while one horse fractured only the medial PSB. Of the fractured sesamoids, 12 were fractured in the basilar region, 7 in the mid-body region, and 1 in the apical region. For the 2014 through 2019 Saratoga race meets, 2-year-old horses represented a mean of 29% of the total number of exercise-related fatalities. During the 2020 Saratoga race meet, 2-year-old horses represented 82% of the total number of exercise-related fatalities. This represents a 183% increase in 2-year-old horse exercise-related fatalities in 2020. 3.2. Exercise Comparisons between Fracture and Control Groups Exercise histories were available for 28 of the 29 horses in this study; training data were not available for one case except for cumulative total furlongs. Exercise history data were similar for fracture cases and controls (Table 1). 3.3. Association between Age and Exercise Exercise variables, including career duration, total weeks of rest, total weeks of work, total furlongs, and the total number of races, were strongly positively correlated with horse age (Supplemental File S5). As a result of the strong correlation between exercise variables and age, the total furlongs measure was selected as the variable to analyze in regression models . 3.4. Intra-Class Correlation Coefficients The majority of intraclass correlation coefficients (ICC) for manual CT parameters identified moderate (0.5 to 0.75 ICC) to good (0.75 to 0.9 ICC) correlation between the two observers. For example, ICCs ranged from a high of 0.91 for frontal plane measurements of the PSB midbody area to a low of 0.35 for the mean area of sclerosis of the MC3 condyle believed to be due to the subjective nature of this area measurement. Complete ICC values are reported in Supplemental Materials (Supplemental File S6). 3.5. Effect of Group: Fracture versus Control 3.5.1. Pathologic Features Palmar osteochondral disease (POD), defined as a gross POD score of >=1, was detected in 19 out of 22 (86.4%) horses in which necropsy POD score data were available. Of these 22 horses, 12 were fracture cases and 7 were controls. Grossly, no differences were noted for POD scores between fracture and control cases (p = 0.20). Discoloration of PSB articular cartilage was present in 7/14 fracture cases and 8/15 control cases (p = 1.0). 3.5.2. Imaging Features DXA bone mineral density measurements of the distal MC3 condyle, palmar distal region of MC3, and the PSBs did not identify any differences between fracture and control groups. However, the CT radiographic density of the basilar PSB region was more homogenous in fracture cases as compared to controls (fracture: 1211.7 +- 61.9, control: 1184.8 +- 70.7 HU; p = 0.049) which could indicate sclerosis. Dorsal cavitation of MC3 at the level of the proximal MCPJ was identified in 7 out of 15 control horses. Interestingly, this feature was not observed in any horses sustaining PSB fracture (p < 0.0001) . No differences in osteophytes, enthesiophytes, or subchondral lysis of MC3, P1, or the PSBs were observed between fracture and control groups (Supplemental File S6). 3.5.3. Ash Fraction Whole bone PSB percent mineral was greater than tuber coxae percent mineral (60.2 +- 1.3% versus 53.4 +- 3.9% (Supplemental File S7). The PSB basilar medullary region had 0.48% less mineral in fracture cases as compared to controls (p = 0.02). Percent mineral measurements for the remaining eight sections did not differ between groups (p = 0.07 to 0.96). Regional comparison of all PSB mid-sagittal sections combined identified 2.44% great mineral in the medullary region compared to subchondral region (p < 0.001) and 4.01% greater mineral that the flexor region (p < 0.001). Additionally, the midbody region had 2.48% greater mineral compared to the apical region (p < 0.001) and 0.96% greater mineral than the basilar region (p < 0.05) (Supplemental File S9). Tuber coxae samples were available for 26 of the 29 horses, including 13/14 fracture cases and 13/15 controls. The percent mineral of the tuber coxae did not differ between cases and controls (p = 0.94). 3.5.4. Raman Spectroscopy When the effect of group (fracture vs. control) was analyzed, all Raman spectroscopic parameters were similar across groups . The absence of differences between groups was consistently observed across all statistical models regardless of how the region was defined (9 sub-regions, 3 dorsal-palmar regions, or 3 proximal-distal regions), (p > 0.05), . 3.6. Effect of Exercise: Total High-Speed Furlongs 3.6.1. Gross Pathologic Features On gross examination, POD lesions were more severe in horses with more accrued total furlongs, with an increase in 1.0 grade per 100 furlongs (p < 0.0001) . POD score data were not recorded in the necropsy reports of the remaining seven horses, including two fracture cases and eight controls. 3.6.2. Imaging Features Both DXA and CT identified increased BMD in horses with greater total furlongs. Distal MC3 condyle bone mineral density was greater by 0.1 g/cm2 per 100 total furlongs, and the palmar distal region of MC3 was greater by 0.2 g/cm2 per 100 total furlongs (p < 0.0001) . Similarly, increased CT radiographic bone density was detected in the lateral condyle of MC3, with an increase of 2.0 HU per 100 total furlongs accrued (p < 0.0001). Radiomics CT analysis identified that distal MC3 condyle BMD was greater by 0.10 g/cm2 per 100 total furlongs (p = 0.005), and the PSB 15 mm bone mineral density was greater by 0.19 g/cm2 per 100 total furlongs (p = 0.03) . CT identified pathological features of the MC3 condyle associated with more total furlongs, including condylar sclerosis (p < 0.0001) and subchondral lysis (p < 0.0001) . The subchondral lysis score was greater by 0.9 per 100 total furlongs (p < 0.0001). A non-significant trend for greater PSB BMD with increased total furlongs on DXA was observed (p = 0.054) . CT identified increased radiographic bone density in the subchondral region of the PSBs by 16.4 HU per 100 total furlongs accrued (p = 0.04). 3.6.3. Ash Fraction Subchondral mineral content (percent mineral) was greater in PSBs from horses with more accrued total furlongs (Supplemental File S7). The midbody subchondral PSB region had 0.47% more mineral for each additional 100 total high-speed furlongs. No other regional differences in mineral content were detected as an effect of group or exercise, though there was a trend for increased mineral content in the mid-body region as an effect of total furlongs (p = 0.07, Supplemental File S7). Tuber coxae percent mineral did not differ as a function of high-speed furlongs (p = 0.43). 3.6.4. Raman Spectroscopy The carbonate:phosphate ratio (p = 0.054, 9-subregion; p = 0.055, dorsal-palmar; p = 0.053, proximal-distal, Supplemental File S4) demonstrated a trend towards significance as a function of total high-speed furlongs. Specifically, a one-unit increase in high-speed furlongs resulted in a 0.04% increase in carbonate:phosphate ratio. However, the effect of high-speed furlongs was not significant for any other Raman parameters (Supplemental File S4). 3.7. Correlation Statistics To assess correlation metrics of PSB bone mineral density across modalities, Pearson's correlation coefficients were calculated. Ash fraction and DXA (r = 0.24), ash fraction and CT (r = 0.23), manual CT and DXA (r = 0.06), and radiomics CT and DXA (r = 0.14) data were only weakly correlated. Manual and radiomics CT values (r = 0.58) were highly correlated. 4. Discussion Cumulative high-speed furlong exercise was strongly predictive of several bone mineral measurements and metacarpophalangeal joint pathologic changes across all modalities, including DXA, CT, Raman spectroscopy, and ash fraction measurements. BMD in the MC3 condyles and PSBs and percent mineral in the subchondral region of PSBs were greater in horses with more total furlongs. MCPJ pathology, including POD scores, MC3 condylar sclerosis, and MC3 subchondral lysis, was greater in horses with more total furlongs. Contrary to our hypothesis, there were no differences in BMD or Raman parameters between fracture and control groups. MC3 dorsal cavitation was detected in 7/15 control cases but was not detected in horses sustaining PSB fracture. Raman spectroscopy and ash fraction measurements revealed regional differences in PSB BMD and tissue composition, including greater mineral-to-matrix ratios in the subchondral and basilar PSB regions and greater carbonate-to-phosphate ratios in the flexor and apical PSB regions. Taken together, these data suggest that cumulative high-speed furlong exercise is a strong predictor of bone mineralization and joint pathology and that these effects predominate over differences between PSB fracture cases and controls. Prior studies have demonstrated that MC3 and PSB morphologic features and fracture risk vary as a function of exercise . For example, BMD was greater in the MC3 condyles in 2-year-old Thoroughbreds with more exercise , and bone volume fraction of the parasagittal groove of MC3 was greater in Thoroughbreds in training than those in rest . Some differences in morphologic features, especially those related to bone mineral content, could be due to adaptive responses to exercise and may not be pathologic, although the degree that these responses are considered adaptive versus maladaptive is currently unknown. In both humans and horses, bone is highly responsive to exercise , with changes occurring as early as 8 weeks of training . Noble et al. identified differences in volumetric bone mineral density and subchondral bone thickness of the proximal phalanx in racehorses with catastrophic proximal phalanx fractures as compared to both raced and unraced controls, including greater variance in bone mineral density, which may indicate a maladaptive response . More advanced osteoarthritic changes and greater POD scores were associated with more accrued total furlongs in New York racehorses . California racehorses sustaining PSB fracture spent more time in active racing and training, exercised for longer periods prior to their last rest period, exercised at higher intensities during the 12 months prior to their death, and accumulated greater distances in their career than non-fracture racehorse controls . In the UK, PSB fractures were also more common in experienced racehorses with more starts than in those in their first season of racing . In the current study, horses sustaining PSB fracture were approximately evenly divided between <=3-year-old horses and >3-year-old horses, with 3 out of 14 horses (21%) sustaining catastrophic PSB fracture prior to their first race. In addition, there were no differences in measures of total exercise between fracture and control horses. While these findings suggest that exercise history may account for some PSB and MCPJ pathologic changes, they suggest that PSB fracture is not always an injury associated with excessive work. It is likely that some PSB fractures occur in horses that have not sufficiently modeled their PSBs prior to their first race. Of significance, in 2020, New York Thoroughbred racetracks were closed due to the COVID-19 pandemic from the first week in March until the first week in June. During this time, horses were not able to complete their regular training schedules in preparation for the 2020 Saratoga race meet (July 16-August 6). This disruption of high-speed exercise training was associated with a disproportionate increase in exercise-associated catastrophic fractures among 2-year-old Thoroughbred racehorses during the 2020 Saratoga race meet compared with that of previous years. This pandemic-associated anomaly in PSB fracture demographics could potentially account for the absence of differences in bone mineralization parameters identified in this cohort of NY racehorses as compared to prior NY studies . Consistent with prior studies , BMD was greater in MC3 condyles from horses with more total furlongs as measured via DXA, CT, Raman spectroscopy, and ash fraction. In addition, quantitative CT bone mineral density measurements were greater in PSBs from horses with more total furlongs. Since high-speed exercise is known to be a strong stimulus for bone remodeling, it is not surprising that total high-speed furlongs was the predominant explanatory variable for MC3 and PSB BMD in our models. Interestingly, no differences were noted in tuber coxae BMD as a function of group or exercise, suggesting that the effects of high-speed exercise are less pronounced at this axial skeletal site. Within PSBs, increased BMD was most notable in the subchondral region as evaluated by CT and ash fraction. However, no differences were found in whole PSB BMD between fracture cases and controls. These results, combined with previous findings of focal decreased bone mineral density in the abaxial subchondral region of racehorses sustaining catastrophic PSB fracture , suggest that regional differences in bone and mineral properties may be better predictors of fracture risk than whole PSB changes. Palmar osteochondral disease (POD) commonly affects Thoroughbred racehorses, with a reported prevalence as high as 80.4% . In the present study, POD was detected on gross examination at necropsy in 19 out of 22 (86.4%) horses, with greater POD scores in horses with more total furlongs. POD scores have previously been associated with greater cumulative racing and training, suggesting that POD is a progressive disease that results from bone fatigue with repetitive loading . POD lesions have previously been associated with microcracks in MC3 condyle subchondral bone and PSB fracture in racehorses. The only gross pathologic finding that differed between fracture and controls in the current study was dorsal MC3 cavitation, which was present in 7 of 15 controls and no fracture cases. Davis et al. previously described MC3 dorsal cavitation as a reliable radiographic marker of POD ; however, in the present study, dorsal cavitation did not correlate with POD scores nor total high-speed furlong exercise. PSB mineral content and crystallinity did not differ as a function of total furlongs or group. Prior studies with higher-resolution micro-CT suggested that horses sustaining PSB fracture had increased bone volume fraction, leading to the hypothesis that tissue mineralization would also be increased in horses with PSB fracture . Contrary to our hypothesis, bone tissue material properties were similar between fracture and control PSBs, consistent with a previous study that revealed similar BMD in whole bone and axial sub-regions of PSBs between fracture and control groups . When examining the effect of region on material properties within all PSBs (fractures and controls combined), lower mineral content, lower advanced glycation end products (AGEs, including pentosidine and carboxymethylysine), and greater carbonate substitution were observed in the flexor region. In PSBs, the flexor region is subjected to high tensile forces, which may promote remodeling activity and decrease average tissue mineralization and AGEs concentration . Lower tissue mineral and AGEs content are both indicators of younger tissue, while greater carbonate:phosphate ratios may be attributed to the loss of mineral or the increase of younger mineral with more substitution . Lower tissue mineral and AGEs content also may contribute to greater toughness in flexor region, which would enable this region to be more durable under tension . The subchondral region had the lowest carbonate:phosphate ratio, an intermediate mineral:matrix ratio, and trended toward the highest collagen maturity. These observations suggest that the subchondral region may have older tissue , consistent with reduced remodeling activity. Focal lesions and microcracks are more frequently observed in subchondral region and may predispose this site to fatigue fractures. Correlation between modalities was considered weak, whereas CT manual and radiomics BMD measurements were highly correlated. CT provides better morphological and texture resolution than DXA or radiography, and access to standing equine CT units is increasing; however, challenges still exist, including image acquisition at high resolution and unifying image analysis across different platforms. Advances in quantitative analyses of large imaging data sets using radiomics hold significant promise to mine the substantial amounts of data provided by volumetric imaging. Raman spectroscopy is not currently employed in vivo for clinical diagnostics, but several groups are investigating the possible future in vivo use of Raman for bone quality assessment in both humans and horses . Ash fraction is the gold standard for measuring bone mineral content and is considered a superior marker of bone strength compared to radiographic measured bone volume fraction in humans ; however, ash fraction measurements are destructive and require an invasive bone biopsy. Here, ash fraction measurements served as a gold standard for comparison to DXA, CT, and Raman measurements of bone mineral content. Study Limitations Paragraph Limitations of this study included the sample size of 29 horses (58 PSBs). While high-speed furlong exercise data were available for horses training and racing on NY racetracks, exercise history was unavailable for time periods where horses were not training at a NY racetrack facility. This study utilized clinical CT with an imaging resolution ranging from 0.296 mm to 0.468 mm pixels rather than micro-CT with resolutions ranging from 2.8 mm to 80 mm. However, clinical CT has practical application in live horses, especially with the increasing availability of standing CT in practice. Of the total 29 samples, Raman spectroscopy was only performed in 16 horses, and both Raman spectroscopy and ash fraction measurements were performed on a single parasagittal section of the proximal sesamoid bone rather than the whole bone. Despite these limitations, our analyses combined complementary analyses across multiple levels of bone structural hierarchy to clinical specimens. The novel application of Raman spectroscopy to study bone tissue from horses with PSB fractures provided information unobtainable from existing clinical imaging modalities, including mineral properties such as carbonate substitution and matrix properties such as AGEs and glycosaminoglycan (GAGs) content which impact bone mechanical properties and are associated with fracture incidence in humans . 5. Conclusions Cumulative high-speed furlong exercise was strongly predictive of several bone mineral measurements and metacarpophalangeal joint pathologic changes across multiple modalities. However, few differences were detected between fracture and control groups, suggesting that whole bone mineral properties or metacarpophalangeal joint pathology are not alone sufficient for identifying horses at risk of PSB fracture. Recent work suggests that focal or regional changes in bone mineral content, such as the presence of osteopenic subchondral PSB lesions, may be associated with fracture risk . Unfortunately, current equine standing imaging modalities may lack the resolution required to identify changes at this length scale. Functional imaging assessments, such as positron emission tomography (PET), may hold more potential for identifying these small defects through metabolic changes which often precede structural changes observed with CT . PET scanning has been shown to detect radiopharmaceutical uptake in the dorsoaxial articular surface and abaxial border of PSBs and palmar subchondral bone of the lateral MC3 condyles in racehorses which was undetectable by CT or MRI . Nonetheless, PET scanning in horses is recommended to be used in conjunction with CT , and identifying parameters correlated with PSB fracture using both modalities could improve the accuracy of identification of horses at risk for fracture. Acknowledgments The authors thank Erica Bender, Manager of the Cornell University Human Metabolic Research Unit, for providing training and access to the dual-energy X-ray absorptiometry (DXA) equipment. Supplementary Materials The following supporting information can be downloaded at: Supplemental File S1: Details of case (unilateral PSB fracture) and control Thoroughbred racehorses; Supplemental File S2: Manual CT measurement descriptions; Supplemental File S3: Raman peak fitting methods ; Supplemental File S4: Raman results table; Supplemental File S5: Multivariate correlation analysis between exercise variables and horse age; Supplemental File S6: Computed tomography results table; Supplemental File S7: DEXA results table; Supplemental File S8: Ash fraction (reported as % mineral by weight) by region; Supplemental File S9: Ash fraction (reported as % mineral by weight) regional comparison results. Click here for additional data file. Author Contributions Conceptualization and methodology, K.J.N., L.C., S.E.P., I.R.P., P.S.B., E.D. and H.L.R.; software, P.S.B.; formal analysis, K.J.N., L.C., E.D., P.S.B. and H.L.R.; data curation, K.J.N., L.C., S.S., B.P., S.P.M., S.E.P., I.R.P., E.D. and H.L.R.; writing--original draft preparation, K.J.N., L.C., B.D.R., D.C.F., E.D. and H.L.R.; writing--review and editing, K.J.N., L.C., B.D.R., D.C.F., S.E.P., E.D. and H.L.R.; supervision, E.D. and H.L.R.; project administration, E.D. and H.L.R.; funding acquisition, E.D. and H.L.R. All authors have read and agreed to the published version of the manuscript. Institutional Review Board Statement All cadaver tissues are property of the New York State Gaming Commission (NYSGC) as part of the requirement for necropsy. Officials of the New York State Gaming Commission approved the study protocols. Informed Consent Statement Not applicable. Data Availability Statement All relevant data are published within the manuscript and Supplemental Materials. Conflicts of Interest The authors declare no conflict of interest. Figure 1 Representative optical micrographs of the mid-sagittal plane of the proximal sesamoid bone showing the 9 regions into which each specimen was subdivided for regional analysis by Raman spectroscopy. Three spectra (represented by solid circles) were collected within each region. (A) nine rectangular regions with a 3 x 3 grid layout; (B) three dorsal-palmar regions; (C) three proximal-distal regions. Figure 2 Evidence of palmar osteochondral disease (POD) was found on both gross pathology and CT analysis of the distal MC3. (A) Sagittal plane CT image of a control case with dorsal cavitation (indicated by the red arrow) of MC3 at the level of the proximal MCPJ. (B) Gross photograph of a fracture case with grade 3 POD of MC3. (C) Sagittal plane CT image demonstrating the region analyzed for condylar sclerosis measurements (dashed line with arrow indicating depth measured). (D) Sagittal plane CT image depicting distal MC3 subchondral lysis (indicated by red arrow). (E) Dorsal cavitation of MC3 at the level of the proximal MCPJ was identified in 7 out of 15 control horses but was not present in any of the fracture horses. (F) Gross POD scores increased with greater total furlongs. (G) The area of condylar sclerosis was larger in horses with greater total furlongs. (H) Distal MC3 lysis scores were higher in horses with greater total furlongs. Figure 3 Box plots with jittered points of comparing the Raman spectroscopic outcomes of the fracture and control groups (n = 8 samples/group) using the nine sub-region model. In each box, red cross marks indicate the estimated mean value, and horizontal lines indicate the median value of each parameter in each group. All Raman spectroscopic outcomes were similar across groups (Abbreviations: CO3:PO4--Carbonate:Phosphate Ratio; CML--Carboxymethyl-lysine; PEN--Pentosidine; GAGs--Glycosaminoglycans; MMC--Mineral maturity/crystallinity). Figure 4 Regional comparison results within a mid-sagittal section of the proximal sesamoid bone of (A) mineral:matrix ratio across 3 dorsal-palmar regions; (B) mineral:matrix ratio across 3 proximal-distal regions; (C) mineral:matrix ratio across 9 anatomic sub-regions; (D) carbonate: phosphate ratio across 3 dorsal-palmar regions; (E) carbonate: phosphate ratio across 3 proximal-distal regions; (F) carbonate: phosphate ratio across 9 anatomic sub-regions; (G) mineral maturity/crystallinity across 3 dorsal-palmar regions; (H) mineral maturity/crystallinity across 3 proximal-distal regions; (I) mineral maturity/crystallinity across 9 anatomic sub-regions; (J) XLR across 3 dorsal-palmar regions; (K) XLR across 3 proximal-distal regions; (L) XLR across 9 anatomic sub-regions; (M) CML across 3 dorsal-palmar regions; (N) CML across 3 proximal-distal regions; (O) CML across 9 anatomic sub-regions. Results in C were shown as percent difference (fracture vs. control) to account for the significant interaction of group with region, others were shown as grouped means from both fracture and control group (Supplemental File S4) (Abbreviations: MMR--mineral:matrix ratio; CP--carbonate:phosphate ratio; MMC--mineral maturity/crystallinity; XLR--collagen maturity; CML--Carboxymethyl-lysine) (***: p < 0.001; **: p < 0.01; *: p < 0.05). In the right column, there are significant differences across regions with no same letter. Figure 5 Dual-energy X-ray absorptiometry (DXA) analysis of the distal metacarpus (MC3) and PSBs. (A) Distal MC3 bone mineral density (BMD) did not differ between fracture and control groups but was greater in horses with more total furlongs. (B) Proximal sesamoid bone (PSB) BMD did not differ between fracture and control groups nor in horses with more total furlongs, though there was a non-significant trend for greater BMD with more total furlongs. Figure 6 Linear regression analysis of CT images analyzed via radiomics. Bone mineral density (BMD) increased with total furlongs in both the distal third metacarpal bone (MC3) (A) and proximal sesamoid bones (PSB) (B) but did not differ between fracture cases and controls. animals-13-00827-t001_Table 1 Table 1 Exercise variables acquired for fracture and control cases as provided by the New York State Gaming Commission, reported as medians followed by ranges in brackets. Exercise History Variable Fracture Control p-Value Age at first start 2 years (2 years) 2 years (2 to 3 years) 0.16 Career duration 64 (0 to 193) weeks 69 (12 to 212) weeks 0.92 Race starts 7.5 (0 to 28) 5 (0 to 40) 0.95 Cumulative total furlongs 190 (0 to 384) 146 (36 to 520) 0.91 Career work weeks 40.5 (5 to 96) 32 (8 to 95) 0.76 Career rest weeks 34 (2 to 112) 35 (4 to 123) 0.95 Career work:rest ratio 1.25 (0.59 to 2.35) 1.07 (0.54 to 2.45) 0.30 Disclaimer/Publisher's Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

PMC10000194

Cattle and water buffalo are the main livestock species that are raised in the Campania region, southern Italy, and they contribute significantly to the regional rural economy. Currently there are limited data on the prevalence of relevant impact infections, such as bovine coronavirus (BCov), an RNA virus that causes acute enteric and respiratory disease. Although these diseases are described primarily in cattle, there have been reports of spillovers to other ruminants, including water buffalo. Here, we determined the seroprevalence of BCoV in cattle and water buffalo in the Campania region of southern Italy. An overall seroprevalence of 30.8% was determined after testing 720 sampled animals with a commercial enzyme-linked immunosorbent assay. A risk factor analysis revealed that the seropositivity rates in cattle (49.2%) were higher than in water buffalo (5.3%). In addition, higher seroprevalence rates were observed in older and purchased animals. In cattle, housing type and location were not associated with higher seroprevalence. The presence of BCoV antibodies in water buffalo was associated with the practice of co-inhabiting with cattle, demonstrating that this practice is incorrect and promotes the transmission of pathogens between different species. Our study found a considerable seroprevalence, which is consistent with previous research from other countries. Our results provide information on the widespread distribution of this pathogen as well as the risk factors that are involved in its transmission. This information could be useful in the control and surveillance of this infection. BCoV betacoronavirus serosurveillance This research received no external funding. pmc1. Introduction The family of coronaviruses (CoVs) includes several positive-sense and single-stranded RNA viruses that commonly infect wild and domestic animals, as well as humans . In addition to the well-known human CoVs (including severe acute respiratory syndrome-related coronavirus, Middle East respiratory syndrome-related coronavirus, and severe acute respiratory syndrome coronavirus 2), the genus Betacoronavirus lists also bovine coronavirus (BCoV), which is closely related to the above mentioned CoVs and belongs to the order Nidovirales, family Coronaviridae, subfamily Orthocoronavirinae . BCoV RNA includes five structural proteins: the spike, the nucleocapsid protein, the haemagglutinin esterase, the integral membrane, and the envelope protein. BCoV was first isolated in respiratory samples by Mebus et al. in 1972, in the United States and represents a primary enteric and respiratory pathogen that is widespread throughout the world. Namely, this virus causes severe gastroenteric syndrome (namely calf diarrhea and winter dysentery in adults, although it can also occur during warmer months) and plays an important role in the bovine respiratory disease complex (BRDC) . It represents a major health problem in calves and is associated with reduced growth performance and increased mortality, resulting in important economic losses for the livestock industry . BCoV is frequently involved in coinfections; indeed, during respiratory disease, it is frequently identified with a variety of pathogens such as parainfluenza virus-3 (PI-3), bovine herpesvirus-1 (BHV-1), bovine viral diarrhea virus (BVDV), Pasteurella spp., and particularly bovine respiratory syncytial virus (BRSV). During enteric disease, rotavirus (BRV), Escherichia coli, Eimeria spp., Cryptosporidium spp., and other pathogens are frequently found . The diagnosis of BCoV infection is based on nucleic acid detection via reverse transcription polymerase chain reaction (RT-PCR) and real-time quantitative polymerase chain reaction (real-time qRT-PCR) using nasal swab or fecal samples as a matrix, whose sensitivity increases when performed during acute stages of infection. Serological assays, such as the enzyme-linked immunosorbent assay (ELISA), virus neutralization (VTN), and immunofluorescence, are also available to detect specific antibodies. ELISA is usually preferred for intra-herd epidemiological surveys because of its rapidity and applicability for large samples (besides being sensitive and specific). These assays mainly detect antibodies against the dominant epitope (the subunit S1 of the spike protein), whose production begins 7-14 days after the infection . Furthermore, the interest in this virus is increasing due to the susceptibility of ruminants to human coronavirus infection, as well as the potential recombination mechanisms for possible CoV dual infections in a single animal or human . In the last decade, several bovine-like coronaviruses have been identified as potential agents of enteric and/or respiratory diseases in a variety of ruminants, suggesting cross-species transmission and the ability to overcome interspecies barriers . These viruses share genomic and antigenic features with BCoV and are widely considered to be host-range variants of BCoV that have emerged from the continuous evolution of the virus. They are inadvertently transmitted to other ruminants mainly by the fecal-oral routes . Bovine-like CoVs have been described in domestic ruminants (water buffalo, sheep, goats, llamas, alpacas, and camels); wild ruminants (deer, bison); zoo ruminants (antelopes, giraffes); and even non-ruminants such as dogs and humans . BCoV and bovine-like coronaviruses have been detected on all continents, and seroprevalence studies have shown high prevalence at both animal and herd levels . However, studies on the prevalence of BCoV and associated risk factors are limited in Europe, and to date no study has been conducted in Italy, although descriptions of the disease in different animal species are regularly reported . This study focused on the seroprevalence of BCoV infection and assessed its associated risk factors among dairy cattle and water buffalo in the Campania region, southern Italy. 2. Materials and Methods 2.1. Sampling and Study Area The current study was carried out in Campania (410000000 N-143000000 E), a region in southern Italy with the largest water buffalo population. There are 158,000 cattle and 306,000 water buffalo that are raised in this area (Banca Dati Nazionale dell' Anagrafe Zootecnica, accessed on 1 November 2022). Given the lack of recent surveys in the study area, we decided to assume an expected prevalence of 0.5 (i.e., 50% for cattle and 25% for water buffalo which is infected by BCoV-like coronaviruses), an absolute precision of 5%, and a 95% confidence interval . Thrusfield's formula was used to calculate the sample size, which was as follows:n = Z2 x P(1 - P)/d2 where Z = 1.96 for a confidence level of 95%, P = expected prevalence, d = 0.05 accepted error, and n = sample size. Sampling procedures started in October 2020 and were completed in April 2021, coinciding with blood collection conducted for previous studies . The study area included 33 different districts and a total of 22 cattle farms and 24 water buffalo farms (randomly selected) distributed in four provinces (at least 12 animals per farm were sampled). Only unvaccinated farms were sampled, and 419 samples from dairy cows and 301 samples from water buffalo were randomly collected. A vacutainer was used to collect blood samples from the tail vein. After each sample was centrifuged at 1000 g for 10 min, aliquots were stored at -20 degC until they were assayed. 2.2. Antibody Detection with Commercial Indirect Enzyme-Linked Immunosorbent Assay (ELISA) (SVANOVIR BCV-Ab) Each sample was tested for BCoV antibodies (IgG) using the "SVANOVIR BCV-Ab" ELISA Test Kit (Boeringer Ingelheim Svanova, Uppsala, Sweden), following the manufacturer's instructions. This assay was already used in other work described in the literature, as well as during the control program against this disease that was carried out in Norway . In a summary, diluted sera were deposited into the plate and incubated for one hour. Following three washes, conjugate antibody was added and incubated for an additional hour. After 15 min, the substrate solution was added, followed by the stop solution. The optical density was then measured at 450 nm with a spectrophotometer (Thermo Scientific, Carlsbad, CA, USA), and the cut-off value that distinguishes between positive and negative samples was calculated. A map representing the spatial distribution of positive foci was created using Epi Info (EPI InfoTM software version 7.2.5.0, Atlanta, GE, USA). 2.3. Statistical Analysis By dividing the number of positive bovines by the overall number of bovines that were tested, prevalence was expressed at the animal level. Risk factor analysis was conducted using data that were gathered during sample selection. For cattle, information about location, housing, age, and origin was evaluated, whereas for water buffalo, information about location, age, origin, and coexistence with cattle was evaluated. Chi-square statistics were used in univariate analysis at the animal level to determine risk factors for BCoV positivity (expressed as binary variables). A p-value of 0.05 or less was considered significant. The statistical analysis was performed using MedCalc Statistical Software, version 16.4.3 (MedCalc Software, Ostend, Belgium; www.medcalc.org, accessed on 5 November 2022, and JMP version 14.1.0 (SAS Institute Inc., Cary, NC, USA). 3. Results A total of 720 samples from 46 farms were tested for the detection of specific antibodies against BCoV (419 cattle and 301 water buffalo). Among them, 134 had less than twenty-four months (18.6%) and 586 had more (81.4%). A total of 381 animals were born on the farm, whereas 339 were purchased from other farms. The distribution among different provinces was as follows for bovine: 26.7% Avellino (112/419), 23.15% Benevento (97/419), 27% Caserta (113/419), and 23.15% Salerno (97/419). Among cattle, 27.7% were partly grazed, and the remaining part were bred to be stall-fed. Water buffalo were sampled in the two provinces where this species is mainly raised, Caserta (141/301) and Salerno (160/301). Among them, 29.7% co-inhabited with cattle. Supplementary Table S1 summarizes specific descriptive information about the collected data. Our results showed an overall seroprevalence of 30.8% (222/720; CI 95% 27.5-34.2) at the animal level. At the herd level, prevalence was 76% (35/46; CI 95% 63.7-88.4), with 100% (22/22) in cattle and 54.2% (13/24) in water buffalo, with only 11 out of 46 herds having no positive animals. A summary of the results is shown in Table 1 while the wide distribution of positive cattle and buffalo herds is provided in Figure 1. Seroprevalence of BCoV varied significantly between species (Table 1), with water buffalo having a seropositivity of 5.3% and cattle having a significantly higher seroprevalence of 49.2% (p < 0.001). Univariate analysis of common risk factors revealed that age and origin were significantly associated with higher seroprevalence. Seroprevalence was higher in animals that were older than 24 months (34.6%) and in purchased animals, which had a higher chance of being positive when compared to farm-born animals (42.5%). As the characteristics of husbandry (and consequently the risk factor involved in the transmission of this disease) differ significantly between the two species, risk analysis was conducted separately for some factors (location and type of housing in cattle and location and cohabitation with cattle in water buffalo, as shown by Table 2 and Table 3). The seroprevalence of BCoV in the selected provinces of Avellino, Benevento, Salerno, and Caserta were 43.7%, 54.6%, 55.6%, and 44.2%, respectively. The results showed negligible and non-significant differences between the different provinces (e.g., Salerno and Avellino). Also, location was not a significant risk factor for buffalo (p = 0.79), while cohabitation with cattle was significantly associated with higher BCoV prevalence (p = 0.003). There were no differences between cattle that were partially grazed (48.3%) and those that were raised for stalling (49.5%) (p = 0.908), nor were there any differences between the different locations. 4. Discussion Despite the knowledge that BCoV is a pathogen with a significant economic impact that has been studied for potential recombination mechanisms that allow it to overcome the species barrier, there are few data in the literature on its spread, particularly in Europe . In Italy, both enteric and respiratory outbreaks are commonly reported, but there is still a lack of information on BCoV spread . We investigated the seroprevalence of BCoV among cattle and water buffalo in the Campania region of southern Italy and observed a very high number of seropositive animals (30.8% seroprevalence at the animal level). Since BCoV is a widespread infection and within-herd transmission is usually rapid, this result was expected and is consistent with results that were obtained in other countries when apparently healthy animals were tested. In Norway, for example, BCoV antibodies were found in 1014 of 1347 herds in a study using an ELISA test on tank milk . Also in Norway, a large-scale survey of respiratory infections in 2009 found a seroprevalence of 39.3% in calves and 80.7% at the herd level . Further studies, using similar approaches in milk samples, reported a herd seroprevalence that was higher than 70% in two different surveys in Sweden, confirming the strong interest of Scandinavian countries in this infection . In Finland, surveillance against this pathogen that was performed on the cattle population also resulted in 89% positive herds and 38% positive animals . Other studies that were published in the literature on different continents found an individual prevalence of 98% in Thailand and 55% in Ghana . A recent meta-analysis study that was carried out in China found 53.3% seroprevalence . The data presented are the result of similar epidemiological scenarios, although the reported prevalence values (which are generally very high) may vary depending on other factors such as the approach, the assay that was used, sampling, etc. In univariate analysis, our results suggest a considerable difference in BCoV exposure between cattle and water buffalo. Similarly, several works that were performed on different continents suggest lower seropositivity in hosts other than cattle, namely small ruminants and wild ruminants . This aspect proposes that natural infections are common in different hosts, while the difference in seroprevalence may be explained by the different tropism of BCoV for cattle and water buffalo. Furthermore, water buffalo are susceptible to infections with bovine-like coronaviruses such as BuCoV, which, may have different transmission dynamics despite high genetic similarity (up to 99%) with BCoV . Another hypothesis concerns the ineffectiveness of the assay that was used for the detection of antibodies against BCoV-like coronaviruses, since point mutations and deletions of the spike gene have been described in these viruses. In fact, the ELISA that was used in this study was validated only in bovine. In the absence of a validated assay for BCoV-like coronavirus antibodies, only adapted kits can be used. The validation of the ELISA is a typical issue for buffalo investigations, although according to the literature, ELISA that was designed for the bovine species may be utilized in the buffalo species (an example is represented by ELISA for the detection of bovine herpesvirus antibodies) . BCoV and BCoV-like coronaviruses share a large part of the genome, up to 99.5%, as, for example, described for BuCoV, and cross-react completely (several examples of extra-species use of antigens, antibodies, or even commercial kits that were validated in bovine and used in other species have been described in the literature). Coronavirus infection in buffalo has already been demonstrated using a BCoV monoclonal antibody in several studies, as well as the cross-reactivity of BuCoV in IFA using a BCoV antiserum . Antibodies against giraffe coronavirus react in hemagglutination with BCoV strains suggesting further evidence of cross-reactivity between BCoV and BCoV-like viruses (the same has been described also for BCoV-like coronaviruses found in camelids, which react with monoclonal antibodies prepared against BCoV antigen) . A commercial BCoV antigen capture ELISA was used to detect antibodies against BCoV-like coronavirus in zoo ruminants in a recent study . Since in vivo and in vitro experiments have shown high levels of cross-protection and cross-neutralization between BCoV and BCoV-like coronavirus, we can assume that antibodies against BCoV-like coronaviruses can still be detected using commercially available assays (pending further studies on the validation of these methods in the buffalo species) . To confirm the presence of antibodies against the different CoVs, the same samples should be tested using specific serum neutralization methods for BCoV and BuCoV. As shown by univariate analysis, different risk factors have been associated with BCoV seropositivity. We found higher seroprevalence in adult animals (> 2 years old); these data are in line with what has been found in other studies and with the persistence of detectable anti-BCoV antibodies (described for over a year from exposure) . Even the one and only meta-analysis study (conducted in China) supports this tendency . BCoV seroprevalence was significantly higher in purchased animals (42.5%) than in animals that were born on the same farm (20.5%). There are some plausible explanations for this outcome. First, the purchased animals may have seroconverted after being exposed to a predisposing factor (transportation) for BRDC and neonatal diarrhea, in which BCoV is an etiologic factor . A recent study found that the prevalence of BCoV (detected by real-time PCR on nasal swabs) increased from 16% to 65.1% after transportation . Numerous studies in the literature describe the higher prevalence of BCoV in only certain regions or countries . Another hypothesis involves purchasing animals from areas with higher prevalence. Concerning the specific bovine risk factors, no differences were found between animals that were kept partly on pasture and fully indoors. A study in Sweden compared the prevalence of BCoV in organic (grazing) and traditional herds and observed no significant differences . Another Swedish study found that conventional (intense) herds have a higher risk of being positive than organic herds . However, as also confirmed by a meta-analysis study that was carried out in China, the animal density in the herds and the size of the farm greatly affect the seroprevalence of this infection . Regarding buffalo specific risk factors, the seroprevalence was higher in animals that lived with cattle as compared to those that did not. Mixed breeding is a common practice in buffalo breeding in Italy, which increases the risk of contagion with common pathogens or allows spillover. Further research has shown coexisting with cattle as a risk factor for BCoV or BCoV-like seropositivity in sheep . In both cattle and water buffalo, however, the prevalence was not affected by location, as observed by Figure 1. This study is the first that describes the presence of detectable antibodies in buffalo in Italy and defines the seroprevalence of BCoV among cattle and buffalo on a large scale. Animals of different species, as well as humans, are constantly in contact with each other, increasing the risk of new and old infections. CoVs pose a persistent threat to humans and animals due to the economic damage that is caused by this virus in livestock, the genetic instability that is responsible for spillover events, and the recently demonstrated susceptibility to SARS-CoV-2 infection in cattle . The information that was obtained in this study could significantly improve BCoV surveillance and is useful for understanding the magnitude of the infection and identifying risk factors that are involved in its transmission. 5. Conclusions BCoV and BcoV-like coronaviruses are prevalent among cattle and water buffalo in southern Italy. This study not only defined regional seroprevalence in this species, but also identified some risk factors that are associated with BCoV seropositivity. This information could be extremely useful for infection control and surveillance. Although our findings are sufficient to warrant nationwide surveillance, more robust research will be required in the future, such as molecular characterization or whole genome sequencing studies to understand the genetic variations at the base of changes in the viral epidemiology. Phylogeographic and phylodynamic approaches could also be interesting to highlight reasons for epidemiological differences among countries and species. Supplementary Materials The following supporting information can be downloaded at: Table S1: Data collected on sampled ruminants. Click here for additional data file. Author Contributions Conceptualization, S.M. and U.P.; methodology, G.F. and E.I.; software, G.F.; formal analysis, S.M.; investigation, G.F.; resources, G.I.; data curation, U.P.; writing--original draft preparation, G.F.; writing--review and editing, U.P.; visualization, V.I.; supervision, S.M.; project administration, G.I. All authors have read and agreed to the published version of the manuscript. Institutional Review Board Statement The animal study protocol was approved by the Institutional Ethics Committee of Department of Veterinary Medicine and Animal production (Centro Servizi Veterinari), University of Naples, Federico II (PG/2022/0093419 20 July 2022). Informed Consent Statement Not applicable. Data Availability Statement Data is contained within the article or supplementary material. Conflicts of Interest The authors declare no conflict of interest. Figure 1 Spatial distribution of positive herds for bovine coronavirus (BCoV). animals-13-00772-t001_Table 1 Table 1 Univariate analysis (chi-square) of common potential risk factors for bovine coronavirus seropositivity. Factor n Positive % 95% CI kh2 p Total 720 222 30.8 27.5-34.2 Species Cattle 419 206 49.2 44.4-53.9 155.8 <0.001 Buffalo 301 16 5.3 2.8-7.8 Age <=24 months 134 19 14.2 8.3-20.1 20.4 <0.001 >24 months 586 203 34.6 30.8-38.5 Origin Born on the farm 381 78 20.5 16.4-24.5 39.7 <0.001 Purchased 339 144 42.5 37.2-47.7 animals-13-00772-t002_Table 2 Table 2 Univariate analysis (chi-square) of potential risk factors for bovine coronavirus seropositivity in cattle. Factor n Positive % 95% CI kh2 p Total 419 206 49.2 44.4-53.9 Province Avellino 112 49 43.7 34.6-52.9 Benevento 97 53 54.6 44.7-64.5 5.2 0.157 Salerno 97 54 55.7 45.8-65.6 Caserta 113 50 44.2 30.1-53.4 Stabulation Partly grazed 116 56 48.3 39.2-57.4 0.05 0.908 Stallfed 303 150 49.5 43.9-55.1 animals-13-00772-t003_Table 3 Table 3 Univariate analysis (chi-square) of potential risk factors for bovine coronavirus seropositivity in water buffalo. Factor n Positive % 95% CI kh2 p Total 301 16 5.3 2.8-7.8 Province Caserta 141 7 4.9 1.4-8.5 0.065 0.79 Salerno 160 9 5.6 2-9.2 Co-living with cattle Yes 119 12 10.1 4.5-15-5 8.9 0.003 No 182 4 2.2 0.1-4.3 Disclaimer/Publisher's Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

PMC10000195

Cancers (Basel) Cancers (Basel) cancers Cancers 2072-6694 MDPI 10.3390/cancers15051403 cancers-15-01403 Correction Correction: Zhu et al. A Feedback Loop Formed by ATG7/Autophagy, FOXO3a/miR-145 and PD-L1 Regulates Stem-like Properties and Invasion in Human Bladder Cancer. Cancers 2019, 11, 349 Zhu Junlan Li Yang Luo Yisi Xu Jiheng Liufu Huating Tian Zhongxian Huang Chao Li Jingxia Huang Chuanshu * Nelson Institute of Environmental Medicine, New York University School of Medicine, New York, NY 10010, USA * Correspondence: huangchuanshu@hotmail.com; Tel.: +1-646-754-9457; Fax: +1-646-754-9471 23 2 2023 3 2023 23 2 2023 15 5 140319 9 2022 02 11 2022 (c) 2023 by the authors. 2023 Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license ). pmcError in Figure In the original article , there was a mistake in Figure 1E (right panel) as published. A sphere image was incorrectly used in Figure 1E (right panel). The corrected Figure 1E is shown below. The authors apologize for any inconvenience caused and state that the scientific conclusions are unaffected. The original article has been updated. Figure 1 PD-L1 acted as an ATG7 downstream mediator being responsible for ATG7-promoted stem-like property, invasion, and anchorage-independent growth in human high invasive BC cells. (A-C) T24T, UMUC3, T24 cells were stably transfected with ATG7 knockdown constructs (#1 & #2), respectively. Western Blot was used to assess the ATG7 protein knockdown efficiency and its effects on other protein expression. (D) The GFP-tagged PD-L1 overexpression plasmid was stably transfected into T24T(shATG7#1) cells. (E,F) The indicated cells were subjected to determination of sphere formation abilities according to the manufacturer's instruction, the number of spheroid formed cells were counted as described in the section of "Materials and Methods". The asterisk (*) indicates a significant decrease in comparison to scramble nonsense transfectant (* p < 0.05), while the symbol (**) indicates a significant increase in comparison to T24T(shATG7#1/pEGFPc1) cells (** p < 0.05). (G,H) The indicated cells were subjected to anchorage-independent soft agar assay using the protocol described in the section of "Materials and Methods". Representative images of colonies of indicated cells were photographed under an Olympus DP71 (G). The number of colonies was counted with more than 32 cells of each colony and the results were presented as colonies per 104 cells, and the bars show mean +- SD from three independent experiments (H). The asterisk (*) indicates a significant decrease in comparison to scramble nonsense transfectant (* p < 0.05), while the symbol (**) indicates a significant increase in comparison to T24T(shATG7#1/pEGFPc1) cells (** p < 0.05). (I) Invasion abilities of the indicated cells were determined using BD BiocoatTM matrigelTM invasion chamber. The migration ability was determined using the empty insert membrane without the matrigel, while the invasion ability was evaluated using the same system except that the matrigel was applied. (J) The invasion ability was normalized to the insert control according to the manufacturer's instruction. The asterisk (*) indicates a significant inhibition in comparison to T24T(Nonsense) cells (* p < 0.05), while the symbol (**) indicates a significant increase in comparison to T24T(shATG7#1/pEGFPc1) (** p < 0.05). Scale bars in (E,I) = 200 mm, Scale bars in (G) = 500 mm. Disclaimer/Publisher's Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content. Reference 1. Zhu J. Li Y. Luo Y. Xu J. Liufu H. Tian Z. Huang C. Li J. Huang C. A Feedback Loop Formed by ATG7/Autophagy, FOXO3a/miR-145 and PD-L1 Regulates Stem-like Properties and Invasion in Human Bladder Cancer Cancers 2019 11 349 10.3390/cancers11030349 30871066

PMC10000196

Obesity and a high-fat diet are risk factors associated with prostate cancer, and lifestyle, especially diet, impacts the gut microbiome. The gut microbiome plays important roles in the development of several diseases, such as Alzheimer's disease, rheumatoid arthritis, and colon cancer. The analysis of feces from patients with prostate cancer by 16S rRNA sequencing has uncovered various associations between altered gut microbiomes and prostate cancer. Gut dysbiosis caused by the leakage of gut bacterial metabolites, such as short-chain fatty acids and lipopolysaccharide results in prostate cancer growth. Gut microbiota also play a role in the metabolism of androgen which could affect castration-resistant prostate cancer. Moreover, men with high-risk prostate cancer share a specific gut microbiome and treatments such as androgen-deprivation therapy alter the gut microbiome in a manner that favors prostate cancer growth. Thus, implementing interventions aiming to modify lifestyle or altering the gut microbiome with prebiotics or probiotics may curtail the development of prostate cancer. From this perspective, the "Gut-Prostate Axis" plays a fundamental bidirectional role in prostate cancer biology and should be considered when screening and treating prostate cancer patients. gut microbiome prostate cancer castration resistant prostate cancer microbiota Japanese Urological Association, KAKENHI18K16693 21K09421 Yakult Bio-Science FoundationThis work was supported by research grants from The Japanese Urological Association, KAKENHI (Grant No. 18K16693 and 21K09421), Yakult Bio-Science Foundation. pmc1. Introduction The incidence of advanced prostate cancer has been increasing in the USA and Japan, although the incidence of all prostate cancer in the USA has decreased since the recommendation of the US preventive service task force for prostate-specific antigen (PSA) screening in 2012 . Prostate cancer develops in patients in their 50 s and is a slow-growing tumor. Prostate cancer develops due to the mutations in driver genes, but several factors, such as genetic background and lifestyle affect the development of prostate cancer. Overall, the incidence of prostate cancer is high in Western countries compared with that in Asia. Japanese who lived in Hawaii had a higher incidence of prostate cancer compared with Japanese who lived in Japan . Obesity is also associated with the incidence of prostate cancer. A meta-analysis showed that obese men had a higher risk of advanced prostate cancer , and dietary habits are one of the major factors affecting prostate cancer. Western-style diets, especially a high-saturated-fat diet, are associated with increased risk of prostate cancer . High-fat diets (HFD) promote local inflammation in prostate tumors which leads to the increased secretion of inflammatory cytokines, such as IL-6 that culminates into increased recruitment and infiltration of myeloid-derived suppressor cells (MDSCs) . Il-6 is a pleiotropic cytokine that in this context, also promotes prostate cancer growth by activating STAT3 signaling. However, the mechanisms as to how consuming a high-fat diet leads to local tumor inflammation remain largely unknown. For some time, the mechanisms that link dietary habits to prostate cancer remained elusive; however, recent advances in technology have allowed us to come closer to unraveling the mystery. Particularly, developments in next generation sequencing have enabled us to probe deeper. For example, we have been able to capture whole profiles of microbes through 16S rRNA sequencing. By having a better understanding of gut microbiota, we are in a better position to discover associations of the gut microbiome with various diseases. The gut microbiome is a dynamic system that is affected by several factors, such as dietary habits, and since prostate cancer and diet are closely linked, it is reasonable to hypothesized that a gut microbiome--affected by diet--could regulate prostate cancer far from the gut, thus creating a gut-prostate axis. We have just begun connecting the dots that exist between the gut microbiome and prostate cancer, and more and more, additional dots are being discovered and linked. In this review, we discuss the current status of the "gut-prostate axis" as regulated by the gut microbiome. 2. The Gut Microbiome Trillions of microorganisms and their genetic material constitute the gut microbiome. The typical gut contains 1013 to 1014 bacteria, and these microorganisms are important as they supply nutrients, such as essential amino acids and vitamins, that cannot be produced by humans . Gut bacteria ferment dietary fiber and produce monosaccharides and short-chain fatty acids (SCFAs), such as butyrate, acetate, propionate, and isopropionate . Humans are then able to utilize absorbed acetate and propionate as substrates for lipid, glucose, and cholesterol metabolism . Herbivores have a long gastrointestinal tract and harbor gut microbes that allow them to digest cellulose and other plant materials to produce short-chain fatty acids as an energy source, and produce amino acids from ammonia by fermentation in the gut lumen . The gut microbiota also coevolves with the host . For instance, the giant panda consumes bamboo as an energy source, but the gastrointestinal tract of the giant panda is short and simple. Genetic sequencing showed that giant pandas have all the genes necessary for digesting meat, but not cellulose, which is the main component of bamboo . Metagenomic analysis of gut microbes of giant pandas revealed putative genes, encoding cellulose-digesting enzymes and hemicellulose-digesting enzyme from gut microbes indicating that this adaptation enabled them to use bamboo as an energy source . Gut microbiota is affected by several factors. Genetic background is an innate factor that affects the gut microbiome and this was demonstrated with a study analyzing the gut microbiome from twins which showed that host genetics do indeed influence the composition of the human gut microbiome . A genome-wide association study of the host genome revealed 31 loci that affect the microbiome, and the lactase gene locus showed a significant age-dependent association with the abundance of Bifidobacterium . In addition to innate factors, perinatal factors contribute to the developing gut microbiome. Everyone's gut microbiome is established early during the neonatal stage. The neonate gut microbiome is affected by the amniotic fluid, maternal lifestyle, and maternal exposure to antibiotics even before birth. After birth, each individual develops a unique gut microbiome based on their sex, race, and lifestyle. Sex differences affect gut microbial composition in mice and humans. Young adult women in Western countries had a higher diversity of gut microbiota than men, but these differences were not found in Chinese individuals . Testosterone also affects the gut microbiome, and the composition of the gut microbiome changes gradually with puberty. In pubertal subjects, the abundance of the genera Adlercreutzia, Ruminococcus, Dorea, Clostridium, and Parabacteroides is associated with the levels of testosterone . Androgen receptor signalling is involved in the maintenance of diversity of gut microbiota; androgen-receptor knock-out mice experienced HFD-induced metabolic syndrome and gut dysbiosis, but intervention of the gut microbiome by antibiotics prevented metabolic syndrome . Lifestyle is one of the main factors affecting gut microbiota. Results from 16S rRNA sequencing of feces has revealed that clusters of individuals with similar lifestyles share similar gut microbiomes. For example, family members share a similar gut microbiome, and obesity is associated with phylum-level changes in gut microbiota . Further evidence of this phenomenon is characterized by differences of the gut microbiome observed between individuals from Western and non-industrialized countries . For instance, the gut microbiome of Japanese is different from that of people in other countries, including Western and even other Asian countries, such as China. The Japanese gut microbiome has a greater abundance of taxa from the Actinobacteria phylum, particularly bacteria from the genus Bifidobacterium, than that in other nations . Interestingly, the gut microbiome also differs among people living in different regions, even within the same country. The gut microbiota of 7009 individuals from 14 districts within one province in China was analyzed, and the individual's geographic location showed an association with microbiota variation. Furthermore, microbiota-based metabolic disease models developed in one location failed when used elsewhere . Japanese people have a unique dietary culture and habits compared to Western people, and given that individuals generally have a lower BMI and longer lifespans, one can surmise that these factors are related. Some Japanese people consume Western-style diets as well as traditional Japanese food, thus this group of individuals with varied Japanese lifestyles could serve as a good model to analyze the association between lifestyle, gut microbiome, and diseases. Additional factors can influence microbial composition such as exercise and physical activity. For instance, gut microbiomes from athletes also differ from those of normal populations. Athletes had a higher diversity of gut microbiota and significantly higher proportions of the genus Akkermansia compared with individuals with high BMI, indicating that exercise can affect the gut microbiome . It should be noted that gut microbes interact with each other, and the overall picture of the microbiome cannot be determined by changes in single bacterial taxa. For example, obesity reduces SCFA-producing Bifidobacteria, but the total amount of SCFAs in the feces of obese people is increased . This discrepancy suggests that SCFA production will be increased in gut microbiome of obese people by SCFA-producing taxa other than Bifidobacteria. In addition to the identification of the intestinal bacterial taxa, functional analysis of the microbiome and measurement of bacterial metabolites may also be useful in understanding the overall picture of the gut microbiota. Lifestyles affecting prostate cancer risk also change the gut microbiome (Table 1). Obesity, well-known risk factors of prostate cancer, decrease the ratio of Firmicutes to Bacteroidetes . High-fat diets also affect gut microbiome, decreasing Bacteroidetes and increasing Firmicutes and Proteobacteria . Dairy products increase prostate cancer risk, although it is still controversial . Dairy products increase Lactobacillus and Bifidobacterium and decrease Bacteroidetes . Gut dysbiosis impairs gut wall integrity, increases gut permeability, and reduces the expression of tight junction proteins such as zonula occludens (ZO)-1 and occludin, causing a "leaky gut." A leaky gut results in the translocation of gut metabolites or bacterial components, such as lipopolysaccharide (LPS), into systemic circulation . Akkermansia muciniphilia is involved in the maintenance of a healthy gut wall by degrading mucin, and its abundance is inversely associated with several diseases . SCFAs are major metabolites of gut microbes, and butyrate plays a role in gut barrier function, and immunoregulations . Obese individuals have higher levels of SCFAs in their feces compared to lean individuals , and the ratio of Firmicutes to Bacteroidetes is decreased in obese subjects. Bifidobacterium, known to improve the gut mucosal barrier and lower intestinal LPS levels, is reduced in the feces of obese people . Interventions to improve dietary habits could change the gut microbiome. A randomized controlled study of obese individuals with metabolic syndrome showed that an energy-restricted Mediterranean diet and increased physical activity changed the gut microbiota. Changes in Lachnospiraceae were positively associated with adherence to the Mediterranean diet . cancers-15-01375-t001_Table 1 Table 1 The lifestyles affecting prostate cancer risk and these effects in gut microbiome. Prostate Cancer Risk Risk Factors Changes in Gut Microbiota References High Obesity The ratio of Firmicutes to Bacteroidetes | Bifidobacterium | High fat diet Bacteroidetes| Firmicutes | Proteobacteria | Dairy product Lactobacillus | Bifidobacterium | Bacteroidetes| Low Mediterranean diet Lachnospiaceae| 3. The Gut Microbiome and Diseases Gut microbes have direct contact with the intestinal wall; consequently, several intestinal diseases are affected by gut microbiota directly or indirectly through the modulation of local immune systems, such as regulatory T cell, dendric cells and CD4+ T cell . In the gut microbiota of IBD patients, the abundance of specific bacteria, such as Enterobacteriaceae and Fusobacteria, are increased, indicating a direct association between gut microbes, local inflammation, and altered local host immunity . Fecal microbiota transplantation, defined as the administration of fecal material containing distal gut microbiota from a healthy donor to the gastrointestinal tract of patients with IBD, has been conducted as a remedial treatment . A systematic review showed that this approach may increase the rate of patients achieving clinical remission in ulcerative colitis (RR = 2.03) . Apart from the gut, diseases of the liver and central nervous system are also reportedly affected by the gut microbiome. The liver receives bacterial components and metabolites that are absorbed in the intestinal tract and delivered via portal circulation . Phenyl sulfate, a metabolite derived from intestinal bacteria, acts as an aggravating compound in diabetic kidney disease and contributes to albuminuria . However, inhibition of sodium-glucose cotransporter 1 (SGLT1) in the gut ameliorates renal failure by altering the gut microbiome and reducing phenyl sulfate concentrations . The increased influx of gut microbiota-derived endotoxins in portal circulation promotes TLR-4 expression and is associated with hepatic inflammation and steatosis . Gut dysbiosis due to a HFD impairs the barrier of the intestinal wall, leading to the leakage of LPS into the systemic circulation. Systemic inflammation due to endotoxemia is implicated with the pathogenesis of insulin resistance and type 2 diabetes mellitus . Endotoxins and amyloids from gram-negative bacteria can penetrate the blood-brain barrier and induce amyloid b aggregation and neuroinflammation in the central nervous system, suggesting that bacterial molecules and metabolites may be involved in the onset and progression of Alzheimer's disease . Gut microbiota-derived SCFAs promote neuroinflammation and tau-mediated neurodegeneration in the hippocampus . These gut microbiome-mediated associations between gut and these distant organs are referred to as the "gut-brain axis" and "gut-liver axis" . However, these associations are not limited to these organs and are likely to include other systems. 4. The Gut Microbiome and Cancer In recent years, it has become evident that gut microbiota affects various types of cancers. Multiple bacterial taxa and their metabolites have contributed to the development and progression of colorectal cancer . Metagenomic and metabolomic studies on feces from participants who underwent colonoscopy showed that the relative abundance of Fusobacterium nucleatum was correlated with cancer progression . Fusobacterium nucleatum has been reported in other studies as a colorectal cancer-associated bacterium that promotes tumor progression in a mouse model of intestinal cancer in a non-inflammatory manner . In the liver, translocated bacterial metabolites and components may be involved in hepatocellular carcinoma (HCC). Obesity-induced hepatic translocation of lipoteichoic acid, a gram-positive intestinal bacterial component, accelerated senescence of hepatic stellate cells, promoting HCC progression through PGE2-mediated suppression of antitumor immunity . It has been suggested that the gut microbiota may influence the development of breast cancer by deconjugating conjugated estrogen excreted in the intestinal tract, and thus allowing the biologically active form to be reabsorbed by the host . In addition, intestinal bacteria can metabolize estrogen-like compounds, such as enterodiol and enterolactone, suggesting that gut microbiota plays a role in breast cancer development . The focus on gut microbiota has not only been directed toward its impact on cancer development and progression, but also on the indirect effects in response to drug therapy, including immune checkpoint inhibitors (ICIs) . In patients with advanced renal cell carcinoma (RCC) treated with anti-PD-L1 therapy, antibiotic use during treatment compared with no use was associated with an increased rate of primary progressive disease (75 vs. 22%, respectively) and shorter progression-free survival (1.9 vs. 7.4 months, respectively HR = 3.1), suggesting that antibiotic-induced dysbiosis reduced the clinical benefit from immune checkpoint inhibitors . Metagenomic data of fecal samples from advanced RCC patients treated with nivolumab showed that the abundance of Akkermancia muciniphila and Bacteroides salyersiae was increased in responders, and that transplantation of these bacteria or feces from responder patients to RCC mouse models rescued responsiveness to anti-PD-1 plus anti-CTLA-4 treatment from mice colonized with the microbiota of non-responder patients . The mechanism by which the gut microbiome influences ICI therapeutic efficacy is not fully understood; however, further studies will lead to innovative methods to enhance ICI treatments. 5. The Gut Microbiome and Prostate Cancer The association between the gut microbiome and prostate cancer has been studied in human samples. In 2018, Golombas et al. examined the gut microbiome in 20 men with either benign prostatic disease or high-risk prostate cancer and reported that Bacteriodes massiliensis was abundant in patients with prostate cancer compared to controls . Sfanos et al. compared the gut microbiome of patients with prostate cancer who received androgen deprivation therapy (ADT) to those of healthy volunteers and found that Akkermansiia muciniphila and Ruminococcaceae were increased in patients treated with ADT . Liss et al. analyzed rectal swabs from 133 men who received prostate needle biopsy and showed that Streptococcus and Bacteroides were increased in patients with prostate cancer. Although 16S rRNA amplicon sequencing alone cannot specifically identify functional genes, it can be used with various computational tools to infer the functional metagenome . Predicted metagenome analysis revealed that the folate and arginine pathways were down-regulated in the gut microbiome of men with prostate cancer therefore implicating these pathways with the pathogenesis of prostate cancer. Bacteroides massiliensis were also found to be increased in the gut microbiome from patients with prostate cancer by another study using a small cohort (8 men with benign prostate hypertrophy and 12 with high-risk prostate cancer) . Matsushita et al. analyzed the gut microbiome of 152 men who underwent prostate biopsy and found that Rikenellaceae, Alistipes, and Lachonospira were increased in patients with high-risk prostate cancer . The predictive value of these bacterial taxa was similar to serum PSA levels for the high-risk prostate cancer group. However, the index comprised from a profile of 18 bacteria taxa identified high-risk prostate cancer cases more precisely. The area under the curve (AUC) of reservoir operating characteristic curve analysis of this index for detecting high-risk prostate cancer was 0.85, while the AUC of serum PSA levels was 0.74. Functional pathway analysis showed that five metabolic pathways (starch and sucrose metabolism, phenylpropanoid biosynthesis, phenylalanine, tyrosine, and tryptophan biosynthesis, cyanoamino acid metabolism, and histidine metabolism) were positively associated with high-risk prostate cancer. The analysis of fecal microbiome of 23 patients with metastatic castration-resistant prostate cancer (CRPC) resistant to enzalutamide prior to treatment with anti-PD-1 showed that the responders have increased levels of Streptococcus salivarius. Interestingly, Akkermansia muciniphila levels were reduced in the fecal samples from responders . The above-mentioned studies suggest that the gut microbiome influences prostate cancer and vice versa. Until recently, the precise mechanisms driving their interactions remained largely undiscovered, however, new findings have shed light on the subject . In 2021, Matsushita et al. reported that SCFAs originating from the gut microbiome promoted the growth of prostate tumors in a transgenic mouse model of prostate cancer . A HFD is considered to be a risk factor for prostate cancer and is implicated in promoting prostate cancer growth by inducing local inflammation . Antibiotics administered to prostate-specific conditional Pten-knockout mice fed a HFD suppressed prostate cancer growth. Interestingly, different types of antibiotics showed different effects on tumor growth--indicating that specific taxa are responsible for this phenomenon. For example, gentamycin suppressed HFD-induced prostate cancer growth, but neomycin did not. Microarray analysis of prostate tumors showed that IGF-1 was significantly down-regulated in HFD-fed Pten-knockout mice treated with antibiotics compared with HFD-fed Pten-knockout mice without antibiotics. Antibiotics also decreased the insulin-like growth factor 1 (IGF-1) levels in serum in wild-type HFD-fed mice. IGF-1 stimulated prostate cancer growth via the mitogen-activated protein kinase (MAPK) and phosphatidylinositol-3 kinase (PI3K) signaling pathways. Short chain fatty acids (SCFAs), such as butyrate, acetate, isobutyrate, and lactate are major metabolites of the gut microbiome, SCFAs play important roles in the regulation of intestinal immune cells with anti-inflammatory effects. SCFAs are also known to suppress colon cancer. Butyrate, the major compound of SCFAs suppress colonic carcinogenesis through this anti-inflammatory effect . On the other hand, it also has a variety of other physiological effects, such as Wnt signal modulation, and promotes colorectal cancer, depending on its concentration . However, in the Pten-knockout prostate cancer mouse model, the administration of antibiotics decreased SCFA levels in the gut. SCFAs from gut microbiota are known to stimulate IGF-1 production, leading to bone growth . In the Pten-knockout prostate cancer mouse model, SCFAs also stimulated the production of IGF-1 in prostate tumors and other organs, such as liver, contributing to augmented prostate cancer growth. Notably, IGF-1 expression was upregulated in prostate cancer of severe obese patients compared with non-obese patients. Patients with benign prostatic hyperplasia (BPH) also have higher levels of fecal isobutyric acid and isovaleric compared to healthy individuals, and isobutyric acid, isovaleric acid, and isocproic acid were associated with the occurrence of metabolic syndrome in patients with BPH . Furthermore, the Firmicutes/Bacteroidetes (F/B) ratio was significantly higher in Japanese men with enlarged prostates than in men with normal-size prostates . SCFAs derived the gut microbiota may be a risk factor for prostate cancer and BPH by a similar mechanism. BPH is a very frequent age-related disease . BPH, characterized by hyperplasia of the transition zone, is associated with inflammation, oxidative stress, and other several biological factors . The relationships between different diets and the development of BPH has been discussed for some time . HFD, in which 60% of kcal consists of lipids, promoted BPH in rat models, and activation of ERK1/2 was likely involved in this process . Matsushita et al. reported that HFD also promoted prostate cancer growth via gut microbiome through a similar mechanism, suggesting that the connection between BPH and diet may be also mediated by the gut microbiome . Fat consumption was reported to increase the incidence of BPH in humans. In a 7-year prospective study of 4770 subjects in The Prostate Cancer Prevention Trial, the highest fat intake group had a significantly increased hazard ratio by 31% compared with the lowest group . It is important to note that the consumption of a HFD causes gut dysbiosis which leads to the development of a "leaky gut" by down-regulating tight junction molecules, such as ZO-1 . Leaky gut leads to the leakage of bacterial components, such as lipopolysaccharide (LPS) or lipoteichoic acids (LTA), into systemic circulation. Leaked LPS and LTA in turn provoke systemic inflammation which have wide ranging implications including cancer promoting effects . In prostate cancer, LPS activates mast cells via toll-like receptor 4 . HFD-fed Pten-knockout mice also demonstrated upregulated levels of histamine decarboxylase (HDC), which plays a crucial role in histamine production. Fexofenadine, an H1 receptor blocker, suppressed the expression of inflammatory cytokines, such as Il-6, IL-10, IL-4, and IL-17, and reduced infiltration of MDSCs into prostate tumors. Furthermore, fexofenadine suppressed prostate cancer growth in HFD-fed mice. This upregulation in HDC levels is due to leaked LPS from dysbiotic gut microbiota of HFD-fed mice, and thus LPS administration to control diet-fed mice leaded HDC upregulation. In addition, inhibition of LPS suppressed the prostate cancer growth of HFD-fed mice. Like obese HFD-fed mice, severely obese patients with prostate cancer exhibited increased tumor-infiltrating mast cells. Gut dysbiosis could also be associated with drug-resistance in prostate cancer . Antibiotics-induced dysbiosis, characterized by the enrichment of Proteobacteria, resulted in the elevation of tumor LPS due to an increase in gut permeability. Intratumoral elevation of LPS activated the NF-kB-IL6-STAT3 axis, leading to prostate cancer growth and docetaxel-resistance . Androgen and its receptor have a pivotal role in the development of CRPC and these studies provide evidence that shows that the gut microbiome is intricately implicated and are further explored in the next section. 6. The Gut Microbiome in Androgenesis ADT has been the gold standard for the treatment of prostate cancer for decades. However, patients eventually develop resistance and progress to CRPC which relies on minute levels of androgens for its growth by amplifying the androgen receptor . Androgens are produced in the testis, adrenal glands, and in prostate tumors by cancer cells, but recent studies suggested that the gut microbiota also plays a role in androgenesis . In the analysis of 31 young Korean men aged 25-65 years using 16SrRNA sequencing, the abundance of Acinetobacter, Dorea, Ruminococcus, and Megamonas correlated significantly with serum testosterone levels . In the analysis of the gut microbiota of 54 Japanese men aged 65 years or older, serum testosterone levels were positively correlated with the abundance of Firmicutes . Several studies have also reported the relationship between the microbiome and polycystic ovarian syndrome (PCOS), a disease caused by high testosterone production in women . Testosterone levels increased when feces from male mice were transplanted into female mice, suggesting that specific intestinal bacteria promoted testosterone production. In addition, Firmicutes synthesize testosterone or promotes its reabsorption through unconjugation . Gut microbiota modulate the enterohepatic circulation of androgens, affecting systemic androgen levels. Gut bacteria can also produce androgens from glucocorticoids . ADT also affects the gut microbiome in both mice and humans and promotes the expansion of specific bacteria . ADT plus abiraterone acetate depletes androgen-utilizing and pro-inflammatory Corynebacterium and increases Akkermansia muciniphila in the gut. Predicted metagenomic analysis from 16S rRNA sequencing suggested that patients with abiraterone acetate increased bacterial biosynthesis of vitamin K2, which is known to be an inhibitor of androgen-dependent and-independent tumor growth . Ruminococcus has genes sharing high sequence homology with human CYP17 and Ruminococcus can convert pregnenolone and hydroxypregnenolone in feces to DHEA and testosterone. Patients with CRPC had a higher abundance of Ruminococcus in their feces and promoted prostate cancer growth via the production of androgens in the gut. Abiraterone acetate, a selective inhibitor of CYP17A1, inhibited the bacterial conversion of pregnenolone to DHEA and testosterone. In contrast, FMT with hormone-sensitive microbiota or administration of Prevotella stercorea can decrease androgens levels in CTX mice and delay the onset of CRPC . In this context, androgenesis by gut microbiota should be considered for patients undergoing ADT for metastatic hormone-sensitive prostate cancer or CRPC. 7. The Association with the Epigenetics Prostate cancer pathobiology is affected not only by genomic mutation, but also by epigenetic modification, namely the acquired regulation of gene expression . Various environmental factors can cause alterations in the epigenome and may be drivers of cancer formation and progression . Aberrant DNA hypermethylation is a prevalent epigenetic modification responsible for the inactivation of tumor suppressor genes in prostate cancer, and GSTP1, a class of Glutathione S-transferases (GSTs), a family of enzymes responsible for processes that protect cells from xenobiotics, was earliest reported to be hypermethylated in human prostate cancer . Similarly, DNA hypermethylation is involved in the expression not only of DNA repair genes but also of the genes involved in cell cycle, apoptosis, and cell adhesion, which have been attempted to be regulated by dietary therapy . The methyl group is extracted from S-adenyl methionine, and bacterial metabolites such as folate and betaine are essential for its synthesis . Certain strains of Lactobacillus and Bifidobacterium used as probiotics have the ability to generate folate, and such strains may enhance prostate cancer risk via DNA hypermethylation . Contrarily, in a gene-based predictive functional analysis of the gut microbiota by Liss et al. the function to synthesize folate was significantly reduced in prostate cancer patients compared to men without cancer . It is possible that the functional analysis is based on gene prediction and does not reflect the actual folate synthesis capacity, but further studies are needed to address this discrepancy. Epigenetic modifications of histones, which give the backbone to chromatin, have been reported in prostate cancer . One type of histone modification is methylation, which may also be influenced by gut microbiota-derived metabolites involved in methyl group donation. The other type, histone acetylation, leads to chromatin loosening, leading to transcription activation. Histone deacetylases (HDACs), which act in transcription inactivation by clearing acetyl groups, are inhibited by short-chain fatty acids (SCFAs) produced by some anaerobic bacteria fermenting dietary fiber . Butyrate, one type of SCFA, at high concentrations, inhibited prostate cancer growth in vitro by altering the expression of cell cycle regulators and AR through the epigenetic histone modification . However, butyrate has contrasting effects on cancer cells depending on its concentration, and we have shown that deficiency of gut microbiota-derived SCFAs rather inhibits prostate cancer growth in vivo . These findings suggest that the gut microbiota may also be involved in epigenetic modifications of prostate cancer cells, although these associations have not yet been directly demonstrated. Future studies are needed. 8. Limitations In these human gut microbiota analyses, the proportion of several intestinal bacteria seems to change according to the prostate cancer status of the host. Most of the human-reported studies have been conducted in the limited regions of Asia and the United States, although the composition of the gut microbiota varies between regions due to the diversity of lifestyles such as dietary habit. In other to achieve the identification of intestinal bacteria that truly work as promotive or preventive factors of prostate cancer linked to lifestyles, extensive global microbiota research of prostate cancer patients will be necessary. 9. Conclusions The gut microbiome is greatly influenced by several environmental factors, such as lifestyle. Change in the gut microbiota can be involved in prostate cancer progression through its metabolites and endotoxins. A greater understanding of the molecular mechanisms underlying these bidirectional interactions have allowed us to establish a "gut-prostate-axis". These interventions could be further incorporated with current treatments as novel strategies for the prevention and management of human prostate cancer. Acknowledgments We thank Daisuke Motooka, Shota Nakamura (Department of Infection Metagenomics, Research Institute for Microbial Diseases, Osaka University), Hisako Kayama and Kiyoshi Takeda (Laboratory of Immune Regulation, Department of Microbiology and Immunology, Osaka University, Graduate School of Medicin), Hiroaki Hase and Kazutake Tsujikawa (Laboratory of Cell Biology and Physiology, Osaka University, Graduate School of Pharmaceutical Sciences) and Shinichi Yachida (Department of Cancer Genome Informatics, Graduate School of Medicine, Osaka University) for the collaborative work. Author Contributions K.F., M.M. and M.A.D.V. wrote manuscript. K.H., T.M., N.N. and H.U. supervised manuscript. All authors have read and agreed to the published version of the manuscript. Institutional Review Board Statement Not applicable. Informed Consent Statement Not applicable. Data Availability Statement Not applicable. Conflicts of Interest K.F., N.N. and H.U. received the honoraria from Jannsen Pharmaceutical K.K. Figure 1 Local effect and gut-distant organ axis via gut microbiome. The endotoxins and the metabolite from the gut microbiome affect the distant organs, SCFA: short-chain fatty acid, LPS: lipopolysaccharides. Figure 2 Gut-prostate axis. Diet and medications affect gut microbiome. Gut dysbiosis results in the leakage of endotoxins into systemic circulation. Gut microbiota also produce androgen affecting prostate cancer progression. Figure 3 Interactions between the gut microbiome and prostate cancer in androgen regulation. Gut microbiome-derived androgen may be involved in prostate cancer growth. Androgen-deprivation therapy conversely change the composition of the gut microbiota. 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PMC10000197

Female hair sheep, 27 Dorper (DOR), 41 Katahdin (KAT), and 39 St. Croix (STC), were used to determine influences of the nutritional plane before breeding and in early gestation on feed intake, body weight, body condition score, body mass indexes, blood constituent concentrations, and reproductive performance. There were 35 multiparous and 72 primiparous sheep, with initial ages of 5.6 +- 0.25 years and 1.5 +- 0.01 years, respectively (average overall initial age of 2.8 +- 0.20 years). Wheat straw (4% crude protein; dry matter [DM] basis) was consumed ad libitum and supplemented with approximately 0.15% initial body weight (BW) of soybean meal (LS) or a 1:3 mixture of soybean meal and rolled corn at 1% BW (HS; DM). The supplementation period was 162 days, with the breeding of animals in two sets sequentially, with the pre-breeding period 84 and 97 days, and that after breeding began at 78 and 65 days, respectively. Wheat straw DM intake (1.75, 1.30, 1.57, 1.15, 1.80, and 1.38% BW; SEM = 0.112) was lower (p < 0.05), but average daily gain (-46, 42, -44, 70, -47, and 51 g for DOR-LS, DOR-HS, KAT-LS, KAT-HS, STC-LS, and STC-HS, respectively; SEM = 7.3) was greater (p < 0.05) for HS than LS treatment during the supplementation period. Additionally, changes in body condition score during the supplementation period (-0.61, 0.36, -0.53, 0.27, -0.39, and -0.18; SEM = 0.058), and changes in body mass index based on height at the withers and body length from the point of the shoulder to the pin bone (BW/[height x length], g/cm2) from 7 days before supplementation (day -7) to day 162 were -1.99, 0.07, -2.19, -0.55, -2.39, and 0.17 for DOR-LS, DOR-HS, KAT-LS, KAT-HS, STC-LS, and STC-HS, respectively; (SEM = 0.297) were affected by supplement treatment. All blood constituent concentrations and characteristics addressed varied with the day of sampling (-7, 14, 49, 73, and 162) as well as the interaction between the supplement treatment and the day (p < 0.05), with few effects of interactions involving breed. Birth rate (66.7, 93.5, 84.6, 95.5, 82.8, and 100.0; SEM = 9.83) and individual lamb birth weight (4.50, 4.61, 4.28, 3.98, 3.73, and 3.88 kg; SEM = 0.201) were not affected by supplement treatment (p = 0.063 and 0.787, respectively), although litter size (0.92, 1.21, 1.17, 1.86, 1.12, and 1.82; SEM = 0.221) and total litter birth weight (5.84, 5.74, 5.92, 7.52, 5.04, and 6.78 kg for DOR-LS, DOR-HS, KAT-LS, KAT-HS, STC-LS, and STC-HS, respectively; SEM = 0.529) were greater (p < 0.05) for HS than for LS. In conclusion, although there was some compensation in wheat straw intake for the different levels of supplementation, soybean meal given alone rather than with cereal grain adversely affected BW, BCS, BMI, and reproductive performance, the latter primarily through litter size but also via a trend for an effect on the birth rate. Hence, the supplementation of low-protein and high-fiber forage such as wheat straw should include a consideration of the inclusion of a feedstuff(s) high in energy in addition to nitrogen. hair sheep reproductive performance supplementation feed intake body condition score USDA Foreign Agricultural Service Borlaug Fellowship ProgramUSDA National Institute of Food and Agriculture (NIFA) Evans-Allen Project OKLUSAHLU20171012650 The project was supported by the USDA Foreign Agricultural Service Borlaug Fellowship Program [BFP19 Algeria (Belkasmi) Animal Hlt] and USDA National Institute of Food and Agriculture (NIFA) Evans-Allen Project OKLUSAHLU2017 (Accession Number 1012650). pmc1. Introduction The nutritional plane is of importance to small ruminants throughout the year, although there are specific periods and stages of production when it is critical. Additionally, of course, nutrient and energy intake at any given time influences needs later to achieve desired levels and efficiencies of production . However, nutritional management and modeling for small ruminants are challenging because of very diverse farming practices, feeding habits, and environmental adaptation, including the unique resilience of different breeds to varying conditions in wide-ranging geographical areas . Before and during the breeding period and early gestation are critical periods for sheep when feeding practices can have a marked influence on reproductive performance resulting from ovulation rate, embryo quality, and uterine environmental conditioning due to metabolic and endocrine balances . Relatedly, 'flushing' for increased conception rate and litter size is a common practice in sheep production systems, particularly with basal dietary forage low to moderate in nutritional value or quality . Additionally, proper nutrition and body condition score (BCS) during gestation influence the performance, health, reproduction, and metabolic responses of progeny . The three most common breeds of hair sheep in the USA are Dorper (DOR), Katahdin (KAT), and St. Croix (STC) . After a project in which the resilience of these breeds to stress factors was expected to increase in importance with anticipated future climate change , a study was conducted with some of these mature females used to ascertain the effects of different supplement treatments before and during the breeding period on body weight (BW) change and composition, reproductive performance, and a number of other variables such as BCS, body mass indexes (BMI), and blood constituent concentrations . Wheat straw was consumed ad libitum and supplemented with 0.16% BW (dry matter [DM] basis) of soybean meal (SBM) or 0.8% BW (DM basis) of a 3:1 mixture of rolled corn and SBM before breeding for 2.25-2.50 months before breeding and the first 17 days of the breeding period. Although the intake of wheat straw was greater for the SBM than for the corn:SBM treatment, partially compensating for the different levels of supplement intake, changes in BW and body energy content, BCS, and BMI were greater for the corn:SBM vs. SBM treatment. However, reproductive performance was similar among supplement treatments, perhaps reflecting the mobilization of body energy stores to support reproductive performance. Moreover, there were no interactions in the reproductive performance between the hair sheep breed and the supplement treatment. Questions that arose from this study include whether results would differ with a slightly higher level of intake of the corn:SBM supplement as well as the potential impact of imposing treatments in the early gestation period. Therefore, the objectives of this study were to determine the effects of different levels of intake and compositions of supplemental concentrate before breeding and in early gestation on the production of Dorper, Katahdin, and St. Croix hair sheep consuming low-quality basal dietary forage. 2. Materials and Methods 2.1. Animals The protocol for the experiment was approved by the Langston University Animal Care and Use Committee. The study occurred in the late summer of 2019 through lambing in the spring of 2020. There were 107 female hair sheep used, 27 DOR, 41 KAT, and 39 STC. The animals included 35 ewes that had previously given birth and 72 that had not, with initial ages of 5.6 +- 0.25 years and 1.5 +- 0.01 years, respectively (average overall initial age of 2.8 +- 0.20 years). The ewes were derived in the summer and early fall of 2015 from four climatic zones and regions of the USA, the Northwest (primarily Oregon), Midwest (parts of Iowa, Wisconsin, Minnesota, and Illinois), Southeast (primarily Florida), and central Texas. The animals were used in a series of trials from the fall of 2015 through the spring of 2018, described by Tadesse et al. and Hussein et al. , evaluating resilience in stress factors projected to become increasingly important and prevalent with future climate change, namely, high heat load conditions, limited water availability, and restricted feed intake. After those studies, some animals were culled for various reasons such as old age, and others were designated for another experiment, with the ones remaining used in the current study. The ewe lambs were progeny from the ewes used in these previous studies. 2.2. Treatments and Housing Before the experiment, sheep were vaccinated against clostridial organisms, and ones with a FAMACHA(c) score greater than 3 were treated for internal parasites. The sheep were allocated to four groups per breed based on body weight (BW) and age, with 6 to 11 animals per group, and two supplement treatments were randomly assigned, with a table of random numbers, to the groups . Each group was housed in a pen with two sections, a 3.66 m x 3.66 m area within a building and a second with the same dimensions located outside but also covered by a roof. The floors were concrete and periodically cleaned. Water was available free-choice in automatic waterers and there was free access to salt blocks. Artificial lighting was provided from 07:00 to 16:00 h. There were two feed troughs (2.44 m and 1.22 m in length) per pen in the front area. There were six pens on one side of the facility and six on the other, with a hallway between the feed troughs and pens. There was one breed x supplement treatment group on each side of the facility. Animals were weighed before supplement treatments were first imposed. Body weight was determined in the morning before refusals from the previous day were collected and new feedstuffs were dispensed. The supplement treatments were soybean meal fed at approximately 0.15% of initial BW and a mixture of 25% soybean meal and 75% rolled corn given at approximately 1% BW (dry matter basis; LS and HS, respectively). Coarsely ground wheat straw with crude protein (CP) content of 3.9% and neutral detergent fiber content (NDF) of 80.2% (Table 1) was consumed ad libitum and offered at approximately 120% of consumption on the preceding few days. After straw refusals were collected and weighed, supplements were fed at 08:00 h, which were completely consumed within a few minutes, and then wheat straw was offered. Wheat straw and supplement samples were collected weekly, ground to pass a 1 mm screen, and analyzed for dry matter (DM), ash , and CP by the Doumas method using a C/N analyzer (Leco TruMac CN, St. Joseph, MI, USA), and NDF with the use of heat stable amylase and containing residual ash , and acid detergent fiber (ADF) content in wheat straw and supplement and acid detergent lignin (ADL) content in wheat straw were analyzed using the filter bag technique (ANKOM Technology Corp., Fairport, NY, USA). With day 1 considered the first day of imposing the supplement treatments, BW was determined on days -7, 14, 49, 73, 120, 162, 198, and 225, with periods 1, 2, 3, 4, 5, 6, and 7 considered 21, 35, 24, 47, 42, 36, and 27 days in length, respectively . The variables in the different periods were analyzed to capture dynamic changes affected by treatment in these periods with periods 1 to 5 for supplement treatment effects and periods 6 and 7 for previous supplements' effects while the animals were on a similar nutritional plane. After the 162-day supplementation phase (i.e., end of period 5), there was an additional 63-day period that ended near the time when lambing began. The animals in periods 6 and 7 received the HS supplement treatment (i.e., 1% of initial BW) and were treated similarly in outside pens, and thereafter given a higher level of a concentrate-based supplement and free-choice access to high-quality forage. 2.3. Breeding One pen of animals of each treatment was assigned to 1 of 2 breeding sets. Four rams of each breed divided into 2 sets were used, which were previously subjected to and passed a breeding soundness examination. The 2 initial breeding periods were 7 days in length on days 84-90 and 97-103 for sets 1 and 2, respectively. Estrus synchronization was employed, with a controlled internal drug release (CIDR) vaginal device (EAZI-breedTM CIDR(r), Pfizer Animal Health, Auckland, New Zealand) inserted on day 0, and 10 mg of PGF2a (Lutalyse, 10 mg dinoprost tromethamine i.m., Zoetis Animal Health, Parsippany-Troy Hills, NJ, USA) was intramuscularly injected 9 days later. On day 10, the CIDR was removed, at which time, 2 rams of each breed fitted with marking harnesses were introduced into a pen of the relevant breeding set. Estrus and breeding markings were checked twice daily at 08:00 and 16:00 h. Non-return to estrus was assumed when females were not marked during a second cycle with a ram at 15-18 days after an observed mating. Pregnancy was also diagnosed at 40 days after breeding using ultrasonography by ventral external examination with a 3.5 MHz linear-array transducer, and 12 open animals were removed from the study. Approximately 1 month before lambing, ewes were vaccinated again against clostridial organisms, and ones with FAMACHA(c) scores greater than 3 were treated for internal parasites. The ewes were moved back to the confinement facility approximately 1 week before lambing. The birth rate was the percentage of females exposed to rams that gave birth (95 of the 107 total, 30 of 35 multiparous, and 65 of 72 primiparous). Fecundity was based on the number of lambs born per female exposed to rams, and other reproduction measures were based on animals giving birth, which occurred in portable lambing pens. The gestation length was based on the last day of observed breeding and the date of birth. 2.4. Other Measures Body condition score (BCS), as described by Ngwa et al. , was assessed by three or four individuals when BW was determined (i.e., on days -7, 14, 49, 73, 120, 162, 198, and 225). Linear measurements of height at the withers (Wither), length from the point of the shoulder to the hook bone (Hook) and pin bone (Pin), and circumference from heart girth (Heart) occurred on days -7, 14, 49, 73, and 162. Of the 13 body mass indexes (BMI) described by Liu et al. , the 4 most meaningful ones as recommended by Wang et al. were calculated as noted below. BMI-WH = BW/(Wither x Hook) [g/cm2](1) BMI-WP = BW/(Wither x Pin) [g/cm2](2) BMI-GH = BW/(Heart x Hook) [g/cm2](3) BMI-GP = BW/(Heart x Pin) [g/cm2](4) Blood samples (10 mL) were collected by jugular venipuncture into three tubes on days 14, 49, 73, and 162. There were two tubes used for plasma, one with sodium fluoride and potassium oxalate, and another with sodium heparin. A third tube without an anticoagulant was used to derive serum. Plasma and serum were harvested by centrifugation for 20 min at 3000x g and frozen at -20 degC. Plasma from the sodium fluoride and potassium oxalate tube was analyzed for glucose and lactate with a USI 2300 Plus Glucose & Lactate Analyzer (YSI Inc., Yellow Springs, OH, USA). Plasma from the sodium heparin tubes and (or) serum was analyzed for constituents such as nonesterified fatty acids (NEFA), triglycerides (TG), cholesterol, urea nitrogen (N), albumin, and total protein with a Vet Axcel(r) Chemistry Analyzer (Alfa Wassermann Diagnostic Technologies, West Caldwell, NJ, USA) according to the manufacturer's instructions. Total antioxidant capacity (TAC) in plasma was determined by measuring the ferric-reducing ability of plasma colorimetrically . Heart rate (HR) was measured in set 1 animals the week preceding days 14, 49, 73, and 162 and on set 2 animals the following week. Heart rate was assessed to predict heat energy (HE) based on the ratio of HE to HR for each animal as employed in many other studies . Heart rate measurement was as described by Puchala et al. . Heart rate was determined for 24 h with animals in 4 pens at a time over a total period of 3 days. Sheep were fitted with stick-on ECG electrodes (Cleartrace, Utica, NY, USA) attached to the chest just behind and slightly below the left elbow and at the base of the jugular groove on the right side of the neck. Electrodes were secured to the skin with a 5 cm-wide elastic bandage (Henry Schein, Melville, NY, USA) and animal tag cement (Ruscoe, Akron, OH, USA). There was the use of ECG snap connecting leads (Bioconnect, San Diego, CA, USA) to connect electrodes to T61coded transmitters (Polar, Lake Success, NY, USA). Human S610 HR (Polar) monitors with wireless connection to the transmitters were used to collect HR data at a 1 min interval. Heart rate data were analyzed using Polar Precision Performance SW software. For the HE to HR ratio, on days 80 to 95, animals were cycled in groups of 5 to 7 into a room with metabolism cages fitted with headboxes of a respiration calorimetry system for 1 day. Emissions of methane and carbon dioxide and oxygen consumption were measured with an indirect, open-circuit respiration calorimetry system (Sable Systems International, North Las Vegas, NV, USA) as described by Puchala et al. . Oxygen concentration was analyzed using a fuel cell FC-1B oxygen analyzer (Sable Systems International), and methane and carbon dioxide concentrations were measured with infrared analyzers (CA-1B for carbon dioxide and MA-1 for methane; Sable Systems International). Before each measurement, analyzers were calibrated with reference gas mixtures. Heat energy was calculated from oxygen consumption and the production of carbon dioxide and methane according to the Brouwer equation without the consideration of urinary nitrogen. 2.5. Statistical Analyses Most data were analyzed with the MIXED procedure of SAS , with fixed effects of breed, supplement treatment, and their interaction and random effect of animal within breed and supplement. Because of the limited number of observations for multiparous animals, parity was not included in the model. However, for discussion purposes, reproduction data were also analyzed separately for multiparous and primiparous animals. The animal group or pen within the breed and supplement treatment was the experimental unit. Day was a repeated measure for the analysis of blood constituent concentrations. For the reproduction variables of birth rate and litter size, the analysis was with the GLIMMIX procedure of SAS . Breed x supplement treatment means are presented regardless of the significance of the interaction. Differences among means were determined by the least significant difference with a protected F-test (p < 0.05). Pearson correlations (r) between BW, BCS, and BMI were determined using SAS . 3. Results 3.1. Feed Intake, BW, and ADG The chemical composition of wheat straw and supplements (Table 1) was similar to that in the earlier experiment of Lourencon et al. . The level of supplement DM intake in g/day and % BW was greater for High than for Low in all periods and overall (p < 0.05; Table 2). The intake of wheat straw DM in g/day and % BW was greater for LS vs. HS in each period (p < 0.05) except for period 1 (p > 0.05) when there was no compensation in wheat straw intake for the higher level of intake of HS. Conversely, there was partial compensation in periods 2 and 3 and overall, with greater total DM intake in % BW for HS than for LS (p < 0.05). In periods 4 and 5, total DM intake in % BW was similar between supplement treatments, as wheat straw intake was considerably higher in these periods for HS. Wheat straw DM intake was 0.29, 0.49, 0.61, 0.66, and 0.43% BW greater for LS vs. HS in periods 2, 3, 4, and 5 and overall, respectively. Likewise, relative to the wheat straw DM intake for HS, the intake for LS was 23.5, 37.5, 43.2, 49.9, and 33.7% greater in periods 2, 3, 4, and 5 and overall, respectively. Although an analysis was not conducted to determine the period effects because of factors such as different lengths and stages of production, it is germane to note that the total DM intake increased as the periods progressed and plateaued in period 4 (1.70, 1.96, 2.09, 2.27, and 2.19% BW in period 1, 2, 3, 4, and 5, respectively). A breed effect (p < 0.05) was noted for total DM intake in g/day in period 4 (DOR > STC) and for overall average supplement intake in % BW (DOR > KAT). There were no interactions between breed and supplement treatment in BW or ADG (p > 0.05; Table 3). Body weight was numerically lower for STC than for DOR and KAT at most times and was lower than for KAT on day 49 and for DOR and KAT on day 225 (p < 0.05). Body weight was similar between supplement treatments on days -7, 14, and 49 (p > 0.05) but was greater for HS than for LS on subsequent days (p < 0.05). The values were 7.0, 7.8, 15.1, 11.2, and 12.0 kg and 13.6, 15.2, 31.3, 21.1, and 20.4% greater for HS than for LS on days 73, 120, 162, 198, and 225, respectively. Average daily gain in periods 1-5 was similar among breeds (p > 0.05) except for a lower mean for STC vs. KAT in period 2 (p < 0.05; Table 3). Additionally, ADG by KAT in period 6, when all animals received HS and had access to a higher quality forage, was the lowest among the breeds (p < 0.05). Supplement treatment affected ADG in all periods (p < 0.05) except periods 7 and 6-7. The values averaged 93, 106, 123, 71, and 113 g greater for HS than for LS in periods 1, 2, 3, 4, and 5, respectively (p < 0.05). In period 6, ADG averaged 110 and 22 g for LS and HS, respectively (p < 0.5), with an 88 g difference. In period 7, ADG was similar between supplement treatments (p > 0.05), resulting in only a numerical difference in ADG of 39 g (p > 0.05) in periods 6-7. Overall, in periods 1-7, ADG averaged 61 g greater for HS vs. LS (p < 0.05; 9 and 70 g for LS and HS, respectively). 3.2. BCS and BMI Body condition score was greatest among the breeds for KAT and greater for HS than for LS on days -7, 49, 73, 120, and 162 (p < 0.05; Table 4). On day 198, BCS was also greater for HS vs. LS (p < 0.05), although the BCS for DOR was greater than for KAT and STC (p < 0.05). There was an interaction between breed and supplement treatment in BCS on day 225 (p = 0.012), which was due in part to similar values for DOR with both LS and HS supplement treatments (p > 0.05) conversely to greater values for HS vs. LS for KAT and STC (p < 0.05). The overall BCS mean did not vary much among days, averaging 3.13, 3.19, 3.10, 3.22, 3.00, 3.03, 3.03, and 3.02 on days -7, 14, 49, 73, 120, 162, 198, and 225, respectively. The only effect of breed on change in BCS without an interaction between breed and supplement treatment was for days 162-198, with means ranking (p < 0.05) KAT < STC < DOR (Table 4). There were breed by supplement treatment interactions (p < 0.05) in changes in BCS on days 120-162, 198-225, and 162-225, which were due to differences between supplement treatments for DOR (p < 0.05) but not KAT or STC. The main effect means of BCS change for supplement treatments were greater for HS vs. LS (p < 0.05) until 120 days, but thereafter, BCS change was similar (p > 0.05) between supplement treatments. The magnitudes of change in BCS between -7 and 162 days and 0 and 225 days were thus greater for HS vs. LS as well as between 162 and 225 days. There were differences (p < 0.05) among breeds in BMI on days -7, 14, 49, 73, and 162, and when not significant, there were tendencies for differences (p < 0.12; Table 5). In many instances, BMI was lowest among breeds for STC and similar among DOR and KAT, although in a small number of cases, the BMI for STC differed only from that of KAT. Supplement treatment did not affect BMI on day -7 as would be expected (p > 0.05), and this was also true for values on days 14 and 49. However, each of the four BMIs was greater for HS vs. LS on day 162 (p < 0.05), and there were similar differences (p < 0.05) or tendencies for BMI on day 73 (p < 0.12). There was only one case (BMI-WH between 73 and 162 days) in which change in BMI differed among breeds (p < 0.05; DOR < KAT), being similar otherwise (p > 0.05). In most time periods, the change in BMI was greater for HS vs. LS (p < 0.05) or tended to differ in this manner. The differences between supplement treatments in change during the entire time when different supplements were given were highly significant (p < 0.01) for each of the four BMIs. Differences for BMI-WH, BMI-WP, BMI-GH, and BMI-GP averaged 3.00, 2.09, 1.51, and 0.97 units greater for HS vs. LS, respectively. Although, it should be realized that BMI-WH and BMI-WP are based on Wither and BMI-GH and BMI-GP are based on Heart, with the latter being greater in magnitude (data not reported). Similarly, BMI-WP and BMI-GP are based on Pin, with BMI-WH and BMI-GH based on Hook, again with the magnitude of the latter being greater. 3.3. HR and HE Breed did not impact HR or any measure of HE on day 14, 49, or 73 (p > 0.05; Table 6). Heart rate was greater for HS than for LS at each time. Heat energy was not affected by supplement treatment on day 14 (p > 0.05), but values in MJ/day were greater for HS on day 49 and 73 and in kJ/kg BW0.75 on day 49. 3.4. Blood Constituent Concentrations All blood constituent concentrations were affected by day and the supplement treatment by day interaction (p < 0.05; Table 7). Moreover, there were some effects of breed and three breed by day interactions (p < 0.05). One variable, the concentration of triglycerides, was affected by a breed by supplement treatment interaction (p < 0.05). The levels of total protein and albumin were lower on days 73 and 162 than earlier for LS, whereas for HS, the levels either decreased only slightly or remained relatively steady on these last 2 days of sampling (Table 8). Additionally, the level of total protein was lowest among the breeds for DOR (p < 0.05). The pattern of change with advancing time for the concentration of urea N also differed between supplement treatments. For LS, the level was highest among days for day -7, but for HS, the level increased and then declined as the day advanced. Additionally, the urea N concentration was greater for STC vs. KAT (p < 0.05), with an intermediate value for DOR (p > 0.05). The TG concentration was similar among days for LS, whereas for HS, the concentration was lowest among the days for day -7 and greatest for day 162 (p < 0.05). Breed by supplement treatment means for triglyceride concentration ranked are (p < 0.05) DOR-HS > KAT-HS > STC-HS, DOR-LS, KAT-LS, and STC-LS (p < 0.05). The concentration of cholesterol for LS increased and then decreased as the day advanced, and that for HS decreased and then increased to the same level as on day -7. The concentration of NEFA for HS was slightly greater on day -7 than on later days, although levels for LS increased markedly from day -7 to 14 and then declined slightly thereafter to levels above that initially on day -7. The glucose concentration for LS decreased markedly from day -7 to days 14 and 49, increasing slightly thereafter (Table 8). Conversely, the concentration for HS decreased slightly from the value on day -7 and was steady thereafter. The concentration of glucose was relatively less for KAT at all times and did not markedly differ among days 14-162, whereas the values on day -7 for DOR and STC were high relative to those on subsequent sampling days. The lactate concentration for LS was considerably less on days 14 and 49 than on day -7, whereas values for HS differed among days relatively less. The lactate concentration for DOR and STC decreased and then increased with advancing time, but the level for KAT was fairly steady over the days. For HS, the TAC was lowest among days for day 162. Conversely, the values for LS on days 49 and 73 were greater than on days 14 and 162. The pattern of change in TAC with advancing time varied considerably among breeds, with values decreasing, increasing, and decreasing for DOR, increasing then decreasing for KAT, and lower on day 162 than on earlier days for STC. 3.5. Reproductive Performance The birth rate was numerically (p = 0.063) greater for HS than for LS (96.3 vs. 78.0%; Table 9). The results were similar for the separate analysis of data from multiparous and primiparous animals, with supplement treatment p-values of 0.036 and 0.082, respectively. Litter size and fecundity were greater (p < 0.05) for HS vs. LS (1.69 vs. 1.37). For the supplement effect, the p-value of litter size for primiparous animals was similar (p = 0.047) to the p-value of overall litter size (p = 0.046), although the p-value (0.398) of litter size for multiparous animals was different. This difference between parity for the supplement effect appeared largely due to values for STC, with means of 1.00, 1.20, 1.50, 2.33, 2.00, and 1.92 for DOR-LS, DOR-HS, KAT-LS, KAT-HS, STC-LS, and STC-HS, respectively (SEM = 0.152). The only reproductive performance variable affected by breed was individual lamb birth weight (p = 0.035), being greater for DOR than for STC (4.56, 4.18, and 3.81 kg for DOR, KAT, and STC, respectively). The p-value for multiparous animals was similar at 0.062, and that for primiparous animals was slightly greater (0.169) because of numerical differences of lesser magnitude (4.43, 4.27, 4.25, 4.06, 3.66, and 3.92 kg for DOR-LS, DOR-HS, KAT-LS, KAT-HS, STC-LS, and STC-HS, respectively; SEM = 0.244). The total litter weight was greater (p = 0.049) as well for HS than for LS (6.68 vs. 5.60 kg). The p-value for primiparous animals was similar at 0.050. Conversely, the p-value for multiparous animals was 0.714, primarily because of values for STC and higher variability (5.03, 6.05, 7.85, 8.49, 7.52, and 7.10 kg for DOR-LS, DOR-HS, KAT-LS, KAT-HS, STC-LS, and STC-HS, respectively; SEM = 1.204). Gestation length was not influenced (p > 0.05) by breed or supplement treatment, and there were no breed by supplement treatment interactions for any reproductive performance variable (p > 0.05). 3.6. BMI Relationships Correlation coefficients between BMI and BW were slightly greater for BMI-WH and BMI-WP on days 49, 73, and 162 and change from day -7 to day 162 relative to those for BMI-GH and BMI-GP, although the r values at earlier days were fairly similar (Table 10). The same was true for r values between BMI and BCS on day 162 and for change from day -7 to day 162, with similar values at earlier days among the four BMI. In all cases, the correlation coefficients between BMI-WH and BMI-WP and BW at different times and change in BW were greater than for BCS, and the same was true for BMI-GH and BMI-GP except for values for day 162 and change from day -7 to day 162. 4. Discussion 4.1. Feed Intake For low-quality forages, digestive capacity including fermentation, the breakdown of feed particles, and digesta passage rate are important determinants of feed intake . Low-quality forages are fermented in the rumen relatively slowly which can result in limited feed intake because of high ruminal NDF fill . The supplementation of low-quality forages is necessary to support microbial growth in the rumen and improve fermentation. Usually, supplemental protein increases voluntary forage intake and digestibility when the forages contain less than 6 to 8% crude protein . However, depending on numerous factors, adding a high-protein feedstuff(s) alone might not be sufficient. The findings regarding feed intake by the three breeds of hair sheep in this study are fairly similar to those of Lourencon et al. , indicating that none was more capable than any other in achieving nutrient and energy intake adequate for higher levels of production with diets based on low-quality forage with supplement treatments varying in the level of feeding and chemical composition. Some of the findings regarding the effects of supplement treatments on feed intake also are similar to those of Lourencon et al. , although there were some interesting differences. In the current experiment, wheat straw DM intake in the first period, only 14 days in length, was similar between LS and HS, which is in contrast to greater wheat straw intake in % BW for LS in the study of Lourencon et al. in each of the four periods. It seems that 14 days was not adequate to establish an optimal ruminal microbial community for improved digestion of this low-quality forage to lessen ruminal digesta fill and increase intake . In the present experiment, wheat straw DM intake was 0.29, 0.49, 0.61, and 0.66% BW and 23.5, 37.5, 43.2, and 49.9% greater for LS than for HS in periods 2, 3, 4, and 5, respectively. However, this compensation in basal forage intake was incomplete or partial in periods 2 and 3, with total intake in % BW greater for HS vs. LS. Conversely, wheat straw intake was fully compensatory for the difference in supplement intake in % BW in periods 4 and 5, with similar total DM intake for each supplement treatment. The degree to which high NDF intake restricts DM intake due to ruminal fill varies with factors such as forage type, animal species, BW, and physiological stage. For example, NDF intake limits have been proposed for different animal types, such as 1.1% BW for dairy cattle and 2.1% and 1.76% BW for ewes of 45 and 90 kg BW, respectively . In the present study, it is likely that daily NDF intake capacity (1.63% BW calculated from the intake and NDF concentration in the diet) reached a maximal level for LS sheep in periods 4 and 5, whereas for HS sheep, the intake was probably not limited by NDF ruminal fill (1.22% BW) but rather by other factors such as nutrient and energy absorption in relation to requirements and potential for efficient metabolism . This latter finding in the present experiment was similar to one of Lourencon et al. in the first three periods when different levels of supplement were offered, with similar total intake between LS and HS. However, it is important to note the difference in the level of offering of HS between the studies, with 0.8% BW used previously by Lourencon et al. and 1.00% BW in the current experiment. This difference appeared responsible for much of that in total intake between supplement treatments, as the degree to which wheat straw intake was greater for LS than for HS noted by Lourencon et al. was fairly similar to that in the current experiment, with differences of 0.44, 0.44, 0.57, and 0.33% BW in periods 1, 2, 3, and 4, respectively. Overall, based on the total DM intake in these two experiments, it would not seem that there were marked differences in negative associative effects of the HS supplement treatment (i.e., 1.00 vs. 0.80% BW DM intake), with greater benefits in terms of nutrient and energy intake from higher intakes of the HS supplement in the present experiment. 4.2. BW and ADG In contrast to the findings of Lourencon et al. and other studies , there were only two measurement days (on days 49 and 225) when differences in BW among breeds were detected, although the values in all periods were numerically lowest for STC. This in part could relate to the limited number of observations and, relatedly, the inclusion of both primiparous and multiparous animals. Because of relatively high variability in BW, even though ADG was greater for HS vs. LS in periods 1, 2, and 3, the differences in BW between the supplement treatments did not occur until day 73. Smaller magnitudes of difference in BW on days 198 and 225 than earlier would relate to imposing the HS treatment on all animals in periods 6 and 7, resulting in greater ADG for LS vs. HS in period 6 and no difference in period 7. That is, higher ADG in period 6 was likely due to compensatory metabolism and tissue accretion as a result of limited nutritional supply in the previous periods . Greater ADG during the higher nutritional plane of previously restricted animals has been attributed to factors such as increased feed intake and efficiency of metabolism in the early segment of the realimentation phase as a function of a reduced maintenance requirement because of the decreased mass of metabolically active organs (e.g., digestive tract and liver) in association with endocrine (e.g., insulin and insulin-like growth factor 1) and metabolic (higher protein sparing) responses . In a study with beef cattle steers, the efficiency of metabolizable energy utilization after a high nutritional plane was elevated for at least 28 days and thereafter decreased steadily to control levels . In period 7, this compensatory response perhaps did not exist, which could also involve the advanced stage of pregnancy. Nonetheless, differences in BW at the end of periods 6 and 7 were substantial (i.e., approximately 21% for HS vs. LS). 4.3. BCS and BMI The differences in BCS (KAT > DOR and STC) at most times despite similar BW suggest considerable disparities in the sites of tissues varying in composition such as lipid. Assuming that BCS is markedly influenced by the presence of subcutaneous fat as well as muscular lean tissue, it would appear that KAT stored relatively more energy in external than internal sites compared with DOR and STC. Breed differences for subcutaneous, carcass, and internal fat deposition that are greatly genetically influenced have been reported in various studies . However, based on the change in BCS during the experiment, it seems that this was due primarily to deposition and maintenance in earlier periods of time. However, these results are considerably different than noted by Lourencon et al. , with no differences among breeds noted at the beginning of the experiment and after 4 wk and then lower BCS for STC than for DOR and KAT after 8 wk. Similarly, the whole body concentrations of water, protein, fat, and energy in that study either were similar among breeds or differed between STC and the other breeds of DOR and KAT. The factors responsible for these differences are unclear, but differences in BW, with higher values for Lourencon et al. vs. the present study, might be one of the factors. The differences in BMI among breeds are quite dissimilar to those in BCS, which again may reflect varying sites and levels of adipose and lean tissues relative to BW and body size. Breed differences in BMI have been consistent because BMIs are functions of body size (e.g., height, length, circumference), which generally vary among breeds of a species at a particular age . Overall, similar BMI for DOR and KAT in most instances, in contrast to greater BCS for KAT, suggests a greater mass of internal adipose tissue stores for DOR. Conversely, the lowest BMI for STC among breeds in most cases, despite similar BCS for STC and DOR, implies minimal tissue energy stores both in subcutaneous and internal sites. However, as suggested earlier for BCS, it would appear that such differences developed and were in place before this study. 4.4. HR and HE Heat energy is associated with basal metabolism, activity, and tissue accretion. Heat energy was 1.234 and 2.430 MJ/day and 15.2 and 21.9% greater for HS than for LS on days 49 and 73, respectively. Greater straw intake by LS sheep could have contributed to higher HE associated with mastication during eating and rumination, but it seems that differences in other conditions such as basal metabolism and tissue deposition had a relatively greater impact. Again, high planes of nutrition elicit increased heat production, at least in part because of the greater mass of metabolically active internal organs . These differences in HE were of considerably lower magnitude than those in total DM intake. Although digestibility and metabolizability, which could be higher in HS vs. LS, may have been affected by supplement treatment, based on these findings, the recovered energy was probably much greater for HS than for LS, in accordance with differences in reproductive performance noted later. 4.5. Blood Constituent Concentrations There were only two interactions between breed and day of sampling, with causal factors unclear. Similarly, interactions between supplement treatment and day of sampling for all variables exemplify the importance of considering the time during which the treatments were implemented on potential differences among nutritional plane treatments. The tendency for a higher total protein concentration for HS than for LS may relate to greater protein intake for the HS supplement treatment. Similarly, the greater blood urea N concentration in HS vs. LS sheep probably resulted from higher protein intake for HS, consequently leading to greater ruminal ammonia N production by ruminal microbes and, thus, elevated ammonia N absorption into the bloodstream . The magnitudes of difference in the total protein and albumin levels for HS sheep were similar throughout periods of the study, but they decreased with advancing time for the LS supplement treatment. This reflects that protein intake for the LS supplement treatment was less than required or that could be efficiently utilized, which was also reflected in ADG as noted before. This is supported by urea N concentrations for LS that were similar among days 14, 49, 73, and 162 and less than on day -7, which contrasts levels for HS greater than for LS on days 14, 49, 73, and 162, though with levels for HS declining from day 49 to 73 and 162. Blood glucose and NEFA concentrations are indicators of energy status in animals. A greater glucose concentration for HS vs. LS at one time and corresponding numerical differences at all others involve conditions such as greater ruminal microbial propionate production because of corn included in the HS supplement , but relatively high variability reflects the influence of many other factors. The concentration of blood NEFA increases when energy demands cannot be met from feed intake and lipolysis occurs to supply precursor molecules for energy , which is in accordance with the substantial differences between supplement treatments at all times except day -7. The greater blood cholesterol levels on days 14 and 49 for LS vs. HS further depict the energy deficit of LS sheep, as cholesterol is involved in the transport of fatty acids from adipose tissue in response to an energy shortfall in absorbed fuels . Opposite differences between supplement treatments in blood cholesterol concentration on days 73 and 162 were somewhat unexpected given differences at earlier times but may reflect the compensatory increased level of wheat straw intake for LS in this latter segment of the supplementation period to lessen maternal tissue mobilization. The greater TG concentration in HS sheep at all times except day -7 reflects an elevated energy status compared with LS sheep. Factors responsible for the greatest level of TG for HS among days on day 163 and the greatest magnitudes of difference between supplement treatments at this time are unclear since total DM intake relative to BW was similar between periods 4 and 5. However, it is possible that a cumulative effect of differences in energy and nutrients absorbed between supplement treatments was somehow involved. 4.6. Reproductive Performance A high plane of nutrition before breeding has been shown to improve the reproductive performance of sheep in many studies via improved reproductive hormonal balance and follicular development . Supplement treatment had a much greater effect on reproductive performance measures than in the previous experiment of Lourencon et al. , which could involve factors such as the higher level of intake of the HS supplement and a longer length of time during early gestation when supplement treatments were imposed in the current experiment. In the previous experiment, there were no significant effects of supplement treatment on these variables. Conversely, in the current study, the tendency for a difference in the birth rate was substantial, with 78.0 and 96.3% for LS and HS, respectively, as was also the case for litter sizes of 1.37 and 1.68 lambs for LS and HS, respectively. The latter difference resulted in a litter birth weight of 1.08 kg and 19.3% greater for HS vs. LS. It has been suggested that BCS during breeding should range from 3 to 3.5 for optimal reproductive performance . The BCS for both HS and LS treatments were in this range. Therefore, it would appear that the difference in reproductive performance was a function of nutritional status during breeding and gestation, which are important for the embryo quality, ovulation rate, uterine response, and reproductive performance of ewes . However, differences in experimental conditions between the present study and that of Lourencon et al. could have had an influence. For example, the trial of Lourencon et al. was with mostly multiparous animals (i.e., 81 of 85), with an average initial age of all females of 4.9 years. In contrast, the current experiment entailed a greater number of relatively young ewe lambs (72; 1.5 years initial age) that had not previously given birth compared with multiparous ewes (35; 5.6 years initial age). A greater number of multiparous ewes would be desirable for a stronger evaluation of the potential effects of parity, although the results of the separate analysis conducted suggest fairly similar effects of supplement treatments. 4.7. BMI and BCS Relationships The correlation coefficients between BMI-WH and BMI-WP and BW were at least slightly greater than those for BMI-GH and BMI-GP, suggesting less variability in measurement or among animals in height than heart girth. Relatedly, higher correlations between change in BMI-WH and BMI-WP and that in BCS from day -7 to 162 relative to BMI-GH and BMI-GP also implies advantages from the use of BMI-WH and BMI-WP. Likewise, as observed in studies such as Lourencon et al. and Wang et al. , the BMI measurements in this study, particularly BMI-WH and BMI-WP, are more highly related to BW than BCS, although, naturally, it should be realized that BMI is calculated from BW and may not be as reflective of sites of different types of tissues relative to BCS. 5. Summary and Conclusions The supplement treatments imposed before breeding and in early gestation caused differences in wheat straw and total feed intake, daily body weight gain, body condition score and mass indexes, litter size, total litter birth weight, and heat energy production, although the effects were generally similar among breeds. Likewise, breed affected many factors such as body weight, condition score, and mass indexes, but reproductive performance was not influenced. The litter size and birth weight for the supplement treatments reflect the importance of considering not only the need for additional protein during breeding and early gestation periods with the consumption of a low-quality basal dietary forage but also the inclusion of a high-energy feedstuff(s) for higher reproductive performance. Author Contributions Conceptualization, A.L.G., R.P. and F.B.; methodology, F.B., R.V.L., R.P., L.J.D. and A.L.G.; validation, R.P., A.K.P. and A.L.G.; formal analysis, F.B., R.P., A.K.P. and A.L.G.; investigation, R.V.L., R.P., L.J.D., L.P.d.S.R., F.E. and A.L.G.; resources, F.B., R.P., L.J.D. and A.L.G.; data curation, F.B., R.V.L., R.P., L.J.D., L.P.d.S.R., F.E. and A.L.G.; writing--original draft preparation, F.B., A.K.P. and A.L.G.; writing--review and editing, A.K.P. and A.L.G.; supervision, R.P., L.J.D. and A.L.G.; project administration, A.L.G.; funding acquisition, F.B., A.L.G. and R.P. All authors have read and agreed to the published version of the manuscript. Institutional Review Board Statement The study was conducted according to the guidelines of the AWA and PHS policy and approved by the Langston University Animal Care and Use Committee (Approval Number: 19-015; 2 August 2019). Informed Consent Statement Not applicable. Data Availability Statement Data are presented in the tables. Conflicts of Interest The authors declare no conflict of interest. Figure 1 Schematic presentation of the experimental design. Figure 2 Two supplements fed to three breeds of sheep during different periods. animals-13-00814-t001_Table 1 Table 1 Composition of wheat straw and supplements (%, dry matter basis) 1. Wheat Straw Low Supplement High Supplement Item Mean SEM Mean SEM Mean SEM Ash 6.4 0.23 8.6 0.12 3.6 0.10 Crude protein 3.9 0.11 47.8 0.19 14.5 0.67 Neutral detergent fiber 80.2 0.52 12.1 0.55 13.5 0.38 Acid detergent fiber 53.9 0.38 8.8 0.36 5.3 0.24 Acid detergent lignin 10.4 0.20 1 Low supplement = soybean meal; High supplement = 25% soybean meal and 75% rolled corn; mean of weekly samples. animals-13-00814-t002_Table 2 Table 2 Effects of breed and supplement treatment on dry matter intake by hair sheep 1. Effect p Value 2 Dorper Katahdin St. Croix Item Brd Sup Brd x Sup Low High Low High Low High SEM Brd 3 Period 1 g/day Supplement 0.266 <0.001 0.332 87 556 83 579 79 494 25.6 Wheat straw 0.648 0.337 0.907 640 618 626 550 679 616 62.8 Total 0.906 <0.001 0.789 727 1174 709 1296 758 1110 70.4 % body weight Supplement 0.588 <0.001 0.290 0.15 0.99 0.14 0.99 0.15 0.98 0.007 Wheat straw 0.211 0.461 0.916 1.13 1.10 1.07 0.94 1.32 1.22 0.131 Total 0.208 <0.001 0.918 1.28 2.09 1.21 1.93 1.47 2.20 0.131 Period 2 g/day Supplement 0.266 <0.001 0.332 87 556 83 579 79 494 25.6 Wheat straw 0.410 0.045 0.927 920 750 801 681 830 700 67.9 Total 0.407 0.002 0.818 1007 1306 884 1261 909 1193 77.0 % body weight Supplement 0.636 <0.001 0.939 0.16 0.96 0.14 0.95 0.16 0.95 0.013 Wheat straw 0.233 0.036 0.951 1.65 1.29 1.39 1.12 1.64 1.35 0.139 Total 0.214 0.005 0.954 1.80 2.25 1.53 2.06 1.80 2.30 0.140 Period 3 g/day Supplement 0.266 <0.001 0.332 87 556 83 579 79 494 25.6 Wheat straw 0.410 0.006 0.338 1031 752 879 773 902 739 54.6 Total 0.312 0.002 0.329 1117 1308 962 1353 981 1233 62.6 % body weight Supplement 0.063 <0.001 0.076 0.16 0.94 0.15 0.92 0.16 0.93 0.009 Wheat straw 0.321 0.005 0.638 1.89 1.27 1.59 1.24 1.88 1.39 0.135 Total 0.292 0.039 0.648 2.04 2.21 1.74 2.17 2.04 2.32 0.135 Period 4 g/day Supplement 0.266 <0.001 0.332 87 556 83 579 79 494 25.6 Wheat straw 0.061 0.002 0.507 1111 870 990 800 938 801 42.1 Total 0.040 <0.001 0.711 1198 1426 1073 1379 1017 1295 46.2 S < D % body weight Supplement 0.063 <0.001 0.117 0.17 0.95 0.16 0.91 0.17 0.91 0.009 Wheat straw 0.253 0.001 0.986 2.12 1.49 1.89 1.27 2.07 1.48 0.130 Total 0.213 0.227 0.988 2.29 2.45 2.05 2.18 2.24 2.39 0.133 Period 5 g/day Supplement 0.281 <0.001 0.315 87 556 83 579 81 494 25.6 Wheat straw 0.441 0.001 0.365 989 814 974 795 910 813 29.5 Total 0.298 <0.001 0.963 1076 1370 1056 1375 992 1306 47.2 % body weight Supplement 0.063 <0.001 0.117 0.17 0.93 0.16 0.86 0.18 0.88 0.011 Wheat straw 0.115 <0.001 0.789 1.96 1.35 1.91 1.18 2.08 1.45 0.088 Total 0.088 0.425 0.643 2.13 2.28 2.07 2.04 2.27 2.33 0.091 Average g/day Supplement 0.269 <0.001 0.328 87 556 83 579 80 494 25.6 Wheat straw 0.350 0.007 0.789 938 761 854 720 852 734 43.7 Total 0.306 <0.001 0.771 1025 1317 937 1299 931 1227 53.9 % body weight Supplement 0.020 <0.001 0.058 0.16 0.96 0.15 0.92 0.17 0.93 0.005 K < D Wheat straw 0.191 0.003 0.992 1.75 1.30 1.57 1.15 1.80 1.38 0.112 Total 0.165 0.009 0.999 1.91 2.26 1.72 2.07 1.96 2.31 0.113 1 Supplement treatments were soybean meal fed at approximately 0.15% of initial body weight (determined 7 days before the start of the feeding period) and a mixture of 25% soybean meal and 75% ground corn given at approximately 1% of initial body weight (dry matter basis; Low and High, respectively); periods 1, 2, 3, 4, and 5 were 14, 35, 24, 47, and 42 days in length, respectively; with estrus synchronization, a 7-day breeding period began for set 1 animals (one of the two pens per treatment) after 83 days of feeding on day 12 of period 4 and for set 2 animals after 96 days of feeding on day 24 of period 4. 2 Brd = breed; Sup = supplement treatment. 3 D = Dorper; K = Katahdin; S = St. Croix; main effect mean differences (p < 0.05) with nonsignificant interactions between breed and supplement treatment (p > 0.05). animals-13-00814-t003_Table 3 Table 3 Effects of breed and supplement treatment on body weight and average daily gain of hair sheep 1. Effect p-Value 2 Dorper Katahdin St. Croix Item 3 Brd Sup Brd x Sup Low High Low High Low High SEM Brd 4 BW (kg), day -7 0.106 0.583 0.969 57.7 55.6 58.5 57.9 51.3 49.4 3.31 14 0.082 0.865 0.974 56.4 56.6 58.2 59.3 51.7 51.6 2.71 49 0.046 0.144 0.838 55.7 59.4 57.1 63.1 49.7 52.3 2.98 S < K 73 0.128 0.039 0.869 53.8 58.8 53.4 62.0 46.7 54.2 3.25 120 0.140 0.020 0.625 51.1 57.6 51.3 65.9 44.2 54.1 3.38 162 0.092 0.002 0.730 50.1 62.7 51.2 69.7 43.5 57.9 3.69 198 0.088 0.004 0.688 55.9 67.3 53.8 67.6 49.8 58.4 3.03 225 0.029 0.004 0.681 63.9 73.7 60.1 75.5 52.6 63.6 3.27 S < D & K ADG (g), period 1 0.279 0.022 0.901 -62 51 -16 64 18 105 37.1 2 0.039 0.001 0.273 -22 76 -32 110 -59 19 19.3 S < K 3 0.247 0.014 0.321 -79 -22 -154 -46 -123 79 43.7 4 0.250 0.046 0.373 -57 -29 -46 83 -56 -1 34.6 5 0.957 0.006 0.739 -24 120 -1 90 -14 90 33.3 6 0.022 0.017 0.258 130 113 48 -59 153 12 33.1 K < D & S 7 0.067 0.501 0.307 295 238 232 273 102 195 43.8 1 to 5 0.175 <0.001 0.256 -46 42 -44 70 -47 51 7.3 6 to 7 0.086 0.172 0.990 200 166 126 84 131 90 30.8 1 to 7 0.221 <0.001 0.770 20 76 6 73 2 62 7.7 1 Supplement treatments were soybean meal fed at approximately 0.15% of initial body weight (determined 7 days before the start of the feeding period) and a mixture of 25% soybean meal and 75% ground corn given at approximately 1% of initial body weight (dry matter basis; Low and High, respectively). 2 Brd = breed; Sup = supplement treatment. 3 BW = body weight; ADG = average daily gain; BW was determined 7 days before the supplementation treatments were imposed (day -7), after 14, 49, 73, 120, and 162 days of feeding, and after additional periods of 36 and 27 days (days 198 and 225, respectively); periods 1, 2, 3, 4, 5, 6, and 7 were 21 (7 days before supplementation began and 14 days thereafter), 35, 24, 47, 42, 36, and 27 days in length, respectively. 4 D = Dorper; K = Katahdin; S = St. Croix; main effect mean differences (p < 0.05) with nonsignificant interactions between breed and supplement treatment (p > 0.05). animals-13-00814-t004_Table 4 Table 4 Effects of breed and supplement treatment on body condition score of hair sheep 1. Effect p Value 2 Dorper Katahdin St. Croix Item 3 Brd Sup Brd x Sup Low High Low High Low High SEM Brd 4 Day -7 0.022 0.014 0.678 3.20 2.90 3.43 3.25 3.16 2.81 0.098 K > D & S 14 0.108 0.398 0.908 3.14 3.11 3.36 3.26 3.16 3.09 0.085 49 0.003 0.031 0.703 2.97 3.15 3.23 3.31 2.89 3.02 0.056 K > D & S 73 0.019 0.018 0.579 3.11 3.27 3.26 3.56 2.90 3.21 0.092 K > D & S 120 0.006 0.001 0.852 2.76 3.19 2.98 3.49 2.55 3.05 0.086 K > D & S 162 0.028 <0.001 0.286 2.59 3.25 2.89 3.52 2.77 3.16 0.083 K > D & S 198 0.007 <0.001 0.598 3.01 3.41 2.67 3.15 2.78 3.15 0.059 D > K & S 225 0.008 <0.001 0.012 3.04 bc 3.23 cd 2.73 a 3.37 d 2.74 a 2.98 b 0.057 Change, day -7 to 14 0.159 0.015 0.375 -0.07 0.22 -0.07 0.01 0.00 0.28 0.077 14 to 49 0.061 0.003 0.962 -0.16 0.03 -0.13 0.05 -0.26 -0.07 0.046 49 to 73 0.749 0.049 0.126 0.14 0.09 0.03 0.25 0.01 0.19 0.058 73 to 120 0.481 0.004 0.725 -0.35 -0.05 -0.29 -0.06 -0.35 -0.16 0.065 120 to 162 0.007 0.082 0.029 -0.17 a 0.07 bc -0.08 ab 0.03 ab 0.22 c 0.11 bc 0.047 162 to 198 <0.001 0.151 0.345 0.30 0.15 -0.25 -0.37 -0.04 -0.01 0.059 K < S < D 198 to 225 0.003 0.115 0.021 0.02 b -0.18 a 0.07 bc 0.20 c -0.03 ab -0.17 a 0.047 -7 to 162 0.334 <0.001 0.384 -0.61 0.36 -0.53 0.27 -0.39 0.34 0.077 162 to 225 0.003 0.020 0.049 0.33 b -0.03 a -0.19 a -0.16 a -0.07 a -0.18 a 0.058 -7 to 225 0.059 <0.001 0.481 -0.28 0.31 -0.71 0.10 -0.42 0.18 0.102 1 Supplement treatments were soybean meal fed at approximately 0.15% of initial BW (determined 7 days before the start of the feeding period) and a mixture of 25% soybean meal and 75% ground corn given at approximately 1% of initial BW (DM basis; Low and High, respectively). 2 Brd = breed; Sup = supplement treatment. 3 Body condition score (1-5) was determined 7 days before the supplementation treatments were imposed (day -7), after 14, 49, 73, 120, and 162 days of feeding, and after additional periods of 36 and 27 days (days 198 and 225, respectively); periods 1, 2, 3, 4, 5, 6, and 7 were 21 (7 days before supplementation began and 14 days thereafter), 35, 24, 47, 42, 36, and 27 days in length, respectively. 4 D = Dorper; K = Katahdin; S = St. Croix; main effect mean differences (p < 0.05) with nonsignificant interactions between breed and supplement treatment (p > 0.05). a-d Means without a common superscript letter within a variable differ (p < 0.05). animals-13-00814-t005_Table 5 Table 5 Effects of breed and supplement treatment on body mass indexes of hair sheep 1. Effect p Value 2 Dorper Katahdin St. Croix Item 3 Brd Sup Brd x Sup Low High Low High Low High SEM Brd 4 Initial, day -7 BMI-WH 0.023 0.396 0.809 14.30 14.33 14.73 14.16 13.06 12.49 0.495 S < D & K BMI-WP 0.044 0.293 0.986 11.62 11.29 11.63 11.16 10.40 9.94 0.443 S < D & K BMI-GH 0.034 0.343 0.695 10.10 10.16 10.62 10.26 9.74 9.34 0.271 S < K BMI-GP 0.069 0.219 0.968 8.20 8.01 8.38 8.09 7.76 7.44 0.234 Day 14 BMI-WH 0.015 0.763 0.643 14.02 13.64 13.55 13.83 12.04 12.46 0.414 S < D & K BMI-WP 0.010 0.964 0.648 11.12 10.80 10.61 10.66 9.42 9.72 0.307 S < D & K BMI-GH 0.031 0.764 0.453 10.42 10.12 10.30 10.56 9.60 9.82 0.218 S < K BMI-GP 0.035 0.943 0.561 8.26 8.01 8.06 8.14 7.52 7.67 0.176 S < D & K Day 49 BMI-WH 0.004 0.087 0.721 13.85 14.16 13.26 14.31 11.51 12.29 0.426 S < D & K BMI-WP 0.005 0.157 0.810 10.99 11.18 10.40 11.04 9.13 9.61 0.330 S < D & K BMI-GH 0.011 0.436 0.565 10.63 10.51 10.32 10.81 9.43 9.62 0.266 S < D & K BMI-GP 0.021 0.802 0.734 8.43 8.30 8.10 8.35 7.48 7.52 0.234 S < D & K Day 73 BMI-WH 0.057 0.039 0.940 13.33 14.70 12.60 13.92 11.01 12.77 0.690 BMI-WP 0.092 0.048 0.922 10.34 11.27 9.81 10.74 8.76 10.05 0.517 BMI-GH 0.064 0.083 0.752 10.07 10.64 10.02 10.43 8.81 9.79 0.384 BMI-GP 0.117 0.115 0.677 7.82 8.15 7.82 8.04 7.02 7.71 0.277 Day 162 BMI-WH 0.034 0.001 0.748 10.94 14.54 12.06 15.09 10.02 12.57 0.663 S < K BMI-WP 0.004 <0.001 0.694 9.63 11.35 9.44 11.71 8.04 10.11 0.305 S < D & K BMI-GH 0.079 0.015 0.810 8.52 10.14 9.34 10.53 8.14 9.16 0.451 BMI-GP 0.006 0.003 0.442 7.50 7.92 7.31 8.18 6.53 7.38 0.176 S < D & K Change, day -7 to 14 BMI-WH 0.647 0.065 0.088 -0.28 -0.70 -1.18 -0.33 -1.14 -0.02 0.280 BMI-WP 0.624 0.077 0.356 -0.50 -0.49 -1.02 -0.51 -1.07 -0.22 0.262 BMI-GH 0.672 0.096 0.074 0.33 -0.04 -0.32 0.30 -0.21 0.48 0.192 BMI-GP 0.599 0.086 0.294 0.066 0.01 -0.31 0.04 -0.29 0.22 0.161 Change, day 14 to 49 BMI-WH 0.155 0.026 0.682 -0.17 0.53 -0.29 0.48 -0.53 -0.17 0.252 BMI-WP 0.217 0.029 0.504 -0.13 0.38 -0.21 0.38 -0.29 -0.11 0.181 BMI-GH 0.111 0.446 0.759 0.21 0.39 0.02 0.25 -0.17 -0.20 0.191 BMI-GP 0.149 0.601 0.580 0.17 0.28 0.03 0.21 -0.05 -0.14 0.140 Change, day 49 to 73 BMI-WH 0.358 0.052 0.592 -0.52 0.52 -0.68 -0.38 -0.51 0.47 0.392 BMI-WP 0.241 0.033 0.624 -0.65 0.07 -0.61 -0.30 -0.37 0.44 0.270 BMI-GH 0.836 0.051 0.209 -0.57 0.12 -0.32 -0.38 -0.63 0.17 0.237 BMI-GP 0.407 0.066 0.320 -0.009 0.004 -0.005 -0.005 -0.007 0.003 0.0039 Change, day 73 to 162 BMI-WH 0.048 0.007 0.390 -2.38 -0.16 -0.55 1.17 -0.99 -0.19 0.486 D < K BMI-WP 0.051 0.002 0.448 -0.71 0.11 -0.36 0.97 -0.72 0.06 0.233 BMI-GH 0.151 0.053 0.335 -1.55 -0.50 -0.68 0.10 -0.68 -0.63 0.318 BMI-GP 0.356 0.063 0.223 -0.32 -0.22 -0.50 0.13 -0.48 -0.33 0.176 Change, day -7 to 162 BMI-WH 0.462 <0.001 0.910 -3.36 0.20 -2.70 0.93 -3.08 0.09 0.579 BMI-WP 0.629 <0.001 0.546 -1.99 0.07 -2.19 -0.55 -2.39 0.17 0.297 BMI-GH 0.709 0.009 0.991 -1.58 -0.03 -1.28 0.27 -1.62 -0.18 0.493 BMI-GP 0.446 0.001 0.345 -0.70 -0.09 -1.06 0.08 -1.24 -0.07 0.189 1 Supplement treatments were soybean meal fed at approximately 0.15% of initial body weight (determined 7 days before the start of the feeding period) and a mixture of 25% soybean meal and 75% ground corn given at approximately 1% of initial body weight (dry matter basis; Low and High, respectively). 2 Brd = breed; Sup = supplement treatment. 3 Body mass indexes (BMI) were determined 7 days before the supplementation treatments were imposed (day -7) and after 14, 49, 73, and 162 days of feeding; Wither = height at withers; Hook = point of the shoulder to hook bone; Pin = point of the shoulder to pin bone; Heart = heart girth; BMI-WH = BW/(Wither x Hook) [g/cm2]; BMI-WP = BW/(Wither x Pin) [g/cm2]; BMI-GH = BW/(Heart x Hook) [g/cm2]; BMI-GP = BW/(Heart x Pin) [g/cm2]. 4 D = Dorper; K = Katahdin; S = St. Croix; main effect mean differences (p < 0.05) with nonsignificant interactions between breed and supplement treatment (p > 0.05). animals-13-00814-t006_Table 6 Table 6 Effects of breed and supplement treatment on heart rate and heat energy by hair sheep 1. Effect p Value 2 Dorper Katahdin St. Croix Item 3 Brd Sup Brd x Sup Low High Low High Low High SEM HE:HR (kJ/kg BW0.75/beat) 0.493 0.778 0.886 6.39 6.16 6.79 6.65 6.52 6.63 0.352 Day 14 HR (beats/min) 0.786 0.043 0.722 72.2 85.8 69.9 85.9 71.5 78.6 5.85 HE (kJ/kg BW0.75) 0.995 0.219 0.959 468 523 458 545 468 526 59.4 HE (MJ/day) 0.729 0.276 0.885 9.87 10.74 9.51 11.65 9.02 10.03 1.610 Day 49 HR (beats/min) 0.282 0.005 0.326 66.8 92.1 63.9 78.4 68.3 77.8 4.67 HE (kJ/kg BW0.75) 0.858 0.023 0.647 423 569 428 519 445 511 40.8 HE (MJ/day) 0.220 0.004 0.302 63.0 84.4 65.3 79.5 81.2 82.6 5.83 Day 73 HR (beats/min) 0.272 0.041 0.302 63.0 84.4 65.3 79.5 81.2 82.6 5.83 HE (kJ/kg BW0.75) 0.287 0.120 0.650 394 510 439 524 528 549 49.8 HE (MJ/day) 0.763 0.039 0.803 7.94 10.83 8.67 11.51 9.39 10.95 1.127 1 Supplement treatments were soybean meal fed at approximately 0.15% of initial body weight (determined 7 days before the start of the feeding period) and a mixture of 25% soybean meal and 75% ground corn given at approximately 1% of initial body weight (dry matter basis; Low and High, respectively). 2 Brd = breed; Sup = supplement treatment. 3 HE = heat energy; HR = heart rate; HR was measured on one set of animals 7 days before days 14, 49, and 73 and 7 days after, with HE based on HR and HE:HR determined after the 162-day supplementation period. animals-13-00814-t007_Table 7 Table 7 p-values for effects of breed, supplement treatment, and day on blood constituent concentrations in hair sheep 1. Source of Variation 2 Item Brd Sup Brd x Sup Day Brd x Day Sup x Day Brd x Sup x Day Total protein (g/dL) 0.009 0.056 0.351 <0.001 0.516 0.012 0.585 Albumin (g/dL) 0.213 0.396 0.665 0.046 0.433 <0.001 0.822 Urea nitrogen (mg/dL) 0.034 <0.001 0.383 <0.001 0.106 <0.001 0.327 Cholesterol (mg/dL) 0.478 0.441 0.934 <0.001 0.569 <0.001 0.872 Triglycerides (mg/dL) 0.004 <0.001 0.021 <0.001 0.323 <0.001 0.761 Glucose (mg/dL) 0.610 0.475 0.644 <0.001 <0.001 0.008 0.056 Lactate (mg/dL) 0.282 0.129 0.205 0.016 0.036 0.011 0.349 Nonesterified fatty acids (mEq/L) 0.002 <0.001 0.072 <0.001 0.800 <0.001 0.330 Total antioxidant activity (mM) 0.350 0.270 0.743 <0.001 0.035 0.007 0.695 1 Supplement treatments were soybean meal fed at approximately 0.15% of initial body weight (determined 7 days before the start of the feeding period) and a mixture of 25% soybean meal and 75% ground corn given at approximately 1% of initial body weight (dry matter basis; Low and High, respectively). 2 Brd = breed (Dorper, Katahdin, and St. Croix); Sup = supplement treatment; Day = 7 days before the supplementation treatments were imposed (day -7) and after 14, 49, 73, and 162 days of feeding. animals-13-00814-t008_Table 8 Table 8 Effects of breed, supplementation treatment, and period on blood constituent concentrations in hair sheep 1. Interaction 2 Breed 3 Sup Day 4 Item 5 Brd Sup DOR KAT STC SEM Low High SEM -7 14 49 73 162 SEM TP (g/dL) 6.66 a 6.95 b 6.99 b 0.052 Low 7.09 e 6.92 c-e 6.91 c-e 6.64 b 6.41 a 0.074 High 7.01 de 7.02 de 7.01 de 6.86 bc 6.79 bc ALB (g/dL) 2.66 2.74 2.66 0.034 Low 2.79 c 2.78 c 2.79 c 2.63 b 2.54 a 0.039 High 2.64 b 2.64 b 2.63 b 2.71 bc 2.73 bc UN (mg/dL) 18.4 ab 17.6 a 19.3 b 0.36 Low 20.7 c 15.6 a 15.7 a 15.3 a 15.4 a 0.52 High 20.2 c 22.4 d 22.5 d 19.3 c 17.1 b TG (mg/dL) 26.5 a 27.5 a 27.6 a 28.8 a 34.9 b 0.89 Low 25.3 a 26.5 a 23.9 a 1.06 26.4 a 24.2 a 24.3 a 25.2 a 26.2 a 5.94 High 37.6 c 33.7 b 27.4 a 26.6 a 30.8 b 30.9 b 32.4 b 43.7 c CHOL (mg/dL) 71.3 67.3 68.8 2.89 70.1 68.1 1.76 Low 71.5 c 80.0 d 80.1 d 62.0 b 57.1 a 2.20 High 70.8 c 64.6 b 64.5 b 70.5 c 69.9 c Glucose (mg/dL) 55.8 48.4 57.6 6.89 50.9 57.0 5.63 Low 62.3 cd 48.7 b 44.3 a 51.0 b 48.4 b 5.69 High 65.0 d 55.4 bc 53.4 bc 55.5 bc 55.6 bc DOR 69.9 f 53.9 d-f 49.3 bc 53.8 d-f 52.2 b-e 6.97 KAT 53.3 c-f 47.0 ab 45.7 a 49.1 b 46.8 ab STC 67.7 ef 55.2 ef 51.6 b-d 56.8 ef 56.9 ef Lactate (mg/dL) Low 18.5 cd 10.9 a 11.3 a 14.3 ab 14.0 ab 1.69 High 15.2 a-d 16.4 b-d 14.9 a-c 17.8 b-d 18.8 d DOR 21.5 e 14.9 a-d 14.4 a-c 15.2 a-d 20.3 de 2.06 KAT 11.8 ab 13.1 a-c 13.7 a-c 16.5 a-e 14.5 a-d STC 17.3 c-e 12.9 ab 11.3 a 16.6 b-e 14.4 a-d NEFA (mEq/L) 0.244 a 0.374 b 0.354 b 0.0185 Low 0.312 c 0.656 e 0.469 d 0.418 d 0.407 d 0.0266 High 0.293 c 0.171 ab 0.132 a 0.173 ab 0.207 b TAC (mM) 251 253 264 6.3 260 252 5.2 Low 264 bc 240 ab 283 c 281 c 234 ab 8.8 High 260 bc 265 bc 263 bc 246 b 225 a DOR 264 cd 223 a 276 cd 258 b-d 235 ab 11.1 KAT 244 a-c 268 cd 263 cd 259 cd 231 ab STC 279 d 267 cd 281 d 272 cd 221 a 1 Supplement treatments were soybean meal fed at approximately 0.15% of initial body weight (determined 7 days before the start of the feeding period) and a mixture of 25% soybean meal and 75% ground corn given at approximately 1% of initial body weight (dry matter basis; Low and High, respectively). 2 Brd = breed; Sup = supplement treatment. 3 D = Dorper; K = Katahdin; S = St. Croix. 4 Blood was sampled 7 days before the supplementation treatments were imposed (day -7), after 14, 49, 73, and 162 days of feeding. 5 TP = total protein; ALB = albumin; UN = urea nitrogen; CHOL = cholesterol; TG = triglycerides; NEFA = nonesterified fatty acids; TAC = total antioxidant capacity. a-f Means within grouping without a common superscript letter differ (p < 0.05). animals-13-00814-t009_Table 9 Table 9 Effects of breed and supplement treatment on reproductive performance of hair sheep 1. Effect p Value 2 Dorper Katahdin St. Croix Item 3 Brd Sup Brd x Sup Low High Low High Low High SEM Brd 4 Birth rate (%) 0.520 0.063 0.738 66.7 93.5 84.6 95.5 82.8 100.0 9.83 Litter size 0.190 0.046 0.184 1.38 1.29 1.38 1.95 1.36 1.82 0.152 Fecundity 0.174 0.021 0.619 0.92 1.21 1.17 1.86 1.12 1.82 0.221 Birth weight (kg) Individual lamb 0.035 0.787 0.329 4.50 4.61 4.38 3.98 3.73 3.88 0.201 S < D Total litter 0.221 0.049 0.294 5.84 5.74 5.92 7.52 5.04 6.78 0.529 Gestation length (days) 0.228 0.739 0.661 145.4 144.6 146.2 147.0 144.9 145.7 0.88 1 Supplement treatments were soybean meal fed at approximately 0.15% of initial body weight (determined 7 days before the start of the feeding period) and a mixture of 25% soybean meal and 75% ground corn given at approximately 1% of initial body weight (dry matter basis; Low and High, respectively). 2 Brd = breed; Sup = supplement treatment. 3 Birth rate and litter size were analyzed with the GLIMMIX procedure and individual lamb and total litter birth weights were analyzed with the MIXED procedure of SAS; gestation length was based on the last day of breeding observed and the date of birth. 4 K = Katahdin; S = St. Croix; main effect mean differences (p < 0.05) with nonsignificant interactions between breed and supplement treatment (p > 0.05). animals-13-00814-t010_Table 10 Table 10 Correlation coefficients (r) between body mass indexes and body weight and body condition score at different times and change during the supplementation phase of hair sheep 1. Body Mass Index 2 Day Item 3 Parameter WH WP GH GP BCS 3 -7 BW r 0.89 0.91 0.82 0.87 0.42 p <0.001 <0.001 <0.001 <0.001 <0.001 BCS r 0.48 0.50 0.43 0.45 p <0.001 <0.001 <0.001 <0.001 14 BW r 0.88 0.88 0.83 0.82 0.50 p <0.001 <0.001 <0.001 <0.001 <0.001 BCS r 0.45 0.47 0.41 0.42 p <0.001 <0.001 <0.001 <0.001 49 BW r 0.88 0.88 0.77 0.75 0.59 p <0.001 <0.001 <0.001 <0.001 <0.001 BCS r 0.58 0.55 0.55 0.52 p <0.001 <0.001 <0.001 <0.001 73 BW r 0.84 0.84 0.77 0.71 0.62 p <0.001 <0.001 <0.001 <0.001 <0.001 BCS r 0.57 0.54 0.53 0.44 p <0.001 <0.001 <0.001 <0.001 162 BW r 0.88 0.92 0.78 0.82 0.77 p <0.001 <0.001 <0.001 <0.001 <0.001 BCS r 0.70 0.70 0.58 0.56 p <0.001 <0.001 <0.001 <0.001 -7 to 162 3 BW r 0.88 0.90 0.74 0.77 0.75 p <0.001 <0.001 <0.001 <0.001 <0.001 BCS r 0.63 0.67 0.45 0.48 p <0.001 <0.001 <0.001 <0.001 1 Body mass indexes (BMI) were determined 7 days before the supplementation treatments were imposed (day -7) and after 14, 49, 73, and 162 days of feeding, and body weight (BW) and body condition score (BSC) were determined at these and other times. 2 Wither = height at withers; Hook = point of the shoulder to hook bone; Pin = point of the shoulder to pin bone; Heart = heart girth; BMI-WH = BW/(Wither x Hook) [g/cm2]; BMI-WP = BW/(Wither x Pin) [g/cm2]; BMI-GH = BW/(Heart x Hook) [g/cm2]; BMI-GP = BW/(Heart x Pin) [g/cm2]. 3 Change in BMI, BW, and BCS. 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PMC10000198

Oxidative stress and in-feed antibiotics restrictions have accelerated the development of natural, green, safe feed additives for swine and poultry diets. Lycopene has the greatest antioxidant potential among the carotenoids, due to its specific chemical structure. In the past decade, increasing attention has been paid to lycopene as a functional additive for swine and poultry feed. In this review, we systematically summarized the latest research progress on lycopene in swine and poultry nutrition during the past ten years (2013-2022). We primarily focused on the effects of lycopene on productivity, meat and egg quality, antioxidant function, immune function, lipid metabolism, and intestinal physiological functions. The output of this review highlights the crucial foundation of lycopene as a functional feed supplement for animal nutrition. antioxidant functional feed additive lycopene poultry swine Jiangxi Provincial Natural Science Foundation20224BAB215035 National Natural Science Foundation of China32102593 the Science and Technology Plan Project of Jiangxi Provincial Department of EducationNo. GJJ2200413 the Natural Science Foundation of Jiangxi Province, China20212BAB215015 This work was supported by the Jiangxi Provincial Natural Science Foundation (No. 20224BAB215035), the National Natural Science Foundation of China (No. 32102593), the Science and Technology Plan Project of Jiangxi Provincial Department of Education (No. GJJ2200413), and the Natural Science Foundation of Jiangxi Province (No. 20212BAB215015), China. pmc1. Introduction Oxidative stress severely threatens the productivity and health status of farm animals, which results in huge economic losses for the livestock industry . In animal production, many factors, such as changes in the environment , physiological stages , and exogenous pathogenic toxins (such as mycotoxins) , can cause oxidative stress, thus disturbing the redox balance in animal bodies. Oxidative stress refers to the imbalance between antioxidants and pro-oxidants . The excessive production of reactive oxygen species (ROS) and reactive nitrogen radicals (RNS) will cause irreversible damage to cell lipids, proteins, and DNA, thus affecting the physiological functions and production performance of the animals . Antioxidation is defined as the process of antioxidant defense against oxidation in organisms . The antioxidant substances in the body are mainly divided into two categories; one is synthesized by the body itself, and the other is obtained from food . When animals are in special circumstances such as high temperatures, weaning, pregnancy, and so on, the supplementation of exogenous antioxidant substances (such as plant-derived polyphenols and carotenoids) can effectively alleviate the oxidative stress status in animals, reduce oxidative damage, and improve their health and production performance . In addition, antibiotic resistance and residues have adversely affected animal production, human health, and environmentally sustainable development . Since the European Union (EU) banned the use of antibiotics as feed additives in animal feed in 2006, researchers have been trying to explore plant-derived feed supplements as safe antibiotic alternatives . After July 2020, China officially entered a new era of in-feed antibiotic bans in livestock production . Therefore, it is urgently necessary to develop natural, green, and safe antibiotic alternatives for the swine and poultry industry. Lycopene (C40H56), a red-colored carotenoid, is a natural pigment found in plants, mainly in fruits and vegetables such as tomatoes, carrots, watermelon, and guava . Lycopene has the greatest antioxidant potential among carotenoids due to its chemical structure . Structurally, it contains 2 non-conjugated double bonds and 11 conjugated double bonds . Lycopene has been listed as a nutrient and food additive in more than 50 countries and is widely used in healthy foods, medicine, cosmetics, agriculture, and other fields . Lycopene is a powerful antioxidant, which is the fundamental basis for its health-promoting effects, including anti-inflammatory, anticancer, and antidiabetic potential , cardiovascular-protecting abilities , and neurobiological, antihypertensive, and anti-aggregative effects . Interestingly, there is an ever-increasing body of evidence indicating that lycopene could be developed as a functional feed additive for swine and poultry. In detail, lycopene has been reported to improve productivity, meat quality, egg quality, antioxidant, immunity, lipid metabolism, and intestinal physiological functions . The biological functions of lycopene are primarily due to its chemical structure . In this paper, we first briefly introduce the sources, physicochemical properties, digestion and absorption, and biological functions of lycopene. Then, we systematically summarize the latest research progress of lycopene in swine and poultry over the last ten years (2013-2022). 2. An Overview of Lycopene 2.1. The Sources of Lycopene Lycopene was first discovered as a red pigment in tomatoes by Millardet in 1876; 27 years later, it was named lycopene by Schunck in reference to the scientific name of tomato (Lycopersicon esculentum) . Lycopene is widely distributed in tomatoes and also in red vegetables and fruits, such as carrots, sweet potatoes, pumpkin, watermelons, apricots, papaya, pink grapefruits, pink guavas, and rosehips . Although lycopene exists in a variety of foods, tomato plays a leading role in lycopene-producing sources because it is the main extraction source, as well as the cheapest raw material for lycopene . It has been reported that the global gross production of tomatoes was 180.8 million tonnes in 2019 . Tomatoes are a rich source of lycopene, accounting for 80% to 90% of all carotenoid food content . The extraction techniques of lycopene from raw materials commonly include conventional methods, ultrasound-assisted extraction, supercritical fluid extraction, and enzyme-assisted extraction . Most importantly, biosynthetic methods have been developed for large-scale lycopene production using biotechnology . Microbial fermentation is a typical traditional biotechnology in lycopene production . Additionally, modern biotechnology, including genetic engineering, protein engineering, and metabolic engineering, has also been applied to lycopene production . 2.2. The Physico-Chemical Properties of Lycopene Lycopene is a form of red-colored carotenoid with the molecular formula of C40H56 . It is a polyolefin chain composed of 13 double bonds, 11 of which are conjugated into a linear array (red color factor) , making it longer than other carotenoids . Owing to the planar symmetry in its structure, lycopene does not have the same activity as vitamin A . Lycopene is a red waxy pigment, which exists in nature in the form of slender needle-like crystals . It is a liposoluble substance that is insoluble in water and easily soluble in benzene, chloroform, and acetone . In nature, lycopene mostly exists in an all-trans configuration, which is relatively stable in terms of thermodynamics. However, at least 50% of its cis isomers are found in the plasma and tissues of humans . The most common forms are 5-cis, 9-cis, 13-cis, and 15-cis isomers, which suggests that cis isomers are more easily absorbed and utilized by both humans and animals . 2.3. The Digestion and Absorption of Lycopene The absorption mode of lycopene is similar to that of lipids, occurring via a passive diffusion pathway . The lycopene in the food matrix is released under the action of gastric acid, bile acid, and enzymes . Upon entering the intestine, lycopene is combined with dietary lipids to form chylomicrons, which are then transported into the mesenteric lymphatic system via diffusion and permeation . Thereafter, the lycopene is finally discharged into portal circulation . This is the main way for lycopene to be absorbed from the gastrointestinal tract . Extrahepatic lipoprotein lipase can partially degrade chyle particles into chyle particle residues. Lycopene and its metabolites are randomly released and transported by low-density lipoprotein (LDL) and very low-density lipoprotein (VLDL) and are finally distributed to the target tissues . The chemical structure of lycopene will affect its distribution process. Through the circulatory system, it will preferentially accumulate in the testis, adrenal gland, liver, and prostate. This uneven distribution indicates its unique biological role in these tissues , such as its regulatory role in hepatic lipid metabolism . 2.4. The Biological Functions of Lycopene The unique double-bond structure of lycopene makes it far better than other carotenoids at scavenging free radicals in humans and animals. The greatest antioxidant carotenoid is lycopene , followed by tocopherol, carotene, cryptoxanthin, zeaxanthin, b-carotene, and lutein . Lycopene is a robust antioxidant, which is the fundamental basis for its health-promoting effects . Furthermore, there is an ever-increasing body of evidence to show that lycopene has anti-inflammatory, anticancer, and antidiabetic potential . Additionally, lycopene has been demonstrated to exert cardiovascular-protecting effects , neurobiological effects, and antihypertensive and anti-aggregative effects . Most recently, lycopene has been developed as an effective feed supplement for swine and poultry due to its potent antioxidant potential and red-colored pigment characteristics. The next section of this review will provide a systematic overview of research progress on lycopene in swine and poultry nutrition during the past decade (2013-2022), mainly focusing on its beneficial effects on production performance, meat quality, egg quality, antioxidant function, immune function, lipid metabolism, and intestinal barrier function. Figure 2 presents an overview diagram of the beneficial effects of lycopene on swine and poultry. 3. Applications of Lycopene in Swine and Poultry Nutrition during the Past Decade (2013-2022) 3.1. Effects on Production Performance During the last ten years, lycopene has garnered extensive research attention in the field of swine and poultry production. Sun et al. reported that 50 mg/kg dietary lycopene supplementation during gestation and lactation improved the reproductive performance of sows, including increased born-alive piglets, weaned piglets, litter birth weight, litter weaning weight, and decreased born-dead piglets. The authors finally concluded that lycopene promoted sow reproductive performance by regulating milk composition, placental immunity, and antioxidant ability . However, it appears that dietary lycopene inclusion does not influence the production performance of farm animals during the finishing period, especially in the case of finishing pigs. For instance, Fachinello et al. pointed out that dietary addition with a variety of lycopene dosages (12.5, 25, 37.5, and 50 mg/kg) did not impact the growth performance of finishing pigs . Likewise, Fachinello et al. indicated that feeding 12.5, 25, 37.5, and 50 mg/kg of lycopene to finishing pigs did not affect their carcass characteristics or relative organ weights . A recent study conducted by Wen et al. also showed no effects on the performance and carcass characteristics of finishing pigs from dietary lycopene supplementation at 100 or 200 mg/kg . In a poultry study, Sun et al. demonstrated that feeding 40 mg/kg lycopene to Xinghua breeding hens for 35 days increased the fertilization rate and hatchability of their eggs . An et al. also found that the dietary addition of 20 mg/kg lycopene or 1.7% tomato paste for 28 days elevated the egg weight and egg production of Hy-line Brown laying hens . The beneficial additive effect of lycopene on productive performance and carcass characteristics was also observed in Japanese quail, according to a study by Al-Jrrah et al. . In addition, Amer et al. noted that dietary supplementation with lycopene at 300 mg/kg increased the relative growth rate of Japanese quail . In a 42-day feeding trial conducted by Wan et al., supplementation with 10, 20, or 30 mg/kg lycopene increased the average daily gain (ADG), feed conversion ratio (FCR), and final body weight of 1-day-old broilers . Similarly, Wan et al. noticed that broilers supplemented with 100 mg/kg lycopene demonstrated greater body weight at day 21 of the feeding trial . Interestingly, Mezbani et al. revealed that the dietary inclusion of lycopene (50, 100, and 200 mg/kg) enhanced growth performance without affecting the carcass performance of Ross 308 broilers, as indicated by an increased ADG, average daily feed intake (ADFI), and decreased FCR . On the contrary, using tomato paste as a lycopene source, Lee et al. observed no effects on the growth performance and relative organ weights of broilers fed diets containing 10 or 20 mg/kg of lycopene, or 17 g/kg of tomato paste . Most importantly, lycopene has been demonstrated to exert growth-promoting effects on farm animals under stressful conditions, such as heat stress, and due to mycotoxin feed contamination . For example, Sarker et al. proved that the supplementation of 200 mg/kg lycopene for 42 days increased the ADG and decreased the FCR of broilers under the aflatoxin B1 (AFB1) challenge condition . Moreover, the results of Sarker et al. showed that the inclusion of lycopene promoted the growth performance of broilers under the AFB1 challenge, as indicated by increased ADG (day1-21: 100 mg/kg lycopene; day22-42 and day1-42: 200 or 400 mg/kg lycopene) and decreased FCR (day22-42 and day1-42: 200 or 400 mg/kg lycopene) . Under heat stress conditions, the supplementation of 0, 200, and 400 mg/kg lycopene for 42 days has also been confirmed to linearly elevate the growth performance of Ross 308 broilers, as reflected by an increased cumulative feed intake, weight gain, and reduced FCR . It should be noted, however, that the high-dosage supplementation of lycopene (500 mg/kg) has been demonstrated to negatively influence the growth performance of Hubbard broilers, including decreased ADFI and ADG during the first week of the feeding trial . Therefore, in light of the observed results, lycopene (or tomato paste) has great potential to be utilized as a growth-promoting supplement (or feedstuff) for swine and poultry. However, further research is needed to confirm its favorable effects on the production performance of pigs and poultry, using feeding trials with a large population of animals. 3.2. Effects on Meat Quality and Egg Quality Along with rapid economic development and livestock production technology improvement, the ever-increasing attention of consumers has been paid to meat and egg quality in place of meat and egg quantity . The quality of meat and eggs is a key criterion for consumers when choosing livestock products . In recent years, lycopene has obtained substantial attention as a natural feed supplement intended to improve meat and egg quality in animal production. As reported by Wen et al., the dietary inclusion of lycopene improved the meat quality of finishing pigs, including reduced L* and b* values, elevated a* value, and intramuscular fat and crude protein contents of the longissimus dorsi (LD) muscle . The thawing loss of the longissimus lumborum (LL) muscle was also found to be linearly reduced when finishing pigs were fed diets supplemented with 0, 12.5, 25, 37.5, and 50 mg/kg of lycopene . Besides this, lycopene was demonstrated to promote muscle fiber type transformation in pigs, which is of great significance in determining meat quality . Specifically, the mRNA levels of Cytc, TFAM, TFB1M, CS, COX1, MyHC1, MyHC IIa, MyHC IIx, and TNNI1 were up-regulated, while the mRNA level of MyHC IIb was down-regulated in the LD muscle by lycopene supplementation . In addition, the protein expression of slow MyHC, Cytc, myoglobin, slow-twitch fiber percentage, succinic dehydrogenase (SDH), and malate dehydrogenase (MDH) was increased, while fast MyHC, fast-twitch fiber percentage, and lactate dehydrogenase (LDH) activity were decreased in the LD muscle of finishing pigs fed lycopene diets (100 or 200 mg/kg lycopene) . Importantly, the oxidative stability of pork has been reported to be elevated by dietary lycopene feeding (20 mg/kg lycopene, 3.4% tomato paste, or 10 mg/kg lycopene with 1.7% tomato paste) . As we know, lipid oxidation negatively influences the color, nutritional value and flavor, and shelf-life of meat . An et al. reported that the oxidative indices in fresh pork belly meats were decreased when finishing pigs were fed lycopene-supplemented diets, including decreased malondialdehyde (MDA) levels and increased lycopene levels . Likewise, Wen et al. noted that dietary lycopene inclusion improved LD muscle antioxidant status, as indicated by increased total superoxide dismutase (T-SOD) and catalase (CAT) activities, decreased MDA level, up-regulated mRNA levels of SOD1, SOD2, CAT, GPX1, GST, GR, and Nrf2, and down-regulated Keap1 mRNA levels . Similar findings were reported by Correia et al., who observed improved oxidative stability of the LL muscle in young pigs fed 5% tomato pomace for 5 weeks . However, An et al. found that fatty acid composition in the fresh belly meat of finishing pigs was unaffected by dietary lycopene supplementation with 20 mg/kg lycopene, 3.4% tomato paste, or 10 mg/kg lycopene with 1.7% tomato paste in a 28-day feeding trial . Regarding egg quality, the study by Shevchenko et al. showed that supplementing High Line W36 laying hens' diets with lycopene (20/40/60 mg/kg) for 90 days resulted in improved egg quality, as indicated by an increased carotenoid level and yolk color in fresh eggs or in eggs undergoing a 4 degC and 12 degC storage period . Orhan et al. also indicated that feeding Lohman LSL laying hens with 20 mg/kg lycopene as a purified powder or tomato powder for 84 days improved egg quality, including increased egg weight, yolk color, yolk weight, yolk ratio, yolk lycopene level, and decreased yolk MDA and cholesterol levels . An et al. also found that the dietary addition of 10 or 20 mg/kg lycopene for 28 days increased yolk color and lycopene levels and decreased MDA levels in the eggs of Hy-line Brown laying hens . A previous study by Sun et al. indicated that feeding 40 mg/kg of lycopene to Xinghua breeding hens for 35 days increased the lycopene levels in the serum, eggs, and liver, and an elevated yolk color score . Additionally, Sahin et al. showed that the supplementation of 200 and 400 mg/kg lycopene for 42 days improved the muscle antioxidant status of Ross 308 broilers under heat stress conditions, including a linearly increased lycopene level, glutathione peroxidase (GSH-Px), superoxide dismutase (SOD) activities, decreased MDA level, down-regulated Keap1, and up-regulated Nrf2 protein expression (400 mg/kg lycopene) . Similarly, Lee et al. noticed that dietary supplementation with 10, 20 mg/kg lycopene, or 17 g/kg tomato paste for broilers decreased the thiobarbituric acid-reactive substance (TBARS) value of LDL isolated from broilers at both 2 weeks old and 4 weeks old . However, in laying quails, Hsu et al. suggested that the supplementation of lycopene (6 and 18 mg/kg lycopene as commercial or bacterial lycopene) for 28 days did not affect laying performance or egg quality, or the serum antioxidant status . Therefore, given the current findings, lycopene has huge development value in terms of being utilized as a green feed supplement for meat and egg production, as well as for producing lycopene-enriched meats and eggs as functional foods for humans . 3.3. Effects on Antioxidant Function The antioxidant potential of lycopene is one of its predominant biological characteristics, as has been proven in swine and poultry by several animal nutritionists. For instance, Sun et al. demonstrated that dietary 50 mg/kg lycopene supplementation during gestation increased total antioxidant capability (T-AOC) and GSH-Px activity and decreased the H2O2 and ROS levels in the placental tissues of sows . Additionally, the mRNA levels of GPX1, GPX4, FABP4, SLPI, ANX1, and APOE in the placenta were also up-regulated by lycopene supplementation at 50 mg/kg . In finishing pigs, the dietary supplementation of 0, 12.5, 25, 37.5, and 50 mg/kg of lycopene linearly reduced the TBARS level and increased the 2,2 diphenyl 1 picrylhydrazyl level in the liver . The gene expression of SOD1 and CAT were also found to be linearly affected by dietary lycopene supplementation (0, 12.5, 25, 37.5, and 50 mg/kg) . Similarly, as reported by Wen et al., the dietary inclusion of lycopene improved antioxidant status, including increased T-AOC, T-SOD, GSH-Px, and CAT activities in the serum and liver, and decreased hepatic MDA levels in finishing pigs . Lycopene was also reported to enhance the antioxidant ability and reduce oxidative damage in piglets. In a study conducted by Meng et al., supplementing 50 mg/kg lycopene to weaned piglets for 28 days increased the activities of CAT in serum and SOD in the jejunum, and decreased H2O2 levels in the serum and jejunum . Lycopene supplementation also up-regulated the mRNA levels of NRF2, SOD2, CAT, GLUT2, GLUT5, CD36, CLDN1, and IL-22, along with the protein levels of NRF2, CD36, and CLDN1, and down-regulated the mRNA and protein levels of KEAP1 . Those results indicate that lycopene could activate the antioxidant signaling pathway (Keap1/Nrf2) and drive the downstream antioxidant gene expression. Using an in vitro model of a pig embryo, Kang et al. noticed that treating the embryo with 0.1 mM lycopene for 6 days increased the blastocyst formation of embryos, the number of total cells, and the trophectoderm . The authors further investigated the underlying mechanisms and finally concluded that the beneficial effects of lycopene on porcine embryos were attributed to its reducing oxidative stress and apoptosis . In an in vitro model with piglet Sertoli cells, lycopene was also demonstrated to alleviate zearalenone-induced oxidative injury via regulating the Nrf2 signaling pathway . In a broiler-feeding trial by Wang et al., supplementing lycopene (especially at 30 mg/kg) to 1-day-old broilers for 42 days markedly elevated the serum antioxidant status, as indicated by increased T-AOC, GSH-Px, and SOD activities, and decreased MDA level on day 21 of the study . Moreover, the hepatic antioxidant status was also improved by lycopene supplementation, as suggested by increased GSH-Px, SOD activities, decreased MDA levels (10, 20, and 30 mg/kg lycopene) at day 21 of the study, and increased GSH-Px activity (30 mg/kg lycopene), decreased MDA level (10, 20, and 30 mg/kg lycopene) at day 42 of the study . Meanwhile, the Nrf2 pathway was activated by lycopene supplementation, which further drove Nrf2-downstream antioxidant gene expression, including SOD2, NQO1, and HO-1 . Feed AFB1 contamination commonly occurs in poultry production and seriously threatens the productivity and health of poultry . It has been confirmed that oxidative stress is one of the key mechanisms of AFB1 toxicity, and the addition of exogenous antioxidants (such as antioxidant polyphenols and carotenoids) in diets can effectively alleviate the adverse effects of AFB1 in poultry . In an experiment by Wan et al., the dietary addition of 100, 200, or 400 mg/kg lycopene all markedly decreased the hepatic activities of cytochrome P450 1A1 (CYP1A1) and cytochrome P450 2A6 (CYP2A6) in broilers fed AFB1-contaminated diets in a 42-day feeding trial . The antioxidant status was also enhanced by lycopene supplementation, as suggested by an increased GSH (200 mg/kg lycopene) level, glutathione s-transferase (100 and 400 mg/kg lycopene), glutamine-cysteine ligase, CAT (100 mg/kg lycopene), GSH-Px activities (100, 200 and 400 mg/kg lycopene), decreased AFB1-8,9-epoxide-DNA and ROS levels (100, 200, and 400 mg/kg lycopene), MDA, 8-hydroxydeoxyguanosine (100, 200 and 400 mg/kg lycopene), 4-hydroxynonenal, and protein carbonyl levels (200 and 400 mg/kg lycopene) . Similarly, Sarker et al. documented the finding that feeding 200 mg/kg lycopene for 42 days to broilers fed AFB1-contaminated feed elevated the hepatic mitochondrial antioxidant status, including greater mGSH, GSH-Px, MnSOD, and ATP levels, along with lower ROS and H2O2 levels, and reduced mitochondrial swelling . Additionally, hepatic mitochondrial function was improved, such as increased complex III and complex V, and an up-regulated mRNA level of MnSOD, Trx2, TrxR2, Prx3, PGC-1a, NRF1, and TFAM . El-Sheshtawy et al. found that the dietary addition of 100 mg/kg lycopene for 10 days in Pekin ducks decreased the hepatic aflatoxin residue and increased hepatic antioxidant status (increased T-AOC, GST, and CAT activities, and decreased MDA content) . Mezbani et al. indicated that the dietary addition of lycopene improved the serum antioxidant status of Ross 308 broilers, which is suggested by an increase in GSH-Px activity, decreased MDA content (100, 200 mg/kg lycopene), and increased CAT activity (50, 100, 200 mg/kg lycopene) . Sahin et al. observed that dietary supplementation with 200 or 400 mg/kg lycopene for 42 days improved the antioxidant status of Ross 308 broilers under heat-stress conditions, including linearly increased serum lycopene content, GSH-Px, and SOD activities, along with decreased MDA content . In laying hens, as observed by Orhan et al., supplementation of 20 mg/kg lycopene as a purified powder or tomato powder for 84 days decreased serum MDA and cholesterol levels in Lohman LSL laying hens . Likewise, An et al. documented that the dietary addition of 10, 20 mg/kg lycopene or 1.7% tomato paste for 28 days all increased hepatic lycopene levels, while supplementation with 20 mg/kg lycopene or 1.7% tomato paste decreased serum MDA levels in Hy-line Brown laying hens . Sun et al. noted that feeding lycopene to Xinghua breeding hens for 35 days increased the antioxidant status, including increased serum SOD (day21, 20, 40, or 80 mg/kg lycopene; day35, 80 mg/kg lycopene), T-AOC (day21, 20, 40, or 80 mg/kg lycopene), GSH/GSSG (day21,28,35, 20, 40, or 80 mg/kg lycopene), GSH-Px (day14, 20, 40, or 80 mg/kg lycopene), and increased hepatic SOD, T-AOC, and GSH/GSSG (day35, 20, 40, or 80 mg/kg lycopene) . Amer et al. observed that dietary 300 mg/kg lycopene supplementation increased SOD activity and decreased MDA levels in both the liver and chest muscle of Japanese quail . Sun et al. evaluated the beneficial effects of lycopene (20, 40, and 80 mg/kg) on breeding hens under lipopolysaccharide challenge conditions and observed higher GSH/GSSG in serum at 24 h post-injection of lipopolysaccharide by lycopene feeding at 20, 40, or 80 mg/kg . Therefore, as a natural antioxidant, lycopene could be developed as a functional supplement for swine and poultry. However, along with in vivo feeding trials, more in vitro or ex vivo studies are required to illuminate the specific mechanism of action of lycopene, which is of great importance for promoting the research and application of lycopene in animal nutrition. 3.4. Effects on Immune Function Inflammation is reported to be the cost paid by livestock productivity, and it is the basis for allocating nutrient resources between growth and survival in livestock production . Inflammatory reactions are implicated in animal diseases (such as diarrhea and enteritis), resulting in compromised productivity and higher mortality levels in swine and poultry . Lycopene has been demonstrated to have health-promoting effects on farm animals due to its immune-regulatory functions . Sun et al. highlighted that feeding gestating sows with 50 mg/kg lycopene improved their placental immunity status, including increased secretory immunoglobulin A (sIgA), immunoglobulin G (IgG), immunoglobulin M (IgM) levels, and decreased interleukin-1b (IL-1b), interleukin-8 (IL-8), tumor necrosis factor-a (TNF-a), and interleukin-12 (IL-12) levels . The authors also noticed that the mRNA levels of IL-1b, IL-8, and TNF-a in the placenta were also down-regulated by lycopene supplementation . In a recent study by Liu et al., the dietary addition of 200 mg/kg lycopene for 70 days notably improved the intestinal immune status of finishing pigs, as indicated by decreased IL-1b, TNF-a, and nuclear factor k-B (NF-kB) levels in the duodenum, down-regulated mRNA levels of IL-1b in the jejunum, and up-regulated interleukin-10 (IL-10) mRNA levels in the duodenum and jejunum . Fachinello et al. investigated the impacts of lycopene on the immune responses of finishing pigs and observed that supplementing 0, 12.5, 25, 37.5, and 50 mg/kg lycopene for 28 days linearly increased the plasma albumin and lymphocyte levels, and linearly and quadratically affected neutrophils and the neutrophil/lymphocyte ratio, and also quadratically affected eosinophils in the blood and anti-bovine serum albumin (BSA) production . The immunomodulatory effects of lycopene have also been reported in poultry. In a study conducted by Shevchenko et al., feeding High Line W36 laying hens with lycopene (20, 40, and 60 mg/kg) for 90 days markedly decreased the leukocytes and erythrocytes, but had no effects on the serum antibody titer of hens vaccinated against Newcastle disease, avian rhinotracheitis, egg drop syndrome, and infectious bronchitis . In a 42-day feeding trial by Alwash et al., supplementing the diet with lycopene increased the blood-packed cell volume, heterophils/lymphocytes, and blood hemoglobin level of Japanese quail under heat-stress conditions . Additionally, Sarker et al. revealed that the supplementation of 200 mg/kg lycopene improved the intestinal immunity of broilers under AFB1 challenge conditions, as suggested by increased IL-10 levels at day 21 and day 42, and decreased the interferon-g (IFN-g) levels in jejunum at day 21 of the study . Furthermore, Sun et al. evaluated the beneficial effects of lycopene on breeding hens under lipopolysaccharide challenge conditions and found that feeding lycopene (20, 40, and 80 mg/kg) to hens for 35 days augmented the immune organ indices of the thymus, spleen, and bursal . However, a high supplemental dosage of lycopene could cause side effects in farm animals. As reported by Pozzo et al., the immune organ weight of the spleen and bursa of Fabricius were reduced by lycopene supplementation at up to 500 mg/kg in the diets of Hubbard broilers . Therefore, further studies are urgently needed to determine the optimal supplemental dosage of lycopene for swine and poultry. Further research is also warranted to clarify the underlying mechanism of action in terms of the immunomodulatory effects of lycopene. 3.5. Effects on Lipid Metabolism Lycopene has been demonstrated to exert modulatory effects on lipid metabolism in swine and poultry. Most recently, Meng et al. reported that supplementing 50 mg/kg lycopene to weaned piglets for 28 days increased their serum total cholesterol (TC) level . In finishing pigs, supplementation with 0, 12.5, 25, 37.5, and 50 mg/kg of lycopene linearly affected plasma cholesterol, high-density lipoprotein (HDL) and LDL levels, and the LDL/HDL ratio of pigs . Wen et al. also proved that the dietary inclusion of lycopene at 100 or 200 mg/kg up-regulated the mRNA levels of AMPKa1, AMPKa2, Sirt1, PGC-1a, and up-regulated the protein levels of P-AMPK/AMPK, NRF1, CaMKKb, Sirt1, PGC-1a in LD muscle of finishing pigs . However, a study by An et al. indicated that serum lipid profiles of finishing pigs were unaffected by dietary supplementation of 20 mg/kg lycopene, 3.4% tomato paste, or 10 mg/kg lycopene with 1.7% tomato paste in a 28-day feeding trial . The placental protein expressions of APOE, ANX1, SLPI, and FABP4 are up-regulated for sows that are fed diets supplemented with 50 mg/kg lycopene during gestation . In broilers, a study by Wan et al. suggested that the dietary inclusion of lycopene at 100 mg/kg for 42 days decreased the abdominal fat weight and abdominal fat percentage of broilers . These results indicated a lipid-lowering property of lycopene. Concurrently, the plasma concentrations of total triglyceride (TG), TC, low-density lipoprotein cholesterol (LDLC), and the hepatic concentrations of fatty acid synthase (FAS) and acetyl-CoA carboxylase (ACC) were reduced in broilers that were fed diets supplemented with 100, 200, or 400 mg/kg of lycopene . Moreover, supplementation of 100, 200, or 400 mg/kg of lycopene all up-regulated the mRNA levels of AMPK-a, and down-regulated the mRNA levels of SREBP-1, FAS, and ACC in the livers of broilers, which are indicative of hepatic lipid metabolism . In an experiment by Wan et al., the dietary addition of 100, 200, or 400 mg/kg lycopene for 42 days decreased the levels of aspartate transaminase (AST) and alanine aminotransferase (ALT) in the serum of broilers fed AFB1-contaminated diets . A study by Mezbani et al. also indicated that feeding lycopene to Ross 308 increased their glucose and HDL (100, 200 mg/kg lycopene) and decreased their cholesterol, triglyceride, VLDL (50, 100, 200 mg/kg lycopene) levels, also decreasing the hepatic activities of ALT and ALP (100, 200 mg/kg lycopene) . Additionally, Lee et al. revealed that supplementing the diet of broilers with lycopene or 17 g/kg tomato paste decreased plasma TG (10, 20 mg/kg lycopene) and LDL cholesterol (20 mg/kg lycopene) levels at 2 weeks old, and increased the lycopene level in the plasma and liver (10, 20 mg/kg lycopene, 17 g/kg tomato paste) at 4 weeks old . Similar results were found in ducks; the supplementation of 100 mg/kg lycopene for 10 days decreased ALT, AST, gamma-glutamyl transferase (g-GT), alkaline phosphatase (ALP), and uric acid levels, and increased albumin and total protein levels in the serum of Pekin ducks . However, Pozzo et al. claimed that feeding a high level of lycopene (500 mg/kg) to Hubbard broilers for 35 days decreased the total protein, gamma globulin, albumin, and alpha globulin levels . The regulatory functions of lycopene on fat deposition and lipid metabolism have also been reported in laying and breeding hens. Shevchenko et al. found that feeding High Line W36 laying hens with lycopene (20/40/60 mg/kg) for 90 days markedly increased the serum levels of glucose, creatinine, and ALT, and decreased the serum levels of cholesterol, AST, alkaline phosphatase at day 31 of the study; increased serum levels of glucose, cholesterol, and ALT, and decreased serum level of AST were recorded at day 61 of the study, and increased serum levels of glucose, creatinine, and cholesterol were recorded at day 91 of the study . Additionally, Orhan et al. fed Lohman LSL laying hens with 20 mg/kg lycopene as a purified powder or tomato powder for 84 days and found that lycopene decreased the protein expression of intestinal NPC1L1, MTP, ACAT2, hepatic SREBP1c, ACLY, and LXRa, and increased the protein expression of hepatic ABCG5 and ABCG8 . In a study with quails, Hsu et al. noticed that feeding laying quail with lycopene for 28 days decreased the yolk triglyceride level (6, 18 mg/kg commercial lycopene; 18 mg/kg bacterial lycopene), serum triglyceride level (18 mg/kg bacterial lycopene), and serum total lipid level (6 and 18 mg/kg commercial lycopene) . Under heat-stress conditions, Japanese quail that were fed lycopene diets for 42 days had increased blood glucose and decreased triglyceride levels (with 150, 200, 250, and 300 mg/kg lycopene), increased cholesterol (with 250 and 300 mg/kg lycopene), total protein, and globulin levels (300 mg/kg lycopene) . In contrast, An et al. did not observe alterations in the serum lipid profile of Hy-line Brown laying hens fed 10 or 20 mg/kg lycopene or 1.7% tomato paste diets for 28 days . In breeding hens, Sun et al. highlighted that supplementing lycopene to Xinghua breeding hens for 35 days decreased the total cholesterol (TCHO) levels (day7,35, 20, 40, and 80 mg/kg lycopene), increased high-density lipoprotein cholesterol (HDLC) (day35, 20, 40, 80 mg/kg lycopene), and T3 level (day14, 20 mg/kg lycopene) in the serum of hens . Tian et al. demonstrated that feeding lycopene (20, 40, and 80 mg/kg) to Xinghua breeding hens for 35 days regulated the gene expression of fat metabolism. Specifically, lycopene up-regulated the hepatic mRNA levels of PPARa, RARa, PGCa (40 and 80 mg/kg lycopene), RXRa (20, 40 and 80 mg/kg lycopene), and down-regulated mRNA levels of FABP10 (40, 80 mg/kg lycopene), FABP1 (20, 40, and 80 mg/kg lycopene), and FATP4 (40 mg/kg lycopene). In the duodenum, the RARa mRNA level was up-regulated by the supplementation of 40 or 80 mg/kg lycopene. In the jejunum, lycopene up-regulated the mRNA levels of PPAPg, RXRg (40 and 80 mg/kg lycopene), and RXRa (20 and 40 mg/kg lycopene), and down-regulated the mRNA levels of FATP4 (40 and 80 mg/kg lycopene) . Sun et al. evaluated the beneficial effects of lycopene on breeding hens under lipopolysaccharide challenge conditions and found that lycopene (at 20, 40, and 80 mg/kg) decreased the levels of TCHO, LDLC, and blood urea nitrogen (BUN), and increased HDLC and T3 levels in serum . Therefore, lipid metabolism regulation by lycopene could partially explain the improvement of meat quality, egg quality, and health promotion in swine and poultry. Given the lowering-lipid ability of lycopene, it also has great potential for development as a functional additive to counteract obesity and diabetes in humans. 3.6. Effects on Intestinal Physiological Functions The intestinal tract is the main site of digestion and the absorption of feed nutrients in farm animals and is also an indispensable line of defense against exogenous pathogenic microorganisms and their toxins . The maintenance of normal intestinal physiological functions and gut homeostasis is a prerequisite for productivity efficiency and well-being in swine and poultry . Lycopene has been confirmed to improve the intestinal morphology of pigs, which is indicative of gut health. Most recently, Meng et al. reported that feeding 50 mg/kg lycopene to weaned piglets for 28 days increased villus height (VH) and the ratio of VH to the crypt depth (CD) of the jejunum . The jejunum's digestive enzyme activities were also increased by lycopene supplementation in piglets, as indicated by elevated lipase, sucrase, maltase, and lactase activities . Consistently, Liu et al. also investigated the beneficial effects of lycopene supplementation on the intestinal morphology of finishing pigs and found that the supplementation of 200 mg/kg lycopene for 70 days significantly augmented the ratio of VH/CD of the jejunum in finishing pigs . In addition, the authors also noticed that the addition of 200 mg/kg lycopene notably up-regulated the mRNA and protein expression of Claudin-1 in the jejunum of pigs, which is indicative of intestinal integrity enhancement via lycopene feeding . The regulatory effects of lycopene on intestinal physiological functions may be attributed to its antioxidant properties. As reported by Liu et al., the supplementation of 100 or 200 mg/kg lycopene for 70 days significantly increased CAT activity in the jejunum and decreased MDA levels in the duodenum of finishing pigs, which suggested that lycopene supplementation promoted intestinal antioxidant capacity . Similar results have been reported in poultry studies. Sarker et al. reported that dietary supplementation with lycopene (at 100, 200, or 400 mg/kg) improved the intestinal morphology of broilers undergoing an AFB1 challenge . The authors also observed elevated digestive enzyme activities in the small intestine, including duodenum amylase and lipase activities (day21: 200, 400 mg/kg lycopene; day42: 200 mg/kg lycopene), and jejunum amylase and lipase activities (day42: 200, 400 mg/kg lycopene), as well as ileum amylase activity (day42: 200 mg/kg lycopene) . According to the findings of Sarker et al., supplementation with 200 mg/kg lycopene enhanced the intestinal integrity of broilers under AFB1 challenge conditions, as suggested by reduced serum diamine oxidase concentrations, with up-regulated jejunum ZO-1 and Claudin-1 mRNA levels at day 42 of the experiment . Additionally, Al-Jrrah et al. noted that feeding Japanese quail with lycopene improved their gastrointestinal and organ development . Similarly to the results in swine, Sarker et al. found that supplementing feed with 200 mg/kg lycopene improved the intestinal antioxidant of broilers under AFB1 challenge conditions, as suggested by decreased jejunum H2O2 (day42) and MDA levels (day21, day42), along with increased GSH, GST, and GR levels, as well as up-regulated mRNA levels of Nrf2, HO-1, Cu/ZnSOD, MnSOD, and CAT (day42) . Hence, the maintenance of intestinal physiological functions is another crucial biological function of lycopene for swine and poultry. A summary of lycopene as a functional feed additive for swine, meat-producing, and egg-producing poultry is presented in Table 1, Table 2 and Table 3, respectively. 4. Conclusions Lycopene is widely considered an effective antioxidant that can capture oxygen free radicals, regulate the cellular expression of related transcription factors, enhance the activities of antioxidant enzymes, and protect cells from free radical damage. As a natural pigment and potent antioxidant, lycopene can be developed as a functional feed supplement for swine and poultry. Several pieces of research have revealed that lycopene has great potential to improve productivity, meat quality, and egg quality, as well as antioxidant activity, immune function, lipid metabolism, and intestinal physiological functions. In view of the current knowledge, lycopene could be supplemented as lycopene products (including commercial lycopene and tomato paste) at 10-400 mg/kg in swine and poultry diets, while a higher supplemental dosage could adversely affect the productivity and health of animals. However, the research on lycopene to alleviate oxidative stress and promote productivity is still in the initial stages, and the research results have not been widely popularized. Therefore, future research directions should further explore the additive effects of lycopene on swine and poultry at different physiological phases and different rearing environments, and, finally, establish the optimum supplemental dosage for the productivity and health of swine and poultry. More importantly, further research is warranted to elaborate the specific mechanism of action of lycopene, which is of great significance in promoting the research and application of lycopene in animal nutrition, or even in human nutrition. Author Contributions Conceptualization, J.C. and J.Y.; methodology, Z.H. and T.Z.; validation, X.C. (Xuehai Cao); writing--original draft preparation, J.C.; writing--review and editing, X.C. (Xingping Chen), and J.Y.; funding acquisition, J.C. All authors have read and agreed to the published version of the manuscript. Institutional Review Board Statement Not applicable. Informed Consent Statement Not applicable. Data Availability Statement Not applicable. Conflicts of Interest The authors declare no conflict of interest. Figure 1 The chemical structure of lycopene. Figure 2 The overview diagram of the beneficial effects of lycopene on swine and poultry. animals-13-00883-t001_Table 1 Table 1 The summary of lycopene (LYC) as a functional feed additive for swine during the past decade (2013-2022). Experimental Subject Supplemental Dosage and Duration Main Findings References Finishing pig Dosage: 100, 200 mg/kg Duration: 70 days | Jejunum morphology: | VH/CD (200 mg/kg LYC); | Intestinal antioxidant status: | duodenum MDA, | jejunum CAT (all LYC); | Intestinal inflammation: | duodenum IL-1b, TNF-a, NF-kB level, | IL-1b level, and mRNA level in the jejunum; | IL-10 mRNA level in duodenum and jejunum (200 mg/kg LYC); | Intestinal integrity: | mRNA and protein expression of Claudin-1 in the jejunum (200 mg/kg LYC). Finishing pig Dosage: 12.5, 25, 37.5, 50 mg/kg Duration: 75.04-100.45 kg BW No effects on carcass characteristics and relative organ weights; | Meat quality: linearly | thawing loss of LL muscle; | Antioxidant status: linearly | TBARS, | DPPH in the liver. Finishing pig Dosage: 12.5, 25, 37.5, 50 mg/kg Duration: 28 days No effects on growth performance; Linearly affected the gene expression of SOD1 and CAT; Linearly affected plasma cholesterol, HDL, LDL, and the LDL/HDL ratio. Finishing pig Dosage: 20 mg/kg LYC, 3.4% tomato paste, 10 mg/kg LYC with 1.7% tomato paste Duration: 28 days No effects on serum lipid profiles and fatty acid composition in fresh belly meat; | Oxidative indices in fresh pork belly meats: | MDA, | LYC level (all LYC). Finishing pig Dosage: 12.5, 25, 37.5, 50 mg/kg Duration: 28 days Linearly | plasma albumin level; Linearly | lymphocyte, along with linearly and quadratically affected neutrophils and neutrophil/lymphocyte ratio, and quadratically affected eosinophils in blood; Linearly | anti-BSA production. Finishing pig Dosage: 100, 200 mg/kg Duration: 70 days No effects on growth performance and carcass characteristics; | Meat quality: | L*, b* values, | a* value, intramuscular fat, crude protein content; | Antioxidant status: serum: | T-AOC, T-SOD, GSH-Px, CAT; liver: | T-AOC, T-SOD, GSH-Px, CAT, | MDA; LD muscle: | T-SOD, CAT, | MDA; LD muscle mRNA level: | SOD1, SOD2, CAT, GPX1, GST, GR, Nrf2, | Keap1; | Muscle fiber type transformation: mRNA level: | Cytc, TFAM, TFB1M, CS, COX1, MyHC1, MyHC IIa, MyHC IIx, TNNI1; | MyHC IIb; protein expression: | slow MyHC, Cytc, Myoglobin; slow-twitch fiber percentage, | fast MyHC, fast-twitch fiber percentage; | SDH, MDH, | LDH activity; Regulated gene expression: | AMPKa1, AMPKa2, Sirt1, PGC-1a; Regulated protein expression: | P-AMPK/AMPK, NRF1, CaMKKb, Sirt1, PGC-1a. Sow Dosage: 50 mg/kg Duration: gestation and lactation | Reproductive performance of sows: | born-alive piglets, weaned piglets, litter birth weight, litter weaning weight, | born-dead piglets; | Placental antioxidant status: | T-AOC, GSH-Px, | H2O2, ROS; | Placental immunity: | sIgA, IgG, IgM, | IL-1b, IL-8, TNF-a, IL-12; Regulated placental gene expression: | GPX1, GPX4, FABP4, SLPI, ANX1, APOE, | IL-1b, IL-8, TNF-a; Regulated placental protein expression: | APOE, ANX1, SLPI, FABP4; | Milk composition: colostrum: | lactose, IgA, IgG; 10-day milk: lactose. Weaned piglet Dosage: 50 mg/kg Duration: 28 days | Intestinal morphology: | jejunum VH, VH/CD; | Jejunum digestive enzyme activities: | lipase, sucrase, maltase, lactase; | Antioxidant status: | SOD, | H2O2 (jejunum), | CAT, | H2O2 (serum); | Serum TC level; Regulated jejunum gene expression: | KEAP1, | NRF2, SOD2, CAT, GLUT2, GLUT5, CD36, CLDN1, IL-22; Regulated jejunum protein expression: | KEAP1, | NRF2, CD36, CLDN1; Regulated gut microbiota. Pig embryo Dosage: 0.1 mM Duration: 6 days | Blastocyst formation of embryos, the number of total cells, and trophectoderm; | Embryos' ROS levels on day2 and day6; | JC-1, | cytochrome C fluorescence intensity on day2 and day6; | Number of CC3-positive cells, apoptosis cells on day2 and day6; | mRNA level of SOD1, SOD2, CAT, and BAX/BCL2L1 on day2 and day6. Piglet Sertoli cells (ZEA challenge) Dosage: 30 mM Duration: 24 h | Cell viability and morphology, | cell apoptosis rate; | Cell antioxidant status: | T-SOD, HO-1, GSH-Px, | ROS, MDA; Regulated gene expression: | Nrf2, GPX1, Bcl-2, | Keap1, LC3, Beclin-1, Bax; Immuno-fluorescence: | Nrf2, | LC3; Regulated protein expression: | Nrf2/Histone-H3, Nrf2, HO-1, GPX1, Bcl-2, | keap1, Bax, Beclin-1. Abbreviations: |, increase; |, decrease; AMPKa1, AMP-activated protein kinase a1; AMPKa2, AMP-activated protein kinase a2; ANX1, annexin A1; APOE, apolipoprotein E; Bcl-2, B-cell lymphoma-2; BSA, bovine serum albumin; CaMKKb, calcium/calmodulin-dependent protein kinase b; CAT, catalase; CC3, cleaved caspase 3; CD, crypt depth; COX1, cyclooxygenase 1; CS, citrate synthase; Cytc, cytochrome c; DPPH, 2,2 diphenyl 1 picrylhydrazyl; FABP4, fatty acid-binding protein 4; GSH-Px, glutathione peroxidase; GPX1(4), glutathione peroxidase 1(4); GR, glutathione reductase; GST, glutathione-stransferase; HDL, high-density lipoprotein; HO-1, heme oxygenase 1; Keap1, Kelch-like ECH-associated protein 1; LD, longissimus dorsi; LDH, lactate dehydrogenase; LDL, low-density lipoprotein; MDA, malondialdehyde; MDH, malate dehydrogenase; MyHC, myosin heavy chain; NF-kB, nuclear factor k-B; NRF1, nuclear respiratory factor 1; Nrf2, nuclear factor E2-related factor 2; P-AMPK/AMPK, phospho-AMP-activated protein kinase/AMP-activated protein kinase; PGC-1a, peroxisome proliferator-activated receptor g coactivator 1a; ROS, reactive oxygen species; SDH, succinic dehydrogenase; sIgA, secretory immunoglobulin A; Sirt1, sirtuin1; SLPI, antileukoproteinase; SOD(1 or 2), superoxide dismutase(1 or 2); T-AOC, total antioxidant capability; TBARS, thiobarbituric acid-reactive substances; TC, total cholesterol; TFAM, mitochondrial transcription factor A; TFB1M, mitochondrial transcription factor B1; TNNI1, troponin I type 1; T-SOD, total superoxide dismutase; VH, villus height. animals-13-00883-t002_Table 2 Table 2 The summary of lycopene (LYC) as a functional feed additive for meat-producing poultry during the past decade (2013-2022). Experimental Subject Supplemental Dosage and Duration Main Findings References Arbor Acres broiler Dosage: 10, 20, 30 mg/kg Duration: 42 days | Growth performance: days22-42 and days1-42: | ADG, final BW, | FCR (all LYC); | Serum antioxidant status: day21: | MDA (all LYC); day42: | GSH-Px (all LYC), SOD (20, 30 mg/kg LYC), T-AOC (30 mg/kg LYC); | Liver antioxidant status: day21: | GSH-Px, SOD, | MDA (all LYC); day42: | GSH-Px (30 mg/kg LYC), | MDA (all LYC); | Hepatic Nrf2-related antioxidant gene expression: day21 and day42: | Nrf2 (20, 30 mg/kg LYC), SOD2 (10, 20, 30 mg/kg LYC), NQO1 (10, 20, 30 mg/kg LYC), HO-1 (20, 30 mg/kg LYC, 10 mg/kg LYC for day21). Arbor Acres broiler (AFB1 challenge) Dosage: 100, 200, 400 mg/kg Duration: 42 days | Serum AST, ALT (all LYC); | Hepatic CYP1A1, CYP2A6 activities (all LYC); | Hepatic AFBO-DNA adducts and ROS levels (all LYC); | Hepatic antioxidant status: | GSH (200 mg/kg LYC), GST (100, 400 mg/kg LYC), GCL, CAT (100 mg/kg LYC), GPx (all LYC), | MDA, 8-OhdG (all LYC), 4-HNE, PC (200, 400 mg/kg LYC). Arbor Acres broiler Dosage: 100, 200, 400 mg/kg Duration: 42 days | Growth performance: | BW at day21 (100 mg/kg LYC); | Abdominal fat: | abdominal fat weight (100 mg/kg LYC), abdominal fat percentage (100, 200 mg/kg LYC); | Plasma lipid level: | TG, TC (all LYC), LDLC (100, 200 mg/kg LYC); | Hepatic enzymatic activity: | FAS (400 mg/kg LYC), ACC (all LYC); Regulation of hepatic lipid-metabolism-related gene expression: | AMPK-a, | SREBP-1, FAS, ACC (all LYC). Arbor Acres broiler (AFB1 challenge) Dosage: 200 mg/kg Duration: 42 days | Growth performance: | ADG, | FCR; | Hepatic mitochondrial antioxidant status: | mGSH, GSH-Px, MnSOD, ATP, | ROS, H2O2, mitochondrial swelling; | Hepatic mitochondrial function: | complex III, complex V; | mRNA level of MnSOD, Trx2, TrxR2, Prx3, PGC-1a, NRF1, TFAM. Arbor Acres broiler (AFB1 challenge) Dosage: 100, 200, 400 mg/kg Duration: 42 days | Growth performance: | ADG (day1-21: 100 mg/kg LYC; day22-42 and day1-42: 200, 400 mg/kg LYC), | FCR (day22-42 and day1-42: 200, 400 mg/kg LYC); | Intestinal morphology: duodenum: | CD, | VH, VH/CD (day21,42: all LYC); jejunum: | CD (day21: all LYC), | VH (day42: 200, 400 mg/kg LYC), VH/CD (day21: all LYC; day42: 200, 400 mg/kg LYC); ileum: | CD (day21: all LYC), | VH, VH/CD (day21,42: all LYC); | Digestive enzyme activities: duodenum: | amylase, lipase (day21: 200, 400 mg/kg LYC; day42: 200 mg/kg LYC); jejunum: | amylase, lipase (day42: 200, 400 mg/kg LYC); ileum: | amylase (day42: 200 mg/kg LYC). Arbor Acres broiler (AFB1 challenge) Dosage: 200 mg/kg Duration: 42 days | Intestinal immunity: | jejunum IFN-g (day21), | IL-10 level (day21, day42); | Intestinal integrity: | serum DAO level, | jejunum ZO-1, Claudin-1 mRNA level (day42); | Intestinal antioxidant status: | jejunum H2O2 (day42), MDA level (day21, day42), | GSH, GST, GR level, mRNA level of Nrf2, HO-1, Cu/ZnSOD, MnSOD, CAT (day42). Pekin duck (Aflatoxin challenge) Dosage: 100 mg/kg Duration: 10 days | Hepatic aflatoxin residue; | Hepatic antioxidant status: | T-AOC, GST, CAT, | MDA; Altered serum indices: | ALT, AST, g-GT, ALP, uric acid, | albumin, total protein. Ross 308 broiler Dosage: 50, 100, 200 mg/kg Duration: 21 days (d21-42) | Growth performance: | ADG: day29-35 (100, 200 mg/kg LYC), day36-42 (all LYC), day21-42 (100 mg/kg LYC); ADFI: | day21-28 (all LYC), | day29-35 (100, 200 mg/kg LYC), day36-42 (200 mg/kg LYC), day21-42 (all LYC); | FCR: day21-28, day36-42, day21-42 (all LYC); No effects on carcass performance; Altered serum biochemical indices: | glucose, HDL (100, 200 mg/kg LYC), | cholesterol, triglyceride, VLDL (all LYC); | Serum antioxidant status: | GSH-Px, | MDA (100, 200 mg/kg LYC), | CAT (all LYC); | Hepatic enzyme activities: | ALT and ALP (100, 200 mg/kg LYC). Ross 308 broiler (heat-stress conditions) Dosage: 200, 400 mg/kg Duration: 42 days | Growth performance: linearly | CFI, WG, | FCR; | Serum antioxidant status: linearly | LYC, GSH-Px, SOD, | MDA; | Muscle antioxidant status: linearly | LYC, GSH-Px, SOD, | MDA; Activated muscle Keap1/Nrf2 pathway: | Keap1, | Nrf2 protein expression (400 mg/kg LYC). Ross broiler Dosage: 10, 20 mg/kg LYC, 17 g/kg tomato paste Duration: 28 days No effects on growth performance and relative organ weight; | Plasma TG (10, 20 mg/kg LYC) and LDL cholesterol (20 mg/kg LYC) at 2 weeks old; | Lycopene level in plasma and liver (10, 20 mg/kg LYC, 17 g/kg tomato paste) at 4 weeks old; | TBARS value of LDL isolated from broilers during 60, 90, 120, 180, or 240 min incubation at 2 weeks old (10, 20 mg/kg LYC, 17 g/kg tomato paste), as well as during 60, 120, 180, 240 min incubation at 4 weeks old (10, 20 mg/kg LYC, 17 g/kg tomato paste) except for 90 min (17 g/kg tomato paste). Hubbard broiler Dosage: 500 mg/kg Duration: 35 days | Growth performance: | ADFI, ADG (day1-8); | Slaughter performance: | bursa of Fabricius, spleen weight; Altered haemato-biochemical indices: | total protein, gamma globulin, albumin, alpha globulin; Degenerative lesions in the bursa of Fabricius and spleen; No effects on chemical composition and MDA level in the breast and thigh; No effects on oxidative stress in the kidney and liver. Abbreviations: |, increase; |, decrease; ACC, acetyl-CoA carboxylase; ADFI, average daily feed intake; ADG, average daily gain; AFB1, aflatoxin B1; AFBO-DNA, AFB1-8,9-epoxide-DNA; ALP, alkaline phosphatase; ALT, alanine aminotransferase; AMPK-a, adenosine monophosphate activated protein kinase a; AST, aspartate aminotransferase; ATP, adenosine triphosphate; BW, body weight; CAT, catalase; CD, crypt depth; CFI, cumulative feed intake; Cu/ZnSOD, copper and zinc superoxide dismutase; CYP1A1(2A6), cytochrome P450 1A1(2A6); DAO, diamine oxidase; 8-OhdG, 8-hydroxydeoxyguanosine; FAS, fatty acid synthase; FCR, feed conversion ratio; 4-HNE, 4-hydroxynonenal; g-GT, glutamyl transferase; GCL, glutamine-cysteine ligase; GR, glutathione reductase; GPx (GSH-Px), glutathione peroxidase; GSH, reduced glutathione; GST, glutathione-stransferase; HDL, high-density lipoprotein; HO-1, heme oxygenase 1; IFN-g, interferon-g; Keap1, Kelch-like ECH-associated protein 1; LDL, low-density lipoprotein; LDLC, low density lipoprotein cholesterol; MDA, malondialdehyde; mGSH, mitochondrial glutathione; MnSOD, manganese superoxide dismutase; NQO1, NAD(P)H quinone dehydrogenase 1; NRF1, nuclear respiratory factor 1; Nrf2, nuclear factor E2-related factor 2; PC, protein carbonyl; PGC-1a, peroxisome proliferator-activated receptor g coactivator 1a; Prx3, peroxiredoxin-3; ROS, reactive oxygen species; SOD(2), superoxide dismutase(2); SREBP-1, sterol regulatory element-binding protein 1; T-AOC, total antioxidant capability; TBARS, thiobarbituric acid-reactive substances; TC, total cholesterol; TFAM, mitochondrial transcription factor A; TG, total triglyceride; Trx2, thioredoxin 2; TrxR2, thioredoxin reductase 2; VH, villus height; VLDL, very low-density lipoprotein; WG, weight gain; ZO-1, zonula occludens-1. animals-13-00883-t003_Table 3 Table 3 The summary of lycopene (LYC) as a functional feed additive for egg-producing poultry during the past decade (2013-2022). Experimental Subject Supplemental Dosage and Duration Main Findings References High Line W36 laying hen Dosage: 20/40/60 mg/kg (Day1-30/Day31-60/Day61-90, different dosages during different phases) Duration: 90 days | Serum metabolic status: day31: | glucose, creatinine, ALT, | cholesterol, AST, alkaline phosphatase; day61: | glucose, cholesterol, ALT, | AST; day91: | glucose, creatinine, cholesterol); | Egg quality: day30-31: | carotenoid level and yolk color of fresh eggs and stored eggs (4 degC or 12 degC for 30 days); day60-61: | carotenoid level and yolk color of fresh eggs and 4 degC-stored eggs, yolk color of 12 degC-stored eggs; day90-91: yolk color of fresh eggs and stored eggs (4 degC or 12 degC for 30 days), carotenoid level of 4 degC-stored eggs. High Line W36 laying hen Dosage: 20/40/60 mg/kg (Day1-30/Day31-60/Day61-90) Duration: 90 days Hematological indices: | leukocytes (day61, day91), erythrocytes (day91); No effects on serum antibody titer of hens vaccinated against Newcastle disease etc. Lohman LSL laying hen Dosage: 20 mg/kg LYC as a purified powder or tomato powder Duration: 84 days | Egg quality: | egg weight, yolk color, yolk weight, yolk ratio, yolk LYC level, | yolk MDA and cholesterol level (all LYC); | Serum MDA and cholesterol level (all LYC); Regulated cholesterol metabolism: | protein expression of intestinal NPC1L1, MTP, ACAT2, hepatic SREBP1c, ACLY, and LXRa, | hepatic ABCG5 and ABCG8 (all LYC). Hy-line Brown laying hen Dosage: 10, 20 mg/kg LYC, 1.7% tomato paste Duration: 28 days | Laying performance: | egg weight (all LYC), egg production (20 mg/kg LYC, 1.7% tomato paste); | Egg quality: | yolk' color (10, 20 mg/kg LYC), LYC level, | MDA level (all LYC); | Oxidative stress: | serum MDA level (20 mg/kg LYC, 1.7% tomato paste), | liver LYC level (all LYC); No effects on serum lipid levels. Xinghua breeding hen Dosage: 20, 40, 80 mg/kg Duration: 35 days Regulated gene expression of fat metabolism: Liver: | PPARa, RARa, PGCa, | FABP10 (40, 80 mg/kg LYC), | RXRa, | FABP1 (all LYC), FATP4 (40 mg/kg LYC); Duodenum: | RARa (40, 80 mg/kg LYC); Jejunum: | PPAPg, RXRg, | FATP4 (40, 80 mg/kg LYC), | RXRa (20, 40 mg/kg LYC). Xinghua breeding hen Dosage: 20, 40, 80 mg/kg Duration: 35 days | Production performance: | fertilization rate, hatchability of eggs set (40 mg/kg LYC); | LYC level: | serum, egg (day7,14,21,28,35, all LYC), liver (day35, all LYC); | Egg quality: | Roche yolk color fan (day7,14,21,28,35, all LYC); | Serum antioxidant status: | SOD (day21, all LYC; day35, 80 mg/kg LYC), T-AOC (day21, all LYC), GSH/GSSG (day21,28,35, all LYC), GSH-Px (day14, all LYC); | Hepatic antioxidant status (day35): | SOD, T-AOC, GSH/GSSG (all LYC); | Serum lipid metabolism: | TCHO (day7,35, all LYC), | HDLC (day35, all LYC), | T3 (day14, 20 mg/kg LYC). Xinghua breeding hen (LPS challenge) Dosage: 20, 40, 80 mg/kg Duration: 35 days | Serum antioxidant status: | GSH/GSSG (0, 24 h post-challenge); | Serum lipid metabolism: | TCHO (0 h post-challenge), | HDLC (0, 6, 24 h post-challenge), | LDLC (6, 24 h post-challenge), | BUN (24 h post-challenge), | T3 (6, 24 h post-challenge); | Organ indices: | thymus, spleen, bursal. Japanese quail Dosage: 100, 200, 300 mg/kg Duration: 28 days | FI (100 mg/kg LYC), RGR (200 mg/kg LYC), | RGR (300 mg/kg LYC); | Antioxidant status: liver: | MDA (200, 300 mg/kg LYC), | SOD (300 mg/kg LYC); chest muscle: | MDA, cholesterol, | SOD (300 mg/kg LYC). Japanese quail Dosage: 50, 100 mg/kg LYC as pure LYC (T1, T2) or tomato powder (T3, T4), or red bell pepper powder (T5, T6) Duration: 49 days | Productive performance: | final BW, BW gain (T1,2,3,5,6), production index, | FCR (T1,2,3,5,6), economic efficiency index (all LYC); | Carcass characteristic: | carcass weight, gizzard yield (T5), liver yield (T1,3,5,6); | Gastrointestinal and organ development: | small intestinal weight (T1,2,4), duodenum weight (T2,4), jejunum and ileum weight (T2,3), cecum weight (T4), cecum length (T2,3,4,6), | abdominal fat weight (all LYC), spleen weight (T3,4,5,6), bursa of Fabricius weight (T1,2,5,6), bursa index (all LYC). Japanese quail (heat-stress conditions) Dosage: 150, 200, 250, 300 mg/kg Duration: 42 days | Blood packed cell volume, heterophils/lymphocytes (200, 250 mg/kg LYC), | blood hemoglobin level (all LYC); | Blood glucose, | triglycerides level (all LYC), | cholesterol (250, 300 mg/kg LYC), total protein, globulin level (300 mg/kg LYC). Laying quail Dosage: 6, 18 mg/kg LYC as commercial LYC or bacterial LYC Duration: 28 days No effects on laying performance, egg quality, and serum antioxidant indices; | Yolk triglyceride level (6,18 mg/kg commercial LYC; 18 mg/kg bacterial LYC); | Serum triglyceride level (18 mg/kg bacterial LYC); | Serum total lipid level (6 and 18 mg/kg commercial LYC). Abbreviations: |, increase; |, decrease; ABCG5/8, ATP binding cassette transporters sub-family G member 5/8; ACAT2, Acyl-CoA: cholesterol acyltransferase 2; ACLY, ATP citrate lyase; ALT, alanine aminotransferase; AST, aspartate transaminase; BW, body weight; BUN, blood urea nitrogen; FABP1/10, fatty acid-binding protein 1/10; FATP4, fatty acid transport protein 4; FCR, feed conversion ratio; FI, feed intake; GSH/GSSG, reduced glutathione to oxidized glutathione ratio; GSH-Px, glutathione peroxidase; HDLC, high density lipoprotein cholesterol; LDLC, low density lipoprotein cholesterol; LXRa, liver X receptor alpha; MDA, malondialdehyde; MTP, microsomal triacylglycerol transport protein; NPC1L1, Niemann-Pick C1-Like 1; RARa, retinoic acid receptor a; PGCa, peroxisome proliferator-activated receptor gamma coactivator 1-a; PPARa/g, peroxisome proliferator-activated receptor a/g; RGR, relative growth rate; RXRa/g, retinoid X receptor a/g; SOD, superoxide dismutase; SREBP1c, sterol regulatory element binding protein-1c; T-AOC, total antioxidant capacity; TCHO, total cholesterol; T3, triiodothyroxine. 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PMC10000199

The kiwi is a highly insect-pollinated dependent crop and is the cornerstone of the Greek agricultural sector, rendering the country as the fourth biggest kiwi producer worldwide, with an expected increase in national production the following years. This extensive transformation of the Greek arable land to Kiwi monocultures in combination with a worldwide shortage of pollination services due to the wild pollinators' decline raises questions for the provision of pollination services, and consequently, for the sustainability of the sector. In many countries, this shortage of pollination services has been addressed by the installation of pollination services markets, such as those in the USA and France. Therefore, this study tries to identify the barriers towards the implementation of a pollination services market in Greek kiwi production systems by conducting two separate quantitative surveys, one for beekeepers and one for kiwi producers. The findings showed a strong basis for further collaboration between the two stakeholders, as both of them acknowledge the importance of pollination services. Moreover, the farmers' willingness to pay and the beekeepers' willingness to receive of the beekeepers regarding the renting of their hives for pollination services were examined. pollination services ecosystem services beekeepers public policy farmer's decision making CIHEAM IAMMUniversity of ThessalyThis research received internal funding from CIHEAM IAMM and the University of Thessaly. pmc1. Introduction Pollination Services (PS) are a vital ecosystem service for agricultural systems, as more than the 75% of the global crops destined for food consumption depend on them . In general, pollination services are provided by a wide range of animals such as bees, butterflies, moths, flies, beetles, birds and mammals , however, bees, which are the predominant and most economically important group of pollinators, are facing a substantial population decline worldwide, and particular, in Europe . This decline has been triggered due to several motives and phenomena attached to agricultural activity, such as intensified pesticide use, monoculture, the deterioration of natural habitats, etc. . Consequently, the continuity of the above practices, in combination with the increasing agricultural land devoted to insect pollinator depended crops , may lead to a pollination crisis according to which crop yields starts to fall due to inadequate pollination services . In this context, the European Union, through the Common Agricultural Policy (CAP), launched a wide range of policy measures in order to protect insect pollinators. These measures include a variety of costly Agri-Environmental Measures (AEM) , a regulation (EU No 485/2013) banning the use of three neonicotinoids in agricultural systems, which are responsible for bees' decline and the Pollinators Initiative. This last measure aims to alter the decline of wild pollinators through a series of actions, focusing mostly on raising knowledge and awareness of the pollinators' importance and addressing the main stressors, such as pesticides . Hence, according to this initiative, every Member State has the opportunity to identify the main issues and design and implement frameworks for the protection of wild pollinators. However, all of the aforementioned policy measures seem ineffective to protect pollinators and safeguard the provision of pollination services in arable crop farms in the long term . For instance, the majority of the implemented AEMs proposed focus only on well-established bee species, such as bumblebees or honey bees and neglect wider pollinators species, such as solitary bees . As a result, these measures may lead to the extinction of a variety of insect pollinators that are valuable for the functioning of ecosystems, and consequently, may negatively affect the pollination services provided by bumblebees and honey bees . In addition, according to the report of the European Court of Auditors, the Pollinators Initiative is not a regulation, but a communication, and will/may consequently fail to mobilize the majority of the Member States to establish a legal framework for the provision of wild pollinators . Therefore, the continuous decline of wild pollinators in combination with the ineffective policies are driving many agricultural systems to depend more and more on renting/buying managed pollinators in order to obtain sufficient pollination services . Indeed, a market of pollination services, such as in the USA , has recently emerged in the south west of France in the Occitanie region . In fact, the creation of pollination services markets may offer a solution to the farmers' needs for pollination services, while at the same time, it can offer alternative sources of incomes for the beekeepers . This is a traditional practice in the USA, especially for almond production . However, in Europe, these markets are quite new, so there is a significant lack of information regarding prices, organization, transaction costs, contracting, etc. Thus, it is of paramount importance to identify the barriers to the creation and the functioning of these markets. For this purpose, it is necessary to understand the decision-making process of beekeepers and farmers. By understanding their perceptions regarding pollination services and their mode of actions, it may be easier to facilitate the collaboration of the two stakeholders, as well as to promote effective policy measures for the protection of wild pollinators. In this study, we are examining the farmers' and beekeepers' perceptions towards pollination services in Greek kiwi production systems. In fact, Kiwi is a highly bee-pollination-dependent crop and is one of the cornerstones of the Greek agricultural sector, rendering the country as the fourth biggest kiwi producer worldwide, with an expected increase in national production in the following five years . This extensive transformation of the Greek arable land to Kiwi monocultures in combination with wild pollinator decline raise questions for the provision of pollination services, and consequently, for the sustainability of the sector. Thus, here we analyze the conditions for the implementation of pollination services markets in Greek kiwi production systems in the region of Kavala, determining the perceptions of the stakeholders. In order to do so, we conducted two separate quantitative surveys, one for beekeepers and one for kiwi producers, in order to collectively explore (i) the possibility of collaboration between the stakeholders, (ii) the stakeholders' perception of pollination services and (iii) the stakeholders' decision making regarding pollination services. The first section justifies the selection of the examined case study and presents a step-by-step analysis of the elaborated methodology. The second section presents the obtained results, while the third section discusses the main findings. The final section summarizes the main conclusions, presents the limitations and discusses the perspectives for future research. 2. Materials and Methods In this section, we present: (i) the selection of the studied area; (ii) the data collection; (iii) the elaborated statistical analysis. 2.1. Study Area As we mentioned above, kiwi is one of the most important and dynamic crops of Greek agriculture, with 250,140 tons being produced in 2019. The most important kiwi regions are Pieria, Kavala and Arta, producing the 31%, 15% and 26% of the national production, respectively. Moreover, in the regions of Arta and Kavala, an increasing number of new kiwi plantations have been observed the last decade in the place of oranges, peaches, apricots, etc. accessed on 1 July 2021). Hence, in both regions, a significant increase in the number of kiwi trees is further anticipated in the near future. This rapid transformation of these two regions to kiwi monocultures in combination with the widespread pollinators' decline in European landscapes raise questions about the future provision of pollination services, and consequently, the sustainability of the sector. Between these two areas, the Kavala region is characterized as one of the most important beekeeping areas in Greece, with more than forty-six thousand registered beehives and a long tradition in beehive products. Moreover, the majority of the professional beekeepers are located in the island of Thasos, moving their hives long distances within the wider region of Eastern Macedonia and Thrace during the year in order to find available nectar resources . Therefore, for all of the aforementioned reasons, the region of Kavala is a suitable case study, as it combines strong kiwi production and a significant presence of nomadic beekeepers, and consequently, it is possible that pollination services market may emerge in the near future. In general, the prefecture of Kavala is located in the wider region of Eastern Macedonia and Thrace in the north east of Greece. It is divided in three municipalities, the municipalities of Kavala, Nestos, and Pangaio, and the minor prefecture of the island of Thasos accessed on 1 July 2021). Despite the fact that kiwi plantations are widespread in this regional unit, the most intensive systems are located in the municipality of Nestos. This municipality has a particular geographical feature: it is located in a valley with numerous agricultural systems and a great variety of agricultural products . Moreover, the River Nestos is traversing this municipality and forms an extensive Delta before it flows to the northern Aegean Sea, irrigating more than 40,000 ha of agricultural land. As a result, the primary economic section of the municipality of Nestos is agricultural activity, with a high rate of production of kiwi fruits, as shown by Sykianakis et al., 2019 . Finally, located in the south of this municipality is the city of Keramoti, which is the main connection port with the island of Thasos and facilitates the movement of beekeepers throughout the year. In this selected case study, through our questionnaires, we identified two geographical areas where the kiwi producers are located . The area (A) is located in the western part of the municipality of Nestos, while the area (B) is on the east is within a close distance away from the Nestos river and a forestry area. 2.2. Data Collection Data have been collected through face-to-face interviews by using two types of questionnaires each for kiwi producers (hereafter, 'farmers') and beekeepers. A small group of 5 kiwi producers and 6 beekeepers were interviewed to test and calibrate the questionnaire, but only minor changes were necessary to be applied. Both questionnaires had a similar format and were inspired and adapted by the study by Breeze et al. (2019) . The first part of the questionnaire was the same for both groups, referring to the demographic characteristics of the respondent (age, gender and educational level), number of hectares or beehives, whether they were amateurs or professional (for beekeepers) and the location of agricultural activity. The following section, regarding farmers, assessed the pollination services deficit attitudes, the implemented strategies to ameliorate pollination services (if any) and the prices for these services. Similar questions were included in the beekeepers' questionnaire about the reasons why they rent their beehives, the price and the crops they prefer or avoid. Moreover, a series of open questions have been used to gain further insights from each group about their beliefs regarding pollination service increases or its perceived limitations. Furthermore, willingness to pay (WTP) and willingness to receive (WTR) were examined to identify any contradiction between the two groups. Finally, the last part had a series of open questions in order to examine the farmers' and beekeepers' perceptions towards the adoption of policy incentives for the provision of pollination services in agricultural systems. Farmers were asked about what types of management strategies or measures they are willing to adopt in order to attract more beekeepers to rent their hives, as well as to identify practices for the provision of wild pollinators within their farmlands. The beekeepers were asked to name what types of policy incentives may encourage them to increase the number of hives in order to offer more pollination services to the farmers. All of the answers were recorded anonymously, without any storage of personal information. The survey was conducted between June and August 2021. Questionnaires were distributed to collaborations of local farmers' cooperatives and beekeepers' cooperatives. The names and basic structure are the following: i. EAS Kavala cooperative (Farmers' cooperative) (250 members, 350 ha); ii. Cooperative Municipality of Nestos (Farmers' cooperative) (60 members, 100 ha); iii. Nespar cooperative (Farmers' cooperative) (51 members, 150 ha); iv. Apiculture cooperative of Kavala (Beekeepers' cooperative) (230 members); v. Beekeeping Cooperative of Thasos (Beekeepers' cooperative) (104 members). Out of the 334 beekeepers in total in the region, 49 questionnaires were collected, representing 14.6% of the total, whereas for the kiwi producers from the Cooperation Municipality of Nestos, which has in total 100 ha of kiwi orchards, 5 members (8.3%) were interviewed, who cultivated, in total, 20 ha of kiwi (20%). In addition, 31 farmers (12.4%) were surveyed from the EAS Kavala cooperative, which had in total 250 members owning 169.8 ha of kiwi orchards or 48.5% of the total surface area of the cooperative. Furthermore, 11 kiwi producers from the Nespar cooperative (21.5%) managing 56.6 ha (37.7%). Overall, 13% of the total kiwi cultivated area in the valley of Kavala, which accounts for 1900 ha, was covered. In total 104 questionnaires were collected, but only 96 were accepted (47 kiwi producers and 49 beekeepers) after the data validation process. Additional information regarding the sample's characteristics can be found in Table 1. Descriptive statistics were extracted from all of the questions, and the most significant ones are visualized in the results section. Additionally, a Pollination Services indicator was created to assess the attitude of farmers towards pollination services. This indicator uses 4 questions from the survey related to (1) the perceived pollination services deficit, (2) the willingness to pay or willingness to increase the amount of money for pollination services, (3) the implementation of measures for encouraging wild pollinators (4) and planning to further support the existing wild pollinators by adopting alternative agricultural practices. Through the above-mentioned variables, the data type was collected, meaning that farmers either have a positive (1) or a negative (0) attitude towards PS. All of the variables were equally treated, without any use of weights since this indicator was used to describe a general overview of the farmers' attitudes. Apart from the apparent grouping between beekeepers and farmers, a new dummy variable was introduced for separating the farmers regarding their proximity to the Nestos river. The farmers near the river or forest areas benefit from the pollination services of existing bees' populations that are situated there due to the increased availability of nesting and foraging habitats, which is contrary to the farmers that are located further away, who do not benefit from these services. In other words, this dummy variable was added to highlight potential differences in the farmers' attitude regarding pollination services between the two groups. Proximity to the Nestos river or forest areas was estimated within a 3 km radius, based on the effective flying distance of bees . 2.3. Statistical Analysis In order to specify the existence of any differences between the medians of the formulated groups, a non-parametric approach has been selected by using a Mann-Whitney test , since the data were not normally distributed according to the Shapiro-Wilk test. The following comparisons have been made for farmers and beekeepers:(a). Number of beehives and provision of pollination services; (b). Farmers' willingness to pay and beekeepers' willingness to receive; (c). Farmers' actual payments and beekeepers' willingness to receive; (d). Current beekeepers' and farmers' prices for pollination services. Moreover, Spearman's rank order correlation coefficient (r) was used for assessing the relationships between the variables . Any statistical correlation coefficient is described using integers ranging from -1 to 1; with -1 indicating a perfect negative relationship between two variables, while 1 indicates a perfect positive relationship. Spearman's correlation was applied between the following variables for the two stakeholders:(a). Years of beekeepers' experience and their perception of being professional or amateurs; (b). Years of beekeepers' experience and the number of beehives; (c). Farmers' management strategies to deal with pollination services deficit; (d). Farmers' perception towards the impact of pollination services on kiwi yields. All of the above statistical analyses were conducted with the use of R software, version 4.0.5 (base and stats package). 3. Results In this section, we present our findings with regard to: (i) the socio-economic characteristics of the two stakeholders, as well as the current situation in the examined area, (ii) the beekeepers' perception towards pollination services, (iii) the farmers' perception of pollination services and collaboration with the beekeepers and (iv) the stakeholders' perceptions towards public policy interventions. 3.1. Actual State of Pollination Services and Socio-Economic Characteristics of Beekeepers and Farmers The research sample consists of 49 beekeepers managing 7402 beehives, distributed in the prefecture of Kavala and the island of Thasos. On average, a beekeeper is 44 years old and owns 218 beehives. Eighty-five percent of the beekeepers interviewed were male, and fifteen percent were female. Sixty-eight percent of the beekeepers have a high school degree, and the rest hold a bachelor's/master's degree. Fifty-nine percent of the beekeepers considered themselves to be amateurs, while the remaining ones are professionals. On average, the beekeepers have 10 years of experience, and the majority (97%) move their hives to another location at least once a year (usually during summer). All of the beekeepers surveyed experienced an increase in demand for pollination services since 2018. Sixty-eight percent of the beekeeper sample rent their hives to local farmers, receiving in return an average of EUR 12 per hive. This price was based mostly on transportation and feed costs. In fact, kiwi orchards offer no nectar to the foraging bees and as a result, the beekeepers need to feed the bees themselves, and as a consequence, the farmers face higher maintenance costs. When they were asked if they were happy with this price, the beekeepers undoubtedly agreed that EUR 12 is not enough, demanding at least double that amount (EUR 24 per hive). Indeed, this finding was further validated by conducting a Mann-Whitney U test (W = 1100, p < 0.001) . Moreover, every beekeeper mentioned that this service takes place with no written contract. Indeed, 53% of these agreements happen orally, and 12% lend their hives to friends or to close relatives . However, the remaining 32% of them who do not provide pollination services had multiple reasons for this choice, such as producing organic honey (12%) or that they are not ready to take this step (6%). As anticipated, the beekeepers are not satisfied with this unofficial agreement, and 65% of them would like to negotiate on the rights and responsibilities of each party in terms of the number of hives per ha, the rental period, the cultivation practices (e.g., lower use of pesticides), the price per hive and the method of payment before proceeding to any exchange. Regarding the kiwi producers, 47 farmers are located in the municipality of Nestos, managing 247 ha in total. One hundred and seventy ha were located in area (A), and seventy-six ha were located in area (B), closer to Nestos river and to the forestry area. The obtained results showed that, on average, a farmer is 46 years old and owns 5.2 ha of kiwi. Eight-seven percent of the farmers interviewed were male, and thirteen percent were female. Additionally, 70% have a high school degree, while the rest have a bachelor's/master's degree. All of the farmers affiliated with cooperatives are full time farmers. The data revealed that 66% of them agreed that they experience a pollination services deficit in their production systems. Fifty-five percent of them were unaware of the reason(s) leading to this deficit. The remaining 34% of the farmers stated that there was no pollination service deficit, and 15% justified their stance by mentioning the presence of beekeepers nearby or their close location to the Nestos river and forestry areas. In order to deal with the shortage of pollination services, 64% of the farmers adopt various strategies, such us renting beehives and buying managed bumblebees, while the remaining 36% do nothing. The farmers take actions against the pollination services deficit, usually by renting hives (44%) for an average price of EUR 12 per hive or by buying bumblebees (34%) for an average price of EUR 64 per hive. It should be stated that private discussions with farmers have revealed that 15-20 hives are needed per hectare, a fact that is also confirmed by the literature . Moreover, we observed two farmers buying "Flying Doctors" hives, a specific category of bumblebees for a price of EUR 200 per hive. In fact, this category of bumblebees is developed in specific hives with a dispenser which contains biopesticides, or previously collected kiwi pollen or even both, that sticks to a premium colony of bumblebees on their way out of the hive, and as a result, the bumblebees can deliver pollen, as well as microbial pesticides, to each flower they visit. Finally, a few farmers (22%) use both honeybee and bumblebee hives, while none of the abovementioned farmers have knowledge of solitary bees or other wild pollinators. 3.2. Beekeepers' Perceptions of Pollination Services As described in the Materials and Methods section, the beekeepers' number of beehives and provision of pollination services were tested. Although non-significant results were obtained (p = 0.291), it is highlighted that there is no clear picture between which strategy should be adopted and whether to provide or not provide pollination services. Spearman's correlation rank was used to identify the relationship between the variables. The beekeepers' years of experience and their perception about being s professional or an amateur do not appear to have a significant correlation, meaning that there are several people with multiple years of experience that still consider themselves as amateurs. However, years of experience have a significant positive relationship (0.338) at significance level of a = 10% (0.051) with the number of self-owned beehives. When it comes to the beekeepers' preferences to crops, 18 crops were listed as ones which are preferable to use, with the most mentioned ones being pine, kiwi, thyme, oak, chestnut and almonds. Similarly, seven crops were listed as the best ones to avoid, with the most averted one being cotton, followed by olives and sunflowers. The main reason for this avoidance is the beekeepers' perception of high use of pesticides in these crops. Surprisingly there was a significant overlap between the two groups, with three out of the seven crops that are commonly avoided also being among the eighteen most favored crops, which are kiwi, sunflowers and almonds . In fact, the beekeepers' main reasons for choosing a crop for hive placement are: (i) the production of higher quality honey, (ii) honey with a better commercial price, (iii) more honey production, (iv) the availability of nectar resources and (v) it being good for colony growth. In general, beekeepers prefer to place their hives close to wild flora, such as pine and thyme, as they perceive that these crops offer honey of better quality with a higher commercial price. However, when it comes to kiwi, 71% of beekeepers stated that the only reason for choosing this crop was the payments provided by farmers in return for their hives . Advancing now to crops that are avoided by beekeepers, cotton obtained the most answers, with 56% of the responses, followed by olives and sunflowers, with 53%, and then kiwi, with 41%. The primary reason for crop avoidance is pesticide use, for example, cotton is widely unaccepted since it is associated with pesticide use (95% of answers) and poor-quality honey (5%). Similarly, sunflower was linked with pesticide danger (52%), and so were olives (28%). However, questions about sunflower and almonds received many controversial answers, as on the one hand, they offer valuable nectar resources for the bees, while on the other hand, they the farmers intensively use pesticides, which increases the bees' mortality. Finally, 41% of them avoided kiwi orchard for two main reasons, the presence of other better pollen sources during this time of the year and the threat of pesticides. 3.3. Farmers Perception of Pollination Services and Collaboration with the Beekeepers As already mentioned in the first part, 66% of the individuals surveyed claimed the presence of a pollination service shortage in their orchard, but when they were asked for the reasons behind this statement, 84% failed to respond, 10% had no idea what was behind this occurrence and only 6% associated this deficit with the lack of insect pollinators. The farmers who denied the presence of a pollination services deficit backed up their statement with more reasons than the farmers who thought there was a deficit did. Likewise, the majority of them (44%) gave no answer, 19% mentioned the presence of beekeepers nearby, another 19% justified their stance with their close location to the Nestos river and forestry areas, where there is beekeeping activity and high abundance of wild pollinators, 13% blamed the structure of the kiwi orchard, which contains many unproductive male plants rather than an insufficient number of pollinators, and the rest (6%) stated ignorance . Indeed, regarding the farmers who responded that they do not experience a pollination service deficit, location seems to be a crucial factor. Hence, we conducted a Mann-Whitney test based on the PS Indicator (mentioned in Section 2) in order to examine the effect of location on the farmers' perception for pollination services deficit (Table 2). With a significance of 99.5% (W = 125, p = 0.005), the results showed that there is a clear difference between the perceptions of kiwi producers situated close to Area (B) compared with those of the other group. This result was expected, since the farmers from Area (B) mentioned that a lot of beekeepers place their hives near the Nestos river, and consequently, they positively benefit from these hives. Moreover, the nearby forestry areas provide a large number of natural habitats, and as a result, wild pollinators are more likely to visit these kiwi systems. The PS indicator was compared to the demographic characteristics and farm size as well, but no statistically significant relationship arose. Regarding the importance of pollination services, our results highlight that the majority of farmers (30%) believe that adequate levels of pollination services increase the kiwi yields by 20-50%, 23% perceive an increase of 10-20%, 28% perceive an increase of 0-10%, 13% believe that pollinators contribute more than 50% to the kiwi yields, and the rest stated that it is not important for production. Hence, the great majority of the farmers believe that pollination services are necessary for improving productivity of their crops (Table 2). The farmers who acknowledged a pollination services deficit adopt several management strategies to address it. In fact, the majority of them (44%) rent beehives from local beekeepers, while others rent managed bumblebees (34%) or use a combination of the two bee species (22%). In assessing the motives behind the utilization of honeybees or bumblebees, the farmers mostly answered that honeybees as pollinators are more effective (34%) than bumblebees are, despite them being recommended by the local agronomists (23%), while completely denying the benefits of wild pollinators. Moreover, in order to further examine the farmers' strategy to deal with pollination services shortage and their perceived increase in kiwi productivity, a Spearman correlation was applied. Between those two variables, a non-parametric correlation with the highest degree of significance was found (p = 0.003). In fact, farmers invest in beehives to acquire a higher yield. In other words, pollination services are adapted not for environmental protection, but primarily for the economic development of local production systems. It should be noted that this could be a way to persuade the beekeepers to provide more beehives to local kiwi producers who will incorporate them as a key input in their kiwi production (Table 3). In general, farmers are willing to increase their purchase of beehives from local beekeepers in order to maintain sufficient pollination services in their production systems. Indeed, 62% of the farmers revealed their willingness to start paying or even pay more than they are currently to receive more hives. For instance, the data show that half of them (52%) were willing to pay between EUR 0-20 per hive, another 38% were willing to pay EUR 20-50 per hive, and lastly only 7% were willing to pay EUR 50-100 per hive. Additionally, farmers have already embraced the importance of pollination services, as the majority of them (78%) have already reduced their current pesticide use in order to encourage beekeepers to supply more hives. After noticing the willingness of farmers to pay or pay more for renting hives from local beekeepers, we conducted a Mann-Whitney test between the willingness of beekeepers to receive and of the farmers to pay EUR 24 per hive. This price has been detected through our questionnaires as the average price which will motivate the beekeepers to continue the provision of pollination services to kiwi production systems. Our findings highlighted no significant difference, and consequently, the value of EUR 24 per hive is commonly accepted by the two stakeholders (W = 155, p = 0.000) (Table 4). 3.4. Stakeholders Perceptions towards Public Policy Interventions Regarding public policy interventions, there is no program focused on the pollination services in the greater region of Eastern Macedonia and Thrace . Hence, in order to examine the possibility of implementing such a measure, we proposed a set of open questions focused on the stakeholders' views towards their participation in public policy incentives, targeting the provision of pollination services. All of the respondents welcomed the implementation of new policy measure, such as Agri-Environmental Schemes (AES) or Payments for Ecosystem Services (PES). When it comes to beekeepers, a premium on the renting price per hive (18%) and the decrease in pesticide use by farmers (21%), including herbicides, or a combination of these two measures (15%) are key elements of any new public policy. Concerning farmers, 87% of them expressed their interest in participating in public policy incentives, such as AESs or PES, to increase the supply of pollination services. Moreover, they responded that they are willing to adopt alternative management strategies in order to increase the farmland biodiversity and benefit from wild pollinators by installing wildflower strips, nesting sites, etc. However, their main obstacles for the adoption of these measures are: (i) 38% labor availability (38%), (ii) a lack of necessary equipment (11%), (iii) a lack of experience (19%) and (iv) possible emerging costs (32%). In addition, they insisted that they are taking of all the necessary precautions regarding pesticides use, something that contradicts the beekeepers' perceptions. In fact, when it comes to pesticides, farmers perceive only the use of insecticide, neglecting the negative impacts of the herbicides on the bee pollinators . In contrast, the regional beekeepers are well aware of these impacts and made a special reference to these active ingredients. 4. Discussion In this paper, we examine the beekeepers' and kiwi producers' perceptions towards the provision of pollination services. Our findings signify the existence of an informal market of pollination services in the region of Kavala, however, both stakeholders seem to be unsatisfied with their current collaboration. In fact, beekeepers usually select crops that offer high-quality honey with a higher commercial value, thus optimizing the production process. Moreover, they also move their hives once per year according to the need for available foraging resources for their colonies. Moreover, regarding crop avoidance, their perceived biggest threat is to place their hive in an area with crops with high pesticides use, such as cotton, sunflower or olives. However, we detected an overlap in the beekeepers' decisions, as many crops, such sunflower or almonds, are also used and avoided by them. This overlap is based on the fact that these crops provide good sources of nectar, especially in periods when other floral resources are not available, suggesting that the beekeepers' actions are mainly based on their own experience regarding pesticides use, rather than based on scientific findings. In addition, the majority of regional beekeepers choose to place their hives next to kiwi production areas only when they receive payment by local producers. However, providing PS to kiwi producers is rather limited as the payments are too low to cover their high traveling and maintenance costs. Consequently, they provide only a small number of hives and only when other pollen resources are not available. Furthermore, despite the fact that farmers demand a higher supply of pollination services, the beekeepers are not willing to provide them due to the high risk of pesticides. The above findings are in accordance with the study by Breeze et al. (2019), who that mentioned that coordination actions and higher prices are needed in order to attract a higher number of beekeepers to offer pollination services. Regarding the farmers' perception, the majority of them acknowledge the importance of pollination services for kiwi production and declared that they experience a pollination services deficit. Moreover, a key element to their perceptions was the location of their production systems. Thus, they are willing to increase the provision of beehives from local beekeepers by increasing the existing prices. Indeed, the willingness of farmers to pay for pollination services is close to the willingness of the beekeepers to receive, and consequently, the basic conditions for the creation of a pollination services market are met. This finding is in accordance with the literature, which proposes that higher payments for pollination services may address the issues of pollination deficit . Furthermore, this is the first case study in Europe where both stakeholders acknowledge the importance of pollination services, and their perceptions of the renting price are the same. Hence, the development of such a market may arise naturally through extensive dialogue between the two stakeholders. However, the collaboration between different stakeholders is not given, as many socio-economic and socio-cultural issues may exist. Several studies suggest that the coordination of different actors, especially on payments for ecosystems services such as pollination, demands the cognitive and normative legitimation of the proposed actions. In other words, both stakeholders have to engage in social interactions in order to share and objectify their beliefs to establish new rules regarding what they can or cannot do in terms of practices and what they should or should not do in terms of moral values . Towards that direction, the biggest problem to overcome is the use of pesticides. According to our findings, on the one hand, the farmers believe that they take all of the necessary measures to protect the bees, however, they do not believe that herbicides are toxic to them. On the other hand, the beekeepers do not trust the farmers and they do not want to offer a larger supply of beehives to kiwi systems. Consequently, a policy measure or a PES scheme should be implemented in order to facilitate the dialogue and mobilize the farmers to take actions for the protection of bees, while at the same time, it provides incentives to the beekeepers in order to reduce their production costs and support the declining apiculture industry . According to the obtained results, both stakeholders are willing to be engaged in such measures, in the form of AESs or PESs, in order to increase the profitability of their sectors. Apart from the economic gains themselves, such a measure may have several environmental outcomes. Providing additional insights on this, the municipality of Nestos has a variety of different production systems, however, in recent years, there had been a tendency for new plantations of kiwi trees. A lack of public intervention will probably lead to a transition to a kiwi monoculture, which will reduce the available nectar resources. Consequently, honey production will decline, while the maintenance costs of the beehives will be significantly increased, as beekeepers will have to feed them more often . Therefore, it is significant that public authorities should intervene and regulate this market in order to guarantee the existence of a diverse agricultural landscape with varied foraging resources. This statement is in accordance with Batary et al. (2015), who support the implementation of AESs for supporting the provision of pollination services in agricultural landscapes . Finally, even though a pollination service market may be beneficial for both farmers and beekeepers, it may also affect the wild pollinators negatively. In fact, the establishment of the market may conclude with the introduction of large numbers of honeybees in the landscape, which in turn, will compete with native wild pollinators for the available foraging habitats, and consequently, drive them to extinction . Hence, the intervention of a public policy is important to regulate the number and the placement of beehives. Moreover, this policy may also motivate the farmers to maintain the natural habitats within their farmlands to provide suitable nesting sites for wild pollinators and profit from the wild pollination services. In fact, wild pollinators are more effective than honeybees are, while the presence of both bee species in the farmland may increase the yield outcomes, and consequently, the economic profit of the farmers . Taking into consideration all of the above-mentioned results, we can state that while base conditions for the establishment of pollination services market exist in the region of Kavala, there is an increased need for public policy intervention. This may be the opportunity for the establishment of the first AES for pollinators in Greece, following the Pollinator Initiative accessed on 1 July 2021). The need for policy intervention is imminent, especially after the wildfires in 2021 on the island of Euboea, which may drive many nomadic beekeepers in the region of Kavala to search for available nesting sites or to offer pollination services to gain an extra income. 5. Conclusions In this study, we examined farmers' and beekeepers' perceptions towards the implementation of pollination services markets in Greek kiwi production systems in the region of Kavala. Hence, two separate quantitative surveys were conducted, one for beekeepers and one for kiwi producers, in order to collectively explore (i) the possibility of collaboration between the stakeholders, (ii) the stakeholders' perception of pollination services and (iii) the stakeholders' decision making regarding pollination services. Our findings indicate that both stakeholders acknowledge the importance of pollination services for kiwi production and that there is indeed an unofficial market for PS in the region. However, the current situation is unpleasant for both stakeholders, as farmers are demanding a higher supply of beehives, while beekeepers are avoiding kiwi systems due to pesticide use and the absence of nectar resources. Moreover, we found that the only reason to rent beehives out to kiwi systems is the received payment. Towards that direction, we presented that farmers are willing to increase their payments in order to ensure that beekeepers place their hives within their production systems. Indeed, the WTP of the farmers overlaps with the WTR of the beekeepers, and consequently, the main principals for the establishment of a sustainable PS markets meet. However, the main obstacle for the establishment of this market is the use of pesticides. Policy implementations could play a major role by initiating the dialogue between the two stakeholders and support the establishment of a PS market, which could positively contribute towards the economic and environmental viability of the regional production systems. Our analyses indicate that both stakeholders are willing to participate in such a policy measure. Nevertheless, it should be noted that our findings depend only on the stakeholders' perception of pollination services, and there is no study regarding the actual provision of pollination services from wild pollinators or honeybees in the landscape. Additionally, there is no information regarding the public sector officials' willingness to formulate and apply the supportive measures that already exist in other EU countries. Acknowledgments This work has been supported by the Mediterranean Agronomic Institute of Montpellier and the University of Thessaly. Author Contributions Conceptualization, G.K., N.G. and S.D.; methodology, G.K.; software, G.K. and L.S.K.; validation, G.K., L.S.K. and C.K.; formal analysis, L.S.K. and T.B.; data curation, E.A.N., L.S.K. and C.K.; writing--original draft preparation, E.A.N.; writing--review and editing, E.AN.; supervision, G.V.; project administration, S.D., G.V. and H.B. funding acquisition, H.B. All authors have read and agreed to the published version of the manuscript. Institutional Review Board Statement Not applicable. Informed Consent Statement Informed consent was obtained from all subjects involved in the study. Data Availability Statement Not applicable. Conflicts of Interest The authors declare no conflict of interest. Figure 1 Location of the two study areas in the municipality of Nestos. Area A is located near urban and road infrastructures, while area B is adjacent to a large water body and woodland, designated as a Natura 2000 protected area. Data sources: Corine Land Cover (2018), EEA (2022) and GADM (2022). Figure 2 Mann-Whitney U test for current price and beekeeper's willingness to receive in exchange for hives supply. Figure 3 Summary of the main crops used (blue) and avoided (black) by beekeepers. Figure 4 Beekeepers' usage and avoidance of different trees and crops. Figure 5 Farmers' perception towards pollination services deficit. animals-13-00806-t001_Table 1 Table 1 Samples' characteristics compared to agricultural/beekeepers cooperatives structure. Samples' Structure Compared to Agricultural/Beekeepers Cooperatives Agricultural Cooperatives' Structure Sample Structure Beekeepers Cooperatives' Structure Sample Structure Farm size (ha) N of farmers (%) N of beehives N of beekeepers (%) 0-3 97 (27) 12 (26) <200 37 (11) 7 (15) 3-6 191 (53) 26 (56) 200-400 224 (67) 35 (71) 6-9 69 (19) 8 (16) 400-600 53 (16) 6 (12) >9 4 (1) 1 (2) >600 20 (6) 1 (2) Total 361 47 Total 334 49 Gender Gender Male 321 (89) 41 (87) 70 (21) 7 (15) Female 40 (11) 6 (13) 264 (79) 42 (85) Total 361 47 Total 334 49 animals-13-00806-t002_Table 2 Table 2 Mann-Whitney U test across farmer's current location and the PS indicator. PS Indicator Scores Across Locations Kavala (N = 32) Scores Nestos River (N = 15) 4 4 0 10 3 0 13 2 10 4 1 3 1 0 2 animals-13-00806-t003_Table 3 Table 3 Spearman's correlation between present strategies and perceived increase in kiwi production. Spearman's Correlation between Present Strategies and Perceived Increase in Kiwi Production Dealing with Shortage Values Perception of increased production with the use of PS Comparison Coefficient 0.425 Sig. (2-tailed) 0.003 N 47 animals-13-00806-t004_Table 4 Table 4 Mann-Whitney U test across farmers and beekeepers' current price in exchange for hives for pollination services. Kiwi Producers WTP vs. Beekeepers WTR Kiwi Producers (N = 16) Price (EUR) Beekeepers (N = 34) 2 20-22 1 0 18-20 0 0 16-18 0 2 14-16 4 1 12-14 4 8 10-12 6 0 8-10 0 1 6-8 0 2 4-6 2 0 2-4 0 0 0-2 0 Disclaimer/Publisher's Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

PMC10000200

Oestrogens treatment is often used to induce oestrus behaviour in anoestrous mares to aid in the collection of stallion semen and as recipient mares to receive embryos when combined with progesterone. However, there are no studies to describe the effect of dose and individual mare on the intensity and duration of the response, in both anoestrous and cyclic mares. In Experiment 1, 13 anoestrous mares were treated with one of five doses of oestradiol benzoate (OB) (1, 1.5, 2, 3 and 4 mg) per mare in five consecutive treatment periods (n = 65), to determine the response in terms of endometrial oedema and oestrous behaviour. Experiment 2 and 3 used 3 mg of OB in cyclic mares to confirm or deny the presence of an active corpus luteum (CL). There was a dose rate of OB and individual mare effect (p < 0.05) on the intensity and persistence of endometrial oedema and oestrous behaviour. A total of 2 mg OB was enough to induce endometrial oedema and oestrous behaviour within 48 h in most mares. Mares with an active CL did not show endometrial oedema following treatment of 3 mg OB. mare oestradiol endometrial oedema oestrous behaviour anoestrus Universidad Cardenal Herrera-CEUINDI22/12 J.C.-A. was supported by Universidad Cardenal Herrera-CEU project ref. INDI22/12. pmc1. Introduction The steroid sex hormone 17-b oestradiol is secreted by ovarian follicles and is responsible for oestrous behaviour and other oestrogenic changes in the uterus of mares (i.e., development of endometrial oedema and cervical relaxation) by binding to oestrogen receptors . The oestrous or sexual behaviour in mares depends on the relationship between circulating follicular oestrogens and basal progesterone: it does not begin until peripheral progesterone concentrations have dropped to less than 2 ng/mL, and oestrogen concentrations are beginning to rise . Circulating concentrations of oestrone sulphate increased from basal concentrations (200 pg/mL) 8 days before ovulation to peak concentrations (850 pg/mL) 2 days before ovulation . Furthermore, exogenous treatment with oestradiol in anoestrous mares induced oestrous behaviour within 8 h, whereas daily treatment of progesterone blocked the oestrus-inducing effect of oestradiol . A similar relationship is observed between oestradiol, progesterone and the development of endometrial oedema . During low/basal circulating concentrations of progesterone, an ovariectomized mare can show prominent endometrial oedema soon after oestradiol treatment, which can last for as long as 7 days , whereas the administration of progesterone 1 day after oestradiol treatment inhibits the development of endometrial oedema. It was concluded that the threshold of circulating progesterone concentration necessary to inhibit endometrial oedema falls between 1 and 2 ng/mL . As a result, the ultrasonographic evidence of endometrial oedema in mares is often used as a clinical sign to confirm that the mare is in oestrus . Most mares follow a common endometrial oedema pattern during oestrus: gradual increase in the oedema score from luteolysis until 2 to 3 days before ovulation, and a rapid decrease thereafter, so that the endometrial oedema at the time of ovulation is returned to the basal score . However, on some occasions a mare may ovulate without showing endometrial oedema and oestrous behaviour in the previous days, despite having basal levels of progesterone and elevated concentration of oestradiol . This is termed as silent oestrus or covert oestrus and is more likely to happen after induction of luteolysis with exogenous prostaglandin, with a reported incidence of 6% . Pregnancy rates of mares which did not show endometrial oedema during silent oestrus were reduced (36.4%) . In field conditions, exogenous oestradiol is used mainly to induce oestrous behaviour in dummy (ovariectomized) mares to facilitate semen collection from stallions, and to prepare anoestrous recipient mares to receive embryos following the administration of long-acting progesterone . For those purposes, esterified oestrogens are preferred over the naturally occurring oestradiol-17b, as they are more potent and have longer half-lives . The objectives of this study were (1) to clinically assess the effect of difference dose rates of oestradiol benzoate on the degree and persistence of oestrous behaviour and on endometrial oedema patterns in individual mares when administered in anoestrus; and (2) to determine whether a single dose of oestradiol benzoate administered to cyclic mares can differentiate between the presence of an active or non-active corpus luteum in the ovary in field conditions. We hypothesized that there would be a dose rate effect of oestradiol benzoate on the magnitude of endometrial oedema and oestrous behaviour in anovulatory mares, and that a single administration of 3 mg oestradiol benzoate would induce endometrial oedema within 24 h of treatment in cyclic mares with no active corpus luteum (CL). 2. Materials and Methods This experiment was carried out in two locations: (1) the controlled studies (Experiment 1 and 2) were performed in the Faculty of Veterinary Medicine, Uberlandia Federal University, MG, Brazil (18deg54' S); and (2) the field study (Experiment 3) was performed in a private standardbred farm in New South Wales, Australia (33deg25' S). 2.1. Experiment 1 A total of 13 crossbred mares from the research herd of the Uberlandia Federal University, with a mean age of 12.4 +- 5.9 years (4 to 22 years old) and mean weight of 465 +- 65 kg (350 to 550 kg) were used for two experiments. In the first experiment, during the non-breeding season (June to September, southern hemisphere), mares were examined weekly by transrectal ultrasonography to confirm their anovulatory status (absence of a CL, lack of endometrial oedema, and follicles of <20 mm in diameter for a period of at least two consecutive weeks). Once in anoestrus, each mare received 1 of 5 different doses of oestradiol benzoate (1 mg/mL, Sincrodiol, Ourofino, Brazil) administered subcutaneously (1, 1.5, 2, 3 and 4 mg per mare), with a period of rest of one week between treatment periods, so that each mare had received all 5 different doses by the end of the study. The order of the oestradiol benzoate was chosen randomly for each of the 13 mares. Overall, 65 treatment periods were analysed (13 mares multiplied by 5 treatments of different doses) in a crossover design. After each treatment, the uterus of the mares was examined daily by transrectal ultrasonography with a scanner Mindray DP-6600 vet (Mindray Brasil, Sao Paulo, Brazil), equipped with a linear array transducer of 7 MHz, to determine the endometrial oedema score for a total of 7 days or until the endometrial oedema had returned to basal. The presence of the endometrial oedema was scored subjectively by the same operator on a scale of 0 (minimum degree of endometrial folding) to 3 (maximum degree of endometrial folding) , as described previously . During the same time periods (before treatment and every 24 h after treatment, and for a maximum of 7 days or until the score had return to 0), each mare was presented to the same familiar active teaser stallion in a head-to-head alignment to evaluate the mare's oestrous behaviour. Initially a distance of at least one metre was maintained between muzzles before the mare was allowed to approach to make contact. This position was maintained for at least one minute and the mare's behaviour observed. The degree of oestrous behaviour was scored according to the following criteria as described previously : Grade 3: oestrous behaviour, approached the stallion willingly and within one minute, with only head to head contact, widened the stance of the hind legs, flexed the hocks and with tail raised, everted the clitoris ('showing'), and passed small or large quantities of urine. Grade 2: oestrus, mares behaved as Grade 3 except the position for urination was not adopted until the mare was either allowed to swing her hindquarters towards the stallion or was turned so that her flank and genitalia could be nuzzled. Grade 1: oestrus, mares approached the stallion positively and allowed nuzzling of their flank and genitalia. Although they continued to evert the clitoris, they did not adopt the urination position within the two-minute teasing period. Most positive signs of oestrous behaviour were shown except hock flexion and urination. Grade 0.5: mares in anoestrus, and often those coming into or going out of oestrus, usually exhibited a combination of positive and negative signs. Eversion of the clitoris did not occur unless accompanied by a definite negative or aggressive attitude, often accompanied by angry tail swishing. This behaviour is often referred to as 'showing in spite'. Mares were often reluctant to approach the stallion until forced or approached willingly as if in oestrus to then turn round and kick out. Others, particularly when in anoestrus, would stand passively whilst the stallion nuzzled and bit the flanks without showing any positive or negative signs. Others would stand and 'show' initially before kicking out. If the mare showed sufficient positive signs that it was considered that she could be mated, providing a twitch or other restraint such as leg strap was used, the behaviour was classified as Grade 0.5. Grade 0: no oestrus. Mares which were not in oestrous, exhibited some, but not necessarily all of the following behavioural characteristics: Reluctance to even approach the stallion; desire to swing hindquarters towards the stallion and at least buck if not kick out at him; tail held either tightly down or swished 'angrily' from side to side; squirting small quantities of urine apparently in anger rather than the static hock flexion position characteristic of Grade 2 or 3 oestrus; striking out at stallion with front legs on approach; squealing. 2.2. Experiment 2 The same mares (n = 13) and location as in Experiment 1 were used for this experiment, once the mares entered the breeding season (following the first ovulation after the anovulatory season), between the months of November and February (southern hemisphere). Ovulation was diagnosed by daily ultrasonography as the disappearance of the pre-ovulatory sized follicle. The Day of ovulation was regarded as Day 0. Once a mare had ovulated, she was allocated randomly into one of three experimental groups according to the stage of dioestrus and Day of ovulation: a) early dioestrus (Day 1; n = 5), mid dioestrus (Day 6, n = 4) and late dioestrus (Day 12, n = 4); Each mare received a single treatment of 3 mg of oestradiol benzoate subcutaneously once. The Day of treatment depended on the experimental group (Day 1 for early dioestrus, Day 6 for mid dioestrus and Day 12 for late dioestrus groups, respectively) Before treatment, each mare was scanned by transrectal ultrasonography and the presence of a recent ovulation or a CL and lack of obvious endometrial oedema (score < 1) was confirmed. Furthermore, a heparinized blood sample was taken from the jugular vein once in each mare to determine serum progesterone concentrations immediately before oestradiol benzoate treatment. Mares were scanned as in Experiment 1 for 3 consecutive days after treatment to record the endometrial oedema score at each examination, using the same endometrial oedema score system. 2.3. Experiment 3 During the breeding season of 2020 to 2021 of the southern hemisphere in Australia, standardbred brood mares presented for routine breeding examinations from a commercial stud farm were used in this study. The farm bred 217 mares and had 5 standing stallions on site during that breeding season. The mean age of the mares from this farm was 10.7 years old (3 to 22). Mares were scanned with an ultrasound scanner (Sonosite Nanomaxx, Sonosite Australia, Melbourne, Australia) equipped with a 7-10 MHz linear probe with a colour-flow doppler and B modes available. The routine breeding management included daily scans in every mare intended for breeding. Foaling mares were scanned daily from 5 days after foaling to diagnose the foal heat ovulation which was not used for breeding. Following foal heat ovulation, mares were administered 250 mg cloprostenol (Estrumate 250 mg/mL, MSD Animal Health, Macquarie Park, NSW, Australia) 6 days after ovulation, and then scanned daily from 3 days after cloprostenol treatment until breeding and once ovulation was confirmed. Pregnancy diagnosis was performed 14 days after ovulation. Mares with a negative pregnancy diagnosis were allowed to return spontaneously to oestrus and were scanned daily from Day 17 after ovulation. Barren or maiden mares brought for first time to the stud farm were examined by transrectal ultrasonography to diagnose the stage of the oestrous cycle. Mares showing endometrial oedema and follicles >25 mm were scanned daily until breeding and ovulation. Mares with the presence of mature CL, classified according to the luteal echogenicity described previously were administered 250 mg of cloprostenol, and scanned daily from 3 days after treatment, as in foaling mares. The breeding management of the farm was as follows: semen was available 3 times a week (Monday, Wednesday, and Friday). Mares in oestrus with endometrial oedema (oedema score >= 1), relaxed cervix and a follicle of 28 mm or larger (the optimal pre-ovulatory size follicle for breeding of each mare was based on previous breeding records of each individual mare) were submitted for induction of ovulation and treated with 1.25 mg of deslorelin (Deslorelin Dechra 1.25 mg/mL, Dechra Veterinary Products, Somersby, NSW, Australia), the day before insemination. Mares were scanned the Day of insemination and 24 h after to confirm ovulation and evaluate the post-breeding endometrial reaction to semen. Mares with free-intrauterine fluid were administered 20 IU oxytocin (Syntocyn 20 IU/mL, Troy Animal Healthcare, Glendenning, Australia) intravenously. Mares that did not ovulate within 48 h of insemination were re-inseminated the next day when semen was available. Mares showing no endometrial oedema despite the presence of a large pre-ovulatory follicle (>35 mm in diameter), which had been treated with cloprostenol or following a negative pregnancy diagnosis, were examined thoroughly, searching the ovaries for the presence of an active CL. An active and visible CL was defined as the presence of an ultrasonographically detectable CL of 20 mm or larger in diameter with the expected echogenicity according to the age of the CL as described previously . A colour-doppler mode was used to evaluate the CL when there were doubts to distinguish between a regressing CL and an active CL, especially in mares that had been treated with cloprostenol. An active CL showed colour-doppler signal, while a non-active CL (regressing CL or corpus albicans) did not. The inclusion criteria for this experiment were: resident mares presented for AI to one of the stallions housed on site, in which the previous Day of ovulation was known (from daily ultrasound examination to diagnose the Day of ovulation) or known interval from the previous treatment with cloprostenol, and presence of a pre-ovulatory sized follicle (>35 mm in diameter) but absence of endometrial oedema. Furthermore, no visible active CL was observed on the ultrasound examination of the ovaries. These mares had been either mares treated with cloprostenol, or scanned 3 days after a negative pregnancy diagnosis. Mares meeting the criteria were treated with a single dose of 3 mg of oestradiol benzoate subcutaneously and scanned within 48 h of treatment to evaluate the presence or absence of endometrial oedema. Mares showing a positive response to oestradiol benzoate treatment were induced to ovulate and inseminated as described previously. 2.4. Hormonal Assay Blood samples from Experiment 2 were taken from the jugular vein in heparinized 10 mL vacutainer tubes and centrifuged at 2000 G for 10 min. Aliquots of plasma were frozen for later analysis. Progesterone concentrations in plasma samples were measured in duplicate using a commercial ELISA assay (DRG Instruments, Marburg, Germany). Dilution curves generated from a pool of equine plasma with a known high concentration of progesterone and the 0 ng/mL standard supplied with the kit, recommended for serial dilution by the manufacturer, were parallel to the curve produced with the standards supplied with the kit. The inter-assay coefficient variations (CV) were 6.4% and 6.6%, respectively. The minimal detectable concentration of the assay was 0.16 ng/mL, calculated by adding two standard deviations to the mean optical density value of 10 zero standard replicates and determining the corresponding concentration of progesterone from the standard curve. Cross reactivities were reported as 0.35% for Pregnenolone, 0.3% for 17alpha OH Progesterone, 1.1% for 11-Desoxycorticosterone and 0.2% for corticosterone. 2.5. Statistical Analyses The overall effect of oestradiol benzoate dose on endometrial oedema score and oestrous behaviour was analysed using a general linear model (GLM) of variance (one model for each variable) with a repeated statement to account for autocorrelation between sequential observations of same mares. Sequential data not normally distributed were ranked (oestrous behaviour and endometrial oedema scores) before computing in the GLM (Systat13, Palo Alto, CA, USA). If an effect of group (dose of oestradiol benzoate) or an interaction of dose and Day was significant in Experiment 1, data were examined further by non-parametric Mann-Whitney test within each Day. Furthermore, the effects of dose, weight of mare, and Mare ID on the (a) Day of peak score, (b) Maximum score, and (c) last Day of score 1, for both endometrial oedema and oestrous behaviour scores, were tested on separate GLMs. Spearman ranked correlation was used to determine the level of association between the endometrial oedema score and the grade of oestrous behaviour expressed by the mare on Days 1 and 2 after treatment (days with maximum scores). In Experiments 2 and 3, data are given in a descriptive way: percentage of mares showing endometrial oedema after treatment. 3. Results 3.1. Experiment 1 The dose rate effects of oestradiol benzoate on endometrial oedema and oestrous behaviour scores in anoestrous mares are depicted in Figure 2 and Figure 3, respectively. For endometrial oedema, there was a significant (p < 0.001) dose effect on Day 1 and Day 2 after treatment but was not significant from Day 3 onwards. The maximum dose (4 mg) gave the highest endometrial oedema score . However, this was not different from 3 and 2 mg on Day 1, and not different either to 1.5 mg on Day 2. By Day 4, all mares failed to show some oedema at some of the dose rates. The highest score seen on Day 4 was two at 3.0 mg. By Day 5 of treatment, all mares had return to basal oedema scores, except one mare that showed oedema score 1 after a treatment of 3 mg. The maximum endometrial oedema score, regardless of dose, was obtained 24 h after treatment, which then decreased significantly (p < 0.01) by the next 24 h (2 days after treatment) and continued to decrease up to 4 days after treatment . The effect of dose on oestrous behaviour score was significant during the first 4 days following treatment . The highest doses (3 and 4 mg) of oestradiol benzoate induced a higher (p < 0.05) grade of oestrous behaviour on Day 2 after treatment than the lowest doses (2, 1.5 and 1 mg). By Day 5 after treatment, in 15% of cycles (10/65), some mares showed a positive teasing behaviour (Grades 1 and 2), regardless of dose (except for the lowest dose, in which all mares had return to basal score by Day 4). The correlation between endometrial oedema score and oestrous behaviour on Day 1 and 2 after treatment was weak (r = 0.33) but significant (p < 0.05). For example, at the extremes, a mare could have an endometrial oedema score of three and non-receptive or low-grade oestrous behaviour (Grades 0 to 1), and vice versa: a Grade 3 oestrous behaviour and lower endometrial oedema score (score of 1 or 1.5). However, no mare with a Grade 2 or 3 of oestrous behaviour showed lower endometrial oedema than a score of 1. There was a significant (p < 0.05) individual mare effect on oestrous behaviour and endometrial oedema score regardless of dose (Table 1). This strong individual mare effect was more marked on the behaviour response to teasing: on the day of treatment (Day 0), three individual mares, on 7 out of 12 occasions, showed weakly positive signs of oestrus. On Day 1, following 1 mg oestradiol benzoate injection, all but 4 mares of the 13 mares showed signs of oestrus which varied in intensity from Grade 0.5 to Grade 2. The number of mares not showing oestrus at any dose rate on any of Days 1, 2, 3, 4 and 5 were 4, 3, 9, 11 and 12 mares, respectively. However, the number of mares showing oestrus following at least one dose rate on Days 1 to 5 was 13, 13, 12, 12 and 8, respectively. One mare never showed oestrus of intensity greater than 0.5 at any dose rate from 1-4 mg while another only showed oestrous intensity of more than 0.5 at the 4 mg dose, and then only for 2 days. By contrast 10 individual mares were still in oestrus (grade 0.5 or more) on Day 5 following doses of 1.5 mg (n = 2), 2 mg (n = 3), 3 mg (n = 6), and 4 mg (n = 2). No effect of body weight on mean behaviour scores was found (p > 0.1; Table 1). The individual mare effect on endometrial oedema was obvious in such that a total of three mares did not show any endometrial oedema on Day 1 or 2 after treatments with 1.5 mg (n = 2) and 2 mg (n = 1). Seven mares failed to show endometrial oedema after treatment of 1 mg of oestradiol benzoate at any of the 5 days after treatment. 3.2. Experiment 2 The mean progesterone concentration (+-S.E.M.) of mares in early, mid, and late dioestrus just before oestradiol benzoate treatment was 1.1 +- 0.2 ng/mL, 11.3 +- 0.9 ng/mL, and 10.7 +- 1.1 ng/mL, respectively. None of treated mares in dioestrus showed any endometrial oedema score within 3 days of treatment (zero endometrial oedema score). Some mares from the group of early dioestrus (Day 1 after ovulation) had slight endometrial oedema at the time of oestradiol benzoate treatment which had returned to basal within 24 h, despite oestradiol treatment. 3.3. Experiment 3 There were 36 mares that met the criteria of having a pre-ovulatory sized follicle without signs of an active CL and no endometrial oedema. These cycles represented 10.2% of all oestrous cycles followed during the breeding season in the farm (36/354). Of those 36 mares, 17 (47.2%) had been given cloprostenol (PGF2a) to induce oestrus 3 to 9 days previously. Another 17 (46.2%) mares were 16 to 21 days after ovulation (following a negative pregnancy diagnosis). The last 2 mares (5.5%) were foaling mares which have not had the foal heat ovulation yet. These 2 mares were treated with oestradiol benzoate 25 and 43 days after foaling. The mean follicular diameter and other reproductive parameters of these mares are shown in Table 2. Following the single administration of 3 mg oestradiol benzoate, 23 mares (63.9%) showed obvious endometrial oedema within 48 h of treatment. The remaining mares (n = 13) did not increase the endometrial oedema score following treatment which remained zero. A total of 9 of these 13 mares were from the prostaglandin group. All 36 mares ovulated between 24 and 120 h after oestradiol benzoate treatment. Only mares which showed an endometrial oedema score >= 1 after treatment were inseminated. The pregnancy rate 14 days after ovulation was 47.8% (11/23). The overall first cycle pregnancy rate in this stud farm was 54% (191/354). 4. Discussion The hypothesis that the magnitude of endometrial oedema score and oestrous behaviour in anovulatory mares would be oestradiol benzoate dose-dependent is substantiated by the results of this study, as larger doses of oestradiol induced a higher score of endometrial oedema and oestrous behaviour which was maintained for longer than lower doses in anoestrous mares. In addition, in cyclic mares, the treatment of oestradiol benzoate only induced obvious endometrial oedema in mares with no active CL. In terms of induction of endometrial oedema, a dose of 2 mg of oestradiol benzoate was as efficient as the largest dose used in the study (4 mg) to induce a maximum endometrial oedema score within 24 h of treatment. For oestrous behaviour induction, however, it appeared that 3 or 4 mg doses were more efficient in inducing a greater grade of positive oestrous behaviour in anoestrous mares. It is interesting that such a small dose (2-3 mg) can elicit an obvious endometrial oedema and oestrous behaviour responses for up to 48 h after treatment. This contrasts with previous studies in which a double dose of 10 mg dose of oestradiol benzoate two days apart was recommended to induce oestrous behaviour and increase circulating levels of oestradiol to 15-20 pg/mL in anovulatory mares . According to the results of this study, a dose of 2-3 mg every 2 to 3 days appears to be sufficient to maintain an anovulatory mare in oestrus, to be used, for example, as a dummy mare to aid in the collection of stallion semen . This positive response in terms of endometrial oedema to only 2 mg of oestradiol benzoate agrees with a previous study in which 2.5 mg administration of oestradiol benzoate to anovulatory mares induced a similar uterine oedema pattern and plasma circulating concentrations of oestradiol to those observed in cyclic mares. Therefore, doses of 2 to 3 mg appear to be the quantity of oestradiol benzoate that better mimics the oestrous behaviour and endometrial oedema patterns observed in cyclic mares. Since no mare from Experiment 1 showed endometrial oedema or positive oestrous behaviour by 6 days after treatment (even with the highest dose), a gap of 7 days between treatments appeared to be enough rest to evaluate accurately the response to the subsequent treatments. The results observed in this study (Experiment 2) following the administration of oestradiol benzoate to cyclic mares (in dioestrus) are consistent with the data showed in the studies from Pelehach and co-workers and confirms the failure of oestradiol benzoate to induce endometrial oedema when the mare has elevated progesterone concentrations from an active CL. Similarly, these results agree with a previous study in which the administration of oestradiol followed by progesterone treatment to anoestrous mares did not result in the development of positive oestrous behaviour . In field conditions, especially in stud farms located remotely, where laboratory facilities to measure progesterone are not available, the use of oestradiol benzoate (i.e., 2 to 3 mg) appears a good method to confirm or rule out that the mare is in dioestrus, so that the clinician can make a rapid judgment on whether to inseminate or not a mare, based on the positive (development of endometrial oedema) or negative response. It is not uncommon, however, that following PGF2a administration, some mares respond to the treatment with a partial luteolysis, especially when the PGF2a is given in early dioestrus . The CL of mares with partial luteolysis is either not obvious, appears regressed or is no longer visible on ultrasound, but it continues producing varying quantities of progesterone sufficient to prevent the development of endometrial oedema despite the growth of a dominant follicle to pre-ovulatory size . In fact, in Experiment 3, the percentage of mares that did not show endometrial oedema after oestradiol treatment was highest in the group that had received PGF2a to induce oestrus. This may account for a greater number of mares with partial luteolysis which may explain the lack of response to oestradiol benzoate treatment. The reason to use this approach (administration of oestradiol benzoate to mares with a large follicle and no endometrial oedema) in a clinical setting (Experiment 3) was double: (1) to determine whether the mare had still some active luteal tissue (which was not diagnosed on ultrasound); and (2) to increase endometrial oedema score before breeding in an attempt to improve the likelihood of obtaining a viable pregnancy, as several studies have shown a positive effect of the duration of endometrial oedema prior to ovulation on subsequent pregnancy rates . Unfortunately, in Experiment 3 there was no control group of mares inseminated but that failed to show endometrial oedema, as these mares were not bred for fear of inducing post-mating endometritis , so that meaningful comparisons on fertility of these mares cannot be made. However, an interesting finding is that approximately half of mares treated with oestradiol benzoate soon before ovulation became pregnant, so the treatment did not apparently impair fertilization and embryo development. In a physiological situation in the mare, the levels of circulating oestradiol begin to decline 2 days before ovulation, so that they are low at the time of oocyte release and fertilization . The main limitation of the study is that Experiment 3 was performed in field conditions, with privately-owned mares, so that additional data could not be obtained (i.e., circulating levels of progesterone and oestradiol). 5. Conclusions In conclusion, a 2 mg dose of oestradiol benzoate in oil given by subcutaneous injection elicits positive oestrus behaviour and obvious endometrial oedema by 24 and 48 h in the majority of mares. Persistence of expression is proportional to the dose and the initial response. Oestrus and endometrial oedema responses may be different due to an individual mare effect, but not weight effect. Maximal oedema response is seen at 24 h but not in all mares at dose rates of less than 3 mg. Mares with an active CL do not show endometrial oedema following treatment of 3 mg of oestradiol benzoate. Author Contributions J.R.N. conceived and designed the study. E.S.M.S. performed Experiments 1 and 2. J.C.-A. performed Experiment 3, data analyses, figure editing and wrote the manuscript. All authors agree to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work have been appropriately investigated and resolved. All authors have read and agreed to the published version of the manuscript. Institutional Review Board Statement Ethical review and approval for this study was issued by the Comissao de Etica na Utilizacao de Animais, Universidade Federal de Uberlandia to Elisa M.S. Silva on 12 July 2019, project licence ref. 028/19. Informed Consent Statement Not applicable. Data Availability Statement Not applicable. Conflicts of Interest The authors declare no conflict of interest. Figure 1 Representative B-mode ultrasonograms of the endometrial oedema scoring system. Figure 2 Mean endometrial oedema score in anoestrous mares treated with different dose rates of oestradiol benzoate in a single administration (Day 0). Probabilities for main effects of dose group (G), Day (D), and the group-by-day interaction (GD) are shown. Within a given Day (*), different letters (a, b, c) indicate a significant difference (p < 0.05) in endometrial oedema score between dose groups. Figure 3 Mean oestrous behaviour score in anoestrous mares treated with different dose rates of oestradiol benzoate in a single administration (Day 0). Probabilities for main effects of dose group (G), Day (D), and the group-by-day interaction (GD) are shown. Within a given Day (*), different letters (a, b) indicate a significant difference (p < 0.05) in oestrous behaviour score between dose groups. animals-13-00938-t001_Table 1 Table 1 Effects of dose, mare and weight on endometrial oedema and oestrous behaviour scores in anoestrous mares administered a single treatment of oestradiol benzoate. Variable Parameter Significance (p Value) Dose Effect Mare Effect Weight Effect Day of peak score Endometrial oedema NS <0.05 NS Oestrous behaviour NS <0.01 NS Last day of score 1 Endometrial oedema <0.1 <0.05 NS Oestrous behaviour <0.05 NS NS Maximum score Endometrial oedema <0.001 NS NS Oestrous behaviour <0.01 <0.05 NS animals-13-00938-t002_Table 2 Table 2 Reproductive parameters of mares from Experiment 3 treated with 3 mg oestradiol benzoate due to lack of endometrial oedema despite of having a large follicle and no CL. Type of Cycle Mean +- S.E.M. Interval Cloprostenol to Oestradiol (Days) Mean +- S.E.M. Follicular Diameter at Oestradiol Treatment Mares with Endometrial Oedema within 48 h (score >= 1) Positive Pregnancy Diagnosis PGF2a-induced (n = 17) 6.1 +- 0.7 38.7 +- 1.1 8/17 (47.1%) 3/8 (37.5%) Spontaneous (n = 17) NA 40.9 +- 0.9 13/17 (70.6%) 7/13 (53.8%) Post-partum (n = 2) NA 40.5 +- 0.5 2/2 (100%) 1/2 (50%) Disclaimer/Publisher's Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

PMC10000201

Background: Hepatocellular carcinoma (HCC) leads to 600,000 people's deaths every year. The protein ubiquitin carboxyl-terminal hydrolase 15 (USP15) is a ubiquitin-specific protease. The role of USP15 in HCC is still unclear. Method: We studied the function of USP15 in HCC from the viewpoint of systems biology and investigated possible implications using experimental methods, such as real-time polymerase chain reaction (qPCR), Western blotting, clustered regularly interspaced short palindromic repeats (CRISPR), and next-generation sequencing (NGS). We investigated tissues samples of 102 patients who underwent liver resection between January 2006 and December 2010 at the Sir Run Run Shaw Hospital (SRRSH). Tissue samples were immunochemically stained; a trained pathologist then scored the tissue by visual inspection, and we compared the survival data of two groups of patients by means of Kaplan-Meier curves. We applied assays for cell migration, cell growth, and wound healing. We studied tumor formation in a mouse model. Results: HCC patients (n = 26) with high expression of USP15 had a higher survival rate than patients (n = 76) with low expression. We confirmed a suppressive role of USP15 in HCC using in vitro and in vivo tests. Based on publicly available data, we constructed a PPI network in which 143 genes were related to USP15 (HCC genes). We combined the 143 HCC genes with results of an experimental investigation to identify 225 pathways that may be related simultaneously to USP15 and HCC (tumor pathways). We found the 225 pathways enriched in the functional groups of cell proliferation and cell migration. The 225 pathways determined six clusters of pathways in which terms such as signal transduction, cell cycle, gene expression, and DNA repair related the expression of USP15 to tumorigenesis. Conclusion: USP15 may suppress tumorigenesis of HCC by regulating pathway clusters of signal transduction for gene expression, cell cycle, and DNA repair. For the first time, the tumorigenesis of HCC is studied from the viewpoint of the pathway cluster. USP15 HCC pathway analysis system biology cell behavior National Natural Science Foundation of China81700551 Science and technology plan of Zhejiang Health Committee2020RC069 This work is supported by the National Natural Science Foundation of China with the number: 81700551. This work was also supported by the Science and technology plan of Zhejiang Health Committee with the number: 2020RC069. pmc1. Introduction Hepatocellular carcinoma (HCC) is one of the most common cancer types worldwide. The development of treatment methods and research on relevant mechanisms is an important topic in current cancer research . HCC accounts for the majority of cases of primary liver cancer, and every year, more than 600,000 people die of HCC . In the focus of current molecular cancer therapies have been protein kinases that mediate antitumor activity primarily through abnormal expression and tumor-dependent or unregulated inactivation of target enzymes. Efforts have increased the number of treatment options . Sorafenib (FDA 2007) and Lenvatinib (FDA 2018) are clinical first-line drugs in the treatment strategy of liver cancer. Post-translational modification, such as ubiquitination, plays an important role in tumorigenesis . Ubiquitination is related to proteasome degradation . Tagging genes such as p27, p53, or NF-kB, ubiquitin (Ub) influences pathways that play an important role in tumorigenesis . Ubiquitin-specific proteases (USPs) remove ubiquitin from proteins and hence may regulate tumor-related pathways . Its simple chemical structure makes ubiquitin a preferable target for anticancer therapy . The adjustment of the balance between ubiquitination and deubiquitination may lead to lesser side effects than targeting the large proteasome complex with drugs such as Bortezomib . Ubiquitin carboxyl-terminal hydrolase 15 (USP15) is a member of the family of ubiquitin-specific proteases. USP15 shows high similarity with the proto-oncogene USP4, which cleaves ubiquitin . USP15 was identified in 1999 and its role in tumorigenesis has been reported recently. A relationship between USP15 and cancer has been proposed by the observation of its stabilization function on the TGF-b receptor in glioblastoma . USP15 has been shown to be elevated in breast and ovarian cancer . USP15 has been shown to repress apoptosis in melanoma and colorectal cancer cell lines through stabilizing the E3 ubiquitin protein ligase MDM2 . USP15 has been shown to suppress glioblastoma cell growth through stabilizing the E3 ubiquitin protein ligase HECTD1 . USP15 has been shown to regulate DNA homologous recombination repair, which has been related to tumorigenesis in the breast cancer cell line MCF7 and human osteosarcoma cell line U2OS . USP15 may be expected to regulate ubiquitination pathways of proteins such as TGF-b, MDM2-p53, Keap1/Nrf2, and Wnt/b-catenin. Ubiquitination pathways may play an important role in HCC. USP15 may regulate tumorigenesis in HCC by multiple pathways. The role of USP15 in HCC is, however, unknown. USP15 has been shown to act as an oncogene or as a tumor-suppressing gene . Systems biology applies network analysis and data integration to explore biomedicine questions. The holistic view of systems biology may help to unravel the complex function of USP15 in the progression of HCC. We applied algorithms of systems biology and experimental methods to investigate the function and mechanism of USP15 in HCC. 2. Materials and Methods 2.1. Tumor-Normal Comparison of Publicly Available Data of GTEx/TCGA We performed a tumor-normal comparison with the database of UCSC Xena accessed on 5 May 2022) and the tool Gene Expression Profiling Interactive Analysis 2 (GEPIA2, accessed on 7 March 2020) . The Xena provided data sources of the Genotype-Tissue Expression Project (GTEx) of The Cancer Genome Atlas (TCGA). We chose the publicly available dataset of liver hepatocellular carcinoma (LIHC) with 369 primary tumor tissue samples (no recurrent tumor) from TCGA and 160 samples from GTEx (dataset: TCGA TARGET GTEx). Boxplot analysis for USP15 expression of RNA-Seq by Expectation-Maximization (RESM) was applied in log scale (dataset: gene expression RNAseq-RSEM norm_count), see Figure 1A. With GEPIA2, we considered mRNA levels of USP15. We split the 369 tissue samples (no recurrent tumor) into two equal-sized groups; one with high expression of USP15 and a second group with low expression of USP15. Before splitting, we normalized the mRNA level of each tissue to the corresponding mRNA level of the control glyceraldehyde 3-phosphate dehydrogenase (GADPH). We used the median value of the 369 normalized USP levels as the threshold for the split between the two groups. We compared the Kaplan-Meier plots of the two groups, see Figure 1B. To compute the hazard ratio, we applied the Cox Proportional-Hazards Model. We chose a 95% confidence interval (CI). We applied the log-rank test to compare two Kaplan-Meier curves. 2.2. Patient Cohort and Tissue Samples We investigated tissues samples of 102 patients that were diagnosed with HCC and underwent liver resection between January 2006 and December 2010 at the Sir Run Run Shaw Hospital (SRRSH). For detailed information on the clinical and pathological characteristics of the patient cohort, we refer to Table 1. We collected a cancerous tissue sample for each of the 102 patient samples. As a control, we took 27 samples from non-cancerous tissue adjacent to the tumor. All specimens were immediately immobilized in formalin and embedded in paraffin block for subsequent pathological testing. We used ten pairs of fresh frozen tissue specimens from the hospital tissue bank of SRRSH for Western blot analysis. We evaluated tumor stages according to the American Joint Committee on Cancer (AJCC) staging system and defined clinical stages according to the Barcelona Clinic Liver Cancer (BCLC) staging system. This study was approved by the Medical Ethics Committee of SRRSH with the number: 20190211-90, and all patients were informed. All the procedures conformed with the Declaration of Helsinki . 2.3. Cell Culture Cell lines HCCLM3, SKhep-1, HA22T, HepG2, Huh1, Huh7, hep3B, QGY-7703, SMMC-7721, Bel-7404, SNU-387, SNU-449, SNU-423, and SNU-745 were provided by the Cang laboratory at Zhejiang University, Hangzhou, China. Cell lines HCCLM3, SKhep-1, HA22T, HepG2, Huh1, Huh7, hep3B, SMMC-7721, Bel-7404, and LO-2 were grown in DMEM (Gibco, Waltham, MA, USA, cat#C11995500BT) supplemented with 10% fetal bovine serum (FBS) (Cellmax, Beijing, China cat#SA102.02). Cell lines SNU-387, SNU-449, SNU-423, SNU-745, and QGY-7703 were grown in RPMI-1640 (Gibco, cat# C11975500BT) supplemented with 10% FBS (Cellmax, cat#SA102.02). All cells were maintained in an atmosphere of 5% CO2 in a humidified 37 degC incubator. 2.4. Plasmid Constructs We designed the single-guide RNA (sgRNA), using the online sgRNA design website accessed on 12 November 2017). The sgRNA was cloned into the lentiCRISPRv2 plasmid (Addgene, Watertown, MA, USA cat#52961, standard protocol). Full-length USP15 cDNA was kindly provided by the Cang laboratory (Zhejiang University). The cDNA was amplified and cloned into the expression vector p3xFlag-CMV-7.1 (Sigma, St. Louis, MO, USA, cat#E4026) using DNA restriction enzymes XbaI and BamHI. We cloned the full-length cDNA of USP15 into the plasmid pXF4H at the restriction sites EcoRI and BamHI; pXF4H was provided by the Cang laboratory. For the sequences of the primers, we refer to Table 2. 2.5. Lentivirus Production and Transduction Lentivirus expressing sgRNA was produced by co-transfecting cells HEK293T with a mixture of plasmid lentiCRISPRv2-sgUSP15 (or plasmid lentiCRISPRv2-sgControl), the Gag-Pol packaging plasmid psPAX2 (Addgene, cat#12260), and the envelope plasmid B19/VSVG (Addgene, cat#88865) at a 4:3:2 ratio. We used transfection reagent Lipofectamine 2000 (lipo2000, Thermo Fisher Scientific, Waltham, MA, USA, cat#11668019). Virus particles were harvested 48 h after transfection. Lentivirus-infected cells were screened with puromycin (Thermo Fisher Scientific, cat#A1113802: Huh1,1mg/mL; HA22T, 1mg/mL). 2.6. Quantitative Real-Time PCR Analysis RNA was purified from liver tissue samples using Trizol (Thermo Fisher Scientific cat#204211) according to the manufacturer's protocol. The reverse transcript was performed using PrimeScript RT Master Mix kit (Takara, Tokyo, Japan, cat#RR037B). Real-time PCR was carried out in SYBR Green PCR Master Mix (Bio-Rad, Hercules, CA, USA, cat#1725270) with ABI PRISM 7500 Sequence Detection System. The USP15 primer sequences were as follows: forward, 5'-CTG CTC AAA ACC TCG CTC C-3' and reverse, 5'-CAA TGG GTC CAG GAT ACA CA-3'. The GAPDH primer sequences were as follows: forward, 5'-AGGTCGGTGTGAACGGATTTG-3' and reverse, 5'-TGTAGACCATGTAGTTGAGGTCA-3'. Each measurement was performed in triplicate, and results were normalized to the expression of the internal reference gene GAPDH. 2.7. Western Blot and Antibodies For protein extraction, tumor tissues or cell pellets were homogenized in the buffer of RIPA (Sigma, cat#R0278), which contained a cocktail of protease inhibitors (Roche, Basel, Switzerland, #4693116001). After incubation on ice for 30 minutes, we centrifuged for 15 minutes at 4 degC at 12,000x g. Protein concentration was quantified using the kit BCA protein assay (Thermo Fisher Scientific, cat#A53227). Equal amounts of protein were separated discontinuously on a 4-12% SDS-PAGE gel and transferred to polyvinylidene difluoride (PVDF) membrane (Merck Millipore, Billerica, MA, USA). Non-specific binding sites on the membranes were blocked for 1 h with 5% non-fat milk (BD, Franklin Lakes, NJ, USA, cat#232100). After blocking, membranes were incubated overnight at 4 degC with primary antibodies, washed three times with TBS/T, and incubated with HRP-conjugated secondary antibodies for 1 h at room temperature. Immunoreactions were visualized using Clarity Western ECL substrate (Bio-Rad, cat#VL001), and the blots were imaged using a luminescent image analyzer (Fujifilm, Tokyo, Japan). The following antibodies were used: anti-USP15 (Santa Cruz, Santa Cruz, CA, USA, cat#sc-100629), anti-b-catenin (Cell Signaling Technology, Danvers, MA, USA, cat#8480), anti-Phospho-Nrf2 (Ser40) (Thermo Fisher Scientific, cat#PA5-67520), anti-Phospho-Rb1 (Ser780) (Cell Signaling Technology, cat#9307), anti-IkBa (Cell Signaling Technology, cat#4814), anti-N-cadherin (Cell Signaling Technology, cat#13116), anti-Phospho-Smad2 (Ser465/467) (Cell Signaling Technology, cat#3108), anti-Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (Cell Signaling Technology, cat#4370), anti-Phospho-AKT (Ser473) (Cell Signaling Technology, cat#4060), anti-C-myc (Cell Signaling Technology, cat#18583), anti-SQSTM1 (P62) (Abcam, Cambridge, UK, cat#ab56416), anti-b-actin (Cell Signaling Technology, cat#3700), and anti-GAPDH (Abcam, cat#ab181602). 2.8. Immunohistochemical Staining Paraffin-embedded sections were provided by the department of pathology of the SRRSH. The tissues were paraffin-embedded using a Tissue-Tek automated processor (Sakura) and cut into 3 mm tissue sections. The tissue sections were deparaffinized and rehydrated. For antigen retrieval, the sections were immersed in 10 mM citrate buffer (pH 6.0) and boiled for 10 min in a microwave oven. Endogenous peroxidase activity was blocked with 3% hydrogen peroxide for 10 min. Non-specific binding sites were blocked with 5% normal goat serum for 30 min. The sections were incubated with an antibody against USP15 (1:100, Santa Cruz, cat#sc-100629) overnight at 4 degC. The sections were incubated with the secondary antibody, and the expression of USP15 in the tissues was observed via microscopy after DAB staining and hematoxylin staining. 2.9. Tissue Scoring By visual inspection, two trained pathologists assigned a score--strong, moderate, weak, or negative--to an immunochemically stained tissue. All decisions were made blinded to the clinicopathologic characteristics. Figure S1 in the supplemental material shows a reference image for each of four staining scores. If the decision was not unanimous, a third pathologist repeated the evaluation and made an independent decision. We denoted tissue with the scores strong or moderate as tissue with high expression of USP15. We denoted tissue with the scores weak or negative as tissue with low expression of USP15. For the comparison of the scores of two patient groups, we replaced the scores negative, weak, moderate, and strong by the numbers 1, 2, 3, and 4, respectively, and applied the Mann-Whitney U test. All tissue slides are accessible on reasonable request. 2.10. Cell Migration Assay The cell lines Huh1 and HA22T cells with USP15-stable overexpression and/or knockout (200 mL FBS-free DMEM medium suspension, 1 x 105/mlHA22T or HUH1) were sucked into the upper chamber of Transwell (PC membrane with 8.0-mm pore size). After 24 h of incubation, the chambers were washed with PBS (pH 7.4) three times to remove the cells in the upper chamber. The cells were fixed with 4% paraformaldehyde for 15 min then stained with crystal violet (0.01% in ethanol) for 25 min followed by washing three times. The cells were counted in an inverted microscope, and five visions were randomly taken at x200. The average number of cells was considered. 2.11. Wound Healing Assay Wound healing assay was carried out by scratching a 6-well dish with a 10-mL pipette tip when the dish was at 80% confluences (including SMMC-7721vec/usp15 overexpression and QGY-7703vec/usp15 overexpression). The widths of the scratches were evaluated at time points 0, 6, 12, 24, and 30 h after scratching. 2.12. Cell Growth Assay We seeded in round numbers 1 x 103 cells per well into 96-well plates. We added cell counting kit CCK8 (5 mg/ml, YEASEN, cat#40203ES60) to the wells. We measured absorbance at wavelength 450 nm. 2.13. Tumor Formation in Nude Mice BALB/c nude mice (n = 10, 4 weeks of age, female) were obtained from ZhiYuan biological company (Hangzhou, China). The nude mice were housed in a pathogen-free facility under controlled temperature (22 +- 2 degC) and lighting (12-h light/dark cycle) conditions. We used two human liver cell lines, Huh7-PLVX and Huh7-PLVX-USP15. Huh7-PLVX was the Huh7 cell line transfected with an empty lentiviral expression vector (PLVX). We used cell line Huh7-PLVX as a control. The second cell line, Huh7-PLVX-USP15, was the Huh7 cell line transfected with a lentiviral expression vector to overexpress USP15. We compared the two cell lines Huh7-PLVX-USP15 and cell line Huh7-PLVX to evaluate the effect of high expression of USP15. Both of the cell lines were suspended in sterilized PBS. Mice were randomized and subcutaneously injected with either cell line Huh7-PLVX or cell line Huh7-PLVX-USP15 (five mice per group, per mouse: 5 x 106 cells suspended in 100 mL of PBS,). After 25 days, the mice were euthanized, and the tumors were excised. The dimensions of the implanted tumors were measured with a vernier caliper. The tumor volume was computed by the formula: volume (cm3) = 0.5 x length (cm) x width2 (cm2). For further analysis, e.g., immunochemical staining, the tumors were embedded in paraffin. All animal experiments were performed under the guidelines reviewed by the Animal Ethics Committee of the Biological Resource Centre of the Agency for Science, Technology and Research at the Sir Run Run Shaw Hospital. 2.14. Protein-Protein Interaction Network Interaction partners of USP15 (layer 1 proteins) were searched in the IntAct database accessed on 15 March 2020) with species restriction to human. We found 106 interaction partners including USP15 (layer 1 proteins). We applied a breadth-first search (BFS) strategy to expand the set of layer 1 proteins. We found 6175 interactions partners (layer 2 proteins) for the layer 1 proteins (IntaAct, human). For visualization of interaction networks, we chose the circle layout of Cytoscape 3.8 . We constructed sub-networks to describe the interaction between USP15 and given small sets of proteins, called target genes. The sub-network had a node for USP15, each target gene, and each of their interaction partners. Each edges denoted an interaction reported in IntAct for human. 2.15. HCC Oncology Gene Collection and USP15 Cancer Gene List We downloaded the list of genes of HCC from the cBioPortal for Cancer Genomics (Dataset: HCC, TCGA, PanCancer Atlas) . We selected genes with a mutation rate > 1%. The HCCgenes were checked to be registered in the OncoKB database , see Table S1 for a list of the 295 identified genes. We determined a set of 143 genes that simultaneously were gene of HCC and interaction partners of USP15 (layer 1 protein or layer 2 protein). We denoted the 143 genes that interact with USP15 as the set of HCC-USP15 genes. For a complete list, we refer to Table S2 in the supplemental material. 2.16. Next-Generation Sequencing The human hepatoma cell lines HA22T sg-control and knockout HA22T sgUSP15 were treated with 1 mL of TRIzol reagent (Thermo Fisher Scientific, cat#204211) per 107 cells and stored at -80 degC and afterwards sent in three replicates to Hangzhou Kaitai Biotechnology Co., Ltd. (Hangzhou, China) to perform mRNA extraction and sequencing. The tool DESeq2 was applied to estimate log-fold changes based on mean counts of triple replicates and false discovery rates (FDR) . The thresholds for the selection of significant genes were set to FDR < 0.1 and log2 fold changes Log2FC > 1. 2.17. Network of Pathway Hierarchies and Mapping of Functions We downloaded the relation of pathway hierarchies from Reactome accessed on 20 July 2020) . Only human pathways were considered. A network represented each pathway hierarchy as a node and each relation between two pathway hierarchies as an arc. The network had 2423 nodes and 2424 directed edges. We imported the network in Cytoscape 3.8 . For the mapping, we extracted a list of Uniprot IDs for the genes from the database Uniprot accessed on 28 July 2020) and downloaded the mapping list "UniProt2Reactome_All_Levels" from Reactome accessed on 28 July 2020) . We selected only the human entries in the mapping list to generate the file "UniProt2Reactome_human.txt". We filtered the Uniprot IDs of the genes in the human mapping list and generated a relevant mapping list "pathway_matching_all.txt". We imported the file "pathway_matching_all.txt" into the network of pathway hierarchies in Cytoscape. For the enrichment analysis, we mapped all the genes extracted from the file "UniProt2Reactome_All_Levels.txt" to the network of pathway hierarchies, applying our Python script for computing the fraction of genes in each pathway. 2.18. Sub-Networks of Pathway Hierarchies The complete network of human pathway hierarchies was analyzed using the tool CentiScape . We identified 28 source nodes with an in-degree of zero to decompose the network into the 28 sub-networks of its connected components. Each sub-network was associated with an individual source node. The sizes of the sub-networks ranged from 606 nodes for the hierarchy "diseases" to only 4 nodes for the hierarchy "circadian clock". 2.19. Statistical Analysis For statistical testing, we applied the tools GraphPad Prism 7 (San Diego, CA, USA), SPSS (V25.0, Chicago, IL, USA), and Microsoft Excel (Office 2021, Redmond, WA, USA). If not indicated otherwise, we give numbers in the form of mean +- standard error. We applied Fisher's exact test for analysis of contingency tables, i.e., to evaluate possible biases in the patient cohort, see Table 1. We applied the Mann-Whitney U test to compare the USP15 levels in tumor and non-tumor tissue in 102 patients. To compare the survival distribution of groups of patients, we produced Kaplan-Meier plots and applied the log-rank test (Mantel-Cox test). For enrichment analysis, we applied the hypergeometric test. We denoted p-values p <0.05 as significant. In graphical representation, we represented significance values as follows: * for p < 0.05, ** for p < 0.01, *** for p < 0.001, and **** for p < 0.0001. 3. Results 3.1. Expression of USP15 Was Low in Tumor Tissue; High Expression of USP15 Correlated with Decreased Risk for Mortality and Cancer Relapse We inspected publicly available data in the Xena database and gene expression profiling interactive analysis tool (GEPIA2) . We chose the publicly available dataset of liver hepatocellular carcinoma (LIHC) with 369 tissue samples from TCGA and 160 samples from GTEx for tumor. The USP15 expression levels in tissue samples of liver cancer were significantly lower than those in normal tissue . We split the patients into two equal-sized groups, one with high expression of USP15 and a second group with low expression of USP15. The Kaplan-Meier plot of the survival data demonstrated a significantly higher survival rate for patients (expression levels above median) with high expression of USP15 than for patients (expression levels below median) with low expression . Figure 1 USP15 is down-regulated in hepatocellular carcinoma (HCC) tissue. High expression of USP15 in HCC patients is associated with significantly decreased risk for mortality and cancer relapse. (A) Box plots of USP15 expression in tumor and non-tumor tissue; liver hepatocellular carcinoma (LIHC) dataset of TCGA and GTEX. The median expression level of USP15 is lower in tumor tissue (N = 369) than in non-tumor tissue (N = 160). (****: p < 0.0001, Mann-Whitney U test). (B) Kaplan-Meier plots of patients in the LIHC dataset of TCGA with high expression of USP15 (red curve) and low expression of USP15 (blue curve). The dotted lines indicate a 95% confidence interval. High expression of USP15 is associated with significantly decreased risk for mortality: hazard ratio 0.6, p-value 0.018 (log-rank test). (C) Two exemplary images of tissue samples that were stained (IHC) for USP15. Right figures are blowups of a part of the left image, see rectangle. By visual inspection, two trained pathologists assigned tumor tissue samples of 102 patient to one of the two classes: high expression of USP15 (n = 26, moderate or strong staining) and low expression of USP15 (n = 76, negative or weak staining). T--tumor tissues; NT--para-HCC tissues. (D) Fractions of tissue samples that were assigned to four staining levels: negative, weak, moderate, and strong. Tumor (n = 102) and non-tumor samples (n = 27) have significantly different distributions (p < 0.001, Mann-Whitney U test). (E) Kaplan-Meier plots for survival of patients with low expression (n = 76, blue curve) and high expression (n = 26, red curve). High expression of USP15 is associated with significantly decreased risk for mortality: hazard ratio 0.379, p = 0.0105 (log-rank test). (F) Kaplan-Meier plots for tumor-free survival of patients with low expression (n = 76, blue curve) and high expression (n = 26, red curve). High expression of USP15 is associated with significantly decreased risk for cancer relapse: hazard ratio 0.420, p = 0.0089 (log-rank test). We retrospectively inspected tissue samples from 102 patients with a diagnosis of HCC at Sir Run Run Shaw Hospital (SRRSH). Information on the cohort of patients is listed in Table 1. Tumor and non-tumor tissue samples were immunochemically stained for USP15 . By visual inspection, we classified tissue samples according to the degree of staining into four levels: negative, weak, moderate, and strong . A total of 59.2% of non-tumor tissue samples were assigned to strong or moderate staining. In contrast, only 25.4% of tumor samples were assigned to strong or moderate staining. (Table S4). The staining of tissue samples differed significantly in tumor samples and non-tumor samples . In accordance with our findings for the publicly available dataset LIHC, USP15 expression tended to be reduced in the tumor tissues of our patient cohort. We split the 102 patients in two groups. Patients with tumor tissue of negative or weak staining were assigned to the group low expression of USP15 (n = 76). Patients with tumor tissue of moderate or strong staining were assigned to a second group, high expression of USP15 (n = 26). We compared the survival data of the two groups by means of Kaplan-Meier plots. High expression of USP15 was associated with significantly decreased risk for mortality: hazard ratio 0.379, p = 0.0105 . We compared data of tumor-free time periods by means of Kaplan-Meier plots. High expression of USP15 was associated with significantly decreased risk for cancer relapse: hazard ratio 0.420, p = 0.0089 . The correlation of high survival rate and reduced cancer relapse with high expression of USP15 might suggest a role of USP15 as a tumor suppressor. 3.2. USP15 Expression Was Increased in HCC Cell Lines We compared the protein level of USP15 in the HCC cell lines Huh1, HA22T, and Huh7 with the protein level of USP15 in cell lines LM3, PLC/PRF/5, HLF, and Hep-G2. To measure the level of USP15, we applied Western blotting. For each cell line, we normalized the luminescence of a USP15 antibody by luminescence of a GAPDH antibody. We found the highest normalized expression levels of USP15 for HCC cell lines Huh1, HA22T, and Huh7 . This finding supported our decision to choose HA22T and Huh1 cell lines to be used as a cell model for researching USP15. 3.3. Overexpressing USP15 Correlated with Slow Proliferation and Slow Migration We transfected USP15 to cell lines Huh1 and HA22T to overexpress USP15. Overexpressing USP15 in cells Huh1 (Huh1-P-F-USP15) had a reduced proliferation index of, in round numbers, 50% of the control . Overexpressing USP15 in cells HA22T (HA22T-P-F-USP15) had a slightly decreased proliferation index compared to the control and the proliferation was reduced only 2.86% . We applied a migration assay (Transwell) to investigate the motility of the overexpressing cell lines. USP15 overexpressing cells Huh1 (Huh1-P-F-USP15) had a reduced migration rate of 45% of the migration rate of the control . On USP15 overexpressing cells HA22T (HA22T-P-F-USP15), the migration rate was nearly completely inhibited compared to the control c . We transfected cell lines QGY-7703 and SMMC-7721 to overexpress USP15. We applied wound healing assays to evaluate the effect of overexpression of USP15. Overexpressing cells, QGY-7703-PXF4H-USP15 and SMMC-7721-PXF4H-USP15, had a lower movement rate than their controls . The USP15 overexpression efficiency in the two cell lines is shown in Figure S4. We also use flow cytometry to detect whether the level of USP15 influences the apoptosis rate of the cell. However, in Huh1 and HA22T cell lines, overexpression or knockout USP15 does not change the apoptosis rate of the two cell lines , which indicated that a high level of USP15 represses the proliferation of these cell lines. 3.4. Knockout of USP15 Correlated with High Proliferation and High Migration We tested whether knockout had reverse effects compared to overexpression of USP15. Hepatocellular cell lines Huh1 and HA22T were modified with CRISPR/Cas9 to knock out expression of USP15. The reduced expression of USP15 was tested for knockout cell lines Huh1-sgUSP15 and HA22T-sgUSP15 using Western blotting . Increased proliferation index was observed for knockout cell lines Huh1-sgUSP15 Figure 2J) and HA22T-sgUSP15 . Migration assays showed increased motility knockout cell lines Huh1-sgUSP15 and HA22T-sgUSP15 . 3.5. Overexpression of USP15 Correlated with Slow Tumor Growth in Mice To investigate a role of USP15 as a possible tumor suppressor, we subcutaneously injected Huh7 cells into BALB/c nude mice. Five mice, the control group, were injected with cells of control cell line, Huh7-PLVX. Huh7-PLVX were Huh7 cells that were transfected by an empty vector. A second group of five mice, OE group, were injected with cells of cell line Huh7-PLVX-USP15. Huh7-PLVX-USP15 were Huh7 cells that were transfected with a lentiviral expression vector to overexpress USP15. Tumors were excised after 25 days . We confirmed a high expression of USP15 by immunostaining of tumor tissue . We found the volume of tumor significantly larger in the OE group (p = 0.0079, two-tailed t-test) , and the weight of tumor was also significantly larger in the OE group (two-tailed t-test) . 3.6. Tumor-Related Proteins Were Indirectly Regulated by USP15 via Interactions Mediated by Other Proteins We selected the proteins from the IntAct database that interact with USP15 (layer 1 proteins). To the set of 105 interaction partners of USP15, we added their interaction partners (layer 2) . For the set of proteins, we constructed the protein-protein interaction (PPI) network. We deleted all non-human genes and compounds. The resulting network had 105 proteins in layer 1 and 6175 proteins in layer 2 . The PPI network had 17,439 edges. In the following, we denote the network as the human USP15 PPI network. With the searching method shown in Figure 3D, we chose a set of the ten genes to see the relations of them to USP15. The ten genes using two conditions: (1) the gene was known to be tumor-related and (2) the availability of an antibody enabled an experimental investigation by Western blotting in our lab. Eight of the ten proteins were members of the human USP15 PPI network . We applied Western blotting to compare gene expression and phosphorylation levels in cell lines Huh1-P-F_USP15 (OE, overexpressing USP15) and Huh1-sgUSP15 (KO, knockout USP15) versus their control cell lines Huh1-PLVX and Huh1-sgControl, respectively, see Material and Methods. We assumed proteins to be regulated by USP15 if overexpression and knockout had reverse effects. We found the eight members of the human USP15 PPI network regulated by USP15 . Also regulated by USP15 was Nrf2, which was not part of the human USP15 PPI network. Nrf2 was, however, an interaction partner of layer 2 proteins. We also tested the level of some downstream proteins of USP15 in the tumor samples from the mouse model. The level of beta-catenin increased, whereas pAKT and pERK decreased . These proteins level changes revealed the mechanism by which USP15 represses tumor growth. 3.7. Regulatory Role of USP15 in HCC: Relevant Pathway Hierarchies We constructed a network of all human pathway hierarchies, see Material and Methods. Nodes and arcs represented pathway hierarchies and hierarchy relations, respectively. The initial network had 2423 nodes and 2424 arcs. Motivated by the experimental evidence of a regulation by USP15 for eight exemplary members of the human USP15 PPI network, we chose all liver cancer genes with a mutation rate of over 1% (295 genes, Table S1) in the database cBioPortal for Cancer Genomics (dataset: HCC, TCGA, PanCancer Atlas) . We identified 143 of the liver cancer genes as members of the human USP PPI network . We mapped the 143 HCC-USP15 genes to the network of human pathway hierarchies. The number of HCC-USP15 genes per node varied between nHCC=1 and nHCC=91 (median 2, mean 3.56 +- 2.82). Using NGS, we measured log-fold change in gene expression after a knockout of USP15 in cell line HA22T . We found 1.4% (n = 203) and 2.0% (n = 278) of the 14,099 identified proteins up-regulated and down-regulated, respectively. We mapped the set of 481 differentially expressed genes to the network of human pathway hierarchies. The number of differentially expressed genes per node varied between ndiff=1 and ndiff=79 (median, 1; mean, 2.55 +- 1.87). To compute an upper bound for the number of genes, we mapped a list of all human genes to the network of human pathway hierarchies. The total number of genes that may be mapped to a node varied between ndiff=1 and ndiff=3443 (median, 17; mean, 56.37 +- 63.63). We ranked the nodes by the score: s=ndiff nHCCntot2 104 . A pathway hierarchy with all associated genes being differentially expressed and simultaneously, being an HCC-USP15 gene, e.g., with ndiff=nHCC=ntot, would reach the theoretical upper bound of s = 104. The score, s, was intended to quantify the specificity of a node for the 624 genes that may contribute to the regulatory role of USP15 in HCC. The values of s varied between 0 and 625 (median, 0; mean, 7.91 +- 8.49; Table S3). For a color-coded representation of the scores, we refer to Figure 5C, and Figure S6 is the original network which is used as the negative control. We selected a subset of 225 nodes (9.2%) with score values above s = 20. Note that a node with a small fraction of differentially expressed genes and a small fraction of HCC genes, such as ndiff/ntot= nHCC/ntot=4.4% , would get a score below 20. Figure S6 was used as blank figure. We denoted the sub-network for the 225 "high score" pathway hierarchies as HCC-USP15 pathways . 3.8. Regulatory Roles of USP15 in HCC Were Cell Proliferation and Migration We downloaded 163, 75, and 143 pathway hierarchies (database: Reactome) that were associated with cell proliferation, cell migration, and lipid processes, respectively. We tested the hypothesis of a random selection of pathway hierarchies for network HCC-USP15 pathways. Since HCC-USP15 pathways had 225 nodes, we would expect to find mean numbers of n=163x225/242315.14, n=75x225/24236.96 , and n=143x225/242313.28 pathway hierarchies associated with cell proliferation, cell migration, and lipid processes, respectively. In the network HCC-USP15 pathways, we found an enrichment of 35 pathways (expected mean 15.14, Table S5-proliferation) associated with cell proliferation (p = 0.0077, hypergeometric test). For a color-coded visualization of pathway hierarchies associated with cell proliferation, we refer to Figure 6B. We found an enrichment of 13 pathways (expected mean 6.96 Table S5-migration) associated with cell migration (p = 2.5 x 10-7, hypergeometric test). For a color-coded visualization of pathway hierarchies associated with cell proliferation, we refer to Figure 6C. We found no enrichment of pathways associated with lipid processes. Only seven pathways (expected mean 13.28, Table S5-lipid processes) were associated with lipid processes (p = 0.035, hypergeometric test). For a color-coded visualization of pathway hierarchies associated with lipid processes, we refer to Figure 6D. The complete network of human pathway hierarchies clustered into 28 connected components, so-called clusters (Table 3). We mapped the 225 "high score" pathway hierarchies, i.e., HCC-USP15 pathways, to the 28 clusters. HCC-USP15 pathways were significantly enriched (hypergeometric test) in 8 of the 28 pathway clusters. We assigned six of the enriched clusters to cancer pathways . For a discussion of cancer-related pathways, we refer to Hanahan and Weinberg . The six tumor-related clusters were "Cellular responses to external stimuli" , "Cell Cycle" , "DNA repair" , "Gene expression" , "Chromatin organization" , and "Signal transduction" . The two clusters "Reproduction" (p = 0.00029321) and "Circadian Clock" (p = 0.00294656) were enriched but not denoted as cancer-related. 4. Discussion Liver cancer is one of the most common cancers in China due to high numbers of infections with the hepatitis B virus, and the mechanism behind liver cancer is still insufficiently understood . Hepatocellular carcinoma (HCC) covers the majority of cases of primary liver cancer. Previous research has indicated that USP15 plays a role in tumor development, but its regulatory function as an oncogene or tumor-suppressing gene has never been demonstrated. We investigated the function of USP15 in HCC through a synergetic application of experimental methods and system biology methods. In a publicly available dataset (LIHC, GEPIA2) of HCC patients, we found the level of USP15 expression reduced in tumor tissue and associated with high survival rate. Additionally, in our own cohort of 102 patients, we found expression of USP15 reduced in tumor tissue. Within our cohort, a high level of USP15 expression was associated with significantly decreased risk for mortality and cancer relapse. However, the mortality rate decreased sharply in the high-USP15 group at late HCC phase in the database and our clinical data. This would indicate that the tumor suppression role of USP15 only happened in the early phase of HCC, and in the end phase, the tumor suppression role disappeared. The results motivated a more detailed investigation of the function of USP15 and its potential role as a tumor-suppressing gene in liver cancer. We investigated the phenotype of overexpression and knockout of USP15. Overexpression of USP15 in HCC cell lines correlated with slow proliferation and slow migration. Knockout of USP15 in HCC cell lines correlated with fast proliferation and fast migration. In a mouse model, overexpression of USP15 correlated with slow tumor growth. The experimental results suggested a preferable role of USP15 in HCC, presumably via a suppression of cell proliferation and motility. To explore the relevant function of USP15, we constructed a PPI network. A set 105 interaction partners of USP15 contained two liver cancer genes, LRRK and NOTCH1. Ubiquitination of one of the 105 interaction partners may affect a larger set of 6175 proteins (layer 2 proteins). To illustrate, we selected eight layer 2 proteins that were known to be related to cancer. In a cell line, overexpression and knockout had effects on the expression of each of these eight proteins. Tumor-related proteins turned out to be regulated by USP15 via indirect interactions mediated by other proteins. Via indirect interactions, USP15 may be able to regulate a rather high number of proteins. From an investigation of a small set of only eight proteins, we were not able to identify the net effect on tumor progression. We found, for example, expressions of oncogene AKT and the tumor-suppressor gene Rb1 negatively correlated with the level of USP15 expression. A similar regulation of oncogene and tumor-suppressor gene may be interpreted as contradictory effects and their concerted effect on tumor progression is difficult to estimate. Metastasis is a complex process that involves a cascade of multiple steps for the successful establishment of clinically impactful metastases. Metastasis involves multiple steps, including epithelial-mesenchymal transition (EMT), migration, matrix degradation, invasion into lymph vascular tissue, extravasation, adhesion, and mesenchymal-epithelial transition (MET) . EMT involves disassembly of cell-cell junctions, actin cytoskeleton reorganization, and increased cell motility and invasion, as characterized by down-regulation and translocation of beta-catenin from the cell membrane to the nucleus and up-regulation of mesenchymal molecular markers such as vimentin, fibronectin and N-cadherin . We found that N-cadherin was not regulated by USP15. USP15 may have no effect on EMT in metastasis. USP15 may, however, regulate a high number of genes that may be relevant for HCC. In our PPI network of USP15, we identified nHCC = 143 known liver cancer genes (mutation rate > 1%). To identify a large set of proteins that may be regulated by USP15, we applied NGS experiments and determined log-fold changes after a knockout of USP in a cell line (Ha22T). We found ndiff = 478 proteins differentially expressed. The investigation of the regulatory effect of USP15 on single individual proteins may not lead to a conclusive picture of its role in HCC. We decided to explore further the function of USP15 not in terms of regulatory effects on individual proteins but in terms of regulatory effects on pathway hierarchies. We scored the 2443 human pathway hierarchies and selected 225 pathway hierarchies that were simultaneously relevant for liver cancer genes and differentially expressed proteins. Note that USP15 may contribute to multiple cellular functions, of which a majority may be irrelevant for tumor progression in HCC. We found functions "cell proliferation" and "cell migration" significantly enriched in the 225 pathway hierarchies that were predicted to be relevant for the role of USP15 in HCC. Note that the relevance of the functions "cell proliferation" and "cell migration" was already indicated by our cell line experiments. Compared to the gene regulation of individual proteins, pathway hierarchies may offer a higher level of viewpoint that can be helpful to understanding the regulation of tumorigenesis. We found the 255 relevant pathway hierarchies enriched in 8 of the 28 clusters, i.e., connected components, of the network of all human pathway hierarchies. Six of the clusters can easily be related to cancer, see Table 3 and Figure 7. Prominent examples were the clusters for signal transduction and gene expression. The cluster signal transduction contained 69 out of 163 proliferation pathways and 35 out of 75 migration pathways, see Table S5 in the supplemental material. The cluster gene expression contained 22 proliferation pathways. The enrichment of these two clusters indicated that USP15 may regulate the two clusters' signal transduction and gene expression. By such a regulation, USP15 may influence cell proliferation and cell migration in tumorigenesis of HCC. The cluster cell cycle contains cell cycling checkpoints which play an important role in cancer . The cluster DNA repair can be related to cancer progression because the accelerated replication of cancer cells induces so-called replication stress . Replication stress in combination with an impaired DNA repair has been associated with cellular senescence and barriers to malignant progression . The role of ubiquitination in DNA damage response has been previously reported by Ramadan and Dikic . Peng et al. reported the involvement of USP15 in regulation of DNA-homologous recombination repair . In particular, Peng et al. demonstrated the recruitment of USP15 to DNA double-strand breaks (DSBs) by the mediator of DNA damage checkpoint protein 1 (MDC1). Recruited to DSBs, USP15 can be deubiquitinated and demobilize a heterodimer of the BRCA1-associated RING domain protein 1 (BARD1) and the BRCA1 C Terminus (BRCT). Note that binding of heterodimer BARD1/BRCA1 to the damaged DNA prevents transcription and restores genetic stability . The independent findings by other groups of a regulatory role of USP15 in DNA repair supports the meaningfulness of our approach. The cluster cellular responses to external stimuli included pathways such as cellular response to heat stress, heat shock factor 1 (HSF1)-dependent transactivation, regulation of HSF1-mediated heat shock response, and HSF1 activation and attenuation phase, see Figure S7B in the supplemental material. HSF1 has been reported to function as a negative regulator of non-homologous end joining (NHEJ) in DNA repair activity . Note that abnormal NHEJ plays an important role in tumorigenesis . HSF1 has been reported to be involved in stress-induced cancer cell proliferation via IER5 . The cluster cellular responses to external stimuli included additional pathways that are cancer-related, such as cellular senescence, DNA damage/telomere stress-induced senescence, and formation of senescence-associated heterochromatin foci, see Figure S7B in the supplemental material. Among the involved proteins were tumor protein P53 (TP53) and retinoblastoma protein (RB1), both of which may act as transcription factors or as a tumor suppressor. TP53 can bind to the promoter of cyclin-dependent kinase inhibitor 1 (CDKN1A) and may trigger transcription . CDKN1A inhibits the activity of cell division protein kinase 2 (CDK2) and leads to an arrest of cell cycle at G1/S phase . In chromatin organization, USP15-related cancer genes enriched in PKMTs and RMTs. Lysine methyltransferases (KMTs) and arginine methyltransferases (RMTs) regulate the Methylation levels of histone by adding methyl groups to histone, which play roles in regulating digestive cancer, including liver cancer . H3K9me2 in the promoter region of RARRES3 gene suppresses its transcription and promotes cancer cell migration in HCC . H3K27me3 enriched in the Kruppel-like factor 2 (KLF2) promoter leads to the tumor cell population in GC and CRC . The decreased H3K27me3 in the promoter of SLUG activates the transcription and promotes migration and invasion in HCC . All this evidence clearly showed that chromatin organization strongly relates to HCC and indicated that USP15 regulates HCC via histone methylation level. To describe phenotypes, we found pathway hierarchies more suitable than a single gene or a small set of genes. Pathway clusters offered a coarser-grained--but still meaningful--viewpoint on regulation. Pathway clusters could be linked phenotypes and helped to understand the relations between USP15 and phenotypes of its regulatory function in HCC. 5. Conclusions We found high level of USP15 expression to be significantly correlated with high survival rate in HCC patients. Relevant cellular phenotypes were slow proliferation and slow migration. We confirmed the role of USP15 as a tumor suppressor in a mouse model. To link the experimentally observed phenotypes to molecular function of USP15 we constructed a protein-protein interaction network. We found the regulatory function of USP15 to be mediated by indirect interactions. Indirect interactions may enable USP15 to regulate thousands of other proteins. The phenotype of complex regulation is hard to predict. We proposed and tested a theoretical approach based on pathway hierarchies. The viewpoint of pathway hierarchies offered a meaningful coarse-grained viewpoint on the regulatory function of USP15. We were able to link our experimentally observed phenotypes to clusters of pathways. Furthermore, in other applications, clusters of pathways may be an appropriate viewpoint to understand the regulatory function of proteins and may offer a starting point to select molecular mechanisms for further experimental investigation. Supplementary Materials The following supporting information can be downloaded at: Figure S1: Reference standards of the classification of tissue; Figure S2: Wound healing assay for cell line SMMC-7721; Figure S3: Wound healing assay for cell line QGY-7703; Figure S4: Overexpression efficiency of the plasmids for PF-USP15 Huh1 and HA22T; Figure S5: (A) Flow cytometery for HA22T SG-control (empty vector transfected into the cells). (B) Flow cytometery for HA22T with USP15 knockout. (C) Flow cytometery for Huh1 SG-control (empty vector transfected into the cells). (D) Flow cytometery for Huh1 with USP15 knockout. (E) Flow cytometery for HA22T OE-control (empty vector transfected into the cells). (F) Flow cytometery for HA22T with USP15 overexpression. (G) Flow cytometery for Huh1 OE-control (empty vector transfected into the cells). (H) Flow cytometery for Huh1 with USP15 overexpression; Figure S6: Global view of the hierarchy relationships of whole human pathways; Figure S7: Original Western blot images; Table S1: 295 HCC related genes; Table S2: 143 USP15-HCC genes; Table S3: 225 HCC-USP15 pathways; Table S4: Pathology scores; Tables S5: Pathways of lipid, proliferation, and migration. Click here for additional data file. Author Contributions Y.R. collected the clinical samples, performed the lab experiments, built the mouse model, collected the experimental data, and drafted the method of the experiment of the manuscript. Z.S. conceived the project performed the bioinformatics analysis, organized the experimental and bioinformatics data, and drafted the manuscript. J.R. wrote the python script for this project. J.A. analyzed the expression profile and revised the manuscript extensively. X.L. cultured the cells. T.J. and X.C. supervised the experimental parts of the project. I.K. supervised the bioinformatics parts of the project. All authors have read and agreed to the published version of the manuscript. Institutional Review Board Statement The study was approved by the Medical Ethics Committee of SRRSH with the number: 20090211-90. Informed Consent Statement Informed consent was obtained from all subjects involved in the study. Data Availability Statement Raw figures were uploaded as manuscript submission. NGS dataset were dropped here: Conflicts of Interest All the authors declared that there is no conflicting interest. Abbreviations AJCC American Joint Committee on Cancer BFS breadth-first search BCLC Barcelona Clinic Liver Cancer CI confidence interval DSBs DNA double-strand breaks EMT epithelial-mesenchymal transition HCC hepatocellular carcinoma IHC immunohistochemical staining KO knockout MET mesenchymal-epithelial transition NHEJ non-homologous end joining NGS next-generation sequencing OE overexpressing PVDF polyvinylidene difluoride PPI protein-protein interaction Ub ubiquitin USPs ubiquitin-specific proteases USP15 ubiquitin carboxyl-terminal hydrolase 15 Figure 2 The expression of USP15 is increased in hepatocellular carcinoma cell lines. Overexpression of USP15 correlates with slow proliferation, slow migration, and slow tumor growth. Knockout of USP15 correlates with fast proliferation and fast migration. (A) Western blot of USP15 protein expression in cell lines LM3, PLC/PRF/5, HLF, Hep-G2, Huh1, HA22T, and Huh7. Glyceraldehyde 3-phosphate dehydrogenase (GAPGH) is the reference. (B) Bar chart of the normalized expression of USP15. We applied GAPGH to normalize the intensity values. HCC cell lines Huh1, HA22T, and Huh7 have the highest expression levels of USP15. (C) The proliferation index (CCK8 assay) of transfected cell line Huh1-P-F-USP15 (red line) versus control Huh1-PLVX (green line), see text. Overexpressing cells, Huh1-P-F-USP15, had a decreased proliferation index (p values at 5 time points all less than 0.0001, two-tailed t-test). (D) The proliferation index (CCK8 assay) of transfected cells line HA22T-P-F-USP15 (red line) versus control HA22T-PLVX (green line), see text. Overexpressing cells, HA22T-P-F-USP15, had a decreased proliferation index (p values at 48 h, 60 h and 72 h are less than 0.05, two-tailed t-test). (E) Images of migration assays (Transwell) for cell lines Huh1-PLVX (left images) and Huh1-P-F-USP15 (right images). Images are shown in two magnifications, 40x (top images) and 100x (bottom images). (F) Counts of migratory cells for transfected cell line Huh1=P-F-USP15 (right bar) versus control Huh1-PLVX (left bar), see text. Overexpressing cells, Huh1=P-F-USP15, had a decreased number of migratory cells (p < 0.01, two-tailed t-test). (G) Images of migration assays (Transwell) for cell lines HA22T-PLVX (left images) and HA22T-P-F-USP15 (right images). Images are shown in two magnifications, 40x (top images) and 100x (bottom images). (H) Counts of migratory cells for transfected cell line HA22T-P-F-USP15 (right bar) versus control HA22T-PLVX (left bar), see text. Overexpressing cells, HA22T-P-F-USP15, had a decreased number of migratory cells (p < 0.0001, two-tailed t-test). (I) Western blot of USP15 protein expression in control cell line, Huh1-sgControl, and knockout cell line, Huh1-sgUSP15, see text. Knockout of USP15 expression by CRISPR/Cas9 is visible for Huh1-sgUSP15 versus control, Huh1-sgControl. (J) The proliferation index (CCK8 assay) of control cell line Huh1-sgControl (green line) and knockout cell line Huh1-sgUSP15 (red line), see text. Knockout of USP15 expression in cells, Huh1-sgUSP15, led to an increased proliferation index (p value at 48 h, 72 h and 96 h are less than 0.0001, two-tailed t-test). (K) Images of migration assays (Transwell) for knockout cell line Huh1-sgUSP15 (right images) and control cell lineHuh1-sgControl (left images). Images are shown in two magnifications, 40x (top images) and 100x (bottom images). (L) Counts of migratory cells for knock cell line Huh1-sgUSP15 (right bar) versus control Huh1-sgControl (left bar), see text. Knockout cells, Huh1-sgUSP15, had an increased number of migratory cells (p < 0.01, two-tailed t-test). (M) Western blot of USP15 protein expression in control cell line, HA22T-sgControl, and knockout cell line, HA22T-sgUSP15, see text. Effect of knockout of USP15 expression by CRISPR/Cas9 is visible for HA22T-sgUSP15 versus control, Huh1-sgControl. (N) The proliferation index (CCK8 assay) of control cell line HA22T-sgControl (green line) and knockout cell line HA22T-sgUSP15 (red line), see text. Knockout of USP15 expression in cells, HA22T-sgUSP15, led to an increased proliferation index (p value at 72 h less than 0.05, two-tailed t-test). (O) Images of migration assays (Transwell) for knockout cell line HA22T-sgUSP15 (right images) and control cell line HA22T-sgControl (left images). Images are shown in two magnifications, 40x (top images) and 100x (bottom images). (P) Counts of migratory cells for knock cell line HA22T-sgUSP15 (right bar) versus control HA22T-sgControl (left bar), see text. Knockout cells, HA22T-sgUSP15, had an increased number of migratory cells (p < 0.01, two-tailed t-test). (Q) Images of tumor growth in an animal model of nude mice. A control group, upper row, was injected by control cells, Huh7-PLVX. A second group, lower row, was injected by transfected cells, Huh7-P-F-USP15, that overexpressed USP15. Tumors were excised 25 days after injection. (R) Two exemplary images of immunochemically staining (dye: 3,3'-diaminobenzidine, DAB) of tumor tissue of nude mice. Upper image shows low yellow staining for USP15 of control cells, Huh7-PLVX. Lower image shows strong yellow staining for USP15 of transfected cells, Huh7-P-F-USP15. H&E counterstaining colors cell nuclear in blue-purple. (S) Box plots of tumor volumes for two HCC cell lines, Huh7-PLVX (control) and Huh7-P-F-USP15 (transfected). Tumors of transfected cells, Huh7-P-F-USP15 (lower row), have smaller dimensions than tumors of the control group, Huh7-PLVX (p < 0.01, two-tailed t-test). OE--overexpressing. (T) Box plots of tumor weights for two HCC cell lines, Huh7-PLVX (control) and Huh7-P-F-USP15 (transfected). Tumors of transfected cells, Huh7-P-F-USP15 (lower row), have less weight than tumors of the control group, Huh7-PLVX. *: p < 0.05; **: p < 0.01; ****: p < 0.0001. Figure 3 Human USP15 PPI network, a network of proteins that interact directly or indirectly with USP15. The role of eight experimentally accessible proteins. (A) Layer 1 proteins interact with USP15, and layer 2 proteins interact with the layer 1 proteins. (B) Visualization (circle layout, Cytoscape) of interactions of 105 proteins of layer 1 (outer circle) with USP15 (center node). (C) Visualization (circle layout, Cytoscape) of indirect interaction of 6175 proteins of layer 2 (outer circle) with USP15 (center). (D) Proteins of layer 2 are linked to USP15 via a protein of layer 1 (inner circle). (E) Choice of ten proteins that are tumor-related and experimentally accessible. (F) Visualization (circle layout, Cytoscape) of indirect interaction of eight preselected proteins (highlighted in yellow, outer circle) with USP15 (center node). (G) Two of the ten preselected proteins were not members of the Human USP15 PPI network (not highlighted in yellow). Figure 4 Tumor-related proteins can be regulated by USP15 via interactions mediated by other proteins. (A) Western blots of protein IKBa and phosphorylated proteins p-Nrf2 and p-AKT in cell lines Huh1-P-F_USP15 (overexpressing USP15) and Huh1-PLVX (control). (B) Western blots of protein b-catenin and phosphorylated proteins, p-Smad2 and p-Erk in cell lines Huh1-P-F_USP15 (overexpressing USP15) and Huh1-PLVX (control). (C) Western blots of proteins IKBa and phosphorylated proteins p-Nrf2, p-Rb1, and p-AKT in cell lines Huh1-sgUSP15 (knockout USP15) and Huh1-sgControl (control). (D) Western blots of proteins C-myc and b-catenin in cell lines Huh1-sgUSP15 (knockout USP15) and Huh1-sgControl (control). (E) Western blots of proteins N-cadherin in cell lines Huh1-P-F_USP15 (overexpressing USP15) and Huh1-sgUSP15 (knockout USP15) versus control. (F) Western blots of proteins SQSTM1 in cell lines Huh1-P-F_USP15 (overexpressing USP15) and Huh1-sgUSP15 (knockout USP15) versus control. (G) Bar plot of intensities, normalized to ss-actin, with (Huh1-P-F_USP15) and without (Huh1-PLVX) overexpression of USP15. Only differentially expressed proteins are shown. (H) Bar plot of intensities, normalized to ss-actin, with (Huh1-sgUSP15) and without (Huh1-sgControl) knockout of USP15. Only differentially expressed proteins are shown. (I) List of investigated proteins. Interaction partners of USP in the human USP PPI network are regulated by USP15 and highlighted in green. Nrf2 and N-caderin are not members of the human USP PPI network. Whereas Nrf2 (highlighted yellow) is regulated by USP15, N-caderin is not. (J) Western blots of downstream proteins of USP15 in tumor samples. The left side is the tumor of HCC cell lines, Huh7-PLVX, which is used as control. The right side is the tumor of Huh7-P-F-USP15 (K). Band densitometry intensity ration of the Western blot in picture (J). **: p < 0.01; ***: p < 0.001. Figure 5 Regulatory role of USP15 in HCC: pathway hierarchies. (A) Sub-network of the human USP15 PPI network. A total of 143 liver cancer genes (highlighted in yellow) interact directly or indirectly with USP15. (B) Volcano map of the effect of knockout of USP15 in cell line HAT22T. The log-fold change of 14,099 identified genes in cell line HA22T sgUSP15 (knockout USP15) versus cell line HA22T sg-control as control, see material and methods. A fraction of 1.4% (203 proteins, highlighted in blue) showed increased expression levels (false discovery rate < 0.1, log-fold change > 1). A fraction of 2.0% (278 proteins, highlighted in red) showed decreased expression (false discovery rate < 0.1, log-fold change < -1). (C). Network of all human pathway hierarchies. Nodes with high score s are highlighted in dark blue and may contribute to the regulatory role of USP15 in HCC, see text. Figure 6 USP15 may repress HCC via genes in cell proliferation and cell migration. HCC-USP15 pathways: network of pathway hierarchies that may contribute to the regulatory role of USP15 in HCC. (A) A sub-network of the human pathway hierarchies. Each node has a score s > 20 and hence may contribute to the regulatory role of USP15 in HCC, see text. (B) Pathway hierarchies associated to the function "cell proliferation" are highlighted in yellow. The function "cell proliferation" is significantly enriched in the network (p =2.5 x 10-7, hypergeometric test). (C) Pathway hierarchies associated to the function "cell migration" are highlighted in yellow. The function "cell migration" is significantly enriched in the network (p = 0.0077, hypergeometric test). (D) Pathway hierarchies associated to the function "lipid processes" are highlighted in yellow. The function "lipid processes" is significantly underrepresented in the network (p = 0.035359066, hypergeometric test). Figure 7 USP15 may repress HCC via six pathway clusters. USP15-HCC pathways are significantly enriched in six cancer-related clusters of the complete network of human pathway hierarchies. (A) Degree of enrichment (as a percentage) of the six cancer-related clusters. (B) Cluster "Cellular responses to external stimuli". (C) Cluster "Cell Cycle". (D) Cluster "DNA repair". (E) Cluster "Gene expression". (F) Cluster "Chromatin organization". (G) Cluster "Signal transduction". UP15-HCC pathways are highlighted in yellow. cancers-15-01371-t001_Table 1 Table 1 Clinical and pathological characteristics of the patient cohort of 102 patients suffering from HCC. A tissue sample of each patient was immunohistochemically stained (IHC) for USP15. By visual inspection, two pathologists classified the stained tissue into low or high expression, see text. All p-values (two-tail Fisher's exact test ) are above 0.05, and hence, no significance differences of USP15 expression exist in the patient subgroups of clinical characteristics, e.g., age (young/old) and gender (male/female). Alpha-fetoprotein--AFP; hepatitis B surface antigen--HBsAg. Total USP15 Expression p-Value Low (%) High (%) Age <53 47 37.25 8.83 0.254 >=53 55 37.25 16.67 Gender Male 85 63.73 19.61 0.363 Female 17 10.78 5.88 AFP (ng/mL) <400 63 45.10 16.67 0.816 >=400 39 29.41 8.82 HBsAg Negative 16 12.75 2.94 0.755 Positive 86 61.76 22.55 Tumor size <5 cm 61 44.12 15.69 1.0 <5 cm 41 30.39 9.80 >=5 cm Tumor number Single 89 63.73 23.53 0.507 Multiple 13 10.78 1.96 Liver cirrhosis No 45 29.41 14.71 0.116 Yes 57 45.10 10.78 cancers-15-01371-t002_Table 2 Table 2 Sequence of primers. F--forward; R--reverse. Name Sequences (5'-3') SgRNA-1 F: 5'-CACCGCGTCGCGATGTCAGACCGC-3' SgRNA-1 R: 5'-AAACGCGGTCTGACATCGCGACGC-3' EcoRI-USP15 F: 5'-ATTACGCTGAATTCATGGCGGAAGGCGGAGCGGCGGAT-3' BamHI-USP15 R: 5'-CGACTCTAGAGGATCCTA TTAGTTAGTGTGCATACAGT-3' cancers-15-01371-t003_Table 3 Table 3 The 28 connected components of the complete network of human pathway hierarchies. HCC-USP15 pathways were significantly enriched in eight connected components, so-called clusters. We denoted six of the enriched clusters (in bold) as relevant to tumor research, highlighted in boldface. Two clusters, "Reproduction" and "Circadian Clock" (in italic), were significantly enriched but not denoted as cancer-related. #: numbers. Cluster #Nodes Hemostasis 39 Intrinsic pathway for Apoptosis 20 Neuronal System 78 Developmental Biology 59 Metabolism 325 Reproduction 8 Extracellular matrix organization 19 Cell-Cell communication 13 Signal Transduction 382 Cell Cycle 122 Disease 517 Immune System 197 Organelle biogenesis and maintenance 13 Transport of small molecules 69 Metabolism of proteins 124 Muscle contraction 11 Circadian Clock 4 Chromatin organization 7 Programmed Cell Death 21 Vesicle-mediated transport 38 DNA Replication 19 DNA Repair 61 Gene expression (Transcription) 125 Metabolism of RNA 51 Cellular responses to external stimuli 24 Digestion and absorption 7 Protein localization 6 Autophagy 10 Disclaimer/Publisher's Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). 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PMC10000202

The Fonni's dog is an ancient Sardinian breed for livestock and property guarding. In recent years, the number of new registrations to the breeding book has slumped and, thus, this breed risks being lost forever. This work refocuses attention to the Fonni's dog, analysing its genomic makeup and comparing different phenotypical and genetic evaluation scores. Thirty Fonni's dogs were ranked by their general accordance to the breed typicality (typicality score) and to the provisional standard by official judges (judges' score). They were genotyped with a 230K SNP BeadChip and compared with 379 dogs of 24 breeds. Genomically, the Fonni's dogs placed themselves near shepherd dogs and showed a unique genetic signature, which was used to create the genomic score. This score better correlated with typicality (r = 0.69, p < 0.0001) than the judges' score (r = 0.63, p = 0.0004), which showed little variability among the included dogs. Hair texture or colour were significantly associated in the three scores. The Fonni's dog is confirmed as a well-distinguished breed, despite being selected mainly for its work abilities. The evaluation criteria used during dog expositions can be improved to increase their variability and include elements typical of the breed. The recovery of the Fonni's dog would be possible only with a shared vision between the Italian kennel club and breeders, and the support of regional programs. Italian dog breed SNPs dog breed conservation This research received no external funding. pmc1. Introduction The Fonni's dog is an autochthonous Italian dog breed that originated in Sardinia in ancient times. Many testimonies come from XIX-century authors . All of them described peculiar local dogs used as property and livestock guardians, but also for hunting hares and boars. They accented their absolute loyalty to their owner, contrasting with their fierce hostility towards strangers, as well as their strength, speed, and good health. The appearance of these dogs was considered unpleasant and coarse, even grim, due to their surly eyes and sharp fangs. However, the origins of Fonni's dogs are much more ancient . It is believed that they were created through the cross between sighthounds and molossers . This hypothesis has been recently supported by a genomic study that revealed that coursing hounds such as Salukis or Pharaoh hounds, as well as molossoid livestock guardians such as Komondors or Neapolitan mastiffs, are plausible ancestors of the Fonni's dog and probably arrived in Sardinia following the peoples from the eastern Mediterranean and North African regions who then populated the isle . Certainly, a large and tail-less dog was already present in Sardinia before the advent of the Romans, but the most ancient evidence of the presence of dogs resembling the modern Fonni's dogs dates back to the Bronze Age (XIX century B.C.-II century A.D.) . The selection of the Fonni's dog has been long based solely on their aptitude for work and behavioural characteristics: they have been long used as livestock and property guardians, which enhanced their strong protectiveness towards their territory and owners. On the other hand, since an appearance-based selection has never been a priority, to this day, this breed shows considerable morphologic variability , although some commonalities can be found, such as the characteristic "monkey face": the oval amber eyes, set in front and close one to each other, together with strongly developed eyebrows, conferring to these dogs a very intense, sad, and authoritative expression. The coat is usually double, with a course outer coat (defined as "goat" hair) and a woolly undercoat, but a short-haired variety exists. Moreover, some dogs are born with a natural bobtail . What is remarkable is that, despite this variability and the absence of a strong selection toward a defined standard, several authors reported that, from a genetic perspective, the Fonni's dog meets all the requirements to be considered a full-fledged breed . It is likely that both a certain degree of geographical isolation and the incredible support of the Fonni's dogs to the prosperity of early Sardinian people contributed to the conservation of this breed through the centuries. At present, the Fonni's dog is in the process of being recognised as a breed; in 2013, it obtained, by the National Agency of the Italian kennel club (ENCI), a specific breeding book (Open Additional Register, RSA) aiming to conserve endangered local dog populations, and a provisional breed standard has been deposited . However, ENCI's data show that, while during the first years of RSA, about 90 dogs--including both founders, enrolled if judged as compelling with the standard by an expertise committee, and their offspring--were annually registered, in the last few years, the breed has seen a slump in registrations to the breeding book, reaching the dramatically low number of 16 subjects enrolled in 2020 (www.enci.it/libro-genealogico/razze/cane-fonnese, accessed on 20 December 2022). At least in part, this was probably due to the disappointment of breeders, who do not feel adequately represented by the morphometric preferences and the subjects' evaluations proposed by ENCI. In light of the above, it is of paramount importance to bring the attention back to the Fonni's dog breed, which otherwise would seriously risk being lost forever, despite the efforts of ENCI. Today, we dispose several genomic tools that can be of help in the management of animal populations; genetics has long been applied to livestock selection, but its use is still limited in pet breeding. However, especially when we consider small, local breeds, the implementation of a genomic evaluation would contribute to their conservation; not only it can be used to choose the best mating schemes and preserve genetic diversity, but it could accompany the traditional phenotypical judgement to identify the most idoneous breeding dogs. Therefore, the aims of the present study are (i) to investigate the genomic makeup of the Fonni's dog with respect to other breeds; (ii) to compare three different evaluation scores, both phenotypical and genetic; and (iii) to identify genomic regions differentiating Fonni's dogs with different physical features. 2. Materials and Methods 2.1. Phenotypical Evaluation In the present study, 30 Fonni's dogs were photographed and judged by breed experts. Enrolled dogs were born between 2007 and 2018. Data were collected about hair colour and texture (goat, woolly, or short), eye colour, tail presence, and type of bite (scissor, pincer, overshot, or undershot). Head (muzzle length, muzzle width, and stop angle), limbs (shoulder and leg angles, and anterior and posterior stance), chest (topline, length, and depth of chest), coat texture, size, height at withers, bone, responsiveness, and predatory behaviour were evaluated using a 20-point rating scale, where 10 corresponded to a perfect accordance with the standard . Each dog was assigned a score according to the % deviation from the perfection ('judges' score'). Moreover, a local researcher with a deep knowledge of the breed ranked the Fonni's dogs according to their general appearance and compliance with the breed typicity (0-100 points 'typicality score'). In this case, the subjects were evaluated as a whole, with particular emphasis given to the typical traits of the breed. The so-called "monkey face" represents a very peculiar element; therefore, great importance was given to the head and its proportions, as well as eye colour, shape, and position. Among the other elements carefully evaluated were also the coat texture and colour. An example of a Fonni's dog is reported in Figure 1. 2.2. Genomic Study Blood samples of the aforementioned 23 unrelated Fonni's dogs were collected in accordance with the Ethics Committee's statement of the University of Messina, number 040/2020bis. DNA was extracted using DNeasy Blood and Tissue Kit (QIAGEN(r), Hilden, Germany) following the manufacturer's instructions and then genotyped via outsourcing with Canine 230K SNP BeadChips (Illumina(r), San Diego, CA, USA). This dataset was merged with publicly available SNP chip data of 7 Fonni's dogs and 379 dogs from 24 Italian and foreign breeds that could be historically or phenotypically related to Fonni's dogs and have been indicated by Sardinian veterinarians as the most diffuse in Sardinia, especially for hunting activities: Pastore Apuano (APUA, n = 19), Bergamasco Shepherd (BERG, n = 15), Bracco Italiano (BRAC, n = 12), Cane Corso (CCIT, n = 22), Cirneco dell'Etna (CIRN, n = 24), Pastore d'Oropa (DORO, n = 15), English Setter (ESET, n = 15), German Shepherd dog (GSD, n = 10), German Shorthaired Pointer (GSHP, n = 10), German Wirehaired Pointer (GWHP, n = 2), Italian Greyhound (IGIT, n = 20), Irish Setter (ISET, n = 9), Lagotto Romagnolo (LAGO, n = 24), Lupino del Gigante (LUGI, n = 23), Lupo Italiano (LUPO, n = 24), Mannara's dog (MANN, n = 12), Maremma and the Abruzzi Sheepdog (MARM, n = 20), Mastiff (MAST, n = 10), Neapolitan Mastiff (NMIT, n = 12), Pastore della Lessinia e del Lagorai (PALA, n = 10), Pastore della Sila (SILA, n = 14), Segugio Italiano a Pelo Forte (SIPF, n = 16), Segugio Italiano a Pelo Raso (SIPR, n = 16), and Spinone Italiano (SPIN, n = 24). A quality control was performed on the genomic data using PLINK 1.9 : only dogs with missingness per individual < 0.1 and SNPs with missingness per marker < 0.05 and minor allele frequency > 0.01 were retained. Related dogs were excluded. The genetic structure of the selected populations was represented with a multidimensional scaling (MDS) analysis, performed with PLINK 1.9. Using ADMIXTURE 1.3 , the individual genetic admixture was investigated for a number of clusters (K) ranging from 2 to 28. For each dog, the probability of being assigned to the different clusters (Q-score) was calculated. The best-fitting model was identified as the one with the lowest cross-validation value (cv-value); according with this model's Q-score of their own cluster, a 'genomic score' was attributed to all the Fonni's dogs. The studied Fonni's dogs were grouped according to the following phenotypical characteristics: (i) Type of bite (n. 24 scissor bite vs. n. 5 pincer bite); (ii) Tail presence (n. 18 brachyure vs. n. 12 tailed); (iii) Hair length (n. 25 longhaired vs. n. 5 shorthaired); and (iv) Hair texture (n. 17 "goat" hair vs. n. 8 woolly hair). Each pair of groups was compared using two genomic analyses: the Wright's fixation index (FST), using PLINK 1.9, and the single SNP cross-population extended haplotype homozygosity (XP-EHH), using SELSCAN 1.1.0 . These analyses identify genomic regions differentiating two groups of individuals according to the amount of differentiation in their allele frequency and the haplotype lengths at each marker , respectively. The SNPs ranking in the top 1% of the empirical distribution of both the analyses were considered relevant and mapped to the reference genome assembly CanFam3.1. The genes containing these relevant markers were further investigated. 2.3. Statistical Analysis Statistical analyses were performed using JMP(r) 15 (SAS Institute Inc., Cary, NC, USA). Descriptive statistics were generated. Pearson correlation coefficient (r) was calculated between the three different scores (genomic, judges', and typicality scores). One-way ANOVA and Chi-squared test were used to compare continuous and categorical variables in different groups, respectively. p-values were considered significant if <0.05. 3. Results 3.1. Genomic Analysis The dataset obtained after the quality check and the exclusion of related subjects consisted of 120853 SNPs and 378 dogs: 30 Fonni's dogs, 18 Pastore Apuano (APUA), 11 Bergamasco shepherds (BERG), 12 Bracco Italiano (BRAC), 20 Cane Corso Italiano (CCIT), 21 Cirneco dell'Etna (CIRN), 15 Pastore D'Oropa (DORO), 15 English Setters (ESET), 10 German Shepherd dogs (GSD), 10 German Shorthaired Pointers (GSHP), 2 German Wirehaired Pointers (GWHP), 14 Italian Greyhounds (IGIT), 9 Irish Setters (ISET), 23 Lagotto Romagnolo (LAGO), 18 Lupino del Gigante (LUGI), 23 Lupo Italiano (LUPO), 12 Mannara dogs (MANN), 16 Maremma and the Abruzzi Sheepdogs (MARM), 10 Mastiffs (MAST), 10 Neapolitan Mastiffs (NMIT), 10 Pastore della Lessinia e del Lagorai (PALA), 14 Pastore della Sila (SILA), 16 Segugio Italiano a Pelo Forte (SIPF), 16 Segugio Italiano a Pelo Raso (SIPR), and 23 Spinone Italiano (SPIN). A representation of the first two components of the MDS analysis is shown in Figure 2. In this plot, LUPO and GSD are both isolated from the other breeds; along the vertical axis, the molossers (MAST, CCIT, and NMIT) are well identifiable and are located close together, far from the other breeds, as well as the hunting dogs (ESET, BRAC, SPIN, ISET, GSHP, GWHP, SIPR, SIPF, and CIRN). Regarding shepherd dogs, a partial differentiation between livestock guardians (SILA, MANN, MARM, and Fonni's dogs) and herding dogs (LUGI, PALA, APUA, DORO, and BERG) is appreciable. At K = 2, the admixture analysis separated LUPO from all the other breeds; at K = 3, the molossers could be identified by a common signature; then, all the following clusters identified single breeds, one at a time. The best-fitting admixture model was identified at K = 18 . In this model, BRAC, ESET, GSD, IGIT, ISET, LAGO, LUPO, MAST, and NMIT were the breeds displaying the least introgression. Fonni's dog showed a unique genetic signature and the dogs included in this study had, on average (+-SD), a Q-score related to their own cluster of 61.7 +- 22.0%, ranging from 19.2 to 100%. In particular, seven subjects were comprised in the fourth quartile (>75%), eleven in the third (50-75%), ten in the second (25-50%, but eight dogs were over 40%), and one in the first (<25%, Table S1). The second most represented clusters, accounting for at least 5% of the Fonni's dogs' genomic background, were: MARM (n = 12, 10.7 +- 0.03%), SIPF/SIPR (n = 5, 9.5 +- 0.03%), NMIT/CCIT (n = 2, 13.4 +- 0.01%), and MAST (n = 1, 9.8%). MARM was also highly represented in the background of other shepherd dogs: MANN, PALA, and SILA. The results of the comparison between groups of Fonni's dogs with different phenotypical characteristics are shown in Table 1. In particular, 23 different genes harboured SNPs in the top 1% of the distribution of both FST (0.16-0.46) and XP-EHH (2.7-4.9) analyses comparing scissor bite and pincer bite groups. Similarly, 21 genes differentiated brachyure and tailed dogs (FST: 0.10-0.39; XP-EHH: 2.7-4.6); 23 genes differentiated longhaired and shorthaired dogs (FST: 0.22-0.53; XP-EHH: 2.9-6.6); and 13 genes differentiated dogs with "goat" hair and woolly hair (FST: 0.15-0.52; XP-EHH: 2.8-5.8). 3.2. Phenotypic Description Of the 30 studied Fonni's dogs, 12 (40%) had a normal tail and 18 (60%) were brachyure. The majority of dogs had a scissor bite (80%) or a pincer bite (17%), both accepted by the breed standard; only one dog (0.3%) had an overshot bite. Grey, in different shades, was the most represented coat colour (50%), followed by brindled (27%) and black (17%); only one subject was found for honey and white colours. Moreover, 63% of dogs did not display white spotting. Most of the dogs had the typical "goat" hair (57%), whereas in eight, the coat was considered "woolly" (27%); the short-coated variety was also well represented (17%). The phenotypic evaluation performed by breed experts was available for 29 out of the 30 studied dogs. Many of the evaluated parameters showed small variability: mean (+-SD) coefficient of variation (CV) was 6.27 +- 3.98%. One parameter (shoulder angle) had a CV = 0; the parameter with the greatest variability was hair texture (CV = 14.1%). On average (+-SD), the judges' score was of 97.6 +- 3.1%; 11 dogs (41%) were evaluated as perfectly in standard (0 faults) and only two dogs had a relatively low score, equal to 86.3 and 91.3%. Individual data about the phenotypical characterisation and the three scores are reported in Table S1. We investigated the correlation between the different scores (genomic, judges', and typicality): the genomic score significantly correlated with both typicality (r = 0.69, p < 0.0001) and judges' score (r = 0.63, p = 0.0004); the two phenotypic scores were less, but still significantly, correlated (r = 0.45, p = 0.02). Statistical analyses did not show any significant association between the genomic or the typicality score and white spotting, tail (tailed or brachyure), and bite. On the other hand, a significant association was found in the hair texture category (short, woolly, or goat), with higher values of Q-score (p = 0.005) and typicality (p < 0.0001) for goat-haired dogs, and a worse typicality score for short-haired ones. Typicality score only was significantly associated with coat colour (p = 0.007), with a preference for ash grey, black, and brindled honey dogs and lower values for brindled dogs, and regarding eye colour (p = 0.01), amber was the preferred colour and black was the least favourite. 4. Discussion Fonni's dogs have long been bred in Sardinia for guarding purposes. However, the recent confrontations between breeders and ENCI contributed to the fall in the number of registrations in the breeding book, putting at risk the survival of this breed. In this work, we aimed to renew the attention to this population, also comparing different morphological and genomic scores and indicating possible tools that might help manage local canine breeds. From a genomic perspective, according to the analyses of the Fonni's dog's population structure and genetic background, this breed appears very distinct from other Italian and foreign populations, as already recorded in Talenti et al. (2018) , where 185 breeds were investigated. As expected, the Fonni's dog shares similarities with other livestock guardian dogs, in particular with the Maremma and the Abruzzi sheepdog, which is the most diffuse livestock guardian. Observing in detail the background of the single subjects, we noticed that there is a remarkable degree of variability in the presence of a genetic signature attributable to the Fonni's dog breed. In this regard, these genomic tools might be of great help in the evaluation of these dogs, particularly of those that do not perfectly adhere to the morphological standard, and, thus, in the selection of the founders to register in the breeding book. Comparing this genomic score with that used by ENCI judges during the expositions and a typicality score based on the general appearance of the individuals, we found a strong and positive correlation between all of them, but with the lowest coefficient between the two morphological scores. Generally speaking, the dogs that showed a very high genomic score also presented a high typicality score, but with some exceptions: this could indicate that, despite the presence of clear typical features, a phenotypical variability is still present in the breed. We propose that the dogs showing the highest genomic scores might be subjected to an accurate phenotypical evaluation to identify what the elements that best define them are, always considering both historical documents and the possible preferences of the single breeders, which are reflected in their dogs. With regard to the judges' score, we found that only a very small number of parameters, such as the hair texture, shows variability among the enrolled dogs, whereas the majority were evaluated as perfect for almost all of them. This, together with the fact that the genomic score better correlated with the typicality score rather than the judges' one, could suggest that including other criteria might be advantageous. For example, some typical features of the breed were not evaluated (e.g., presence or absence of the tail) or indicated as open answer (e.g., eye colour), making it difficult to collect data about them in the population. The breed, indeed, shows some peculiar traits, such as frontal amber eyes, hostile expression, and goat hair texture, that should be carefully reported in the evaluations in order to understand their variability in the population and considered in the decision to register a dog into the breeding book. Furthermore, since the selection of these dogs has historically been based on their behaviour and performance in work, it would be appropriate to also include these elements in their evaluation, in order not to lose the guarding attitude of this breed. For example, distrust towards strangers and territoriality are necessary characteristics for guard dogs, and therefore are traits to be rewarded. The second part of our study focused on the identification of selection signatures of Fonni's dogs with different phenotypes, including the bite, the presence of tail, and the hair length and texture. From the comparison of longhaired and shorthaired subjects, the latter composing 17% of the sample, we identified RSPO2, a gene known to be responsible for the presence of furnishing and wire hair in the canine species . Moreover, the evidence of different combination of mutations in RSPO2 and FGF5 associated with the longhair phenotype in several dog breeds suggests an epistatic interaction between the two and, thus, that RSPO2 plays a role in the determination of the hair length in this species . CREM, another gene we identified, is involved in the regulation of AP-1 , a dimer of FOS and JUN that seems to regulate the composition of dog undercoat . In addition, a recent study involving over 20000 dogs reported an association between fur texture and EXT1 and ANGPT1 genes . Consistently, mice harbouring mutations in EXT1 were characterised by densely distributed hair follicles which only presented anagen phase, as well as accelerated hair regrowth . Interestingly, two of the identified genes are, instead, associated with coat colour: USH2A is related to roan and ticked colour in dogs , whereas LYST is known to determine beige, aleutian, and diluted colour in mice, minks, and bovines, respectively , and also to cause Chediak-Higashi syndrome in several species . Among the Fonni's dogs enrolled in the present study, 60% presented a short tail, a feature that is accepted by the provisional breed standard. This phenotype in many, but not all, dog breeds has been associated to a mutation in the T gene, which is homozygous lethal . The comparison of brachyure and normal-tailed Fonni's dogs did not identify it, but the genes that differentiated these two groups included DISC1. This gene acts in the neuroectoderm, regulating somite and tail formation, but its main role regards brain formation. A study on zebrafish showed that DISC1 mutations can also lead to the development of an abnormally shaped and truncated tail . Lastly, we compared Fonni's dogs presenting the two accepted bites: scissor and pincer (17%). Interestingly, we found the COL13A1 gene, which has been shown to regulate bone growth and resorption. An overexpression of COL13A1 can abnormally increase bone formation and, therefore, it is supposed to influence bone modelling, in particular in relation with its mechanical use . The same study also highlighted a probable interaction between COL13A1, IGF-II, and RUNX2, the latter being one of the candidate genes accounting for the differences in muzzle length in dogs and class II malocclusions in humans . 5. Conclusions Our study focused on a local dog breed that, due to management reasons, might be lost in the near future. Indeed, the zootechnical management of the Fonni's dog population, like several other breeds with limited diffusion, sees "livestock keepers" choosing the breeding dogs through a morphological and functional evaluation, often driven by their experience or that of their family. In this context, it frequently occurs that the selected dogs deviate from the morphological criteria of the provisional breed standard provided by ENCI. As a consequence, litters are not regularly registered and cannot obtain a pedigree, not contributing to the official breed statistics. To limit this problem, it is of great importance to solve the disagreements between breeders and the kennel club, for example, improving the role and the responsibility of the breed club and the livestock keepers, which actually deal with the preservation activities over time, and creating accurate mating plans and exchanging of dogs between breeders that share a common vision of the breed. Another suggestion might be to follow the successful example of other breed clubs, including the Mannara dog breed club , establishing a roving commission of ENCI judges who personally visit the farms of the rural areas where the possible founder dogs live, giving the opportunity to increase the total and breeding population size. This direct contact with the breeders would also allow them to collect their concerns about the management and vision of the breed, and thus improve the breed standard like it was a "participatory project", it being, in fact, a dynamic document that should evolve accordingly with the population. Moreover, we propose to implement the use of genomics, which demonstrated to be useful in avoiding excessive inbreeding and in distinguishing the subjects that, from a genomic perspective, should be highly representative of the breed. To leverage these tools, the development of a reference dataset including updated data from the highest number of breeds possible is relevant, thus filling the gaps recorded for the small Italian local populations, such as Fonni's dogs. In our opinion, this would pave a way toward more informed and ethical dog breeding. The process of recovery and conservation of local breeds certainly demands great efforts and time, especially in the peculiar pastoral reality of southern Italy and isles. However, letting a population die out would also mean losing the related history, tradition, and biodiversity, which is not an acceptable option. Acknowledgments The authors thank all those who collaborated in the collection of samples, in particular Elaine Ostrander and all dog breeders and owners. Supplementary Materials The following supporting information can be downloaded at: Table S1: Scores and description for all the enrolled Fonni's dogs. Click here for additional data file. Author Contributions Conceptualisation, P.C., A.B., R.C. and L.L.; formal analysis, M.C. and A.B.; investigation, P.C., A.B. and M.C.; resources, R.C., S.S. and P.C.; data curation, P.C., A.B. and M.C.; writing--original draft preparation, P.C., A.B., M.C. and L.L.; writing--review and editing, all authors; supervision, P.C.; project administration, P.C. All authors have read and agreed to the published version of the manuscript. Institutional Review Board Statement The animal study protocol was approved by the Ethics Committee of the University of Messina (protocol code number 040/2020bis). Informed Consent Statement Informed consent was obtained from all subjects involved in the study. Data Availability Statement Genomic data can be downloaded from the Gene Expression Omnibus database under accession number GSE121027 accessed on 20 December 2022), GSE96736 accessed on 20 December 2022), and GSE90441 accessed on 20 December 2022). Conflicts of Interest The authors declare no conflict of interest. Figure 1 An example of brachyure Fonni's dog. Figure 2 Multidimensional scaling analysis plot showing the first two principal components (PC). Each point represents a subject and each colour a breed: Fonni's dog (FONN), Pastore Apuano (APUA), Bergamasco Shepherd (BERG), Bracco Italiano (BRAC), Cane Corso Italiano (CCIT), Cirneco dell'Etna (CIRN), Pastore D'Oropa (DORO), English Setter (ESET), German Shepherd dog (GSD), German Shorthaired Pointer (GSHP), German Wirehaired Pointer (GWHP), Italian Greyhound (IGIT), Irish Setter (ISET), Lagotto Romagnolo (LAGO), Lupino del Gigante (LUGI), Lupo Italiano (LUPO), Mannara dog (MANN), Maremma and the Abruzzi Sheepdog (MARM), Mastiffs (MAST), Neapolitan Mastiff (NMIT), Pastore della Lessinia e del Lagorai (PALA), Pastore della Sila (SILA), Segugio Italiano a Pelo Forte (SIPF), Segugio Italiano a Pelo Raso (SIPR), and Spinone Italiano (SPIN). Figure 3 Admixture analysis for the best-fitting model. Each bar represents the genetic composition of a subject, according to the Q-value of the 18 clusters (K) included. Breeds included: Fonni's dog (FONN), Pastore Apuano (APUA), Bergamasco Shepherd (BERG), Bracco Italiano (BRAC), Cane Corso Italiano (CCIT), Cirneco dell'Etna (CIRN), Pastore D'Oropa (DORO), English Setter (ESET), German Shepherd dog (GSD), German Shorthaired Pointer (GSHP), German Wirehaired Pointer (GWHP), Italian Greyhound (IGIT), Irish Setter (ISET), Lagotto Romagnolo (LAGO), Lupino del Gigante (LUGI), Lupo Italiano (LUPO), Mannara dog (MANN), Maremma and the Abruzzi Sheepdog (MARM), Mastiffs (MAST), Neapolitan Mastiff (NMIT), Pastore della Lessinia e del Lagorai (PALA), Pastore della Sila (SILA), Segugio Italiano a Pelo Forte (SIPF), Segugio Italiano a Pelo Raso (SIPR), and Spinone Italiano (SPIN). animals-13-00818-t001_Table 1 Table 1 Genes identified by both FST and XP-EHH analyses comparing Fonni's dogs with different phenotypes. Compared Groups Gene Name CFA Start End Complete Name Scissor bite (n = 24) vs. Pincer bite (n = 5) PLXDC2 2 12609638 13056951 Plexin domain-containing 2 COL13A1 4 20838674 20985200 Collagen type XIII alpha 1 chain TYSND1 4 21106873 21132547 Trypsin domain-containing 1 PPA1 4 21132550 21181653 Pyrophosphatase (inorganic) 1 EIF4EBP2 4 21338907 21357830 Eukaryotic translation initiation factor 4E binding protein 2 ADAMTS14 4 21534016 21612585 ADAM metallopeptidase with thrombospondin type 1 motif 14 NUDT13 4 23695064 23711400 Nudix hydrolase 13 ECD 4 23708872 23740243 Ecdysoneless cell cycle regulator USP54 4 24030697 24094273 Ubiquitin specific peptidase 54 DYNC2H1 5 28387793 28727334 Dynein cytoplasmic 2 heavy chain 1 STX8 5 33834271 34087011 Syntaxin 8 SLC9A2 10 40338284 40560356 Solute carrier family 9 member A2 SEMA6A 11 6116854 6177817 Semaphorin 6A CCDC192 11 16794360 16983758 Coiled-coil domain-containing 192 ALLC 17 2197913 2215973 Allantoicase SEMA3C 18 20457610 20635275 Semaphorin 3C EEFSEC 20 2155781 2407022 Eukaryotic elongation factor, selenocysteine-trna-specific DIAPH3 22 15572575 16079207 Diaphanous-related formin 3 PTPRT 24 30000993 30764482 Protein tyrosine phosphatase, receptor type T XKR4 29 6640126 7064029 XK-related 4 CTNND2 34 2521662 3439858 Catenin delta 2 MARCH6 34 3884154 3944135 Membrane-associated ring-CH-type finger 6 STK39 36 12866818 13307782 Serine/threonine kinase 39 Brachyure (n = 18) vs. Tailed (n = 12) ADGB 1 37752823 37915540 Androglobin C19orf81 1 106012709 106306177 Chromosome 19 open reading frame 81 SHANK1 1 106086132 106128643 SH3 and multiple ankyrin repeat domains 1 NEBL 2 11891943 12229221 Nebulette DISC1 4 7520420 7822558 Disrupted in schizophrenia 1 WAPL 4 34219462 34536265 WAPL cohesin release factor DOCK2 4 41778325 42177874 Dedicator of cytokinesis 2 MAD1L1 6 14888955 15236462 MAD1 mitotic arrest deficient like 1 NEGR1 6 74164157 74634072 Neuronal growth regulator 1 LAMA3 7 64405533 64655273 Laminin subunit alpha 3 LONRF1 16 36217906 36257537 LON peptidase N-terminal domain and ring finger 1 NCKAP5 19 35633416 36498816 NCK associated protein 5 ESD 22 4548798 4571383 Esterase D LRCH1 22 4573064 4781189 Leucine rich repeats and calponin homology domain-containing 1 TRPM8 25 45261107 45353285 Transient receptor potential cation channel subfamily M member 8 PITPNB 26 21304452 21369641 Phosphatidylinositol transfer protein beta B4GALT4 33 22902375 22928826 Beta-1,4-galactosyltransferase 4 POGLUT1 33 23122291 23152469 Protein O-glucosyltransferase 1 PARP14 33 25763335 25801465 Poly(ADP-ribose) polymerase family member 14 FARS2 35 5436316 5889563 Phenylalanyl-trna synthetase 2, mitochondrial CCDC148 36 4084608 4342485 Coiled-coil domain containing 148 Longhaired (n = 25) vs. Shorthaired (n = 5) GLIS3 1 92278216 92724554 GLIS family zinc finger 3 CREM 2 1751312 1828948 Camp-responsive element modulator LYST 4 4106784 4294142 Lysosomal trafficking regulator PCNX2 4 6341243 6699442 Pecanex homolog 2 (Drosophila) HK1 4 20449974 20519488 Hexokinase 1 TSPAN15 4 20556860 20607961 Tetraspanin 15 COL13A1 4 20838674 20985200 Collagen type XIII alpha 1 chain SGPL1 4 21666395 21718699 Sphingosine-1-phosphate lyase 1 MCU 4 23309391 23511184 Mitochondrial calcium uniporter ANXA11 4 29250164 29266002 Annexin A11 SH2D4B 4 29651821 29734014 SH2 domain-containing 4B WAPL 4 34219462 34536265 WAPL cohesin release factor RGS6 8 45791392 45975087 Regulator of G-protein signalling 6 OXR1 13 7220958 7581210 Oxidation resistance 1 ABRA 13 7587713 7599641 Actin binding Rho-activating protein ANGPT1 13 8068880 8312114 Angiopoietin 1 RSPO2 13 8610233 8755897 R-spondin 2 EXT1 13 17172851 17456469 Exostosin glycosyltransferase 1 HDAC9 14 32788493 33304272 Histone deacetylase 9 KIAA1549 16 9696592 9834339 KIAA1549 RAD54L2 20 37966800 38084854 RAD54-like 2 (S. Cerevisiae) WSCD2 26 18574098 18691280 WSC domain-containing 2 USH2A 38 11063439 11750372 Usherin "Goat" hair (n = 17) vs. Woolly hair (n = 8) PHLPP1 1 14027635 14233389 PH domain and leucine-rich repeat protein phosphatase 1 RHOJ 8 37802907 37880272 Ras homolog family member J PPP2R5E 8 37947434 38085846 Protein phosphatase 2 regulatory subunit b'epsilon SGPP1 8 38193018 38228404 Sphingosine-1-phosphate phosphatase 1 GALNT16 8 43099310 43187969 Polypeptide N-acetylgalactosaminyltransferase 16 SIPA1L1 8 45110084 45260233 Signal-induced proliferation-associated 1 like 1 CHN2 14 42217570 42513386 Chimerin 2 ZSWIM5 15 15110474 15311001 Zinc finger SWIM-type containing 5 TMTC2 15 24629162 25019828 Transmembrane and tetratricopeptide repeat-containing 2 C2CD3 21 24127598 24407702 C2 calcium-dependent domain-containing 3 DNAJB13 21 24314400 24327205 DnaJ heat shock protein family (Hsp40) member B13 PAAF1 21 24338678 24381829 Proteasomal atpase-associated factor 1 B3GLCT 25 8817641 8938884 Beta 3-glucosyltransferase Disclaimer/Publisher's Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). 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PMC10000203

Much research has been conducted in an attempt to decrease skeletal injuries in athletic horses. The objective of this literature review is to compile the findings of over three decades of research in this area, make practical recommendations, and describe how research can develop over the years. An initial study investigating the role of bioavailable silicon in the diets of horses in race training produced the unexpected finding of decreased bone mineral content of the third metacarpus subsequent to the onset of training. Further studies revealed this decrease to be associated with stall housing eliminating high-speed exercise, leading to disuse osteopenia. Only relatively short sprints (between 50 and 82 m) were necessary to maintain bone strength and as few as one sprint per week provided the needed stimuli. Endurance exercise without speed fails to elicit the same benefits to bone. Proper nutrition is also required for optimal bone health, but without the right exercise, strong bone cannot be maintained. Several pharmaceuticals may have unintended consequences capable of impairing bone health. Many of the factors influencing bone health in horses also exist in humans including a sedentary lifestyle, improper nutrition, and pharmaceutical side-effects. equine horse bone skeleton exercise injury sprint confinement silicon nutrition This review received no external funding. pmc1. Introduction For many decades, the high incidence rate of dorsal metacarpal disease (commonly known as bucked shins ) has been of concern to those in the horse racing industry, with 70% of two-year-old Thoroughbreds developing the problem, as reported by Norwood . This ailment is similar to shin splints which has commonly plagued human athletes, and, in particular, runners . In 1989, as an exercise rider of racing Thoroughbreds, the author often engaged in conversations with the trainer for whom he rode about the latest research into how to prevent bucked shins. At that time, there was limited research into how equine bone responds to training, with some of the most prominent work being presented by Dr. David Nunamaker of the New Bolton Center. His research provided some guidance and understanding on how training techniques affect this problem . In October of 1990, during the running of the Breeders' Cup Distaff, the catastrophic breakdown of the filly "Go For Wand" in front of the grandstands at Belmont Park in New York emphasized the need for greater research into preventing bone-related injuries . Coincidentally, that same month, the author had commenced research for his graduate work at Texas A&M University focused on trying to find ways to strengthen bone and prevent skeletal injuries in performance horses. The following is a review of over 30 years of that research. Besides demonstrating how research evolves and how one project can lead to another, the three decades of research provides recommendations, supported by science, in how to decrease injuries in athletic horses, with implications for humans as well. 2. Bioavailable Silicon The initial project began with the examination as to whether supplementing a bioavailable source of silicon (Si) could decrease injury rates in equine athletes . The work was inspired by earlier reports on the essentiality of dietary Si and the implications for new bone development . With 53 Quarter Horses in race training in the blinded and placebo-controlled study, benefits from supplementing sodium aluminosilicate (SZA), the Si source, were documented . First, all three supplemented groups (low, medium, and high dosages) had more horses complete the required race program (nine races scheduled two weeks apart) than were injured compared to the control group which had more horses injured than were able to complete the study without injury. (It should be noted, injuries were not of catastrophic nature but simply were injuries that required the horses to miss days of training.) Further, the medium and high dosage groups completed substantially greater distances in training before experiencing an injury or completing the project if no injury occurred (90 and 83 km, respectively) compared to the control group (50 km). Finally, the medium treatment group had a faster average race time (20.3 s) than the control and low Si groups (20.7 s) at the race distance of 320 m. While supplementation was not believed to make horses faster, it was concluded that faster individuals within the medium treatment group were able to better withstand the rigors of race training without experiencing injury, contributing to the faster overall race time of that group compared to the control and low Si groups. The specific mode of action for the benefits reported in that study could not be determined. Thus, several future studies used markers of bone turnover not readily available at the time of the initial study to elicit possible reasons for the decrease in injury rates. Lang et al. found that supplemented yearling horses had decreased markers of bone resorption compared to unsupplemented controls . Likewise, with broodmares, trends (p < 0.10) for altered bone resorption were observed in postpartum supplemented mares compared to controls . Combined, the two studies by Lang and colleagues suggest that an altered rate of bone turnover may have influenced injury rates by allowing for a more rapid rate of bone repair that would prevent subclinical issues from becoming clinical. This was supported, to some degree, by work from Turner et al., who used 20 calves beginning at three days of age to test whether Si supplementation could impact their skeleton . No differences in bone architecture or mechanical properties could be detected, but the rate of bone turnover again appeared to be altered. While the change in bone turnover could be deemed positive, there was an accompanying increase in the aluminum (Al) content of cortical bone and articular cartilage. The Si concentration was increased in the aorta, spleen, lung, muscle, and kidney of Si-supplemented calves, but Al was also increased in all tissues . Only benefits from Si supplementation were noted in the prior studies with horses, but there were concerns about the amount of Al that was present in the SZA supplement (up to 130 g/kg). This prompted the evaluation of another bioavailable Si source without Al; that being oligomeric orthosilicic acid (OSA) . Both SZA and OSA altered calcium (Ca) retention and boron metabolism compared to controls, but only OSA was able to alter Si retention, digestibility, and plasma concentration. Thus, it appeared to be a viable option to provide dietary bioavailable Si without adding substantial amounts of Al to the equine diet. Another option may be a mineral supplement from a marine source that was tested against limestone (to provide a similar amount of Ca). In that study, yearling horses given the marine mineral supplement had enhanced bone turnover. Perhaps not coincidentally, that mineral supplement also provided a similar amount of Si as did SZA, suggesting the benefits may have come from Si . With the prospect of bioavailable Si aiding in bone health, products claiming to contain bioavailable Si became available commercially. While research had shown some benefits, anecdotally people made claims that providing sources of bioavailable Si also aided in resorption of certain joint lesions. As work by Reynolds and colleagues had previously documented spontaneous resorption of lesions without any outside intervention in young, growing horses , skepticism accompanied these claims. To investigate this, 44 two-year-old Standardbreds were radiographed to identify osteochondrotic defects in the fetlock and hock joints . From that group, eight horses met the inclusion criteria and were pair-matched and assigned to a control group (receiving a placebo) or a treatment group (receiving 200 g of a bioavailable Si source). Horses were kept on their respective treatment for 120 days at which point they were radiographed again. No treatment differences were observed, though it was acknowledged that the number of horses completing the project was small, limiting the ability to conclusively say treatment had no effect. However, no findings in the study supported the anecdotal reports of lesion regression with supplementation. It was acknowledged that supplementation may need to occur at a younger age to prevent the development of osteochondrosis. The concern regarding age of horse when supplementing was echoed by Pritchard et al. Retired Standardbred racehorses were supplemented with a source of bioavailable Si to examine whether it could affect lameness, particularly through its potential role in collagen synthesis . In this 84-day study, 10 horses were pair-matched and assigned to a Si-supplemented group or control. No treatment differences in lameness examination scores, radiographic scores of joints, or other indices of collagen degradation or synthesis were observed. With the mean age of horses being over 10 years, this study questioned whether supplementation provides little benefit in the aged animal. It also questioned whether the dosage provided in a commercially available form (0.3 g supplement/100 kg BW per d) was insufficient to elicit a response. Pritchard et al. also examined supplementation in broilers supplemented from day 1 after hatching until 42 days of age . No differences were seen in bone density, morphology, and strength measures between treatments, though supplementation altered serum mineral concentrations. Like with the previous Standardbred study , the dosage recommended by the manufacturer was below the intakes previously utilized in studies that reported positive influences on bone. Combined, these studies suggest the importance of careful scrutiny of commercial products that make claims based on studies that were not performed on their products, or that recommend dosages not shown to produce benefits in published research. 3. Bone Loss in Early Training Associated with Stall-Housing In an attempt to find a dietary supplement that can prevent skeletal injuries, arguably a more important finding occurred by happenstance. In the initial study utilizing 53 horses in race training, radiographs were taken of the third metacarpus throughout the study. This was done to examine differences in bone mineral content using radiographic photodensitometry measured in radiographic bone Al equivalences (RBAE) . This technique allows the optical density of the various steps of an Al stepwedge penetrometer attached to the radiographic cassette to be equated to the peak optical density of each cortex of the third metacarpal (reported in mm Al) . The bone optical density is influenced by both the thickness of the bone and the density of the bone. This technique was later refined to allow the total bone mineral content of a cross-section of the third metacarpus to be assessed . While not as precise as techniques such as computed tomography , which has been shown to be strongly correlated to bone ash weight , it is a non-invasive technique that has the advantage of not requiring sedation and being relatively inexpensive to use. In recent years, this technique has been modified for use with digital radiographs and, when used with digital radiographs, the need to use unprocessed images has been recognized . Using this technique, the initial racehorse study revealed a surprising result. When horses commenced race training, the RBAE (again, an estimate of bone mineral content) had decreased by day 62 of training . The RBAE remained low through day 104 of the study but had begun to increase by the conclusion of the study at day 244. These findings were surprising as most physiological systems are typically believed to increase in strength when athletic training commences (cardiovascular, muscular, respiratory). Thus, to find the skeletal system losing bone mass was confusing and alarming. Further, it should be noted that most bone-related injuries happened between days 60 and 120 of training when the bone mass was at its lowest. Accompanying this, horses began racing during week 9 of the study. While it was surprising that horses were losing bone mass during the initial stages of training, it was not surprising that the greatest injury rates were occurring at the time when bone was the weakest and horses were beginning to race. The combination of fast speeds and weak bones logically results in injuries. At the time of the study, most discussions about modifications of equine bone focused on bone remodeling--the process by which old or damaged bone is replaced by new bone . In theory, the loss of bone at the start of training could be explained if equine bone was not sufficiently strong to withstand the rigors of training resulting in damaged bone that needed to be removed to allow new bone to replace it. If that were to occur, it raised the question as to whether the amount of dietary mineral recommended by the 1989 Horse NRC would be sufficient once new bone began to be deposited. Given that shin soreness in humans had been linked with low Ca intake , there was concern that insufficient Ca in the diet might leave horses susceptible to bone-related injuries. To address this, 10 previously untrained Quarter Horses were put into race training for 112 days . They were fed diets balanced to meet NRC requirements . Radiographs revealed a decrease in bone mineral content of the third metacarpus by day 56 of training, prior to the initiation of speedwork being introduced during the second half of the study. This decrease in bone mass was similar to the decrease seen in the prior study using 53 horses. In the second half of the study, bone mass increased, and was accompanied by greater Ca retention. This inspired a follow-up study using 12 previously untrained horses, divided into one group that received Ca and P similar to the NRC recommended concentrations and another group that received them at higher concentrations . Of note, in the study utilizing only 10 horses, there was an adaptation period of 19 days in which horses were walked on a mechanical walker for one hour per day. In contrast, in the later study using 12 horses, the adaptation period was decreased to 9 days and horses were walked on a mechanical walker twice a day. This was done to minimize potential changes associated with a decrease in load being placed on the skeletal system due to stall confinement prior to training as there was increasing evidence that restriction of activity could be associated with loss of bone mass . While the increased allocation of dietary mineral did result in increased Ca retention for young horses in training, another observation was that the changes in RBAE of the third metacarpal did not appear as great in the study with the shortened adaptation period as they did in the study with the longer adaptation period . Reflecting on the initial study in which bone loss had been observed in the 53 horses in race training , it was noted that prior to entering race training, the young horses had been moved from pasture housing and placed into box stalls. By eliminating access to free exercise and having no fast exercise during the early part of training, it was hypothesized that the loss of bone may have been caused by the lack of loading on the skeleton. At the time, it was not commonly believed among horsemen that confinement had anything to do with bone loss. Fortuitously, one of the researchers in the latter two studies had no previous horse experience that could bias her, though she had extensive experience with bone loss in human bed-rest patients. When the idea was presented to her, she responded that it was logical. Lacking horse experience kept her from having any preconceived notions based upon how things have traditionally been done with horses. To test the hypothesis that stalling of horses with no access to high-speed exercise was responsible for bone loss, 16 Arabian yearlings, previously housed together on pasture, were randomly divided into two groups . Half of the horses remained on pasture while the other half were moved into box-stall housing with one hour of walking daily on a mechanical walker. By day 28 of the study, the RBAE of the third metacarpus of box-stalled horses had decreased and remained low throughout the 140-day study. Even when horses were started under saddle and began race training after 12 weeks on the study, no increase in bone mass was seen in the stalled horses during 8 weeks of slow racetrack training without speed. The concentration of serum osteocalcin (a marker of bone formation) was lower and urinary deoxypyridinoline (a marker of bone resorption) was higher in the confined horses at days 14 and 28, respectively, compared with the pastured horses, and both markers subsequently returned to baseline in the confined horses. Those alterations suggest bone formation decreased and bone resorption increased in the confined horses, thus, explaining the loss of bone. With this study closely mirroring how yearling racehorses are managed while being prepared for sales and during the early training, it was concerning to note that horses kept in stalls had lower bone mass at the end of the nearly five-month study than they did when they started it. The results suggested a probable cause for the high injury rates observed in young horses managed in that fashion. At the same time, a study examined bone mineral content of the third metacarpus in 11 mature Arabian horses (ages 4 to 7 years) that had been previously conditioned but were then placed into box stalls for 12 weeks . Despite being walked on a mechanical walker daily in two 30 min exercise bouts, and being fed dietary Ca at twice the 1997 NRC-recommended amounts , horses lost bone mass. While those studies showed that having horses housed in box stalls without access to high-speed exercise (either forced or voluntary if housed on pasture) resulted in bone loss, it raised the question as to whether having horses housed on pasture completely (as opposed to partially) was necessary to avoid bone loss. To test this, 17 weanling Arabian horses were randomly assigned to three treatment groups: (1) housed on pasture, (2) housed in stalls, and (3) housed in stalls for 12 h per day and housed on pasture for 12 h per day . After 56 days, greater increases in bone mass of the third metacarpal were observed in both groups allowed access to pasture, as opposed to the group confined completely to stalls. Thus, it appeared that even partial turnout could prevent bone loss associated with disuse. At the end of that study, all weanlings were returned to pasture housing. Close to a year later, horses were radiographed again as part of a follow-up study . Being returned to pasture allowed the horses subjected to complete stalling to have a similar bone mineral content of their third metacarpus as did their counterparts that had pasture housing. The results of that study suggest that short-term stall housing of young horses does not doom them to lower bone mass throughout life, assuming the return to pasture occurs while the horses are young and experiencing relatively fast bone growth. However, this study did not answer whether the same holds true for mature horses. A later study that included mature horses was able to detect alterations in bone metabolism markers indicative of enhanced bone formation when stalled horses were returned to pasture after 4 weeks though it is unclear if these alterations would result in fully restoring any bone lost due to stalling . 4. Role of Exercise in Bone Development While it was assumed that the differences in bone associated with stall-housing were due to lack of high-speed exercise experienced by the stalled horses, other factors could have played a role including such things as differences in nutrition or exposure to sunlight. To determine if stalling caused disuse osteopenia (the loss of bone associated with lack of mechanical loading), exercise needed to be the sole factor that was altered in a research study. To test this, 18 juvenile bull calves were used to allow for a terminal study in which actual bone strength could be tested, as opposed to only using an indirect measure of bone mineral content . They were assigned to one of three treatment groups: (1) group-housed, (2) confined with no exercise, and (3) confined with exercise consisting of running 50 m on a concrete surface once daily, 5 d/week for the duration of the six-week study. At the conclusion of the study, calves were humanely sacrificed, and the third and fourth metacarpal bone was scanned using computed tomography to determine cross-sectional geometry and bone mineral density. The bones were then subjected to a three-point bending test to failure. Exercised calves had increased cortical thickness and decreased medullary cavity area, as well as increased cortical bone density. There was also a trend for higher fracture force compared to the confined calves. The changes were considered quite remarkable given they were the result of running a cumulative distance of only 1500 m during the six-week study. Despite the obvious benefit such a small amount of sprinting produced on bone, it was recognized that individuals in the horse industry might be reluctant to believe the results, obtained with calves, could be applicable to horses. Thus, we also performed a study utilizing 18 weanlings divided into three groups: (1) group-housed, (2) box stall-confined with no exercise, and (3) box stall-confined with a daily sprint of 82 m, 5 d/week for the duration of the 8-week study . Using the RBAE technique , increased bone mineral content and altered bone geometry were noted in the confined horses that were allowed to sprint short distances (a cumulative distance of 3280 m over 8 weeks), compared to the confined horses that experienced no sprinting. Exercise also produced beneficial changes in exercising swine . Gestating gilts were divided into treatment groups receiving (1) no exercise, (2) low exercise (122 m/d, 5 d/week), or (3) high exercise (122 m two d/week and 427 m three d/week). All animals were stall housed during gestation and the study took place between d 35 and 110 of gestation. Bone density and breaking force were greater in the exercised gilts compared to the gilts receiving no exercise, with the additional benefits of exercise being shown in piglet survivability. While both of those studies evaluated the response of bone to exercise performed five days per week , the question remained as to whether that frequency was needed, or whether fewer times per week would result in the same benefits. To examine this, 24 juvenile bull calves were divided into four groups: a control group receiving no exercise, or groups receiving a 71 m sprint either once, three times, or five times per week for the duration of the six-week study . Calves were humanely euthanized and the left fused third and fourth metacarpal bones were scanned using computed tomography and tested for fracture force using four-point bending. All exercised groups had greater dorsal cortical widths compared to control animals and the fracture force was greater for all exercised groups than the control group. There were no differences between exercised groups, indicating that only one short sprint per week was required to enhance bone strength. With one 71 m sprint per week conducted over six weeks, the cumulative distance sprinted in a month and a half was 426 m, resulting in over a 20% increase in bone strength compared to calves that received no sprinting. This dramatic difference, brought about by such a small amount of high-speed exercise, should be alarming to those in the horse industry that do not afford any opportunity for their horses to run at speed and, are thus, developing weakened bone. 5. Speed (Load) versus Strides (Cycles) It should be noted that not all exercise produces similar benefits. As bone responds to mechanical loading, any alterations in how strides are taken can impact bone formation. For instance, circular exercise such as lunging is commonly done with horses. While believed to be detrimental to joint health, various bone parameters have been shown to be altered between inside and outside legs of juvenile bull calves exercised in only one direction five days per week in a study lasting seven weeks . Further, bone responds more to mechanical strain (the amount of bending) than it does the number of times it is bent . This means the force which is exerted on the bone has more influence than does the number of cycles of bending or, for instance, strides the animal takes. In rats, just 36 cycles of bending three times per week was sufficient to prevent disuse osteopenia associated with immobilization . In an avian model, it was determined that only four consecutive cycles prevented bone loss, and 36 cycles increased new bone formation . Increasing the number of cycles from 36 to 1800 resulted in no additional strengthening of bone. With bone responding to the magnitude of strain (how much it bends) as opposed to how often it bends, it was not surprising that, during a conditioning study lasting 78 days, carrying weight (progressively increasing the amount of weight carried up to 45 kg) resulted in increased RBAE in young horses exercised in a walker compared to control horses that were exercised similarly, but without carrying supplemental weight . Prior to entering the conditioning period, all horses were stall-confined for 108 days, during which time they had lost bone mineral content of the third metacarpal, also reaffirming the detrimental effects that stalling without access to proper exercise has on bone. Likewise, while it has long been believed that months of slow training will increase bone strength and prevent injuries , science does not support this. Using 11 two-year-old Arabians that were split into two groups, the influence endurance exercise has on bone mineral content of the third metacarpus was examined . One group was trained on a high-speed treadmill for 90 days, with training consisting of walking (1.6 m/s), trotting (4 m/s), and cantering (8 m/s) at increasing distances until the target of 60 km/d was met. Starting on day 90, the exercised horses were placed on a regular exercise schedule including a 60 km endurance test every three weeks. The other group served as controls and lived on pasture without any forced exercise. Radiographs of the third metacarpal were taken at the start of the study and at day 162. No differences were seen between treatments in RBAE, suggesting endurance exercise does little to alter bone optical density compared with free-choice exercise on pasture. However, it would be interesting to repeat this study with greater numbers. The total RBAE of the endurance-exercised group was 493 +- 48 mm Al2 at the start of the study and finished at 424 +- 44 mm Al2. The total RBAE of the control group (pasture-housed with no forced exercise) started at 394 +- 48 mm Al2 and finished at 429 +- 48 mm Al2. In previous studies , it was noted that although animals had access to exercise because they were group-housed, it did not guarantee they did any sprinting. In the endurance study , it is likely that after the horses had their daily endurance training bouts and were returned to a group-housed setting, they likely opted to eat, drink, and rest as opposed to doing any additional fast exercise. By contrast, those in the control group likely occasionally played, including sprinting, which would have a greater impact on bone strength than would the endurance exercise. Similarly, no treatment differences in RBAE and biomarkers of cartilage turnover were reported between yearling horses conditioned at a walk on an aquatic treadmill, on a dry treadmill, and those receiving no forced exercise . However, all horses had access to turnout for about 10 h per day where voluntary exercise was allowed, and it was believed that this access to turnout had a greater effect on bone and cartilage than did the forced exercise at a slow speed. Voluntary activity can play an important role in maintaining or increasing bone strength if the opportunity is allowed and if the animal takes advantage of such. Given that some of the previous studies have suggested group-housed animals allowed free access to exercise (as opposed to confinement-housed animals not allowed to exercise at speed) do not always take advantage of this opportunity, it is critical for horse caretakers to take horse behavior into consideration. In a recent study, the impact of weather was investigated on activity of horses while on pasture . An interesting finding was a difference in activity between horses housed on farms located 3.5 km apart. That suggested that factors other than weather played an important role. The farm reporting greatest activity had horses primarily bred for racing. However, one of the horses in that group was a 33-year-old Spotted American Saddle Horse with pituitary pars intermedia dysfunction. Despite her age, disease status, and breed, it is likely her increased activity was due to being kept with horses bred for racing and who often ran. When they ran, she did also. The desire to be with other horses is strong and has been reported before . This can increase the frequency of sprinting if kept with other horses inclined to sprint or can decrease the frequency if kept with horses not inclined to do so. Thus, housing horses on pasture does not guarantee they will perform exercise necessary to enhance bone strength, but it does increase the likelihood of it. By contrast, if confined to a stall and never afforded the opportunity to run, it can be assured that skeletal strength will be compromised. 6. Pharmaceutical Factors Besides nutritional and biomechanical influences on bone, pharmaceutical influences also exist, some with unintended consequences. In recent years, there has been growing concern regarding the use of bisphosphonates. Approved in 2014 to treat navicular disease in horses over the age of four years, bisphosphonates have also been used extra-label for other skeletal issues. With bisphosphonates inhibiting osteoclasts, whose function is to resorb bone, there is concern regarding whether this will impair bone healing, leaving horses, particularly young horses in training, more susceptible to injury . In humans, long-term use has been linked to increased risks of some types of fractures. Furosemide is commonly given to racehorses in North America in an attempt to decrease the incidence of exercise-induced pulmonary hemorrhage. As the usage of such has been shown to negatively impact Ca balance for several days after administration, concern existed as to whether it could have detrimental effects on bone if this effect persisted beyond that. Using a crossover design with ten horses, serving as both controls and furosemide-treated, during two 8-day periods of total collection of urine and feces, it was shown that Ca balance returned to baseline in three days after furosemide administration and does not present much risk to bone, particularly since there seemed to be a compensatory effect on of having lower fecal Ca loss in treated horses by the end of the collection period . Likewise, omeprazole has been commonly provided to aid in healing or preventing gastric ulcers in horses, but concerns have existed whether the suppression of gastric acid may inhibit absorption of Ca and thus impair skeletal health. Using a preventative dose (1 mg/kg BW daily) for up to two months provided no indication of such . Usage for longer periods or at the treatment dose (4 mg/kg BW daily) could still pose a risk. Incidental findings of alterations in markers of bone formation, likely associated with the stalling of horses near the end of the study, reaffirm the concern with stalling of horses. In particular, many athletic horses are stalled, and these would often be the same horses that have a greater propensity to develop ulcers. These findings reaffirm the concern surrounding the stalling of horses without access to exercise capable of supporting optimal skeletal health. 7. Conclusions Many without research experience believe solutions to problems can often be achieved through a single study. By contrast, this paper highlights how it can sometimes take decades, utilizing dozens of studies, to be able to make solid, research-based recommendations. Often a single study will inspire other studies to answer new questions that arise. Additionally, sometimes research provides incidental findings that may prove to be more important than are the findings for which the study was designed. While originally looking for a nutritional approach to preventing skeletal injuries, a loss of bone mass of the third metacarpal of horses in race training was shown. Trying to find ways to prevent that bone loss led to the realization that the loss was due to stalling of horses without access to speed. Future studies showed that pasture turnout prevented that bone loss and as little as one short sprint per week made dramatic differences in bone strength. Failure to provide athletic horses with such an opportunity would seemingly put them at increased risk of injury--particularly once high-speed work resumes. Exploring whether stalling of horses caused the loss of bone mass was inspired by a mentor who had no horse experience and, thus, was not biased by how things have traditionally been done in the horse industry. Currently, many in the horse industry believe that training of young, growing horses is detrimental and should not be done. However, scientists working in this area realize that is not the case . Bone modeling, the process by which changes in the size, shape, and strength of bone occur, happens primarily in the juvenile animal. Once skeletally mature, changes primarily occur through bone remodeling--the process which involves simply replacing old or damaged bone. Thus, little change in size, shape, and strength can occur once skeletal maturation is complete. Nutrition does play an important role in bone health. Having the necessary nutrients in proper balance is crucial to bone development, particularly in the growing horse . However, examining studies using markers of bone metabolism to detect treatment differences, those studies involving changes in nutrition usually failed to show differences, whereas studies involving altering exercise usually resulted in significant differences in those biochemical markers . Likewise, in young female humans, only childhood physical activity had a significant positive on bone density, as opposed to nutritional and other lifestyle factors that had no influence . Granted, this is based upon the assumption of adequate Ca intake and other nutrients, and this is especially true after menopause . These studies emphasize the benefit of an active lifestyle on the skeletal system, particularly when young and growing to achieve a higher peak bone mass upon maturity. However, activity is still crucial when older as a sedentary lifestyle hastens bone loss and is permissive to osteoporosis, regardless of diet. These findings do not lessen the importance of proper nutrition on bone health, it simply suggests that once requirements are met, providing additional nutrients cannot assure good bone health. It also suggests it is not possible to have optimal bone health if no exercise, or the wrong type of exercise, is provided, regardless of what is consumed through the diet. While the temptation by some is to fault nutrition for skeletal injuries, more often it is improper training, management, or lifestyle that is to blame. Research also suggests caution be taken when providing pharmaceuticals that may inhibit mineral absorption or enhance mineral loss, though proper exercise and nutrition may mitigate potential negative effects. More concerning are pharmaceuticals that inhibit normal bone metabolism, such as by reducing osteoclastic activity, as this can impair normal bone healing . Additionally, potential analgesic effects should be of great concern whether with pharmaceuticals such as bisphosphonates or corticosteroids . Masking pain when an injury is still present increases the chance for greater injury, potentially even catastrophic in nature, to occur. Though the intent of much of the afore-mentioned research was to improve the lives, health, and well-being of horses, implications also exist for humans. A sedentary lifestyle, without sufficient mechanical loading of bone, will lead to a weakened skeleton. With the lifespan of humans being longer than horses, failure to strengthen the skeleton when young, and failure to maintain skeletal strength when mature, likely will increase the risk of skeletal injury throughout life and increase the chance of developing osteoporosis when older. Improper nutrition will increase the risk, though proper nutrition, without the correct form of exercise, will not prevent it. Fortunately, as with horses, the proper exercise does not require long bouts of exercise. Short bouts (only about 20 cycles) of bone-centric exercise can increase bone strength without the damage that repeated cycles can cause . Again, like with horses, having a skeleton adapted for high load requires loading while young, continuing through maturity. Attempts to improve skeletal health once mature is more difficult to accomplish and much greater care needs to be given to managing loads placed upon it and preventing bone loss when aging. With both horses and humans, many of the skeletal injuries that develop are the result of bone being ill-prepared for high loads due to sedentary periods without loading. As quoted by Dr. Gary D. Potter, the author's major professor in graduate school at Texas A&M University, when an old horse trainer was asked what he believed to be the major cause of injuries in his racehorses, his answer was respiratory problems. The trainer claimed he did not know why, but it seemed like every time one of his horses became sick and needed to be rested for a while, it became injured when he put it back in training. With decades of research to support it, the answer is clear. Acknowledgments Dozens of mentors, colleagues, graduate students, and undergraduate students all played critical roles in the development and implementation of the research that helped to form the conclusions drawn by these studies. To borrow a quote, "it takes a village", and this work could not have been completed without them. Most notably though, for over two decades, C.I. Robison has served as my research assistant and has played an instrumental role in many of these studies. Beyond that, the preparation of this manuscript was aided by the efforts of one student who digitized the author's three decades of printed journal articles and whose efforts made the preparation of this manuscript much easier. I am indebted to B.J. Emmert for her assistance! Institutional Review Board Statement Not applicable. Informed Consent Statement Not applicable. Data Availability Statement Not applicable. Conflicts of Interest The author declares no conflict of interest. Figure 1 Total radiographic bone Al equivalences (RBAEs) of all horses and periods during which bone-related injuries occurred . a,b,c Days lacking a common superscript differ (p < 0.05). Disclaimer/Publisher's Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

PMC10000204

Segmental ureterectomy (SU) is an alternative to radical nephroureterectomy (RNU) in the treatment of upper-tract urothelial carcinoma (UTUC) of the ureter. SU generally preserves renal function, at the expense of less intensive cancer control. We aim to assess whether SU is associated with inferior survival compared to RNU. Using the National Cancer Database (NCDB), we identified patients diagnosed with localized UTUC of the ureter between 2004-2015. We used a propensity-score-overlap-weighted (PSOW) multivariable survival model to compare survival following SU vs. RNU. PSOW-adjusted Kaplan-Meier curves were generated and we performed a non-inferiority test of overall survival. A population of 13,061 individuals with UTUC of the ureter receiving either SU or RNU was identified; of these, 9016 underwent RNU and 4045 SU. Factors associated with decreased likelihood of receiving SU were female gender (OR, 0.81; 95% CI, 0.75-0.88; p < 0.001), advanced clinical T stage (cT4) (OR, 0.51; 95% CI, 0.30-0.88; p = 0.015), and high-grade tumor (OR, 0.76; 95% CI, 0.67-0.86; p < 0.001). Age greater than 79 years was associated with increased probability of undergoing SU (OR, 1.18; 95% CI, 1.00-1.38; p = 0.047). There was no statistically significant difference in OS between SU and RNU (HR, 0.98; 95% CI, 0.93-1.04; p = 0.538). SU was not inferior to RNU in PSOW-adjusted Cox regression analysis (p < 0.001 for non-inferiority). In weighted cohorts of individuals with UTUC of the ureter, the use of SU was not associated with inferior survival compared to RNU. Urologists should continue to utilize SU in appropriately selected patients. upper-tract urothelial carcinoma ureter cancer comparative effectiveness segmental ureterectomy nephroureterectomy This research received no external funding. pmc1. Introduction Upper-tract urothelial carcinoma (UTUC) is a rare cancer that accounts for 5-10% of all urothelial carcinomas, with an estimated annual incidence of less than two cases per 100,000 in western countries . Currently, radical nephroureterectomy (RNU) with bladder cuff excision is the standard of care in patients with high-risk upper-tract urothelial carcinoma (UTUC), while nephron-sparing treatments are preferred for patients with low-risk disease. Previous studies have demonstrated that RNU has a significant and enduring detrimental impact on renal function, putting the patient at risk of chronic kidney disease (CKD) and related sequelae . The changes in renal function might affect eligibility for adjuvant cisplatin-based therapy in 30-60% of patients who were eligible for chemotherapy before surgery . Lastly, given the advanced age of presentation and its associated comorbidities, many patients have contraindications to RNU . Segmental ureterectomy (SU) can be utilized in selected cases as an alternative for tumors confined to the ureter. Although SU has been generally demonstrated to have superior renal functional outcomes without compromising oncologic control , concerns regarding selection criteria, oncological safety, and efficacy remain. Several studies have reported higher rates of local tumor recurrence after the kidney-sparing approach , including with segmental ureterectomy . However, oncological safety data of SU have mainly been generated from small institutional databases, with underpowered analyses due to the small sample sizes. In this setting, we sought to compare the OS of patients with localized UTUC based on whether they received SU vs. RNU with bladder cuff excision to better elucidate long-term survival outcomes associated with each surgical approach. We hypothesized that SU would be non-inferior to RNU in terms of OS recognizing that the NCDB may overcome some of the issues with sample size. 2. Materials and Methods 2.1. Data Source and Study Population The National Cancer Database (NCDB) is a joint effort between the Commission on Cancer, the American Cancer Society, and the American College of Surgeons. It is a national cancer registry that compiles data for more than 1500 commission-accredited cancer programs in the United States . With more than 30 million records across the country, NCDB is an essential source of standardized data for cancer surveillance providing standardized data on patients, hospitals, and therapies . We captured all adult patients (>18 years old) with a primary diagnosis of urothelial carcinoma of the ureter (ICD-10-CM code C66) between January 2004 and December 2015. Patients with non-urothelial histology were excluded. In order to focus on patients with localized disease, we also excluded patients with clinical or pathologic evidence of nodal or distant metastases; moreover, we excluded patients with missing histology, follow-up, or local therapy information. 2.2. Exposure of Interest The exposure of interest was the type of surgery. Specifically, patients undergoing SU were compared with those undergoing RNU with bladder cuff excision. We excluded patients subjected to other types of surgery or with missing information regarding local therapy. 2.3. Definition of the Variable of Interest The outcome of this study was OS, defined as the duration of elapsed months from the date of diagnosis to the date of death or censor at last follow-up. Specifically, OS, calculated as the number of months between the date of diagnosis and the date on which the patient was last contacted or died, of patients undergoing SU and RNU was compared; moreover, we reported five-year OS of the two groups. 2.4. Covariates Baseline characteristics included age at diagnosis, gender, race (defined as "White", "Black", or "other"), and year of diagnosis. The Charlson comorbidity index (classified as 0, 1, 2, or >=3) provided by the NCDB was used to account for underlying disease status. We abstracted sociodemographic and hospital-level covariates including county-level median household income (defined as "high" (>=$48,000) or "low" (<$48,000)), residence type, county-level median education level (defined as "high" (<13% of adults in the patient's zip code who did not graduate from high school) or "low" (>=13% of adults in the patient's zip code who did not graduate from high school)), insurance status, facility type, facility county, and facility US region. We used the TNM classification system to categorize clinical tumor classification (<=T1, T2 T3, T4, or Tx), according to the American Joint Committee on Cancer, 8th Edition . Other tumor-specific variables included tumor grade (defined as "low", "high", or "unknown"), tumor size (categorized as "<2 cm", ">2 cm", or "unknown"), and presence of lymph vascular invasion. We included chemotherapy as a treatment-related covariate. 2.5. Statistical Analyses Means and standard deviations or median and interquartile ranges (IQRs) were reported for normally or nonnormally distributed continuous variables, respectively. Categorical variables were presented as frequencies and proportions. Statistical significance in differences between groups was assessed using a t-test for continuous variables and a chi-square test for categorical variables. To assess for balance between groups, we used the standardized-differences approach to compare observed covariates between RNU and SU patients . This quantitative method allowed us to assess the balance in baseline characteristics between treatment groups. A standardized difference >=10% for a given covariate indicated a significant imbalance. Multivariable logistic regression analysis was performed to identify independent predictors for undergoing SU compared to RNU in the unweighted population. To account for potential selection bias, a propensity score-overlap weighting (PSOW) method was used to balance baseline characteristic differences between RNU and SU groups. Each patient was assigned a statistical weight proportional to the probability of that patient belonging to the opposite treatment group. PSOW improves the balance and precision at the baseline relative to other conventional propensity score methods . We used the standardized differences approach to assess the balance of the covariates between groups after weighting. PSOW-adjusted Kaplan-Meier curves were generated to compare overall survival (OS) between patients who received SU and those who received RNU. Equality of survivor functions was tested using the log-rank test and Cox test of equality in unweighted and weighted population, respectively; then, we performed a univariable Cox regression analysis to calculate the PSOW-adjusted hazard ratio (HR) of SU . Finally, we ran a non-inferiority test with a priori upper-bound for inferiority set as HR > 1.1 with a one-sided alpha of 0.05. All analyses were performed using Stata IC 16.0 (Stata Corp LLC, College Station, TX, USA). An institutional review board waiver was obtained from the Brigham and Women's Hospital. 3. Results A total of 13,061 men and women diagnosed with localized urothelial carcinoma of the ureter were included in this study. Median follow-up was 38.1 (18.2-70.1) months, in which 9016 underwent RNU with bladder cuff excision, while 4045 underwent SU. A flowchart describing cohort selection is shown in Figure 1. A comparison between 11,049 (45.8%) patients with urothelial carcinoma of the ureter who did not meet the study criteria and 13,061 (54.2%) patients who were included in the study is reported in Supplementary Table S1. 3.1. Baseline Patient Characteristics and Predictors of Receiving Segmental Ureterectomy Baseline characteristics of the study population pre-weighting are shown in Table 1. The mean age at diagnosis was 71.9 +- 10. Prior to weighting, most of the covariates had a standardized difference smaller than 10% between the two groups. In our multivariable logistic regression analysis, independent factors associated with the decreased likelihood of receiving SU were female gender (OR, 0.81; 95% CI, 0.75-0.88; p < 0.001), advanced clinical T stage (cT3: OR, 0.67; 95% CI, 0.56-0.80; p < 0.001; cT4: OR, 0.51; 95% CI, 0.30-0.88; p = 0.015), and high-grade tumor (OR, 0.76; 95% CI, 0.67-0.86; p < 0.001). Having an age at diagnosis greater than 79 years was associated with increased probability of undergoing SU (OR, 1.18; 95% CI, 1.00-1.38; p = 0.047). Remaining independent predictors of receiving SU are summarized in Table 2. 3.2. Patient Characteristics and Survival Analyses After overlap weighting, the distribution of baseline patient and tumor characteristics were well balanced between the groups; indeed, all standardized differences of weighted comparisons indicated differences smaller than 0.001 in the observed covariates between the two groups (Table 1). The five-year OS in patients undergoing SU was 53.1% (95% CI: 51.3-54.8) and 52.6% (95% CI: 51.0-53.8). The total number of events (i.e., death) observed in the whole population was 6518 (1982 and 4536 in patients undergoing SU vs. RNU, respectively). The PSOW HR for SU is 0.98 (95% CI, 0.93-1.04; p = 0.538). The HR met the non-inferiority criterion with a threshold HR of 1.1 and a one-sided alpha of 0.05 (p < 0.001 for non-inferiority) rejecting the null hypothesis of SU conferring inferior survival compared RNU. The PSOW survival curves are shown in Figure 2. The PSOW-adjusted Cox regression analysis is shown in Table 3. The survival curves of patients who received SU and RNU according to the T stage group and the grade of the disease are shown in Figure 3 (unweighted population) and Figure S1 (weighted population). 4. Discussion In this retrospective analysis, we aimed to investigate whether SU is associated with inferior long-term survival compared to RNU. Among 13,061 men and women receiving extirpative surgery for localized UTUC with a median follow-up of 38.1 months, we found that OS following SU was non-inferior to RNU. Current guidelines by the European Association of Urology for UTUC recommend kidney-sparing surgery such as SU for: (1) low-risk localized tumors, (2) high-risk distal ureteral tumors, or (3) patients with impaired renal function and/or solitary kidney. Ultimately, RNU is the standard for high-risk UTUC regardless of location . Patients who undergo RNU may experience a mean decrease in the estimated glomerular filtration rate (eGFR) by 24% and are at a higher risk of renal insufficiency . Consequently, other groups have investigated an expanding role for more conservative therapies, with SU becoming the most common alternative surgical procedure to RNU. In this study, we compared the OS of patients who received SU vs. RNU for localized UTUC using NCDB and we identified demographic and clinical predictors of underdoing SU. Given the rarity of this disease, retrospective observational data provide a key perspective on real-world outcomes. This study included more than 13,000 men and women, of whom more than 4000 have undergone SU, and this group is the largest in terms of population size that compared SU vs. RNU. In order to minimize confounding, we used overlap weighting, a propensity score method that mimics important attributes of a randomized clinical trial, such as a clinically relevant target population, covariate balance, and precision . This statistical technique improves baseline balance and precision in comparison to other methods such as standard inverse probability weighting or matching . We reached a full balance between the study groups for a large number of covariates, including patient, hospital, and tumor level characteristics. After adjusting for confounding, we found no difference in OS between patients undergoing SU compared to those who received RNU. Our results are consistent with the most recent systematic review and meta-analysis. Indeed, that survey included 18 studies for a total of 4797 patients undergoing either RNU or SU and found no statistically significant difference in the five-year metastasis-free survival nor the cancer-specific survival between the two surgical interventions . Similar conclusions were reached by other authors on retrospective data . However, our study included the largest population and used a robust study design to compare the two procedures. We identified several factors associated with the utilization of SU vs. RNU, as expected tumor characteristics associated with the use of SU included low cT stage, low-grade pathology, and tumor size <2 cm; additionally, low education attainment and being treated at a non-academic institution were also associated with a lower probability of undergoing SU; on the other hand, we found that age >79 and male gender were more likely to receive SU compared to RNU. Altogether, our findings corroborate the idea that this is a complex surgery for an uncommon disease, and it should, therefore, be performed in high-volume centers on well-selected patients. Due to NCDB limitations, we could not retrieve pre-operative and post-operative data on renal function; however, several studies have shown that nephron-sparing surgeries are associated with higher post-operative eGFR, and a lower risk of developing CKD . Thus, kidney-sparing surgeries such as SU are indicated in patients with a functional or anatomical solitary kidney, bilateral disease, or severe renal insufficiency . The superior renal functional outcomes of SU could not only ameliorate the downstream sequelae of chronic kidney disease and dialysis when compared to RNU, but also guarantee a better chance for patients to be eligible for platinum-based chemotherapy. This is of particular importance given the increasing role of peri-operative systemic therapy for UTUC, supported by high-quality evidence . Given the survival benefit conferred by adjuvant chemotherapy and the superior renal functional outcomes conferred by kidney-sparing surgery, SU may maximize the chance of receiving downstream chemotherapy compared to RNU in those very well selected HR UTUC patients who are candidates for kidney-sparing surgery. Lastly, while pre-existing renal function may be a confounder, we would generally expect men and women with worse pre-operative renal function to receive nephron-sparing surgery. Because impaired renal function is likely associated with worse overall survival, this should bias our non-inferiority analysis towards the null hypothesis (inferior overall survival in the SU group). While our analysis adjusted for clinical T stage, there are doubtless technical factors such as tumor location and prior surgeries which could tend to lead a surgeon to preferentially offer a SU versus an RNU. Some of these factors relate to patient selection and cannot be captured in this type of analysis; there will always be an element of surgical decision-making around whether to offer an RNU. Despite these limitations, our study confirms that in appropriately selected patients, SU confers no worse survival than RNU and should be offered to men and women who have tumors suitable for this approach. UTUC is usually a lethal disease with a bad prognosis, therefore it is crucial to avoid under-treatment. With that said, recent data have emphasized the importance of multimodality treatment and the combination of surgery and systemic therapy is often required . Thus, the dual aims of nephron sparing and maximal disease excision need to be balanced, our paper supports that the judicious use of nephron-sparing approaches does not meaningfully sacrifice survival. As a result of the advancements in the endoscopic diagnostic optic instruments, utilization of diagnostic upper-tract endoscopy for UTUC increased. Adaptation of improved diagnostic technologies led to better risk stratification of the disease, resulting in a trend towards more conservative therapy. Future advancements in the diagnosis of UTUC will allow for better patient selection for kidney-sparing surgeries to mitigate side-effects and risks associated with RNU. Our study is not devoid of limitations. Firstly, our study is limited by its observational retrospective design and the inherent limitations associated with such study designs. Secondly, although we corrected for possible selection bias using PSOW, there may be underlying differences between the SU cohort and the RNU cohort that were not detected by the available covariates. We believe that many of these unobserved factors are non-differential between RU and SNU and may, in fact, bias towards the null hypothesis of inferior survival in SU (e.g., worse renal function or comorbidities in men or women who received SU). Thirdly, NCDB data is also limited in terms of granularity of relevant data. For example, we were not able to identify pre-operative hydronephrosis, location of the ureteral tumor (proximal or distal), focality of the tumor, and pre-operative and post-operative renal function, as well as smoking status. Finally, we had no information about the follow-up schedule, and we could not assess cancer recurrence and cancer-specific survival. 5. Conclusions In appropriately selected UTUC of the ureter patients treated in a real-world setting, SU provides non-inferior OS compared to RNU. Given the potential renal functional benefits of SU and the increased probability of requiring adjuvant chemotherapy in UTUC, the judicious use of SU is likely a valid surgical approach which does not meaningfully sacrifice survival outcomes. Supplementary Materials The following supporting information can be downloaded at: Table S1: Comparison of the baseline characteristics of patients affected by urothelial carcinoma of the ureter who were included in the study vs. those who were excluded; Figure S1: Overlap weighting-adjusted Kaplan-Meier analysis of overall survival in patients who received Radical Nephroureterectomy vs. Segmental Ureterectomy, according to pathological T stage. SU = Segmental Ureterectomy, RNU = Radical Nephroureterectomy, pT stage = pathological T stage. Click here for additional data file. Author Contributions Conceptualization, M.P. and A.P.C.; methodology, M.P., S.R.L. and A.P.C.; validation, M.P., S.R.L. and A.P.C.; formal analysis, M.P., S.R.L. and A.P.C.; resources, Q.-D.T. and A.P.C.; data curation, M.P., S.R.L. and A.P.C.; writing--original draft preparation, M.P., K.Y.A. and A.P.C.; writing--review and editing, K.Y.A., D.-D.N., K.Y., M.M., P.C., P.M.P., A.S.K., Q.-D.T., N.M.B., G.L. and A.P.C.; supervision, Q.-D.T., G.L. and A.P.C. All authors have read and agreed to the published version of the manuscript. Institutional Review Board Statement An institutional review board waiver was obtained from the Brigham and Women's Hospital. Informed Consent Statement The National Cancer Database (NCDB) is a joint project of the Commission on Cancer (CoC) of the American College of Surgeons and the American Cancer Society. The CoC's NCDB and the hospitals participating in the CoC NCDB are the sources of de-identified data used herein; they have not verified and are not responsible for the statistical validity of the data analysis or the conclusions derived by the authors. Data Availability Statement Restrictions apply to the availability of these data. Data were ob-tained from the National Cancer Database and are available from the authors with the permission of the National Cancer Database. Conflicts of Interest QDT reports personal fees from Astellas, Bayer and Janssen, outside the submitted work. QDT reports research funding from the American Cancer Society, the Defense Health Agency, and Pfizer Global Medical Grants. APC reports research funding from the American Cancer Society and Pfizer Global Medical Grants. ASK reports personal fees from Janssen, Insightec, Merck, Profound, and Bristle Meyers Squib. Figure 1 Flowchart that describes the selection of patients who received Radical Nephroureterectomy with bladder cuff excision (RNU) versus patients who received Segmental Ureterectomy (SU) for localized urothelial carcinoma of the ureter in the National Cancer Database, 2004 to 2015. Figure 2 Overlap weighting-adjusted Kaplan-Meier analysis of overall survival in patients who received Radical Nephroureterectomy with bladder cuff excision vs. Segmental Ureterectomy for localized ureteral cancer. Number at risk from the non-adjusted population. Figure 3 Kaplan-Meier analysis of overall survival in patients who received Radical Nephroureterectomy vs. Segmental Ureterectomy, according to grade and clinical T stage. SU= Segmental Ureterectomy, RNU= Radical Nephroureterectomy, LG= Low-Grade, HG= High-Grade, cT= Clinical T Stage. cancers-15-01373-t001_Table 1 Table 1 Baseline characteristics of patients treated with Radical Nephroureterectomy with bladder cuff vs. Segmental Ureterectomy for localized ureteral carcinoma in unweighted and weighted population. Characteristic Unweighted Population, No. (%) Weighted Population, % Overall n = 13,061 RNU n = 9016 (69%) SU n = 4045 (31.0%) Standardized Difference (%) Overall RNU SU Standardized Difference (%) Mean (SD) Age, Years 71.9 (10.0) 71.8 (10.0) 71.9 (10.0) 1.1 71.9 71.9 72.0 0.8 Age categories, years <60 1559 (11.9) 1078 (12) 481 (11.9) -0.2 11.8 11.8 11.8 0 60-69 3441 (26.4) 2371 (26.3) 1070 (26.5) 0.4 26.4 26.4 26.4 0 70-79 4852 (37.2) 3379 (37.5) 1473 (36.4) -2.2 36.8 36.8 36.8 0 >80 3209 (24.6) 2188 (24.3) 1021 (25.2) 2.3 25.0 25.0 25.0 0 Gender, n (%) Male 8239 (63.1) 5544 (61.5) 2695 (66.6) 10.7 65.0 65.0 65.0 0 Female 4822 (36.9) 3472 (38.5) 1350 (33.4) -10.7 35.0 35.0 35.0 0 Race, n (%) White 12,104 (92.7) 8345 (92.6) 3759 (92.9) 1.4 92.8 92.8 92.8 0 Black 443 (3.4) 314 (3.5) 129 (3.2) -1.6 3.3 3.3 3.3 0 Other 399 (3.1) 271 (3.0) 128 (3.2) 0.9 3.1 3.1 3.1 0 Unknown 115 (0.9) 86 (1.0) 29 (0.7) -2.6 0.8 0.8 0.8 0 Comorbidity index, n (%) 0 8377 (64.1) 5791 (64.2) 2586 (63.9) -0.6 64.0 64.0 64.0 0 1 3281 (25.1) 2253 (25.0) 1028 (25.4) 1.0 25.2 25.2 25.2 0 2 1028 (7.9) 714 (7.9) 314 (7.8) -0.6 7.9 7.9 7.9 0 >=3 375 (2.9) 258 (2.9) 117 (2.9) 0.2 2.9 2.9 2.9 0 Insurance status, n (%) Private 3218 (24.6) 2180 (24.2) 1038 (25.7) 3.4 25.1 25.1 25.1 0 Medicaid 364 (2.8) 256 (2.8) 108 (2.7) -1.0 2.7 2.7 2.7 0 Medicare 9170 (70.2) 6351 (70.4) 2819 (69.7) -1.6 70.1 70.1 70.1 0 Uninsured 146 (1.1) 110 (1.2) 36 (0.9) -3.2 1.0 1.0 1.0 0 Unknown 163 (1.3) 119 (1.3) 44 (1.1) -2.1 1.2 1.2 1.2 0 Income, n (%) High 8054 (61.7) 5476 (60.7) 2578 (63.7) 6.2 62.6 62.6 62.6 0 Low 4932 (37.8) 3494 (38.8) 1438 (35.6) -6.6 36.8 36.8 36.8 0 Unknown 75 (0.6) 46 (0.5) 29 (0.7) 2.6 0.6 0.6 0.6 0 Education, n (%) High 8103 (62.0) 5491 (60.9) 2612 (64.6) 7.6 63.2 63.2 63.2 0 Low 4891 (37.5) 3483 (38.6) 1408 (34.8) -7.9 36.2 36.2 36.2 0 Unknown 67 (0.5) 42 (0.5) 25 (0.6) 2.1 0.5 0.5 0.5 0 Facility type, n (%) Academic 4563 (34.9) 2962 (32.9) 1601 (39.6) 14.0 37.1 37.1 37.1 0 Non-academic 8467 (64.8) 6035 (64.9) 2432 (60.1) -14.2 62.6 62.6 62.6 0 Unknown 31 (0.2) 19 (0.2) 12 (0.3) 1.7 0.3 0.3 0.3 0 Facility location, n (%) East 5766 (44.2) 4018 (44.6) 1748 (43.2) -2.7 43.7 43.7 43.7 0 Central 5546 (42.5) 3877 (43.0) 1669 (41.3) -3.5 41.8 41.8 41.8 0 West 1718 (13.2) 1102 (12.2) 616 (15.2) 8.7 14.3 14.3 14.3 0 Unknown 31 (0.2) 19 (0.2) 12 (0.3) 1.7 0.3 0.3 0.3 0 Facility county (%) Metro 10,531 (80.6) 7239 (80.3) 3292 (81.4) 2.8 80.9 80.9 80.9 0 Urban 1928 (14.8) 1348 (15.0) 580 (14.3) -1.7 14.7 14.7 14.7 0 Rural 255 (2.0) 190 (2.1) 65 (1.6) -3.7 1.7 1.7 1.7 0 Unknown 347 (2.7) 239 (2.7) 108 (2.7) 0.1 2.7 2.7 2.7 0 Facility distance (%) First 7158 (54.8) 5011 (55.6) 2147 (53.1) -5.0 54.1 54.1 54.1 0 Second 4194 (32.1) 2878 (31.9) 1316 (32.5) 1.3 32.2 32.2 32.2 0 Third 1653 (12.7) 1094 (12.1) 559 (13.8) 5.0 13.1 13.1 13.1 0 Unknown 56 (0.4) 33 (0.4) 23 (0.6) 3.0 0.5 0.5 0.5 0 Clinical T stage (%) <=T1 5158 (39.5) 3454 (38.3) 1704 (42.1) 7.8 40.9 40.9 40.9 0 T2 1079 (8.3) 741 (8.2) 338 (8.4) 0.5 8.3 8.3 8.3 0 T3 827 (6.3) 640 (7.1) 338 (4.6) -10.6 5.3 5.3 5.3 0 T4 92 (0.7) 74 (0.8) 18 (0.4) -4.7 0.5 0.5 0.5 0 Unknown 5905 (45.2) 4107 (45.6) 1798 (44.5) -2.2 45.0 45.0 45.0 0 Tumor size (%) <=2 cm 3675 (28.1) 2258 (25.0) 1417 (35.0) 21.9 32.4 32.4 32.4 0 >2 cm 6505 (49.8) 5065 (56.2) 1440 (35.6) -42.2 41.8 41.8 41.8 0 Unknown 2881 (22.1) 1693 (18.8) 1188 (29.4) 25.0 25.7 25.7 25.7 0 Tumor grade (%) Low grade 1975 (15.1) 1291 (14.3) 684 (16.9) 7.1 15.8 15.8 15.8 0 High grade 4392 (33.6) 3138 (34.8) 1254 (31.0) -8.1 32.3 32.3 32.3 0 Unknown 6694 (51.3) 4587 (50.9) 2107 (52.1) 2.4 51.8 51.8 51.8 0 Lymph vascular invasion (%) Not present 4488 (34.4) 3160 (35.1) 1328 (32.8) -4.7 33.6 33.6 33.6 0 Present 954 (7.3) 693 (7.7) 261 (6.5) -4.8 6.8 6.8 6.8 0 Unknown 7619 (58.3) 5163 (57.3) 2456 (60.7) 7.0 59.5 59.5 59.5 0 Chemotherapy (%) Not received 10,997 (84.2) 7530 (83.5) 3467 (85.7) 6.1 85.1 85.1 85.1 0 Received 1568 (12.1) 1142 (12.7) 426 (10.5) -6.7 11.1 11.1 11.1 0 Unknown 496 (3.8) 344 (3.8) 152 (3.8) -0.3 3.8 3.8 3.8 0 The standardized differences >=10% are indicated in bold. The standardized difference is indicated as "0" when <0.1% and > -0.1%. cancers-15-01373-t002_Table 2 Table 2 Multivariable logistic regression analysis examining factors associated with undergoing segmental ureterectomy compared to radical nephroureterectomy. Variable Hazard Ratio 95% Confidence Interval p-Value Age <60 Ref 60-69 1.06 0.92-1.22 0.433 70-79 1.04 0.89-1.21 0.637 >79 1.18 1.00-1.38 0.047 Gender Male Ref Female 0.81 0.75-0.88 <0.001 Race White Ref Black 1.00 0.81-1.25 0.970 Other 0.990 0.79-1.24 0.922 Unknown 0.76 0.49-1.17 0.209 Comorbidity index 0 Ref 1 1.09 1.00-1.19 0.061 2 1.03 0.89-1.19 0.656 >=3 1.06 0.84-1.33 0.632 Insurance status Private Ref Medicaid 0.88 0.69-1.13 0.315 Medicare 0.95 0.86-1.06 0.364 Uninsured 0.76 0.51-1.13 0.171 Unknown 0.79 0.55-1.15 0.217 Annual income High Ref Low 0.95 0.86-1.05 0.297 Unknown 2.87 0.68-1.21 0.150 Education High Ref Low 0.89 0.81-0.98 0.014 Unknown 0.15 0.02-1.28 0.083 Facility type Academic Ref Non-academic 0.75 0.69-0.82 <0.001 Unknown 1.39 0.66-2.95 0.386 Facility location East Ref Central 1.02 0.94-1.12 0.570 West 1.37 1.22-1.54 <0.001 Facility county Metro Ref Urban 0.96 0.84-1.09 0.488 Rural 0.77 0.57-1.05 0.098 Unknown 0.97 0.75-1.25 0.807 Distance First Ref Second 1.08 0.99-1.19 0.077 Third 1.16 1.01-1.33 0.040 Unknown 3.51 0.66-1.85 0.139 Year of diagnosis 2004/6 Ref 2007/9 1.08 0.97-1.21 0.157 2010/12 1.27 0.99-1.61 0.056 2013/15 1.18 0.92-1.51 0.190 Clinical T stage <= T1 Ref T2 0.99 0.85-1.15 0.885 T3 0.67 0.56-0.80 <0.001 T4 0.51 0.30-0.88 0.015 Unknown 0.93 0.86-1.02 0.120 Tumor grade Low grade Ref High grade 0.76 0.67-0.86 <0.001 Unknown 0.76 0.61-0.96 0.023 Tumor size <2 cm Ref >2 cm 0.45 0.42-0.50 <0.001 Unknown 1.12 1.01-1.24 0.027 Lymph vascular invasion Not present Ref Present 1.11 0.94-1.31 0.239 Unknown 1.32 1.15-1.51 <0.001 The p-values <= 0.05 are indicated in bold. cancers-15-01373-t003_Table 3 Table 3 Univariate Cox proportional hazards regression examining the risk of all-cause mortality in patients diagnosed with localized ureteral carcinoma after overlap weighting. Heading Hazard Ratio 95% Confidence Interval p-Value Type of Surgery Radical Nephroureterectomy 1 [REF] --- --- Segmental Ureterectomy 0.98 0.93-1.04 0.538 Disclaimer/Publisher's Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

PMC10000205

Large mammals can perceive humans as predators and therefore adjust their behavior to achieve coexistence with humans. However, lack of research at sites with low hunting intensity limits our understanding of how behavioral responses of animals adapt to different predation risks by humans. At Heshun County in North China, where hunting has been banned for over three decades and only low-intensity poaching exists, we exposed two large ungulates (Siberian roe deer Capreolus pygarus and wild boar Sus scrofa) to the sounds of humans, an extant predator (leopard Panthera pardus) and a control (wind), and examined their flight responses and detection probabilities when hearing different type of sounds. Both species showed higher flight probabilities when hearing human vocalization than wind, and wild boar were even more likely to flee upon hearing human vocalization than leopard roar, suggesting the behavioral response to humans can equal or exceed that of large carnivores in these two ungulates even in an area without hunting practices. Recorded sounds had no effect on detection probability of both ungulates. Additionally, with repeated exposure to sounds, regardless of treatment, roe deer were less likely to flee and wild boars were more likely to be detected, indicating a habituation-type response to sound stimuli. We speculate that the immediate flight behavior rather than shifts in habitat use of the two species reflect the low hunting/poaching pressure at our study site and suggest further examination of physiological status and demographic dynamics of the study species to understand human influence on their long-term persistence. human disturbance fear ecology acoustic cues Automated Behavioral Response (ABR) system playback experiment National Key Program of Research and Development2022YFF1301500 This research was funded by the National Key Program of Research and Development, Ministry of Science and Technology of China (grant number 2022YFF1301500). pmc1. Introduction Humans can function as an apex predator in ecosystems, killing wildlife at rates that equal or exceed that of non-human predators . Fear of humans is thought to be an important mechanism mediating human effects on wildlife behavior, including enhanced vigilance , reduction of animal movements and reduced activities in daylight . These changes in wildlife behavior have the potential of influencing the community structures and ecological functions within ecosystems through a trophic cascade . Despite a large body of evidence demonstrating humans' negative effects on wildlife habitat use , diurnal activity and foraging intensity , whether these effects result from animals' fearful response to humans as predators or response to general disturbances (e.g., sudden noise, quick-approaching object) still requires examination . Playback experiments, which expose animals to acoustic cues, provide a powerful and relatively easily-implemented method to investigate inter-specific interactions by quantifying an animal's response to acoustic stimuli . Playback experiments have been utilized to explore anti-predator responses of prey species, especially ungulate responses to large carnivores , and also wildlife responses to humans . Recent experiments have incorporated camera traps, which automatically record animal behavior, into playback experiments to overcome the potential bias induced by the presence of the observer in the traditional methods . This integrated method has been implemented on several mammal species including Puma concolor , Meles meles , Lynx rufus, Mephitis mephitis, and Didelphis virginiana , Odocoileus virginianus , and Alces alces . These studies have documented behavioral responses usually associated with fear of a predator, and substantiate that the presence of humans can mediate wildlife behavioral changes by creating a landscape of fear . The behavioral responses of wildlife to human activities can be highly variable , implying that the strength of the response is context-dependent. Animals' perceived risks from humans may be correlated to their probabilities of being killed by humans, as hunting can intensify their responses to human activities . For example, brown bears (Ursus arctos) in Scandinavia reduced diurnal activities only after the start of annual bear hunting season , and African elephants (Loxodonta africana) exhibit stronger responses to voices of adult men than women and youth . Animals may be able to assess the lethality of human activities and reduce their intensity of response to non-lethal activities. Caribou (Rangifer tarandus) in Svalbard reduced flight distances with increased number of approaches by the researcher , and flight probability of guanacos (Lama guanicoe) near the roads decreased progressively once poaching ceased in Argentina . Animals' ability to assess predation risks from humans can help them cope with human encounters, and avoid excessive costs of unnecessary behavioral responses . However, experimental tests of wildlife fear responses to humans have been largely limited to regions where human hunting is common (e.g., Suraci et al. , for mesocarnivores in Santa Cruz Mountains under predator control; Crawford et al. , for white-tailed deer in southwestern Georgia as game species), and studies in areas with low hunting intensity where wildlife may respond to humans differently are lacking. To document whether wildlife perceive humans as predators at sites with low hunting intensity, and how they behaviorally respond to human presence, we conducted a playback experiment by exposing two large ungulates (Siberian roe deer Capreolus pygargus and wild boar Sus scrofa) to sounds of humans, an extant large carnivore (leopard Panthera pardus), and wind as a control sound. Hunting has been banned for over three decades since the enactment of the Wildlife Protection Law, but poaching still exists in some parts of China. Our study site in Shanxi Province supports a single native large carnivore, the leopard, with a relatively high density (4.23 individuals/100 km2, 95% CI 2.82-5.64; ) compared to leopard populations in other parts of China. The landscapes here are also highly modified by human activities and densely occupied by humans, with poaching activity of low intensity. Both roe deer and wild boar are common (occupancy > 0.9) and are prey of leopards , showing no spatial avoidance to sites frequented by humans . Based on camera trap data recorded during the playback experiment, we examined the flight behaviors and detection probabilities of the two ungulates in response to continued broadcasting of the three types of sounds. We compared the relative strength of the behavioral responses of the two species to different sound stimuli and their gradual changes as the experiment progressed. We hypothesized that even at a very low poaching intensity and a high leopard density, fear of humans may outweigh fear of leopards in both ungulate species. 2. Materials and Methods 2.1. Study System We conducted this study in a temperate forest in Heshun County of Shanxi Province, China. Located in the central Taihang Montains of North China, the study area is surrounded by highly modified landscape . This area is characterized by rolling hills with elevation ranging from 1200-1700 m, and is mainly covered by secondary mixed forest consisting of coniferous and broad-leaved deciduous tree species. About 2000 residents from 9 villages live in this area, and engage in farming, cattle raising, and collecting of non-timber forest products (e.g., medical herbs, mushrooms, etc.). Residents from the county town (about 15 km from the study site with a total population about 55,000) also frequently visit this area. The poaching intensity is low, with less than 20 detections of hunters (e.g., people with shotgun or hunting dogs) recorded per year during the camera-trapping survey from 2017-2019 in a more extensive area encompassing our study site (56,000 camera-days from 123 sites in over 1500 km2) . 2.2. Playback Experiment We used an Automated Behavioral Response (ABR) system to measure the behavioral response of ungulates to a suite of sounds present within the landscape . The ABR system is composed of one infrared-triggered camera and one waterproof loudspeaker, powered by a 10,000 mAH lithium battery and a solar panel. The loudspeaker is connected to the camera and can be triggered by the heat sensor of the camera. Thereby, warm-blooded animals passing by the ABR trigger the heat sensor of the camera, and the speaker then plays a random 30-s sound clipping that is stored in the ABR while the camera simultaneously records a video of the same length. We extracted 8 clippings for each sound type (e.g., the sound of man reading, leopard roaring, or wind blowing) from videos downloaded from YouTube (www.youtube.com [accessed on 5 December 2021]; searched by "Chinese reading" in Chinese, "leopard roaring" and "wind blowing", respectively) and edited all for consistency of amplitude and clipped them to 30 s. The sound clips were broadcasted at a consistent mean sound pressure of 70 dB (measured at 1 m from the ABR system using a decibel meter app on a iPhone 12 [Apple Inc., Copertino, CA, USA]), following the settings used in previous studies . We selected 36 experimental sites in mixed forests and placed one ABR (supplied by Qingdao Yequ Nature Technology Co., Ltd., Qingdao, China) at each site . Each ABR was attached to a tree at height of 60-80 cm above ground along the trail to maximum the detection of the two study species. Before being set, each ABR was tested to assure that they could be triggered and broadcasted the sound clips at the same amplitude. To eliminate the potential bias induced by variation among sites, we employed a repeated-measure design following Suraci et al. . The 36 sites were evenly split into three groups, each group receiving a different type of treatment (human, leopard or wind) in one experimental period, and switched to the other two treatments for the next two experimental periods. Thus, three types of sounds were broadcasted at all sites by the conclusion of the three experimental periods, while the sounds played at each group of sites differed during the same experimental period. Sites from different treatments were separated by at least 1 km to avoid mutual interference. The average home range of roe deer and wild boar at similar sites were estimated (95% minimum convex polygons) as 1.9 km2 and 1.7 km2, respectively . Thus, site separation of 1 km also reduced the probability of the same individual being subjected to different sound treatments in the same experimental period. Within a treatment group, cameras were separated by at least 250 m. The experiment was conducted from December 2021 to June 2022, with a variable time length among experimental periods (40, 82 and 83 days for period 1, 2 and 3, respectively) due to COVID-19 restrictions. 2.3. Statistical Analysis We used a logistic regression model with mixed effects to examine the effects of different types of recorded sounds on the immediate behavior and habitat use of the study species during the study period. For the immediate behavior, we measured the flight probability of the individual after hearing the sound. We grouped detections of the same species at the same site within 30 min into one independent detection. For each independent detection, we determined whether the ungulate fled away from the experimental site (denoted as 1 and 0, respectively) in response to the broadcast. A social group was defined as fleeing if any individual demonstrated a flight response. We measured the habitat use of the study species by dividing the experiment period into sequential one-week periods from each site when the ABR system was operational (a site-week, hereafter) and assessed whether roe deer or wild boar were detected or not in each site-week (denoted as 1 and 0, respectively). We hypothesized that repeated exposure to the recordings would alter a species' use of the experimental site, leading to changes in their overall detection probability. We used the logistic regression for our analysis as formal occupancy model did not meet convergence due to the high naive occupancy rate of the study species (1.00 and 0.97 for roe deer and wild boar, respectively). For our dependent variable we used detection probability during each site-week, rather than the count of independent detections, as the study species were rarely detected more than once during a site-week. For both analyses (i.e., flight probability and detection probability), we incorporated three variables as fixed effects, including type of recorded sounds (Treatment), number of days (for flight response) or weeks (for site-week detection probability) from the beginning of each experimental period (Length), and the ID of the experimental period (Period). We used leopard call rather than wind sound as the reference level for Treatment to compare whether human effects differed from leopard effects. As the univariate additive models (GAMM) indicated no nonlinear correlation between Length and the probability of flight and detection (estimated degree of freedom = 1), we did not incorporate any higher order of terms for Length in the subsequent analysis. For each analysis, we formulated five candidate models, each with a different hypothesis (Table 1), and also included one model incorporating the interaction term between Treatment and Period to examine whether the potential effect of the treatment varied across experimental periods. Following Crawford et al. , the experimental site was used as the random effect for each candidate model to account for potential correlations among independent detections or site-week detections at the same site. We fitted all candidate models for both flight and detection probability in the two study species separately using the package "lme4" in the R environment and ranked them with Akaike information criteria (AICc) . Models with ^AICc < 2 were considered as top models with similar performance, and we defined the top model with least variables as the best model following the principle of parsimony . We validated the predictive capacity of the models using area under the curve (AUC), and examined potential spatial and temporal autocorrelation of the Pearson residuals using the graphical diagnostic for autocorrelation function, following Zuur et al. . 3. Results Three ABR units was lost during the experiment (two in period 2, one in period 3), and we also excluded records when the ABR unit malfunctioned during the site-week. In total, we obtained data from 5909 camera-days in which the ABR was operational (36, 34, and 33 operational sites with 1287, 2665 and 1957 camera-days in period 1, 2 and 3, respectively). During the entire study period we obtained 255 and 224 independent detections for roe deer and wild boar, respectively. For the flight response, roe deer were more likely to flee upon hearing sounds of leopards than wind (b +- se = -1.52+- 0.37, p < 0.001), and exhibited a similar probability of flight upon hearing sounds of human and leopards (b +- se = 0.72 +- 0.42, p = 0.083) . The flight probability of roe deer reduced as the experiment progressed (b +- se = -2.21 +- 0.58, p < 0.001) . For wild boar, three top models were considered equivalent (^AIC < 2), with the best model incorporating only Treatment (Model 1) indicating that wild boar were more likely to flee upon hearing human sounds than the sounds of a leopard (b +- se = 0.94 +- 0.43, p = 0.031), and also were more likely to flee upon hearing sounds of a leopard than the sounds of wind (b +- se = -1.36 +- 0.43, p = 0.002) . For shifts in habitat use during the course of the study, no model was a better predictor of detection of roe deer than the null model. Model 2 had similar performance to the null model (^AIC = 1.87), and indicated that Length had no significant effect . For wild boar, the best model (model 2) indicated that their detection probability increased as the experiment progressed (b +- se = 1.00 +- 0.36, p = 0.006) . Model 3 that incorporated Treatment had similar performance with the best model, but the effect of treatment was not significant (p = 0.20 for human, p = 0.49 for wind). The experimental period and its interaction term with treatment had no significant effect on either the flight or detection probability of roe deer and wild boar (Table 2 and Table 3), indicating that the results were stable among the different periods. AUCs indicated adequate predictive capacity of the best logistic regression models (ranging from 0.71 to 0.84), and we found no significant pattern in the auto-correlation functions for the residuals , indicating no evidence for spatial or temporal autocorrelation in the best models. 4. Discussion Despite numerous studies focusing on human effects on ungulate spatio-temporal behaviors, as well as vigilance and foraging intensity , experimental evidence for their fear and behavioral responses to humans as predators is rare (but see Crawford et al. , Bhardwaj et al. and Widen et al. ). Our results showed that both ungulates had higher flight probabilities upon hearing human sounds than wind, suggesting that the two species exhibit anti-predator responses and actively avoid encounters with humans in our study area. These results complement the hypothesis that presence of humans can create a landscape of fear in some components of the mammal community, manifested in widespread behavioral changes . We also found that roe deer and wild boar had similar or higher flight probabilities upon hearing human than leopard sounds at our study site. This result indicates that humans can trigger equal or even stronger behavioral responses in the two ungulates than large carnivores even with low levels of hunting, similar to the ability of large carnivores to induce significant anti-predator behaviors at low population densities . Our study also showed that flight response to human cues was a more sensitive response than spatial avoidance of humans as we found no variation in site use between treatments for the two ungulates, which is also consistent with our previous findings that roe deer and wild boar exhibit no spatial avoidance to sites frequented by humans but avoid direct encounters with humans in our study area . Anti-predator behaviors that animals exhibit are results of tradeoffs between energy expenditure, resource acquisition and predation risks . Shifts in habitat use come at the cost of giving up potential resources from suitable habitats (e.g., food), so they are weighed against the benefits of giving up human-occupied sites to reduce human-caused mortalities . Previous studies have shown that in areas where humans are the leading predator, such as Scandinavia and Southwest Georgia, U.S.A., ungulates significantly reduce the use of sites with human sound broadcasting . At our study site where hunting intensity was low, flight response seems to be a more cost-efficient behavior in response to human cues, allowing the ungulates to use human-frequented sites while reducing their probability of being hunted. Combined with findings from previous experiments in areas with hunting practices, our results demonstrate the ability of ungulate species to assess the predation risks by humans and respond accordingly, which may be a pivotal trait of species to sustain themselves in human-dominated landscapes. Roe deer and wild boar also exhibited increased flight probability when hearing leopard sounds as compared to hearing wind sounds. Moreover, wild boar were more likely to flee when they heard human sounds than when they heard leopard sounds, while roe deer exhibited similar flight probabilities when they heard both sounds. Since there was no evidence of different hunting pressure by poachers on these two species in our study area, we speculate that this difference was due to wild boar experiencing higher hunting pressure from humans than leopards, as wild boars are not a preferred prey of leopards compared to roe deer . Additionally, although the amplitude of the sound clips were controlled in the experiment, the perception of the sounds could still differ between roe deer and wild boar because of their different frequency range of hearing, which may also lead to bias in the comparison between behavioral responses of roe deer and wild boar. Neither of the two ungulates avoided sites with leopard sounds. This is contrast to our previous studies based on camera-trap data showing that roe deer avoid sites frequented by leopards , possibly due to the lack of synergy between sounds and other cues (e.g., olfactory and visual) in this experiment as ungulates rely on multiple senses to detect predators . The potential synergy and relative importance of different types of cues (audio, olfactory and visual) in triggering prey's behavioral responses to predators is still unclear, and further study examining ungulates' responses to different combinations of cues would help to fill this gap. Both ungulates fled when hearing wind sounds, although the flight probability was lower than when hearing human and leopard sounds. Similar results (i.e., behavioral responses to sounds as control) have been widely found in previous playback experiments , which may be due to the suddenness of the sound appearance, though it does not convey any risk-related information. Similarly to a previous experiment , our study also showed reduced responses with repeated exposure to the playback. These habituation-type responses in the two ungulates may be due to increased endurance to the sounds or the expulsion of low-tolerant individuals . The use of predator cues has been used as a proxy for predator presence to examine cascading effects mediated by anti-predator behaviors and to reduce human-wildlife conflict . Our results emphasized the necessity of accounting for habituation in long-term experiments aimed at quantifying demographic changes at population level or wildlife management through the use of predator cues. 5. Conclusions As human disturbances within ecosystems increase , understanding the underlying mechanisms that mediate wildlife behavioral responses is pivotal to achieving human-wildlife co-existence. Our results suggest that human acoustic cues triggered flight responses, but did not change spatial use in our two study ungulate species that shared habitats with humans, which is different from previous findings in areas where the human is a major predator. Our previous study has also shown that roe deer and wild boar exhibit spatial-temporal segregation to avoid direct encounters with humans . These changes in behavior could be a reflection of the low-intensity hunting activities in our study area; whether they affect the fitness of either animal is unclear. Direct comparison of how wildlife perceive risks from humans in areas with and without hunting activities could help to understand the potential mechanisms that mediate the human effect on wildlife behaviors. We suggest further research that integrates an examination of behavioral responses, physiological status, and demographic dynamics of wildlife persisting in shared habitats with humans to understand human impacts on their future sustainability. Acknowledgments We sincerely thank the local government of Heshun County for providing permission and assistance for this study. We thank all the volunteers and local field staff, especially Li Yudong, and Chinese Field Conservation Alliance (CFCA) for their great contributions to the experiments. We thank all the staff from Qingdao Yequ Nature Technology Co., LTD, especially Yi Haiping, Fei Fan and Deng Zhimin for their manufacturing and maintenance of the ABR systems. We also want to thank Kong Yueqiao for the painting in Figure 1b. Supplementary Materials The following supporting information can be downloaded at: Figure S1: Spatial and temporal auto-correlation functions (ACF) of the residuals of the best logistic regression models for the flight probability; Figure S2: Spatial and temporal auto-correlation functions (ACF) of the residuals of the best logistic regression models for the detection probability. Click here for additional data file. Author Contributions Conceptualization, M.L.; methodology, M.L.; software, M.L.; validation, S.L. and X.S.; formal analysis, M.L.; investigation, M.L., Y.W. and F.X.; resources, M.L., Y.W. and F.X.; data curation, M.L., Y.W. and F.X.; writing--original draft preparation, M.L.; writing--review and editing, M.L., W.J.M., S.L. and X.S.; visualization, M.L.; supervision, W.J.M., S.L. and X.S.; project administration, W.J.M., S.L. and X.S.; funding acquisition, S.L. All authors have read and agreed to the published version of the manuscript. Institutional Review Board Statement We did not obtain ethical approval because neither of the study species are listed as protected species in China and the experiment was conducted outside of any protected area, only involving simulation of disturbances that actually exist on a regular basis. Informed Consent Statement Not applicable. Data Availability Statement The dataset used for generating this analysis is openly available in ResearchGate (DOI: 10.13140/RG.2.2.13631.38560; available at: upload on 21 January 2023). For details about the dataset, please contact M.L. at liumingzhang@pku.edu.cn. Conflicts of Interest The authors declare no conflict of interest. Figure 1 Location of the study area (a), experimental procedure (b) and the 36 experimental sites of three groups within the study area (c). Human modification index in Figure 1 (a) (obtained from Kennedy et al. ) ranges from 0 to 1 (very low: 0-0.1; moderate: 0.1-0.4; high: 0.4-0.7; very high: 0.7-1.0). Figure 2 (a) Roe deer: flight probability across treatments and correlations between flight probability and time length of experiment; (b) wild boar: flight probability across treatment. Shaded areas and vertical bars indicate the 95% confidence intervals of the predicted values. Figure 3 Correlations between occurrence probability of roe deer and wild boar and time length of experiment. Predicted values were obtained by model 2 for roe deer which had comparable performance to the null model (the best model), but indicating a non-significant effect of Length on detection probability. Shaded areas indicate the 95% confidence intervals for the predicted values. animals-13-00845-t001_Table 1 Table 1 Formula and corresponding hypothesis for the candidate model. ID Formula Hypothesis 1 ~Treatment Ungulates exhibit different behavior in response to sounds of human, leopard and wind. 2 ~Length Ungulates exhibit progressive changes in their intensity of response to the sounds with increased exposure. 3 ~Treatment + Length 1 + 2; The intensity of the response does not vary among type of sounds with increased exposure. 4 ~Treatment x Length 1 + 2; The intensity of response varies among type of sounds with increased exposure. 5 ~Treatment x Period 1; The intensity of response varies among experimental periods. 6 ~1 Null model animals-13-00845-t002_Table 2 Table 2 Rank of logistic regression models for flight probability of roe deer and wild boar based on AICc. ID Formula df logLik AICc Delta Weight Roe deer 3 Treatment + Length 5 -141.51 293.25 0.00 0.80 4 Treatment x Length 7 -140.81 296.07 2.81 0.20 1 Treatment 4 -149.69 307.54 14.28 0.00 5 Treatment x Period 10 -146.53 313.96 20.70 0.00 2 Length 3 -162.40 330.89 37.63 0.00 6 Null 2 -168.05 340.14 46.88 0.00 Wild boar 1 Treatment 4 -128.90 265.99 0.00 0.46 4 Treatment x Length 7 -126.30 267.11 1.13 0.26 3 Treatment + Length 5 -128.74 267.76 1.77 0.19 5 Treatment x Period 10 -124.16 269.35 3.36 0.09 6 Null 2 -144.16 292.37 26.39 0.00 2 Length 3 -144.16 294.42 28.43 0.00 animals-13-00845-t003_Table 3 Table 3 Rank of logistic regression models for detection probability of roe deer and wild boar based on AICc. The best model is shown in bold. df logLik AICc Delta Weight Roe deer 6 Null 2 -412.02 828.06 0.00 0.53 2 Length 3 -411.95 829.93 1.87 0.21 1 Treatment 4 -411.24 830.52 2.47 0.15 3 Treatment + Length 5 -411.13 832.34 4.28 0.06 5 Treatment x Period 10 -406.59 833.46 5.41 0.04 4 Treatment x Length 7 -410.58 835.30 7.24 0.01 Wild boar 2 Length 3 -354.57 715.18 0.00 0.59 3 Treatment + Length 5 -353.34 716.76 1.58 0.27 4 Treatment x Length 7 -352.43 718.99 3.82 0.09 6 Null 2 -358.36 720.73 5.55 0.04 1 Treatment 4 -357.25 722.55 7.37 0.01 5 Treatment x Period 10 -353.07 726.42 11.24 0.00 Disclaimer/Publisher's Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

PMC10000206

Previous studies have demonstrated that the strains Enterococcus gallinarum L1, Vagococcus fluvialis L21 and Lactobacillus plantarum CLFP3 are probiotics against vibriosis or lactococosis in sea bass or rainbow trout. In this study, the utility of these bacterial strains in the control of saprolegniosis was evaluated. For this purpose, both in vitro inhibition studies and competition for binding sites against Saprolegnia parasitica and in vivo tests with experimentally infected rainbow trout were carried out. In the in vitro tests, the three isolates showed inhibitory activity upon mycelium growth and cyst germination and reduced the adhesion of cysts to cutaneous mucus; however, this effect depended on the number of bacteria used and the incubation time. In the in vivo test, the bacteria were administered orally at 108 CFU g-1 in the feed or at 106 CFU ml-1 in the tank water for 14 days. None of the three bacteria showed protection against S. parasitica infection either through water or feed, and the cumulative mortality reached 100% within 14 days post infection. The obtained results show that the use of an effective probiotic against a certain disease in a host may not be effective against another pathogen or in another host and that the results obtained in vitro may not always predict the effects when used in vivo. biocontrol probiotics saprolegniosis inhibition assays Oncorhynchus mykiss Spanish "Ministerio de Economia y Competitividad"AGL2014-54683-R European Regional Development Fund (ERDF)This study was funded through research project AGL2014-54683-R of the Spanish "Ministerio de Economia y Competitividad" and cofinanced through the European Regional Development Fund (ERDF). C.G.P. was awarded a predoctoral contract by the "Consejeria de Educacion" of the regional government "Junta de Castilla y Leon" cofinanced through the European Social Fund. pmc1. Introduction Saprolegniosis is a fungal disease, the main pathogenic agent of which is Saprolegnia parasitica, that can cause heavy economic losses in inland fish farms due to depletion of fish stock and eggs . This disease affects freshwater fish at any stage of development and is characterized by skin lesions such as patches with a cotton wool-like appearance. Current prevention and treatment measures for saprolegniosis rely on the use of chemicals products that may cause damage to the environment and humans . In addition, none of the available compounds offers enough protection after the rearing period . Recently, metal-based nanoparticles have attracted attention as a potential material for prevention and control of saprolegniosis . Among the biological control alternatives to chemotherapy is the use of plant extracts or probiotics . In this sense, a number of in vitro studies have evidenced that some bacterial strains can inhibit Saprolegnia spp., including Pseudomonas fluorescens , Alteromonas sp., Pseudomonas alcaligenes, Pseudomonas saccharophila, Aeromonas caviae and Aeromonas eucrenophila , Serratia marcescens , Bacillus subtilis , Aeromonas media , Aeromonas sobria, Pantoea agglomerans, Serratia fonticola, Xhantomonas reflexus and Yersenia kristensenii , Lactobacillus plantarum and Pseudomonas aeruginosa . However, their action in vivo has only been studied in eel Anguilla australis , silver perch Bidyanus bidyanus and eggs of rainbow trout Oncorhynchus mykiss or eggs of gourami Osphonemus gouramy . In previous studies by our research group, various bacterial isolates with an in vitro ability to inhibit the growth of S. parasitica were obtained . Their harmless nature with respect to rainbow trout (Oncorhynchus mykiss) and their ability to adhere the cutaneous mucus and reduce the adhesion of zoospores and cysts of S. parasitica were also demonstrated . It was also found that of the fifteen investigated isolates, under experimental conditions, two isolates of Pseudomonas fluorescens reduced infection by S. parasitica in rainbow trout when these bacteria were added to the water in the tanks , and the mode of action of these bacteria was likely associated with the production of siderophores . Enterococcus gallinarum L1 (obtained from Dicentrarchus labrax gut content) and Vagococcus fluvialis L21 (obtained from Solea solea gut content) demonstrated high levels of protection against Vibrio anguillarum in European sea bass (Dicentrarchus labrax) after an experimental challenge when these bacteria were added to feed for 20 days . In the same way, Lactobacillus plantarum CLFP-3 (obtained from O. mykiss cutaneous mucus) added to the diet of rainbow trout for 36 days was demonstrated to improve protection against Lactococcus garvieae . In the present study, the in vitro inhibitory activity of these probiotic bacteria against S. parasitica was determined, and in vivo tests with experimentally infected rainbow trout were carried out in order to investigate their potential use for the biocontrol of saprolegniosis. 2. Materials and Methods 2.1. Collection of Isolates Used in the Study Three probiotic bacteria (Enterococcus gallinarum L1, Vagococcus fluvialis L21 and Lactobacillus plantarum CLFP3) previously obtained by our research group that were stored at -80 degC in our laboratory facilities were used in this study (Table 1). The first two have shown their efficacy against infection by Vibrio aguillarum in sea bass (Dicentrarchus labrax), and L. plantarum reduced mortality in rainbow trout (O. mykiss) infected with Lactococcus garvieae. After thawing, the bacterial isolates E gallinarum L1 and V. fluvialis L21 were cultured overnight at 20 degC in 3 mL of brain-heart infusion broth (BHIB, Pronadisa, Condalab, Madrid, Spain), while L. plantarum CLFP3 was cultured in Man, Rogosa and Sharpe broth (MRS, Pronadisa). An aliquot of 500 mL of the culture was inoculated into 25 mL of BHIB or MRS broth and incubated at 20 degC and 200 rpm on a rotary orbital shaker (Innova(r) 44, New Brunswick Scientific, Edison, NJ, USA) until the middle of the exponential growth phase (based on previously established growth curves). Bacteria were pelleted and washed twice by centrifuging at 1000x g for 15 min and resuspended in sterile saline solution. The number of bacteria was adjusted to the desired concentration according to the experiment in which they were used (see below) by counting in a hemocytometer chamber (Improved Neubauer Type, Albert Sass, Germany). The strain Saprolegnia parasitica TRU12 isolated from a wild brown trout (Salmo trutta) with saprolegniosis and maintained refrigerated in our laboratory was used. It was cultured for 3 d at 20 degC on glucose peptone (GP) agar; then, autoclaved half hemp seeds of Cannabis sativa were placed on the edges of the colony, followed by an additional 24 h of incubation at 20 degC. To obtain zoospores, the half hemp seeds colonized by hyphae were placed in Petri dishes with filtered autoclaved river water. After 36-48 h at 20 degC, the water was filtered through Whatman 541 cellulose filter paper. The number of zoospores was then estimated using a Hawksley Cristalite BS 748 counting chamber. 2.2. In Vitro Inhibition Assays of the Probiotic Bacteria against S. parasitica To determine the in vitro inhibition of the probiotic bacteria strains against S. parasitica, different assays were performed. All these experiments were conducted following the procedures described in a previous study . 2.2.1. Inhibition of Hyphal Growth on Solid Media (Plate Assay) This test was performed in with BHI agar (Cultimed, Panreac Quimica SLU, Barcelona, Spain), whereby each bacterium was streaked twice. This assay was performed in triplicate with incubations at 3, 5 or 7 d at 20 degC. Then, a 3 mm diameter block of agar with young hyphal tips of S. parasitica was placed between the 2 bacterial streaks and incubated for 3 d at 20deg C, measuring the diameter of the S. parasitica colony. A negative control plate was used without bacterial streaks under the same conditions. 2.2.2. Inhibition of Hyphal Growth from Colonized Hemp Seeds and Cyst Germination in Liquid Media Bacterial dilutions, hemp seeds colonized by Saprolegnia and zoospore suspensions were prepared following the methods described above. The bacterial concentration was adjusted to 2 x 105 cells mL-1, and, taking this as the first dilution, four 10-fold dilutions of bacterial suspension were prepared. The concentration of zoospores was adjusted to 4 x 104 zoospores mL-1. For the inhibition of hyphal growth, duplicate wells of a 24-well tissue culture plate (Falcon, Corning, Glendale, AZ, USA) filled with 1 mL of each bacterial dilution, 1 mL of BHIB and a half hemp seed colonized by S. parasitica were dispensed into each well. For the inhibition of zoospores/cyst germination, the same procedure was followed, with the only difference being that 0.5 mL of the zoospore suspension (4 x 104 zoospores mL-1) was added in place of a colonized hemp seed, and 0.5 mL of each bacterial dilution was used instead of 1 mL. In both assays, plates were incubated for 3 d at 20 degC, and bacteria and S. parasitica zoospore negative controls were included on each plate. The presence or absence of macroscopic or microscopic hyphal growth and germination of cysts were observed by a Nikon Diaphot inverted microscope. 2.2.3. Assay to Evaluate Fungicidal Effects The initial step was the same as that for the hemp seed test, but the plates were placed in an incubator at 20 degC for 1, 2 or 3 d. Following each incubation period, the hemp seeds from the wells where the growth of S. parasitica was inhibited were removed, washed 3 times with sterile distilled water and transferred to a Petri dish containing 20 mL of filtered and autoclaved river water with streptomycin (2 x 102 mg L-1) and penicillin (2 x 105 UI L-1). The plates were incubated for 20 d at 20 degC. To avoid false-positive results due to inhibitory effects of viable bacteria in the mycelium, only the results with no bacterial growth in water samples collected after 24 h were accepted. A negative control was achieved by incubating a hemp seed colonized by S. parasitica in water both with and without antibiotics. The bacteria were considered to have a fungicidal effect if no mycelial growth was observed after 20 d. 2.2.4. Inhibition Assays with Bacterial Culture Supernatants The supernatants used to perform this assay were prepared according to Lategan et al. using BHIB for L1 and L21 or MRS for CLFP3 strains. This supernatant and three 2-fold serial dilutions were tested to analyze the ability of the supernatants to inhibit hyphal growth (hemp-seed test) and to evaluate the capacity of the supernatants to inhibit cyst germination (cyst test). In the hemp-seed test, 2 mL of each supernatant dilution and a half hemp seed colonized by S. parasitica were dispensed in duplicate into each well of a 24-well tissue culture plate. In the cyst test, 1 mL of each supernatant dilution and 1 mL of the zoospore suspension (4 x 104 zoospores ml-1) were added to each well. Incubation times and observation procedures were the same as those described in Section 2.2.2. Negative controls with only culture medium were included in both assays. 2.3. Adhesion to Skin Mucus of the Probiotic Bacteria and Inhibition of S. parasitica Cyst Adhesion The adhesion capacity of the probiotic strains L1, L21 and CLFP3 to cutaneous mucus of trout and its potential to hinder the adhesion of S. parasitica cysts under conditions of exclusion, competition and displacement were investigated using the methods described by Carbajal-Gonzalez et al. . Cutaneous mucus was obtained from four male brown trout (Salmo trutta) with an average body weight of 196.7 +- 39.5 g from a hatchery belong to the Regional Government of Castile and Leon by scraping the surface with a plastic spatula. The mucus was centrifuged twice at 12 000x g for 5 min at 4 degC to eliminate particulate and cellular debris. The protein concentration was adjusted to 0.05 mg ml-1 in PBS using Bradford reagent (Sigma-Aldrich, Merck Life Science, Madrid, Spain). The resulting mucus suspension was finally sterilized by UV light exposure for 30 min and stored in aliquots at -20 degC until use. Bacteria and cysts of S. parasitica were stained with Syto 9(r) green fluorescent nucleic acid stain (Invitrogen, Fisher Scientific, Madrid, Spain) at a concentration of 1 mL per 109 bacteria in 1 mL and 1 mL per 105 cysts in 1 mL. 2.3.1. Adhesion to Skin Mucus of the Probiotic Bacteria This assay was performed in 96-well black polystyrene plates (Costar, ImmunoChemistry Technologies, Davis, CA, USA) coated with cutaneous mucus. In the adhesion assay with the probiotic strains, wells without coating or coated with BSA (0.05 mg ml-1, Sigma-Aldrich) or mucin from swine stomach type II (MSS; 0.05 mg ml-1, Sigma-Aldrich) were used as controls for non-specific adhesion. To coat the plates, 25 mL of mucus, BSA or mucin and 75 mL of coating buffer (16.8 g sodium hydrogen carbonate, 21.2 g sodium carbonate per liter, pH 9.6) were added to each well and left overnight at 4 degC. Subsequently, the wells were washed with PBS-Tween 20 (0.1%), the fluorescently labelled bacterial solution (25 mL of 109 bacterial cells ml-1) was added and plates were centrifuged at 163x g for 12 s to promote the adherence of bacteria to the mucus. Bacterial fluorescence was measured with a spectrophotometer (Synergy HT Multi-Detection Microplate Reader, Bio-Tek(r), Winooski, VT, USA) at 485 nm excitation and 535 nm emission after 30 min of incubation in darkness and at room temperature (time 0). Non-attached bacteria were removed by washing with saline solution (50 mL/well), and plates were placed upside down on absorbent paper and centrifuged to remove any remaining liquid. Then, 50 mL of saline solution was added per well, and a new measurement was performed. Adhesion was expressed as the percentage of fluorescence recovered in the second measurement relative to the fluorescence at time 0. 2.3.2. Inhibition of S. parasitica Cyst Adhesion Seven serial 10-fold dilutions were performed from non-stained bacteria (2 x 109 cells mL-1) in saline solution. The cysts were stained using the method mentioned previously using a concentration of 105 cysts mL-1. In the exclusion test, each dilution of the bacterial isolates was added to the wells, and after being incubated and washed, the labelled cysts were added. In the competition test, both bacterial dilution and stained cysts were added simultaneously, and in the displacement test, the labelled cyst suspension was first added, incubated and washed; then, each dilution of the bacterial suspension was added. Plates were washed with saline solution after 60 min of incubation at room temperature for each condition; then, the fluorescence was recorded. Wells with saline solution instead of bacterial cells were used as controls in this assay. The percentage of cysts bound to these control wells was considered the reference value (100%), and the percentage of cyst adhesion in the presence of bacteria was compared to this reference value. 2.3.3. Statistical Analysis In the adhesion assays, statistical analyses were carried out using SPSS for Windows version 26 (IBM SPSS Statistic, Armonk, NY, USA) by the Student's t-test (p <= 0.05). Data were shown as the mean +- standard deviation of three separate trials. 2.4. Pathogenicity for Rainbow Trout The probiotic bacterium CLFP3 (L. plantarum), which has previously been used successfully to prevent lactococcosis in trout , was obtained from rainbow trout. However, the L1 (E. gallinarum) and L21 (V. fluvialis) bacteria were obtained from marine fish, and although they were previously reported to be non-pathogenic for sea bass , their potential pathogenicity for rainbow trout is unknown. Therefore, prior to being used in in vivo tests, experimental inoculations with L1 and L21 isolates were conducted to verify their lack of pathogenicity for rainbow trout. Rainbow trout (O. mykiss) from a commercial fish farm with an average body weight of 37.24 +- 7.56 g were acclimatized for 10 days in a 120 L tank and observed daily to ensure that they did not show clinical signs of disease. Trials were conducted using groups of 20 fish kept in 40 L tanks filled with chlorine-free well water with a renewal rate of 1.5 L per h at 12 degC with constant aeration and a photoperiod of 12/12 h. Effluent water was disinfected with ozone. The experimental protocol was approved by the Subcommittee for Experimentation and Animal Welfare of the University of Leon, Spain (protocol number ULE_04_2015). The bacterial isolates were grown in BHIB as described in Section 2.1 to obtain a suspension of 107 cells mL-1. Fish were anaesthetized with tricaine methanesulfonate (MS-222, 50 mg mL-1) and injected with 0.1 mL intraperitoneally (ip) and intramuscularly (im) in two separate groups of 20 trout each. Control groups of 20 trout were inoculated with 0.1 mL of saline solution at the same time points. Fish were observed for 10 days and euthanized by overdosing them with MS-222 (100 mg mL-1) at the end of the observation period. They were subsequently examined for evidence of disease (i.e., gross lesions). Additionally, loopfuls of material from the kidney, liver and spleen were spread over plates of BHI agar with incubation at 20 degC for 3 days to determine the presence or absence of the inoculated bacterial isolates in the fish. The recovered bacterial isolates were examined by means of Gram-stained smears and basic biochemical tests such as catalase and oxidase tests. Temperature and other physicochemical parameters of water were measured daily, and no significant differences were observed between the different groups (temperature, 12.47 +- 0.24 degC; pH 7.92 +- 0.06; dissolved oxygen, 9.72 +- 0.16 mg L-1; nitrites (NO2), 0.034 +- 0.005 mg L-1; and non-ionized ammonia (NH3), 0.059 +- 0.005 mg L-1). 2.5. Biocontrol of Saprolegniosis by Adding Bacteria to Water or Feed 2.5.1. Biocontrol by Adding Bacteria to Water For each of the tested bacteria, 60 rainbow trout with an average body weight of 28.79 +- 10.01 g in three 40 L tanks were used: 20 rainbow trout for treatment with the probiotic and 2 as controls for infection and ami momi treatment, respectively. Fish were infected with S. parasitica TRU12 following the method described previously by Fregeneda-Grandes et al. . Briefly, the fish were slightly scarified on the skin using a variation of ami momi treatment . Groups of 10 fish were shaken for 2 min in a cylindrical stainless-steel colander with a mesh of 7 mm before adding a suspension of S. parasitica zoospores to the water to obtain a final concentration of 3 x 102 spores mL-1 in the tank water. At the same time, a bacterial suspension of 106 cells ml-1 in PBS was added. During the next 48 h, the flow of water was halted and then resumed. The bacterial treatment was repeated every 24 h for 14 days, halting the flow of water for 6 h. The bacterial suspension was replaced by the same volume of sterile PBS in the infection control tank, while neither bacteria nor zoospores were added to the tank to control for death caused by ami momi treatment. The fish were monitored under observation for 14 days to detect the emergence of signs of saprolegniosis. 2.5.2. Biocontrol by Adding Bacteria to Feed To conduct these experiments, three groups of 20 rainbow trout with an average body weight of 29.29 +- 10.33 g were maintained in 40 L tanks: one tank for the bacterial treatment and two tanks as controls. Fish were fed for 14 days with T2 Optiline 1P (Skretting(r), Burgos, Spain) feed containing 108 bacteria g-1 feed, with a daily administration of 2% of tank biomass, followed by a fast of 48 h and 24 h before and after infection with S. parasitica, respectively. After infection, the feed with the bacteria continued to be administered for another 10 days. To prepare the feed with the probiotics, 10 mL of a bacterial suspension with a concentration of 109 cells mL-1 was added to 90 g of feed, and the mixture was homogenized with an electric hand mixer (HM 3100, Braun GmbH, Germany) for 1 min. Subsequently, the feed was allowed to dry for 30 min at room temperature in a laminar flow cabinet. Infection with S. parasitica zoospores was performed as described above, and water flow was halted for the subsequent 24 h. The fish were maintained under observation to detect the emergence of signs of saprolegniosis. The origin of the fish, their acclimation, experimental conditions and the quality of the physicochemical parameters of the water during these biocontrol tests were similar to those detailed in Section 2.4. 2.5.3. Statistical Analysis Survival analysis was performed by the Kaplan-Meier method, and the survival probability of the groups treated or not with the probiotics was compared using the log-rank test, considering that the differences were statistically significant at p <= 0.05. All calculations were performed using SPSS for Windows version 26. 3. Results 3.1. In Vitro Inhibition Assays of the Probiotic Bacteria against S. parasitica 3.1.1. Assays with solid medium The results of the plate assay in solid medium are shown in Table 2. On the negative control plates, the colony of S. parasitica reached more than 5 cm in diameter . All three bacteria showed a higher mycelial growth inhibition capacity when S. parasitica was tested on BHI medium with bacterial strains previously grown for 7 days. L. plantarum CLFP3 showed the highest fungistatic activity since it was already observed with a 3-day bacterial culture. 3.1.2. Assays with Broth Medium (Hemp Seed Test and Cyst Test) Both E. gallinarum L1 and V. fluvialis L21 isolates partially inhibited the growth of the mycelium of S. parasitica with the dilution containing 2 x 104 bacteria mL-1 and with higher concentrations . On the contrary, L. plantarum CLFP3 did not inhibit the growth of the mycelium with any of the concentrations used. E. gallinarum L1 and V. fluvialis L21 also inhibited cyst germination at concentrations greater than 4 x 103 bacteria mL-1, but L. plantarum CLFP3 only controlled germination with the highest bacterial concentration of 4 x 105 mL-1. 3.1.3. Fungicidal Effect of the Bacteria The three isolates showed to be lethal for S. parasitica, but the effect varied according to the concentration and incubation time of the bacteria. A lethal effect on the mycelium was achieved at lower bacterial concentrations as the incubation time increased. Thus, with one or two days of incubation, a lethal effect was observed only with the highest concentration (2 x 105 bacteria mL-1), while with 3 days of incubation, a lethal effect was observed up to the third dilution (2 x 103 bacteria mL-1). V. fluvialis L21 was the bacterium that presented the greatest fungicidal effect. An atrophied and small mycelium was observed after a period of incubation if a lethal effect was produced, while the mycelium grew and produced zoospores on the control plate and on the plate with a non-lethal concentration of bacteria. 3.1.4. Assays with Supernatants Culture supernatants only inhibited mycelium growth when they were undiluted, as well as in the case of E. gallinarum L1 with the first double dilution (1:2). Regarding the inhibition of cyst germination, none of the supernatants of the three bacteria was inhibitory. 3.2. Adhesion to Skin Mucus of the Probiotic Bacteria and Inhibition of S. parasitica Cyst Adhesion Table 3 summarizes the results for the adhesion of the three probiotic strains to cutaneous mucus of brown trout. The adhesion was generally low (between 14.60% for V. fluvialis L21 and 7.54% for L. plantarum CLFP3), but it was higher than the adhesion of bacteria to bovine serum albumin and swine gastric mucus, although there were no significant differences between the adhesion of cysts to the mucus and that observed on the other substrates (p > 0.05, Student's t-test). The results of the exclusion, competition and displacement of S. parasitica cysts are shown in Table 4. In general, the three bacteria required lower concentrations to reduce the adhesion of cysts in the competition test (bacterial isolates and cysts were added at the same time). On the contrary, for the displacement of the cysts (bacterial isolates were added after the cyst), higher bacterial concentrations were required. L. plantarum CLFP3 was the bacterium that best reduced the adhesion of S. parasitica cysts in the three tests, despite presenting lower percentages of adhesion to mucus than L1 and L21 isolates (Table 3). 3.3. Pathogenicity for Rainbow Trout Both E. gallinarum L1 and V. fluvialis L21 isolates were non-pathogenic for rainbow trout. No signs of disease or lesions were observed in any of the inoculated fish, and no mortality occurred during the days that the fish were kept under observation. In the microbiological study of the internal organs of rainbow trout, only the injected strain was reisolated in two fish inoculated with E. gallinarum L1. In one of the fishes, which was inoculated intramuscularly, it was reisolated from the kidney, and in the other, which was injected intraperitoneally, it was detected in the spleen. 3.4. Biocontrol of Saprolegniosis by Adding the Bacteria to Water or Feed None of the three probiotic strains, i.e., E. gallinarum L1, V. fluvialis L21 or L. plantarum CLFP3, was able to prevent S. parasitica infection in rainbow trout when added to tank water or administered through feed. Between 24 and 48 h after infection, the first macroscopic S. parasitica lesions were observed, while the first deaths were noted on day 3 post infection, reaching 100% mortality between days 6 and 9 post infection both in the groups treated with the probiotic strains and in those not treated , with no statistically significant differences observed. In the control groups (not infected with S. parasitica), 100% survival was observed, except in the experiment with the E. gallinarum L1 strain, in which one trout died 24 h post infection, probably due to the ami momi treatment. 4. Discussion Previous studies have shown that E. gallinarum L1, V. fluvialis L21 and L. plantarum CLFP3 can be used as potential probiotics against vibriosis or lactococosis for sea bass or rainbow trout. In the present work, the possible utility of these bacteria in the control of saprolegniosis was studied. For this purpose, both in vitro inhibition studies and competition for binding sites against S. parasitica and in vivo tests with experimentally infected rainbow trout were carried out. The three bacteria inhibited the growth of S. parasitica in solid medium, with the most pronounced inhibition in the case of L. plantarum CLFP3, since it appeared after three days of incubation of the bacteria. The greatest inhibition was observed in bacterial cultures that already had 7 days of growth. Our results agree with those obtained by Hussein and Hatai , although they found differences depending on the culture medium used; they observed the greatest inhibition when they used BHI agar with longer incubation times. Regarding the inhibition of S. parasitica in liquid culture with hemp seed, the strains E. gallinarum L1 and V. fluvialis L21 prevented mycelial growth, while L. plantarum CLFP3 was unable to control it. However, the three bacteria inhibited the germination of cysts, although L. plantarum CLFP3 required higher concentrations. In all cases, the concentration of bacteria necessary to inhibit mycelial growth was greater than that required to prevent cyst germination. These data agree with those obtained by Bly et al. , who found that small amounts of different bacteria of the Pseudomonas genus prevented the germination of cysts. Carbajal-Gonzalez et al. suggested that the need for lower concentrations of bacteria to inhibit cysts may be due to the fact that a hyphal thallus already exists in hemp seeds, while the cysts have to germinate and form the mycelium. Inhibition of cyst germination in vitro could indicate that these bacteria hinder germination on the external surface of the fish, thereby helping to control S. parasitica infection. In addition, the three bacterial isolates were lethal to S. parasitica. This fungicidal effect is of interest since the three bacteria are able to inactivate S. parasitica and help control infection, acting not only as fungistatics. None of the supernatants of the three bacteria inhibited the germination of S. parasitica cysts, in agreement with ; however, in our case, the supernatants did stop the growth of the preexisting mycelium in the hemp seed. The greater inhibition exhibited by E. gallinarum L1 may be due to the fact that this bacterium produces lactic and acetic acid, as well as small amounts of ethanol . Epithelium surface colonization and adhesion, interfering with the pathogen's adhesion, are two positives for the selection of candidate probiotics . Adhesion to the cutaneous mucus of brown trout for the three bacterial strains tested in the present work was found to be low. The limited adhesion of bacteria to fish mucus agrees with results reported by other authors, with greater adhesion on the intestinal mucus of rainbow trout and other fish species than on cutaneous mucus . Comparing the adhesion reported by Sorroza et al. for E. gallinarum L1 and V. fluvialis L21 to the intestinal mucus of various marine fish and to the cutaneous mucus of brown trout obtained in the present study, both bacteria presented a higher percentage of adhesion to intestinal mucus. These results agree with those of Balcazar et al. who, using three bacteria from the intestinal microbiota of rainbow trout, observed a greater adhesion to intestinal mucus than to cutaneous mucus. The greater adhesion of E. gallinarum L1 and V. fluvialis L21 bacteria to intestinal mucus may be related to the origin of these bacteria, since they were isolated from intestinal mucus; however, L. plantarum CLFP3, which was originally isolated from cutaneous mucus, presented percentages of adherence to skin mucus lower than the other two bacteria. Chabrillon et al. indicated no specific adhesion of bacteria to a specific host or substrate but that the adhesion capacity depends more on the strain. This is in agreement with the data obtained in the present study for E. gallinarum L1, V. fluvialis L21 and L. plantarum CLFP3, since there were no significant differences in adhesion to the different substrates used, although adhesion to skin mucus was greatest. Although adhesion to skin mucus was low, the three bacteria isolates significantly reduced adhesion of S. parasitica cysts, although higher bacterial concentrations were required for displacement of S. parasitica cysts. This agrees with the results obtained by Carbajal-Gonzalez et al. , who indicated that the greater need for bacteria for displacement may be due to a high adhesion of S. parasitica cysts to mucus, making it difficult for bacteria to subsequently eliminate them. The lowest concentrations of bacteria to significantly reduce attachment of S. parasitica cysts occurred under competitive conditions. An essential characteristic of a potential probiotic is its safety and lack of pathogenicity. It has previously been shown that L. plantarum CLFP3 is safe for rainbow trout and that E. gallinarum L1 and V. fluvialis L21 are safe for sea bass . In the present study, found that L1 and L21 are also non-pathogenic for rainbow trout. The genera Lactobacillus, Enterococcus and Vagococcus are included in the group of lactic acid bacteria, which are mostly non-pathogenic and considered part of the commensal microbiota in the gut of several fish species. Thus, Gonzalez et al. reported the presence of L. plantarum and V. fluvialis as a part of the microbiota of several species of freshwater fish, including rainbow trout, and Petersen and Dalsgaard isolated various Enterococcus spp., including E. gallinarum, from the intestine of fish from integrated chicken-fish farms. However, Osman et al. found signs of septicemia in Nile tilapia (Oreochromis niloticus) associated with two isolates of E. gallinarum. In addition, other species of these genera, such as V. salmoninarum, have been isolated from diseased fish and are capable of causing mortalities up to 50% in rainbow trout farmed at low water temperature . Regarding the genus Enterococcus, three species (E. faecalis, E. faecium and E. hirae) have been associated with enterococcosis/streptococcosis both in freshwater and marine fish . Another Enterococcus species, E. seriolicida, initially described as pathogenic for yellowtail (Seriola quinqueradiata) in Japan was later shown to be really identical to Lactococcus garvieae . Although together, the three probiotic strains used in the present study achieved acceptable results in vitro, inhibiting the growth of S. parasitica and decreasing the ability of cysts to adhere to the cutaneous mucus, none of the three strains showed effective results in vivo to prevent experimental saprolegniosis in rainbow trout either through the water or administered in feed. Nevertheless, the beneficial effects against S. parasitica, even if limited to in vitro responses, must be considered since the combination of these bacteria with other strains could ensure a sufficient synergistic effect, with the capacity to prevent the presentation of the disease. Gram et al. pointed out that the selection and application of probiotics must be tested for each individual host-pathogen combination and that in vivo activity cannot be predicted based on in vitro testing. These authors found that a strain of Pseudomonas fluorescens AH2 was strongly inhibitory against Vibrio anguillarum in vitro and that it was also able to reduce mortality in rainbow trout against V anguillarum infection via addition to the tank water . Later, they found that this probiotic strain also inhibited A. salmonicida in vitro but was unable to prevent furunculosis in salmon (Salmo salar) in an in vivo model . On the contrary, Nurhajati et al. managed to prevent infection by S. parasitica in catfish (Pangasius hypophthalamus) by adding different concentrations of L. plantarum FNCC 226 to the water and found that the effect was dose-dependent, with higher concentrations of S. parasitica necessary to achieve inhibition. The best concentration treatment was between 4.2 x 105 cfu mL-1 and 8.4 x 105 cfu mL-1 of L. plantarum FNCC 226, which can inhibit infection with a concentration up 4 x 107 zoospores mL-1 of S. parasitica. In our case, the concentration of L. plantarum CLFP3 used was very similar (106 bacteria mL-1), but the concentration of zoospores was much lower (3 x 102 zoospores mL-1), although the results are not necessarily comparable since a different experimental infection method was used, as well as different species and sizes of fish. In previous works by our research group using the same experimental conditions, we observed that two Pseudomonas fluorescens strains (LE89 and LE141) were able to prevent infection by S. parasitica in rainbow trout when added to tank water but not when administered with feed . Subsequently, we investigated the possible mechanisms of action of these two isolates and verified that they seems to be related to activity of competitive exclusion and to the production of some siderophores . In addition, Lategan et al. found that administration of Aeromonas media strain A199 in water significantly decreased the incidence of saprolegniosis in eel (Anguilla australis) and silver perch (Bidyanus bidyanus) and that the substance associated with the inhibitory activity was indole production . In another study , the application of supernatant from a culture of a bacterium identified as Burkholderia sp. Reduced the rate of infection by Saprolegnia sp. in grass carp (Ctenopharyngodon idella). The substance involved with that antifungal activity was thermostable and was identified as 2-pyrrolidone-5-carboxylic acid, a derivate of glutamic acid. Competitive exclusion is generally considered a major strategy by which probiotic bacteria prevent the adhesion of pathogenic microorganisms to fish mucous membranes. Although in vitro studies have frequently reported this effect, studies with animals have failed to reproduce the same results with in vivo models on many occasions , which could partly elucidate the results obtained in the present study. Probiotics have been shown to have the capacity to increase innate and adaptive immunity of fish, with the effects exerted on the fish innate immune system as the main desirable characteristics . Perez-Sanchez et al. investigated the expression of immune-related genes in rainbow trout feeding with L. plantarum CLFP3 (106 CFU g-1 f for 36 days) and found that mRNA levels of IL-10, IL-8 and IgT were significantly higher in the L. plantarum group compared to the control group after Lactococcus garvieae infection, suggesting that protection conferred by L. plantarum was mediated by the stimulation of the immune response. In the present study, the possible mechanism of action of the three isolates used was not investigated, so we do not know whether these isolates could increase the immune response of rainbow trout under the tested conditions. The mechanisms by which probiotics stimulate the immune system are not yet well understood, but it is known that factors such as the type of strain, dose, duration and mode of administration or environmental conditions can affect the immunomodulating potency of probiotics . In this work, a dose of 106 bacteria mL-1 in a water tank or 108 bacteria g-1 for two weeks was used, which may not have been sufficient to stimulate the immune system. However, in the studies in which the usefulness of probiotic strains in the prevention of saprolegniosis has been confirmed, their beneficial effect seemed to be related to the production of inhibitory substances or competition for iron and not to the increase in the immune response . 5. Conclusions The obtained results show that the use of an effective probiotic against a certain disease in a host may not be effective against another pathogen or in another host and that the results obtained in vitro may not always predict the effects when used in vivo. These results highlight the importance of selecting the most appropriate probiotic and its mechanism of action depending on the species of fish and the disease to be prevented. Acknowledgments We are grateful for the assistance provided by the laboratory technician Gloria Fernandez-Bayon; the fish farm Los Leoneses in Leon, which supplied the rainbow trout; the "Servicio Territorial de Medio Ambiente de Leon" of the "Junta de Castilla y Leon", which supplied the brown trout; and Skretting Espana S.A., which donated some of the feed. Author Contributions Conceptualization, J.-M.A.-G. and F.R.; Formal analysis, C.G.-P. and J.-M.A.-G.; Funding acquisition, J.-M.A.-G. and F.R.; Investigation, C.G.-P. and J.-M.F.-G.; Methodology, C.G.-P. and J.-M.F.-G.; Project administration, J.-M.A.-G. and F.R.; Resources, J.-M.A.-G.; Supervision, J.-M.F.-G. and J.-M.A.-G.; Validation, J.-M.A.-G.; Writing--original draft, J.-M.F.-G.; Writing--review and editing, T.P.-S., D.P. and F.R. All authors have read and agreed to the published version of the manuscript. Institutional Review Board Statement The animal study protocol was approved by the Subcommittee for Experimentation and Animal Welfare of the University of Leon, Spain (Protocol number ULE_04_2015). Informed Consent Statement Not applicable. Data Availability Statement The data presented in this study are available upon request from the corresponding author. Conflicts of Interest The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses or interpretation of data; in the writing of the manuscript; or in the decision to publish the results. Figure 1 In vitro growth inhibition tests of the bacterial strains against S. parasitica on BHI agar (a,b) and in 24-well tissue culture plates with a colonized hemp seed and BHI broth (c,d): (a) S. parasitica after 3 days of incubation at 20 degC on the control plate; (b) high level of inhibition of S. parasitica by Lactobacillus plantarum CLFP3 strain after 3 days (bacterial strain was previously grown for 7 days at 20 degC); (c) S. parasitica after 3 days of incubation at 20 degC used as a control well; (d) partial growth inhibition of S. parasitica produced by Enterococcus gallinarum L1 strain (2 x 105 cells mL-1) after 3 days at 20 degC. Figure 2 Kaplan-Meier survival curves in rainbow trout (Oncorhynchus mykiss) treated with the probiotics strains Enterococcus gallinarum L1, Vagococcus fluvialis L21 and Lactobacillus plantarum CLFP3 administered in water or in feed and infected with Saprolegnia parasitica. animals-13-00954-t001_Table 1 Table 1 Probiotic bacterial strains tested in the present study for biocontrol of saprolegniosis in rainbow trout (Oncorhynchus mykiss). Bacterial Isolate Origin Probiotic Activity Against Reference Enterococcus gallinarum L1 Gut content of sea bass (Dicentrarchus labrax) Vibrio angillarum in D. labrax Vagococcus fluvialis L21 Gut content of sole (Solea solea) Vibrio angillarum in D. labrax Lactobacillus plantarum subsp. plantarum CLFP3 Cutaneous mucus of rainbow trout (O. mykiss) Lactococcus garvieae in O. mykiss animals-13-00954-t002_Table 2 Table 2 Inhibition of Saprolegnia parasitica in solid medium (plate assay) shown as mean +- standard deviation (n = 3) colony diameter (cm) after culturing for 3 days at 20 degC on a BHI plate with a 3-, 7-day bacterial culture. Incubation Time of the Bacteria (Days) Enterococcus gallinarum L1 Vagococcus fluvialis L21 Lactobacillus plantarum CLFP3 Control Plate 3 4.25 +- 0.05 5.45 +- 0.04 0 5.70 +- 0.11 5 3.46 +- 0.06 2.90 +- 0.05 0 5.17 +- 0.07 7 0 2.35 +- 0.04 0 5.65 +- 0.14 animals-13-00954-t003_Table 3 Table 3 Percentage of adhesion (mean +- SD of three experiments) of Enterococcus gallinarum L1, Vagococcus fluvialis L21 and Lactobacillus plantarum CLFP3 strains to cutaneous mucus of brown trout (CM), bovine serum albumin (BSA), mucin from swine stomach (MSS) and plate polystyrene (PP). Bacterial Isolate CM BSA MSS PP Enterococcus gallinarum L1 9.92 +- 7.59 4.57 +- 3.48 3.28 +- 3.48 4.39 +- 2.97 Vagococcus fluvialis L21 14.60 +- 5.48 7.87 +- 4.56 8.48 +- 3.89 7.98 +- 0.15 Lactobacillus plantarum CLFP3 7.54 +- 4.77 4.13 +-1.31 4.20 +- 1.45 4.51 +- 0.22 animals-13-00954-t004_Table 4 Table 4 Percentage of reduction in the adhesion (mean +- SD of three experiments) of Saprolegnia parasitica cysts to cutaneous mucus of brown trout tested with different bacterial strain concentrations of Enterococcus gallinarum L1, Vagococcus fluvialis L21 and Lactobacillus plantarum CLFP3 under conditions of exclusion, displacement and competition. Bacterial Strain Bacterial Concentration Exclusion * Displacement * Competition * Enterococcus gallinarum L1 2.5 x 107 28.55 +- 0.01 26.68 +- 2.92 65.03 +- 1.28 2.5 x 106 19.05 +- 6.73 24.93 +- 5.97 27.08 +- 3.05 2.5 x 105 7.50 +- 0.01 18.40 +- 14.77 6.33 +- 1.39 2.5 x 104 6.87 +- 1.89 0 6.26 +- 1.22 Vagococcus fluvialis L21 2.5 x 107 83.11 +- 1.31 93.24 +- 18.73 73.86 +- 5.55 2.5 x 106 62.81 +- 3.72 25.09 +- 9.21 40.03 +- 1.76 2.5 x 105 55.45 +- 35.84 13.59 +- 9.88 7.39 +- 3.14 2.5 x 104 17.67 +- 22.71 6.45 +- 4.13 6.49 +- 4.15 Lactobacillus plantarum CLFP3 2.5 x 107 87.50 +- 0.01 33.32 +- 8.14 33.33 +- 2.09 2.5 x 106 76.01 +- 6.48 22.30 +- 8.37 7.78 +- 1.45 2.5 x 105 70.98 +- 8.39 11.05 +- 8.33 5.44 +- 1.44 2.5 x 104 46.49 +- 17.63 0 7.29 +- 0.68 * Values in blod show the percentage of reduction in the adhesion of S. parasitica cysts significantly different (p < 0.05, Student s t-test) from the control (no bacteria added) determined by testing the minimum effective numbers of bacteria. Disclaimer/Publisher's Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

PMC10000207

Dystocia is a multifactorial, life-threatening condition commonly affecting pet reptiles. Treatment for dystocia can be either medical or surgical. Medical treatment usually involves the administration of oxytocin, but in some species or, in some cases, this treatment does not work as expected. Surgical treatments such as ovariectomy or ovariosalpingectomy are resolutive, but invasive in small-sized reptiles. In this paper, we describe three cases of post ovulatory egg retention in three leopard geckos (Eublepharis macularius) successfully treated through a cloacoscopic removal of the retained eggs, after a non resolutive medical treatment. The intervention was fast, non-invasive, and no procedure-related adverse effects were noted. The problem relapsed six months later in one animal, and a successful bilateral ovariosalpingectomy was performed. Cloacoscopy should be considered a valuable, non-invasive tool for egg removal in dystocic leopard geckos when the egg is accessible to manipulation. Recrudescence or complications such as adhesions, oviductal rupture, or the presence of ectopic eggs should recommend surgical intervention. Eublepharis macularius dystocia endoscopy reproductive disorders This research received no external funding. pmc1. Introduction Dystocia (egg binding) in reptiles is a common, multifactorial disease . Different factors of captivity are often related to the occurrence of dystocia, such as inadequate husbandry (improper temperature gradients or humidity levels, inadequate nesting sites, overcrowding), poor physical conditions (illness, dehydration, malnutrition), reproductive apparatus disorders (infectious diseases, traumatic injuries, misshapen or large eggs), and metabolic diseases (hypocalcemia secondary to nutritional or renal hyperparathyroidism) . Two common forms of reproductive disorders in reptiles are recognizable: preovulatory follicular stasis and postovulatory stasis or egg binding. The first form occurs when egg development stops prior to ovulation after vitellogenesis, resulting in persistent follicles and leading to inflammation and eventually rupture, coelomitis, and death . The second form, the postovulatory stasis, occurs when the eggs are not laid and retained inside the oviduct for an indefinite period of time, or ectopic . In both conditions, the animal can refuse to eat and its conditions can deteriorate rapidly, leading it to death . The diagnosis of postovulatory stasis is usually made by X-ray due to the radiopacity of the eggshell. Retained eggs can become overcalcified and appear more radiopaque on radiographic images . Egg presence can also be assessed through careful palpation of the caudal coelom. Vitellogenic follicles during preovulatory stasis are better visualized and even measured through ultrasound or CT scan . Treatment for dystocia can be either medical or surgical. Medical treatment involves the administration of oxytocin (1 to 10 UI/kg) intramuscularly (IM) or the administration of arginine vasotocine . Oxytocin seems to work better in chelonians than in snakes and lizards . It is generally used one hour after IM calcium gluconate administration, especially if the blood calcium level is low . Arginine vasotocine seems to be more effective in reptiles than oxytocin, but the short storage life, high cost, and unlicensed use make this medical treatment option problematic in clinical practice . Percutaneous ovocentesis is often reported by some authors as a treatment of dystocia in geckos . However, it carries a high risk of organ rupture, with consequent coelomitis. If the eggs can be visualized through the cloacal opening, ovocentesis can be performed to make the eggs collapse and therefore be removed more easily . Hormonal therapy, such as deslorelin implants, seems to be ineffective in suppressing ovarian activity in leopard geckos and its use is still under debate in other reptiles . In cases where medical treatment fails or is not applicable, surgery remains the elective choice. Ovariectomy or ovariosalpingectomy is resolutive . 2. Clinical Cases 2.1. Histories and Clinical Examination 2.1.1. Case 1 A 2-year-old, 45 g captive bred female leopard gecko (Eublepharis macularius) was presented for clinical examination due to one week of anorexia and weight loss. The animal was kept in a 60 (length) x 50 (height) x 30 (depth) cm glass terrarium without substrate, with only paper towels at the bottom that were changed weekly. The temperature inside the cage was 30 degC (86 degF) during the day and 25 degC (77 degF) at night. The diet was consistent and consisted of insects with calcium powder given three times a week. A UVB light bulb with 5.0 spectrum was provided and changed every 6 months. A plastic box with peat moss was present in the terrarium and misted daily. The owner indicated that the gecko was acquired two weeks earlier from another breeder. Once introduced in the new environment, the gecko spent the most of time burrowed in the substrate. At clinical examination, the gecko was lethargic and mildly to moderately dehydrated. The coelomic cavity was distended, and the presence of a large egg was palpable and visible in transparency through the skin in the caudal half of the coelom on the right half of the sagittal plane. 2.1.2. Case 2 A 4-year-old, 50 g captive bred female leopard gecko (Eublepharis macularius) was presented for clinical examination due to dysecdysis, lethargy, anorexia, and distention of the coelomic cavity . The animal was housed with a two-year-old male in a 50 (length) x 50 (height) x 50 (depth) cm glass terrarium with paper towels as a substrate and maintained at 30 degC (86 degF) during the day and 24 degC (75.2 degF) at night. The diet was consistent and consisted of insects dusted with calcium powder given twice a week. A UVB light bulb with 5.0 spectrum was provided and changed every 6 months. The owner also reported that a box with a wet mixture of peat moss and coconut fiber was added to the terrarium to facilitate deposition, and the female spent most of the time burrowing inside the box. No eggs were found during the daily check by the owner. At clinical examination, the gecko was lethargic and moderately dehydrated. The abdomen was swollen, and the presence of two voluminous masses was palpable in the caudal half of the coelom on the left and right halves of the sagittal plane. 2.1.3. Case 3 A 4-year-old, 52 g captive bred female leopard gecko (Eublepharis macularius) was presented for a veterinary second opinion. The owner indicated that the gecko was kept with an adult male of unknown age; both were kept in an 80 (length) x 40 (height) x 40 (depth) glass terrarium with paper towels as a substrate at 32 degC (89.6 degF) at day and 24 degC (75.2 degF) at night. In the cage, a box with a wet mixture of peat moss and coconut fiber was present as a wet nest to facilitate molting, and after the suggestion of the original breeder, deposition. The female spent most of the time burrowing inside without eating for two weeks. The diet was consistent and consisted of insects dusted with calcium powder given twice a week. No UVB light was provided. The animal was first presented due to lethargy and anorexia, and a diagnosis of egg binding was made by the first veterinarian based on the anamnesis and by the dorsoventral X-ray taken the same day. Oxytocin at 10 UI/kg was administered by the previous veterinarian. IM was performed twice with a one-hour interval without any results. At clinical examination, the gecko was lethargic and mildly dehydrated. The coelomic cavity was distended, and the presence of a large egg was palpable in the caudal half of the coelom on the right half of the sagittal plane. 2.2. Diagnostic Procedures 2.2.1. Case 1 Complete blood work, X-rays, and ultrasounds were performed. The CBC count and PCV were unremarkable. The biochemistry tests showed a moderate increase in AST and the CK X-rays showed the presence of large eggs in the caudal half of the coelomic cavity on the right half of the sagittal plane, and the presence of radiopaque foreign material was interpreted as ingested sand in the intestinal tract . Ultrasound examination confirmed the egg presence and the presence of hyperechoic material inside the intestinal lumen; no oviduct rupture was noted. Oxytocin at 5 UI/kg (10 UI/mL neurofisin, FATRO S.p.a, Ozzano dell'Emilia, Italy) was administered in the right tricep after IM administration of calcium gluconate at 100 mg/kg (200 mg/mL Calcio PH, FATRO S.p.a, Ozzano dell'Emilia, Italy) 1 h before, without success. Given the inefficacy of the treatment, cloacoscopy was planned to examine any potential oviduct abnormalities and determine whether the egg could be removed through endoscopic grasping forceps, avoiding a more invasive celiotomy. The animal was anaesthetized using alfaxalone (10 mg/mL Alfaxan, Dechra Veterinary Products Srl, Torino, Italia) at a dosage of 5 mg/kg delivered intravenously in the right jugular vein . The heart rate and respiratory rate were monitored during all procedures. The animal lost the rightening reflex after approximately 40 s but experienced apnea, so it was intubated and mechanically ventilated (one breath every 10 s). A 2.7 mm x 18 cm, 30deg oblique telescope (within a 4.8 mm operative sheath) (Storz Telepack TP100 EN, Karl Storz Endoscopia Italia Srl, Verona, Italy) was used for cloacal inspection, and showed one egg protruding from the right salpinx to the cloaca . During the procedure, the operator held the animal in ventral recumbency with the left hand, while the right hand maneuvered the telescope. The eggshell was broken using grasping forceps, and the content was removed without any difficulties . 2.2.2. Case 2 Complete blood work, X-rays, and ultrasounds were performed. The CBC count showed an increased WBC count with moderate heterofilia and monocytosis, mild basophilia, mild eosinopenia, and lymphocytosis (Table 1). The PCV was mildly increased. Biochemistry tests showed a moderate increase in creatinine kinase (Table 2). X-rays were performed in dorsoventral and latero-lateral projections, showing the presence of two large eggs in the caudal coelom . Ultrasound examination confirmed the presence of eggs; no oviduct rupture was noted. The use of oxytocin as medical treatment was not performed due to the severe weakness of the gecko. Due to the mild dehydration, warm fluids (ringer solution) were administered subcutaneously at 10 mL/kg/die and stabilized at 28 degC for 12 h. Cloacoscopy was planned for the day after to verify whether the eggs were visible and removable using endoscopic grasping forceps, avoiding a more invasive celiotomy. The animal was anesthetized using alfaxalone (10 mg/mL Alfaxan, Dechra Veterinary Products Srl, Via Agostino da Montefeltro, 2, Torino, Italia) at a dosage of 5 mg/kg delivered intravenously in the right jugular vein . The animal lost the rightening reflex after approximately 60 s and maintained spontaneous breathing during the entire procedure. A 2.7 mm x 18 cm, 30deg oblique telescope (within a 4.8mm operative sheath (Storz Telepack TP100 EN, Karl Storz Endoscopia Italia Srl, Verona, Italy) was used for cloacal inspection and showed the surface of the two eggs protruding from the left and right salpinx to the cloaca . During the procedure, the operator held the animal in ventral recumbency with the left hand, while the right hand maneuvered the telescope, as described in the Case 1. The eggshell was broken using grasping forceps, and the content was removed by performing cloacal lavages with warm sterile NaCl solution. The empty eggshells were removed as described for case 1. 2.2.3. Case 3 Complete blood work, X-rays, and ultrasounds were performed. The CBC count showed a mildly increased WBC count with mild basophilia. The PCV was mildly increased. Biochemistry tests were unremarkable. X-rays were repeated and showed the presence of one large hypocalcified egg on the left half of the coelomic cavity and a high radiopaque area on the left side, measuring approximately 0.5 cm x 1 cm in diameter. Ultrasound examination confirmed the egg presence in the right oviduct and the presence of a hyperechoic foreign body in the left oviduct. No oviduct ruptures were noted. Cloacoscopy was planned for the day after. As described for the case 2, warm fluids (ringer solution) were administered subcutaneously at 10 mL/kg/die and the gecko stabilized at 28 degC for 24 h. The animal was anesthetized using the same protocol described for cases 1 and 2. The animal lost the rightening reflex after approximately 40 s and maintained spontaneous breathing during the entire procedure. A 2.7 mm x 18 cm, 30deg oblique telescope (within a 4.8 mm operative sheath) (Storz Telepack TP100 EN, Karl Storz Endoscopia Italia Srl, Verona, Italy) was used for cloacal inspection and showed the surface of the egg protruding from the right salpinx to the cloaca. During the procedure, the operator held the animal as described in case 1 and 2. The eggshell was broken, and the content was removed by performing cloacal lavages with warm sterile NaCl solution. The empty eggshells were removed as described for cases 1 and 2. A subcutaneous deslorelin implant (4.7 mg, Suprelorin(r), Milano, Italy) was applied in the neck region upon the owner's request despite the owner having been informed that there is no scientific evidence of its efficacy in leopard geckos. 2.3. Postoperative Care and Follow-Up 2.3.1. Case 1 The gecko recovered 15 min after the end of the procedure, and no adverse effects were noted. Warm fluids (ringer solution) were administered subcutaneously at 15 mL/kg/die at the end of the endoscopic procedure to support the renal function after the anesthesia. Assisted feeding (Emeraid IC critical care formula, Emeraid LLC, A Division of Lafeber, Cornell, IL, USA) was administered once a day as nutritional support. Five days after the procedure, the gecko started to eat, so it was discharged. One month later, the gecko's weight had increased by 5 g, and she was active and in good nutritional status. One year later, the gecko laid six infertile eggs, but dystocia had not occurred. 2.3.2. Case 2 The gecko recovered 20 min after the end of the procedure, and no adverse effects were noted. Fluid therapy and nutritional support were administered as described in case 1. Two days after the procedure, the gecko started to eat, so it was discharged. The owner was instructed to separate the male gecko from the female gecko and the problem had never occurred at the two-year follow-up. 2.3.3. Case 3 The gecko recovered 18 min after the end of the procedure, and no adverse effects were noted. Warm fluids (ringer solution) were administered subcutaneously at 15 mL/kg/die at the end of the endoscopic procedure. Fluid therapy and nutritional support were administered as described in case 1. Five days after the procedure, the gecko started to eat, so it was discharged. The owner was instructed to separate the male gecko from the female gecko. The leopard gecko did not lay any eggs in the next 6 months, and then a new episode of dystocia occurred; a large egg was evident in the right oviduct again, and the calcification in the left oviduct was still present. A bilateral ovariosalpingectomy was proposed and accepted by the owner. The gecko was premedicated with a mixture of dexmedetomidine at a dosage of 0.1 mg/kg (0.5 mg/mL Dexdomitor(r), Vetoquinol Italia S.r.l, Bertinoro, Italy) and ketamine at a dosage of 10 mg/kg (100 mg/mL Ketavet(r), Intervet production Srl, Aprilia, Italy) delivered in the right triceps . The animal was intubated, maintained under gaseous anesthesia with 2% isoflurane (IsoFlo(r), Zoetis Italia s.r.l, Roma, Italy) and mechanically ventilated (one breath every 15 s). A paramedian incision was performed to avoid the abdominal venous sinus. The cutis, abdominal muscles, and the coelomic membrane were cut, and the right oviduct with a voluminous egg inside was rapidly identified and isolated. A right salpingotomy was performed to remove the egg, making the access to the ovaries easier and improving the visibility of the near organs and blood vessels. Both the right and left ovaries and the respective oviducts were removed after ligature of the main vessels with a 7/0 absorbable monofilament (Monosyn(r) Braun Avitum Italy S.p.A. Mirandola, Italy). The hyperechoic structure visible on ultrasound examination revealed a residual hypercalcified eggshell inside the left salpinx. The coelomic membrane was closed with a simple continuous suture through the abdominal muscles with a 6/0 absorbable monofilament (Monosyn(r) Braun Avitum Italy S.p.A. Mirandola, Italy). The cutis was closed with an everted simple interrupted suture with a 6/0 absorbable monofilament (Monosyn(r) Braun Avitum Italy S.p.A. Mirandola, Italy). Atipamezole at a dosage of 0.5 mg/kg (Atidorm(r) Fatro Industria Farmaceutica Veterinaria S.p. A, Ozzano dell'Emilia, Italy) was administered to the left triceps muscles at the end of the surgical procedure. The animal awakened 4 min after administration of the anesthesia reversal agent. A single 5 mg/kg dose of tramadol was administered IM, and 0.2 mg/kg meloxicam was administered SC once a day (SID) postoperatively. The antimicrobial ceftazidime was administered SC every 3 days at a dose of 20 mg/kg . The gecko started to eat after 2 days of therapy and was discharged after one week of hospitalization in good condition. At the 3-month follow-up, the clinical examination was unremarkable. 3. Discussion Rigid endoscopy is a useful tool for the direct and also indirect evaluation of the reproductive tract in reptiles . Coelioscopy is used as a direct method for the evaluation of the reproductive tract in chelonians, snakes, and lizards . In tortoises, cystoscopy permits an indirect visualization of the coelomic organs in transparency through the urinary bladder wall . This technique also permits the detection of retained eggs in the urinary bladder as well as their removal . To the authors' knowledge, this is the first report of endoscopic-assisted egg removal through cloacoscopy in leopard geckos. All three cases presented here were diagnosed as obstructive postovulatory stasis in three female leopard geckos. As stress can be a predisposing factor for dystocia , it is possible that the already pregnant female, recently acquired and recently housed in a new environment, developed postovulatory stasis due to a high level of stress. As sexually active males can attempt mating persistently, their constant presence in the same cage, together with the female, could have been a predisposing factor to dystocia in case 2 and case 3 . In case 1, PCV% and CBC count were unremarkable. In case 2, PCV%, RBC, WBC, heterophils, basophils, monocytes, and lymphocytes were slightly increased (Table 1), and in case 3, PCV%, WBC, RBC and basophils were slightly increased. These alterations were considered to be related to the dehydration status and not related to any reproductive disorder. In cases 1 and 2, CK was mildly augmented (Table 2). In reptiles, CK levels can vary with variations in environmental temperature . Given the above, in case 1 and 2, the increase in CK was considered to not be related to any disease. AST, total protein, and uric acid were slightly increased in case 2 (Table 2). Increases in AST and total protein in reptiles with follicular development have been reported ; however, in this case, the mild increases in AST and total protein were thought to be related more to dehydration, as uric acid was also augmented. Oxytocin, as a promoter of muscle contractibility , was administered in cases 1 and 3 without success. It is possible that in geckos, oxytocin is less effective than in chelonians, as has been described for snakes and some lizards . Deslorelin acetate is a synthetic nonapeptide analogue of the natural gonadotrophin-releasing hormone (GnRH). It acts as a contraceptive by temporarily suppressing the hypothalamic-pituitary-gonadal axis (HPG axis), inhibiting the production of pituitary hormones such as follicle-stimulating hormone (FSH) and luteinizing hormone (LH) . In reptiles, the use of GnRH agonists has been poorly investigated; the use of deslorelin implants in leopard geckos seems to be less effective or ineffective at suppressing gonadal activity . In lizards, suppression of gonadic activity was reported only in iguanas . In chelonians, a desloreline implant was successfully used to treat chronic ovodeposition in a Greek tortoises (Testudo graeca) up to 24 months after application . In a male Chelonia mydas, an annual application of deslorelin implants decreased serum testosterone levels after the fourth treatment . In case 3, the gecko did not lay any eggs in the 6 months after treatment. A mature female leopard gecko can lay four to five clutches of two eggs per season with a one-month interval between clutches . In this case, we do not know whether the gecko was at the end of the reproductive season or whether the ovarian activity was suppressed by the implant. Available scientific data suggest that the latter hypothesis is unlikely. In all three cases, cloacal endoscopy permitted not only egg visualization but also egg removal through the cloacal opening under general anesthesia. Only one gecko (case 1) underwent apnea, and it was intubated and mechanically ventilated. Apnea has been reported in leopard geckos with the protocol used in case 1 . In case 3, the recrudescence of egg binding required ovariosalpingectomy intervention. No procedure-related complications were noted. The underlying causes of dystocia in these three cases remain unclear: it is likely that the voluminous eggs were too large to pass through the cloaca openings, or the oviduct was hypocontractile. Even the total calcium was considered normal according to the reference values , the ionized calcium was not measured. It is possible that an underlying hypocalcemia caused a decreased oviductal smooth muscle contractility . The authors find this hypothesis unlikely since calcium supplementation was regularly provided by the owners. Moreover, no clinical signs of hypocalcemia such as seizures or skeletal deformities were noted at the physical examination. No evident fractures or skeletal demineralizations were radiographically detected. Diminishing egg pressure, leaking the shell with grasping forceps, and removal were performed easily. However, this procedure should be performed carefully to avoid the oviduct damage or rupture. A rude egg manipulation with the grasping forceps or a powerful cloacal flush could tear the oviductal or cloacal walls, predisposing to coelomitis. Even if ultrasonography is a useful tool in diagnosing dystocia in reptiles , we were unable to visualize the distal part of the oviduct in all three geckos. In fact, the distal part of the reproductive tract is often difficult to image in lizards due to its location within the pelvic canal . In these three described cases, the eggs protruding from the salpinx to the cloaca were visible and directly approachable during the cloacoscopy. This procedure should be critically evaluated whenever the eggs are not directly accessible or when adherences between the eggshell and the oviduct is suspected. Cloacoscopic egg removal should be considered before performing more invasive techniques, such as explorative celiotomy, when medical treatment fails. In fact, wound dehiscence and herniation are common post-surgical complication in leopard geckos, in relation to the thinness and the softness of the coelomic membrane and body wall musculature, which causes the muscles to tear more easily when tension is applied during the coelomic closure . Percutaneous ovocentesis is contraindicated in reptiles and is not recommended because it increases the risk of yolk coelomitis and oviduct adherence. . Many reptiles that are routinely presented for medical examinations are used for breeding and commercial purposes. In particular, leopard geckos have been bred for many years and today there are selections or "morphs" with a high commercial value on the market . It is therefore of paramount importance to consider the owner's desire to maintain functionality and the integrity of the reproductive system. 4. Conclusions Considering the predisposition that leopard geckos have to dystocia, the use of the rigid endoscope as a minimally invasive instrument to reduce soft tissue trauma, duration, and post pain surgery, is a valuable option in all subjects affected by dystocia secondary to malformations of the egg, as in the cases described herein. Endoscopic egg removal could be a valuable tool in dystocic leopard geckos when the egg is accessible to manipulation, or in subjects in which it is essential to preserve the possibility of future reproduction. Adhesions, oviductal rupture, or the presence of ectopic eggs should recommend surgical intervention. However, the veterinarian must understand and correct the underlying causes of reproductive disorders to prevent a recurrence. Acknowledgments Not applicable. Supplementary Materials The following supporting information can be downloaded at: Video S1. Click here for additional data file. Author Contributions A.V. was the major contributor to the article's writing. F.D.I., M.M., M.R. and E.L. contributed to the acquisition, analysis and interpretation of the data and contributed to methodology acquisition and validation of the methods. E.B. supervised the procedures. All authors have read and agreed to the published version of the manuscript. Institutional Review Board Statement Ethical review and approval were waived for this study due to the spontaneous pathologies of all reptiles. Informed Consent Statement Not applicable. Data Availability Statement Data sharing is not applicable. No new data were created or analyzed in this study. Data sharing is not applicable to this article. Conflicts of Interest The authors declare no conflict of interest. Figure 1 Case 2: The gecko presented with distenction of the coelomic cavity, dysecdysis, and poor nutritional status. Figure 2 Case 1: Dorso-ventral X-ray. A large egg was visible in the caudal coelom (white arrows). Figure 3 Case 1: Cloacoscopy. Endoscopic view of the right egg protruding from the oviduct to the cloaca. Figure 4 Case 1: End of the procedure. The egg was aspirated and easily extracted through the cloacal opening. Figure 5 Case 2: Dorsoventral X-ray. Two large eggs were visible in the caudal coelom (white arrows). Figure 6 Cloacoscopy. Endoscopic view of the right (black asterisk) and the left (yellow asterisk) egg protruding from the vagina to the cloaca. Figure 7 Case 3: Dorso-ventral X-ray. A large egg was visible in the caudal half of the coelomic cavity (white arrows). Figure 8 Case 3: Surgery. Left ovarian stalk (black asterisk) ligature during the ovariosalpingectomy intervention. Cr: cranial. Cd: caudal. Lfb: left fat body. animals-13-00924-t001_Table 1 Table 1 Comparison between CBC (complete blood count) parameters of the cases and the reference values. Abnormal values are in bold. Case 1 Case 2 Case 3 Normal Values (Female) PCV% 40 45 45 21-40% WBC x 103/mm3 8 13 10 6-9.4 RBC x 106/mm3 0.7 1.3 1.1 0.43-0.89 Heterophils (103/mL) 2.9 3.5 2 1.08-2.73 Eosinophils (103/mL) 0.4 0 0.8 0.15-1.95 Basophils (103/mL) 1 3 3 0.00-2.26 Monocytes (103/mL) 1 4 2 0.60-2.16 Lymphocytes (103/mL) 3 4 3 1.67-5.39 animals-13-00924-t002_Table 2 Table 2 Comparison between biochemical values of the cases and the reference values. Abnormal values are in bold. Case 1 Case 2 Case 3 Reference Values AST (U/L) 12 78 11-65 Total protein (g/dL) 7 9 4.2 2.4-8.0 Albumin 18 22 15 13-23 Creatinkinasis (U/L) 4.897 4.000 1861 0-3.701 Phosphorous (mg/dL) 14.5 8.4 14 1.5-16.4 Calcium (mg/dL) 18 22 31 14->37 Potassium (mmol/lt) 6.8 5.5 6.1 4.50-7.0 Uric acid (mg/dL) 3.3 6.7 4 0.5-6.6 mg/dL Biliary acids 0.6 3.2 1.1 0.8-21 mmol/L Disclaimer/Publisher's Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

PMC10000208

Analysis of the efficacy/pharmacology of long-term/lifetime medical treatment of acid hypersecretion in a large cohort of ZES patients in a prospective study. This study includes the results from all 303 patients with established ZES who were prospectively followed and received acid antisecretory treatment with either H2Rs or PPIs, with antisecretory doses individually titrated by the results of regular gastric acid testing. The study includes patients treated for short-term periods (<5 yrs), patients treated long-term (>5 yrs), and patients with lifetime treatment (30%) followed for up to 48 years (mean 14 yrs). Long-term/lifelong acid antisecretory treatment with H2Rs/PPIs can be successfully carried out in all patients with both uncomplicated and complicated ZES (i.e., with MEN1/ZES, previous Billroth 2, severe GERD). This is only possible if drug doses are individually set by assessing acid secretory control to establish proven criteria, with regular reassessments and readjustments. Frequent dose changes both upward and downward are needed, as well as regulation of the dosing frequency, and there is a primary reliance on the use of PPIs. Prognostic factors predicting patients with PPI dose changes are identified, which need to be studied prospectively to develop a useful predictive algorithm that could be clinically useful for tailored long-term/lifetime therapy in these patients. gastrinoma Zollinger-Ellison syndrome PPI omeprazole acid hypersecretion neuroendocrine tumor NIH#DK053200-26 #NCT0000-1254 This research was partially supported by the intramural program of NIDDK (NIDDK #DK053200-26; #NCT0000-1254) of the NIH. pmc1. Introduction Zollinger-Ellison syndrome (ZES) was characterized in its first description in 1955 by a triad of findings, including the presence of severe gastric acid hypersecretion due to a non-beta islet cell tumor of the pancreas, which resulted in refractory peptic ulcer disease/gastroesophageal reflux disease (GERD) that only responded to total gastrectomy . Later studies demonstrated that the acid hypersecretion was secondary to the ectopic release of gastrin by a neuroendocrine tumor (i.e., gastrinoma) , the gastrinomas were more frequently located in the duodenum than in the pancreas , and that 20-25% of all patients had ZES as part of the autosomal dominant disorder, Multiple Endocrine Neoplasia-type 1 (MEN1), which is characterized by multiple endocrine tumors/hyperplasia of the pancreas and the parathyroid and pituitary glands . From the very beginning, it became apparent that the major cause of the long-term morbidity and mortality in these patients was not the gastrinomas, which were malignant in a majority of cases and generally slow-growing , but instead the presence of uncontrolled gastric acid hypersecretion . Acid secretory studies showed that the untreated basal gastric acid hypersecretory rate averaged 4-fold higher than the upper limit of normal in these patients and could reach almost 10-fold higher than the normal upper limit of basal acid secretion in some patients . Furthermore, the tumor induced-hypergastrinemia caused a stimulatory effect on the cells of the gastric mucosa , resulting in a 4-6 fold increase in parietal cell mass , which secretes gastric acid, and was manifested by a marked increase in maximal acid output capacity . This degree of acid hypersecretion in almost all patients could not be controlled by the limited medical therapies initially available (antacids, anticholinergics) and it was not until the late 1970s/early 1980's, with the introduction of increasingly potent histamine H2-receptor antagonists (metiamide, cimetidine, ranitidine, famotidine, nizatidine, etc.), that a possible medical alternative to routine total gastrectomy became possible to control the acid hypersecretion . Subsequently, even more potent and longer-acting acid antisecretory drugs, gastric H+K+ ATPase inhibitors (also called proton pump inhibitors [PPIs], i.e., omeprazole, lansoprazole, esomeprazole, pantoprazole, rabeprazole), have become available and are now considered the antisecretory drugs of choice for ZES . There are several important current controversies in the treatment of ZES, but one central issue to the management of all ZES patients is that the acid hypersecretion needs to be controlled at all times and it is unclear whether this is feasible long-term (>5 years). This uncertainty occurs for a number of reasons. First, only 5-20% of ZES patients are surgically curable by complete resection of the gastrinoma . This is because of the frequent multiplicity of the tumors in MEN1/ZES patients ; the fact that gastrinomas may be microscopic in size and not localizable by even the most sensitive imaging techniques ; gastrinomas frequently (>50%) metastasize to adjacent lymph nodes and resections are incomplete ; and 20-40% of all ZES patients present with unresectable hepatic metastases . Second, there are an increasing number of reports of failure of long-term H2R maintenance therapy and even difficulty with the ability of long-term PPI treatment to continue to successfully control the acid hypersecretion . The uncertainty is also primarily due to the lack of data from any long-term/ lifetime treatment studies of acid antisecretory control in ZES patients. This is in marked contrast to an abundance of studies of acute acid control and short-term (<5 yrs) control with small numbers of ZES patients . This lack of information about the long-term efficacy of antisecretory drugs in ZES is particularly disturbing because the unique constant hypersecretory drive of the unresected gastrinoma requires constant inhibition of its effects on acid secretion, which is unique to ZES and can only be addressed by long-term/lifetime study data. Whereas long-term PPI studies in non-ZES patients, especially in patients with advanced idiopathic GERD, are providing evidence for the safety of lifetime PPI treatment , which is applicable to chronic treatment of ZES patients, unfortunately this is not the case with long-term/lifetime efficacy data in ZES. This is because of the marked variation in the dose requirements between individual ZES patients as well as in each patient, which has been well-examined in short-term ZES acid antisecretory studies . The current study aimed to address these issues. The NIH prospective study on all aspects of ZES has been in effect since 1974, and part of the study involves the long-term medical management of the acid hypersecretion of all patients . In the current study, we analyzed data from this prospective database related to all issues of the long-term efficacy of both H2Rs and PPIs in these patients. This includes a detailed analysis of initial patient drug dosing for each class of acid antisecretory drug, the final drug dose after long-term/lifetime treatment, including how drug use and dosing has changed with time, as well as the dosing frequency and effects of dosing frequency on total daily dosing. Additionally, we compared acid control with the two classes of antisecretory drugs and finally, we analyzed factors that could be used to predict the need for a possible dose change in long-term PPI-treated patients. Our results demonstrate long-term/lifetime treatment of acid hypersecretion is possible in all patients with each class of acid antisecretory drug; however, PPIs are preferable because of their greater potency and long duration of action. We also comment on a number of aspects of the comparative pharmacology of these two antisecretory drug classes in long-term treatment of ZES patients and provide important guidelines on how similar results can be obtained at other treatment centers. 2. Materials and Methods The patients in this study included a cohort of all patients who have been entered into an ongoing NIH prospective study of various aspects Zollinger-Ellison syndrome (ZES) since 1974, as approved by the clinical research committee of the National Institute of Diabetes, Digestive and Kidney Diseases of the National Institutes of Health, the characteristics of which have been previously described . This cohort includes all patients with established ZES who received acid antisecretory treatment with either H2Rs or PPIs, with antisecretory doses set by the results of gastric acid testing, as previously described and reviewed briefly below . 2.1. Initial Investigations The diagnostic criteria for Zollinger-Ellison syndrome were previously described and included an elevated fasting serum gastrin level in the presence of an elevated basal acid output, positive provocative testing with secretin or calcium , a positive histological diagnosis of gastrinoma, or a combination of these criteria. Basal acid output (BAO) was measured after discontinuing all oral antisecretory medication (greater than 30 h for histamine H2-receptor antagonists and greater than 7 days for PPIs) by collecting four consecutive 15-min samples of gastric fluid and titrating them to pH 7.0 with 0.01 N sodium hydroxide . Maximal acid output was measured by collecting four 15 min samples following subcutaneous administration of pentagastrin . Fasting serum gastrin (FSG) values were determined by Bioscience Laboratories (New York, NY, USA) and all samples were diluted to the normal range for accurate determination of higher values . All gastrin determinations were made during a period when the patient was known to produce gastric acid in order to limit the likelihood of obtaining falsely elevated levels due to drug-induced achlorhydria . Upper gastrointestinal endoscopy was performed following sedation with meperidine (Demerol; Winthrop Pharmaceuticals, New York, NY, USA) and midazolam (Versed; Roche Laboratories, Nutley, NJ, USA) as previously described . The upper gastrointestinal mucosa was carefully examined, and biopsies were taken if any abnormalities were detected . Tumor imaging studies included selective abdominal arteriography , CT scan , ultrasonography , magnetic resonance imagining , and since 1995, somatostatin receptor imaging . Selected patients with negative imaging underwent functional imaging by assessing gastrin tumor gradients using either portal venous sampling or selective intra-arterial secretin injections . Patients with potentially resectable disease underwent surgical exploration, which included particular attention to the duodenum (duodenotomy, palpation, intraoperative transillumination) since 1987 . Patients with unresectable liver disease (e.g., multiple liver metastases) underwent percutaneous liver biopsy to confirm the nature of their metastatic disease as well as in patients with distal metastases . An exploratory laparotomy was not generally performed . Patients were placed in the disease-free category post-operatively only if their fasting serum gastrin concentration in the presence of known gastric acid secretion was not elevated, if they had a negative secretin provocative test, if all imaging studies were negative for recurrent tumor, and if there was a significant reduction in symptoms of gastric acid hypersecretion as well as antisecretory drug requirements , i.e., they were assessed as having no evidence of ZES . Patients who were rendered disease-free were generally not treated with PPIs unless they had significant acid-peptic symptoms that were not easily controlled with histamine H2-receptor antagonists but responded to PPIs. Surgical patients who were not rendered disease-free (>60%) or who relapsed were treated with PPIs for long-term control of gastric acid hypersecretion. The possible presence of MEN-1 was considered in all patients with ZES and was evaluated by obtaining a careful family and personal history as well as by performing an endocrine evaluation of the pituitary and parathyroid glands and the pancreas . The diagnosis of MEN1 directly affected the management of both the tumor itself and the gastric acid hypersecretion. Specifically, patients with MEN1/ZES are more resistant to acid antisecretory medications if hyperparathyroidism is not controlled ; thus, these patients are usually treated with higher and more frequent drug dosing . Patients with MEN-1 were not routinely subjected to exploratory laparotomy unless they had an imageable tumor (>1.5-2 cm) as studies show the likelihood of rendering them disease -free is low (<5%) without aggressive surgery . Consequently, almost all MEN1 patients required long-term medical control of the gastric acid hypersecretion . 2.2. Determination of the Antisecretory Drug-Dose Prior to 1983, the acid hypersecretion of all patients was treated with histamine H2-receptor antagonists [H2Rs] (cimetidine, ranitidine) either alone or with anticholinergic agents , and afterwards, in almost all cases by either more potent H2R's (famotidine) or PPIs (omeprazole, lansoprazole) in almost all cases . In all cases, the initial and subsequent dosing was determined by acid titration with gastric acid output measurement for the hour prior to the next dose. The usual criteria was to identify the drug dose that reduced the acid output to <10 mEq/h for this time with an absence of acid-peptic symptoms (<5 mmol/h for patients with severe GERD or prior partial gastrectomy ). These criteria were established to induce peptic ulcer healing, control non-severe GERD symptoms, and prevent further mucosal damage . Initially, H2R dosing frequency was every 6-8 h starting with 300/600 of cimetidine, 150/300 mg of ranitidine, or 20/40 mg of famotidine and adjusting the dosage upward or downward as previously described to reduce the acid hypersecretion to the control criteria above. A similar approach was used for PPIs, initially starting with a once daily dose of 60 mg/day omeprazole/lansoprazole and increasing the dose by 20 mg/day omeprazole or 15 mg/day lansoprazole until a dose of 120 mg/day was reached, and then the daily dose was split into 60 mg twice per day and subsequently increased as needed . Because initial studies suggested that patients with complicated ZES (MEN1, previous Billroth 2 surgery, or with advanced GERD) generally required higher dosing, new patients with these features in later studies were initially started on twice per day 40-60 mg/day PPI. After PPIs were licensed for general use in the USA, many new ZES referrals to the NIH were already taking PPIs at the time of their initial evaluation. In these instances, after assessing the BAO to establish the diagnosis off all acid antisecretory drugs, the initial dose of PPI at the NIH was not re-titrated, provided that gastric acid output was effectively controlled as defined above unless this dose induced total achlorhydria. Because studies suggested prolonged drug treatment could induce achlorhydria and result in low vitamin B12 levels in these patients , the daily referral PPI doses were reduced to levels allowing acid values in the control range without total achlorhydria in uncomplicated cases showing total achlorhydria. Once the initial acid antisecretory dose requirement was determined for each patient, patients were reevaluated at least once a year to assess both tumor status and confirm that control of gastric acid output remained effective, as previously described . During this reassessment, all patients had a complete history and physical examination, fasting serum gastrin levels and other standard blood biochemical/hematological parameters (i.e., electrolytes, BUN, CBC, etc.) were measured, gastric acid output control was assessed, upper gastrointestinal endoscopy was performed with routine biopsies of the gastric body in the region of the greater curvature, and repeat tumor imaging studies were performed, as previously described . During follow-up evaluations, long-term antisecretory drug maintenance doses were titrated upwards in patients with inadequate control or were titrated downwards in patients who were rendered achlorhydric, as previously described . However, after the completion of a successful formal dose-reduction protocol in a subgroup of the patients, long-term maintenance doses were reduced in amenable patients who were well controlled but not achlorhydric, provided that the criteria for adequate control of gastric acid hypersecretion were not violated. In the results, to enable comparative drug dose analyses with individual patients treated with different types of H2Rs (cimetidine, ranitidine, famotidine) or different PPI's (omeprazole, lansoprazole) at the time of initial acid antisecretory treatment and final treatment, the doses for H2Rs were converted to ranitidine-equivalent doses, as previously described , or to omeprazole-equivalent doses, as described below. A previous study in ZES patients established that the relative potencies of famotidine: ranitidine: cimetidine are 1: 9:32, with ranitidine being 3.6-fold more potent than cimetidine . To calculate an omeprazole-equivalent dose, results were used from a recent study which demonstrated that 20 mg omeprazole = 40 mg esomeprazole, 30 mg lansoprazole, 40 mg pantoprazole, and 20 mg of rabeprazole . In the analysis, we separated the patients into two different groups based on the length of follow-up. The short-term group was treated for <5 years at NIH, whereas the long-term group was treated for >5 years. Five years was chosen for this division because almost all previously reported ZES patients had only been treated with acid antisecretory agents for <5 years . We wanted to compare our patients to these previously reported patients, while we know there would be no comparative group for the long-term treatment group. We also wanted to divide the patients into these two groups because we thought the length of follow-up could affect the results, and this allowed us to perform that comparison. 2.3. Statistics Values are expressed as means +- S.E.M. The statistical methods employed for all comparisons were the Fisher's exact test or Mann Whitney U test. p-values of <0.05 were considered statistically significant. 3. Results The clinical, laboratory, and tumor features of the 303 patients included in the current study are listed in Table 1 and Table 2. The results are generally similar to those in other large series of patients with ZES in regard to age at either onset or diagnosis of ZES, gender, percentage with MEN1, presentation of symptoms, disease duration, frequency of prior gastric acid reduction surgery, fasting gastrin levels, basal and maximal acid secretory rates, as well as tumor location and extent. The percentage of patients with duodenal gastrinomas was lower than reported in recent surgical series because the present series included all ZES patients, both medical and surgical, who had their acid hypersecretion controlled in the NIH prospective study. In this prospective study, 28% of ZES patients with advanced metastatic disease (unresectable liver or distant metastases), MEN1/ZES with imaged pancreatic tumors <=1.5-2 cm, or an accompanying serious medical illness that limited life expectancy did not undergo routine surgical exploration , so the exact site of the primary tumor was not established surgically and the imaging studies were inclusive. cancers-15-01377-t001_Table 1 Table 1 Patient clinical characteristics. Characteristic Number (%) Patient number Total treated 303 (100%) Number followed long-term (>=5 yrs) (a) 260 (86%) Number not followed long-term (<5 yrs) (a) 43 (14%) Age at ZES onset (yrs) (b) Mean +- SEM 40.0 +- 0.7 (Range) (11.0-65.8) Age at ZES Diagnosis (yrs) (c) Mean +- SEM 45.8 0.7 (Range) (11.0-65.8) Gender Male 170 (56%) Female 133(44%) MEN1 present 89 (29%) Presenting clinical symptoms/features (d) Pain 233 (77%) Diarrhea 220 (72%) GERD (moderate/severe) 133 (44%) Ulcer history 205 (67%) Bleeding 70 (23%) Other GERD/PUD Complication (e) 39 (13%) Prior gastric acid reduction surgery 37 (12%) Vagotomy-pyloroplasty/Selective vagotomy 14 (4.6%) Billroth I resection 6 (2.0%) Billroth 2 resection 17 (5.6%) Duration of medical acid treatment started (yrs) (f) From disease onset Mean +- SEM 3.7 +- 0.3 (Range) (0-26.1) Prior to ZES diagnosis (yrs) (g) Mean +- SEM 3.5 +- 0.3 (Range) (0.01-26.0) From time of diagnosis (yrs) Mean +- SEM 1.2 +- 0.3 (Range) (0-18) Abbreviations: ZES, Zollinger-Ellison syndrome; MEN1, Multiple Endocrine Neoplasia type 1; yrs, years; GERD, gastro-esophageal reflux disease; PUD, peptic ulcer disease; PMD, patient's private medical doctor. (a). Patients not followed long-term (i.e., <5 yr.) at NIH because of early death, surgical cure , or returned to PMD after diagnosis, stabilization, and evaluation. (b). Onset defined as onset of recurrent symptoms compatible with ZES, as previously described . (c). Criteria for diagnosis of ZES required assessment of BAO/fasting gastrin/secretin tests, as previously described . (d). Clinical symptoms were determined as previously described . (e). Other PUD complications included non-bleeding complications such as stricture [esophageal, duodenal, small intestine], perforation, penetration, obstruction, and advanced Barrett's esophagus, as previously defined . (f). Time to any medical acid treatment included time from onset/diagnosis to start of treatment with histamine H2-receptor antagonists or PPIs. (g). A total of 193 patients were started on medical treatment prior to the established diagnosis of ZES and the remainder were started on treatment at the time of diagnosis or after diagnosis. cancers-15-01377-t002_Table 2 Table 2 Patient laboratory/tumor characteristics. Characteristic Number (%) Fasting serum gastrin (FSG) (pg/mL) Mean +- SEM 4932 +- 1272 (Range) (72-286,800) Median 644 Hi FSG (>=644 pg/mL) (>=median) 149 (49%) BAO (mEq/h) (no gastric surgery) (a) Mean +- SEM 43.0 +- 1.5 (Range) (1.8-159) BAO (mEq/h) (previous gastric surgery) (b) Mean +- SEM 26.3 +- 3.0 (Range) (2-94) Hi BAO (>=36.8 mEq/h) (median output of 276 with BAO) 127 (46%) MAO (mEq/h) (no gastric surgery) (b) Mean +- SEM 65.0 +- 2.0 (Range) (13-159) MAO (mEq/h) (previous gastric surgery) (b) Mean +- SEM 36.9 +- 4.3 (Range) (9.0-113) Hi MAO (>=59.7 mEq/h) (median output of 227 with MAO) (b) 112 (49%) Primary tumor location (c) Pancreas 78 (26%) Duodenum 114 (38%) Other (d) 36 (12%) Unknown (d) 86 (28%) Tumor extent (e,f) Primary only 113 (37%) Primary and lymph node metastases 82 (27%) Primary and liver metastases 84 (28%) Abbreviations: BAO, Basal acid output; MAO, Maximal acid output; (a). A total of 276 patients had a BAO (241 had no gastric surgery and 35 had previous gastric acid reduction surgery) determined, as previously described . (b). A total of 227 patients had a MAO (202 had no gastric surgery and 25 had previous gastric acid reduction surgery) determined, as previously described . (c). Patients with diffuse liver metastases, with MEN1/ZES, or severe co-morbidities did not undergo routine surgical exploration, as previously described , and primary location, if not seen on imaging, was not localized. (d). Non-pancreaticoduodenal primary sites occurred in lymph nodes , hepato-biliary tract , ovary, jejunum, mesentery, heart, lung, and gastric antrum , as previously described. (e). Tumor extent was determined as described in the Section 2. (f). In 24 patients, it was not possible to determine whether primary only or primary plus lymph nodes were present via imaging and no surgical exploration was performed. Among the 330 patients included in this study, 260 (86%) were followed at NIH in the prospective study and the gastric acid hypersecretion was controlled for more than 5 years (long-term follow-up group), whereas the remaining 43 (14%) patients were followed at NIH for <=5 years (short-term follow-up group) (Table 1). The short-term follow-up group included early death of the patient after entering the study, early surgical cure of the patient , or the patient requested a return to their private medical doctor after the diagnosis had been established, the disease was stabilized, and/or the initial treatment plan was defined. No patient in either the short-term or long-term treatment groups treated with PPIs or second-generation H2Rs (ranitidine, famotidine) ended treatment because of drug side effects, even with some patients receiving very high daily doses. In our early studies in the 1980s when only cimetidine was initially available, some patients treated with very high doses of cimetidine developed anti-androgen side effects (impotence, gynecomastia) and were switched to ranitidine as soon as it was available. This has been previously reported . Initially, all ZES patients entering the NIH prospective study after 1978 had their gastric acid hypersecretion treated with H2R's (first-cimetidine, later ranitidine) , which were the only effective, generally available, approved gastric acid antisecretory drugs able to control acid hypersecretion in ZES patients at that time . This continued until 1983, when other gastric antisecretory drugs became available and were used in the NIH patients, with the PPI omeprazole first becoming available , followed by the more potent H2R, famotidine, in 1987 , and a second PPI, lansoprazole, becoming available in 1989 . The increased potency and longer duration of action of PPIs resulted in them being used for long-term maintenance by >90% of patients in this study by the late 1990s . In Table 3, the overall schedule for the temporal order of acid drug treatments is summarized for the entire time of treatment, as well as for the duration of various treatments. At some time, the majority (81%) of the 303 patients were treated with an H2R; however, this was only for a limited time in most patients because only 13.5% were only treated with an H2R (Table 3, Part I). cancers-15-01377-t003_Table 3 Table 3 Acid treatment: Schedule and Duration. Characteristic Number (% Total) I. Treatment schedule Total acid treatment drugs (n = 303) (a) H2R (cimetidine, ranitidine, famotidine, nizatidine) at any time 245 (81%) Only H2R without any PPI treatment at any time 41 (13.5%) H2R with anticholinergeric agent at any time 50 (16.5%) H2R without anticholinergeric agent at any time 195 (64.4%) H2R then PPI (b) 204 (67%) PPI (omeprazole, lansoprazole, pantoprazole) at any time 262 (86%) PPI only 58 (19.1%) PPI then H2R (c) 0 (0%) First medical acid treatment drug (n = 303) (a) H2R 245 (81%) PPI 58 (19%) Pts with only short-term acid medical treatment <5 yrs (n = 42) (d) H2R only without any PPI treatment at any time 18 (6.6%) H2R with anticholinergic agent at any time 7(2.3%) H2R without anticholinergic agent at any time 35(11.6%) PPI only 7 (2.3%) H2-R then PPI 17 (6.9%) Pts with any long-term acid treatment (>=5 yrs) (n = 261) (d) H2R only without any PPI treatment at any time 22 (7.3%) H2R with anticholinergic agent at any time 19 (6.2%) H2R without anticholinergic agent at any time 93 (30.7%) PPI only 149 (49%) H2R, then PPI 90(30%) II. Treatment duration (yrs) All acid treatment (n = 303) Mean +- SEM 13.7 +- 0.5 Range (0.08-48.1) Only Short-term acid medical treatment <5 yrs (n = 42) Mean +- SEM 2.8 +- 0.2 Range (0.08-4.9) Any Long-term acid treatment >=5 yrs (n = 261) 15.5 +- 0.5 Mean +- SEM (5.1-48.1) Range Any treatment with H2R (n = 245) Mean +- SEM 6.4 +- 0.45 Range (0.07-29.2) Any treatment with PPI (n = 262) Mean +- SEM 9.8 +- 0.4 Range (0.1-48.1) (a). In the NIH perspective trials, histamine H2-receptor antagonists were the first effective acid antisecretory medical therapy starting with cimetidine in in 1978, ranitidine in 1982, and famotidine in 1983. PPIs were first used in 1983 with omeprazole, and then lansoprazole in 1989 . Thus, all patients initially enrolled in this study were first treated with H2Rs (cimetidine, ranitidine, famotidine) and later, most changed to PPIs (omeprazole, lansoprazole), while new patients generally started treatment with PPPIs . (b). Patients with active disease were initially treated with H2Rs and then switched to PPIs. (c). Patients with active disease were initially treated with PPIs and then switched to H2Rs. (d). For explanation of long-term treatment, see Table 1 footnote (a). Before the availability of PPIs, almost 1 in 5 patients treated with an H2R required the addition of anticholinergic agent to control their acid hypersecretion, which corroborated with previous reports that the addition of an anticholinergic agent can potentiate the acid inhibitory effectiveness of an H2R in a small number of ZES patients . When an H2R was used in a patient with active ZES, it was used prior to the use of PPIs in all cases, primarily due to the time span of the availability of these drugs . Similar to treatment with H2Rs, most (86%) of the 303 patients were treated with a PPI at some time; however, only a minority (19.1%) of patients were treated only with a PPI during their disease course (Table 3). In general, the acid treatment sequences for the 261 patients who had long-term follow-up (i.e., >5 yrs) were similar to those who underwent only short-term follow-up (<5 yrs), with only 6-7% being only treated with an H2R and 2-6% requiring an anticholinergic agent in addition to the H2R (Table 3, Part I). However, for the 86% of patients who had long-term follow-up, a higher percentage of patients were treated with only a PPI or had the initial treatment with an H2R changed to a PPI (30.7% vs. 2.3% and 30% vs. 6.9%, respectively) (Table 3 Part I). In contrast to previous acid antisecretory studies with ZES patients, the duration of acid treatment in the patients in this study was long (>2-6.5 yrs) , with a maximum follow-up time of 48 years, a mean for all patients, including those treated with long-term follow-up, of 13.7 years, and 33% of patients were treated for longer than 15 years (Table 3, Part II). For the 261 patients in the long-term follow-up group, the mean duration of treatment was 15.5 years with a range of 5.1-48 years (Table 3, Part II). For all patients, the mean duration of treatment with a PPI was more than 50% longer than that with an H2R (9.8 vs. 6.4 yrs) (Table 3, Part II). During the prospective study, 99 (33%) patients died, none from acid-related problems; therefore, life-time control of the acid hypersecretion was shown in 33% of all ZES patients. The initial and final acid antisecretory doses for all patients who received H2Rs (n = 245) at any time or PPIs (n = 262) are compared in Table 4 and Figure 2 and Figure 3. The initial H2R daily ranitidine-equivalent dose for all patients was 946 mg/day, but it showed a wide range from 80 to 4800 mg/day for individual patients and the final dosage, which was a mean of 6.4 years later, was 3.6-fold higher (2440 mg/day) (Table 4). With PPIs, the mean initial dose was 72 mg/day, also with a wide range (20-240 mg/day) ; however, in contrast to the rise in final mean dose with H2Rs, the final dose of PPIs decreased to 58 mg/day (Table 4). A more detailed analysis showed the pattern of dose changes with H2Rs and PPIs varied significantly and in the opposite directions, with an increase in dosage required in 5.6 times more patients with H2Rs than with PPIs (p < 0.0001), a decrease in dose occurring 22 times more frequently in patients with PPIs than with H2Rs (0.0001), and no change in dose occurring 1.5 times more frequently in patients with PPIs than with H2Rs (p = 0. 0037) . These results were comparable to the distribution of the lowest and highest doses of H2Rs or PPIs at the initial and final dosing . This analysis showed a marked decrease (i.e., 2.5-fold) in the percentage of patients with the lowest final dose and a marked increase (i.e., 4.6-fold) in the percentage of patients with the highest dose for patients treated with H2Rs, whereas the opposite pattern was observed in patients treated with PPIs (i.e., 2.5-fold increase in lowest, 1.3-fold decrease in highest) . Similar results in the total patient group were also observed in both the long-term treatment groups . Numerous studies have shown that in addition to the dosage of H2R or PPI given, the frequency of dosing can also have a marked effect on acid suppression efficacy in both ZES patients and patients with acid peptic disorders and/or GERD . To further assess this possibility in ZES patients, the change in dosing frequency between the initial and final dosage for H2Rs and PPIs was analyzed in all patients, as well as those receiving long-term acid suppression by each class of antisecretory drug . Overall, in the 241 patients taking H2Rs at some time, the initial daily dosing frequency was 2-fold higher per day (2.8 times/day) than that for patients taking PPIs (1.5 times per day) and this difference was further increased at the final dosing (mean interval, 6.4 years for H2Rs and 9.8 years for PPIs) to 2.4-fold higher in patients taking H2Rs than PPIs (3.5 vs. 1.48 times/day) . A patient-by-patient comparison of dosing frequency changes between the initial and final H2R and PPI doses demonstrated that an increase in dosing frequency occurred in almost half of the H2R-treated patients and that a decrease in dosing frequency was uncommon (i.e., 5.6%) . In contrast, an increase in dosing frequency was uncommon in patients treated with PPIs (15%), as was a decrease in PPI dosing frequency . Overall, this resulted in an increased dosing frequency occurring in almost 3-fold more patients taking H2Rs than in those taking PPIs, a 2.3-fold higher percentage of PPI-treated patients having a decreased dosing frequency, and 1.4-fold higher percentage of PPI-treated patients not showing a change in dosing frequency (i.e., 72% vs. 51%) (Table 5). In patients with idiopathic peptic ulcer disease/GERD, a number of studies reported that splitting the daily dose of PPI may have greater efficacy than increasing a single dose, and splitting the PPI dose could enable use of a lower total daily PPI dose. Similar conclusions have been proposed from studies of small numbers of patients with ZES . To further investigate the relationship between increasing the drug dosing frequency and its effect on the total daily dose in ZES patients, we compared the effect on daily drug dosage of increasing the dosing frequency in 99 ZES patients treated with H2Rs and 39 patients treated with PPIs . In H2R-treated patients, an increase in drug dosing frequency almost always (93%) resulted in an increased total daily dose of the drug, which was in marked contrast to what occurred with PPIs . In contrast to H2Rs, splitting the daily PPI dose resulted in a decreased dose in 13 of patients and an increase in daily dose of only 46% of patients (Table 4). These results supported previous proposals that splitting the dose will frequently allow control of the acid hypersecretion with a lower daily total PPI dose in ZES patients with unsatisfactory acid control, rather than increasing the daily dose . In contrast, with H2Rs, splitting the daily dose may be necessary to control the acid hypersecretion in ZES patients because of the shorter duration of action of these drugs , but it will not generally result in a lower total daily dose . The data showed the percentage of ZES patients treated with either a histamine H2-receptor antagonist or proton pump inhibitor at any time, during short-term (<5 yrs) or long-term (>=5 yrs) treatment who required an increase in daily drug frequency, no dosage frequency change, or had a decrease in dosage frequency, determined as described in the Section 2. The durations between the first and last daily doses used for comparing daily dosing frequency were the same as listed for the daily dose comparison in the legend of Figure 2. Table 6 shows the treatment outcomes with both H2Rs and PPIs for acid output and symptom control. In both PPI-treated patients, the initial mean untreated BAO was almost 4-fold higher than normal (40-42.2 mEq/h), and the mean acid output was reduced to the therapeutic treatment level of <10 mEq/h prior to the next dose of antisecretory drug (or lower for patients with GERD/Billroth 2 surgery as described in the Section 2) in both the initial and final dosing. For both treatment acid assessments (i.e., initial/final acid control), the mean acid outputs were 2-3-fold higher when H2Rs were used than when PPIs were used (4.2/3.3 vs. 2.2/0.97 mEq/h for initial/final acid control, respectively), although both were well within the therapeutic control range (i.e., <10 mEq/h) (Table 6). In both PPI-treated patients, symptoms (i.e., pain, diarrhea, GERD-symptoms, etc.) due to the acid hypersecretion were controlled in all patients, all peptic ulcers/esophagitis or other mucosal abnormalities due to the acid hypersecretion healed if initially present, and new lesions were prevented. At present, in >95% of all ZES patients, gastric acid hypersecretion is controlled by PPIs . Identifying possible predictors of which patients might require daily dose changes with PPI continued treatment could be of major clinical value in allowing more tailored follow-up of acid control in these patients. To attempt to identify such predictive factors, we analyzed the possible predictive value of various clinical, laboratory, and tumoral features and previous acid control values for identifying which patients required a subsequent daily PPI dose change (Table 7). cancers-15-01377-t007_Table 7 Table 7 Predictive value of various clinical, laboratory, tumor, or acid features for identifying patients requiring at least one dose change during PPI treatment between the first and final doses (a). Variable Number (Percent) Change Daily PPI Dose (n = 123) Same PPI Dose (n = 139) p-Value Clinical GERD Any (b) 77/156 (49%) 53/106 (50%) 0.99 Severe (b) 24/50 (48%) 11/212 (5.2%) <0.0001 Diarrhea at onset (c) 121/156 (78%) 78/106 (73%) 0.47 PUD/GERD complication (d) 31/156 (65%) 17/106 (19.8%) 0.52 >6.4 yrs from ZES onset to treatment (c) 76/156 (49%) 53/106 (50%) 0.90 Age > 40 yrs at ZES onset (c) 74/156 (47%) 51/106 (48%) 0.99 Male gender 91/156 (58%) 60/106 (57%) 0.80 MEN1 present (c) 43/156 (27.6%) 35/106 (33%) 0.41 Prior gastric acid reduction surgery (d) Any acid reduction surgery 18/156 (11.5%) 7/106 (6.6%) 0.21 Prior Billroth2 10/148 (6.3%) 0/104 (0%) 0.007 Lab High BAO > 36.8 mEq/h (e) 78/156 (50%) 39/106 (37%) 0.043 High MAO > 62 mEq/h (e) 99/216 (89%) 12/12 (100%) <0.0001 High FSG > 644 pg/mL (e) 78/156 (50%) 48/106 (45%) 0.53 Tumor features Primary tumor size (f) >=3 cm 42/156 (26.9%) 34/105 (32.3%) 0.41 <1 cm 98/156 (63%) 60/105 (57%) 0.37 Localized Disease (g) 110/156 (70%) 80/206 (75%) 0.40 Liver Metastases 46/156 (29.5%) 26/106 (24.5%) 0.40 Pancreatic primary 24/108 (53%) 21/67 (31.3%) 0.21 Duodenal primary 64/109 (59%) 33/67 (49%) 0.27 Acid treatment Length PPI Tx >= 9.5 yrs 84/156 (54%) 46/106 (43%) 0.10 Acid control 1st PPI dose (h) 0 mEq/h 7/91 (7.5%) 29/144 (32%) 0.0095 >=5-9.9 mEq/h 23/143 (16%) 10/92 (10.9%) 0.34 PPI daily dose (1st PPI dose) QD: Daily PPI dose > 55 mg/day (i) 54/80 (68%) 26/80 (32%) 0.0267 BID: Daily PPI dose >= 80 mg/day (i) 60/71 (84%) 31/47 (15%) 0.0252 Frequency 1st PPI dose: >1x/day 71/156 (46%) 47/76 (62%) 0.90 H2R dose day > 600 mg prior to PPI 59/124 (48%) 35/83 (42%) 0.48 Abbreviations: GERD, gastro-esophageal reflux disease; PUD, peptic ulcer disease; yrs, years; MEN1, Multiple endocrine Neoplasia type 1; OM, omeprazole; ZES, Zollinger-Ellison syndrome; Tx, treatment; 1st PPI dose, initial dose patient treated with PPI determined by acid titration, as described in the Section 2 and previously in ref. ; UGI, upper gastrointestinal; for other abbreviations, see legends for Table 1 and Table 2. (a). The median duration with PPI treatment between first and last dose was 9.8 +- 0.4 years, during which time patients were seen yearly and PPI dosage was checked by assessing acid suppression and adjusted, if needed, as described in the Section 2. (b). GERD was determined by history, UGI endoscopy, and the presence of heartburn and dysphagia; severity was determined as previously described . (c).Presence of diarrhea, time of ZES onset, presence of MEN1, and length of time from ZES onset to diagnosis/treatment were determined as previously described . (d). PUD/GERD complications and previous gastric acid reduction surgery were determined as described in Table 1 legend. (e). High BAO, MAO, or FSG were determined as values exceeding the median values for all patients. (f). Tumor size was determined at surgery or by detailed imaging studies, as previously described . (g). Localized disease was defined as occurring in patients with proven ZES with no evidence of liver or distant metastases via detailed imaging studies or surgery, as detailed in the Section 2. (h). The first PPI acid control was assessed by measuring gastric acid output 1 h prior to the next dose of PPI and is reported in mEq/h, which was determined initially and then later assessed yearly, as described in the Section 2. (i). PPI dose is expressed as omeprazole-equivalent dose, as described in the Section 2. Significant factors for predicting a PPI dose change were the presence of severe GERD (not mild/moderate GERD); previous history of Billroth 2 surgery (but not other acid reduction surgery, such as vagotomy, pyloroplasty, or Billroth 1 surgery); the presence of a high BAO (i.e., >36.8 mEq/h) or high MAO (i.e., >62 mEq/h); or the presence of achlorhydria on the initial acid control evaluation (Table 7). A number of other factors reported in small numbers of ZES patients with possible predictive value for drug dose changes or correlations with higher acid secretory rates were found not to correlate with PPI dose changes. This latter group included the presence of MEN1; presence of acid secretory complications; moderate GERD; large tumor size; more advanced disease; or the presence of higher acid control levels (Table 7). 4. Discussion The primary purpose of this study was to determine whether long-term/lifelong gastric acid antisecretory control was possible and effective in patients with ZES and to study the pharmacology of the antisecretory drugs during this prolonged treatment period. This study was undertaken for several reasons. First, lifelong treatment of gastric acid hypersecretion continues to be required by most ZES patients. This continues to occur because only 5-20% of all ZES patients are cured surgically . This low overall cure rate is due to multiple causes, including the fact that 20-40% of all ZES patients present with unresectable hepatic metastases ; 25% have ZES/MEN1 and a 0-5% long-term cure rate because of the characteristic presence of multiple, metastatic, and small duodenal gastrinomas that cannot be cured without aggressive resection, such as Whipple resection, which is not generally recommended ; and in the remaining 30-40% of ZES patients with possible resectable gastrinomas or with sporadic ZES, the long-term cure rate is only 20-40% . Second, the lifelong period of maintenance acid treatment eventually needed by most ZES patients is long because the average age of ZES onset is between 27-44 years and the mean survival rate is 90% at 25 years after onset in MEN1/ZES patients and >60% in sporadic ZES patients, thus most patients require acid secretory control for >25 years . Third, while numerous studies have reported the effectiveness of both different H2Rs and PPIs in initially controlling acid hypersecretion in ZES patients , numerous studies have raised questions about the long-term efficacy of histamine H2-receptor antagonists (H2Rs) to fulfil the need for life-long therapy for these patients because of their lower potency/duration of action, resulting in the requirement for high, frequent doses that increase with time . Furthermore, more recently, increasing numbers of reports have raised similar concerns about the long-term effectiveness, as well as safety, of PPIs for controlling lifelong acid hypersecretion in these patients , even though this class of acid antisecretory drugs is more potent and longer-acting than H2Rs in ZES patients . Fourth, there is minimal data on the pharmacology of long-term (i.e., >5 yrs) H2R or PPI treatment in ZES patients. Short-term maintenance studies provide evidence that maintenance dose adjustments may be frequently required in ZES patients treated with H2Rs or PPIs, including the need for increased dosing, decreased doses, or no dosing change in some patients; however, the frequency of these changes in long-term treated patients remain unclear. Furthermore, the development of drug tolerance has been proposed in both PPI-treated ZES patients , but the possible frequency or severity remain unknown. Some studies, but not others , have suggested that acid antisecretory dose reduction may be possible with prolonged antisecretory treatment in ZES patients, but its frequency or durability remain unclear. In addition, some studies suggest that, pharmacologically, it may be better to alter the antisecretory drug dose interval rather than altering the drug dose at a specific time for optimum antisecretory control . However, relatively few patients have been studied to fully resolve this issue. Finally, if antisecretory doses need to be altered in some ZES patients with time, this can only be established at present by performing periodic acid secretory testing, which is invasive and needs to be performed in all patients because there is currently only minimal information about who may require a drug dose adjustment at some point. Hence, a systematic study attempting to identify patients who may require a dose adjustment could be of marked clinical importance in patient management by helping to better tailor acid antisecretory control during maintenance. Unfortunately, with the data available at present, it is not possible to address the issues raised in the previous paragraph about the long-term/lifelong efficacy/pharmacology of acid antisecretory treatment in ZES patients. The primary reason for this conclusion is that there is a lack of studies on long-term/lifelong acid antisecretory treatment in ZES patients. With both different H2Rs and PPIs for ZES patients, all maintenance acid antisecretory studies have been short-term (<1-5 yrs), with only small numbers of patients followed for longer durations, thereby limiting the ability to perform detailed analyses or pharmacologic studies . The present study has none of these limitations and allows the question of efficacy /pharmacology of long-term/lifelong acid antisecretory treatment in these patients to be systematically addressed. In this study, the data was collected prospectively from a large number of ZES patients (i.e., 303 patients) as part of the NIH prospective study on ZES, alterations in drug dosing/frequency were performed according to a set protocol, both the efficacy of H2Rs and PPIs was studied to allow comparisons, the study had a long follow-up providing data for up to 48 years of antisecretory treatment with a mean treatment period of 14 years, and treatment was lifelong for 33% of the patients. The most important conclusion of the present study is that long-term/lifelong medical treatment of acid hypersecretion in ZES patients is possible and can be successful in controlling peptic acid symptoms and preventing the development of acid-related complications in all patients. While this statement includes long-term/lifelong treatment with either a PPI or H2R, for all practical purposes, the findings with PPIs will be of primary interest to most clinicians. Presently, because of the greater potency and longer duration of action of PPIs, which allows once or twice a day dosing, PPIs have become the drugs of choice for acid treatment in ZES . PPIs have almost completely replaced the use of H2Rs, as shown in this study, with PPI use almost completely replacing of H2R use, which is similar to their relative use in other recent studies of acid control in ZES patients . These results differ from several recent case reports and small series that have suggested long-term treatment with PPIs did not satisfactorily control the gastric acid hypersecretion in some ZES patients. They also differ, in the case of long-term treatment with H2Rs, from a number of studies that have reported poor results maintaining acid secretory control in ZES patients . The primary difference between this study and others that have also successfully controlled acid hypersecretion in ZES patients for shorter periods (i.e., 1-6 yrs) and those that report a high rate of drug failure in maintenance control of acid hypersecretion, is whether systematic acid secretory testing to establish efficacious criteria was routinely used to appropriately titrate both the initial and all follow-up H2R/PPI doses. Numerous acid secretory control studies in patients with ZES support the conclusion that acid antisecretory drug dose requirements for both PPIs and H2Rs can vary greatly among patients and thus need to be individually titrated using established criteria for acid suppression in these patients . In ZES patients with intact stomachs, without moderate to severe GERD or MEN1, antisecretory drugs inducing acid suppression to <10 mEq/h for the hour prior to the next drug dose is the generally accepted criteria . In contrast, in patients with complicated ZES (moderate-severe GERD, previous Billroth 2 surgery, or MEN1) greater acid inhibition to <1 mEq/h may be needed depending on the UGI endoscopic findings . Numerous short-term acid antisecretory studies with PPIs or H2Rs in ZES patients have reported that not only can the daily total dose vary markedly between individual patients, but it can also change considerably in different directions for different patients over time. In our study, the initial daily dose determined by upward or downward drug-dose titration based on acid secretory results (expressed as omeprazole-equivalent dose) for the 262 patients treated with PPIs was 72 mg/day and the initial daily dose for the 245 patients taking H2Rs (expressed as a ranitidine-equivalent dose) was 968 mg/day. However, for both PPIs and H2Rs, the daily dose varied markedly for individual patients with a range of 20-240 mg/day for PPIs and 80-4800/mg/day for H2Rs. The mean and range of doses for initial treatment for both PPIs and H2Rs were similar to those reported in various short-term studies in ZES patients using similar methods to establish initial drug dosing. The marked difference in daily doses required by individual patients (12-fold for PPIs, 60-fold for H2Rs) demonstrate why individual dose titration is required to successfully manage the acid hypersecretion in these patients, because at present, the required dose needed by each patient cannot be established by any other method. On the final acid assessment after a mean of 9.8 years (range 0.1-48.1 yrs) for the PPI-treated patients, the mean daily omeprazole-equivalent dose had significantly decreased (p < 0.001) by 20% to 58.5 mg/day (range 20-240 mg/day). The direction of the change in the mean PPI dose between the initial and final daily doses was in the opposite direction of the final daily dose seen in patients treated with H2Rs, which was significantly (p < 0.001) higher (i.e., 2440 mg/day) than that seen with the initial dosing. These results are consistent with short-term studies of ZES patients treated H2Rs in which patients generally required increasing H2Rs dosing with time, with an average of at least one dose adjustment per year, which was generally an increased dosage . These results of long-term PPI treatment were similar to some short-term studies with PPIs in ZES patients ; however, they differed from other studies generally reporting that increased PPI dosing was needed with time, although it occurred at a much slower rate of 0.13 changes per year than the 1 dose per year generally seen with H2Rs in various studies . To help resolve this difference between what was previously reported in short-term PPI studies with the PPI results determined in our study, we analyzed the total direction of dose changes for our 303 patients overall and patients undergoing short-term (<5 yrs) or long-term (mean 15.5 yrs, range 5.1-48.1 yrs) treatment. Our results showed there was a large difference between the distribution of overall drug dose changes over time (mean total treatment duration = 13.7 +- 0.5 yrs) between patients treated with PPIs or H2Rs. A 6-fold higher percentage of patients treated with H2Rs required an increased drug dose (i.e., 70% vs. 12.6%). In contrast, a 22.5-fold higher percentage of PPI-treated patients required a dose reduction compared to patients treated with H2R's (47% vs. 2.1%). Meanwhile, the percentage of patients who did not need a dose change was generally similar in PPI-treated patients (28% vs. 40%). The comparison of our results from the long-term treatment groups showed that the major difference was a lower percentage of dose reduction in the patients in the short-term treatment group, suggesting this may contribute to the above difference in the frequency of dose changes between previous short-term PPI studies in ZES patients and the results of the long term/lifetime results in this study. The daily dose change results reported in the above paragraph from our study raise both important pharmacological considerations, as well as important clinical aspects in the long-term treatment of these patients. First, the exact pharmacological basis for the 6-fold higher rate of daily dose increase that is required with H2R long-term/lifetime treatment compared to that seen in ZES patients treated with PPIs is unclear. A number of pharmacological studies in patients treated with H2Rs, primarily with GERD, report that there is a high rate of developing tachyphylaxis with continued treatment , whereas most , but not all studies , report a lack of tachyphylaxis with continued PPI treatment. In our study, the failure to find any correlation between markers of either tumoral function, aggressiveness, or tumor growth and the increasing H2R dose requirement suggests that the increased H2R daily dose requirement is not a direct result of tumor growth/activity and is compatible with the possibility of developing H2R tachyphylaxis. This difference in the development of tolerance between H2Rs and PPIs in ZES patients has the important clinical consequence that once the initial daily dose is adequately set in ZES patients with a PPI, the likelihood of needing early dose escalation is a much less than with an H2R. Furthermore, it raises the possibility that if predictors for PPI dose change can be identified, the requirement during follow-up for repeat acid assessment can be much better tailored toward certain patients. Second, the finding that dose reduction was possible with time in almost half of all PPI-treated patients, but in only 2% of H2R-treated patients, has important pharmacological and clinical considerations for the treatment of ZES patients. The entire subject of whether dose reduction of either PPIs or H2Rs should be routinely attempted during maintenance treatment in ZES patients is controversial at present. Some short-term studies report that the daily dose of PPIs can be reduced in up to 58-83% of ZES patients and that patients were safely maintained on these lower doses, whereas another study reported that the daily PPI dose could be reduced in only 27% of patients and concluded that it could not be generally recommended until further studies were performed. There are no prior comparable studies of attempted dose reduction in ZES patients treated long-term with H2Rs. Previous pharmacological time-course studies of acid inhibition with different doses of H2Rs (cimetidine/ranitidine), demonstrated that their action was maximal at 5-6 h, followed by a rapid rebound of acid secretion . Furthermore, as seen in the present study and reported previously, in contrast to PPIs, H2Rs rarely reduce acid secretion to very low levels in ZES patients (i.e., <2 mEq/h) . No prior studies of systematic dose reduction with H2Rs have been reported, likely because of the above pharmacological findings with H2Rs. It is generally thought that an H2R dose reduction would, in most cases, result in inadequate acid control. The current study supports this conclusion in that dose reduction during maintenance treatment in H2R-treated patients was rarely (i.e., 2%) successful. The resolution of the controversy of PPI dose reduction during maintenance therapy with ZES patients remains a pressing issue primarily for three reasons. First, both recent reviews and numerous papers (primarily epidemiological studies), have raised increasing concerns about the possible long-term/lifetime side effects of PPI treatment. These include PPIs being associated with increased bone fractures/bone problems (dental implants); nutritional/drug malabsorption (vitamin B12, magnesium, calcium, iron); increased chronic renal disease; lung disease (pneumonia); cardiovascular disease (CVS) (CVS mortality, myocardial infarction, stroke); CNS abnormalities (dementia); increased infections (Clostridia, bacterial infections with liver disease); increased development of carcinoids and other tumors; and interference with metabolism of important therapeutic agents/drugs . Although causality or possible mechanisms of action by which PPIs cause these changes are not clear in most cases, the daily dose of PPI could be a contributing factor in some cases . Because of this, in most guidelines for chronic treatment with PPIs in various diseases increasingly recommend using the lowest doses of PPIs, or even stopping PPI treatment for periods of time, and that PPIs only be used for established indications . A number of these potential PPI side effects have been reported in ZES with long-term PPI treatment , particularly, decreased nutrient absorption (Mg2+, vitamin B12), and development of carcinoid tumors and other neoplasms, which have led to difficulties in long-term acid control and even the need for total gastrectomy. Second, because of the recommended manner of establishing the initial individual drug dose requirement by serial upward dose titration for both PPIs and H2Rs, subsequent PPI dose reduction becomes a particularly important issue. This occurs because in most cases when patients are first evaluated for diagnosis and tumor localization/extent, their initial maintenance dose requirement is usually established starting with an omeprazole-equivalent dose of 60 mg/day (or 60 mg twice a day in patients with complicated diseases such as severe GERD, Billroth 2 resection, MEN1) . This approach is recommended and generally used because many patients, when initially seen, have acute mucosal disease/marked symptoms that need to be rapidly controlled to avoid the rapid development of life-threatening peptic-ulcer/GERD complications . The lowest dose of PPI equivalent to omeprazole (20 mg or 20 BID) could be used with subsequent upward titration, but this may delay adequate dosing and disease control because of the mechanism of action of PPIs . This conclusion was supported by one prospective study in ZES patients where this recommended low dose of PPI (20 mg/day omeprazole) was used in 49 patients to determine whether this method satisfactorily identified the initial dose needed to control the acid hypersecretion. Using this method, one third of all patients failed to reach satisfactory acid control during the duration of the study, and the patients that failed at a low dose could not be clearly predicted , leading the authors to recommend that this approach should not generally be used. The mechanism of action of PPIs is via inactivation of the final common pathway of acid secretion by directly binding to and inactivating the activated hydrogen-potassium ATPase pump The active drug only has a short plasma half-life (<60 min) and although it irreversibly inactivates the active pumps, its short half-life and subsequent pump biosynthesis result in the final duration of effect of these drugs with the efficacy of PPIs increasing over the first 3-5 days of use . Therefore, the current recommended initial dosing method allows rapid control of acid secretion, but a proportion of these patients who cannot be predicted will be able to subsequently have their acid controlled on lower PPI doses with time, as shown in our study. The third reason for dose reduction, if possible, is monetary. Most insurance companies do not pay the full amount of the drug cost, thus placing a burden on some patients at various times. In contrast to issues related to changes in the magnitude of the antisecretory drug dose in ZES patients, which has been well-studied in numerous short-term studies and reviewed in the previous paragraphs, the issue of the frequency of antisecretory daily drug dosing and its possible effect on total daily drug dose has not been well-studied in ZES patients. Specifically, the effects of antisecretory drug frequency have neither been systematically nor well-studied in short-term studies of ZES patients, nor has it been studied in any manner in long-term studies. This is an important area of study both for clinical concerns and pharmacological insights. In studies of patients primarily with GERD, it has been reported that the frequency of administration of both H2Rs and PPIs can have a marked effect on their therapeutic efficacy, even to the extent that symptoms and esophageal pH can be controlled with greater ease by regulating the frequency of drug administration, rather than only increasing the dose . Because of their pharmacological shorter duration of action, this type of clinical response would be predicted particularly with H2Rs; however, it is also reported in some studies of GERD patients treated with PPIs at different frequencies and dosages . A number of our results support the conclusion that altering the antisecretory drug dosing frequency of both H2Rs and PPIs in ZES patients can achieve similar results as those reported above in GERD patients due to similar drug pharmacological effects on gastric hypersecretion in ZES patients, thus resulting in similar positive clinical results. Our results showed that the frequency of both the initial drug dosing as well as the frequency of rate of change in daily dosing during long-term maintenance antisecretory therapy varied markedly with long-term treatment in ZES patients and that the frequency varied markedly between ZES patients treated with long-term maintenance H2Rs or PPIs. Specifically, we found that the initial daily dose frequency was 2-fold higher with H2Rs than with PPIs, which reflected the known greater duration of action of PPIs over H2Rs in both peptic/ulcer/GERD and ZES patients . Overall, in our study, one half of all patients on long-term maintenance with H2Rs demonstrated a change in daily dosing frequency (either increase or decrease), whereas with long-term PPI treatment, almost one third of patients required a change in dosing frequency over the treatment period of a mean of 9.8 years (range 0.1-48.1 yrs). Furthermore, the percentages of patients were similar in both the short-term (<5 yrs) (H2Rs = 60 pts, PPIs = 128 pts) or the long-term (H2Rs = 117 pts, PPIs = 202 pts) treatment groups, demonstrating that this change in drug dosing frequency was not a function of the length of the follow-up. Not only did the overall frequency of the daily antisecretory dose vary between PPI-treated patients during long-term maintenance, the direction of the dosing frequency changes differed markedly with a 3-fold high percentage of patients showing an increase in dose with H2Rs over PPIs, while a 2.5-fold higher percentage of PPI-treated patients showed a decrease in the daily dosing frequency compared to H2R-treated patients. Lastly, in a subgroup analysis of 99 patients, we found that increasing the frequency of PPI dosing resulted in a proportion of patients requiring a lower total daily PPI dose, whereas this rarely occurred in H2R-treated patients with increased dosing frequency, which resulted in an increased total daily dose in 95% of patients. The above findings from our long-time/lifetime study are consistent with the results from a few short-term studies on ZES , supporting the conclusion that dividing the dose can be a better strategy for patients requiring a high daily dose of PPIs, rather than further increasing the daily dose, and may allow a lower effective total daily dosage. As pointed out in the initial paragraph of the discussion and frequently mentioned in various short-term studies of acute initial control and longer maintenance control of acid hypersecretion in ZES patients, the key to successfully managing acid hypersecretion is the careful assessment of gastric acid hypersecretion control to establish suppressive values, which have been shown to allow mucosal healing and prevent addition mucosal damage . As shown in this study and others , both the initial and subsequent assessments of acid secretory control in ZES patients show marked dose requirement variability from patient to patient, hence, the approach needs to be individualized for each patient with appropriate titration of their antisecretory dose . This requires repeated gastric analysis, which is labor intensive, uncomfortable for many patients, and may require travel to one of the few centers that offer such a service. Because, as shown in the present study and other short-term studies , gastric acid hypersecretion is currently treated with PPIs in more than 95% of all ZES patients, both acutely and for maintenance, any predictor that could identify patients who will or will not need a PPI drug dose change could be of marked clinical value in allowing patients to be selected for assessment and better tailoring of care. In the present study, we found a number of clinical and laboratory features that significantly correlated with the subsequent need for PPI dose change during long-term treatment extending over a mean of 10 years (range 1-48 yrs). These variables included the presence of severe GERD, but not mild/moderate GERD; the history of a previous Billroth 2 procedure, but not a previous vagotomy with other drainage procedure; the presence of a high initial BAO (i.e., >36.8 mEq/h) or MAO (i.e., >62 mEq/h); the presence of achlorhydria on the initial acid assessment (i.e., acid output = 0 mEq/h); and two variables related to the initial PPI dosing, including a high PPI daily dose for those taking QD PPI dosing (i.e., >55 mg/day) or a high daily dose for those taking BID/TID dosing (i.e., >80 mg day of omeprazole). These prognostic results overlap with some but not all features previously described in short-term studies in ZES patients that correlate with final drug PPI drug doses, the subsequent ability to reduce PPI drug doses, or the subsequent need to increase PPI drug doses either because of continued/new peptic symptoms or the development of acid-peptic related mucosal changes during maintenance PPI treatment, after first setting the initial dose by acid titration . Our results disagree with previous short-term studies that reported no predictive value of the BAO/MAO or fasting serum gastrin level for the need for a higher/lower dose of lansoprazole during maintenance . However, our predictive results agreed with previous short-term studies in which PPI maintenance doses did not correlate with tumor extent ; patients less frequently had dose changes when they had previously required lower doses of antisecretory drugs ; increased dosing changes were required for patients with two of the three features of complicated ZES (i.e., advanced GERD, Billroth 2, but not the presence of MEN1) ; there was a lack of predictive value for most clinical factors, (i.e., age, gender, duration of disease, or symptoms) ; there was a lack of effect of the occurrence of prior peptic ulcer complications on subsequent dosing changes ; and with other studies that showed increased PPI doses/dose changes occurred more frequently with prior high BAO . One of these predictive features that caused some confusion is the importance of MEN1/ZES. MEN1/ZES occurs in 20-25% of all ZES patients and numerous studies have clearly established the fact that the presence of hyperparathyroidism in these patients (>95% at some point) results in increased BAO, MAO, fasting gastrin levels, and increased resistance to antisecretory drug therapy . If hyperparathyroidism is recognized and successfully corrected, which can be difficult in some patients , these changes were reversed post-parathyroidectomy . The problem is that hyperparathyroidism has a high recurrence rate and can be missed, so this can affect the PPI maintenance dose . In our patients who were assessed yearly for the recurrence of MEN1 features, recurrences were rapidly detected and corrected, with the result being that we saw no effect of the presence of MEN1 (i.e., hyperparathyroidism) on drug maintenance doses. The result of identifying these predictive factors for PPI daily maintenance dose changes will need to be prospectively studied to determine whether they can be safely applied in order to enable structured tailoring of gastric analysis, thereby more easily managing lifelong acid hypersecretion in these patients. It could be argued that this study's results cannot be extrapolated to most clinical situations because it was carried out as part of the NIH prospective study on ZES, which is a different clinical scenario that that seen in many centers. We think that this is not the case for a number of reasons. First, it is recommended by almost all guidelines that patients with ZES should be treated in specialty centers where there is expertise in all aspects of this disease and with other neuroendocrine tumors. A number of these centers exist in the US and have this capability. This is best demonstrated by the fact that many of these centers have the capability of carrying out a treatment course such as outlined in this study, including the measurement of gastric acid outputs in response to drug dosing, attested by the numerous, short-term, and successful acid inhibitory studies in the recent literature. These reports include the successful treatment of acid hypersecretion in ZES patients with various acid antisecretory drugs, including esomeprazole , lansoprazole , pantoprazole , rabeprazole , and omeprazole . With the availability of PPIs, which are both more potent and longer acting than H2Rs, the ability to control acid hypersecretion in these patients has become more manageable in different centers. While this study investigated the effectiveness of long-term medical control of the lifelong acid hypersecretion that is present in most ZES patients, it is important to remember that all ZES patients have two problems: control of the acid hypersecretion and treatment of gastrinomas, which are malignant in 60-90% of ZES patients. Surgery remains the only possibility of cure and treatment for both of these problems. With the increased sensitivity of imaging modalities, the possibility of rendering a patient disease-free is increasing and thus, systematic reassessment of possible surgical resection in addition to control of the acid hypersecretion need to be performed. 5. Conclusions The key findings of this study are summarized in Table 8. The most important overall conclusion of the current study was that long-term/lifetime medical treatment of gastric acid hypersecretion in uncured ZES patients (>70-90% of all cases) was possible in all patients included in this study. These results should be applicable to almost all series of ZES patients in different centers. The study included a large number of patients (n = 303), they resembled most smaller series in the literature in all characteristics, and the cohort had all aspects of the disease, including different tumor extents, with or without MEN1, with or without previous gastric acid reduction surgery, with and without different degrees of GERD, all of which have been reported to affect the success of medical acid antisecretory therapy in ZES patients. Furthermore, the present study was perspective, with a fixed protocol, and extended up to 48 years for one patient (mean 15 yrs), allowing it to be carried out for lifetime treatment in 30% of all patients. The conclusion of this study is that the lack of efficacy of gastric acid antisecretory therapy in ZES patients does not have to be a major factor in deciding the course of treatment (for example, medical vs. surgical) in these patients. The development of increasingly sensitive imaging methods for localizing these tumors (i.e., somatostatin receptor imaging, etc.) is increasing the possibility of surgical cure; therefore, improved understanding of the natural history of patients with different aspects of ZES, especially those with MEN1/ZES, and the development of novel and effective methods for the treatment of ZES patients with advanced disease can now become the main area of attention for therapeutic approaches. Author Contributions Conceptualization, T.I., I.R.-A. and R.T.J.; Methodology, T.I., I.R.-A. and R.T.J.; Validation, I.R.-A.; Formal analysis, T.I. and R.T.J.; Investigation, R.T.J.; Writing--original draft, T.I., I.R.-A. and R.T.J.; Writing--review and editing, R.T.J.; Supervision, R.T.J. All authors have read and agreed to the published version of the manuscript. Institutional Review Board Statement This research has been approved by the clinical research committee of the National Institute of Diabetes, Digestive and Kidney Diseases of the National Institutes of Health: the protocol for the use of histamine H2 receptor antagonists was approved on 20 August 1978 (89-DK-15) and the protocol for the PPI omeprazole on 15 January 1983. Informed Consent Statement Informed consent was obtained from all subjects involved in the study. Data Availability Statement The data presented in this study are available in this article. Conflicts of Interest The authors declare no conflict of interest. Figure 1 Time course of acid antisecretory drug treatment in 303 patients with ZES. Shown are the percentages of ZES patients with acid hypersecretion during different time periods treated with either histamine H2-receptor antagonists (cimetidine, ranitidine, famotidine, nizatidine) or proton pump inhibitors (PPIs) (omeprazole, lansoprazole, pantoprazole). The time periods analyzed were 1/76-1/83 (90 patients), 2/83-1/85 (117 patients), 2/85-1/87 (147 patients), 2/87-1/90 (177 patients), 2/90-1/93 (196 patients), 2/93-1/96 (216 patients), 2/96-1/99 (210 patients), 2/99-1/2002 (188 patients), 2/2002-1/2006 (166 patients), and 2/2006-2020 (91 patients). Data are reported as percentage of patients during the given time period that were treated with either group of drugs. Figure 2 Pie charts showing distribution of changes in drug dosage with short- (<5 yrs) or long-term (>=5 yrs) treatment with either a histamine H2-receptor antagonist or proton pump inhibitor. Data shows the percentage of ZES patients treated with either a histamine H2-receptor antagonist or proton pump inhibitor at any time, during short-term (<5 yrs) or long-term (>=5 yrs) treatment, who required an increase in daily drug dosage, no dosage change, or a decrease in dosage determined as described in the Section 2. For PPI users, the mean time between the first and last dose for the total was 9.78 +- 0.35 years, the mean time for the short-term treatment group was 2.45 +- 0.17 years, and the mean time for the long-term treatment group was 12.00 +- 0.32 (range 5.2-32.2) years. For H2R users, the mean time from first to last dose time for all users was 6.38 +- 0.32 years, the mean time of the short-term treatment group was 2.32 +- 0.13 years, and the mean time of the long-term treatment group was 10.86 +- 0.50 (range 5.0-29.2) years. Abbreviations: Inc.: increased dose, Dec.: decreased dose. Figure 3 Pie charts showing distribution of changes in drug daily frequency with long-term treatment with either a histamine H2-receptor antagonist or proton pump inhibitor. Figure 4 Pie charts showing distribution of changes in total drug daily dose in patients who had an increased frequency of dosing with long-term treatment with either a histamine H2-receptor antagonist or proton pump inhibitor. Data shows the percentage of ZES patients having an increased daily dosing frequency treated with either a histamine H2-receptor antagonist or proton pump inhibitors at any time, during short-term (<5 yrs) or long-term (>=5 yrs) treatment, which resulted in an increase in total daily drug frequency, no total daily dose change, or had a decrease in total daily drug dose, determined as described in the Section 2. The duration between the first and last daily doses used for comparing daily dosing frequency were the same as listed for the daily dose comparison in the legend of Figure 2. Figure 5 Distribution of the first and final total daily drug dosages in 303 ZES patients with long-term treatment with either a histamine H2-receptor antagonist or proton pump inhibitor. In the top panel, the distribution of the first and final total daily drug doses are separated for patients taking PPIs once, twice, or three times per day. In the bottom panel, the first and last total daily doses of H2Rs are reported. Distributions at different drug doses are reported as the percentage of the total number of patients taking the given drug class at the listed total daily dosage. In the top panel, the drug dosages are reported as omeprazole-equivalent doses for the different PPIs, determined as described in the Section 2. In the bottom panel, the total daily H2R doses are reported as ranitidine-equivalent daily doses, determined as described in the Section 2 and previously in ref. . The mean times between doses for both classes of drugs are shown above in the legend of Figure 2. cancers-15-01377-t004_Table 4 Table 4 Acid treatment: comparison of initial and final daily H2R and PPI drug doses. Characteristic Number (%) H2R (a,b) PPI (a,c) p-Value I. Dosing (a) Overall (n = 303) Initial daily dose (mg/day) # of patients 241 262 Mean +- SEM 968 +- 62 71.7 +- 2.3 (Range) (80-4800) (20-240) Final daily dose (mg/day) Mean +- SEM 2440 +- 141 58.5 +- 2.4 (Range) (233-14400) (20-240) % Patients with change in daily dose Increase 70.0% 12.6% <0.0001 Decrease 2.1% 46.9% <0.0001 No change 27.9% 40.5% 0.0037 % of patients with different daily doses Lowest initial dose (i.e., H2-R <= 900 mg/day/PPI 20 QD/20 BID) 154/229 (67%) 143/261 (22%) <0.0001 Lowest final dose (i.e., H2-R <= 900 mg/day/PPI 20 QD/20 BID) 61/229 (27%) 128/261 (49%) <0.0001 Higher initial doses (i.e., >=2300 mg/day/PPI-80 mg/day) 31/229 (13%) 125/261 (49%) <0.0001 Higher final doses (i.e., >=2300 mg/day/PPI-80 mg/day) 138/229 (60%) 99/261 (38%) <0.0001 Short-term acid treatment (<5 yrs) with any drug (c) Initial daily dose (mg/day) # of patients 124 60 Mean +- SEM 1112 +- 96 63.3 +- 3.8 (Range) (80-4800) (20-120) Final daily dose (mg/day) Mean +- SEM 2534 +- 204 62.7 +- 5.0 (Range) (90-4800) (20-200) Long-term acid treatment (>=5 yrs) with any drug (d) Initial daily dose (mg/day) # of patients 114 202 Mean +- SEM 812 +- 75 74.2 +- 2.7 (Range) (100-4800) (20-240) Final daily dose (mg/day) Mean +- SEM 2341 +- 196 57.3 +- 2.7 (Range) (200-12000) (20-240) (a). Both the H2R and PPI control drug doses were established by determining the minimum drug dose that reduced the gastric acid output in mEq/h for the hour prior to the next drug dosage to the endpoint, as previously described and in the Section 2. (b). The total daily H2R doses are reported as ranitidine-equivalent daily doses, determined as previously described and in the Section 2. (c). PPI drug dosages are reported as omeprazole-equivalent doses, as described in the Section 2. (d). For explanation of short and long-term treatment, see Table 1 footnote (a). cancers-15-01377-t005_Table 5 Table 5 Acid treatment: comparison of initial and final daily H2R and PPI dosing frequencies. Characteristic Number (%) H2-R (a,b) PPI (a,c) p-Value I. Dosing (a) Overall (n = 303) Initial daily dosing frequency # of patients 241 262 Mean +- SEM 2.85 +- 0.07 1.47 +- 02.3 <0.0001 (Range) (1-6) (1-3) Final daily dosing frequency Mean +- SEM 3.53 +- 0.07 1.48 +- 0.3 <0.0001 (Range) (1-6) (0-3) % Patients with change in daily dosing frequency Increase 99/229 (43%) 39/262 (15%) <0.0001 Decrease 13/229 (5.6%) 33/262 (13%) <0.0001 No change 117/229 (51%) 190/262 (72%) 0.0037 % of patients with different daily doses Lower initial frequency (i.e., H2-R <= 3x/day/PPI <= 1x/day) 104/241 (43%) 142/262 (54%) <0.0001 Lower final frequency (i.e., H2-R <= 3x/day/PPI <= 1x/day) 44/229 (19%) 134/262 (51%) <0.0001 Highest initial frequency (i.e., >=4x/DAY-H2-R/ day/ > 2/day PPI) 86/241 (36%) 2/262 (0.76%) <0.0001 Higher final frequency (i.e., >=4x/H2-R/ day/ > 2/day PPI) 150/229(66%) 2/262 (0.76%) <0.0001 Short-term acid treatment (<5 yrs) with either H2R or PPI (c) Initial daily dosing frequency # of patients 126 60 Mean +- SEM 2.76 +- 0.10 1.28 +- 0.6 (Range) (1-6) (1-2) Final daily dosing frequency Mean +- SEM 3.54 +- 010 1.32 +- 0.07 (Range) (1-6) (1-2) Long-term acid treatment (>=5 yrs) with either H2R or PPI (c), (d) Initial daily dosing frequency # of patients 117 202 Mean +- SEM 2.93 +- 0.09 1.52 +- 0.4 (Range) (1-5) (1-3) Final daily dosing frequency Mean +- SEM 3.52 +- 0.10 1.52 +- 0.4 (Range) (1-6) (1-2) (a). Both the H2R and PPI control drug doses were established by determining the minimum drug dose that reduced the gastric acid output in mEq/h for the hour prior to the next drug dosage to the endpoints previously described and in the Section 2. (b). The total daily H2R doses are reported as ranitidine-equivalent daily doses, determined as previously described and in the Section 2. (c). PPI drug dosages are reported as omeprazole-equivalent doses for the different PPIs, determined as described in the Section 2. (d). For an explanation of short and long-term treatment, see Table 1 footnote (a). cancers-15-01377-t006_Table 6 Table 6 Treatment outcomes-acid/symptoms. Characteristic Number (%) H2R (a,b) (n = 245) PPI (a,b) (n = 262) p-Value I. Acid control on treatment (a) A. All -no drug (mEq/h) Mean +- SEM 40.1 +- 1.54 42.2 +- 1.6 (Range) (1.8-159) (6-159) B. All /PPI control (a) (mEq/h) Mean +- SEM 4.16 +- 0.46 2.16 +- 0.16 <0.0001 (Range) (01-10.5) (0-9.8) Final acid control (mEq/h) (a,b) Mean +- SEM 3.31 +- 0.40 0.97 +- 0.12 <0.0001 (Range) (0-10.4) (0-7) C. Level of acid control in all /PPI (a) (% total at initial/final treatment) <1 mEq/h (c) 39/130 (30%) 207/377 (55%) <0.0001 1-5 mEq/h (c) 53/130 (22%) 138/377 (37% 0.40 >=8-10 mEq/h (c) 23/130 (14%) 11/377 (2.9%) <0.0001 D. Outcome of acid control-all patients Control symptoms/acid-peptic mucosal disease 100% 100% Not control symptoms/acid acid-peptic mucosal disease 0% 0% (a). Both the H2R and PPI control drug doses were established by determining the minimum drug dose that reduced the gastric acid output in mEq/h for the hour prior to the next drug dose to the endpoint, as previously described and in the Section 2. (b). Included all patients treated with either H2R or PPI at any time. (c). For the <1, 1-5, and 8-10 mEq/h acid control groups for both the PPI-treated patients, the results are expressed as the number of patients with acid control levels within this range for either initial or final acid control over the total number of patients that were included in this assessment. cancers-15-01377-t008_Table 8 Table 8 Summary of key findings. Nr. Summary of Key Findings 1. Long-term control (mean 14 yrs)/lifelong control (30%) as well as acute control of gastric acid hypersecretion by antisecretory medications was successful in all in 303 ZES patients. 2. This was only possible by individually regulating antisecretory drug doses using proven acid inhibitory secretory criteria and adjusting doses accordingly, combined with assessments of acid-peptic/GERD symptoms and UGI endoscopic findings. 3. Both H2Rs and PPIs were successfully used for both acute and maintenance control of acid hypersecretion; however, because of their greater potency and longer duration of action, PPIs are now routinely used in almost all patients. 4. During the initial drug dosing, the mean PPI dose was 72 mg/day, whereas with long-term treatment, half of ZES patients could have their daily PPI total dose reduced and 13% required an increase in their daily PPI dose. In contrast, with H2Rs, the mean initial dose was 969 mg/day of a ranitidine-equivalent dose (RED), with 70% of patients requiring increased daily doses and only 2% tolerating a total H2R daily dose decrease, resulting in an almost 3-fold increase in final mean final H2R daily dose (i.e., 2440 mg RED/day). 5. During long-term control of acid hypersecretion, total PPI daily use could be reduced to the lowest dose level of 20 mg QD/20 mg BID, with a 2-fold higher percentage of patients than during initial treatment, whereas the opposite occurred with H2Rs, with a 2.6-fold lower percentage of patients requiring the lowest dose. 6. H2Rs also differed from PPIs in dosing frequency. At the initial acid control, the mean frequency of H2Rs was almost 3 times/day, whereas it was 1.5 times/day for PPIs. On the final dose, the frequency of H2Rs had increased further to 3.5/day and the PPI mean frequency remained unchanged. These changes were amplified by individual patient drug frequency changes, which showed an average 3-fold greater increased dose frequency required for H2Rs compared to no change with PPIs. 7. The use of PPIs at both the initial and final drug dosing demonstrated markedly better (p < 0.001) acid control than with H2Rs. 8. Some clinical (i.e., severe GERD), laboratory (BAO, MAO), and initial acid treatment variables (presence of achlorhydria, PPI dosing frequency) correlated with the need for subsequent PPI dose changes, raising the possibility of selectively identifying patients needing dose changes and tailoring acid testing based on these variables. Disclaimer/Publisher's Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

PMC10000209

Internal egg and eggshell quality are often deteriorated in aging laying hens, which causes huge economic losses in the poultry industry. Selenium yeast (SY), as an organic food additive, is utilized to enhance laying performance and egg quality. To extend the egg production cycle, effects of selenium yeast supplementation on egg quality, plasma antioxidants and selenium deposition in aged laying hens were evaluated. In this study, five hundred and twenty-five 76-week-old Jing Hong laying hens were fed a selenium-deficient (SD) diet for 6 weeks. After Se depletion, the hens were randomly divided into seven treatments, which included an SD diet, and dietary supplementation of SY and sodium selenite (SS) at 0.15, 0.30, and 0.45 mg/kg to investigate the effect on egg quality, plasma antioxidant capacity, and selenium content in reproductive organs. After 12 weeks of feeding, dietary SY supplementation resulted in higher eggshell strength (SY0.45) (p < 0.05) and lower shell translucence. Moreover, organs Se levels and plasma antioxidant capacity (T-AOC, T-SOD, and GSH-Px activity) were significantly higher with Se supplementation (p < 0.05). Transcriptomic analysis identified some key candidate genes including cell migration inducing hyaluronidase 1 (CEMIP), ovalbumin (OVAL), solute carrier family 6 member 17 (SLC6A17), proopiomelanocortin (POMC), and proenkephalin (PENK), and potential molecular processes (eggshell mineralization, ion transport, and eggshell formation) involved in selenium yeast's effects on eggshell formation. In conclusion, SY has beneficial functions for eggshell and we recommend the supplementation of 0.45 mg/kg SY to alleviate the decrease in eggshell quality in aged laying hens. selenium yeast eggshell quality transcriptomic analysis aged laying hens the National Key R&D Program of Intergovernmental Key Projects in China2018YFE0101700 the National Key R&D Program in China2016YFD0501202 This work was supported by the National Key R&D Program of Intergovernmental Key Projects in China (2018YFE0101700) and the National Key R&D Program in China (2016YFD0501202). pmc1. Introduction Maintaining egg quality and production performance in aged laying hens for longer laying cycles has become an increasing concern. In general, the peak production of commercial laying hens lasts until around 60 weeks of age, and the laying rate and egg quality begin to decline thereafter . Aging of hens leads to deterioration in internal and shell quality of eggs, which may lead to breakage during collection and transport . Thus, overcoming the negative effects of age on egg quality is important in the late phase of the laying cycle. Some previous studies suggested that the decline in egg quality is associated with nutrient status and oxidative stress in aged laying hens . Selenium (Se) is an essential micronutrient for humans and animals which plays a vital role in antioxidant defense, redox state regulation, reproduction, and a wide variety of specific metabolic pathways . The pleiotropic aspects of this essential nutrient have raised the interest of worldwide geneticists and nutritionists in the last few decades. In general, Se functions through inserting the amino acid selenocysteine (Sec) into a multitude of proteins, which are known as selenoproteins. To date, 25 human selenoprotein genes and 24 confirmed avian selenoprotein genes have been identified. The health impact of upregulation of antioxidant selenoenzymes and cytoprotective properties mediated by Se has been well researched. Dietary Se intakes can affect the synthesis and expression of selenoproteins by modulating status of Se . Appropriate Se status leads to health benefits, such as decreased mortality and enhanced immune function. Moreover, early research demonstrated that both Se deficiency and excess can cause adverse health effects. Impairments in antioxidant protection, redox regulation, immunity response, energy production, infertility, and reproduction are associated with Se deprivation . Se deficiency has been demonstrated to impair growth performance and lead to exudative diathesis , which causes economic loss in chicken. Oversupplementation of Se may also have adverse effects on people or animals, commonly called selenosis. It causes decreased laying rate and skin and liver damage in poultry . Se is widely used as a feed additive for enhancing the antioxidants, immunity, and health of chickens, pigs, and other livestock, as well as to obtain Se-enriched byproducts. As the organic form of Se, selenium yeast is commonly used because it is well absorbed and biologically safe . Some studies on broilers and laying hens showed that selenium yeast supplementation aids in maintaining poultry health, maintaining a desirable egg shape index, and supporting shell thickness , as well as enhancing antioxidant status and organs Se deposition . Previous studies also demonstrated that dietary supplementation of selenium yeast can improve the productive performance in aging hens . Selenium as a trace element plays an important role in the production performance and antioxidant capacity in laying hens. Higher absorption and deposition efficiency of selenium yeast may be more conducive to the utilization of selenium in aged laying hens, helping to extend the laying cycle of hens and reduce the breeding costs. We hypothesized that selenium yeast has beneficial effects on egg and shell quality, plasma antioxidants, and selenium deposition in aged laying hens. Moreover, the potential molecular mechanisms of selenium yeast on eggshell strength were explored by using RNA-seq-based global transcriptome analysis. 2. Materials and Methods The experimental animal protocols for this study were approved by the Animal Care and Use Committee of China Agricultural University (No. AW05060202-1). 2.1. Animals, Experimental Design, Diets, and Husbandry A total of 525 76-week-old commercial Jing Hong laying hens were housed in the Poultry Experiment Base of China Agricultural University (Hebei, China) and fed with an average basal Se content of 0.056 +- 0.012 mg/kg corn-soybean diet (the composition and nutrient level of the basal diet are shown in Table 1) for 6 weeks (from 76 to 82 weeks of age) to induce Se depletion in aged laying hens before the selenium yeast (SY) feeding period. After Se depletion, Jing Hong laying hens with similar laying rate (average laying rate = 74.2%) were randomly allocated to 7 treatment groups with 5 replicates (15 chickens per replicate) each. All of the chickens were raised in cages, with three chickens per cage. One group was fed the basal diet only (SD), three groups were supplemented with 0.15, 0.30, or 0.45 mg/kg selenium yeast (SY) (Alltech, Nicholasville, KY, USA), and the other three groups were supplemented with 0.15, 0.30, or 0.45 mg/kg sodium selenite (SS) for 12 weeks (from 83 to 95 weeks of age). The supplemental and analyzed Se levels of each diet are shown in Table 2. The different dose of Se was premixed into the diets, and the corn-soybean diet was formulated to satisfy the Chinese Feeding Standard of Chicken (NY/T33-2004). Batches of the experimental diets were produced every 4 weeks to prevent the feed from mildewing. The hens were housed in an environmentally controlled room maintained at 25 degC and had a daily lighting schedule of 16 h light (from 5 am to 9 pm), 8 h dark (from 9 pm to 5 am). 2.2. Data and Sample Collection Blood samples (8 mL per laying hen) were taken from the main wing vein and collected into an ethylene diaminetetraacetic acid (EDTA) anticoagulant tube every two weeks (at 76, 78, 80, 82, 85, 87, 89, 91, 93, and 95 weeks of age) during the whole 18-week experiment. Plasma was separated by centrifugation at 4 degC, 3000 rpm, for 10 min and stored at -30 degC for further analysis. After Se supplementation for 12 weeks (experimental weeks 18), 2 random laying hens per replicate group were slaughtered 15-20 h post-ovulation. Ovary, magnum, isthmus, and shell gland were sampled and frozen in liquid nitrogen immediately. All samples were stored at -80 degC prior to analysis. In addition, shell gland organs were fixed in 4% paraformaldehyde for hematoxylin-eosin staining. 2.3. Measurement of Egg Quality and Translucent Egg Egg quality was measured during the Se depletion and Se feeding periods. In the Se depletion periods, egg quality was measured at the start and end of this period. In the Se feeding period, egg quality was measured at 6-week intervals; an average of 6 eggs from each replicate in one day were selected randomly for eggshell strength, egg weight, egg yolk color, Haugh unit, and albumen height measurement using a digital egg tester (NABEL, DET-6000) immediately. An average of 6 eggs from each replicate in one day were used for the measurement of the translucent egg at 6-week intervals. The eggs were stored for 0, 7, and 14 days at 20 degC and classified into 4 score levels: 1 means the best shell translucence degree, 4 means the worst shell translucence degree, and the scoring method was referenced in . 2.4. Determination of Se Content To determine the Se content of the feed, plasma, ovary, magnum, isthmus, and shell gland, all samples (0.5-1 g for feed, ovary, magnum, isthmus, and shell gland and 0.5-1 mL for plasma) were digested in a mixture of concentrated nitric acid and perchloric (2 HNO3:1 HClO4) for about 2 h at 200 degC until white fumes appeared. Then, 5 mL hydrochloric acid (1 HCl: 4 H2O) solution was added in the mixture and heated until white fumes appeared. After the mixture cooled, 20 mL EDTA was added in the digested sample and pH was adjusted to 1.5-2.0. After this, 3 mL 2,3 DiAminoNaphthalene (DAN) was added in the mixture and heated in the boiled water for 5 min. After the mixture cooled at room temperature, 4 mL cyclohexane was added and the mixture was shaken for 8 min. The supernatant was measured by fluorescence method using a Hitachi 850 fluorescence spectrophotometer (Tokyo, Japan) . 2.5. Measurement of Antioxidant Enzyme Activity The activities of glutathione peroxidase (GSH-Px) and total superoxide dismutase (T-SOD) in plasma samples were determined using the detection kits of GSH-Px and T-SOD (Nanjing JianCheng Bioengineering Institute, Nanjing, China). The activity of total anti-oxidation capacity (T-AOC) in plasma samples was measured by T-AOC Kit (KeyGEN BioTECH, Nanjing, China) following the manufacturer's protocol. 2.6. Hematoxylin-Eosin Staining For histological assessment, three-micrometer sections were stained with hematoxylin and eosin (H&E) to assess whether prolonged selenium supplementation in aged laying hens can lead to selenosis or pathological status in shell gland. The entire images were captured by APERIO CS2 (Leica, Germany). 2.7. RNA Extraction, Library Preparation, Sequencing, and RNA-Seq Data Analyses Total RNA was isolated from the shell gland of 4 laying hens using the RNA Isolated Kit (RN4402, Aidlab Biotechnologies, Beijing, China) according to the manufacturer's protocol. Quality and quantity measurements of the extracted RNA were performed using a nanophotometer (IMPLEN, Westlake Village, CA, USA) and a qubit fluorometer (Life Technologies, Carlsbad, CA, USA), respectively. RNA integrity numbers (RINs) were determined using the 2100 RNA Nano 6000 Assay Kit (Agilent Technologies, Santa Clara, CA, USA). Paired-end transcriptome sequencing was performed using an Illumina Novaseq6000 sequencing platform at Annoroad Biotechnology Co. Inc. (Beijing, China). 2.8. Transcriptome Bioinformatic and Statistical Analysis Raw transcriptome reads were quality controlled by filtering out low-quality and adaptor sequences by fastp Version 0.20.0 to provide clean data for downstream analysis. Clean reads were mapped using Hisat2 to the reference genome downloaded from Ensembl accessed on 5 September 2020). Quantification of gene expression level was conducted by FPKM (fragments per kilobase of exon per million parts mapping) using the featureCounts package, and differentially expressed genes (DEGs) were accessed using DESeq2 R package. The DEGs were identified with the following parameters: p-values < 0.05 and the fold change value |log2Ratio| >= 1. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis were further analyzed in the website DAVID accessed on 13 October 2020). Short Time-series Expression Miner (STEM) was performed (default parameters) to investigate the dynamic changes in gene expression in shell gland related to different selenium yeast supplementation levels. Weighted gene coexpression network analysis (WGCNA) was performed to identify candidate genes affecting eggshell quality regulated by selenium yeast. Pearson correlation (p < 0.05 and |r| > 0.4) was constructed for the relationship between modules and Se content. Common genes in significant modules and DEGs were performed with GO enrichment analysis. Sankey charts exhibited the common pathways between pathways and DEGs to investigate the molecular processes affecting eggshell quality by selenium yeast. The genes in common pathways were investigated for protein-protein interaction (PPI) analysis on the STRING accessed on 14 November 2020) website (confidence = 0.7). 2.9. Quantitative Real-Time PCR (qRT-PCR) Analysis Expression of mRNA from the transcriptomic analysis was verified by qRT-PCR with cDNA from shell gland samples. Primer Premier (v. 5.0) with default parameters was used to design primers for selected genes (Supplement Table S1). b-actin gene served as a housekeeping gene. qRT-PCR analysis was conducted with a reaction volume of 20 mL containing 10 mL Premix (FP209-2, Tiangen, Beijing, China), 0.6 mL forward and reverse primers, 1 mL cDNA, and 7.8 mL DNase/RNase-free deionized water. The reaction conditions followed the protocols and instructions. The 2-DDCT method was used to quantify the relative changes in gene expression versus those of b-actin from qRT-PCR experiments. 2.10. Statistical Analysis Data are presented as mean +- standard error of the mean (SEM), and data were analyzed with one-way ANOVA, followed by Duncan test (SPSS for Windows, version 25; IBM). Statistical differences were considered significant at p < 0.05. The linear or quadratic effect of different level of sodium selenite and selenium yeast effects on egg quality were analyzed using SPSS (SPSS for Windows, version 25; IBM), and orthogonal polynomial contrasts were used to examine whether the responses to different Se levels were linear or quadratic. Phenotypic data presentation and linear correlation analysis was carried out using GraphPad Prism (version 7.0, GraphPad Software Inc, San Diego, CA, USA). Every experiment was repeated at least three times. 3. Results 3.1. Plasma Se Status and Antioxidant Enzyme Activity In our study, plasma Se status and antioxidant enzyme activities were measured in aged laying hens during the whole experimental period (Table 3 and Table 4). During the depletion period (experimental weeks 0-6), the plasma Se content significantly declined from 0.237 mg/mL to 0.068 mg/mL (experimental weeks 0-4) (p < 0.01) and then remained at a stable low status (experimental weeks 4-6) (Table 3). Moreover, the redox state in plasma was altered by dietary Se deficiency (Table 4). There were concomitant decreases in plasma T-AOC activity, T-SOD activity, and GSH-Px activity in the first two weeks of Se depletion (experimental weeks 0-2) (p < 0.01, p < 0.01, and p < 0.05, respectively), then from experimental weeks 2-6, plasma antioxidant status remained relatively stable. In the supplementation period (experimental weeks 6-18), Se content increased drastically in the first two weeks (experimental weeks 6-8) (p < 0.01), with a peak after approximately 6 weeks of supplementation (experimental week 12) for SY supplemented. From experimental weeks 12-18, plasma Se content plateaued in all supplementation groups (Table 3). Se content had a better deposition effect in SY supplementation than SS supplementation. Interestingly, the plasma Se content in the SY0.45 group recovered to the level before depletion after 8-week supplementation (experimental weeks 14), but other treatments did not. Similarly, the plasma antioxidant capacity rose when the Se supplementation diets were fed. There were no significant differences in T-AOC activity after 4 weeks of Se supplementation (experimental weeks 10), but it was higher in the SS and SY groups than the SD group after 8 weeks of supplementation (p < 0.01). The T-SOD and GSH-Px activity in plasma after 12 weeks of supplementation (experimental week 18) were significantly higher (p < 0.05) in the SY0.45 group than the SD group (Table 4). Compared with the SS and SD groups, SY supplementation groups had a better antioxidant activity in plasma. 3.2. Egg Quality and Organs Se Levels The changes in the egg quality during the selenium deficiency over time are shown in Table 5. With the increasing week of age and Se depletion, egg weight has no significant changes; however, eggshell strength was significantly lower (p = 0.04). Albumen height, Haugh unit, and yolk color were also significantly lower (p < 0.01). During the supplementation period, eggshell strength was significantly higher in the SY0.45 group than in the SD group at 6 weeks and 12 weeks post supplementation start and higher than the SY0.15 group after 12 weeks of SY supplementation (p < 0.05) (Table 6). Eggs collected after 6 and 12 weeks of supplementation (experimental weeks 12 and 18) were classified into four grades according to the degree of shell translucence . After 6 weeks of SY supplementation, shell translucence score was generally lower in the supplemented groups at all three lengths of egg storage . In addition, eggs from Se supplemented hens had lower egg weight, albumen height, and Haugh unit after 12 weeks of supplementation (Table 6). Se status of organs associated with laying performance including shell gland , eggshell , magnum , isthmus , and ovary which were measured after 12 weeks of selenium yeast supplementation (experimental week 18). The results showed that dietary selenium yeast levels correlated in a dose-dependent manner with organs Se levels. Shell gland H&E staining showed no degeneration or necrocytosis of the mucosal epithelium cell layer, and shell gland edema in the uterus for all treatment . 3.3. Gene Expression in the Shell Gland A total of 1308 DEGs were assessed in the SY groups compared with the SD group (fold change > 1.00, p < 0.05) (Supplement Table S2). The upregulated and downregulated DEGs are displayed in Figure 2a. In comparison with the SD hens, 442 DEGs were shared among the three SY supplemented groups, and 296, 144, and 130 specific DEGs were found for the SY0.15, SY0.30, and SY0.45 groups, respectively . The biological functions of these DEGs were classified by GO term and KEGG pathway enrichment analysis . The GO analysis identified DEGs enriched in 44 biological progress, 8 molecular functions, and 18 cellular compartments relevant to the current work. These include cell cycle processes (regulation of cell proliferation, regulation of epithelial cell proliferation, and extrinsic apoptotic signaling pathway), female organ development (response to estrogen), and developmental processes affected by calcium ions (osteoblast development, positive regulation of bone mineralization, and voltage-gated calcium channel activity). Based on the KEGG pathway enrichment analysis, ECM-receptor interaction, MAPK signaling pathway, TGF-beta signaling pathway, calcium signaling pathway, glycosaminoglycan biosynthesis, and glutathione metabolism were obtained. The dose effects of Se on shell gland gene expression were performed by STEM. Total DEGs were clustered into 20 profiles, of which three trend profiles (1, 2, and 17) showed significant enrichment (p < 0.05) with the colored block , and 17 profiles without color represented the nonsignificant trends (data not shown). Profile 2 showed that the expression of 155 genes displayed a reducing trend during the low Se level phase. The expression of 136 genes displayed an opposite (rising) trend during the low Se level phase in profile 17. In profile 1, the expression of 46 genes showed an initial decrease but a subsequent increase with the SY0.15 treatment. 3.4. Shell Gland Transcriptome Analysis Egg quality traits are important for laying hens, especially in the late laying period. In our study, eggshell strength in the SY0.45 group was significantly higher than that in the SD and SY0.15 groups. Thus, RNA-seq and bioinformatics were used to explore the potential key genes associated with eggshell strength after selenium yeast supplementation. The Venn diagram showed DEGs from comparisons of SD vs. SY 0.45 and SY0.15 vs. SY0.45 ; 78 DEGs were shared in both two comparisons, including cell migration inducing hyaluronan binding protein (CEMIP), syndecan 3 (SDC3), solute carrier family 6 member 17 (SLC6A17), ovalbumin (OVAL), period circadian clock 2 (PER2), and proenkephalin (PENK). A total of 73 DEGs were unique in the SY0.15 vs. SY0.45 including solute carrier family 13 member 5 (SLC13A5), and proopiomelanocortin (POMC), and 697 DEGs were unique in the SD vs. SY0.45, including epiregulin (EREG), otopetrin 2 (OTOP2), Wnt family member 11 (WNT11), pleiotrophin (PTN), and carbonic anhydrase 2 (CA2). To validate the transcriptomic analysis results, the relative gene expression of the candidate gene (OVAL, CEMIP, SLC6A17) was performed by quantitative real-time PCR (qRT-PCR). The qRT-PCR results showed a similar expression pattern compared with RNA-seq results, indicating that these genes were validated . Functional enrichment analysis results showed that all DEGs were enriched in cell cycle processes (regulation of cell proliferation, p53 signaling pathway, cell cycle, and apoptosis), follicle development (progesterone-mediated oocyte maturation and response to estrogen), eggshell mineralization (calcium signaling pathway, glycosaminoglycan biosynthesis, negative regulation of BMP signaling pathway, and cellular zinc ion homeostasis), and glutathione metabolism . Trend analysis was performed to reveal the expression patterns of candidate DEGs related to eggshell strength at different selenium yeast supplementation levels. Total DEGs were clustered into eight profiles, of which two were significant trend profiles (p < 0.05), including profiles 1 and 6 (colored block) . The expression of 158 genes including CA2, SLC13A5, and OTOP2 decreased with higher supplementation levels of SY in profile 1. In profile 6, the expression of 123 genes including PENK, WNT7A, and EREG exhibited an obvious increase with the higher SY supplementation levels. Network analysis is useful to identify genes that are putative hubs of gene coregulation. Here, WGCNA was used to explore hub genes related to eggshell strength affected by selenium yeast intake. Due to that the eggs collected did not correspond to laying hens for transcriptome sequencing, eggshell strength from each group was not used for WGCNA analysis. Thus, linear correlation was used to analyze the relationship between shell gland Se content with eggshell Se content and eggshell strength , and Se content in shell gland had a positive relationship with egg strength (p = 0.0013) and eggshell Se content (p < 0.05). The results of WGCNA analysis showed that these genes were segmented into three significant modules (p < 0.05), and for each of these modules, the correlation of the eigengene with shell gland Se content was computed . The expression of gene in the Memagenta and MEgreen module was positively correlated with the shell gland Se content, and in MEpurple it was negatively correlated (Supplementary Table S4). Common genes both in significant modules and DEGs were selected to explain the effect of selenium yeast on eggshell strength. Functional enrichment analysis showed that common pathways were implicated in the regulation of cell cycle, cell migration, eggshell mineralization, reproductive organ development, and shell gland development . The PPI result of genes in common GO pathways showed that candidate genes in previous results included PTN, SDC1, WNT11, and PENK . 4. Discussion Avian eggshell is made of columnar calcite crystals which protect the eggs from physical damage and microbial contamination and provide a calcium source for the developing embryo . Nevertheless, eggshell quality can begin to deteriorate over time with the characteristics of decreasing eggshell strength, increasing ratio of translucent eggs, and increasing numbers of abnormal eggs, which cause substantial economic losses. Numerous studies have identified that Se is beneficial for health, egg, and shell quality of laying hens . However, the effect of selenium yeast on egg quality and antioxidant activity in aged laying hens has not been elucidated. Se deficiency and supplementation are closely related to Se status and antioxidants capacity. In general, plasma Se content and antioxidant enzyme are generally considered as useful biomarkers of both Se status and dietary intake . Antioxidant enzyme activity including T-AOC and T-SOD serves as an important index for redox states in livestock and poultry. GSH-Px is a Se-dependent enzyme which has abilities for protecting organs from oxidative damage. In our study, Se deficiency for 6 weeks (experimental weeks 0-6) led to a significant decrease of Se status (71.31% of Se content) and antioxidants capacity (49.60% of T-AOC, 58.31% of GSH-Px, and 16.71% of T-SOD) in aged laying hens. Consistent with other studies, dietary Se deficiency led to a significant decrease of organs Se content, GSH-Px, T-AOC, and SOD activities in plasma. Based on the percent decline of these measured variables, Se content in plasma decreased the most, suggesting that it was more sensitive to Se status than the antioxidant enzymes. In the Se supplementation period, both selenium yeast and sodium selenite supplementation also increased the antioxidant capacity and organs Se status. Consistent with other studies , Se content in plasma and organs increased with a dose-dependent trend after Se supplementation. However, different sources of Se supplementation do not have the same effect on plasma Se deposition in aged laying hens. Overall, selenium yeast is more effective in supplementing plasma indicators in aged laying hens. After 6 weeks of supplementation (experimental week 12), plasma Se content in the SY0.45 group was recovered, but other groups did not, suggesting that higher dose and better-absorbed Se may be required by aged laying hens. Meanwhile, the activities of GSH-Px, T-AOC, and T-SOD in plasma increased in the Se supplementation period. The data are in agreement with the results previously published showing that antioxidant enzyme activities exhibited a dose-dependent increase with increasing Se supplementation , but in our study, some deeper understanding of Se supplementation in aged laying hens was obtained; supplementation with 0.30 mg/kg sodium selenite more consistently restored plasma antioxidant enzyme activity affected by depletion, and in the selenium yeast groups, 0.45 mg/kg selenium yeast supplementation achieved better effects. Overall, selenium yeast supplementation was more beneficial in mitigating the loss of antioxidant enzyme activity in aged laying hens, probably due to the better utilization of organic selenium. Interestingly, it was noticed that the Se deposition hierarchy in organs 15-20 h post-ovulation associated with laying were isthmus, magnum, ovary, and shell gland, successively. In the late stage of the laying period, changes in nutrient intake have a great impact on laying performance. Studies clearly indicated that selenium yeast plays crucial roles in poultry nutrition and production. Eggshell strength is vital in ensuring the integrity and safety of the egg contents, and it tends to deteriorate with the increasing of bird age . In our study, Se deficiency decreased eggshell strength in aged laying hens. It has been reported that Se deficiency is detrimental to bone microarchitecture, possibly through decreasing antioxidant capacity , and bone microarchitecture plays an essential role in eggshell formation. Thus, these results suggest that oxidative stress caused by Se deficiency may decrease eggshell strength in conjunction with age. Moreover, 0.45 mg/kg selenium yeast supplementation for 6 and 12 weeks increased eggshell strength and decreased egg weight and shell translucency; however, the eggshell strength was not found to be improved in sodium selenite supplementation groups in aged laying hens. Consistent with previous studies, eggshell breaking strength was significantly increased after high selenium yeast supplementation . Translucent eggshells are a problematic issue that affects eggshell appearance and decreases the commercial value of eggs. The cause of eggshell translucency is still unknown but is inferred to be associated with variations of the eggshell membrane . The observed decrease in translucent eggshell suggests that Se supplementation for 6 weeks may ameliorate the decline of egg quality. However, after 12 weeks of both sodium selenite and selenium yeast supplementation, there was no significant improvement in translucent eggshell, and these results also suggest that translucent eggshell may not be influenced by different selenium sources. Increasing egg weight in the last phase of a laying cycle is another problematic issue because large eggs are more difficult to handle and are more prone to breaking during transport and collection. The results in our study showed that with increasing weeks of supplementation, high dose of sodium selenite and selenium yeast supplementation decreased the egg weight, suggesting that high dose of sodium selenite and selenium yeast supplementation in the late laying period may play a beneficial role in egg quality. The yeast in selenium-enriched yeast was mainly to provide the organic source of selenium, which was not in an activated state. It did not produce ingredients such as Xylanase that improve bone strength and eggshell quality . The Se intake range which is beneficial for humans and animals is fairly narrow and has been described as a U-shaped curve. A low feeding level was chosen in this experiment, but whether this dose will cause the shell gland of aged laying hens to be in a toxic state still needs to be measured by H&E staining. The results indicated that 0.45 mg/kg selenium yeast supplementation for 12 weeks (experimental week 18) did not cause pathological lesion of aged laying hens. In our study, the effect of selenium yeast supplementation on shell gland of aged laying hens was obtained by whole transcriptome analysis. The results of transcriptome analysis revealed several novel genes and biological pathways regulating ion transport, eggshell calcification, and, consequently, the eggshell formation. Based on the enrichment pathways, molecular functions, and gene expression trends, thirteen genes were identified as potential candidate genes during eggshell formation, including CEMIP, SDC3, OVAL, secreted phosphoprotein 1 (SPP1), SLC6A17, SLC13A5, OTOP2, CA2, POMC, PTN, PENK, WNT11, and EREG. Calcium carbonate crystals interact with the shell organic matrix to form a highly ordered microstructure during shell calcification. Genes involved in shell mineralization have attracted our attention, including CEMIP, SDC3, OVAL, and SPP1. CEMIP is involved in glycosaminoglycan metabolism and calcium release from the endoplasmic reticulum . The upregulated CEMIP expression (log2 fold change > 2 for SD vs. SY0.45 and SY0.15 vs. SY0.45) showed that selenium yeast supplementation promoted gene expression for calcium release needed for eggshell formation. SDC3 protein possesses domains containing some potential glycosaminoglycan attachment sites . Glycosaminoglycan is widely thought to regulate mineral deposition and determine the properties of eggshell . Thus, it is inferred that the upregulation of SDC3 may play an active role in eggshell formation. OVAL is an abundant eggshell matrix protein binding calcium, and it plays an active role in carbonate formation . In coherence with our results, the upregulation of OVAL might improve eggshell formation. SPP1, also known as osteopontin, has mineral-binding domains which are involved in calcium metabolism and calcium carbonate precipitation for eggshell calcification. Consistent with other studies, the upregulation of SPP1 occurred during eggshell calcification . Similarly, DEGs and enriched pathways, including calcium signaling pathway, glycosaminoglycan biosynthesis and positive regulation of bone mineralization, were also closely associated with eggshell calcification and formation. Hence, the results suggest that selenium yeast supplementation has a role in eggshell calcification by regulating the expression of CEMIP, SDC3, OVAL, SPP1, and other candidate genes beneficial for eggshell formation. The eggshell formation process requires a large amount of calcium (Ca2+) and bicarbonate (HCO3-); thus, ion transport plays a crucial role. Some DEGs found in molecular pathways associated with ion transport were screened in our study, including voltage-gated calcium channels and calcium signaling pathways. SLC6A17 and SLC13A5 genes are part of the solute carrier family and play an important role in transporting ions across cell membranes for synthesis of eggshell. It has been reported that SLC6A17 may regulate alanine transport during eggshell formation, while SLC13A5 plays an important role in the initiation of eggshell synthesis . The carbonate ions required for eggshell synthesis are produced by carbonic anhydrase (CA) reactions, and the uterine glandular cells possess the carbonic anhydrase activity sites. There is growing evidence that high expression levels of CA2 expression play a pivotal role in the conversion of intracellular CO2 to chickens . As a member of the otopetrin gene family, the OTOP2 gene may have similar functions to OTOP1, which is regarded as a modulator of cellular calcium influx , to transport calcium across the uterine epithelium for eggshell calcification . Our findings inferred that SLC6A17, SLC13A5, OTOP2, and CA2 are regulators of ion transport in the shell gland and are affected by selenium yeast supplementation. The eggshell formation also depends upon numerous physiological adaptations and processes by the uterine cells, as well as reproductive hormones. In our study, functional enrichment analysis showed that selenium yeast supplementation may affect the response to estrogen, regulation of cell proliferation, regulation of epithelial cell proliferation, and extrinsic apoptotic signaling pathway. POMC plays a role in stimulating the release of cortisol hormone and its upregulated expression during the mineralization period . Consistent with other studies, a higher expression of POMC may result in a hard eggshell . PTN is a developmentally-regulated growth factor and its expression is induced by estrogen ; PTN and estrogen were reported to have a pivotal role in eggshell formation . These findings suggest that selenium yeast supplementation may affect the expression of reproductive hormones and, subsequently, eggshell formation. Indeed, WGCNA analysis also identified some candidate genes which affected the eggshell formation regulated by selenium yeast supplementation, including PENK, WNT11, and EREG. These upregulation DEGs were reported in previous studies to have a vital role in eggshell formation . Calcium (Ca) is one of the key nutrients for bone formation and eggshell quality , but all ingredients except Se were consistent in our treatment; thus, the content of Ca was the same for each treatment. The effect of different Se levels on the utilization and deposition of calcium deserves further discussion. Previous studies also found that trace elements manganese and zinc enhanced eggshell strength by improving biosynthesis of glycosaminoglycan and affecting carbonic anhydrase activity , respectively. An increasing number of studies have explored the fact that selenium yeast has a positive effect on eggshell quality of laying hens . However, studies on the molecular mechanism of selenium yeast affecting eggshell quality are limited. To date, it is hypothesized that selenium exhibits beneficial effects on eggshell quality and may be directly involved in the process of regulating eggshell formation or the interaction between trace elements. Overall, based on biological functions of the DEGs, it is hypothesized that the molecular pathways impacted by selenium supplementation on aged laying hens are those related to eggshell mineralization, specifically regulation of shell calcification and ion transduction. Despite that, additional studies are needed to understand the molecular mechanisms modulated by selenium yeast supplementation in the various phases of eggshell formation. 5. Conclusions Dietary selenium yeast supplementation at a dose of 0.45 mg/kg improved eggshell strength and reduced eggshell translucence in aged laying hens. Moreover, observed increases in plasma Se content, plasma antioxidant enzyme activity, and reproductive organs Se content were dependent on dietary selenium yeast supplementation level. Transcriptomic analysis revealed that selenium may play a role in eggshell quality through regulating the expression of genes involved in eggshell mineralization and ion transport. CEMIP, OVAL, SLC6A17, SLC13A5, POMC, and PENK were identified as candidate genes affecting eggshell formation via regulation by selenium. Supplementary Materials The following are available online at Figure S1: The different degrees of shell translucence in eggs of aged laying hens. The degree was classified into 4 grades; 1 means the best shell translucence degree, 4 means the worst shell translucence degree. Table S1: Primers used in this study. Table S2: DEGs in different comparisons. Table S3: GO term and KEGG pathway function analysis. Table S4: WGCNA results. Click here for additional data file. Author Contributions Conceptualization, B.Z., K.W., Y.G. and H.H.; Methodology, Z.L., B.Z., Y.G. and H.H.; Validation, H.H.; Formal analysis, Y.C.; Investigation, Z.L., Y.A., X.Y., L.W. and M.W.; Resources, X.Y.; Data curation, Z.L.; Writing-original draft, Z.L.; Writing-review & editing, Z.L., G.L. and K.W.; Visualization, Z.L. and Y.C.; Supervision, H.H.; Funding acquisition, H.H. All authors have read and agreed to the published version of the manuscript. Institutional Review Board Statement The experimental animal protocols for this study were approved by the Animal Care and Use Committee of China Agricultural University (No. AW05060202-1). Informed Consent Statement Not applicable. Data Availability Statement The datasets generated and analyzed in the current study are available in the Sequence Read Archive (SRA) database at NCBI under BioProject ID PRJNA716513. Conflicts of Interest The authors declare no conflict of interest. Figure 1 Eggshell quality and organs Se status in aged laying hens during the whole experiment period. (a) The changes of translucent eggs during selenium yeast supplementation, the average score is shown in the middle of the bar. The data in the horizontal row were detected on different days when eggs were stored; similarly, the data in the vertical row were detected in different weeks of age. (b) Selenium content in the shell gland after selenium yeast supplementation for 12 weeks. (c) Selenium content in eggshell after selenium yeast supplementation for 12 weeks. (d) Selenium content in magnum after selenium yeast supplementation for 12 weeks. (e) Selenium content in isthmus after selenium yeast supplementation for 12 weeks. (f) Selenium content in ovary after selenium yeast supplementation for 12 weeks. Data are expressed as mean +- SEM. Within each panel, means without a common letter differ at p < 0.05. (g) H&E staining of representative in shell gland after selenium yeast supplementation for 12 weeks (20x), (i) the mucosal epithelial cell layer, (ii) shell gland, (iii) lymphocyte. Scale bar = 200 mm. Figure 2 Transcriptome analysis revealed the effect on gene expression of shell gland in aged laying hens. (a) Scatter plots of DEGs (SD vs. SY0.15, SD vs. SY0.30, and SD vs. SY0.45). Red points represent upregulated genes with a log2 (fold change) > 1 and p < 0.05. Blue points represent downregulated genes with a log2 (fold change) < -1 and p < 0.05. Gray points represent genes showing no significant difference. Fold change = normalized gene expression in the SY0.15, SY0.30, and SY0.45 group/normalized gene expression in the SD group. (b) Venn diagram showing the distribution of DEGs in three comparisons of Se supplementation. The number of DEGs is indicated in the diagram. (c) Gene Oncology enrichment analysis of DEGs identified among the three conditions (SD vs. SY0.15, SD vs. SY0.30, and SD vs. SY0.45). (d) KEGG pathway enrichment analysis of DEGs identified among the three conditions (SD vs. SY0.15, SD vs. SY0.30, and SD vs. SY0.45). (e) Identification of 3 significant gene cluster profiles by STEM. Colored block trend: significant enrichment trend (p < 0.05). The number of genes in each significant cluster is shown after the cluster number. Figure 3 The potential molecular mechanism of selenium yeast affecting the eggshell strength in aged laying hens. (a) Venn diagram showing the distribution of DEGs associated with eggshell strength. (b) Validation of DEGs associated with eggshell strength using qRT-PCR, * means p < 0.05 and ** means p < 0.01. (c) Gene Oncology enrichment analysis of DEGs associated with eggshell strength (SY0.15 vs. SY0.45 and SD vs. SY0.45). (d) KEGG pathway enrichment analysis of DEGs associated with eggshell strength (SY0.15 vs. SY0.45 and SD vs. SY0.45). (e) Trend analysis of DEGs associated with eggshell strength expression. Colored block trend: significant enrichment trend (p < 0.05). (f) The linear correlation between shell gland selenium content with eggshell strength and eggshell selenium content. (g) WGCNA analysis identified specific transcriptional modules related to eggshell strength in aged laying hens after selenium yeast supplementation. (h) Sankey charts were performed among differential expressed genes in modules and pathways. (i) The PPI network of common genes in modules and DEGs. animals-13-00902-t001_Table 1 Table 1 Composition and nutrient of the basal diet. Items Content (%) Nutrient Level Content (%) Corn 60.50 AME, Mcal/kg 2.57 Soybean meal 21.60 Crude protein (%) 15.0 Wheat 4.10 Lysine (%) 0.74 Cottonseed meal 2.00 Methionine (%) 0.3 Soybean oil 0.50 Methionine + Cystine (%) 0.55 Calcium carbonate (GR) 9.50 Calcium (%) 3.7 Calcium phosphate (GR) 1.00 Available phosphorus (%) 0.31 DL-methionine 0.08 Total phosphorus (%) 0.54 Phytases 0.015 Selenium c (mg/kg) 0.056 Vitamin premix a 0.035 Mineral premix b 0.15 Sodium chloride (GR) 0.30 50% Choline chloride 0.10 Experimental additives 0.12 Total 100.00 Abbreviations: GR = guaranteed reagent; a provided per kilogram of diet: vitamin A, 12,500 IU; vitamin D3, 2500 IU; vitamin E, 18.75 mg; vitamin K3, 2.65 mg; vitamin B1, 2 mg; vitamin B2, 6 mg; vitamin B12, 0.025 mg; biotin, 0.325 mg; folic acid, 1.25 mg; niacin, 50 mg; b provided per kilogram of diet: Cu, 8 mg; Fe, 80 mg; Zn, 80 mg; Mn, 60 mg; I, 1.2 mg. Selenium in each treatment group is shown in the experiment design in Table 2; c selenium in the basal diet is measured value. animals-13-00902-t002_Table 2 Table 2 Dietary selenium levels. Control Selenium Yeast Sodium Selenite Group SD SY0.15 SY0.30 SY0.45 SS0.15 SS0.30 SS0.45 Measured values 1 0.056 0.211 0.377 0.522 0.205 0.267 0.480 SEM 0.012 0.015 0.049 0.030 0.016 0.049 0.030 Abbreviations: SD = selenium deficiency; SY0.15 = 0.15 mg/kg selenium yeast group; SY0.30 = 0.30 mg/kg selenium yeast group; SY0.45 = 0.45 mg/kg selenium yeast group; SS0.15 = 0.15 mg/kg sodium selenite group; SS0.30 = 0.30 mg/kg sodium selenite group; SS0.45 = 0.45 mg/kg sodium selenite group; 1 the values were measured by fluorescence method, and the unit is mg/kg. animals-13-00902-t003_Table 3 Table 3 Selenium content of plasma during the whole experiment period (experimental weeks 0-18). Items SD SS0.15 SS0.30 SS0.45 SY0.15 SY0.30 SY0.45 SEM p-Value Se content 0 week 0.24 0.01 Se content 2 week 0.10 0.01 Se content 4 week 0.07 0.01 Se content 6 week 0.05 0.01 Se content 8 week 0.05 a 0.12 b 0.18 d 0.19 d 0.15 c 0.20 d 0.23 e 0.01 <0.01 Se content 10 week 0.04 a 0.15 b 0.18 bc 0.17 bc 0.15 bc 0.19 cd 0.24 d 0.01 <0.01 Se content 12 week 0.05 a 0.16 b 0.19 bc 0.19 bc 0.16 b 0.22 cd 0.24 d 0.01 <0.01 Se content 14 week 0.07 a 0.17 b 0.18 b 0.19 bc 0.18 b 0.22 c 0.26 d 0.01 <0.01 Se content 16 week 0.09 a 0.18 b 0.18 b 0.22 c 0.20 bc 0.22 c 0.26 d 0.01 <0.01 Se content 18 week 0.08 a 0.16 b 0.19 b 0.20 bc 0.19 b 0.23 cd 0.26 d 0.01 <0.01 Abbreviations: SD = selenium deficiency; SY0.15 = 0.15 mg/kg selenium yeast group; SY0.30 = 0.30 mg/kg selenium yeast group; SY0.45 = 0.45 mg/kg selenium yeast group; SS0.15 = 0.15 mg/kg sodium selenite group; SS0.30 = 0.30 mg/kg sodium selenite group; SS0.45 = 0.45 mg/kg sodium selenite group; a-e values within a row with different superscripts differ significantly at p < 0.05, and the unit of the selenium content is mg/mL. animals-13-00902-t004_Table 4 Table 4 Antioxidants capacity of plasma during whole experiment period (experimental weeks 0-18). Items SD SS0.15 SS0.30 SS0.45 SY0.15 SY0.30 SY0.45 SEM p-Value GPx 0 week 3550.15 96.41 GPx 2 week 2082.02 155.19 GPx 4 week 1605.42 253.39 GPx 6 week 1480.00 208.94 GPx 10 week 2102.40 2323.20 3316.00 2904.00 2720.00 2893.33 3184.00 139.45 0.31 GPx 14 week 2111.60 a 2874.58 b 3430.51 c 3486.15 c 3480.65 c 3458.10 c 3502.11 c 105.73 <0.01 GPx 18 week 2340.10 a 2791.19 ab 3421.85 b 3151.45 ab 3210.45 ab 3377.27 b 3545.17 b 123.47 0.10 T-SOD 0 week 270.61 10.44 T-SOD 2 week 236.91 8.79 T-SOD 4 week 250.81 6.39 T-SOD 6 week 225.37 11.42 T-SOD 10 week 248.73 240.42 253.51 230.07 269.56 247.52 253.91 6.51 0.83 T-SOD 14 week 251.36 252.82 257.31 257.60 267.29 244.79 255.16 6.22 0.99 T-SOD 18 week 257.87 261.67 269.36 294.32 270.94 275.39 299.94 5.17 0.62 T-AOC 0 week 3.72 0.49 T-AOC 2 week 2.64 0.37 T-AOC 4 week 1.88 0.16 T-AOC 6 week 1.87 0.13 T-AOC 10 week 1.91 2.72 3.15 2.67 2.52 2.92 3.13 0.20 0.23 T-AOC 14 week 1.48 a 3.17 b 3.84 b 2.93 b 3.18 b 3.50 b 5.02 c 0.21 <0.01 T-AOC 18 week 2.09 a 3.44 ab 4.04 bc 3.94 bc 4.41 bc 5.34 c 5.12 c 0.27 <0.01 Abbreviations: SD = selenium deficiency; SY0.15 = 0.15 mg/kg selenium yeast group; SY0.30 = 0.30 mg/kg selenium yeast group; SY0.45 = 0.45 mg/kg selenium yeast group; SS0.15 = 0.15 mg/kg sodium selenite group; SS0.30 = 0.30 mg/kg sodium selenite group; SS0.45 = 0.45 mg/kg sodium selenite group; a-c values within a row with different superscripts differ significantly at p < 0.05, and the unit of the selenium content is U/mL. animals-13-00902-t005_Table 5 Table 5 Egg quality of aged laying hens fed selenium-deficient diets over time. Items Egg Weight (g) Eggshell Strength (kg Force) Albumen Height (mm) Haugh Unit (U) Yolk Color Before depletion 61.78 3.26 6.53 79.10 6.89 After depletion 63.83 2.89 5.63 71.46 5.90 SEM 1.07 0.17 0.22 2.01 0.99 p-value 0.06 0.04 <0.01 <0.01 <0.01 animals-13-00902-t006_Table 6 Table 6 Egg quality of aged laying hens after selenium supplementation. Items SD SS0.15 SS0.30 SS0.45 SS-Linear p-Value SS-Quadratic p-Value SY0.15 SY0.30 SY0.45 SY-Linear p-Value SY-Quadratic p-Value SEM Treatment p-Value Experimental weeks 12 Egg weight (g) 65.30 64.09 64.02 62.56 0.05 0.90 63.59 63.94 62.27 0.39 0.44 0.36 0.38 Eggshell strength (kg force) 2.70 a 2.80 ab 3.16 ab 2.99 ab 0.08 0.38 2.96 ab 2.95 ab 3.28 b 0.08 0.26 0.06 0.13 Albumen height (mm) 6.25 5.89 6.05 5.54 0.26 0.88 6.27 5.97 5.54 0.10 0.39 0.09 0.47 Haugh unit (U) 76.09 73.01 74.36 70.64 0.15 0.01 76.51 73.69 70.74 0.08 0.98 0.86 0.43 Yolk color 5.80 5.87 5.76 6.04 0.27 0.37 5.82 5.97 6.12 0.09 0.98 0.05 0.31 Experimental weeks 18 Egg weight (g) 67.82 a 65.59 abc 66.25 ab 63.76 c <0.01 0.77 63.76 c 65.40 bc 65.13 bc 0.22 0.31 0.30 <0.01 Eggshell strength (kg force) 2.98 a 3.15 ab 3.16 ab 2.84 a 0.05 0.07 2.88 a 3.08 ab 3.48 b <0.01 0.57 0.05 0.03 Albumen height (mm) 7.15 a 6.62 b 6.30 bc 5.97 c <0.01 0.45 6.29 bc 6.70 b 6.28 bc 0.97 0.06 0.06 <0.01 Haugh unit (U) 81.66 a 79.26 ab 78.31 ab 74.22 c <0.01 0.42 76.83 bc 78.27 ab 77.31 bc 0.81 0.49 0.47 <0.01 Yolk color 5.90 6.06 5.88 6.15 0.19 0.60 5.96 5.98 6.18 0.15 0.49 0.04 0.26 Abbreviations: SD = selenium deficiency; SY0.15 = 0.15 mg/kg selenium yeast group; SY0.30 = 0.30 mg/kg selenium yeast group; SY0.45 = 0.45 mg/kg selenium yeast group; SS0.15 = 0.15 mg/kg sodium selenite group; SS0.30 = 0.30 mg/kg sodium selenite group; SS0.45 = 0.45 mg/kg sodium selenite group; a-c values within a row with different superscripts differ significantly at p < 0.05. 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PMC10000210

During cold storage, boar spermatozoa undergo oxidative stress, which can impair sperm function and fertilizing capacity. The objective of the present study was to assess the effects of Schisandrin B (Sch B) in semen extenders on the quality of boar semen stored at hypothermia. Semen was collected from twelve Duroc boars and diluted in extenders supplemented with different concentrations of Sch B (0 mmol/L, 2.5 mmol/L, 5 mmol/L, 10 mmol/L, 20 mmol/L, and 40 mmol/L). Here, we demonstrated that 10 mmol/L Sch B provided the best effects on motility, plasma membrane integrity, acrosome integrity, sperm normality rate, average movement velocity, wobbility, mitochondrial membrane potential (MMP), and DNA integrity of sperm. The results of Sch B effects on antioxidant factors in boar sperm showed that Sch B significantly elevated the total antioxidant capacity (T-AOC) and markedly decreased the reactive oxygen species (ROS) and malondialdehyde (MDA) content of sperm. The expression of catalase (CAT) and superoxide dismutase (SOD) mRNA was increased, while the expression of glutathione peroxidase (GPx) mRNA demonstrated no change compared to non-treated boar sperm. Compared to the non-treated group, Sch B triggered a decrease in Ca2+/protein kinase A (PKA) and lactic acid content in boar sperm. Similarly, Sch B led to a statistically higher quantitative expression of AWN mRNA and a lower quantitative expression of porcine seminal protein I (PSP-I) and porcine seminal protein II (PSP-II) mRNA. In a further reverse validation test, no significant difference was observed in any of the parameters, including adhesion protein mRNA, calcium content, lactic acid content, PKA, and protein kinase G (PKG) activity after sperm capacitation. In conclusion, the current study indicates the efficient use of Sch B with a 10 mmol/L concentration in the treatment of boar sperm through its anti-apoptosis, antioxidative, and decapacitative mechanisms, suggesting that Sch B is a novel candidate for improving antioxidation and decapacitation factors in sperm in liquid at 4 degC. schisandrin B sperm quality boar oxidative stress National Natural Science Foundation of China31660700 This research was funded by the National Natural Science Foundation of China (31660700). pmc1. Introduction At present, the preservation of pig semen mainly includes chill storage (4 degC) and ultra-cryopreservation (-196 degC). Cryopreservation results in the low fertility of pig semen, which has a serious impact on research in the boar breeding industry. The pregnancy rate and litter size are similar at 4 degC fluid storage compared with natural mating. Therefore, liquid storage at 4 degC is an effective method to preserve boar semen. In mammals, boar sperm is susceptible to temperature due to its special membrane structure. Especially under low-temperature conditions, the sperm membrane structure is easily destroyed, resulting in ultrastructural changes in the acrosome and plasma membrane, including deterioration of sperm motility, viability, and DNA integrity, resulting in reduced sperm quality and thus reduced sperm longevity and fertility. Even if appropriate chilling and cryopreservation protocols have already been developed for many species, as shown in bull , ram , goat , and mouse studies. There are limited data on the successful freezing, liquid storage, and cryopreservation of boar sperm. The cryopreservation of boar semen is still in the research and improvement stage; it is more commonly used in the preservation of sperm of threatened species and has not been widely used, which makes the cryopreservation research of boar semen of very important practical significance. Oxidative stress is an important factor affecting sperm quality during the liquid storage of boar semen in vitro. Adding various protective components to the diluent is an effective method to maintain the survival and function of sperm. Schisandrin B (Sch B) is mainly derived from Schisandra chinensis (Turcz) Baillon and has the highest content in Schisandra chinensis. Sch B, C23H28O6 with a molecular weight of 400.6, is a natural antioxidant that not only enhances the antioxidant status of cells but also has been found to prevent the structural and functional degradation of cell mitochondria, both of which are critical to cell survival. Experimental investigations have demonstrated that Sch B has multiple pharmacological properties, including anti-oxidation, anti-inflammation , anti-apoptosis , and anti-tumor effects. Meanwhile, research suggests that Sch B may be a potential therapeutic agent against anxiety associated with oxidative stress through reduced malondialdehyde (MDA) levels, production of reactive oxygen species (ROS), and neuronal damage . In terms of reproductive function, Sch B has spermatogenic effects and can repair damaged seminiferous tubules and spermatogenic cells in the testis, providing a novel way to treat male infertility . Based on its antioxidant and reproductive function protection properties, we speculate that Sch B could be used for sperm protection during liquid preservation of boar semen at 4 degC. To date, the antioxidative effects of Sch B in the preservation of boar sperm and the mechanism of protective effects by Sch B have not been determined. In this context, Sch B is used for the first time in this study to explore its effects on boar sperm liquid preservation, revealing the underlying mechanism(s) of Sch B in sperm quality parameters, antioxidant capacity, and sperm fertilization ability during liquid preservation of boar semen at 4 degC. We further evaluated the effects of Sch B on sperm capacitation parameters before and after capacitation. 2. Materials and Methods 2.1. Chemicals Unless otherwise stated, all chemicals used in this study were purchased from Solarbi (Beijing, China). Sch B was obtained from Victory Biological Technology (Cat. No. 61281-37-6, Sichuan Province, China). 2.2. Collection and Processing of the Ejaculates Mixed semen from 12 mature Duroc boars aged 2-4 years was collected through the hand-gloved technique. Boars were fed at the Keda Livestock and Poultry Improvement Co., Ltd. (Guangxi Province, China). An entire ejaculation was collected twice a week. Only ejaculates that satisfied the sperm quality requirements to produce commercial semen AI-doses (namely, sperm concentration >200 x 106 sperm/mL, sperm motility >70%, and morphologically normal sperm >75%) were included in the study. For seminal plasma harvesting, entire ejaculates were centrifuged twice (1500x g for 10 min at room temperature (Rotofix 32A, Hettich Centrifuge UK, Newport Pagnell, Buckinghamshire, UK)). 2.3. Experimental Designs In this study, three experiments were carried out. 2.3.1. Experiment I: Effects of Different Concentrations of Sch B on Sperm Quality Parameters Boar semen samples were diluted in the extender (glucose 3 g, sucrose 4 g, and egg yolk 20 mL in double-distilled water and volume made up to 100 mL) to a concentration of 80 million sperm/mL. Sch B was added to yield six different final concentrations: 0, 2.5, 5, 10, 20, and 40 mmol/L. The extended sperm were incubated at 4 degC for 24 h. The sperm motility, abnormal sperm morphology, average movement velocity, wobbility (wobbility is the measure by which the sperm head wobbles along the average spatial path of its actual trajectory), acrosome integrity, plasma membrane integrity, mitochondrial membrane potential (MMP), and DNA integrity were assessed. Each experiment was repeated three times. 2.3.2. Experiment II: Effects of Sch B on Antioxidant Capacity of Boar Sperm after Low Temperature Preservation In experiment 2, we measured sperm superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx) mRNA expression, total antioxidant capacity (T-AOC) ability, ROS content, MDA content, Ca2+ content, lactic acid content, the mRNA expression of AWM, AQN1, AQN3, porcine seminal protein I (PSP-I), porcine seminal protein II (PSP-II), protein kinase A(PKA), and protein kinase G (PKG) activity before capacitation. Each experiment was repeated three times. 2.3.3. Experiment III: Effects of 10 mmol/L Sch B Supplementation to Boar Semen Dilution on Sperm Quality after Sperm Capacitation We measured Ca2+ content, lactic acid content, the mRNA expression of AWM, AQN1, AQN3, PSP-I, PSP-II, PKA, and PKG activity after sperm capacitation. Each experiment was repeated three times. 2.4. Methods 2.4.1. Motility, Abnormal Sperm Morphology, Average Movement Velocity and Wobbility Analyzed with Computer-Assisted Semen Analysis (CASA) After rewarming, 1 mL sperm was centrifuged at a speed of 900x g/min in a cryogenic centrifuge at 4 degC for 5 min, washed with PBS, repeated 3 times, and then suspended with PBS to adjust the sperm density to 1 x 106~2 x 106 sperm/mL. Then, 20 mL of the sperm resuspended in PBS was applied to a slide, covered with a coverslip, and the sperm motility, abnormal sperm morphology, average movement velocity, and wobbility were examined by CASA (CEROSII, IMV, Shanghai, China) after low-temperature preservation. 2.4.2. Plasma Membrane Integrity of Spermatozoa by HOST A sample of 1 mL of rewarmed sperm was taken and centrifuged with phosphate-buffered saline (PBS) at 900x g/min in a low-temperature centrifuge at 4 degC for 5 min. This was repeated three times and added to the pre-configured hypotonic solution. The sperm density was adjusted to 1 x 106~2 x 106 sperm /mL, and incubated at 37 degC for 30 min. Then, 15 mL of the mixture was put on a slide, covered with a cover slide, and the tail bending rate of sperm was observed under a microscope at 400 times. At least 200 sperm were counted, and this was repeated 3 times. 2.4.3. Acrosome Integrity of Sperm by Coomassie Brilliant Blue After sperm rewarming, the sperm density was adjusted to 1 x 106~2 x 106 sperm /mL, and the semen was washed by PBS centrifugation 3 times. The sperm were fixed with formalin and sodium citrate in the EP tube. The fixed sperm 2000x g/min was centrifuged for 5 min and then the supernatant was discarded. The sperm were washed once with PBS. Then, 100 mL of PBS was added to the sperm to make a suspension. A 10 mL mixed smear was taken and dried naturally. The air-dried slides were stained in a staining jar with Coomassie brilliant blue, and then the staining solution was slowly rinsed off with water and placed on a staining rack to dry naturally. The acrosome integrity was observed under the microscope (acrosomes in intact sperm were stained with Coomassie brilliant blue to a smooth and uniform blue surface). The experiment was repeated 3 times. 2.4.4. Detection of Sperm DNA Damage Rate The operation was carried out under dark conditions according to the instructions of the AO fluorescent staining kit (Cat. DA0037, Leagene, Beijing, China). After staining, the same samples were examined under a fluorescence microscope (the stained yellow-green fluorescence was the nucleus of normal sperm, and the green granules were the nucleus of DNA damage). At least 200 spermatozoa were counted per smear. The experiment was repeated 3 times. 2.4.5. MMP in Spermatozoa The sperm density was adjusted to (1-2) x 106 sperm /mL. The sperm were rewashed with PBS by centrifugation (900x g; 5 min), and the supernatant was discarded. The operation was performed according to the MMP detection kit (JC-1) (Cat. CA1310, Solarbio, Beijing, China), and the fluorescence values of each group were detected by a multi-functional microplate tester. The experiment was repeated 3 times. 2.4.6. Measurement of Oxidant-Antioxidant Status in Boar Sperm After being rewarmed, the sperm density was adjusted to (1-2) x 106 sperm /mL. The semen was centrifuged 3 times with PBS. Oxidant-antioxidant parameters (MDA (Cat. BC0025, Solarbio, Beijing, China), ROS (Cat. S0033S, Beyotime, Shanghai, China), and sperm T-AOC (Cat. BC1310, Solarbio, Beijing, China)) were identified in fresh, Sch B-treated, and non-treated sperm. Sperm MDA (lipid peroxidation marker) concentration was detected by a fluorescence enzyme labeler according to the manufacturer's instructions, following the Tecan protocol. The absorbance was determined at 450 nm, 532 nm, and 600 nm. MDA measurements were presented in nmol/g protein. In addition, the activity of ROS enzymes and sperm T-AOC was measured in boar sperm, as previously analyzed. The experiment was repeated 3 times. ROS: relative fluorescence unit (RFU); MDA: nmol/mg; T-AOC: mmol/mg. 2.4.7. Quantitative Real-Time PCR Analysis for SOD, CAT, GPx and Adhesion Protein Gene in Spermatozoa The total RNA was extracted using the TRIzol reagent (Vazyme, Nanjing, China) for the boar sperm. The first-strand cDNA was synthesized from the total RNA using a reverse transcription kit (Cat. R211-02, Vazyme, Nanjing, China). Amplification reactions were conducted in triplicate using gene-specific primers designed from the clone sequences shown in Table 1. The PCR products in each cycle were monitored using a fluorescence quantitative PCR instrument (PRISMR 7500, ABI, Los Angeles, CA, USA). The data were analyzed using the comparison Ct (2-DDCt) method and were expressed as fold changes relative to the respective controls. Each sample was analyzed in triplicate. The experiment was repeated 3 times. 2.4.8. Measurement of Sperm Free Ca2+ Influx Boar sperm was taken from each group after being cryopreserved and centrifuged with PBS 3 times (4 degC, 900 g/min, 5 min); then, the sperm density was adjusted to 2 x 106/mL and centrifuged with 1/5 (4 degC, 900x g/min, 5 min). After the supernatant was abandoned, 300 mL PBS was added to the reinserted sperm. A 20 mL 0.1 mmol*L-1 Ca2+ fluorescent probe Fluo-3 (Cat. IF0150, Solarbio, Beijing, China) was added into the probe, which was fully blown with a pipette gun, and then incubated in a constant temperature incubator at 37 degC for 30 min. After centrifugation for 5 min (1500x g/min), the supernatant was abandoned and resuspended in 500 mL PBS. Then, the 200 mL sperm suspension was placed in a 96-well plate, and the fluorescence values of each experimental group were detected by a multifunctional enzyme marker . The experiment was repeated 3 times. Ca2+: relative fluorescence unit (RFU). 2.4.9. Lactic Acid Content in Spermatozoa Lactic acid content was evaluated by using the lactic acid content assay kit (Cat. No. BC2230, Solarbio, Beijing, China). Samples were centrifuged at 3000x g for 5 min and the sperm pellets were re-suspended to a final concentration of 1 x 106 cells in 0.5 mL of PBS. After ultrasonic fracturing in an ice bath, the samples were centrifuged at 8000x g/min for 10 min at 4 degC, and the supernatant was taken to measure the protein concentration and placed on ice until use. Then, 0.02 mL of distilled water, a standard tube, and the sample were added to the blank tube, standard tube, and determination tube, respectively, and then 1 mL enzyme working solution and 0.2 mL chromogenic agent were added, respectively, to mix evenly and react accurately at 37 degC for 10 min. Finally, 2 mL terminating solution was added. We mixed this evenly in a colorimetric dish with a 1 cm optical diameter and measured the absorbance of each tube at 530 nm . The experiment was repeated 3 times. Lactic acid: nmol/g. 2.4.10. Assessment of PKA and PKG in Boar Sperm by ELISA Both PKA and PKG were identified in boar sperm using commercially available ELISA kits for PKA (Cat. MM-77570O1, Meimian, Jiangsu, China) and for PKG (Cat. MM-77540O1, Meimian, Jiangsu, China). We established the standard curve according to the kit's instructions. Each test was performed in triplicate. 2.4.11. Sperm Capacitation As described in a previous study , after sperm collection, samples were centrifuged with PBS (4 degC, 900x g/min, 5 min) 3 times to wash away impurities, and then the sperm density was adjusted to 1 x 106~2 x 106 sperm/mL with Biggers-Whitten-Whittingham (BWW) capacitive medium, which was placed in a 5% CO2 cell incubator and incubated at 37 degC for 2 h. 2.4.12. Data Analysis Data were analyzed using one-way ANOVA (version 13.0, SPSS Inc., Chicago, IL, USA). When a significant difference (p < 0.05) was observed among treatments, Tukey's studentized range test was used for post-test comparisons. Percent motility was arcsine-square root transformed, and the means of three straws per treatment were used for analysis. Values presented are mean +- SD. 3. Results 3.1. Experiment I 3.1.1. Effects of Sch B on the Motility, Abnormal Sperm Morphology, Average Movement Velocity and Wobbility of Boar Sperm after Chilling The effects of Sch B on motility, abnormal sperm morphology, average movement velocity, and wobbility at 4 degC were initially investigated in this sperm quality parameters analysis. Sperm motility is shown in Figure 1A. Sperm motility in 10 Sch B was significantly higher (p < 0.01), while 40 Sch B was markedly decreased (p < 0.001) compared to the 0 Sch B group. There was no significant difference between the 0 Sch B group and the Sch B-treated group (2.5, 5, and 20 Sch B) (p > 0.05). The differences in abnormal sperm morphology are shown in Figure 1B. Compared to 0 Sch B, 5 Sch B and 10 Sch B significantly decreased the abnormal sperm morphology level in a dose-dependent manner. On the contrary, 40 Sch B significantly increased the abnormal sperm morphology level compared with 0 Sch B. The effects of hypothermic injury on the average movement velocity of boar spermatozoa are shown in Figure 1C. There was no significant difference in the average movement velocity (p > 0.05) in 2.5 Sch B and 20 Sch B compared with 0 Sch B. The average movement velocity in 10 Sch B and 5 Sch B was significantly higher than 0 Sch B (p < 0.001 and p < 0.01, respectively) on the hypothermic liquid preservation. A significantly lower average movement velocity was observed in 40 Sch B than in 0 Sch B (p < 0.01) The results of sperm wobbility after chilling are shown in Figure 1D. Our results showed that the addition of 10 mmol/L Sch B to the base solution can significantly improve the wobbility compared to 0 Sch B (p < 0.05), but there was no significant difference between the other Sch B-treated groups (2.5, 5, 20, and 40 Sch B) and 0 Sch B (p > 0.05). 3.1.2. Effects of Sch B on the Acrosome Integrity and Plasma Membrane Integrity of Boars Sperm After treatment with Coomassie bright blue, the acrosomal reaction rate of sperm was detected, as shown in Figure 2A. All Sch B-treated groups significantly (p < 0.01) enhanced plasma membrane integrity, except for 40 Sch B. When the Sch B concentration exceeded 10 mmol/L, the sperm acrosome integrity showed a decreasing trend with increasing concentration. The percentage of plasma membrane integrity of sperm in the hypothermia treatment groups is presented in Figure 2B. The plasma membrane integrity in 2.5, 5, and 10 Sch B was significantly higher than in 0 Sch B (p < 0.01). It is worth noting that the plasma membrane integrity of boar sperm was significantly (p < 0.05) lower when the concentration of Sch B was 40 mmol/L. 3.1.3. Effects of Sch B on the DNA Damage Rate and MMP Changes of Boars Sperm The sperm DNA damage rate from representative semen samples of all six groups at the chilled-thawed stage is presented in Figure 3A. The DNA damage rate in 5, 10, and 20 Sch B was significantly lower than in 0 Sch B (p < 0.01). The difference between 0 Sch B and 2.5 Sch B was not significant (p > 0.05). When the concentration of Sch B was 40 mmol/L, the DNA damage rate showed an increasing trend. Sperm MMP at the chilled-thawed stage are presented in Figure 3B. The percentage of sperm with MMP was higher (p < 0.01) in 10 Sch B than in 0 Sch B, while no significant difference was observed between 20 Sch B, 40 Sch B, and 0 Sch B after chilling (p < 0.05). The MMP was significantly lower in the 2.5 and 5 Sch B than in 0 Sch B (p < 0.01). 3.2. Experiment II 3.2.1. Effects of Sch B on the SOD, CAT and GPx mRNAs Expression of Boar Sperm The expression level of SOD and CAT was significantly increased in sperm with Sch B compared to 0 Sch B (p < 0.05, p < 0.01). The 0 Sch B showed a significant decrease (p < 0.001) in the mRNA level of sperm SOD and CAT compared to the fresh sperm . The differences in the quantitative expression of GPx mRNA were not significant in the fresh and Sch B-treated sperm compared to the non-treated sperm (p > 0.05). 3.2.2. Effects of Sch B on the MDA, ROS and T-AOC Level of Boar Sperm The 0 Sch B samples had significantly higher activity in sperm ROS compared to fresh sperm . The Sch B-treated samples revealed lower activity in sperm ROS (p < 0.01) compared to the 0 Sch B samples. When compared to fresh, 0 Sch B had a significantly lower T-AOC level . Compared to 0 Sch B, the Sch B-treated sperm had a significant increase (p < 0.001) in the T-AOC level. Compared to fresh, the 0 Sch B sperm had a significant increase in the sperm lipid peroxidation marker MDA , whereas the Sch B-treated sperm had a lower MDA content than the 0 Sch B sperm (p < 0.001). 3.2.3. Effects of Sch B on The Ca2+ and Lactic Acid Content of Boars Sperm Compared to fresh, 0 Sch B had significantly higher sperm Ca2+ and lactic acid contents (p < 0.01 and p < 0.001, respectively). Compared to 0 Sch B, Sch B-treated samples revealed significantly lower sperm Ca2+ and lactic acid contents . 3.2.4. Effects of Sch B on the mRNA Expression of AWM, AQN1, AQN3, PSP-I and PSP-II of Boar Sperm The mRNA level of AWM was significantly reduced in 0 Sch B sperm compared to fresh (p < 0.001). However, boar sperm treated with Sch B at a dose of 10 mmol/L showed a significant increase (p < 0.01) in the mRNA level of AWM compared to 0 Sch B . The mRNA level of AQN1 was identified in fresh, Sch B-treated, and non-treated sperm. Both the non-treated and Sch B-treated sperm showed a significant reduction in the level of AQN1 mRNA compared to the fresh samples (p < 0.001) . No significant difference was observed between the non-treated and Sch B-treated samples (p >= 0.05). The mRNA levels of AQN3 in both the 0 Sch B and Sch B-treated sperm showed significant decreases compared to the fresh sperm (p < 0.01). AQN3 mRNA levels between the 0 Sch B and Sch B-treated sperm showed no significant differences (p >= 0.05). Quantitative expression of both PSP-I and PSP-II mRNA showed significant increases in 0 Sch B compared to the fresh sperm (p < 0.001 and p < 0.01, respectively). When sperm were treated with Sch B at a dose of 10 mmol/L, a significant decrease in the quantitative expression of both PSP-I and PSP-II mRNA was identified compared to 0 Sch B (p < 0.01 and p < 0.05, respectively). 3.2.5. Effects of Sch B on PKA and PKG Levels of Boar Sperm PKA was increased in sperm without any additives compared to fresh (p < 0.05). However, boar sperm treated with 10 mmol/L Sch B showed a decrease in the PKA level compared to 0 Sch B as measured by ELISA immunoassay techniques . Compared to the fresh, the 0 Sch B and 10 Sch B sperm both had no significant differences in PKG levels. PKG level between the 0 Sch B and Sch B-treated sperm showed no significant differences . 3.3. Experiment III 3.3.1. Effects of Sch B on Ca2+ and Lactic Acid Content in Capacitated Boar Sperm Compared to 0 Sch B, the capacitation groups demonstrated significantly higher (p < 0.01) sperm Ca2+ and lactic acid contents. The Ca2+ and lactic acid contents in the 10 Sch B sample were not significantly different compared with the 0 Sch B capacitation sample . 3.3.2. Effect of Sch B on mRNA Expression of AWM, AQN1, AQN3, PSP-I, and PSP-II in Capacitated Boar Sperm mRNA levels of both AWM and AQN1 were significantly reduced in the capacitation groups compared to 0 Sch B (p < 0.01). However, 10 Sch B showed no significant differences (p >= 0.05) in the AWM and AQN1 mRNA levels compared to 0 Sch B capacitation as measured by quantitative PCR . The mRNA levels of AQN3 in 0 Sch B capacitation and 10 Sch B showed no significant differences compared to 0 Sch B, as did 10 Sch B compared to 0 Sch B capacitation (p >= 0.05). Quantitative expression of both PSP-I and PSP-II mRNA was significantly increased in 0 Sch B capacitation compared to 0 Sch B (p < 0.01 and p < 0.001, respectively). The 10 Sch B sample did not show significant differences with regard to the parameters of the two genes compared to the 0 Sch B capacitation sample. 3.3.3. Effects of Sch B on PKA and PKG Levels in Capacitated Boar Sperm The results of Sch B affecting sperm PKA and PKG levels of capacitated boar spermatozoa are shown in Figure 11. After 24 h of liquid storage, the sperm PKA levels of 0 Sch B capacitation and 10 Sch B were higher than 0 Sch B (p < 0.05), and the sperm PKA levels were not significantly different between 0 Sch B capacitation and 10 Sch B. Sperm PKG levels are shown in Figure 11. The PKG level in 0 Sch B was not significantly different compared with 0 Sch B capacitation (p > 0.05), and 0 Sch B capacitation showed no significant difference compared to 10 Sch B (p > 0.05). 4. Discussion Sch B, isolated from Schisandra chinensis, has been documented to possess diversified pharmacokinetic properties, among them neurogenesis , anti-inflammatory , anti-tumor , and liver-protective effects. The present study validated the effect of Sch B on male infertility , suggesting that Sch B is a potential natural active ingredient for reproduction protection. A previous study determined that oral administration of Sch B at 2 weeks not only increased the sperm number and average number of births but also increased sperm motility parameters in mice. These results suggest that Sch B may have positive effects on reproduction and improve sperm quality. Our present sperm quality parameters show that Sch B triggered an increase in the motility, average movement velocity, wobbility, plasma membrane integrity, and acrosome integrity of boar sperm, while decreasing abnormal sperm morphology, indicating that Sch B could directly act on sperm to enhance its motility. Notably, the plasma membrane integrity and acrosome integrity were increased after treatment with Sch B. Suggesting that Sch B has potential improvement functions on sperm quality parameters equally in in vitro models and at the cellular level, a previous study demonstrated that Sch B can modulate DNA damage through inhibition of MAPK/p53 signaling . Our results are consistent with previous research on rodents in that administration with Sch B can decrease DNA damage of boar sperm in vitro. Subsequently, the results of the MMP changes showed that the MMP increase may be responsible for the decreased apoptosis and increased quality in Sch B-treated sperm. Our results correspond with a previous study in that the mitochondrial functional and structural integrity were improved by long-term Sch B treatment in rat kidneys . These data corroborate the previous suggestion that Sch B increased the quality parameters by causing changes in anti-apoptotic ability, DNA damage reduction, and increased MMP. Sch B in concentrations of 0-10 mmol/L had a dose-dependent positive effect on sperm characteristics, while the higher levels of Sch B supplementation (20 and 40 mmol/L) had either a negative or no effect on sperm characteristics. Accordingly, we selected 10 mmol/L Sch B as the optimal concentration for a follow-up study. Oxidative stress is a leading factor contributing to the poor overall quality of spermatozoa after the freezing and thawing process since the spermatozoa are rich in polyunsaturated fatty acids and are an easy target for ROS, which results in lipid peroxidation of the spermatozoan plasma membrane . It has been well established in sperm that ROS content is inversely correlated with sperm motility . Meanwhile, MDA is also involved in lipid peroxidation, which is an important indicator of cellular oxidative damage. The results of our analysis showed that the MDA and ROS contents were notably down-regulated and that the expression levels of GPx had no significance, accompanied by a significantly improved SOD and CAT mRNA expression and T-AOC levels after Sch B treatment in sperm, which is consistent with other studies performed in rodents, except for GPx . Supplementation of extenders with CAT, SOD, and GPx can preserve sperm motility and DNA integrity, thereby improving cooled storage, especially with poor semen quality . These results suggest that Sch B may improve sperm quality by increasing the antioxidant capacity of sperm. Dona et al. reported that spermatozoon ROS content directly influences the levels and locations of tyrosine phosphorylation and then enables the spermatozoa to undergo an acrosome reaction . In boars, the majority of seminal plasma proteins belong to the sperm-adhesin family, a group of (glyco) proteins built by a single CUB domain architecture . Previous research has shown that the addition of seminal plasma attenuates in vitro and cooling-induced capacitation-like changes in boar spermatozoa . Premature capacitation-like changes lead to spontaneous acrosome reactions and cell death, with a subsequent decrease in sperm fertilizing ability. The sperm adhesion protein gene family plays an important role in the processes of spermatogenesis, ejaculation, and sperm-egg binding. AQN-1, AQN-3, AWN, PSP-I, and PSP-II adhesion protein genes are the main members of the sperm adhesion protein gene family. The AQN-1 and AWN proteins are involved in the formation of the acrosomal reaction inhibition receptor complex before sperm-egg binding and premature sperm capacitation can be prevented by them . AWN and AQN-3 also mediate sperm binding to the pellucida during fertilization . It could be argued that the PSP-I/PSP-II heterodimer is responsible for the stabilization of the sperm membrane, thus counteracting the destabilizing phenomena that lead to membrane destabilization, impaired acrosome integrity, and reduced cell viability. Several studies have demonstrated a significantly higher expression of both genes in low-litter size boar spermatozoa than in high-litter size spermatozoa . In addition, in another study, it was reported that the expression of PSP-I protein in seminal plasma was negatively correlated with the motility and morphology of boar sperm and litter size . Our results showed that Sch B could significantly increase the expression of AWN, whereas it reduced the expression of and decreased PSP-I and PSP-II in the boar sperm. However, the results of the expression of AQN-1 and AQN-3 were not significant. This is the first evidence revealing that the molecular mechanism of Sch B is involved in sperm capacitation by regulating the expression of the sperm adhesion protein gene family, suggesting that adding Sch B to a semen extender can improve sperm quality in the cryogenic liquid state by reducing capacitation. Ca2+ signaling is of particular significance in sperm, where it is a central regulator in many key activities (including capacitation, hyperactivation, chemotaxis, and acrosome reaction). Previous studies have indicated that the increase in Ca2+ is related to sperm death. An excessive Ca2+ level affects capacitation during the fertilization of sperm, which leads to sperm death . Fluorometric recordings of sperm loaded with fluo3-AM revealed that low-temperature preservation evoked elevations of intracellular Ca2+. Therefore, the high Ca2+ concentration during the cryopreservation of sperm is an important reason for its quality decline. In contrast, when boar spermatozoa were treated with Sch B in our study, decreases in the spermatozoa's membrane instability and calcium concentration were observed. It has been reported that lactic acid is a sperm motility inactivation factor . Carr concluded that the low pH of the cauda epididymal fluid from bulls and dogs, plus the presence of lactate, is sufficient to cause inhibition of sperm motility . In our study, we found that the lactate content of sperm decreased after the addition of Sch B, which explained the possible factor of the previously detected increase in sperm motility, and we speculated that lactate content might be an important factor affecting sperm capacitation. Sperm-capacitated processes shorten their lifespan and result in premature death . Ejaculated spermatozoa are unable to fertilize the oocyte; on entry to the female reproductive tract, maturational changes occur in sperm that render them competent for fertilization in a process known as capacitation . Even though the molecular basis of this process remains unclear, it correlates with a series of cellular and biochemical changes, including cholesterol efflux from the sperm plasma membrane , ion influx, membrane hyperpolarization , cAMP production , kinase activation and protein phosphorylation . Enzymes such as members of the PKA are directly regulated by cAMP . Thus, this nucleotide orchestrates several downstream events with critical outcomes in cell physiology. Activation of PKA enzymes is essential for capacitation, and thus, cAMP levels are tightly regulated during this process. cAMP, an important second messenger, activates PKA, leading to potassium channel opening and calcium channel closing, which can decrease the concentration of calcium in the cytoplasm. Previous results suggest that Sch B was able to significantly improve erectile dysfunction in rats with nerve injury through the cAMP-PKA pathways. Similar results were found in our present capacitation investigation in that Sch B triggered an increase in the PKA level, resulting in a Ca2+ concentration decrease. PKG inhibitors have been reported to inhibit sperm, hyperactivation, and the acrosome reaction. It has also been reported that increased PKG in the cGMP/PKG signaling pathway can increase Ca2+ and tyrosine-phosphorylated proteins, promote hyperactivation, and induce the acrosome reaction, which ultimately facilitate sperm capacitation . Our study showed that adding Sch B had no effect on the level of PKG in boar sperm. We believe that adding Sch B in a semen extender can regulate sperm Ca2+ concentration through the cAMP-PKA pathway, which leads to the decrease of sperm number with capacitation. These findings suggest that Sch B was identified as a primary candidate for a decapacitation factor. Since our data showed that Sch B could affect the capacitation state of sperm by regulating sperm adhesion proteins and the CAMP-PKA pathway, we therefore hypothesized that Sch B may be involved in decapacitation. To test this hypothesis, we further assessed Ca2+ influx, lactic acid content, the quantitative expression of sperm adhesion protein gene family mRNAs, and the PKA/PKG activity of boar spermatozoa after capacitation. However, no significant difference (p > 0.05) was observed in any of the capacitation-related parameters (Ca2+ and lactic acid content, expression of sperm adhesion protein gene family, PKA and PKG levels) evaluated in samples with Sch B compared to 0 Sch B. Current studies on sperm decapacitation factors have shown that they can prevent premature capacitation and the acrosome reaction but have no decapacitation effect on sperm that has been capacitated . In other words, Sch B does not reverse capacitation when spermatozoa are highly capacitated. Therefore, we suspected that Sch B acted only on non-capacitated sperm, as it did not reverse capacitated sperm. This selectivity allows its decapacitating action only in non-capacitated sperm, without affecting capacitated sperm. Collectively, our study based upon sperm quality parameters, antioxidant capacity, and sperm fertilization ability during liquid preservation of boar semen at 4 degC significantly supports the effect of Sch B at a dose of 10 mmol/L as a semen storage protectant. This was evidenced by downregulation of Ca2+, reduced PKA, and regulated sperm adhesin family, reduced amounts of capacitated sperm, elevated antioxidative levels (SOD, CAT, and T-AOC increased, ROS, and MDA decreased), lowered proapoptotic properties of DNA damage, increased MMP, and, ultimately, the protected quality of boar sperm. Based on the above results, Sch B was identified as a primary candidate for an antioxidant and decapacitation factor in sperm in liquid at 4 degC. 5. Conclusions In conclusion, our findings demonstrated that 10 mmol/L is the optimal concentration of Sch B supplement in boar semen extender. Sch B enhanced sperm quality by increasing oxidation resistance and reducing the capacitation-like changes in spermatozoa stored at 4 degC. However, it did not have reversing effects on the capacitation status of capacitated spermatozoa. Acknowledgments We wish to thank Chuanhuo Hu, Xun Li and Lei Chen for reagents and technical support. Author Contributions Conceptualization, methodology, software and investigation, Y.X., Z.C. and C.H.; validation and formal analysis, X.W.; Investigation, X.W., X.P., C.X. and Y.T.; data curation, Y.W.; writing--original draft preparation, Z.C.; writing--review and editing, X.L.; visualization, N.F.; supervision, project administration and funding acquisition, C.H. All authors have read and agreed to the published version of the manuscript. Institutional Review Board Statement All of the experiments were performed in accordance with the guidelines of the regional Animal Ethics Committee and were approved by the Guangxi University Ethical Committee (GXU2022-212). Informed Consent Statement Not applicable. Data Availability Statement The data presented in this study are available on request from the corresponding author. Conflicts of Interest The authors declare no conflict of interest. Figure 1 (A-D): Effect of Sch B on the motility, abnormal sperm morphology, average movement velocity and wobbility of boar sperm: (A) Sperm motility. (B) abnormal sperm morphology (C) Average movement velocity. (D) Sperm wobbility. Data are expressed as mean +- SD (n = 12). *** p < 0.001, ** p < 0.01 and * p < 0.05 vs. 0 Sch B. Figure 2 (A,B): Effects of SchB on the plasma membrane integrity and acrosome integrity of boar sperm: (A) Sperm motility. (B) abnormal sperm morphology. Data are expressed as mean +- SD (n = 12). *** p < 0.001, ** p < 0.01 and * p < 0.05 vs. 0 Sch B. Figure 3 Effects of Sch B on the DNA damage rate and MMP changes in boar sperm: (A) DNA damage rate. (B) MMP changes in sperm. Data are expressed as mean +- SD (n = 12). *** p < 0.001 and ** p < 0.01 vs. 0 Sch B. Figure 4 Effect of Sch B on mRNA and of SOD, CAT, and GPx in boar sperm: Data are expressed as mean +- SD (n = 12). ## p < 0.01 and # p < 0.05 vs. 0 Sch B, *** p < 0.001 vs. fresh. Figure 5 Effect of Sch B on boar sperm ROS (A), T-AOC (B), and MDA level (C): Data are expressed as mean +- SD (n = 12). ### p < 0.001 and ## p < 0.01 vs. 0 Sch B, *** p < 0.001 and * p < 0.05 vs. fresh. Figure 6 Effects of Sch B on boar sperm Ca2+ (A) and lactic acid (B) content: Data are expressed as mean +- SD (n = 12). ## p < 0.01 and # p < 0.05 vs. 0 Sch B, *** p < 0.001 ** p < 0.01 and * p < 0.05 vs. fresh. Figure 7 Effects of Sch B on boar sperm mRNA expression of AWM, AQN1, AQN3, PSP-I, and PSP-II in boar sperm: Data are expressed as mean +- SD (n = 12). ## p < 0.01 and # p < 0.05 vs. 0 Sch B, *** p < 0.001 ** p< 0.01 and * p < 0.05 vs. fresh. Figure 8 Effects of Sch B on PKA (A) and PKG Levels (B) in boar sperm: Data are expressed as mean +- SD (n = 12). # p < 0.05 vs. 0 Sch B, * p < 0.05 vs. fresh. Figure 9 Effects of Sch B on Ca2+ (A) and lactic acid (B) content in capacitated boar sperm: Data are expressed as mean +- SD (n = 12). ** p < 0.01 vs. 0 Sch B. Figure 10 Effects of Sch B on mRNA expression of AWM, AQN1, AQN3, PSP-I, and PSP-II in capacitated boar sperm: Data are expressed as mean +- SD (n = 12). *** p < 0.001 and ** p < 0.01 vs. 0 Sch B. Figure 11 Effects of Sch B on PKA (A) and PKG Levels (B) in capacitated boar sperm: Data are expressed as mean +- SD (n = 12). * p < 0.05 vs. 0 Sch B. animals-13-00848-t001_Table 1 Table 1 List of primer sequences used in real-time PCR analysis. Gene Primer Sequences SOD2 F:GCTGGAAGCCATCAAACG R:TTAGAACAAGCGGCAATCTG CAT F:TGCCCATACTTCCCGTCC R:GGTCCAGGTTACCGTCAG GPx F:CAGGTACAGCCGTCGCTTTC R:AAAATCCCGAGAGTAGCACT AQN1 F:GCTGCTCAGTACAGCTACAC R:ATTACCAGCACCTGGCAGTC AWN F:CAATACCGCCTCTCAACCTC R:GGCCTTCTGGATCAGCATAG AQN3 F:AACGGGCAGGACCTGATAAC R:CCCTCAGACACGAGTCTAAG GAPDH F:CCCCAACGTGTCGGTTGT R:CTCGGACGCCTGCTTCAC Disclaimer/Publisher's Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

PMC10000211

In this prospective study (NCT01595295), 272 patients treated with azacitidine completed 1456 EuroQol 5-Dimension (EQ-5D) questionnaires. Linear mixed-effect modelling was used to incorporate longitudinal data. When compared with a matched reference population, myeloid patients reported more pronounced restrictions in usual activities (+28%, p < 0.0001), anxiety/depression (+21%, p < 0.0001), selfcare (+18%, p < 0.0001) and mobility (+15%, p < 0.0001), as well as lower mean EQ-5D-5L indices (0.81 vs. 0.88, p < 0.0001), and lower self-rated health on the EuroQol Visual Analogue Scale (EQ-VAS) (64 vs. 72%, p < 0.0001). After multivariate-adjustment, (i) the EQ-5D-5L index assessed at azacitidine start the predicted time with clinical benefit (TCB) (9.6 vs. 6.6 months; p = 0.0258; HR = 1.43), time to next treatment (TTNT) (12.8 vs. 9.8 months; p = 0.0332; HR = 1.42) and overall survival (OS) (17.9 vs. 12.9 months; p = 0.0143; HR = 1.52); (ii) Level Sum Score (LSS) predicted azacitidine response (p = 0.0160; OR = 0.451) and the EQ-5D-5L index showed a trend (p = 0.0627; OR = 0.522); (iii) up to 1432 longitudinally assessed EQ-5D-5L response/clinical parameter pairs revealed significant associations of EQ-5D-5L response parameters with haemoglobin level, transfusion dependence and hematologic improvement. Significant increases of the likelihood ratios were observed after addition of LSS, EQ-VAS or EQ-5D-5L-index to the International Prognostic Scoring System (IPSS) or the revised IPSS (R-IPSS), indicating that they provide added value to these scores. health-related quality of life (HRQoL) patient-reported outcome (PRO) EuroQol 5-Dimension 5-Level questionnaire (EQ-5D-5L) azacitidine Austrian Registry of Hypomethylating Agents prognosis mixed-effects linear models acute myeloid leukaemia myelodysplastic syndromes chronic myelomonocytic leukaemia Austrian Group for Medical Tumor Therapy (AGMT)The Austrian Group for Medical Tumor Therapy (AGMT) is the sponsor for the Austrian Registry of Hypomethylating Agents and received funding from Celgene/BMS, AbbVie, and Janssen Cilag. The AGMT is a non-for-profit organisation and an academic study group. The group performed administrative and legal management, as well as funding acquisition. No pharmaceutical company and no other funding source were involved in any way and had no role in the study design, data collection, data analysis, data interpretation, or writing of the report. No medical writer or editor was involved. pmc1. Introduction Azacitidine is the first treatment to be associated with improved overall survival (OS), and to be approved by both US and European regulatory authorities for the treatment of patient subgroups with myeloid neoplasms. In patients with myelodysplastic syndromes (MDS) and chronic myelomonocytic leukaemia (CMML) , it remains the only approved disease-modifying therapeutic substance, whereas several new drugs have recently been approved for certain patient subgroups with acute myeloid leukaemia (AML). Globally, there has been a distinctive shift towards taking patient perspectives into account when making (regulatory) healthcare and treatment decisions. Traditional clinical ways of measuring health and the effects of treatment are thus increasingly being accompanied by patient-reported outcome measures. In the broad field of the latter, the generic EuroQol 5-Dimension (EQ-5D) questionnaire is multiply validated and globally has been the most used tool in many areas of medicine, including oncology, for over three decades. Although regulatory agencies offered guidance for the use of patient-reported outcome measures to support labelling claims as early as 2005 , the European LeukemiaNet pointed to the importance of assessing HRQoL in the clinical management of patients with MDS in 2013 , and the EQ-5D has been the preferred measure of HRQoL for the UK National Institute for Health and Care Excellence since 2008, published reports on HRQoL data in MDS, CMML and AML are scarce. There are 4 publications in MDS, and 10 in AML, 13 of which only report on the mean EQ-VAS and/or the mean or median EQ-5D-5L index value. Only one report assessed the impact of the EQ-5D-5L index on a time-to-event endpoint , and only one publication provided details on non-composite results , both in patients with lower-risk MDS (Table 1). Publications correlating EQ-5D-5L measures with treatment outcomes in general, and with azacitidine-related outcomes in particular, are lacking to date. The only detailed EQ-5D-5L data on this topic stem from this report. In this prospective study, we compared EQ-5D-5L responses between patients with MDS, CMML and AML and a population norm (i.e., a representative sample of the German general adult population without myeloid (or other) neoplasias) from a similar geographic region, matched by age, sex and number of comorbidities. In myeloid patients treated with azacytidine within the Austrian Registry of Hypomethylating Agents, we performed more detailed analyses and assessed (1) whether EQ-5D-5L composite variables provided added value to the (R)-IPSS; (2) there might be a predictive value of EQ-5D-5L composite variables (including LSS, EQ-VAS and EQ-5D-5L index value) on the response to azacitidine and several time-to-event endpoints; and (3) performed longitudinal assessments of EQ-5D-5L response/clinical parameter pairs. 2. Materials and Methods 2.1. Study Design and Participants In this prospective cohort study, data from non-selected, consecutive patients were provided by seven Austrian centres (Supplementary p. 1) participating in the Austrian Registry of Hypomethylating Agents of the Austrian Group for Medical Tumour Therapy (AGMT) Study Group . The EQ-5D consists of five questions (also known as dimensions (5D): mobility, selfcare, usual activities, pain/discomfort, anxiety/depression) with 5 levels (5L) of problem severity in the responses, as well as a Visual Analogue Scale (EQ-VAS) aiming to capture a respondents' rating of their 'health today' on a scale from 0-100. The composite scores Level Sum Score (LSS) and EQ-5D-5L index are explained in Supplementary p. 2. The EQ-5D questionnaires were assessed at the start of azacitidine treatment cycles. The EQ-5D-5L results of patients diagnosed with MDS, CMML or AML were compared with those of a German population norm (i.e., a representative sample of the German general adult population without myeloid (or other) neoplasias) . The EQ-5D-5L German value set and the reverse crosswalk tool provided by EuroQol on November 16, 2020 were used for calculation of the EQ-5D-5L indices (Supplementary p. 3). Patients with an EQ-5D available at azacitidine treatment start were stratified according to their LSS, EQ-VAS or EQ-5D-5L index being </>= the respective group median. 2.2. Statistical Analyses Only observed values were analysed. Baseline and treatment-related factors were compared using the kh2 test for categorical variables and the Wilcoxon test for continuous variables. Patient subgroups were compared using the log-rank test. All p-values and 95% CIs are two-sided. The threshold for statistical significance was 0.05. Time-to-event endpoints were analysed using the Kaplan-Meier method. Proceeding in analogy to Efficace et al. who demonstrated that self-reported fatigue provided added value to the IPSS and R-IPSS in patients with MDS, the likelihood ratio (LHR) test was used to determine whether EQ-5D-5L response parameters provided added value to the IPSS or R-IPSS. The prognostic information provided by LSS, EQ-VAS and EQ-5D-5L index, with regards to whether a patient is likely to respond to azacitidine or not was assessed by univariate and multivariate-adjusted logistic regression analyses. Cox regression models for time-to-event endpoints were applied. To identify variables that might be associated with patient-reported outcomes, linear mixed-effect modelling was utilised, with patient identity as the grouping variable. p-values were visualised using heatmaps. Sensitivity analyses were performed to check the general conclusions by assessing different endpoints (response subtypes, OS, TCB, TTNT), and assessing both continuous and dichotomised variables. The definition of outcomes and further statistical details are given in Supplementary pp. 4-7. Assign Data Management and Biostatistics GmbH performed statistical analyses with SAS(r) 9.4. The Life & Medical Sciences Institute, University of Bonn performed statistical analyses including mixed-effect linear modelling with Python 3.8.12. 3. Results 3.1. Myeloid Patient Characteristics Data from 272 patients diagnosed with MDS, CMML or AML who were treated with azacitidine between 21 May 2007 and 21 December 2020 were prospectively analysed . Of these, 205 had filled out an EQ-5D at azacitidine treatment start . This subset was used for time-to-event endpoint analyses. Myeloid patient characteristics at azacitidine treatment start by EQ-5D group are shown in Supplementary p. 8. In the group, 129 (47%), 33 (12%) and 110 (40%) of 272 patients had MDS, CMML or AML, respectively. A total of 168 (62%) of 272 patients were male, the median age was 74.0 (IQR 69.0-79.0) years, 33 (12%) had treatment-related disease, 51 (19%) had an ECOG performance score of >=2 and median bone marrow blasts were 12% (IQR 5-35%). Differential blood count and other lab values of the EQ-5D group are shown in Supplementary pp. 9-10. A further 86 (32%) and 35 (13%) of 272 patients were red blood cell and/or platelet transfusion dependent, respectively (Supplementary pp. 9-10). Finally, 205 (75%) of 272 patients had at least one additional comorbidity (Supplementary p. 11). Azacitidine treatment and response characteristics are shown in Supplementary pp. 12-13. Median follow-up duration from diagnosis was 23.4 months (IQR 12.3-40.9) and from azacitidine treatment start 14.7 months (7.8-26.7). 3.2. Patients Treated with Azacitidine Reveal Profound Impairments in HRQoL Supplementary pp. 14-15 show the most frequent response patterns for questionnaires filed out at azacitidine treatment start and for all EQ-5D questionnaires. Supplementary p. 16 gives an overview of the EQ-5D responses by patient group, response status and number of azacitidine treatment cycles. The mean number of filled-out EQ-5D questionnaires per patient was 5.4 (SD 6.2), the median number was 3.0 (IQR 1.0-3.0). The myeloid cohort (n = 272) was characterised by mean (SD) LSS, EQ-5D-5L index value and EQ-VAS of 9.1 (3.9), 0.807 (0.232) and 63.9 (21.7), respectively, in their first available EQ-5D-5L questionnaire; results were similar when focusing on patients who had filled out an EQ-5D at azacitidine treatment start (n = 205) (Table 2). In this subgroup, problems (slight, moderate, severe or extreme) were self-reported in the dimensions of mobility (104 (51%) of 205), selfcare (46 (22%)), usual activities (120 (59%)), pain/discomfort (102 (50%)) and anxiety/depression (100 (49%)). The following parameters at azacitidine treatment start significantly correlated with adverse EQ-5D-5L responses: monocytes >=10%, haemoglobin levels <10 g/dL, >3 red blood cell transfusions prior to azacitidine start, Eastern Cooperative Oncology Group Performance Status (ECOG-PS) of >=2, high risk Hematopoietic Cell Transplantation-specific Comorbidity Index (HCT-CI) (Table 2). For example, patients with an ECOG-PS >=2 experienced more significantly problems in the dimensions of mobility (+30%, p = 0.0008), selfcare (+34%, p < 0.0001), usual activities (+28%, p = 0.0012) and anxiety/depression (+32%, p = 0.0003), and had significantly reduced EQ-VAS (-10%, p = 0.0092) . 3.3. Comparison of HRQoL with a Reference Population Matched by Age, Sex and Number of Comorbidities We compared HRQoL of the myeloid cohort with that of a German population norm (i.e., a representative sample of the German general adult population without myeloid (or other) neoplasias) with a similar ethnical and socioeconomic background . Myeloid patients reported more pronounced restrictions in mobility (51 vs. 35%, p < 0.0001), selfcare (25 vs. 7%, p < 0.0001), usual activities (56 vs. 28%, p < 0.0001) and anxiety/depression (+15%, p < 0.0001), as well as lower mean EQ-5D-5L indices (0.81 vs. 0.88, p < 0.0001) and lower self-rated health on EQ-VAS (64 vs. 72%, p < 0.0001) than the German population norm (Table 3). These significant differences could also be observed after stratification by age group, sex or number of comorbidities (Table 3). 3.4. IPSS and R-IPSS Prognosticate OS and TTNT Myeloid patients with lower-risk IPSS had significantly longer unadjusted survival than patients with higher-risk IPSS (21.0 months [95% CI 14.6-30.3] vs. 12.8 months [10.2-16.9]; HR = 0.62 [0.44-0.88]; LHR 7.32; p = 0.0068). Similarly, patients with lower-risk R-IPSS had significantly longer unadjusted survival than patients with higher-risk R-IPSS (30.3 months [11.2-39.3] vs. 14.6 months [11.9-17.8]; HR = 0.561 [0.3320.949]; LHR 5.37; p = 0.0205) (Table 4, first four columns). Patients with lower-risk IPSS showed a trend towards longer TTNT (p = 0.0578), and patients with lower-risk R-IPSS showed significantly longer TTNT than their higher-risk counterparts (17.6 months [6.9-37.7] vs. 10.8 months [9.3-12.6]; HR = 0.615 [0.379-1.000]; LHR 4.31; p = 0.0379) (Table 4, first 4 columns). 3.5. EQ-5D-5L Composite Scores at Azacitidine Start Provide Added Value to the (R)-IPSS For the endpoint OS, significant increases of the likelihood ratio (LHR) were observed after addition of (i) the LSS to the IPSS (LHR increased from 7.32 to 10.69; p = 0.0048) or the R-IPSS (LHR increased from 5.37 to 9.05; p = 0.0108); (ii) the EQ-VAS to the IPSS (LHR increased from 7.32 to 11.56; p = 0.0031) or the R-IPSS (LHR increased from 5.37 to 10.28; p = 0.0058); (iii) the EQ-5D-5L index to the IPSS (LHR increased from 7.32 to 13.02; p = 0.0015) or the R-IPSS (LHR increased from 5.37 to 13.48; p = 0.0012), indicating that they provided added value to the IPSS and R-IPSS (Table 4, grey shaded columns). For the endpoint TTNT, significant increases of the LHR were observed after addition of (i) the LSS to the R-IPSS (LHR increased from 4.31 to 7.74; p = 0.0209); (ii) the EQ-VAS to the R-IPSS (LHR increased from 4.31 to 6.85; p = 0.0327); (iii) the EQ-5D-5L index to the IPSS (LHR increased from 3.60 to 6.38; p = 0.0411) or the R-IPSS (LHR increased from 4.31 to 6.55; p = 0.0378), indicating that they provided added value to the IPSS and R-IPSS (Table 4, grey shaded columns). 3.6. EQ-5D-5L Composite Scores at Azacitidine Start Impact Time-to-Event Endpoints Myeloid patients with an EQ-5D available at azacitidine treatment start (n = 205) were stratified according to their LSS, EQ-VAS or EQ-5D-5L index being </>= the respective group median. In unadjusted analyses, patients with (i) an LSS < 8.0 at azacitidine treatment start had significantly longer OS and showed a trend for longer TCB and TTNT; (ii) an EQ-VAS < 65 at azacitidine treatment start had significantly longer OS; (iii) an EQ-5D-5L index >=0.8845 had significantly longer OS, longer TCB and longer TTNT (Table 5, first four columns) . After multivariate adjustment (for ECOG-PS, number of comorbidities, platelet count <=30 G/L or transfusion dependence, peripheral blood blasts, azacitidine treatment line and azacitidine dose in cycle one) patients with an EQ-5D-5L index above the group median (i.e., >=0.8845) had significantly longer OS (17.9 months [95% CI 14.0-21.0] vs. 12.9 months [10.3-16.8]; HR 1.52 [1.09-2.13]; p = 0.0143), longer TCB (9.6 months [95% CI 6.8-12.1] vs. 6.6 months [4.9-8.5]; HR 1.43 [1.04-1.95]; p = 0.0258) and longer TTNT (12.8 months [95% CI 10.5-20.2] vs. 9.8 months [8.5-11.9]; HR 1.42 [1.03-1.96]; p = 0.0332) (Table 5, last three columns) . 3.7. EQ-5D-5L Composite Scores at Azacitidine Start Prognosticate the Likelihood of Response to Azacitidine In univariate logistic regression, the LSS (p = 0.0009), EQ-VAS (p = 0.0237) and EQ-5D-5L index (p = 0.0110) were significantly correlated with response to azacitidine. After multivariate adjustment, LSS remained significantly predictive of response to azacitidine (p = 0.0160; OR 0.451 [95% CI 0.235-0.852]), and the EQ-5D-5L index showed a trend (p = 0.0627; OR 0.522 [0.296-1.032]) (Table 6). An LSS of >=8 at azacitidine treatment start thus indicates a significantly lower chance of responding to azacitidine as expressed by the OR of 0.45. 3.8. Longitudinal Assessment of EQ-5D-5L Responses and Clinical Parameters Multivariate-adjusted mixed-effect linear models of up to 1432 longitudinally assessed EQ-5D-5L response/dichotomised clinical parameter pairs revealed significant associations for haemoglobin level, red blood cell transfusion dependence, platelet count, platelet transfusion dependence, levels of ferritin, bilirubin, albumin, cholinesterase, the occurrence of adverse events, number of days with azacitidine treatment, and haematologic improvement (HI-any, HI-E, HI-P) with at least two EQ-5D dimensions, and at least one of the EQ-5D composite variables (LSS, EQ-VAS, EQ-5D-5L index) . Sensitivity analyses for continuous clinical parameters yielded similar results (Supplementary pp. 17-18). 3.9. Minimally Clinically Important Differences Of the statistically significant associations found in the dichotomised analyses, the following exhibited an effect size equal to or larger than the minimally clinically important difference: platelet transfusion dependence (LSS), ferritin >=1000 mg/L (LSS), albumin >=3.4 mg/dL (LSS), adverse events grade 3-4 (LSS, EQ-5D-5L index) and cholinesterase >=2.5 U/L (EQ-VAS). These findings were corroborated in sensitivity analyses using continuous parameters. 4. Discussion To our knowledge, our group is the first to compare EQ-5D-5L data of patients with MDS, CMML or AML with data from a reference population from a similar ethnic, socioeconomic and geographic background. In this prospective cohort analysis, we found that patients treated with azacitidine had significantly worse HRQoL than the German population norm (i.e., a representative sample of the German general adult population) matched by sex, age group and number of comorbidities. In contrast to observations by Stauder et al. who used the EQ-5D-3L, all significant differences observed for the EQ-5D-5L index and the EQ-VAS fulfilled the definitions of the minimally clinically important difference used by that group (>0.03 on the index and >3.0 on the EQ-VAS). The current gold standards of prognostication in patients with MDS/CMML and low blast count AML are the International Prognostic Scoring System (IPSS) and the revised IPSS (R-IPSS) . The clinical relevance of these scores is underscored by the fact that approval of azacitidine for MDS patients in Europe is restricted to those with higher-risk IPSS (i.e., intermediate-2 and high risk categories). To our knowledge, our data are the first to indicate that LSS, EQ-VAS and EQ-5D-5L index at azacitidine treatment start provided added value to the IPSS and R-IPSS for the endpoints OS and TTNT. Other groups have prominently shown that patient-reported outcomes (other than EQ-5D) may predict OS and/or add value to the (R)-IPSS in elderly patients with MDS or AML . However, these questionnaires/indices incorporate 30 , 42 and 44 items , many of which are not routinely assessed in patients with myeloid neoplasms, thus hampering the clinical everyday utility outside of clinical trials. Our data are the only information on the impact of HRQoL, as assessed by the EQ-5D-5L, on time-to-event endpoints of patients treated with azacitidine. After multivariate adjustment (for ECOG-PS, number of comorbidities, platelet count <=30 G/L or transfusion dependence, peripheral blood blasts, azacitidine treatment line and azacitidine dose in cycle one) an EQ-5D-5L index <0.8845 at azacitidine start indicated a significantly shorter median survival (-5.0 months), an increased risk of death (+52%), significantly shorter azacitidine treatment duration (-3.0 months), shorter TTNT or death (-3.0 months) and a significantly higher risk of requiring a next treatment or dying (+42%). Our data further show that an LSS of >=8 at azacitidine start indicates a significantly lower chance of responding to the drug (OR 0.45). This is the first report on the longitudinal assessment of EQ-5D-5L responses with clinical parameters. Multivariate adjusted mixed-effect linear modelling revealed significant associations for EQ-5D-5L response parameters with clinical parameters associated with haematologic improvement, disease progression, or the occurrence of adverse events. Thus, these data show that quality of life ameliorated in responding patients and deteriorates in patients experiencing disease progression or grade 3-4 adverse events. It is difficult to interpret these findings compared with the wider literature as longitudinal analyses of HRQoL data on patients with MDS, CMML or AML are scarce, performed with questionnaires other than EQ-5D-5L and are often without multivariate adjustment. Efficace et al. found no association between ferritin levels and HRQoL as assessed by EORTC QLQC30 both at baseline and during the study period in heavily transfused patients with MDS treated with iron chelation therapy using linear mixed-effect models . We observed significant associations of ferritin, bilirubin and albumin levels with problems in six of eight EQ-5D-5L dimensions/composite variables. This is the first indication that these clinical variables, two of which (hypalbuminaemia and hyperferritinaemia) have been shown to be associated with adverse prognosis in patients with MDS , CMML or AML correlate with HRQoL. Little is known of the longitudinal effect of azacitidine on patients' HRQoL, but recent publications demonstrating significant improvements of EQ-VAS and/or EQ-5D-5L index in patients responding to treatment in other malignancies highlight the contemporality and clinical relevance of the topic. The mean (SD) EQ-VAS and EQ-5D-5L index values of our cohort were similar to those previously reported in patients with MDS/CMML or AML. Problems (slight, moderate, severe or extreme) were most commonly self-reported in the dimensions of usual activities (59%), mobility (51%), pain/discomfort (50%), anxiety/depression (49%) and selfcare (22%). Similar to the lower-risk MDS population reported by Stauder et al. , (i) MDS, CMML and AML patients in our cohort had the least problems in the dimension of selfcare, (ii) no correlation could be found between IPSS or R-IPSS risk group at azacitidine treatment start and EQ-5D responses, and (iii) patient-related factors such as haemoglobin <10 g/dL, red blood cell transfusion dependence, ECOG-PS >= 2 and high-risk HCT-CI were found to be associated with significantly more problems in several dimensions and/or significantly worse EQ-5D-5L composite variables. We could, however, not find a significant difference in EQ-5D-5L response by sex or age group. A limitation of this study is that we cannot speculate what the HRQoL would have been without azacitidine therapy. Furthermore, this question cannot be addressed by real-world evidence or by future randomised clinical trials due to ethical reasons. A further limitation is that we do not have EQ-5D-5L questionnaires for all patients for all treatment cycles. However, to impose mandatory pre-specified required time-points for filling out EQ-5D-5L questionnaires would be against the non-interventional nature of non-interventional studies in general, and of the Austrian Registry of Hypomethylating Agents in particular. Furthermore, these results cannot, eo ipso, be generalised to other treatments of patients with MDS, CMML or AML, as we exclusively studied HRQoL of patients treated with azacitidine. In the future, we aim to analyse EQ-5D-5L responses in myeloid patients irrespective of treatment type within the Austrian Myeloid Registry (NCT04438889; Ethics committee approval was provided by the Ethikkommission fur das Bundesland Salzburg (415-E/2581/Feb-2020)), which is a disease-specific (rather than a drug-specific) registry, once sufficient data have been accumulated, and are open for collaborations with other study groups in this regard. The strengths of this study are that we report the first evidence-based data on all of the above; the prospective nature of data collection; the proven quality of our database in direct patient-level comparison with randomised phase-3 clinical trial data ; few missing data; calculation and validation of diagnosis, cytogenetic risk groups, and prognostic scores; response to reduce human errors; multivariate adjustment; longitudinal analyses; correction for multiple testing; and that additional sensitivity analyses confirmed the robustness of our results. 5. Conclusions In conclusion, the current findings support the use of EQ-5D-5L instruments in future clinical trials and real-world evidence databases, in order to fully consider all factors that can be potentially associated with treatment outcomes. They also extend knowledge on the safety and efficacy of azacitidine by showing that clinical benefits such as improvement of laboratory values associated with haematologic improvement, as well as haematologic improvement itself, correlate with improved HRQoL. Acknowledgments We would like to express our gratitude to EurQol for granting us the permission to use both the 3L and the 5L versions of the EuroQol 5-Dimension (EQ-5D) questionnaire, for the provision of the reverse crosswalk and for answering questions pertaining to the analyses and the presentation of EQ-5D-related results. Special thanks to Thomas Grochtdreis et al. for providing additional, non-published EQ-5D-5L data of the German population norm for cohort comparisons. Supplementary Materials The following supporting information can be downloaded at Supplemental Table S1. Contributions of participating centres; Supplemental Table S2. Information on EQ-5D composite scores; Supplemental Table S3; Choice of appropriate value set region, value set and population norm; Supplemental Table S4. Further statistical details; Supplemental Table S5. Missing data; Supplemental Table S6. Univariate Cox regression analyses of baseline parameters present at azacitidine treatment start in patients with an available EQ-5D in cycle 1 or 2; Supplemental Table S7. Variables remaining in the final multivariate Cox model; Supplemental Table S8. Patient characteristics at azacitidine start; Supplemental Table S9. Lab values assessed at azacitidine start; Supplemental Table S10. Comorbidities assessed at azacitidine start; Supplemental Table S11. Azacitidine treatment and response characteristics; Supplemental Table S12. Most frequent response patterns of EQ-5D questionnaires at azacitidine treatment start; Supplemental Table S13. Most frequent response patterns of all EQ-5D questionnaires; Supplemental Table S14. Overview of EQ-5D responses by patient group and responder status; Supplemental Table S15. Multivariate-adjusted longitudinal analyses of EQ-5D-5L results and continuous parameters per azacitidine treatment cycle using mixed-effects linear models; Supplemental Figure S1. Heatmap of p-values resulting from multivariate-adjusted mixed-effect linear models of longitudinally assessed EQ-5D-5L responses and concomitantly assessed continuous clinical parameters; The Ethics Commottee Approval statement; The protocoll of the Austrian Registry of Hypomethylating Agents; The signed sponsor approval page; The informed consent of the Austrian Registry of Hypomethylating Agents. (References are cited in the Supplementary Materials). Click here for additional data file. Author Contributions Conceptualisation: L.P., M.D., J.L.-S., M.V., T.G., J.H. and R.S.; formal analysis: L.P., M.D., J.L.-S., M.V., T.G. and J.H.; funding acquisition: R.G.; investigation: all co-authors; methodology: L.P., M.D., J.L.-S., M.V., T.G. and J.H.; project administration: L.P., N.Z. and R.G.; resources: L.P., S.H., C.T., S.V., M.S., T.M., N.S., K.T.F.Q., M.L., A.E., L.S., D.W., T.G., N.Z., R.G. and R.S.; software: L.P., M.D., J.L.-S., M.V., T.G. and J.H.; supervision: L.P.; validation: L.P., M.D., J.L.-S., M.V., T.G. and J.H.; visualisation: L.P., M.D., J.L.-S. and M.V.; writing--original draft preparation: L.P.; writing--review and editing: all co-authors. All authors have read and agreed to the published version of the manuscript. Institutional Review Board Statement Ethics committee approval was provided by the Ethikkommission fur das Bundesland Salzburg (415-EP/39/Feb-2009). The Ethics Committee Approval, the study protocol, the signed Sponsor Approval Page, and the Informed Consent form of the AGMT and the Austrian Registry of Hypomethylating Agents have been made available in the Supplementary pp. 19-30. Informed Consent Statement The Informed Consent form of the Austrian Registry of Hypomethylating Agents have been made available in the Supplementary pp. 31-32. Data Availability Statement The datasets supporting the conclusions of this article are included within the article and the Supplementary Materials. Data sharing of patient level data collected for the study is not planned. However, we are open to research questions asked by other researchers, and we are also open to data contributions by others. Participation requests or potential joint research proposals can be made at any timepoint to the corresponding author via email (dr.lisa.pleyer@gmail.com) and are subject to approval by the AGMT and its collaborators. Conflicts of Interest L.P.: Honoraria from AbbVie, BMS and Novartis; S.H.: Honoraria from AbbVie, AOP, BMS, Janssen Cilag, Novartis and Roche; CT: No potential conflicts of interest; S.V.: Honoraria from Bristol Myers Squibb, Merck, MSD and Pfizer; consultancy fees from Roche, MSD, EUSA Pharma and Merck; Travel support from Pfizer, Roche, Pierre Fabre and Angelini; M.S.: Honoraria and consultancy BMS/Celgene; T.M.: Honoraria from AbbVie and Celgene/BMS; NS: No potential conflicts of interest; K.T.F.Q.: No potential conflicts of interest; M.L.: Honoraria from BMS, Celgene, Gilead, Takeda and Novartis; Travel support: Celgene and Novartis; AE: Honoraria, consultancy and travel support from AbbVie and BMS/Celgene; L.S.: No potential conflicts of interest; D.W.: Research Funding: BMS/Celgene, MSD, Novartis, Pfizer and Roche; Honoraria: BMS/Celgene, GEMOAB, Gilead, Incyte, MSD, Novartis, Pfizer and Roche; R.B.: No potential conflicts of interest; M.D.: No potential conflicts of interest; J.L.-S.: No potential conflicts of interest; T.G.: No potential conflicts of interest; M.V.: No potential conflicts of interest; J.H:. Research Funding: Bohringer-Ingelheim; N.Z.: No potential conflicts of interest; R.G.: Honoraria from AbbVie, Amgen, AstraZeneca, BMS/Celgene, Daiichi Sankyo, Gilead, Merck, Novartis, Roche, Takeda, BMS, MSD, Sandoz and Gilead; Research funding from Celgene, Roche, Merck, Novartis, MSD, Sandoz and Takeda; Consulting: AbbVie, Astra Zeneca, BMS/Celgene, Novartis, Roche, Takeda, Janssen, MSD, Merck, Gilead and Daiichi Sankyo; Travel support from AbbVie, Amgen, Astra Zeneca, BMS/Celgene, Daiichi Sankyo, Gilead, Janssen Cilag, MSD, Novartis and Roche; R.S.: Honoraria from BMS/Celgene. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results. Figure 1 Consort diagram. The data collection and cleaning period was from 2 February 2012 to 3 March 2022. Database lock (last patient in) was on 13 December 2020. The inclusion criteria were (1) the diagnosis of MDS, CMML or AML, which was independently and centrally verified on the basis of submitted data; (2) treatment with azacitidine; (3) inclusion in the Austrian Registry of Hypomethylating agents; (4) the presence of a written informed consent for all patients alive at the time of data entry; (5) age >= 18 years; (6) the completion of at least one EQ-5D questionnaire. No data from patients <18 years were received. No patients fulfilling these criteria were excluded from the analyses. A total of 6 of 1456 (0.4%) of EQ-5D questionnaires were excluded (empty questionnaire). Permissions to use the German version of EQ-5D questionnaires was obtained from EuroQol. All data for this study were collected prospectively. This study has been reported according to the Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) statement. To ensure uniformity, composite variables based on provided data were allocated for each individual patient at the start of azacitidine treatment, including diagnosis of MDS, CMML or AML according to the WHO 2016 diagnostic criteria , cytogenetic risk group according to the International Prognostic Scoring System (IPSS) and the revised IPSS (R-IPSS) and the IPSS and R-IPSS risk categories themselves. Figure 2 EQ-5D-5L responses available at azacitidine treatment start (n = 205), stratified by ECOG-PS. Figure 3 Impact of the EQ-5D-5L index at azacitidine treatment start on time-to-event endpoints. (A) Endpoint overall survival (OS), unadjusted. (B) Endpoint OS, adjusted 1. (C) Endpoint time with clinical benefit (TCB), unadjusted. (D) Endpoint TCB, adjusted 1. (E) Endpoint time to next treatment (TTNT), unadjusted. (F) Endpoint TTNT, adjusted 1. (1 Adjusted for the following characteristics at azacitidine treatment start: ECOG-PS, number of comorbidities, platelet count <=30 G/L or platelet transfusion dependence, peripheral blood blasts, azacitidine treatment line and azacitidine dose in cycle 1). Figure 4 Heatmap of p-values from multivariate-adjusted mixed-effect linear models of longitudinally assessed EQ-5D-5L response/clinical parameter pairs. The individual boxes contain the p-values (red coloured p-values denote significant values <=0.05, orange denotes a trend and is used for p-values between >0.05 and <=0.065) of the corresponding multivariate-adjusted mixed-effect linear models using EQ-5D-5L responses as endogenous variables (x-axis), and various clinical measurements as exogenous variables (y-axis). Multivariate adjustment was performed by admitting the following variables remaining in the final Cox model as covariates: ECOG-PS, number of comorbidities, platelet count/platelet transfusion dependence, peripheral blood blasts, azacitidine treatment line and azacitidine dose in cycle one. cancers-15-01388-t001_Table 1 Table 1 Comparison of published EQ-VAS and EQ-5D index values in patients with MDS and AML. First Author Year Published Patients, n Disease EQ-5D, Type EQ-VAS, Mean (SD) Index Value, Mean (SD) Index Value, Median (IQR) Impact on Time-to-Event Endpoint MDS Szende A. 2009 47 MDS 3L NR 0.78 (NR) NR NR Oliva E. 2012 148 MDS 3L 60 (20) NR 0.74 (0.62-0.85) NR Stauder R. 2018 1683 Lower-risk MDS 3L 69.6 (20.1) 0.74 (0.23) NR NR de Swart L. 2020 NR Lower-risk MDS 3L 70.5 (19.7) NR NR EQ-5D-3L index was significantly associated with progression-free survival in univariate analysis Pleyer L. (this article) 2023 162 MDS/CMML 5L 64.4 (21.2) 0.79 (0.3) 0.88 (0.73-0.95) EQ-5D-5L index, LSS and EQ-VAS were significantly associated with overall survival and the likelihood to respond to azacitidine in univariate analysis; EQ-5D-5L index was significantly associated with overall survival, time with clinical benefit and time to next treatment in multivariate-adjusted analyses. LSS was significantly associated with the likelihood to respond to azacitidine in multivariate analysis. AML Uyl-de Groot C.A. 1998 NR NR AML 3L 70.6 (NR) 64.8 (NR) NR NR NR NR NR Slovacek L. 2007 NR AML 3L 67.5 (NR) NR NR NR Leunis A. 2014 88 AML 3L 74.6 (17.4) 0.82 (17.4) NR NR Kurosowa S. 2015 392 AML 3L NR NR NR NR van Dongen-Leunis, A. 2016 111 AML 5L NR 0.81 (0.22) 0.87 (NR-NR) NR Mamolo C. 2019 NR AML 3L 61.2 (NR) 0.74 (NR) NR NR Horvath Walsh L. 2019 75 AML 3L 61.2 (NR) 0.74 NR NR Yu H. 2020 NR/168 NR/168 AML 3L 5L 76.9 (15.1) 0.829 (0.16) 0.786 (0.25) NR NR NR Peipert J. 2020 307 AML 5L 61.9 (20.1) 0.67 (0.26) NR NR Pratz K.W. 2022 642 AML 5L NR NR NR NR Pleyer L. (this article) 2023 110 AML 5L 64.7 (21.7) 0.83 (0.2) 0.89 (0.76-0.98) EQ-5D-5L index, LSS and EQ-VAS were significantly associated with overall survival and the likelihood to respond to azacitidine in univariate analysis; EQ-5D-5L index was significantly associated with overall survival, time with clinical benefit and time to next treatment in multivariate-adjusted analyses. LSS was significantly associated with the likelihood to respond to azacitidine in multivariate analysis. NR indicates not reported. cancers-15-01388-t002_Table 2 Table 2 Prevalence of problems in patients with myeloid neoplasias (assessed by EQ-5D-5L at azacitidine treatment start (n = 205) 1) by disease-related and patient-related parameters. Mobility Problem 2 Selfcare Problem 2 Usual Activities Problem 2 Pain/Discomfort Problem 2 Anxiety/Depression Problem 2 Level Sum Score 3 Index Value 4 EQ-VAS n/n (%) p 5 n/n (%) p 5 n/n (%) p 5 n/n (%) p 5 n/n (%) p 5 n Mean (SD) p 5 n Mean (SD) p 6 n Mean (SD) p 6 Total cohort 1st available EQ-5D 136/272 (50.0) NA 68/72 (25.0) NA 150/272 (55.1) NA 138/272 (50.7) NA 125/272 (46.0) NA 266 9.1 (3.9) NA 266 0.807 (0.232) NA 263 63.9 (21.6) NA EQ-5D in cycle 1 or 2 104/205 (50.7) NA 46/205 (22.4) NA 120/205 (58.5) NA 102/205 (49.8) NA 100/205 (48.8) NA 200 9.2 (3.9) NA 198 0.810 (0.229) NA 200 64.5 (21.4) NA Disease-related parameters 1 Azacitidine >=2nd line: No Yes 75/145 (52.1) 29/59 (49.2) 0.7045 35/143 (24.5) 11/59 (18.6) 0.3688 86/141 (61.0) 34/59 (557.6) 0.6577 76/143 (53.1) 26/59 (44.1) 0.2406 72/143 (50.3) 28/59 (47.5) 0.7085 141 59 9.3 (4.0) 8.7 (3.5) 0.3288 141 59 0.800 (0.243) 0.831 (0.192) 0.4282 141 57 63.3 (22.0) 67.5 (19.7) 0.2136 Diagnosis: MDS or CMML AML 59/112 (52.7) 45/91 (49.5) 0.6472 28/111 (25.2) 18/91 (19.8) 0.3585 66/109 (60.6) 54/91 (59.3) 0.8619 66/111 (59.5) 36/91 (39.6) 0.0049 53/111 (47.4) 47/91 (51.6) 0.5812 109 91 9.4 (4.0) 8.8 (3.6) 0.2921 109 91 0.788 (0.256) 0.835 (0.192) 0.2160 110 88 64.4 (21.2) 64.7 (21.7) 0.9440 Treatment-related disease: No Yes 89/175 (50.9) 12/24 (50.0) 0.9372 39/174 (22.4) 6/24 (25.0) 0.7769 102/172 (59.3) 14/24 (58.3) 0.9279 84/174 (48.3) 15/24 (62.5) 0.1914 79/174 (45.4) 17/24 (70.8) 0.0194 172 24 9.1 (3.9) 9.5 (3.7) 0.4741 172 24 0.810 (0.238) 0.809 (0.182) 0.4869 170 24 64.7 (21.8) 64.4 (19.9) 0.7998 IPSS: Low or intermediate-1 Intermediate-2 or high 39/72 (54.2) 62/125 (49.6) 0.5369 17/71 (23.9) 27/125 (21.6) 0.7055 40/69 (58.0) 75/125 (60.0) 0.7830 39/71 (54.9) 58/125 (46.4) 0.2510 32/71 (45.1) 64/125 (51.2) 0.4093 69 125 9.3 (4.2) 8.9 (3.5) 0.7663 69 125 0.789 (0.274) 0.836 (0.169) 0.7950 70 122 65.6 (20.7) 64.6 (21.8) 0.6783 R-IPSS: Very low or low Intermediate, poor, very poor 11/26 (42.3) 90/169 (53.3) 0.2984 6/26 (23.1) 39/168 (23.2) 0.9877 13/26 (50.0) 102/166 (61.4) 0.2682 15/26 (57.7) 82/168 (48.8) 0.3992 12/26 (46.2) 84/168 (50.0) 0.7151 26 166 9.3 (5.0) 9.1 (3.6) 0.5927 26 166 0.758 (0.369) 0.821 (0.188) 0.7551 25 165 64.6 (21.8) 64.7 (21.1) 0.9609 IPSS cytogenetic risk: good Intermediate or poor 60/125 (48.0) 34/56 (60.7) 0.1135 29/124 (23.4) 14/56 (25.0) 0.8143 67/123 (54.5) 35/55 (63.6) 0.2534 62/124 (50.0) 29/56 (51.8) 0.8244 64/124 (50.0) 27/56 (48.2) 0.6729 123 55 9.0 (3.9) 9.5 (3.8) 0.2706 123 55 0.814 (0.228) 0.806 (0.216) 0.3255 122 54 65.7 (21.4) 64.1 (21.1) 0.6006 Peripheral blood blasts: <10% >=10% 78/156 (50.0) 26/47 (55.3) 0.5225 34/155 (21.9) 12/47 (25.5) 0.6065 94/153 (61.4) 26/47 (55.3) 0.4539 83/155 (53.5) 19/47 (40.4) 0.1150 77/155 (49.7) 23/47 (48.9) 0.9291 153 47 9.3 (4.0) 8.8 (3.3) 0.7245 153 47 0.798 (0.246) 0.847 (0.162) 0.4270 153 45 64.8 (20.9) 63.8 (23.1) 0.7879 Monocytes: <10% >=10% 56/121 (46.3) 44/75 (58.7) 0.0918 23/121 (19.0) 22/74 (29.7) 0.0846 61/119 (51.3) 52/74 (70.3) 0.0091 60/121 (49.6) 41/74 (55.4) 0.4301 52/121 (43.0) 43/74 (58.1) 0.0402 119 74 8.5 (3.5) 10.1 (4.3) 0.0053 119 74 0.850 (0.193) 0.752 (0.267) 0.0052 118 73 67.7 (19.8) 61.5 (22.5) 0.0626 Haemoglobin: <10.0 g/dL >=10.0 g/dL 81/142 (57.0) 23/61 (37.7) 0.0115 37/141 (26.2) 9/61 (14.8) 0.0739 89/139 (64.0) 31/61 (50.8) 0.0792 73/141 (51.8) 29/61 (47.5) 0.5807 71/141 (50.4) 29/61 (47.5) 0.7135 139 61 9.5 (4.0) 8.3 (3.5) 0.0295 139 61 0.790 (0.242) 0.855 (0.191) 0.0429 137 61 62.8 (21.0) 68.5 (21.8) 0.0545 Red blood cell transfusions: <=3 >3 62/138 (44.9) 22/26 (84.6) 0.0002 31/137 (22.6) 8/26 (30.8) 0.3724 75/135 (55.6) 20/26 (76.9) 0.0425 73/137 (53.3) 16/26 (61.5) 0.4384 64/137 (46.7) 15/26 (57.7) 0.3045 135 26 9.0 (4.0) 9.9 (3.2) 0.0723 135 26 0.809 (0.247) 0.864 (0.163) 0.1412 134 25 65.6 (21.7) 55.2 (17.6) 0.0147 Platelet count: <100 G/L >=100 G/L 36/65 (55.4) 68/138 (49.3) 0.4165 17/65 (26.2) 29/137 (21.2) 0.4299 39/63 (61.9) 81/137 (59.1) 0.7092 34/65 (52.3) 68/137 (49.6) 0.7226 30/65 (46.2) 70/137 (51.1) 0.5117 63 137 9.4 (4.0) 9.0 (3.8) 0.4665 61 137 0.797 (0.254) 0.815 (0.218) 0.4980 64 134 65.8 (19.9) 63.9 (22.1) 0.6100 Patient-related parameters 1 Sex male: No Yes 45/81 (55.6) 59/122 (48.4) 0.3152 21/81 (25.9) 25/121 (20.7) 0.3819 49/79 (62.0) 71/121 (58.7) 0.6366 44/81 (54.3) 58/121 (47.9) 0.3735 47/81 (58.0) 53/121 (43.8) 0.0475 79 121 9.6 (4.0) 8.9 (3.7) 0.1644 79 121 0.786 (0.261) 0.825 (0.206) 0.2445 77 121 66.3 (21.9) 63.4 (21.1) 0.2408 Age >=75 yrs: No Yes 47/105 (44.8) 57/98 (58.2) 0.0563 19/104 (18.3) 27/89 (27.6) 0.1159 64/103 (62.1) 56/97 (57.7) 0.5252 44/104 (42.3) 59/98 (59.2) 0.0165 51/104 (49.0) 49/98 (50.0) 0.8913 103 97 8.7 (3.4) 9.6 (4.2) 0.2478 103 97 0.832 (0.191) 0.785 (0.263) 0.2429 103 95 66.9 (21.0) 60.0 (21.6) 0.1083 ECOG-PS: 0-1 >=2 74/163 (45.4) 30/40 (75.0) 0.0008 26/162 (16.0) 20/40 (50.0) <0.0001 87/160 (54.4) 33/40 (82.5) 0.0012 79/162 (48.8) 23/40 (57.5) 0.3224 70/162 (43.2) 30/40 (75.0) 0.0003 160 40 8.4 (3.4) 12.0 (4.3) <0.0001 160 40 0.847 (0.185) 0.659 (0.315) <0.0001 159 39 66.5 (20.8) 56.6 (22.3) 0.0092 HCT-CI: Low risk Intermediate risk High risk 31/77 (40.3) 33/65 (50.8) 40/61 (65.6) 0.0127 13/77 (16.9) 12/65 (18.5) 21/60 (35.0) 0.0259 40/75 (53.3) 36/65 (55.4) 44/60 (73.3) 0.0406 38/77 (49.4) 26/65 (40.0) 38/60 (63.3) 0.0324 36/77 (46.8) 30/65 (46.2) 34/60 (56.7) 0.4155 75 65 60 8.3 (3.3) 8.9 (3.7) 10.4 (4.4) 0.0133 75 65 60 0.849 (0.186) 0.822 (0.224) 0.748 (0.271) 0.0189 75 64 59 67.8 (20.1) 65.4 (21.0) 59.5 (22.7) 0.0750 No. of comorbidities: 0-1 >=2 52/116 (44.8) 52/87 (59.8) 0.0350 23/116 (19.8) 23/86 (26.7) 0.2464 66/114 (57.9) 54/86 (62.8) 0.4841 57/116 (49.1) 45/86 (52.3) 0.6541 52/116 (44.8) 48/86 (55.8) 0.1225 114 86 8.7 (3.5) 9.8 (4.3) 0.0689 114 86 0.839 (0.183) 0.770 (0.276) 0.0703 113 85 66.8 (21.0) 61.6 (21.6) 0.0829 IPSS, International Prognostic Scoring System; IPSS-LR, IPSS lower-risk; IPSS-HR, IPSS higher-risk; R-IPSS, revised IPSS; ECOG-PS, Eastern Cooperative Oncology Group Performance Score; HCT-CI, Haematopoietic Stem Cell Comorbidity Index; MRC, Medical research Council. 1 EQ-5D in cycle 1 or 2 with a non-missing value for the respective parameter (hence patient numbers may vary slightly for each parameter analysed). 2 Problems were defined as answer options 2, 3, 4 or 5 for EQ-5D-5L and answer options 2 or 3 for EQ-5D-3L. 3 Represents the numerical sum of all EQ-5D responses. 4 The EQ-5D-5L index is measured on a scale from 0 to 1, whereby 0 indicates death and 1 perfect health. 5 Baseline parameters and EQ-5D-5L results were compared using the Chi-squared test (based on non-missing observations) for EQ-5D-5L problems (=2,3,4,5) vs. EQ-5D-5L no-problems (=1). 6 Baseline parameters and EQ-5D-5L results were compared using the Wilcoxon rank-sum test (also called Mann-Whitney U-test or Mann-Whitney-Wilcoxon Test) for Level Sum Score, EQ-5D-5L index value and EQ-VAS. Font color is red for all significant p-values <0.05. cancers-15-01388-t003_Table 3 Table 3 Comparison of HRQoL (as assessed by first available EQ-5D-5L) 1 between myeloid patients (n = 269) and a German population norm without myeloid neoplasias (n = 5001) 2 matched by age group, sex or number of comorbidities. Mobility Problem 3 Selfcare Problem 3 Usual Activities Problem 3 Pain/Discomfort Problem 3 Anxiety/Depression Problem 3 Index Value EQ-VAS n/n (%) p 4 n/n (%) p 4 n/n (%) p 4 n/n (%) p 4 n/n (%) p 4 n Mean (SD) p 5 n Mean (SD) p 5 Total cohort Austrian Registry German Norm 136/269 (50.6) 1772/5001 (35.4) <0.0001 68/268 (25.4) 360/5001 (7.2) <0.0001 150/266 (56.4) 1417/5001 (28.3) <0.0001 138/268 (51.5) 2847/5001 (56.9) 0.0802 125/269 (46.5) 1256/5001 (25.1) <0.0001 266 5001 0.81 (0.23) 0.88 (0.18) <0.0001 260 4997 63.9 (21.6) 71.6 (21.4) <0.0001 >=75 years Austrian Registry German Norm 74/130 (56.9) 399/593 (67.3) 0.0245 39/130 (30.0) 111/593 (18.7) 0.0041 71/129 (55.0) 281/593 (47.4) 0.1151 75/130 (57.7) 418/593 (70.5) 0.0046 59/131 (45.0) 160/593 (27.0) <0.0001 129 593 0.79 (0.25) 0.80 (0.28) 0.7547 127 590 61.7 (22.4) 60.9 (26.2) 0.7662 65 < 75 years Austrian Registry German Norm 50/105 (47.6) 324/654 (46.1) 0.7146 22/105 (21.0) 69/654 (10.6) 0.0023 60/104 (57.7) 198/654 (30.3) <0.0001 49/105 (46.7) 411/654 (62.8) 0.0016 49/105 (46.7) 158/654 (24.2) <0.0001 104 654 0.84 (0.19) 0.85 (0.240 0.5650 102 654 66.8 (19.3) 66.1 (25.5) 0.7777 <65 years Austrian Registry German Norm 12/34 (35.3) 1049/3754 (27.9) 0.3420 7/33 (21.2) 180/3754 (4.8) <0.0001 19/33 (57.6) 938/3754 (25.0) <0.0001 14/33 (42.4) 2017/3754 (53.7) 0.1948 17/33 (51.5) 938/3754 (25.0) 0.0005 33 3754 0.77 (0.26) 0.90 (0.15) <0.0001 34 3753 63.5 (24.0) 74.2 (19.1) 0.0011 Females Austrian Registry German Norm 59/103 (57.3) 980/2584 (37.9) <0.0001 30/103 (29.1) 203/2584 (7.9) <0.0001 63/101 (62.4) 789/2584 (30.5) <0.0001 60/103 (58.3) 1497/2584 (57.9) 0.9487 56/103 (54.5) 734/2584 (28.4) <0.0001 101 2584 0.78 (0.26) 0.86 (0.20) <0.0001 98 2581 64.6 (21.8) 71.1 (22.2) 0.0048 Males Austrian Registry German Norm 77/166 (46.4) 791/2417 (32.7) 0.0003 38/165 (23.0) 157/2417 (6.5) <0.0001 86/165 (52.7) 628/2417 (26.0) <0.0001 78/165 (47.3) 1350/2417 (55.9) 0.0319 69/166 (41.6) 522/2417 (21.6) <0.0001 165 2417 0.83 (0.21) 0.90 (0.16) <0.0001 165 2416 63.5 (21.5) 72.1 (20.5) <0.0001 One comorbidity Austrian Registry German Norm 24/66 (36.4) 455/1432 (31.8) 0.4344 9/66 (13.6) 74/1432 (5.2) 0.0033 31/64 (48.4) 361/1433 (25.2) <0.0001 32/66 (48.5) 813/1432 (56.8) 0.1843 31/67 (46.3) 317/1432 (22.1) <0.0001 64 1432 0.87 (0.17) 0.90 (0.15) 0.0861 64 1432 66.3 (22.8) 73.0 (19.2) 0.0067 Two comorbidities Austrian Registry German Norm 42/85 (50.6) 378/820 (46.1) 0.4295 23/85 (27.1) 74/821 (9.0) <0.0001 49/85 (57.7) 294/821 (35.8) <0.0001 43/85 (50.6) 570/821 (69.4) 0.0004 31/85 (37.7) 245/821 (29.8) 0.1370 85 821 0.82 (0.21) 0.85 (0.18) 0.1154 83 821 65.7 (20.6) 65.1 (21.9) 0.7841 >=Three comorbidities Austrian Registry German Norm 69/118 (58.5) 627/870 (72.1) 0.0024 36/117 (30.8) 179/871 (20.6) 0.0119 70/117 (59.8) 536/870 (61.6) 0.7104 63/117 (53.9) 748/871 (85.9) <0.0001 62/117 (53.0) 374/871 (42.9) 0.0398 117 871 0.77 (0.27) 0.72 (0.28) 0.0944 116 871 61.3 (21.4) 55.2 (24.0) 0.0093 EQ-VAS indicates EuroQol Visual Analogue Scale. 1 First available EQ-5D with a non-missing value for the respective parameter (hence patient numbers may vary slightly for each parameter analysed). 2 Published and unpublished data provided by Grochtdreis et al. . 3 Problems were defined as answer options 2, 3, 4 or 5 for EQ-5D-5L and answer options 2 or 3 for EQ-5D-3L. 4 The prevalence of EQ-5D-5L problems (=2,3,4,5) vs. EQ-5D-5L no-problems (=1) were compared using the Chi-squared test. 5 EQ-5D-Indices and EQ-VAS were compared between the Austrian Registry of Hypomethylating Agents and the German Norm cohorts using Student's T-test. Font color is red for all significant p-values <0.05. cancers-15-01388-t004_Table 4 Table 4 Prognostic value of the IPSS and R-IPSS with or without baseline Level Sum Score (LSS), EQ Visual Analogue Scale (VAS) or EQ-5D-5l index value, by time-to-event endpoint (patients with EQ-5D-5L responses available at azacitidine treatment start (n = 205)). (R)-IPSS (R)-IPSS + LSS (R)-IPSS + EQ-VAS (R)-IPSS + Index Months [95% CI] 1 LHR p 6 LHR p 6 LHR p 6 LHR p 6 Overall survival IPSS: Lower-risk 2 Higher-risk 3 21.0 [14.6-30.3] 12.8 [10.2-16.9] 7.3195 0.0068 10.6911 0.0048 11.5552 0.0031 13.0219 0.0015 R-IPSS: Lower-risk 4 Higher-risk 5 30.3 [11.2-39.3] 14.6 [11.9-17.8] 5.3691 0.0205 9.0542 0.0108 10.2840 0.0058 13.4753 0.0012 Time with clinical benefit IPSS: Lower-risk 2 Higher-risk 3 8.9 [5.6-13.1] 7.9 [5.2-9.6] 1.0693 0.3011 3.6196 0.1637 1.9171 0.3835 3.6196 0.1637 R-IPSS: Lower-risk 4 Higher-risk 5 7.8 [3.4-14.9] 8.0 [6.4-9.6] 0.0757 0.7832 4.0208 0.1339 1.5603 0.4583 4.0208 0.1339 Time to next treatment IPSS: Lower-risk 2 Higher-risk 3 14.6 [9.5-19.3] 11.3 [8.9-12.6] 3.5998 0.0578 5.7236 0.0572 4.7933 0.0910 6.3834 0.0411 R-IPSS: Lower-risk 4 Higher-risk 5 17.6 [6.9-37.7] 10.8 [9.3-12.6] 4.3114 0.0379 7.7372 0.0209 6.8408 0.0327 6.5489 0.0378 IPSS, International Prognostic Scoring System; R-IPSS, revised IPSS; LHR, likelihood ratio test. 1 Estimated via univariate Cox proportional hazards regression. 2 IPSS lower-risk comprises IPSS low and intermediate-1 risk categories. 3 IPSS higher-risk comprises IPSS intermediate-2 and high risk categories. 4 R-IPSS lower-risk comprises R-IPSS very low and low risk categories. 5 R-IPSS higher-risk comprises R-IPSS intermediate, high and very high risk categories. 6 Estimated via multivariate Cox proportional hazards regression. cancers-15-01388-t005_Table 5 Table 5 Time-to-endpoint results for patients with EQ-5D-5L results available at azacitidine treatment start (n = 205). Univariate (n = 205) Multivariate 4 (n = 205) Months [95% CI] p HR [95% CI] Months [95% CI] p HR [95% CI] Overall Survival Level Sum Score: <median 1 >=median 19.3 [14.6-21.5] 12.4 [8.7-15.0] 0.0407 1.408 [1.013-1.956] 16.9 [12.9-37.4] 14.2 [11.7-17.8] 0.2286 1.234 [0.876-1.737] EQ-VAS (health today): >=median 2 <median 17.9 [13.8-21.3] 12.8 [8.7-16.8] 0.0141 1.511 [1.084-2.106] 16.9 [12.9-30.6] 14.0 [11.4-24.7] 0.2293 1.242 [0.872-1.769] EQ-5D-5L index: >=median 3 <median 18.5 [15.0-21.0] 11.9 [8.5-14.9] 0.0093 1.536 [1.109-2.127] 17.9 [14.0-21.0] 12.9 [10.3-16.8] 0.0143 1.523 [1.088-2.131] Time with Clinical Benefit Level Sum Score: <median 1 >=median 10.2 [6.6-13.2] 6.1 [4.3-8.2] 0.0573 1.340 [0.989-1.815] 8.7 [6.5-11.8] 6.8 [5.2-8.8] 0.2174 1.221 [0.889-1.677] EQ-VAS (health today): >=median 2 <median 9.6 [6.6-12.1] 6.7 [4.6-8.5] 0.1841 1.227 [0.906-1.662] 8.4 [6.4-11.4] 7.7 [5.6-9.6] 0.5233 1.111 [0.998-1.012] EQ-5D-5L index: >=median 3 <median 10.2 [7.2-12.8] 6.1 [4.0-8.2] 0.0134 1.456 [1.078-1.966] 9.6 [6.8-12.1] 6.6 [4.9-8.5] 0.0258 1.425 [1.044-1.945] Time to Next Treatment Level Sum Score: <median 1 >=median 13.5 [9.8-17.6] 9.4 [7.6-11.9] 0.0633 1.347 [0.982-1.846] 12.6 [10.2-16.5] 10.8 [8.9-12.6] 0.1144 1.302 [0.938-1.806] EQ-VAS (health today): >=median 2 <median 12.6 [9.4-16.8] 11.1 [8.5-12.8] 0.1034 1.305 [0.946-1.801] 11.9 [9.7-14.6] 11.1 [9.0-20.2] 0.4197 1.150 [0.819-1.614] EQ-5D-5L index: >=median 3 <median 13.1 [10.8-17.4] 9.2 [6.7-11.9] 0.0414 1.383 [1.011-1.890] 12.8 [10.5-20.2] 9.8 [8.5-11.9] 0.0332 1.420 [1.028-1.962] EQ-VAS indicates EuroQol Visual Analogue Scale. 1 Median for Level Sum Score: 8.0. 2 Median for EQ-VAS: 65. 3 Median for EQ-5D-5L index: 0.8845. 4 Adjusted for the covariates remaining in the final Cox model: ECOG-PS, number of comorbidities, platelet count/transfusion dependence, peripheral blood blasts, azacitidine treatment line and azacitidine dose in cycle one. cancers-15-01388-t006_Table 6 Table 6 Prognostic value of baseline Level Sum Score, EQ visual analogue scale (VAS) or EQ-5D-5L index value for the likelihood to respond to azacitidine (patients with EQ-5D-5L responses available at azacitidine treatment start (n = 205)). Univariate p Multivariate 4 p Multivariate 4 OR [95% CI] Level Sum Score: >= vs. < median 1 0.0009 0.0160 0.451 [0.235-0.852] EQ-VAS: < vs. >= median 2 0.0237 0.1065 0.590 [0.321-1.116] EQ-5D-5L index: < vs. >= median 3 0.0110 0.0627 0.522 [0.296-1.032] 1 Median for Level Sum Score: 8.0. 2 Median for EQ-VAS: 65. 3 Median for EQ-5D-5L index: 0.8845. 4 Adjusted for the covariates remaining in the final Cox model: ECOG-PS, number of comorbidities, platelet count/transfusion dependence, peripheral blood blasts, azacitidine treatment line and azacitidine dose in cycle one. cancers-15-01388-t007_Table 7 Table 7 Multivariate-adjusted 1 longitudinal analyses of EQ-5D results and dichotomised parameters per azacitidine treatment cycle using mixed-effects linear models. Mobility Selfcare Usual Activities Pain/Discomfort Anxiety/Depression Level Sum Score 2 EQ-VAS EQ-5D-5L Index Differential blood count n 3 p n p n p n p n p n p n p n p Peripheral blood blasts< vs. >=5% 1425 0.9897 1417 0.2548 1417 0.1447 1421 0.9703 1416 0.8775 1395 0.2930 1365 0.0996 1395 0.3916 White blood cell count< vs. >=30.0 G/L 1429 0.1502 1421 0.5278 1421 0.2869 1425 0.0801 1420 0.2674 1399 0.1371 1368 0.7712 1399 0.1272 Absolute neutrophil count< vs. >=1.0 G/L 1415 0.2206 1407 0.1586 1407 0.8529 1411 0.6784 1406 0.6362 1385 0.5171 1355 0.1329 1385 0.9389 Monocytes< vs. >=1.0 G/L 1417 0.2559 1409 0.9738 1409 0.4770 1413 0.5203 1408 0.8287 1387 0.6366 1357 0.2476 1387 0.9439 Lymphocytes< vs. >=1.0 G/L 1402 0.4021 1394 0.5043 1394 0.6879 1398 0.5349 1393 0.0941 1372 0.8871 1343 0.5429 1372 0.6557 Haemoglobin< vs. >=10.0 g/dL 1429 <0.0001 1421 0.0227 1421 <0.0001 1425 0.9289 1420 0.7871 1399 <0.0001 1368 <0.0001 1399 0.0110 Red blood cell transfusions: Yes vs. No 1429 0.0003 1421 0.7072 1421 <0.0001 1425 0.1935 1420 0.6996 1399 0.0003 1368 <0.0001 1399 0.0161 Platelet count< vs. >=50 G/L 1429 0.0122 1421 0.0647 1421 0.0248 1425 0.3142 1420 0.9574 1399 0.0212 1368 0.0006 1399 0.0156 Platelet transfusions: Yes vs. No 1429 0.0257 1421 0.0047 1421 0.0044 1425 0.0002 1420 0.2067 1399 0.0002 1368 <0.0001 1399 <0.0001 Comorbidity/toxicity Ferritin< vs. >=1000 mg/L 723 0.0006 720 0.0598 720 0.0020 722 0.0785 718 0.5635 709 0.0024 703 0.0053 709 0.0163 Creatinine< vs. >=1.5 mg/dL 1417 0.7976 1409 0.8133 1409 0.6386 1413 0.7286 1408 0.7550 1387 0.9162 1356 0.5338 1387 0.8874 Lactate dehydrogenase, U/L 1399 0.4066 1391 0.1095 1392 0.7977 1395 0.0642 1390 0.9778 1370 0.3834 1337 0.3343 1370 0.3673 Glutamate oxaloacetate transaminase, U/L 1406 0.7039 1398 0.8181 1399 0.5276 1402 0.2078 1397 0.4316 1377 0.6822 1345 0.5734 1377 0.9119 Glutamate pyruvate transaminase, U/L 1348 0.0867 1340 0.9662 1340 0.6501 1344 0.4822 1339 0.8201 1318 0.4770 1288 0.7212 1318 0.7369 Bilirubin< vs. >=1.2 mg/dL 1407 0.0149 1399 0.0066 1399 0.0451 1403 0.9600 1398 0.4338 1377 0.0158 1346 0.0494 1377 0.0170 Albumin< vs. >=3.4 mg/dL 583 0.0052 579 <0.0001 578 0.0412 580 0.0942 576 0.0454 567 0.0034 565 0.2309 567 0.0355 Cholinesterase< vs. >=3.7 U/L 584 0.0108 581 0.0437 580 0.6728 582 0.1706 580 0.5751 567 0.0992 567 0.0216 567 0.7691 Adverse events 4 Grade 0-2 vs. 3-4 1429 0.0208 1421 0.0616 1421 0.0229 1425 0.0028 1420 0.0179 1399 0.0005 1368 0.0074 1399 <0.0001 Azacitidine dose/regimen Azacitidine< vs. >=7 days 1429 0.1648 1421 0.0129 1421 0.4369 1425 0.0964 1420 0.0158 1399 0.0096 1368 0.4788 1399 0.0288 Azacitidine< vs. >=75 mg/m2/day 1426 0.1485 1418 0.1155 1418 0.0249 1422 0.0168 1417 0.0001 1396 0.0003 1365 0.0040 1396 0.0013 Haematologic improvement (HI) HI-any 5: Yes vs. No 1275 0.0004 1268 0.0130 1270 0.0003 1272 0.6473 1266 0.1747 1248 0.0005 1221 <0.0001 1248 0.0048 HI-Erythrocytes: Yes vs. No 1296 0.0008 1289 0.0163 1291 <0.0001 1293 0.2981 1287 0.7419 1269 0.0084 1239 <0.0001 1269 0.1645 HI-Platelets: Yes vs. No 1317 0.0025 1310 0.0011 1311 0.0008 1315 0.0951 1310 0.2232 1288 0.0005 1262 <0.0001 1288 0.0003 HI-Neutrophils: Yes vs. No 1362 0.4299 1355 0.7016 1354 0.2083 1358 0.1326 1353 0.4239 1333 0.2837 1303 0.0012 1333 0.6162 1 Adjusted for the covariates remaining in the final Cox model: ECOG-PS, number of comorbidities, platelet count/transfusion dependence, peripheral blood blasts, azacitidine treatment line and azacitidine dose in cycle one. 2 Represents the numerical sum of all EQ-5D-5L responses. 3 Number of parameter/EQ-5D-5L response pairs. 4 Assessed according to CTCAEv4.0. 5 Includes HI-Neutrophils and/or HI-Erythrocytes and/or HI-Platelets. 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