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10.3390/ani11040955

PMC8066815

Herbal products as feed supplements show beneficial effects on the productive performance and health of non-ruminants, but there is limited information about the effects in ruminants. The objective of this study was to evaluate the effects of a polyherbal mixture on growth performance, carcass characteristics, meat quality, and concentration of blood metabolites in lambs during the fattening period. Polyherbal mixture supplementation improved dry matter intake and increased the live weight of lambs without affecting carcass characteristics or meat quality. Polyherbal mixture supplementation was associated with lower blood creatinine concentration suggesting beneficial effects on the renal health condition of lambs. The results suggest that the use of herbal mixtures as additives in diets of finishing lambs can improve productivity without affecting carcass characteristics and meat quality.

The objective of this study was to determine the effects of the supplementation of a polyherbal mixture (HM) on the productive performance, carcass characteristics, meat quality, and the profile of blood metabolites of lambs fed a high-concentrate diet. Thirty-six male Pelibuey lambs (25.21 ± 0.96 kg BW) were housed in individual pens during a 56-day feeding period and were randomly assigned to four treatments: (1) Control (CON): Basal diet without HM; (2) HM1: CON + 1 g of HM kg−1 dry matter (DM); (3) HM2: CON + 2 g of HM kg−1 DM; and (4) HM3: CON + 3 g of HM kg−1 DM. Data were analyzed using the GLM (General Linear Model) procedure of statistical analysis system (SAS), and linear and quadratic effects were tested to evaluate the effects of the HM level. A quadratic increase was observed in the dry matter intake and in daily weight gain (p < 0.05) of lambs fed with HM2 and HM1, respectively. However, final body weight, body condition, carcass characteristics, and meat quality were similar among treatments (p > 0.05). It was observed a linear increase (p < 0.05) in the mean corpuscular hemoglobin concentration. Lymphocytes in blood from lambs supplemented with the HM1 diet increased and segmented neutrophils decreased compared to lambs receiving the CON treatment (p < 0.05). The concentration of uric acid in the blood had a linear increase (p < 0.05) and the serum creatinine level decreased (p < 0.05) as the HM dietary dose increased. In conclusion, dietary inclusion of 2 and 1 g of HM kg−1 of DM improves feed consumption and daily weight gain, respectively, without affecting carcass characteristics, meat quality, and health status on finishing lambs.

1. IntroductionGrowth promoters have been widely used to increase the productivity of ruminants and non-ruminants. However, in several countries, the use of these products has been prohibited, which has led the industry and researchers to search for dietary supplementation with herbal additives as an alternative [1]. Some herbal products that contain phenols or flavonoids have shown promising effects when used as food additives for lambs and cattle [2,3]. Animunin Powder® is a polyherbal mixture (HM) composed of parts from various plants such as Solanum xanthocarpum and Hedychium spicatum, which contain a high concentration of phenolic and flavonoid compounds [4,5].Previous studies have shown that herbal products (7.8 and 16% kg−1 of DM) containing flavonoids stimulate muscle protein synthesis [6] and increase serum levels of the growth hormone in sheep [7]. In addition, these compounds have been successfully used to increase the duodenal flow of amino acids and microbial protein [8] and to reduce methane production in cattle [9]. Other phenol-containing products (10 g kg−1 of DM) have shown a positive impact on immune response, rate of rumen fermentation, and rumen microbial activity [10]. Diverse sources of phenolic compounds (3.2 mg d−1) have also been used to improve the quality of meat in lambs and goat kids [6,11]. The inclusion of phenols and flavonoids in the diet (3.4 and 10 g kg−1 of DM) could increase weight gain in lambs through changes in the rumen microbiome [12]. Nevertheless, the effects of both secondary metabolites on the animal response appear to be dependent on the dose and the source from which they are derived. [2,6,7,13]. Due to the beneficial effects of herbal products and their secondary metabolites, it has been hypothesized that supplementation with HM as a source of phenols and flavonoids can contribute to improving the productivity of the lambs during the final fattening period without affecting the quality of the meat or the health of the animals. However, there is little information on the effects of herbal products containing phenols and flavonoids on the productivity of fattening lambs. The objective of this study was to evaluate the effect of increased doses of HM containing phenols and flavonoids on the productive performance, carcass characteristics, meat quality, and concentration of blood metabolites of lambs fed high-concentrate diets.2. Materials and Methods2.1. LocationThe experiment was conducted at the Teaching and Research Unit of Small Ruminants located at the Experimental Farm of the Universidad Autónoma Chapingo, Mexico, located at the 19°22′ north latitude 98°35′ west longitude, with an altitude of 2250 m. The climate is temperate subhumid with rain during the summer and dry during the winter, with average annual temperature and precipitation of 15.2 °C and 665 mm, respectively [14]. The care and handling procedures for the lambs were carried out following the guidelines of the Official Mexican Standard (NOM-062-ZOO-1995).2.2. Diet Composition and ManagementThe lambs were fed a finishing diet comprised of 19.4% oat straw, 24.1% ground sorghum, 8.1% soybean meal, 30.3% ground corn, 7.1% wheat bran, 7.4% corn gluten, 2.3% bypass fat, 0.3% calcium carbonate, 0.5% salt, 0.5% vitamin and mineral supplement (DM basis). The nutrient composition of the basal diet was 15.69% crude protein, 2.64% ether extract, 26.04% neutral detergent fiber, 13.75% acid detergent fiber, 5.52% ash, and 2.8 Mcal of metabolizable energy (DM basis). HM was fed to the lambs through diets formulated to have weight gains of 300 g d−1 [15]. The HM was mixed with the few hundreds of corn grams then they were mixed the total diets.The feed was provided at 08:00 and 16:00 h, and the drinking water was supplied ad libitum. The lambs were weighed after 12 h post fasting on 2 consecutive days, at the beginning (day 0 and 1), and weighed at the end of the experimental period (day 55 and 56). The variables measured were: Initial body weight (IBW) measured after the adaptation period, final body weight (FBW), dry matter intake (DMI), and the daily weight gain (DWG) calculated as (FBW − IBW)/56. The body condition scoring (BCS) was determined on day 56 of the experiment using a 5 category scale (1 to 5), assigning BCS = 1 to an emaciated animal, BCS = 3 to an animal in average body condition, and BCS = 5 to an obese animal [16].2.3. Polyherbal Mixture CharacteristicsThe HM used was Animunin Powder® (Nuproxa S. de RL. de CV. Querétaro, México), which was a labeled commercial herbal formula that came from the same batch, composed of plant parts from Solanum xanthocarpum, Hedychium spicatum, Curcuma longa, Piper longum, and Ocimum sanctum. S. xanthocarpum, which contains flavonoids and phenols with antioxidant activity [4]; H. spicatum contains phenolic and flavonoid compounds with antimicrobial, anti-inflammatory, and antioxidant activities [5]; C. longa contains curcumin, a polyphenolic substance with antioxidant and anti-inflammatory effects [17]; P. longum contains phenols and flavonoids such as quercetin and naringenin [18]; and O. sanctum contains phenols with antifungal activity [19].2.4. Experimental DesignThirty-six male Pelibuey lambs (25.2 ± 0.9 kg BW, 4–5 months old) were randomly distributed to 4 treatments: (1) Basal diet without HM (CON); (2) HM1, CON + 1 g of HM kg−1 dry matter (DM); (3) HM2, CON + 2 g of HM kg−1 DM; and 4) HM3, CON + 3 g of HM kg−1 DM. The lambs were placed in individual pens (2.6 m × 0.8 m) equipped with individual feeders and automatic drinkers. Prior to the start of the experimental phase, the lambs were dewormed through an oral administration of Koptisin ovine® (Chinoin Labs, México City, México, 10 mg kg−1 BW), and vaccinated against Pasteurella and Clostridium (Bobact® 8 MSD-Merck, Kenilworth, NJ, USA, 2.5 mL lamb−1). The lambs had an adaptation period to the basal diet of 14 days, and the experimental phase lasted 56 days. Figure 1 shows the experimental procedure.2.5. Sampling and Analyses of FeedsSamples of feed provided and rejected were collected daily to determine the chemical composition. Prior to the analysis, the food samples were dried at 55° C in a forced-air oven and then ground in a Wiley mill (model 4, Arthur Thomas Co., Philadelphia, PA, USA). The variables determined were: Dry matter, ether extract, crude protein, and ash [20]. Neutral detergent fiber and acid detergent fiber were determined using the procedures described by Van Soest et al. [21].2.6. Carcass Characteristics and Meat QualityThe backfat thickness (BFT) and the longissimus muscle area (LMA) located between the 12th and 13th ribs of the lamb were measured on day 54 of the experiment using a Sonovet 600 (Medison, Inc., Cypress, CA, USA) with a 7.5 Mhz transducer [22]. After the last weighing (day 56 of the experiment), the lambs were fasted for 16 h before being slaughtered. The slaughter process was conducted in a commercial slaughterhouse in accordance with standard procedures of the Official Mexican Standard (NOM-033-SAG/ZOO-2014). Immediately after the slaughter, the hot carcass weight was registered (HCW), and subsequently, the carcasses were left to repose at 4 °C for 24 h to register the cold carcass weight (CCW). The hot carcass yield (HCY) and the cold carcass yield (CCY) were determined through: HCY = (HCW/FBW) × 100 and CCY = (CCW/FBW) × 100, as it was described by Zimerman et al. [23]. The body morphometry, external length of the carcass (ELC), internal length of the carcass (ILC), length of the leg (LL), perimeter of the leg (PL), and the carcass compactness index (CCI), were obtained based on the methodology described by Yañez et al. [24], where: CCI = CCW/ILC, in kg cm−1. In addition, the head, legs, skin, rumen (full and empty), liver, spleen, kidneys, heart, lungs, small intestine, and large intestine were each weighed separately.Muscle samples were collected from the cold carcass longissimus dorsi (approximately 400 g) and frozen at −20 °C for later analysis of meat quality. The meat color was measured in cuts of the muscle 24 h after slaughter using a spectrophotometer Minolta CM-2006d (modelo Konica, Minolta Holdings Inc., Osaka, Japan). Lightness (L*), redness (a*), and yellowness (b*) were evaluated as attributes of meat quality using the procedure proposed by Ripoll et al. [25]. With the values of a* and b*, the Croma (C*) and Hue (H*) indexes were calculated using the equations: Croma = (a*2 + b*2)0.5 and Hue = tan−1(b*/a*) × 57.29 both expressed in degrees [25]. The color coordinate values were calculated using the average of 3-color measurements for each sample. The pH of the meat was obtained following the procedure described by Negrete et al. [26]. This was measured 3 times in 3 g of muscle longissimus dorsi homogenized with deionized water (20 mL) with a blender Waring 51BL32 (model 700, Torrington, CT, USA), using a pH meter Hanna® (Model HI 98127, Waterproof Tester, EE. UU.). Previous to the proximate analysis of the meat, the samples were defrosted at 4 °C for 24 h. The subcutaneous fat and connective tissue were removed from the muscle with a scalpel, and the meat was ground and then homogenized for 5 min in a blender. Subsequently, 180 g of sample was taken in triplicate to determine protein, fat, moisture, and collagen (g kg−1) using a near-infrared spectrophotometer FOSS FoodScan™, as described by Anderson [27].2.7. Blood MetabolitesOn day 55 of the experimental phase, blood samples were taken from the jugular vein of the lambs, before the morning feeding (08:00 h) with the purpose of determining hematological and biochemical parameters. For determining the hematological parameters, the methodology described by Ayala et al. was followed [28]. For this, 5 mL of blood was extracted from each lamb in tubes BD Vacutainer® K2 EDTA with anticoagulant. An additional 5 mL of blood was collected in tubes BD Vacutainer® without anticoagulant to obtain blood serum. The samples were stored at 4 °C to measure the red blood cells, hematocrit, platelets, hemoglobin, and leukocyte, with an automated hematology analyzer (Sysmex, XS-1000i™, Kobe, Japan). The samples without anticoagulant were centrifuged (centrifuge Sigma, 2–16k, Osterode am Harz, Germany) at 3500 rpm for 20 min, and the serum obtained was stored in tubes Eppendorf at −20 °C. The concentration of blood metabolites: Cholesterol, glucose, total protein, albumin, globulin, creatinine, bilirubin, urea, uric acid, alkaline phosphatase, and lactate dehydrogenase, were determined using a blood auto-analyzer (EasyVet, KontroLab ES-300, Michoacán, México) and using Spinreact kits (Barcelona, Spain).2.8. Statistical AnalysisFirst, the normality test was performed on all variables using the SAS UNIVARIATE procedure [29]. Afterward, the data were analyzed according to the SAS GLM (General Linear Model) procedure [29]. Both linear and quadratic orthogonal polynomials were used to evaluate the effects of the level of the polyherbal mixture in the diet on each of the target variables. Initially, IBW was included as a co-variable to adjust the FBW, DWG, and DMI variables. However, this co-variable was removed from the model because it was not significant (p > 0.05). Significant differences were considered when p ≤ 0.05, and a trend was considered when p >0.05 but ≤0.10.3. Results3.1. Productive PerformanceFBW showed a tendency of quadratic increase (p = 0.07), and the lambs that consumed the HM1 diet presented a higher body weight at the end of the experimental trial compared to the lambs assigned to the other treatments (Table 1). DMI, DWG, and TWG also showed quadratic increases (p ≤ 0.05). The lambs assigned to the control group (CON) and to the HM3 treatment had lower averages (p = 0.05) compared to the lambs that consumed the HM1 and HM2 diet. On the other hand, BCS showed a tendency of quadratic increase (p = 0.10), and the lambs that were supplemented with HM1 performed higher than the lambs fed with the other diets. However, the feed conversion ratio was not affected by the level of HM added to the diet (p = 0.15).3.2. Carcass Characteristics and Non-Carcass ComponentsThe carcass characteristics and the body morphometry are shown in Table 2. The hot carcass weight, the cold carcass weight, the hot carcass yield, the cold carcass yield, losses due to carcass cooling, the backfat thickness, and the muscle area longissimus dorsi, were similar in the lambs of all treatments (p > 0.05). Similarly, the external length of the carcass and the internal length of the carcass, the perimeter and the length of the leg, and the carcass compactness index were also not affected by the addition of HM to the diet (p > 0.05).On the other hand, a tendency of linear increase (p = 0.08) was observed in the average weight of the liver, and a tendency of quadratic increase (p = 0.09) in the weight of the heart after slaughter, while for other internal organs (rumen, small intestine, large intestine, lung, kidney, spleen), skin, head, and feet, the weights were similar (p > 0.05) in the lambs across all treatments (Table 3).3.3. Meat QualityThere were no significant effects (p > 0.05) and no trends (p > 0.10) of the supplementation HM on the chemical composition (protein, fat, moisture, and collagen), pH, and color of the meat (L*, a*, b*, Chroma, and Hue°; Table 4).3.4. Hematological VariablesThe results of the blood metabolite analysis on lambs supplemented with HM are reported in Table 5. Most blood components were not affected by the level of HM in the diet. However, a positive linear increase (p = 0.01) was observed in the mean corpuscular hemoglobin concentration as the dose of HM increased. In contrast, a tendency of linear decrease in basophil concentration (p = 0.06) was observed. Lymphocytes in blood from lambs supplemented with the HM1 diet increased by 35% (p = 0.004), and segmented neutrophils decreased by 13% compared to lambs receiving the CON treatment (p = 0.001).3.5. Blood BiochemistryTable 6 shows the effects of increasing doses of HM on the serum biochemical parameters. Apart from urea, uric acid, and creatinine, all other metabolites were not affected by the level of HM in the diet (p >0.05). Urea concentration showed a tendency of quadratic increase (p = 0.09), while uric acid concentration increased linearly (p = 0.04) and creatinine concentration decreased linearly (p = 0.02) as the dose of MH in the diet increased.4. DiscussionIn all treatments, lambs showed DWG and FBW within the range reported in the literature for hair lambs of similar age fattened on high concentrate rations [30]. The lambs fed with the HM1 diet were 6% heavier at the end of fattening compared to lambs fed with the CON diet. In addition, DMI was 11% higher in lambs fed with HM2 compared to lambs assigned to CON. In a similar study, Razo et al. [31] examined the effects of HM (0, 1, 2, and 3 g kg−1 DM for 60 days) based on Withania somnifera, Ocimum tenuiflorum, Tinospora cordifolia, and Emblica officinalis containing polyphenols and flavonoids, on lambs fed high-concentrate diets. In their investigation, lambs supplemented with low doses of polyphenols and flavonoids had higher DWG, DMI, and FBW compared to the other treatments. However, as the dose of polyphenols and flavonoids increased, the growth performance worsened, similar to the results observed in our study. In another study by Lobo et al. [2], the effects of supplementing lambs were evaluated using leaf extracts from Illex paraguariensis (0, 1, 2, and 4% for 53 days) containing phenolic compounds. It was observed that DWG, DMI, and FBW increased at doses up to 2% of the extract in the diet, however, these decreased at doses of 4%. These results suggest that the effects of phenols and flavonoids can improve the productive performance at low doses, but at high doses, they could affect the growth rate, probably due to toxic effects. The higher DMI observed in lambs that consumed HM1 was potentially associated with higher digestibility, as previously reported by Ma et al. [32] in lambs supplemented with 0.25 mg d−1 of resveratrol (a natural polyphenol). The inclusion of flavonoids in the diet can reduce the concentration of propionate in the rumen by changes in the fermentation carried out by the rumen microbiome [3,10]. Additionally, supplementation with flavonoids has the potential to modify gene expression involved in the regulation of DMI [3]. Similar effects from the consumption of flavonoids seen in our study would partially explain a higher DMI in the lambs supplemented with 1 and 2 g of HM kg−1 DM.On the other hand, Du et al. [12] reported that supplementation with flavonoids extracted from the plant Allium mongolicum increased DWG by increasing the presence of bacteria (Tenericutes and Mollicutes) positively correlated with DWG in the rumen microbiome. It is suggested that the presence of flavonoids in the diet consumed by lambs stimulates the protein synthesis in muscle and inhibits proteolysis [6], also increasing the duodenal flux of amino acids and microbial protein [8]. In addition, a recent study in beef cattle showed that a 0.04% flavonoid dietary supplementation extracted from Citrus aurantium improves rumen health [3,8]. This results in higher absorption of propionate through the rumen wall—a higher metabolic availability of nitrogenous compounds—which, in our case, explains the higher DWG in lambs supplemented with HM.Results from other studies suggest that the addition of phenolic and flavonoid compounds in the diet acts by increasing the serum levels of growth hormone [7], reducing the production of enteric methane [9], increasing digestibility [31], and improving energy utilization [33]. These findings could explain the increases in DMI and DWG observed in lambs that consumed HM in the present studyFeed conversion ratio and BCS were similar for all treatments. These results are congruent with the findings of Lobo et al. [2], who used leaf extracts of Illex paraguariensis (0, 1, 2, and 4% for 53 days) containing phenolic compounds to feed lambs. In their study, CA and BCS were not affected by the supplementation of the extract in the diet. However, it was observed that TWG and carcass weight were higher in lambs supplemented with 2% of the extract. Additionally, Odhaib et al. [10] also observed no differences in CA of lambs fed with high-concentrate diets supplemented with Rosmarinus officinalis leaves, Nigella sativa seeds and a combination of both plants, containing 12.35, 19.08, and 34.86 mg of total polyphenols kg−1 DM, respectively. Although DWG increased significantly in lambs that consumed HM, in our study, DMI also increased, which explains the absence of significant changes in CA.Regarding the carcass characteristics, the results of the present study agree with those reported by Simitzis et al. [34] in lambs that consumed diets with a high proportion of concentrate, supplemented with a pure flavonoid (hesperidin) at dietary concentrations of 1500 and 3000 mg kg−1 of feed for 35 days. In their study HCW, CCW, and HCY were similar among treatments, perhaps as a consequence of the low impact of flavonoids supplementation on the FBW of the lambs and on the nutritional composition of the diet consumed, similar to what was observed in this study. Cimmino et al. [11] also observed no differences in HCW, CCW, HCY, and CCY of goat kids fed with high-concentrate diets and supplemented with polyphenols (3.2 mg d−1 for 78 days) taken from residual water after the olive oil extraction. However, polyphenols had positive effects on the fatty acid profile of the meat. The similarity of BFT in carcasses across all treatments could also partially explain the lack of changes in the carcass performance observed in the present study.BFT and LMA values were also similar among treatments. Similar results were reported by Valero et al. [35] in bulls supplemented with 0.162 g d−1 of flavonoids extracted from propolis. On the contrary, Lobo et al. [2] used incremental doses (0, 1, 2, and 4% for 53 days) of extracts from Illex paraguariensis leaves, which contain phenols, in a 60% concentrate diet. Their results showed that BFT decreased as the extract dose increased. However, LMA was higher in lambs receiving the 2% extract dose, resulting in a higher lean muscle production, thus suggesting that the effects of phenols are dose-dependent. The mechanism of action of phenols and flavonoids on lipogenesis has not been studied in lambs. However, in beef cattle fed with high concentrate diets, the inclusion of isoflavone in the diet increased BFT, and it enhanced the synthesis of triglycerides on subcutaneous adipocytes by changing the differential expression of genes involved in lipid metabolism [36]. In the present study, BFT was not affected by the inclusion of HM despite the fact that it contained flavonoids, which indicates that the source of flavonoids has an influence on the changes in BFT. Although genotype, age, sex, and weight of lambs show positive correlations with carcass fat deposition [37] and with carcass physical and chemical characteristics [38,39], the homogeneity of these characteristics in the lambs used in the treatments partially explains the absence of changes in BFT and LMA.Similar results on body morphometry and carcass compactness index were previously reported by Lobo et al. [2] in lambs supplemented with extracts (0, 1, 2, and 4%) from Illex paraguariensis leaves, containing phenolic compounds. However, there is limited information about the effects of phenolic and flavonoid compounds derived from herbal mixtures on morphometric measurements of ruminant carcasses. Perhaps, slaughter weight, cold carcass weight, muscle mass, and adipose tissue deposition are related to these variables [24].Regarding the organs, similar results were observed by Simitzis et al. [13,34] in lambs fed with high concentrate diets enriched with 2500 mg of purified flavonoids (hesperidin or naringenin) and with 1500 and 3000 mg of hesperidin kg−1, respectively. Information about the effects of phenol and flavonoid consumption on the size and weight of internal organs of lambs is still limited, which complicates the explanation of the results observed in this and other studies. However, according to Riley et al. [40], differences in weight and size of internal organs in ovines are influenced by genotype, sex, and age of the animals, and by the dietary restrictions [41]. In addition, the tendency for liver and heart weights to increase in lambs supplemented with HM could be explained by their higher feed consumption [42].The average values of the chemical composition of meat are in the range reported in the literature for lambs fed high-concentrate diets [30]. Similar results were previously reported by Qin et al. [6] on lambs fed with pomace (0, 7.8, and 16% for 80 days) obtained from the Hippophae rhamnoides fruits, containing 0.69 and 1.02% of flavonoids, respectively. In their study, the dose of flavonoids did not affect the moisture, protein, and ash content of the meat. However, the high dose of flavonoids increased the fat content, which according to the authors, could result in a juicier and softer meat. Cimmino et al. [11] also observed higher fat content, but similar moisture content, protein, ash, and collagen in meat from goat kids fed with high-concentrate diets supplemented with polyphenol extracts. (3.2 mg d−1 for 78 days) taken from residual water after the olive oil extraction. These results suggest that the effects of phenols and flavonoids on the composition in lamb meat may be dependent on the dose, the botanical origin, and the duration of the experimental phase. On the other hand, considering that the chemical composition of meat varies according to the feeding regime [43] and by the slaughter weight [39], in our study, it is likely that the lack of effect on the chemical composition of meat was because HM had minimal effect on the nutritional composition and the FBW of the lambs.The pH value of meat was similar among treatments but only the pH value of meat from lambs fed with HM was in the considered normal range between 5.5 and 5.8 [44]. These results suggest that supplementation with HM during the final fattening period could improve the meat quality compared to the meat of lambs not supplemented with HM. Our findings are largely congruent with the results of Qin et al. [6] in lambs supplemented with incremental levels (7.8 and 16% for 80 days) of pomace from Hippophae rhamnoides fruits, containing 0.69 and 1.02% of flavonoids, respectively. In addition, with the results of Simitzis et al. [13] in lambs supplemented with hesperidin or naringenin at dietary feed concentrations of 2500 mg kg−1; and results of Cimmino et al. [11] in goat kids supplemented with 3.2 mg d−1 of polyphenol extracts taken from residual water after the olive oil extraction. A pH value below 5.8 is important for the preservation of meat during storage as it has a bacteriostatic effect, while higher pH values favor the growth of proteolytic microorganisms [45]. This indicates that supplementation with HM during the final fattening period can increase the shelf life of lamb meat.Additionally, in the present study, there were no effects on meat color attributes (L*, a*, b*, Chroma, and Hue°) associated with the inclusion of HM in the diet. Similar results were previously reported by Qin et al. [6] on meat from lambs fed with pomace from Hippophae rhamnoides fruits in the diet (7.8 and 16% for 80 days), containing 0.69 and 1.02% of flavonoids, respectively, and by Simitzis et al. [13] in lambs supplemented with hesperidin or naringenin at dietary concentrations of 2500 mg kg−1 of feed for 35 days. In addition, Muela et al. [46] did not observe changes in meat color in lambs supplemented with a commercial extract (150 mg kg−1 DM for 40 days) taken from whole fruits of Citrus paradisi, Citrus aurantium bergamia, Citrus sinensis, and Citrus reticulata, plants, containing 3.5% polyphenols and 0.8% bioflavonoids (naringenin, quercetin and rutin). These results suggest that the effects of phenolic and flavonoid compounds on the color of lamb meat are not dependent on the dose, the botanical origin, or the period of administration.Color is an important attribute of meat quality because it is the first aspect that attracts consumers when choosing fresh meat [11]. Color stability depends on the oxidation of myoglobin and the formation and accumulation of metamyoglobin [11]. However, several studies [11,46] have reported that the inclusion of polyphenols and flavonoids in the diet does not affect myoglobin, oxymyoglobin, and metamyoglobin contents in lamb meat, which would explain the absence of changes in meat color attributes observed in the lambs that were fed with HM in the present study.The evaluation and validation of a new feed additive require the assessment of the health status of the animals after its consumption. In our study, the hematological parameters of lambs were similar among treatments and showed values within the normal physiological range [47]. In a similar study, Razo et al. [31] were investigated the effects of supplementation with a polyherbal mixture (0, 1, 2, and 3 g kg−1 DM for 60 days) containing polyphenols and flavonoids on blood metabolites in lambs that were fed high-concentrate diets. In their study, they observed the highest platelet concentration in lambs consuming 1 g of HM, however, platelets decreased as the dose of HM increased, thus indicating that phenols and flavonoids can stimulate the immune response at low dose, but at high doses they may depress the immune system. On the other hand, Morsy et al. [48] evaluated the effects of supplementation with red propolis (3 g d−1 for 21 days) containing 43% of isoflavonoids in pregnant ewes fed with 50% of roughage in the diet and observed that the presence of isoflavonoids improved the total leucocyte concentration, total protein, and globulin in the blood. These results suggest that flavonoids can stimulate the immune system regardless of the physiological stage of the animal or the source of the metabolites, even when flavonoids are administered for short periods.In our study, it was observed a quadratic reduction in the concentration of segmented neutrophils in lambs that consumed 1 and 2 g of HM. Similar results were reported by Molosse et al. [32] in suckling lambs supplemented with curcumin, and by Odhaib et al. [10], where lambs were fed high-concentrate diets supplemented with 1% of Rosmarinus officinalis leaves, Nigella sativa seeds, or a combination of both plants containing 12.35, 19.08, and 34.86 mg of total polyphenols kg−1 DM, respectively. Their results also showed that polyphenols improved the immune response without affecting hematological parameters or blood biochemistry in all treatments. Generally, the reduction of neutrophils occurs in the presence of bacterial infections, however, there are other factors that can decrease the number of neutrophils in healthy individuals [49].The blood concentration of lymphocytes often increases in animals under stress, excitement, and fear [50] or during bacterial infections [47]. Lobo et al. [2] observed no changes in the concentration of lymphocytes in lambs supplemented with 1, 2, and 4% extract of Illex paraguariensis leaves as a source of phenols. On the other hand, in the present study, the concentration of lymphocytes increased in lambs supplemented with HM. However, lambs supplemented with HM showed no signs of disease during the experimental phase and even had better productive performance compared to lambs assigned to the control treatment.According to Braun et al. [51], biochemical parameters in ovines are mainly used for the diagnosis of liver, muscle, and nutritional disorders. In the present study, the concentration of these blood metabolites was in the normal range for ovines [52], suggesting that the HM used did not cause liver, muscle, or nutritional disorders in the lambs. Similar results were previously reported by Lobo et al. [2] on lambs supplemented with 1, 2, and 4% extract of Illex paraguariensis leaves containing phenols; by Qin et al. [6] on lambs supplemented with 7.8 and 16% of pomace from Hippophae rhamnoides fruits, containing 0.69 and 1.02% of flavonoids; and by Razo et al. [31], where lambs were supplemented with incremental doses (0, 1, 2, and 3 g kg−1 DM for 60 days) of a polyherbal mixture of Indian plants containing polyphenols and flavonoids, and observed that the blood chemistry values of all treatments were within the ranges reported as normal for ovines.Regarding serum uric acid and urea concentrations, similar results were previously reported by Zhong et al. [53] in lambs supplemented with polyphenols extracted from the green tea plant at concentrations of 2, 4, and 6 g kg−1 of feed for 56 days, and by Razo et al. [31], in lambs supplemented with a polyherbal mixture (1, 2, and 3 g kg−1 DM for 60 days) containing polyphenols and flavonoids. Their results showed that uric acid rose as the dose of polyphenols and flavonoids increased, however, the urea was similar among treatments. It has also been reported an increased amino acid and microbial protein duodenal flux in ruminants supplemented with flavonoids [8], which would partially explain the increase in urea and serum uric acid in lambs supplemented with HM.In our study, lambs supplemented with HM showed low serum creatinine levels, but these were in the normal range for healthy ovine [52], while concentrations of that blood metabolite in the lambs assigned to the control group were up to 27% above the normal range. By contrast, Lobo et al. [2] reported an increase of serum creatinine in lambs as the dietary level of phenolic compounds was increased. On the other hand, it is known that serum creatinine increases in cases of chronic and acute renal failure [54], which suggests that the HM used in the present study did not affect renal health.There is limited information about the use of herbal mixtures and their effects on the mineral status of ruminants. Calcium and phosphorus in blood serum are valuable indicators of the nutritional status of animals due to the low variability of its concentration in the blood [55]. The serum concentrations of calcium and phosphorus in our study were similar among treatments (p > 0.05) and were in the normal range for ovines [53], indicating that the consumption of HM did not affect the mineral balance in the diet or the nutritional status of the lambs. However, in a similar study, Razo et al. [31] investigated the effects of supplementation with a polyherbal mixture (0, 1, 2, and 3 g kg−1 DM for 60 days) containing polyphenols and flavonoids on blood metabolites of lambs fed high-concentrate diets. In that study, serum calcium was also not affected by the dose of polyphenols and flavonoids, however, serum phosphorus concentration increased as the dose of polyphenols and flavonoids increased. These results suggest that the effects of HM containing polyphenols and flavonoids on the mineral status of lambs depend on the dose used and the botanical origin.5. ConclusionsThe results of this study indicate that the inclusion of 2 and 1 g of HM kg−1 DM in the diet improves the feed consumption and weight gain, respectively, without affecting the carcass characteristics, meat quality, and health status of the lambs. HM Animunin® can be used to improve the productivity of lambs fed high-concentrate diets. However, further research is required to test the effects of other doses of this HM in ovines at different concentrate proportions, experimental periods, and physiological stages.

animals : an open access journal from mdpi

10.3390/ani11082299

PMC8388528

At the beginning of the productive life of rabbit does, there must be a balance between ensuring at least a minimal degree of bodily development to guarantee a successful reproductive life, and the minimization of the unproductive rearing period, but nowadays there is no clear recommendation about the optimal moment for the first artificial insemination (AI). A better body condition at the first AI (higher body protein, fat and energy), that indicates a higher degree of maturity of the rabbit doe, did not influence fertility at the first AI (that is usually very high), but improved it at the second AI (that is usually lower than the first one). The percentage of kits born alive at the first and at the second AI also were positively influenced by the body protein content at the first AI. We can conclude that the degree of maturity at the first AI is a key point to optimize the does reproductive success, with body fat and body protein content being relevant factors.

The relationship among live weight, chemical body composition and energy content (at artificial insemination (AI) and three days before parturition), estimated by bioelectrical impedance with fertility rates and the percentage of kits born alive, was studied during the first three AI. The first AI was conducted at 16 weeks of age in 137 rabbit does that weighted 3.91 ± 0.46 kg. Their body chemical composition was 17.4 ± 0.50%, 16.1 ± 2.6%, 1067 ± 219 kJ/100 g body weight, for protein, fat and energy, respectively. An increase in body protein, fat and energy content at the first AI did not affect fertility at the first AI but improved it at the second AI (p ≤ 0.030). Moreover, an increase in body fat and energy content at the second AI improved fertility at the second AI (p ≤ 0.001). Fertility at the third AI was positively influenced by body protein at the third AI and the increase in body protein and fat between the second parturition and the third AI (p ≤ 0.030). The percentage of kits born alive at the first and at the second AI improved with the increase in body protein at the first AI (p ≤ 0.040). In conclusion, a minimal body protein and fat content is required at the first AI to optimize the reproductive performance in young does.

1. IntroductionIn the last four decades, rabbit production underwent a noticeable change from a traditional and familiar organization to industrial and intensive systems. Consequently, genetic selection programs and new breeding management systems were established, improving the production of the new hybrid lines used. This has led to an increase in female nutritional requirements [1,2], health problems [3,4,5,6,7] and welfare necessity [8,9,10,11]. Nutritional strategies in reproductive females need to be global and consider both the short-term productive factors (litter size, milk production, or fertility) as well as the long-term factors (body condition or health). Therefore, the expected improvement in nutritional management should be based on an accurate analysis of the requirements of the doe, its evolution during successive reproductive cycles and the identification of crucial moments in the life of rabbit does to optimize productivity and longevity.Two of the key points of the reproductive success of rabbit does are their birth weight and maturity at the first artificial insemination (AI). There is an optimal threshold for birth weight (>57 g) to optimize the initial reproductive performance, which is associated with an increase in the live weight and fat reserves at the first AI [12,13,14]. The latter are the traits used to define the maturity of rabbit does at the first AI, and both are also related to the nutritional rearing strategy and the time of AI [13,15]. However, there is no clear recommendation indicating the weight and/or body condition of the nulliparous rabbit doe at the onset of its reproductive life [16]. In the current management systems, does are inseminated at a fixed age with minor or no consideration of their weight and chemical body composition. As in other species, there must exist a balance between ensuring at least the minimal degree of body development needed to guarantee a successful reproductive life, and minimizing the unproductive rearing period. Accordingly, the study of the body chemical composition of rabbit does seems to be a useful tool not only to improve feeding, but also for general rabbit doe management [17]. The final aim is to extend the lifespan of rabbit does, which is limited by the relatively high early mortality and culling rate in intensive production systems [5].A new non-destructive technique to estimate rabbit doe body chemical composition (moisture, ash, protein, fat, and energy content) based on the bioelectrical impedance measurement, live weight and physiological status of rabbit does, was developed by Pereda [18]. This method is easier and cheaper than TOBEC [19], and in both methods, the variations of gut contents are included in the error term. Furthermore, it allows the prediction of total fat and energy content (not only the perirenal fat content, as it is with the case with the ultra-sound technique [20]), as well as the body protein, which is not usually estimated with other methodologies to evaluate body condition.The aim of this work was to establish the relationship between chemical body composition (at AI and parturition), determined using bioelectrical impedance, and both fertility and the percentage of kits born alive during the first three inseminations, and to identify the most important moments to record the body chemical composition.2. Materials and Methods2.1. Animal Husbandry and ManagementData of the estimated body chemical composition, the fertility and percentage of kits born alive recorded in two different farms in 2010 were used in order to obtain a wide variation in chemical body composition. In farm A, 106 crossbred (New Zealand White × Californian) rabbit does from the UPV hybrid line (genetic line selected for prolificacy in Polytechnic University of Valencia in Spain using the crossline A × Line V) were used. After the first parturition, does were submitted to three different breeding systems, defined by the parturition–AI and parturition–weaning intervals (4/32, 11/35 and 14/42, Table 1) to obtain a wide variability of body condition. In farm B, 37 crossbred (New Zealand White × Californian) rabbit does from the Hyplus hybrid line (prolific hybrid maternal line selected by Hypharm in France) were used and they were inseminated 11 d after parturition and litter weaned at 35 d of age. Rabbit does were inseminated for the first time between 16 and 18 weeks of age. Rearing period of rabbit does was not controlled and they were fed ad libitum after the first insemination. Non pregnant-non lactating rabbit does were restricted to around 150 g/d. Two commercial diets were used, one in farm A containing 17.5% crude protein, 32.0% neutral detergent fibre and 9.95 MJ digestible energy/kg (based on dehydrated alfalfa, wheat bran and barley), and another in farm B containing 16.5% crude protein, 30% neutral detergent fibre, and 10.8 MJ digestible energy/kg (based on dehydrated alfalfa, sugar beet pulp, sunflower meal, rye and wheat). All rabbit does were submitted to a cycle of 16 h light/8 h dark, and heating, cooling and forced ventilation systems allowed the building’s temperature to be maintained between 18 and 24 °C. Animals were handled according to the principles for the care of animals in experimentation published by the Spanish Royal Decree 53/2013 [21] and favourably assessed retrospectively by the Ethics Committee of the Polytechnic University of Madrid.The relationship between body chemical composition and fertility was studied during the first three cycles as the number of replicates decreased from the third parturition onwards (Table 2). The percentage of kits born alive was only studied at the first and second parturition (N = 121 and 83, respectively) due to the reduction in replicates in the third parturition. The range of variation in chemical body composition of these does at the moments of insemination and parturition is shown in Table 2.Seminal doses with more than 20 million spermatozoa in 0.5 mL of a commercial diluent (Magapor S.L.) were made using a pool of fresh heterospermic semen from bucks selected for growth performance. In order to synchronize the oestrus in the second, third and fourth insemination, 48 h before insemination, the does were injected with 25 IU of eCG (Equine Chorionic Gonadotropin, Segiran, Lab. Ovejero, León) [22]. On the day of insemination, the does received an intramuscular injection of 10 µg of buserelin Suprefact® (Hoechst Marison Roussel, S.A., Madrid). Buserelin is a Gonadotropin-releasing hormone agonist (GnRH agonist), which is used to induce ovulation in rabbit does [23].2.2. Experimental Procedures and Data RecordingIn order to determine the chemical body composition of the does, a bioelectrical impedance analysis (BIA) technique was used [18,24]. Measurements were taken with the four-terminal analyzer Quantum II (Model BIA-101, RJL Systems, Detroit, MI, USA). This device generates an alternating current of 425 µA of intensity at a frequency of 50 kHz. It is provided with 2 black electrodes to conduct the electrical current through the doe’s body, and 2 red electrodes to register the resistance and reactance resulting from the passing current. When it is used with rabbit does, a needle (Terumo, 21G × 1 ½′, 0.8 × 40 mm, nr 2) is inserted in the extremity of each electrode. The needles must pass through the skin of the rabbit doe at four reference points along the loin (two near the scapula and other in the distal part of the loin). Impedance is defined by the equation: Impedance = (Resistance2 + Reactance2)1/2. These three parameters, in addition to the physiological status, live body weight and parturition order were used further to estimate the body composition of the rabbit does. Rabbit does were weighed and their body composition estimated on the days of artificial insemination and parturition (three days before parturition, both pregnant and non-pregnant does) during the first three reproductive cycles. The regression equations developed and validated by Nicodemus et al. [24] and Pereda [18] enabled the prediction of the doe’s body content in moisture, protein, fat and ash expressed as percentages or as g/100 g of body weight. Furthermore, energetic body content expressed in MJ or in kJ/100 g of body weight could be also estimated.2.3. Data Treatment and Statistical AnalysisIn order to establish correlations among parameters of chemical body composition (moisture, fat, protein, ash and energy) at the moments of insemination and parturition during the first three parities, we used Pearson’s correlation coefficient (CORR procedure of the SAS system) (SAS Inst., Cary, NC, USA). The GENMOD procedure of the SAS system [25] was used to study the relationship between chemical body composition, fertility during the first three parturitions and the percentage of kits born alive in the two first parturitions, as the logistic regression [26,27] is an adequate tool for modeling proportions [28]. In the generalized linear models used, the link function employed was ‘logit’ (Equation (1)) and we considered that both fertility and the percentage of kits born alive were proportions arising from a binomial distribution. In equation 1, ‘p’ is the mean of the proportion of fertility and kits born alive. Logit (p) = ln (p/1 − p); p ∈ (0,1)(1)Our first aim was to determine whether the variations in fertility and percentage of kits born alive (dependent variables) were significantly affected by the body chemical composition and its variation between insemination and parturitions (independent covariates). The model also included, as fixed effects, the farm and the breeding system (except for nulliparous does). Afterwards, only significant covariates were retained for the interpretation of coefficient estimates (β) and the linear predictor (η = x’β). In order to display the covariates’ effects, three levels (low, medium and high), representative of the range of variation of each covariate, were fixed for each significant covariate (body constituent) based on the data used in this investigation. To predict the means of fertility and percentage of kits born alive expected for each level (the mean or predicted values, ‘p’) the inverse logit function was used (Equation (2)). p = eη/(1 + eη)(2)Among the criteria of the goodness of fit, we used the likelihood ratio chi-square, ‘QL’, also called ‘deviance’, to establish whether models fitted appropriately [29]. The inverse function of the logit transformation was also used to calculate a confidence interval for the expected percentages of fertility and kits born alive for each level [29]. In fact, in the SAS output and for each estimator calculated, there are the values of lower (Lη) and upper (Uη) limits for its 95% confidence interval. In this case, the method consists in transforming the linear values of the limits to the log-link scale using Equation (3). Upper limit p = eUη/(1 + eUη); Lower limit p = eLη/(1 + eLη)(3)To calculate a standard error for the mean of predicted values, ‘p’, for each level of the covariates retained, we used the Delta method [30], which involves the standard error of the coefficient estimate (SEη) as well as the predicted value, as explained in Equation (4). SEp ≈ p(1 − p) SEη(4)The results were transformed from the logit scale.3. Results3.1. Correlation among Does’ Corporal Constituents during the First Three ParturitionsThe initial live weight and chemical composition of rabbit does had a wide range of variation, which was more important for the fat than for the protein content (16 vs. 3%, respectively, for coefficient of variation (Table 2)). Rabbit does showed an important degree of growth between the first and the second AI (3906 vs. 4187 g, weight of does at 1st and 2nd AI, p < 0.05 (Table 2)). Live body weight was positively correlated (p < 0.001) with fat (r = 0.43 to 0.83) and energy body content (r = 0.43 to 0.71), and negatively with moisture (r = −0.35 to −0.72) (expressed on % and MJ/100 g body weight, respectively) from the first AI to the third parturition (Table 3). In this period, body fat was inversely and closely correlated with moisture (r = −0.95 to −0.99), and positively with body energy (r = 0.79 to 0.98), while body protein was positively correlated with ash content (r = 0.30 to 0.83). Regarding moisture, a negative correlation was observed with energy (r = −0.43 to −0.71).Protein content at the first AI was not correlated with protein content at any other moment of the cycle (Table 4). However, protein content was positively correlated at parturition and the subsequent AI (both at the first and second parturition; p < 0.001). In contrast, the body fat content was mainly positively correlated in the period between the first AI and the third parturition, and the same was observed for body energy content (p < 0.05 (Table 4)).3.2. Relationship between Body Condition and Fertility during the First Three Parturitions: Nulliparous, Primiparous and Multiparous Rabbit DoesThe fertility rate registered in the first parturition was 93.4%. There were no differences between data from the two farms used for the analysis (p > 0.05). Neither live body weight, nor its chemical composition at the first AI, affected the fertility rate at this moment. In fact, there were no differences in the initial body composition and live weight at the first AI between pregnant and non-pregnant does (Table 5), although these results have to be taken with caution due to the small number of non-pregnant rabbits.As expected, in the second parturition, fertility was lower than in the first parturition (56.2 vs. 93.4%, respectively; p < 0.05). Breeding system and farm did not have any effect on fertility in the second parturition. Recorded fertility means were 54.0%, 50.8% and 65.8% for the studied breeding systems R4, R11, and R14, respectively. Live body weight at the moments of first AI, first parturition, and second AI did not affect the fertility rate in the second parturition. Fertility in the second AI was related to chemical body composition at the first AI, in contrast to that observed at the first parturition. Body protein content (p = 0.007), fat (p = 0.030), and energy (p < 0.001) at the first AI positively affected the fertility rate in the second AI (Table 6). The relationship between these parameters and fertility is lineal in the logit scale. Consequently, the model was fitted using three fixed levels of body protein, fat, energy, and protein/energy ratio. These levels were chosen to cover the data range used for the analysis. Fat and energy were positively correlated at the first AI (r = 0.79, p < 0.001), and with their values at the second AI (r = 0.33 and 0.74; p < 0.001 (Table 3 and Table 4)), while at the first AI protein was negatively correlated with fat but did not have any correlation with energy, and was not related to protein at the second AI. The three levels determined for each constituent and used in the model are shown in Table 6. The increase in body protein from 16 to 18% and body fat from 10 to 20% increased fertility from 19.9 to 72.0% and from 31.3 to 69.5%, respectively. The results showed that does with higher energy content (1400 vs. 700 kJ/100 g) underwent improvements in fertility from 19.1 to 83.6%.A higher body protein/energy ratio at the moment of the first AI negatively influenced the fertility rate in the second AI (p < 0.001 (Table 6)). This ratio reflects again the importance of the energetic content, since high ratios (30 g/MJ) corresponded to low corporal energy (800 kJ/100 g) rather than to high body protein content. In fact, the latter showed lower variability (2.9 vs. 20.5%, coefficient of variation of body protein and energy at the first AI, respectively (Table 2)).Chemical body composition at the second AI was also related to fertility in the second AI, as detailed in Table 7. Fat and energy contents at the second AI were positively related to fertility at this moment (p < 0.001) in a similar way to their relation at the first AI. These two variables were positively correlated both at the first and at the second AI (p < 0.001 (Table 3)), and were also positively correlated with fat and energy content at the first AI (p < 0.001; Table 4), although their coefficient of variation increased at the second AI (Table 2).Body protein at the second AI did not exert any effect on fertility at the second AI, in contrast with its observed effect at the first AI. It was positively correlated with fat and energy content at the second AI. Protein contents at the first and at the second AI were not correlated (Table 4), although their average values and variability were similar (Table 2), and were lower than body protein values observed from second parturition onwards. A higher body protein/energy ratio at the second AI again negatively affected the fertility rate at the second AI, reflecting the effect of energy content at the second AI.Moreover, to study the factors that influenced the fertility at the second AI, multiple linear regressions were fitted considering two independent variables: live weight or body composition at the first AI combined with their respective variations between the first and second AI (Table 8). Live body weight at the first AI and weight gain during the interval between the first AI and the first parturition as well as during the interval between the first and second AI were positively related to fertility at the second parturition.The fertility rate recorded at the third AI was 73.9%. No effect of breeding system or farm was found. Recorded fertility means were 56.2.0%, 90.3% and 75.8% for the studied breeding systems R4, R11, and R14, respectively, showing differences in the first two values (p = 0.007). The fertility rate recorded at the third AI was not affected by body composition (fat and energy) at the moments of the first and second AI. Unlike the fertility at the first and second AI, we registered a positive effect of protein content on fertility at the third AI (p = 0.030 (Table 9)). Body protein content at the third AI and second parturition were positively correlated (Table 4), and none of them were correlated with body fat or energy content (Table 3). Moreover, we studied the effect of the changes in chemical components between consecutive AI and parturitions. A positive effect of body protein gain between the first and third AI (p = 0.020), and between the second parturition and third AI (p < 0.001 (Table 9)), was observed. Similarly, body fat gain between the second parturition and third AI was also positively related to fertility rate at the third AI (p = 0.020 (Table 9)).The effect of the previous reproductive success (or not) is reflected in the strong effects of both protein and fat gain before the third AI on the fertility at the third AI. In fact, pregnant does at the third AI showed lower fertility at the second AI, which allowed them a better body reserve recovery (especially protein) at the third AI compared to non-pregnant does (that showed higher fertility in the previous AI (Table 10)). This is confirmed by the positive relationship between fertility rates at the third and first AI (p = 0.050), and the negative relationship between fertility at the third and second AI (p = 0.050). We observed that does that gave birth at the second parturition had lower chances of becoming pregnant at the third AI (p = 0.043; Table 10).3.3. Relationship between Body Condition and Percentage of Kits Born Alive during the First Two Parturitions: Nulliparous and Primiparous DoesThe percentage of kits born alive out of the total born in the first parturition was 93.4%, and the number of kits born alive per doe was 8.00 ± 2.97. Among chemical body constituents, body protein (p = 0.040) and energy contents (p = 0.010) at the first AI increased the percentage of kits born alive (Table 11). Both variables were not correlated at the first AI (Table 3). The relationship between these parameters and the percentage of kits born alive is linear in the logit scale. Consequently, the model with logit link was made using three fixed levels of body protein and energy contents. These levels were chosen to cover the data range used for the analysis. When protein content at the first AI increased from 16 to 18%, the percentage of kits born alive increased from 88.7% to 95.2%, respectively. Furthermore, when body energy at the first AI increased from 900 to 1300 kJ/100 g, the percentage of kits born alive increased from 92.4 to 95.0, respectively.Body fat content was negatively correlated with body protein and positively correlated with energy content at the first AI (r = −0.21 and 0.79, respectively; p < 0.05 (Table 3)), and no effect on the percentage of kits born alive was observed. Live weight at the first AI was also correlated with body protein, energy and fat contents (r = −0.46, 0.65 and 0.83, respectively; p < 0.001 (Table 3)) but was not related to the percentage of kits born alive. Furthermore, a higher percentage of kits born alive was observed in farm B compared to farm A (98.1 and 88.7%, respectively; p < 0.001), but the number of kits born alive per doe was not different between the two farms.The percentage of kits born alive recorded in the second parturition was 87.5% and the number of kits born alive per doe was 10.3 ± 4.46. This rate is lower than that observed in the first parturition (93.4%), but the number of kits born alive increased (8.00 ± 2.96 kits born alive/doe in the first parturition). No effect of the breeding system or farm on the percentage of kits born alive at the second parturition was reported. Weight and body composition at the first AI were not related to this trait. However, a higher body protein at the second AI (p < 0.001) was associated with an increase in the percentage of kits born at the second parturition (Table 12).4. DiscussionA general problem observed in rabbit does is their low fertility rate in the second parturition [31]. It is usually explained by the energy deficiency observed before the second insemination [16]. In this period, it might be difficult for the rabbit doe to meet the requirements for both pregnancy and growth due to the limited feed intake [1,32,33,34,35]. However, when the energy supply was increased, it was dedicated to milk production with no limitation of reserve mobilization [36,37]. In this way, Pascual [13] hypothesized that this negative energetic balance would be a natural adaptation to optimize evolutionary success. In this context, it is interesting to study the relationship between the factors related to body chemical composition and their influence on fertility and kit survival at birth, in order to identify the threshold to be met by rabbit does at the beginning of their reproductive life.The evolution of the live weight and chemical composition of rabbit does from their first AI onwards indicated that they were still growing when inseminated the first two times, which agreed with recent data [38]. Live body weight, body energy and fat were closely and positively correlated, which was similar to the correlation between perirenal fat thickness and body energy content reported previously [39,40]. In contrast, body protein had a minor or no correlation with the latter traits, but a negative one with live weight from the second parturition onwards. These results agree with rabbit doe maturation in this period, which would depend on maturity at the first AI and on reproductive success. Once maturity is reached (or nearly reached), the changes in body weight might be mainly associated with fat mobilization and/or deposition. This would agree with the moderated and positive correlation between live weight and body condition score [41].The differences in chemical body composition and live weight between the first AI and the first parturition seemed to be related to the success in the first insemination, which was not influenced by body condition or live weight. This effect was reported by other authors [2,34,42]. It may be explained by the specific situation of non-pregnant does, which would use the entire intake for body protein and fat accretion, and accordingly, energy accretion, towards the completion of the final step to reach their maturity, where the fat deposition is much higher than the protein deposition (24 vs. 6% increment, respectively). Meanwhile, pregnant does have to supply gestation requirements that are especially important during the last 10 days of gestation, and which can impair not only fat content [17,43,44], but also protein balance [45], with respect to non-pregnant does. It must be taken into account that rabbit does inseminated later show a higher feed intake capacity [46] and lower growth requirements. Furthermore, rabbit does reduce feed intake in the days before parturition, contributing to the impairment of their nutrient balance [43].The impairment of fertility in the second insemination reflects the specific situation of primiparous rabbit does, which suffer a negative energetic balance during their first pregnancy and lactation, compared to non-pregnant does, which seems to negatively influence reproductive performance and especially fertility [2,32,34], although this could be considered a natural adaptation, as commented before [13]. Live body weight at the moments of first AI, first parturition and second AI did not affect the fertility rate in the second parturition. This agrees with the findings of Rommers et al. [46], who did not observe any effect of body weight at the first AI on fertility rate in the first two parturitions. Nevertheless, the combination of a high live weight and high weight gain between the first two AI was also related to better fertility. Rabbit does that lose weight between the first two AI, regardless their initial body weight, were unable to present an acceptable reproductive performance in the second cycle. In this period, live weight was positively correlated with body fat and energy content, but not with protein, as does are finishing their protein accretion. The relationship between the fertility in the second AI and the chemical body composition at the first AI confirms the importance of the rearing management of rabbit does. Diets used [2,14,42,47] and the time of first insemination [15,48,49,50] influenced body composition, which consequently affected fertility. Therefore, at the end of the rearing period, reproductive does should reach an optimal body condition (minimal body protein, fat and energy content), assuring an adequate feed intake and body development, which enable high fertility rates during first parturitions [16]. In this sense, Pascual et al. [13] stated that body data at the first AI are a sign of doe soma and might be related to its productive potential. In this context, the supplementation of reproductive sows with certain daily amounts of amino acids enabled an adequate retention of nitrogen that led to an acceptable reproductive performance [51]. In this study, the threshold in the body composition at the first AI to avoid a sharp reduction in fertility in the second insemination might be set at 18% protein and 20% fat, but few does met this condition (12 and 7%, respectively). Other studies where the time of first insemination was delayed (to 18.4 or 19.5 weeks of age) rendered an increase in the body protein at that moment, as well as higher fertility values (83–87%) that were also associated with a higher fat content [38], although this was not always the case [52]. When the latter two studies were considered together, the does that were successful in the first five AI were lighter and had less body fat than the average (although their mean and range were similar to the current study), and the same body protein (although it was in the upper threshold that was previously mentioned: 17.9%) [53], suggesting the potential relevance of body protein at the beginning of the productive life. Another difference between these extraordinary does and the average population was their higher fat mobilization between the second AI and the first weaning (this was recovered between weaning and the third AI) [52]. These results partially differed from those of Theilgaard et al. [54], who indicated that there was no positive effect of perirenal fat at the first AI on reproductive life. In fact, a higher risk of culling was associated with high fat mobilization, although does that were too lean also seemed to increase their risk of culling. Similarly, Castellini et al. [50], using perirenal fat, found that does that were too fat and too lean (at AI) showed the poorest fertility. Recent results confirmed the negative effect of fatness at the first AI on the risk of being culled and litter size [14]. The disagreement among these studies and the current one might be related to the different fatness range (probably the absence of does that are too fat at the first AI in our work: maximal fat content at the first AI: 22.1%; Table 2), which might be associated with the time of the first AI (in the latter studies, nulliparous does were inseminated later than in the current one). Body fat and energy at the second AI was also related to fertility in the second AI, which might also reflect the observed positive influence of initial body condition on fertility at the second AI.Quevedo et al. [55] suggested that the success of AI 11 days after parturition was conditioned by the rabbit doe’s body condition at parturition rather than at insemination. This fact was not observed in this work, probably due to the fact that in our work, we measured body condition at parturition three days before birth (to avoid disturbing the doe) instead of after birth. This prevented the recording of an important proportion of fat mobilization, which was described by Savietto et al. [17]. Once confirmed, the BIA measurement did not alter the doe immediately after parturition (unpublished results); subsequent studies recorded it just after parturition of the doe [38,52].The reproductive success at the beginning of reproductive life also influenced the fertility of the third AI. In fact, rabbits that are reproductively successful during the first two parturitions were more vulnerable, and consequently, their reproductive performance was impaired in the third AI if they did not have the opportunity to recover. In this sense, Castellini et al. [50,56] proposed the delaying of the second AI after weaning in order to allow rabbit does to recover properly from the first gestation lactation and continue their growth. Otherwise, reproductive success at the beginning of the reproductive life of rabbit does, when they cannot recover their body reserves, might worsen the rabbit doe’s productivity and shorten its life span [13].The absence of effects of the breeding system and farm may be explained by the synchronization of rabbit does at the moment of AI. Rebollar et al. [57] did not register a rhythm effect on fertility in the second parturition when using controlled lactation as the does’ synchronization tool. However, in experiments without any synchronization method [56,58], it was concluded that the reproductive rhythm was related to fertility rate. Anyway, it must be stressed that the current study was not designed specifically to study the effect of breeding system on fertility rate. Besides, an influence of the breeding systems on the body condition could not be ruled out and further studies would be required to figure out the nature of their relationship.There were also a positive influence of body protein and energy at the first AI on the percentage of kits born alive at the first parturition. Rommers et al. [15,59] also related a higher protein content at the first insemination, and lower fat content, with a trend towards increasing numbers of kits born alive and percentages of kits born alive at the first parturition. They observed that restricted nulliparous rabbit does inseminated at 17.5 weeks, compared with those fed ad libitum and inseminated at 14.5 weeks, showed higher protein and lower fat content with a similar live weight, and tended to increase the number of kits born alive and the percentage of kits born alive at the first parturition. In primiparous rabbit does, a better body condition (higher body protein, lipid, and energy contents) was also related with changes in metabolic signals (increase in serum protein and leptin concentrations) that might influence ovarian follicle and gamete quality and might be associated with an improved reproductive outcome [60]. Similarly, in sows, ovarian activity and oocyte quality were influenced by the body protein content [61,62]. Another explanation may involve body protein being related to fetal survival, as the increase in litter size has been related to a higher fetal survival, independently of ovulation rate [63].Live weight and body fat content were also positively correlated with body energy content at the first AI, but negatively with body protein, and had no effect on the percentage of kits born alive. In contrast, Rommers et al. [46] reported that heavier females at the first AI (>4.0 kg) decreased the percentage of kits born alive, although this was combined with an improvement in the litter size at the first parturition. They related it to the development of the reproductive apparatus (larger uterine horns and more corpora lutea in the ovaries). Moreover, these results did not agree with those of Quevedo et al. [44], where rabbit does with higher perirenal fat thickness at 3 months of age tended to increase their percentage of kits born alive at the first parturition.The reduction in the percentage of kits born alive in the second parturition, and the increase in the number of kits born alive, agreed with the results reported by Rommers et al. [46], who also observed, at the second parturition, a higher number of kits born alive and a lower percentage of kits born alive with respect to the first parturition. The different percentage of kits born alive observed in the two farms might be due to the different hybrids used and/or the different environmental management conditions in each farm. No effect of the breeding system or farm on percentage of kits born alive at the second parturition was detected. Weight and body composition at the first AI were not related to this trait. However, higher body protein at the second AI increased the percentage of kits born alive. This result is similar to that recorded at the first AI (on the percentage of kits born alive at the first parturition) and again suggests a positive role of nitrogen content on conception success and fetus viability.5. ConclusionsAn adequate body chemical composition at the first AI (around 18% protein and 20% fat) allowed a better fertility at the second AI. However, the consecutive reproductive success at the first and second AI did not allow rabbit does to recover body reserves and impaired fertility at the third AI. Body composition also affected the percentage of kits born alive at the first AI and at the second AI that increased with body protein content.Consequently, rearing management (e.g., time of first AI) is key to avoiding low fertility rates in primiparous rabbit does. Furthermore, when reproductive success is reached, rabbit does may require alternative management strategies to recover their body condition. Finally, determining body composition at moments of AI may be an adequate tool to anticipate the reproductive success possibilities of rabbit does. Further studies considering different ages at the first AI and breeding systems are warranted to confirm these conclusions and to clarify the relationship between breeding systems and body conditions.

animals : an open access journal from mdpi

10.3390/ani12070880

PMC8996880

Fat deposition capacity greatly impacts the production capacity of sheep. The production of high amounts of fat affects the economic benefits of animals. Gastrointestinal microorganisms play an important role in the characteristics of host fat deposition. This study compares differences in gastrointestinal microorganisms in sheep with different body mass indices. Results showed that there were different microflora compositions among different groups. This provides a new idea for the regulation of fat deposition traits in sheep.

Fat deposition is the key factor affecting the efficiency of animal husbandry production. There are many factors affecting fat deposition, in which the gastrointestinal microbiota plays an important role. Therefore, the body mass index (BMI) was introduced into the evaluation of sheep fat deposition, and the different microbiota and functional pathways of the sheep gastrointestinal tract in different BMI groups were analyzed. We selected 5% of individuals with the highest and lowest BMI from a feed test population (357 in whole group). Microorganisms in 10 sites of the gastrointestinal tract in 36 individuals (18 in each group) were evaluated by 16S rRNA V3–V4 region sequencing. There were differences (p < 0.05) in fat deposition traits between different BMI groups. In the 10 parts of the gastrointestinal tract, the diversity and richness of cecal microflora in the high-BMI group were higher than those in low-BMI Hu sheep (p < 0.05). Principal coordinate analysis (PCoA) showed that there was separation of the cecum between groups, and there were differences in the cecal microbial community. Linear discriminant analysis effect size (LEfSe) showed that most biomarkers were in the cecum. On the basis of an indepth study of cecal microorganisms, 26 different bacterial genera were obtained (p < 0.05). Correlation analysis between them and the characteristics of fat deposition in sheep showed that Colidextribacter, Alloprevotella, and Succenivibrio were positively correlated with fat deposition, while Lachnospiraceae_ND3007_Group was negatively correlated (p < 0.05). The above results show that the cecum may be an important part leading to the difference of BMI in sheep, and its microorganisms may affect the level of fat deposition.

1. IntroductionSheep (Ovis aries) belong to the Artiodactyla cattle family, which is a major economic livestock species. They digest and absorb nutrients through the gastrointestinal tract of ruminants. Sheep is one of the earliest domesticated livestock, widely distributed, especially in China, and there are many local subspecies [1]. Mutton has high nutritional value and low cholesterol content, which is popular on the market [2]. The fat deposition characteristic of sheep is one of the important indices to evaluate its meat production performance. Therefore, excessive fat deposition negatively affects the quality and economic benefits of mutton [3].In sheep, fat deposition is a complex trait, which is composed of, for example, back, mesenteric, perirenal, and tail fat. Body mass index (BMI) can be utilized to reflect the body’s fat and thin degree, and it is also a predictor of energy reserve [4]. By using the ratio of body weight to body height squared to evaluate the degree of obesity, it is not easy to be affected by a change in height [5]. Compared with fat content measured by expensive instruments, the body mass index calculated by using simple body size data is simpler and more economical to reflect the fat content of the body [6,7]. However, correlation between BMI and fat deposition traits in sheep has not been reported. Many studies have shown that BMI is largely affected by heredity. Through genomewide association analysis, more than 300 single-nucleotide polymorphisms were identified to be associated with obesity traits such as BMI [8]. Moreover, microorganisms in the body affect BMI. Gastrointestinal microorganisms play an important role in the normal development, digestion, and metabolism of the host. Studies showed that it has an effect on human obesity and BMI [9,10,11]. Yang et al. reported that intestinal microbiota had an effect on fat deposition in sheep tail, and Lachnospiraceae and Akkermansia may be the key microbiota [12]. Lin et al. believe that microorganisms act on host physiology through metabolites, resulting in mitochondrial fragmentation and lipid accumulation [13].In ruminants, most researchers pay more attention to the rumen [14,15], and less to the intestinal microflora and its impact on the host phenotype. However, the digestive tract of ruminants is a complex system in which different parts play different roles, and microorganisms have spatial heterogeneity. Wang et al. showed that microorganisms change along the gastrointestinal tract of sheep, in which the community composition of stomach, and the small and large intestines is obviously separated [16]. Xie et al. studied the microbial composition of 10 gastrointestinal parts of 7 ruminants and reached a consistent conclusion [17]. Therefore, microorganisms in different gastrointestinal tracts may play different functions.In this study, the composition and functional changes of bacteria in 10 parts of the gastrointestinal tract of Hu sheep with different BMIs were compared and analyzed, aiming to find the key parts and microorganisms that affect the BMI of sheep, so as to determine the key differences of gastrointestinal microbes and reveal the microbiota related to fat deposition in sheep.2. Materials and Methods2.1. Animals and GroupsThe experiment sheep (357 male Hu sheep) were purchased from a commercial sheep farm (Gansu Zhongsheng Huamei Sheep Industry Development Co. Ltd. In Qingyang City, Gansu Province, China). Immunization was conducted according to standardized procedures before weaning, and the sheep were weaned when they reached 56 days of age. All lambs were kept indoors in separate enclosures (0.8 × 1 m) until they were 180 days old. In short, all lambs were exposed to a period of adaptation of 14 days; during this period, the dietary proportion of pellet feed (Gansu Sanyangjinyuan Husbandry Co. Ltd. In Jinchang City, Gansu Province, China) was gradually increased by 7.1% every day, and the silage alfalfa proportion was simultaneously decreased until the pellet feed proportion had become 100%. The pellet feed consisted of 27% barley straw, 44% corn, 2.2% soybean meal, 2.60% rapeseed meal, 4.20% cottonseed meal, and 20% concentrate, containing 16.28% crude protein, 28.48% starch, 36.54% neutral detergent fiber, 14.12% acid detergent fiber, 0.60% calcium, and 0.30% phosphorus. All animals had ad libitum access to water and pellet feed. They were weighed using a calibrated electronic scale before feeding in the morning. Growth traits such as body weight and height, body length, and cannon circumference at 180 days old were recorded. Feeding methods and the environment were kept consistent at all times during the experiment.BMI is calculated as body weight/body length2. Mean ± 3× standard deviation was used to eliminate abnormal BMI values in the test population. The population was sorted according to BMI, and 5% of the highest and lowest BMI in the test population were selected and divided into two groups. Low-BMI individuals were Group 1, and high-BMI individuals were Group 2.2.2. Sample Collection and Character DeterminationAfter 180 days of age determination, 36 sheep in the 2 experimental groups were slaughtered by carotid bloodletting, and the intact gastrointestinal tract of the experimental animals was taken out and ligated at the boundaries of different parts to prevent contamination caused by the flow of the contents. Content samples were collected at corresponding locations in 10 sites: rumen (L), reticulum (W), omasum (B), abomasum (Z1), duodenum (S), jejunum (K), ileum (H), cecum (M), colon (J) and rectum (Z2). Samples were temporarily stored in liquid nitrogen (−196 °C). After the slaughter test, samples were transported back to the laboratory and transferred to a −80 °C ultralow-temperature refrigerator for storage.The fat deposition characteristics of sheep were determined during the slaughter test. Backfat thickness: fat thickness of the posterior edge of scapula, the rib end, and the front edge of hip tubercle was measured, and the average value was taken [18]. Carcass fat content (GR) [19]: tissue thickness between the 12th and 13th ribs and 11 cm from the midline of the spine was measured. Perirenal, mesenteric, and tail fat of the test individual was taken out, weighed with an electronic scale, and the relative weight of fat was calculated with premortem live weight.2.3. DNA Extraction and AmplificationCetyltrimethylammonium Bromide (CTAB) was used to extract DNA from the samples. Agarose gel electrophoresis was used to detect the purity and concentration of DNA. Some samples were diluted to 1 ng/µL with sterile water (TransGen Biotech, Beijing, China). The remaining DNA samples were stored at −20 °C. The extracted DNA was used as a template for PCR amplification, and primers were 314F (CCTAYGGGRBGCASCAG) and 806R (GGACTACNNGGGTATCTAAT). The amplified region was the V3–V4 region of microbial 16S ribosomal RNA. The PCR used a 30 μL system: 15 μL Phusion® High-Fidelity PCR Master Mix (New England Biolabs), 0.2 μm upstream and downstream primers, 10 ng template DNA and 2 μL sterile water. Cycling conditions were as follows: 1 min at 98 °C; 10 s at 98 °C, 30 s at 50 °C, and 30 s at 72 °C for 30 cycles; lastly, 5 min at 72 °C. The same volume of 1X loading buffer (contained SYB green) was mixed with PCR products and electrophoresis was operated on 2% agarose gel for detection. Samples with a bright main strip in the range of 400–450 bp were chosen for further experiments. PCR products was mixed in equidensity ratios. Then, the mixture of PCR products was purified with GeneJET Gel Extraction Kit (Thermo Scientific. Waltham, MA, USA). 2.4. Library Construction and Data ProcessingThe library was constructed by using the library building kit (TruSeq DNA PCR-Free Library Preparation Kit, Illumina, San Diego, CA, USA). Library quality was assessed on a Qubit @ 2.0 Fluorometer (Thermo Scientific) and Agilent Bioanalyzer 2100 system. Lastly, the library was sequenced on an Illumina NovaSeq platform, and 250 bp paired-end reads were generated.Sequenced DNA fragments were paired-end reads using FLASH (version 1.2.7) [20]. Paired-end reads were assigned to each sample according to the unique barcodes. Sequences were analyzed using QIIME [21] software (version 1.9.1) (Quantitative Insights into Microbial Ecology), and inhouse Perl scripts were used to analyze alpha (within-samples) and beta (among-samples) diversity. First, reads were filtered with QIIME quality filters. Then, we used pick_de_novo_otus.py to choose operational taxonomic units (OTUs) by producing an OTU table. Sequences with ≥97% similarity were assigned to the same OTUs. We chose representative sequences for each OTU and used the RDP classifier [22] to annotate taxonomic information for each representative sequence. In order to compute alpha diversity, we rarified the OTU table and calculated three metrics: Chao1, which estimates species abundance; Observed Species, which estimates the number of unique OTUs found in each sample; and the Shannon index. Rarefaction curves were generated on the basis of these three metrics. QIIME (Version 1.9.1) calculates unweighted UniFrac, which are phylogenetic measures of beta diversity. We used unweighted UniFrac for principal coordinate analysis (PCoA) and unweighted pair group method with arithmetic mean (UPGMA) clustering. OTUs were compared and annotated with the SILVA138 database with the Mothur method [23]. To mine deeper data of the microbial diversity of the differences between samples, significance tests were conducted with some statistical analytical methods, including the t-test, LEfSe, rank sum test, MetaStat, and MRPP. Results were visualized using R software (Version 2.15.3).Tax4Fun function prediction is achieved by the nearest-neighbor method based on minimal 16S rRNA sequence similarity. The specific method is to extract the KEGG database prokaryotic whole genome 16S rRNA gene sequence and use the BLASTN algorithm to align it to the SILVA SSU Ref NR database (BLAST bitscore > 1500) to establish a correlation matrix, and map the prokaryotic genomewide functional information of the KEGG database annotated by the UProC and PAUDA methods to the SILVA database to realize the functional annotation of the SILVA database. Sequenced samples were clustered with the SILVA database sequence as the reference sequence to cluster OTUs and obtain functional annotation information.3. Results3.1. Baseline Characteristics of Test PopulationWe used Spearman correlation to analyze the correlation between sheep BMI and various fat deposition traits, as shown in Table 1. Results showed that BMI was significantly correlated with various fat deposition traits (p < 0.01), indicating that BMI can effectively assess the level of fat deposition in sheep.In total, 36 Hu sheep were enrolled in the present cross-sectional study. The trait characteristics of the test population are shown in Table 2. When p-value < 0.05, the difference was statistically significant. As shown in Table 2, the fat deposition traits in the high-BMI group were higher than those in the low-BMI Hu sheep group except for the relative weight of tail fat (p < 0.05). The difference in body length between the two groups was not statistically significant (p > 0.05).3.2. Sequencing Data OverviewSamples were taken from the digestive tract of 36 Hu sheep divided into high- and low-BMI groups. We successfully amplified the 16S rRNA sequence from contents collected from 10 different gastrointestinal regions of Hu sheep. All 339 result samples were sequenced, and 30,842,807 raw tags were generated after splicing (Table S1). After filtering the low-quality sequences and chimeras, generated clean tags and effective tags are represented in Supplementary File S1. The average length of each sequence was 414 bp. A total of 20,928,585 sequences were used for follow-up study after quality control. These sequences were clustered into 7788 operational taxonomic units (OTUs) using the MOTHUR method. In order to confirm whether the sequencing depth and sample size could meet the analytical requirements, we conducted dilution and species accumulation analysis. With the increase in sequencing depth and sample size, the dilution curve and species accumulation curve tended to be flat, indicating that this condition met the analytical requirements (Figure S1).3.3. Diversity AnalysisIn order to study the microbial community composition in different areas of the gastrointestinal tract of two groups of sheep, we evaluated the alpha diversity differences in ten test areas (Table S2). Results showed differences in the alpha diversity of digestive tract microbes among different BMI Hu sheep groups (p < 0.05). The cecal microbial Shannon index and Chao1 index were different between the two groups (p < 0.05), and the cecal microbial diversity and richness of high BMI Hu sheep were higher (p < 0.05).On the basis of the phylogenetic relationship between OTUs, UniFrac distance was calculated. Principal coordinate analysis (PCoA) based on unweighted UniFrac distance showed that the cecum were significantly separated between the two groups, and other parts overlapped between the two groups (Figure 1).We also used MRPP analysis to evaluate differences in the community composition between the two groups (Table 3). In the microbial community structure of the rumen (A = −0.0005) and duodenum (A = −0.0032) between the two groups, difference within a group was greater than that between the groups. There were significant differences in the community structure of the reticulum, flap stomach, cecum, colon, and rectum between the two groups (p < 0.05), which confirmed the PCoA results.3.4. Analysis of the Gastrointestinal Tract Microbiota Composition20,928,585 effective tags were annotated by the 16S Silva database, which was divided into 47 phyla, 115 classes, 248 orders, 358 families, and 634 genera. Unallocated taxa accounted for 2.94% of the total OTUs. At the phylum level, the abundance of Firmicutes and Bacteroidetes in the gastrointestinal tract of Hu sheep in high- and low-BMI groups was higher. Actinobacteriota also had a high abundance in the duodenum, jejunum, and ileum (Figure 2).We performed LEfSe analysis on high- and low-BMI Hu sheep groups using LDA score = 4 (Figure S2). There were biomarkers in gastrointestinal microorganisms among different groups that were statistically different. Among the 10 sites of microbes in the 2 groups of sheep, the large intestine (cecum, colon, rectum) had the most differential biomarkers, and differential biomarkers were similar in the three sites. We used a Venn diagram to analyze cecum and colorectal biomarkers, in which Firmicutes and Oscillospirales were common, and the cecum contained the most biomarkers (Figure 3). Firmicutes, Clostridia, Oscillospirales, Oscillospiraceae, and UCG_005 are biomarkers in the cecum of high-BMI Hu sheep, while Verrucomicrobiota, Verrucomicrobiae, Verrucomicrobiales, Akkermansiaceae, Akkermansia, Bacteroidota, Bacteroidia, Bacteroidales, Prevotellaceae, and Prevotellaceae_UCG_001 are biomarkers in the cecum of low-BMI Hu sheep.3.5. Cecal Microorganisms Affect Fat DepositionWe used the Metastat [24] method to deeply study microbiota with significant differences in the cecum between the two groups (Figure 4A). Fibrobacterota and Halobacterota numbers in the cecum of low-BMI Hu sheep were significantly higher than those of high-BMI Hu sheep (p < 0.05), Firmicutes and Proteobacteria were significantly lower than those of high BMI Hu sheep (p < 0.05). The abundance ratio of Firmicutes to Bacteroidetes in the high-BMI Hu sheep population was significantly higher than that in the low-BMI Hu sheep population (p < 0.05) (Figure 4B). At the genus level (Table 4), 12 bacterial genera such as Saccharofermentans, Prevotella and Erysipelotrichaceae in the cecum of low-BMI Hu sheep were significantly higher than those of the high-BMI Hu sheep (p < 0.05). The numbers of 14 bacterial genera, such as Oscillospiraceae_UCG-005, Colidextribacter and Agathobacter, were significantly lower than those in the cecum of high-BMI Hu sheep (p < 0.05).We took 26 different bacterial genera in different groups of the cecum as the research object to analyze the correlation between different bacterial genera and fat deposition traits of Hu sheep (Figure 5A). The different bacterial genera in the cecum were related to the characteristics of fat deposition in Hu sheep, including Oscillospiraceae_UCG-005, Butyricicoccaceae_UCG-009, Alloprevotella, Dorea, Succinivibrio, Prevotellaceae_UCG-003, Colidextribacter, Parabacteroides, Oscillibacter were all positively correlated with BMI (p < 0.01). Moreover, Candidatus_Saccharimonas, Fibrobacter, Lachnospiraceae_NK3A20_group, Lachnospiraceae_ND3007_group, Methanocorpusculum, [Ruminococcus]_gauvreauii_group were negatively correlated with BMI (p < 0.01). While Colidextribacter, Alloprevotella, Succenivibrio and Lachnospiraceae_ND3007_Group were associated with more than 80% of the fat deposition traits (p < 0.05).On the basis of the Spearman correlation coefficient, the correlation network of the top 50 genera of cecum of Hu sheep with different BMI was analyzed. Bacteroides was negatively correlated with Syntropococcus in the cecum samples of low-BMI Hu sheep, while Syntropococcus and Firmicutes were positively correlated with the genera of multiple Firmicutes (Figure 5B). However, this was not observed in the genus level network of cecum of Hu sheep with high BMI (Figure 5C).3.6. Functional Prediction AnalysisThe functional clustering heat map showed that similar functional pathways existed in the stomach, and small and large intestine groups. The cecum, colon, and rectum showed higher clustering in the same group (Figure S3). In order to determine the functional pathways related to BMI, gastrointestinal tract microorganisms of Hu sheep with high and low BMI were compared. The functional pathway of cecal flora was evaluated using Tax4Fun. At Level 1, the cecum was composed of the most differential functional pathways, mainly in metabolism, human diseases, and systems (Figure S4). Therefore, the cecal functional pathways were analyzed at Level 2 (Figure 6), and 23 differential pathways were obtained. Among them, the enrichment degree of low BMI Hu sheep in translation, energy metabolism, glycan biosynthesis, and metabolism was significantly higher than that of the high-BMI Hu sheep (p < 0.05). Eight pathways, such as membrane transport, signal transmission, and cell mobility, were significantly enriched in the high-BMI Hu sheep population (p < 0.05). These findings clearly show that, when BMI is different, there are great differences in the functional pathways of cecal microbial enrichment.4. DiscussionA number of studies were conducted in humans to show the degree of obesity through BMI [25,26]. To our knowledge, it is rare to use BMI to evaluate sheep fat deposition. Previous studies on the sheep gastrointestinal tract mainly focused on rumen samples [27]. However, there is spatial heterogeneity in microbial colonization in the digestive tract. The evaluation of BMI on fat deposition is carried out in humans. Results show that BMI can effectively judge the content of subcutaneous adipose tissue. Its description of body fat is similar to waist circumference, waist body ratio, and other indicators, which can effectively reflect the proportion of fat content [28,29].Similar results were obtained in the study of the sheep cohort. This study found that most correlation of fat deposition in the high-BMI group was significantly higher than that in the low-BMI group, which showed that high-BMI Hu sheep had higher fat deposition ability. A cross-sectional correlation study showed that the higher the height is, the higher the risk of subsequent obesity [30]. We used body length instead of height to calculate BMI, and there was no significant difference in body length between different groups, which reduces the impact of body length on BMI calculation, verifying our previous speculation that BMI can reflect the obesity level of Hu sheep.Along the gastrointestinal tract of sheep, microbial community structure and composition changed. There were significant differences in the alpha diversity index between the high- and low-BMI groups in the reticulum and cecum, including the Shannon and Chao1 indices. The diversity index showed the same trend in the cecum, which was higher in the high-BMI group. Our results showed that the cecal microorganisms of high-BMI Hu sheep showed higher diversity and richness. Our results are consistent with those of previous studies, which found that the intestinal alpha diversity of overweight people is higher than that of normal-weight people, and the bacterial diversity of obese subjects is higher [30,31]. Obesity leads to the dysbiosis of the gut microbiota, with increased types and abundances of obesity-related microbiota [10,32]. Principal coordinate analysis (PCoA) based on UniFrac distance showed a separation of the cecum and colon between different groups and aggregation within the group. This shows that there are differences in the microbial community composition of cecum and colon samples in different groups. We verified this with the MRPP method, and confirmed that there are significant differences between cecum and colon in different groups. These results were consistent with those of other species that found that there was separation in the principal coordinate analysis of cecal samples of normal and obese mice [33].According to our findings, there are differences in the relative abundance of microorganisms along the gastrointestinal tract of Hu sheep. These results are consistent with previous research results in humans [34], rats [35] and donkeys [36]. Similar to previous reports, the duodenum and jejunum are mainly composed of Firmicutes and Actinobacteriota [37], while the rest of the gastrointestinal tract is composed of Firmicutes and Bacteroidetes. However, the relative abundance of Bacteroidetes is the highest in rumen, reticulum, omasum and abomasum [38], while the relative abundance of Firmicutes is the highest in ileum, cecum, colon and rectum [39]. Combined with the UPGMA cluster tree, the stomach, and small and large intestines form three clusters. Only clusters between the high- and low-BMI groups in the large intestine had differences, and the enrichment of functional pathways also showed consistent results. This may be related to the fermentation degree of the large intestine because microorganisms provide nutrition and energy to the host by fermenting undigested components, but there may be a risk of obesity [40,41].Combined with LEfSe analysis, biomarkers in different parts of the gastrointestinal tract of Hu sheep were determined. Firmicutes and Bacteroidetes are biomarkers in the cecum, colon, and rectum. Many previous studies reported that obesity is related to an increase in Firmicutes and a decrease in Bacteroidetes [42,43]. Akkermansiaceae in the cecum was observed to be negatively correlated with overweight and obesity [44]. These are consistent with our results. In addition, the cecum comprises the most biomarkers in the gastrointestinal tract of different groups, suggesting that the cecum may be an important site related to BMI in the gastrointestinal tract of sheep.In order to determine the key microbiota related to BMI, differences in cecal microorganisms in different groups were analyzed. We obtained a phylum consistent with LEfSe results, namely, Firmicutes. In high-BMI Hu sheep, the relative abundance ratio of Firmicutes and Bacteroidetes was significantly higher than that of the low-BMI Hu sheep. This is consistent with results obtained in other reports in both humans and rats [45,46]. The increase in Proteobacteria was observed to be related to obesity and intestinal microbiota imbalance in animals, and the level of Proteobacteria decreased in the experiment of reversing obesity [47,48].In this study, we identified 19 bacterial genera significantly related to BMI, of which Colidextribacter, Alloprevotella and Succenivibrio were significantly positively correlated with more than 80% of fat deposition traits, while Lachnospiraceae_ND3007_Group showed significantly negatively correlated. In this study, negative correlation between Lachnospiraceae_ND3007_Group with BMI and fat deposition was identified for the first time. In previous reports, Colidextribacter and Alloprevotella were positively correlated with hyperlipidemia in rats and daily gain in pigs, respectively [49,50], and we found them in the cecum of high-BMI Hu sheep. In particular, Saccharivibrio belonging to Proteobacteria presented high relative abundance in the cecum of high-BMI Hu sheep. Succinivibrio can ferment to produce a variety of sugars. The main metabolic end products are acetic acid and succinic acid, and small amounts of formic acid and lactic acid. A high abundance of Succinivibrio produces more acetic acid, and acetic acid promotes lipid synthesis; we can speculate that a high concentration of acetic acid is absorbed by the intestinal epithelium, which improves the efficiency of fat deposition [51]. In conclusion, the four bacterial genera above may be key bacterial genera related to the host BMI, which suggests the need for further research.Changes in intestinal microorganisms may reshape intestinal barrier function, host metabolism, and signaling pathways [52]. Studies showed that transplanting gut microbiota from normal mice into sterile mice increases body fat in sterile mice without increasing food intake [53]. We, therefore, analyzed the microbial function of the sheep gastrointestinal tract. The cecum is composed of the most differential functional pathways. Membrane transport and cell motility are highly enriched in the cecum of Hu sheep with high BMI, which indicates that microorganisms affect the epithelial cells of the gastrointestinal tract and improve energy absorption. In contrast, energy metabolism, transport and catabolism, folding sorting, and the degradation pathway are enriched in the cecum of low-BMI Hu sheep, suggesting that microorganisms may reduce fat deposition by accelerating the rate of metabolism. However, further research is needed to describe the specific functional pathway differences of different microorganisms. At present, analysis of functional differences is predictive.5. ConclusionsThis study introduced BMI into the evaluation of fat deposition trait levels in Hu sheep, and revealed the different microbial and functional pathways in the gastrointestinal tract of Hu sheep with different BMI levels. We found significant positive correlation between BMI and fat deposition traits in Hu sheep. Among the gastrointestinal tracts of Hu sheep with different BMI levels, cecal microbial composition and function presented the greatest differences, which suggested that the cecum might be a key site for differences in BMI. Among the differential microbes in the cecum of the two groups, Colidextribacter, Alloprevotella, Succenivibrio, and Lachnospiraceae_ND3007 Group were significantly associated with BMI and 80% fat deposition traits in Hu sheep, which can be used as biomarkers affecting BMI in Hu sheep. The relationship between BMI and gastrointestinal microbiota provides a new direction for the regulation of fat deposition traits. In future work, we aim to integrate multiomics methods to study the specific functional pathways of different microbiota on fat deposition in Hu sheep, so as to provide a theoretical basis for the regulation of fat deposition traits.

animals : an open access journal from mdpi

10.3390/ani11103005

PMC8532677

Natural montmorillonite (NM) is the most common clay used as a feed additive in ruminant diets. Under normal pH conditions, it can adsorb hydrogen and may affect methane (CH4) formation; however, it possesses less efficiency than other clays. Due to NM’s negative charge flat surface and positive charge edges, its physicochemical properties can be modified by cationic or anionic surfactants. Therefore, two types of modified nano-montmorillonite (MNM) were developed by ion-exchange reactions using cationic and anionic surfactants. Comparisons were made with monensin as a rumen modulator to reduce CH4 emission from ruminants. The results indicated that the physicochemical properties of both MNM types were enhanced (e.g., cation-exchange capacity and zeta potential). All MNM clays and monensin successfully reduced rumen CH4 production and ammonia concentration; however, clay modified by cationic surfactant was more efficient than what was modified by anionic surfactant in modulating in vitro rumen fermentation properties

Two types of modified nano-montmorillonite (MNM) were developed by ion-exchange reactions using two different surfactants; sodium dodecyl sulfate (SDS) and cetyltrimethylammonium bromide (CETAB), to prepare MNMSDS and MNMCETAB, respectively. Both MNM types were on the nano-scale and had higher cation-exchange capacity values than NM clay. The MNMCETAB had the highest zeta potential (−27 mV) compared with the other clays. Effects of MNM types on in vitro ruminal batch culture fermentation, nutrient degradability, and methane (CH4) emission compared with monensin were evaluated in vitro using a semi-automatic gas production system. The experimental treatments were the control (0 supplementations), monensin (40 mg/kg DM), and NM (5 g NM/kg DM), and two levels of MNMSDS and MNMCETAB were supplemented at 0.05 (low) and 0.5 (high) g/kg DM to the control basal feed substrate. Among the experimental treatments, the high dose of both MNM types reduced (p < 0.01) CH4 production and ammonia concentrations compared with the control, while only MNMCETAB treatment tended to increase (p = 0.08) the truly degraded organic matter compared with monensin. All MNM treatments increased (p < 0.01) acetate molar proportions compared with monensin. The high MNMCETAB increased (p < 0.01) the in vitro ruminal batch culture pH compared with the control and monensin. The MNMCETAB supplemented at 0.5 g/kg DM is the most efficient additive to reduce CH4 emission with the advantage of enhancing the in vitro nutrient degradability of the experimental feed substrate. These results indicated that MNM could modulate the in vitro ruminal fermentation pattern in a dose- and type-dependent manner.

1. IntroductionEnteric fermentation is considered an actual cause of climate change and environmental pollution due to the emissions of greenhouse gases (e.g., methane (CH4) and carbon dioxide (CO2)) [1]. Methane is 28 times more powerful as a potent greenhouse gas than CO2; it is directly produced by ruminal methanogens, while other hydrogen-producing microorganisms (e.g., protozoa) can indirectly contribute the CH4 formation through a synergistic association relationship with methanogens [1,2,3]. Moreover, the CH4 emission from ruminants represents a significant loss of dietary energy, which could be redirected towards valuable animal products [2]. Therefore, various rumen fermentation modifiers have been applied for ruminants to inhibit CH4 emission; however, the dietary antibiotic ionophores (e.g., sodium monensin) seem to be the most successful ones [3]. Ionophores are polyether antibiotics acting as inhibitors to deamination and H+ producing bacteria. They mainly disturb the bacterial cell wall membrane through ion exchange capacity, specifically H+/Na+ and H+/K+ antiport activity [1]. Nevertheless, the current global scenario has shifted the interest towards natural and safe feed supplements instead of antibiotics for protecting the environment and producing safe animal products [2,3].Geophagy (eating clays) is a common natural habit of ruminants. Therefore, several clay classes are recognized as safe for animal and human consumption [4,5]. Natural montmorillonite (NM), also named microcrystalline kaolinite, has an advantage over other clays because of its high availability, low cost, large surface area, small particle size, and high ion exchange activity characteristics [5,6]. Moreover, it acts as a buffering agent to regulate acidosis. Additionally, it works against bloat and diarrhea and can adsorb heavy metals and aflatoxins [7]; therefore, NM was widely used as a feed additive for ruminants. Natural montmorillonite has lower antibacterial effects than other nano or organically modified clays [5,6]. Tate et al. [5] reported the first investigation on using NM as a rumen modifier to reduce rumen CH4 production in vitro through a direct inhibition effect on methanogens. They found that NM was less effective in inhibiting methanogenesis than other kaolinite clays.Montmorillonite platelets have a unique ionic composition: a negative charge flat surface and positive charge edges [5,6]. Thus, inorganic ions in NM can be effectively exchanged with both cationic and anionic surfactants through ion exchange reactions [8]. This phenomenon was exploited to modify and enhance the cation exchange capacity (CEC) and antibacterial properties of NM. Compared with NM, modification of montmorillonite using cationic surfactants (e.g., quaternary ammonium salts) leads to damage to the cellular membrane of Gram-positive bacteria cells (e.g., Staphylococcus aureus) [6]. In addition, montmorillonite modified by anionic organosulfur surfactants that have antimicrobial properties (e.g., sodium dodecyl sulfate (SDS)) possesses high CEC, which in turn increased the uptake of heavy metal ions [8]. Additionally, modifying NM by mechanical grinding improved the clay’s stability and physicochemical properties while exhibited intense antibacterial activity against Escherichia coli [4].Recently, grinding the natural clays (e.g., zeolite) in the nano-scale (1–100 nm) enhanced the clay’s chemical stability and physicochemical properties [9]. At the same time, it reduced CH4 and ammonia production while improved the fiber or organic matter rumen degradability in a dose and particle-size-dependent manner [9]. It can be speculated that, if nano-scale dispersion for modified montmorillonite would be achieved, new exceptional physicochemical properties might appear for the modified clays and/or the lowest effective supplementation dose could be obtained. Our working hypotheses are as follows: (1) The actively modified nano montmorillonite (MNM) can be prepared by chemical and nano grinding modifications. (2) The prepared MNM can modulate ruminal in vitro batch culture fermentation patterns, including methanogenesis. Therefore, we developed two different types of MNM using anionic (sodium dodecyl sulfate (SDS)) and cationic (cetyltrimethylammonium bromide (CETAB)) surfactants. This study is the first investigation to evaluate the effects of modified clays compared with antibiotic monensin on in vitro fermentation, protozoal count, and nutrient degradability.2. Materials and MethodsThis study was carried out at the Advanced Laboratory of Animal Nutrition, Faculty of Agriculture, Alexandria University, Alexandria, Egypt. All procedures and experimental protocols were carried out according to the guidelines for the care and use of animals in research of Alexandria University (AU 08-200415164).2.1. Preparation of MNM ProductsNatural montmorillonite clay (NM) was commercially supplied (Egypt Bentonite and Derivatives Co., Alexandria, Egypt) in powder form. The NM clay contained 544 g/kg dry matter (DM) of silicon dioxide, 190 g/kg dry matter of aluminum oxide, 135 g/kg dry matter of Iron(II) + iron(II, III), 52 g/kg dry matter of sodium oxide, 18.1 g/kg dry matter of titanium dioxide, 31 g/kg dry matter of magnesium oxide, 16 g/kg dry matter of calcium oxide, 12 g/kg dry matter of potassium oxide, and 1.9 g/kg dry matter of phosphorus pentoxide. The method of Bujdáková et al. [10] was followed to obtain the experimental MNM types, using two different surfactants, SDS and CETAB (Sigma Aldrich Co., Irvine, Scotland), to prepare the modified nano montmorillonite by SDS (MNMSDS) and the modified nano montmorillonite by CETAB (MNMCETAB), respectively. To obtain the nano-scale of MNMCETAB and MNMsds, the resulting materials were ground using High-Energy Laboratory Planetary Ball Miller (Retsch PM, VERDER SCIENTIFIC, North Rhine-Westphalia, Haan, Germany) for six hours with a reverse rotation speed of 300 rpm and vial rotation speed of 600 rpm with the ball to powder ratio of 9:1 mass/mass.2.2. Physicochemical Properties of NM, MNMsds, and MNMCETABThe particle size and the surface charge of the experimental clays were measured by zeta potential analysis using a Malvern ZETASIZER Nano series (Malvern, Worcestershire, United Kingdom) with a range of particle size detection from 0.3 nm to 10 microns at 25.0 ± 1 °C temperature, count rate (kcps) 347.4, measurement position (mm) 2.0, and attenuator 7.0.The pH and electrical conductivity (EC) of the experimental feed additives were determined in a suspension of clay and distilled water (pH = 6.80) in a ratio of 1:2.5 by a multi-parameter pH meter (GLP 21 model; CRISON, Barcelona, Spain). The cation-exchange capacity was measured according to [11] using 1 M sodium acetate−0.1 M sodium chloride.The transmission electron microscope (TEM) was used to provide dimensional images for the experimental NM, MNMsds, MNMCETAB feed additives to detect the size and shape of their nanoparticles. Clay samples were coated with gold to improve the imaging of the clay sample and scanned using a TEM (JSM1400 plus-JEOL, Los Angeles, CA, USA) operated at a vacuum of the order of 10, and the accelerating voltage of the microscope was kept in the range of 10–20 kV.The functional groups of the experimental feed additives were identified by Fourier Transform Infra-Red Spectroscopy (FTIR) using an infrared spectrometer (Shimadzu FTIR-8400S, Osaka, Japan) equipped with a deuterated triglycine sulfate (DTGS) KBr detector and purge gas generator.2.3. In Vitro Gas Production (GP)2.3.1. Basal Feed Substrate and Experimental DesignA basal feed substrate consisted of 500 g/kg dry matter of concentrate and 500 g g/kg dry matter of berseem hay (Trifolium alexandrinum) of the 3rd cut; this feed substrate was formulated to fulfill the national research council [12] nutrient requirements of growing cattle. The basal feed substrate was chemically analyzed according to the Association of Official Analytical Chemists [13] for DM, organic matter (OM), crude protein (CP; as 6.25 × nitrogen), and ether extract (EE). Neutral detergent fiber (NDF) and acid detergent fiber (ADF), and lignin were analyzed according to Van Soest et al. [14]. All plant cell well fractions were sequentially determined using the semi-automatic fiber analyzer (ANKOM, model A2001, Macedon, New York, NY, USA) using the same sample in a filter bag (F57-ANKOM Technology Corporation, Macedon, New York, NY, USA). Primary ingredients and chemical analyses of the basal feed substrate are shown in Table 1.The experimental treatments were the control (basal feed substrate without supplementations), monensin (basal feed substrate supplemented with 40 mg/kg DM sodium monensin (Rumensin®, Elanco, Itapira, São Paulo, Brazil)), and NM (basal feed substrate supplemented with 5 g NM/kg DM), and four MNM treatments were tested using two doses (low and high) supplemented to the basal feed substrate. The low dose was 0.05 g MNMSDS or MNMCETAB/kg DM, and the high dose was 0.5 g MNMSDS or MNMCETAB/kg DM. The experimental dose of NM was tested according to Maki et al. [7]. Monensin was evaluated because it is one of the most common ionophore feed additives used to reduce ammonia and CH4 emissions [1,3]. The experimental dose of monensin was the manufacturer’s recommendation; this dosage (with the same source) was previously found to decrease CH4 production and ammonia concentration in vitro while exerting minimal effects on the in vitro degradation of OM and total short-chain fatty acids (SCFAs) concentrations [3]. Therefore, a dose of 40 mg/ kg DM of monensin was used in the current study.2.3.2. Procedure of GPThe experimental treatments were evaluated using the semi-automatic GP system according to Bueno et al. [15] and adapted to Soltan et al. [3]. The ruminal contents were collected separately from three fasted, slaughtered crossbred cow calves (440 ± 5 SE kg body weight) from the slaughterhouse that belongs to the Department of Animal and Fish Production, Faculty of Agriculture, Alexandria University, to avoid any unusual individual rumen environmental conditions [16]. These slaughtered calves were fed a local diet for beef production consisting of 500 g berseem hay (Trifolium alexandrinum) and 500 g commercial concentrate mixture (145 g crude protein/kg DM) ad libitum. The ruminal contents were transferred into pre-warmed thermo-containers (40 °C) under CO2 flushing. To prepare the ruminal inocula (n = 3) to the in vitro incubation, the ruminal contents of each calf were blended for 10 s, squeezed by four layers of cheesecloth, and kept in water bath (39 °C) under continuous flushing of CO2.For each ruminal inoculum, six in vitro incubation flasks (Arab Pharmaceutical Glass Company S.A.E., Suez, Egypt) as analytical replicates were prepared for each experimental treatment in addition to blank flasks (containing Menke’s buffered medium and ruminal inoculum) [15] that were used to obtain the net gas production values and internal standard flasks (containing Menke’s buffered medium, ruminal inoculum, and berseem clover hay) to correct for sensitivity variations induced by the inocula; variations above 10% were rejected [16].A sample of 500 mg of each experimental feed substrate was accurately weighed into an incubation flask and incubated with 30 mL of the buffer solution and 15 mL of the inoculum while leaving a headspace of 75 mL [3,16]. The flasks were closed with 20 mm butyl rubber stoppers, sealed with aluminum seals, and incubated at 39 °C in a forced air incubator (FLAC STF-N 52 Lt, Treviglio, Lombardy, Italy) for 24 h. The headspace gas pressure of the flask was determined at 3, 6, 9, 12, and 24 h from the incubation start using a pressure transducer and a data logger (Pressure Press Data GN200, Piracicaba, Sao Paulo, Brazil). The volume of GP (mL) was calculated as 4.97 × measured pressure (psi) + 0.171 (n = 500; r2 = 0.99) [3].For CH4 determination, one mL of the headspace gas was sampled at each pressure measuring time by a 3 mL syringe (Dawliaico, Assiut, Egypt) and was accumulated in 5 mL vacutainer tubes (BD Vacutainer® Tubes, Jersey, NJ, USA). Methane concentrations were determined by gas chromatography (GC, Model 2014, Drawell Scientific Instrument Co., Ltd., Shanghai, China) equipped with a Molesieve 5A micro packed column (1 m, 2 mm ID, Ref no. 80440-800; Restek, Bellefonte, PA, USA). The GC separation conditions were reported in detail by Sabry et al. [17].2.3.3. In Vitro Ruminal Batch Culture Fermentation, Protozoal Count, and Nutrient DegradabilityAfter 24 h of incubation, all flasks were placed on ice to stop the microbial fermentation. Values of pH were determined by a portable pH meter (the same model that was used to measure the pH of the clays). The ammonia concentrations were determined calorimetrically using a commercial kit (Biodiagnostic kits, Giza, Egypt). The concentrations of SCFAs were measured according to Palmquist and Conrad [18] and adapted by Soltan et al. [3] using gas chromatography (GC; Thermo TRACE 1300, Rodano, Milan, Italy) equipped with a capillary column (TRFFAP 30 m × 0.53 mm ID × 0.5 μm film (thermo-part No: 260N225 P). The GC separation details have been reported by Salama et al. [16]. Protozoa were counted microscopy following the method described by Dehority et al. [19] using Neubauer improved bright-line hemacytometer counting chamber (Paul Marienfeld GmbH & Co. KG., Baden-Württemberg, Germany).To determine the truly degraded organic matter (TDOM), the contents of the flasks were treated with the neutral detergent solution for three hour at 90 °C. The residuals non-degraded of the contents of the flasks were filtered in pre-weighed crucibles, washed with hot distilled water and acetone, dried, and allowed to be turned into ash. The TDOM was estimated by the difference between the incubated and non-degraded organic matter amounts, while the truly degraded neutral detergent fiber (TDNDF) was calculated by the difference between the amount of incubated NDF and the non-degraded NDF amounts [3]. The partitioning factor (PF) was calculated as the ratio of TDOM and net gas volume for 24 h [20].2.4. Statistical AnalysisThe in vitro assay was completed in one run (one day) for all experimental treatments. The actual statistical replications (n = 3) were the average of the analytical replicates (n = 6/inoculum). The experimental unit was the mean of the six analytical replicates formed one statistical replicate. All data were analyzed by one-way ANOVA using the MIXED procedure of SAS (SAS Institute Inc., Cary, NC, USA, version 9.0). Orthogonal contrast statements (contrast 1 and contrast 2) were designed to test each experimental parameter’s linear and quadratic responses to increasing concentrations (0, 0.05, and 0.5 g/kg feed substrate) of MNMSDS and MNMCETAB, respectively. Comparisons among treatments were performed using Tukey’s test, the effects were declared significant at p ≤ 0.05, and the trends were accepted if p < 0.10.3. Results3.1. Physicochemical of NM, MNMsds, and MNMCETABPhysicochemical characteristics of the experimental clays are shown in Table 2. Negligible variations in pH were detected among all clay products.The modification of NM either by CETAB or SDS resulted in a numerical reduction in the values of EC but enhanced CEC compared with the NM, MNMCETAB had the lowest EC values, and MNMSDS had the highest CEC values compared with other clays. The Zeta potential of NM clay was negative and became more negative after modifications by CETAB or SDS; MNMCETAB had the highest zeta potential compared with the other clays (Figure 1).The average size of both MNM products was on the nano-scale; MNMCETAB had the smallest nanoparticle size compared with the other clays. The TEM images of the size and size distribution of the experimental MNM particles are shown in Figure 2. TEM images confirmed the formation of nano nanoparticles of both MNM products; it also indicated the high quality of the synthesis method for producing similar nanoparticles. The images showed that most of the nanoparticles are within the 26.9–63.7 and 28.2–98.2 nm ranges for MNMCETAB and MNMSDS, respectively.Figure 3 shows the results of the FTIR analysis to investigate the characteristics of MNM products compared with NM clay. In the high-frequency range, well-defined peaks (OH-group) were shifted from 3417 cm−1 in NM to higher frequencies at 3435 cm−1 in both MNMCETAB and MNMSDS, and the bands frequency-shifted from 1633.7 cm−1 in NM to 1644 cm−1 in MNMCETAB and 1640.03 cm−1 in MNMSDS. In the lower frequency range (750–1300 cm−1), a band at 778 cm−1 (attributed to the Si–O stretching vibrations) appeared only for both modified clays, while it was absent in NM. Three bands at 450 and 550 cm−1 corresponding to the bending mode of Si–O and Si–O–M bonds appeared in MNMSDS, while just two bands were observed in NM and MNMCETAB.3.2. Effect of MNM on In Vitro Ruminal Batch Culture GP, CH4, Nutrient Degradability, and Partitioning FactorTable 3 shows that NM treatment had the highest (p < 0.01) GP compared with antibiotic monensin and all MNM treatment except the low level of MNMSDS, while no differences were observed between the NM and the control. The contrast tests were significant for both MNM products. A linear decrease (p < 0.01) in GP values was observed by increasing the supplemental level of the modified clays. Similar CH4 reductions (p < 0.01) were observed by all MNM products and monensin treatments compared with the control. The high dose of both MNM treatments resulted in the highest reduction (p < 0.01) in CH4 production among all the experimental treatments. The contrast analysis showed that the decrease in CH4 was in a dose-dependent manner by MNM products; MNMSDS reduced CH4 in linear (p < 0.01) and quadratic (p = 0.02) trends, while MNMCETAB declined CH4 in a linear (p < 0.01) trend.Monensin tended to reduce (p = 0.08) TDOM compared with MNMCETAB treatments and decreased (p < 0.01) TDNDF compared with all MNM treatments. The contrast analysis showed that TDOM and TDNDF were not affected by MNMSDS supplementation, while quadratic increases (p = 0.01) were recorded with the increasing doses of MNMCETAB supplementation. All MNM treatments (except MNMSDS low) enhanced (p < 0.01) the PF compared with the control. Increasing dosages of MNMsds showed linear increases (p < 0.01) in PF values, while MNMCETAB showed both linear (p < 0.01) and quadratic (p = 0.01) increases in PF values.3.3. Effect of MNM Supplementation on In Vitro Ruminal Batch Culture pH, NH3-N and SCFAsThe results of the effects of the experimental montmorillonite types on in vitro pH, NH3-N, and SCFAs are shown in Table 4. The high MNMCETAB treatments increased (p < 0.01) ruminal pH compared with the control and monensin treatments. The contrast test showed that MNMCETAB quadratically reduced (p = 0.01) in vitro ruminal pH while MNMsds did not affect the pH values. The high doses of both MNM types, NM and monensin, decreased in (p < 0.01) NH3-N compared with the control. Both MNM types resulted in linear reductions (p < 0.01) in the NH3-N concentrations. The high MNMSDS and all MNMCETAB treatments increased (p < 0.01) the protozoal count compared with the monensin, and both MNM types linearly (p < 0.05) increased the protozoal count. The experimental treatments did not affect the total SCFAs concentrations, while modifications of molar proportions of individual SCFAs were observed. Increases in the acetate molar proportions and the acetate-to-propionate ratio were observed (p < 0.01) in the MNM treatments compared with monensin. Linear and quadratic increases (p < 0.01) in acetate molar proportions were marked by increasing levels of both MNM types. Monensin followed by MNMCETAB treatment had the highest (p < 0.01) propionate molar proportions compared with other treatments. Treatments with MNMSDS and MNMCETAB had increased (p < 0.05) the propionate molar proportions linearly and quadratically. All MNM treatments and monensin presented similar reductions (p < 0.01) in butyrate compared with NM and control treatments. Linear and quadratic declines (p < 0.05) in butyrate were observed in MNM treatments. All of the experimental feed additives reduced isovalerate compared with the control (p < 0.01), while the high MNMCETAB treatment had higher (p = 0.05) isobutyrate than monensin. Linear and quadratic decreases (p < 0.01) were observed by both MNM types, while MNMCETAB presented quadratic increase (p = 0.03) in isobutyrate molar proportions.4. DiscussionNatural montmorillonite is a 2:1 phyllosilicate clay and has a unit crystal lattice formed by one alumina octahedral sheet sandwiched between two silica tetrahedral sheets; its interlayer contains water molecules and inorganic cations [4]. Due to this unique form, NM has a high CEC and surface area compared with other 1:1 clays. The mechanical grinding and modification of the natural montmorillonite can lead to the interlayer’s collapse and can affect the swelling capacity and surface charge of the modified clays [4]. In this work, the physicochemical properties of the resultant MNM products were affected by the used surfactants compared with NM. The decrease in EC after CETAB and SDS modifications indicated that few electrons could move from the valence band to the conduction band [4]. Electrical conductivity is an indicator of salinity to measure a substrate’s ability to allow for the transport of electric charges; thus, the EC of clays may affect ruminal passage rate of the digesta, dilution of feed particles, and microbial degradability [21].A high CEC was observed for the experimental NM (77.5 mmol/100 g), which became higher by CETAB and SDS modifications. This high CEC of both MNM types can indicate the high number of metal hydrolysates and ions that can be intercalated into the clay interlayer space [22], which in turn improves the clay activity compared with NM. This can be indicated by the frequency shifts and intensity of the hydroxyl H–O–H bond detected by FTIR analysis in the MNM clays compared to NM. Additionally, at the medium frequency range, a new band related to Si–O–Si bond [4] appeared only for both MNM clays, while it was absent in NM. These frequency shifts indicate the higher hydrophobicity of the resultant MNM clays than NM [22]. The most likely explanation for the differences between MNMCETAB and MNMSDS in CEC is the way in which the experimental surfactants bonded the clay interlayer space, which affected the structure and surface affinity of the resultant MNM products. Anionic surfactants as SDS have weaker interactions with the montmorillonite interlayer than cationic surfactants as CETAB [8]. Anions (SO3−) of SDS can be adsorbed on the edges of montmorillonite and compensated for its positive charges [8], while cations (NH4+) of CETAB can be adsorbed on the flat surface of the clay. This partly explains the higher negative charge of MNMCETAB than MNMSDS.Three bands at the low-frequency range corresponding to the bending mode of Si–O and Si–O–M bonds [4] appeared by FTIR analysis in MNMSDS, while just two bands were observed in MNMCETAB. This result could be due to the functional sharing group of SDS with the structure R-O-SO3−. The increase of negative charges of MNMCETAB might be favorable to enhance its affinity with cationic matters, which might improve the adsorption capacity. These results confirm our first hypothesis that the physicochemical properties of MNM can be enhanced by the mechanical nano grinding and modification of NM. Thus, we evaluated their effects on rumen fermentation properties.Rumen microbial fermentation is associated with the formation of greenhouse gasses (mainly CO2 and CH4). Montmorillonite is a potential adsorbent to capture CO2 through a reaction between CO2 molecules and its interlayer –OH groups by forming –HCO3− species, which in turn can react with other interlayer cations [23]. The reduction in GP caused by MNM types (especially MNMCETAB) may suggest that MNM had a higher absorbance capacity to capture CO2 than the NM. The literature reported that the modified montmorillonite has a higher CO2 reversible retention capacity than the NM due to increases in hydrophobic surface, interlayer spacing, and intercalation of organic cations between the base –OH sites of the clay and the CO2 molecules [23]. High CEC, shifts of the frequency and intensity of the hydroxyl H–O–H bonds detected by FTIR analysis, and increased negative charge of the MNM clays compared with NM may enhance the absorptive efficiency of MNM to capture CO2. It may be speculated that the CO2 adsorption is also varied by the solvated cations situated in the MNM interlayer spacing; hence, MNMCETAB was the most effective clay to reduce GP.Reductions in GP and CH4 were consistent with enhancements in protozoal count and degradability of OM and NDF by MNM types, while this phenomenon did not appear after monensin treatment in the present study, in which CH4 inhibition by monensin was consistent with adverse effects on nutrient degradability and protozoal count. These results suggested that monensin had a different CH4 reduction mechanism from that of MNM. The antibacterial activity of sodium monensin against H2 producing bacteria (including methanogens and cellulolytic bacteria) arises from disrupting the cell membranes through the ion transport of H+/K+ and Na+/H+ cations [1,24]. Monensin is also known for the inhibition effects of ruminal fungi and protozoa, which contribute to fiber degradation [1,3]; thus, it partly explained the decreased TDOM combined with CH4 reduction by monensin therein. On the other hand, enhancing the protozoal count, TDOM, and TDNDF by MNM would promote H+ production. Hydrogen is the major intermediary metabolite in the ruminal degradation of NDF and OM that Archaea mainly use to reduce CO2 into CH4. Thus theoretically, enhancing the OM and NDF degradability promotes CH4 formation [1,25]. Therefore, CH4 reduction caused by MNM would indicate that it may bind not only CO2 but also H+. The increase in the intensity on the absorption bands of the OH group detected by FTIR analysis rather than the high negative charge zeta potential of the experimental MNMCETAB would indicate the increased ability to bind the acidic H+. Increases in ruminal in vitro batch culture pH observed by MNMCETAB may confirm such speculation, which in turn was favorable for microbial NDF degradation and may prevent ruminal acidosis.Although the effect of MNM on the bacterial community was not evaluated (this has to be kept in consideration with MNM future studies), it can speculate that MNM has antibacterial effects against specific communities. However, both clay surfaces and bacterial cells have negatively charged sites; but the literature confirmed the ability of modified montmorillonite clays to bind them [5]. This is because of the presence of positively charged interlayer ions of the clay. In the current study, the changeable cations in MNMSDS and MNMCETAB in the clay edge or surface sites may affect the binding of rumen microbes to MNM surfaces through extracellular polysaccharides of the bacterial cell wall and, as a consequence, may affect the in vitro fermentation, including CH4 formation [5]. It seems that both MNM types can affect methanogenesis by possessing direct antibacterial activity since the protozoal counts and TDNDF were enhanced [25]. The literature confirmed the synergistic relationship between protozoa and methanogens. Protozoa can provide them with their end metabolites, including H2 [1]; thus, the protozoal count can indicate whether the treatments affected directly or indirectly the CH4 emission [1,3]. The anionic organo-sulfate surfactants (e.g., SDS) possess antibacterial and anti-inflammatory properties by sharing R–O–SO3− functional groups [26]; thus, it may affect the antibacterial activity of the prepared MNMSDS. The more substantial reduction in CH4 caused by MNMCETAB may be due to the quaternary positively charged ammonium group that can interact with Gram-positive bacterial cells, disrupt their cell membranes, and finally causes cell lysis [27]. Moreover, nano-clays have higher anti-methanogenic activity without adverse effects on the TDOM compared with their natural form [9]; this can partly explain the low effectiveness of NM to affect GP and CH4 compared with MNM in the current study.Enhancements in PF values may also contribute to the CH4 reduction observed by MNMCETAB [28]. Removing H+ from the rumen ecosystem is known to increase ruminal pH and to stimulate ruminal microbial activity; thus, when CH4 decreases, H+ may be used for producing SCFAs to ensure optimal ATP yield for the microbial mass production [2]. Increasing ruminal pH may increase protein solubility and generate branched-chain volatile fatty acids (BCVFA) production as isovalerate and isobutyrate [29]. Thus, it partly explains the increase in isobutyrate molar proportions consistent with high protozoal numbers and PF by MNMCETAB treatment. A puzzling finding of the current study was the decrease in the isovalerate molar proportion found by all clay treatments compared with the control. No clear explanation for this finding can be presented. Branched-chain volatile fatty acids (BCVFA) such as isovalerate and isobutyrate can be produced from leucine and valine degradation, respectively [30]. Consequently, rumen microbes utilize the produced BCVFA to promote protozoa and microbial protein synthesis [31,32]. Thus, it can be assumed that clay treatments may likely be incorporated differently into the rate of microbial degradation of these amino acids and/or BCVFA utilization. Apajalahti et al. [30] found that not all BCVFA produced are incorporated similarly to the microbial protein synthesis.The typical mode of action to reduce CH4 emission by monensin has occurred in this study by enhancing the redirections of the SCFAs pattern towards more propionate molar proportions and by reducing the acetate-to-propionate ratio [24]. The declines in ruminal in vitro batch culture pH, protozoal abundance, and TDNDF caused by monensin were favorable conditions for propionate producers [33]. On the other side, the associative enhancements in ruminal in vitro batch culture pH, protozoal numbers, and TDNDF were favorable conditions to acetate producers [3]; thus, acetate proportions were enhanced by MNM clays. Monensin inhibits Gram-positive bacteria, which are involved in protein degradation [24]. Therefore, further indications that monensin reduced the diet protein degradation can be provided by low TDOM, BCVFA, and ammonia values. Theoretically, enhancing TDOM may increase ammonia production; thus, it seems that ammonia reduction caused by MNM treatments was not a result of inhibition of protein degradation. Even NM treatment poetically reduced ammonia concentration without affecting TDOM. These results could be related to the presence of the acidic functional groups of the montmorillonite rather than the clay pore structure, which can enhance ammonia capture capacity to the clay. This function might be improved after the SDS or CETAB modifications because of the increases in CEC and shifts in the hydroxyl H–O–H bonds in addition to the more negative charge of MNM clays. These results may confirm our second hypothesis that MNM clays can modify the in vitro microbial fermentation, including CH4 emission, and this effect was type- and dose-dependent.5. ConclusionsTwo different feed additives of MNM have been developed at the nanoscale using cationic (CETAB) and anionic (SDS) surfactants. The modification and the mechanical nano grinding enhanced the physicochemical properties of the natural montmorillonite clay. Both MNM types had lower EC and higher CEC values than the natural clay. The MNMCETAB showed a more significant negative charge than the other clays. All MNM clays and monensin successfully reduced the in vitro ruminal batch culture CH4 production and ammonia concentration, while MNMCETAB enhanced TDOM, TDNDF, and pH compared with monensin. The experimental feed additives differently modified the SCFAs pattern. All MNM clays increased the acetate molar proportions, while only monensin increased propionate molar proportions. Under the conditions of this study, clay modified by cationic surfactant was more efficient than the anionic surfactant to modify rumen fermentation properties. The MNMCETAB supplemented at 0.5 g/kg can be used as a novel natural feed additive to reduce CH4 without adversely affecting rumen fermentation or fiber degradability. These results emphasized that MNM clays can modulate in vitro microbial fermentation patterns in different pathways from that of monensin.

animals : an open access journal from mdpi

10.3390/ani12040441

PMC8868349

Horseback safari rides, where tourists are led by experienced guides on horseback to find and observe wildlife, are a popular activity in Africa. However, close encounters between horses and wildlife on safari rides may be stressful for both types of animals. In this study we looked at the behaviour of horses and wildlife during close encounters on horseback safari rides, focusing on their behaviour at the start and end of each encounter, and the most extreme behaviour seen. Encounters with seven wildlife species were observed, all large herbivores. The seven species differed in their behaviour towards the horses. The horses also differed in their behaviour towards the different wildlife species, being more wary of giraffe. Horses generally approached the wildlife species at walk and few flight behaviours were observed. Further studies, including those incorporating physiological measures of stress, are recommended.

In Africa, wildlife-watching experiences create substantial revenue from tourists that can finance wildlife conservation. Horseback safaris, where an experienced guide takes guests through the bush on horseback to observe plains game species, are a popular activity. Close encounters between ridden horses and game species are unnatural and potentially stressful situations, and horseback safaris may have adverse impacts on both the horses and the wildlife they have come to observe. This study aims to provide a preliminary insight into the behavioural responses of horses and herbivorous plains game species, including giraffe, zebra and impala, as a proxy measure of the potential welfare implications of horseback safaris. Seventeen group safari rides were observed encompassing 72 encounters with plains game species. Game species differed in their response to encounters with the horseback safari ride. Equine response behaviour appeared to be influenced by the species of game encountered. Horses seemed more wary of giraffe than other species, with a higher percentage of horses showing stationary and retreat behaviour at the start of giraffe encounters. They were also most likely to shy at giraffe. The behavioural responses suggest that game encounters can elicit a stress response in both animal groups, although it is not usually extreme, potentially indicating that some degree of habituation has occurred. Balancing the welfare of both the horses and the plains game species along with tourist preferences may be challenging in this context.

1. IntroductionIn Africa, the financial cost of wildlife conservation is significantly underpinned by income from tourists [1]. Safari adventures are a major part of this, and there are a plethora of wildlife watching experiences available, from luxury hot air balloon rides to guided off-road drives in 4 × 4 s and horseback safaris. The direct and indirect impact of wildlife tourism on the resident wildlife can be considerable [2], but is typically balanced against the potential benefits such as raised public awareness of conservation issues and the generation of revenue to finance conservation work [3,4,5]. Tourist activity can damage natural ecosystems [6] and has been associated with an increase in physiological indicators of stress in a range of species, including elephants [7], gorillas [4], howler monkeys (Alouatta pigra) [8], and capercaillie (Tetrao urogallus) [9], often with corresponding changes to their behaviour, e.g., in mountain hares (Lepus timidus) [10] and elephants (Elephas maximus) [11].Horseback safari rides are a popular activity in Africa, with a range of different lengths and difficulties available to suit tourist requirements. During these rides, an experienced guide takes the guests through the bush on horseback, to find a range of plains game species. The term “plains game” is commonly used in the safari industry (Cumming 1989) to refer to herbivorous plains dwelling species, including zebra (Equus quagga) and species of antelope. Safari rides typically focus their encounters on plains game species which are herbivorous, not predators. These are more suitable for close tourist encounters on horseback, particularly for beginner riders, than the so-called dangerous game species such as the ‘Big Five of Africa’ (lion (Panthera leo), leopard (Panthera pardus), rhinoceros (Ceratotherium simum), elephant (Loxodonta africana) and African buffalo (Syncerus caffer)), as well as crocodile (Crocodylus niloticus) and hippopotamus (Hippopotamus amphibius).Close encounters between ridden horses and game species are unnatural and potentially stressful situations, and horseback safaris may have adverse impacts on both the domestic horses being used to carry tourists and the wildlife they have come to observe. Horses and herbivorous plains game species typically use locomotion as their anti-predation strategy, meaning they are highly vigilant, sensitive to the behaviour of other individuals within their group and have a well-developed flight response [12,13,14,15]. Grazing ungulates are also known to respond to behavioural unease of other herbivorous prey species within their proximity [16,17]. The potential for two-way emotional contagion [18] between horses and game species during horseback safari rides may significantly impact the welfare of both the horses and the game species they encounter.While the welfare of equines involved with the tourist industry is increasingly prioritised by customers and operators [19,20,21], there has yet to be any research published into the welfare implications of use as a horseback safari horse. Indeed, intrinsic welfare costs have not been investigated within many sectors of the equestrian tourism industry, although it has in other working horse roles, such as police horses [22], working equids in developing countries [23,24], racehorses and endurance horses [25]. Björlinger and Johansson [26] and Giampiccoli [27] investigated welfare standards in horse and mule tourism, respectively, identifying key issues such as poorly fitting tack, overwork, and limited access to water and good quality feed. However, neither evaluated the impact of the specific activity the horses and mules were being asked to perform.The welfare consequences of human disturbance on plains game species has received less research attention than the more charismatic Big Five species. In impala in the Serengeti, faecal glucocorticoid metabolite (FGM) concentration is affected more by forage quality than human disturbance [28], and although impala may be more vigilant in tourist areas, there is some evidence of habituation to human presence [29]. Other research has reported diurnal changes in African ungulate behaviour as a result of human disturbance: in areas where human hunting is allowed, three large African ungulates (impala (Aepyceros melampus), greater kudu (Tragelaphus strepsiceros) and sable antelope (Hippotragus niger)) are more likely to drink at night [30], presumably due to hunting pressures. Diurnal and seasonal changes in grazing behaviour were observed by Schuette et al. [31] as ungulates selected a foraging strategy that was optimal for the level of human disturbance, the risk of predation and the vegetation quality. While Yamashita et al. [15] identified an influence of human settlements on the antipredator behaviour exhibited by African ungulates, they highlight the lack of detailed information on how different human activities may affect wildlife behaviour. The immediate, acute, impact of an encounter with tourists does not appear to have been considered previously.Our primary hypothesis was that horseback safari rides would be stressful for both the horses and the plains game species they encounter, with a secondary hypothesis that that response will vary with game species. Therefore, this study aims to provide a preliminary insight into the behavioural responses of horses and plains game species as a proxy measure of the potential welfare implications of horseback safaris.2. Materials and Methods2.1. EthicsHuman and animal ethical approval for this study was granted by the following University of Bristol ethics committees: the Animal Welfare and Ethical Review Body (AWERB) and the Faculty of Health Science Student Research Ethics Committee (HSSREC; Ref: 65602). All riders participating in the study were provided with a Participant Information Sheet and gave informed consent to participate via reading and signing consent forms. Riders were assigned a Participant Identification Number to ensure anonymity.2.2. Study AreaThe study was conducted at an equestrian centre set in a popular private game reserve in Gweru, Zimbabwe. The reserve is 1214 hectares of savannah grassland and houses a variety of plains game species ranging freely including blue wildebeest (Connochaetes taurinus), impala (Aepyceros melampus), South African giraffe (Giraffa camelopardalis giraffa), Burchell’s zebra (Equus quagga burchellii), greater kudu (Tragelaphus strepsiceros), waterbuck (Kobus ellipsiprymnus) and red hartebeest (Alcelaphus buselaphus caama). All of the aforementioned species were observed during the study and are hereafter referred to collectively as plains game species for the purposes of this study.It should be noted that captive lions and elephants also existed within the reserve. However, these species were both part of conservation projects and not free-ranging. Their enclosures were located in a completely separate area of the reserve away from the safari ride routes and horses. Therefore, neither species would have been directly encountered by the horses or game species, although they may have been aware of their presence through auditory and olfactory means. No hunting by humans was permitted within the reserve.2.3. HorsesThe centre was home to 30 horses at the time of the study which are used for a variety of disciplines including horseback safaris, where a guide takes guests around the park on horseback in search of game. The horses were either owned by the centre or kept on working livery and were all used regularly for horseback safari rides. However, due to illness and lameness, only 20 horses were involved in the study (see Table 1 for details of horses). Of these 20, 25% (n = 5) were safari horses, native African bush ponies which are bred and ideally suited for safari riding. The remaining 75% (n = 15) were Thoroughbred ex-racehorses. The Thoroughbreds were mainly used for polocrosse and show jumping but were also used for safari rides. Several had temperaments suitable for riding by beginners; however, many were only used by experienced riders. The horses’ management remained consistent for the duration of the study. All horses were fed a concentrate feed twice daily at 8:00 a.m. and 4:00 p.m. with appropriate quantities for their body condition and workload. The frequency and intensity of each horse’s exercise regime was also kept the same. All horses spent the day in small paddocks with grass and supplemented hay as it was dry season when they were not working. The Thoroughbred horses were kept individually stabled at night, each with a hay net, and the safari horses were allowed to roam the game reserve.2.4. RidersHorses were allocated to the riders by the safari organisers based on rider height, weight and self-reported riding experience. The researcher had no influence over horse allocation, or the specific horses used on each ride.2.5. Data CollectionPrior to formal data collection, a week-long reconnaissance study was conducted for method refinement. Data collection was carried out on all rides occurring throughout June and July 2018. An encounter was defined as the interaction between at least one game animal (of the seven herbivorous plains game species described earlier) and the group of horses on the ride, starting and ending when the guide leading the ride began to purposefully approach or retreat from the game, respectively.Due to the observational nature of the study, it was only possible to collect data when a ride was scheduled. The species of game encountered during each ride was purely due to chance.Before each ride, the horse and rider combinations, time and weather conditions were recorded. The researcher then joined each ride on horseback, remaining at the rear of the ride for the best view. The researcher and their horse were included as a rider and subject, respectively. Video footage was recorded throughout the ride on a GoPro HERO (2018) Action Camera (GoPro Inc, California, USA), which was attached to the researcher’s riding hat via a head strap.Although before the safari ride, riders had described their riding experience to the safari organisers to aid allocation of the most suitable horse, for study purposes their riding ability was scored by the researcher during the ride and each rider assigned a riding ability score from 1 (Total Beginner) to 7 (Professional) (see Table 2 for scores).Following each ride, the video footage was analysed. For each encounter, the game species and number of individuals in the group were recorded, as well as the duration of the encounter, in minutes and seconds, from the start when the guide began to purposefully approach the game, and the end when the guide began to purposefully retreat from the game.2.6. Game BehaviourThe response of the game to the approaching horses was recorded by continuous sampling using a predefined ethogram consisting of three categories of behaviour: stationary behaviour, approach behaviour and retreat behaviour with ordinal options defined within each category (Table 3). Behavioural scores were taken at the start and end of each encounter (initial score and final score), and the most extreme behaviour seen in each behavioural category during the encounter was also recorded (extreme score).For encounters where there was more than one game animal present, one initial, one final and one extreme score was assigned to the whole group of that game species for the encounter, according to the mode, or most common behaviour displayed. This is because individuals within herds generally perform the same or similar behaviours as each other; this is the ‘mood’ of the herd [34].animals-12-00441-t003_Table 3Table 3Ethogram of game response behaviours to encounters with horses on safari rides.BehaviourMeasurementDefinitionStationaryScale1Lying downSternal or lateral recumbency [35]2Grazing/Browsing(As relevant for the species)Grazing: Standing with head down eating grassy vegetation. Vegetation is gathered and broken off with the lips and tongue [35,36]Browsing: Standing with head up eating from trees and shrubs. Vegetation is gathered and broken off with the lips and tongue [35].3Standing at restUpright stance in a relaxed posture with head slightly lowered and eyes partly closed. Inactive and may be weight bearing on 3 legs [35].4Standing alertUpright stance with a rigid body position and neck elevated and head upright. Ears stiffly upright and pointing forwards. Focused eyes open and alert. Nostrils may be dilated [36,37].ApproachScale1Approach at walkForward movement towards the horses at a walk; a slow 4 beat gait [35,38].2Approach at trotForward movement towards the horses at a trot; a 2 beat gait with diagonal pairs [35,38].3Approach at canterForward movement towards the horses at a canter; a 3 beat medium speed gait [35,38].4Approach at gallopForward movement towards the horses at a gallop; a 4 beat fast gait [35,38].Retreat1Back upBackward movement to maintain or increase distance from the horses, by reversing at a walk [35].2Retreat at walkForward movement to maintain or increase distance from the horses at a walk; a slow 4 beat gait [35,36,38].3Retreat at trotMovement to maintain or increase distance from the horses at a trot; a 2 beat gait with diagonal pairs [35,36,38].4Retreat at canterMovement to maintain or increase distance from the horses at a canter; a 3 beat medium speed gait [35,36,38].5Retreat at gallopMovement to maintain or increase distance from the horses at a gallop; a 4 beat fast gait [35,36,38].2.7. Horse BehaviourAs the video footage was recorded from the researcher’s position behind the ride during the encounters, behavioural scores could be taken for each individual horse using focal sampling and an ethogram (Table 4). The ethogram was comparable to that developed for the game species, with the addition of the category ‘ear position’ and shying, bucking and rearing behaviour. The footage was analysed continuously throughout the encounter. Three behavioural responses for each horse were recorded: at the start (initial score) for the first behaviour displayed, and end (final score) for the last behaviour displayed, as well as the most extreme score observed throughout the encounter for each category (extreme score). If a behaviour was not observed, e.g., a stationary score could not be given because the horse was showing an approach behaviour, that category was recorded as zero. There were also additional event behaviours (shy, rear and buck) in the equine ethogram which were recorded as frequencies.While pilot testing indicated that reporting a group mood for game species using the behavioural mode adequately captured the response of the game to encounters with the horseback safari, the individual horses on a ride showed a wider range of behaviours in the same situation. Reporting the mode for each behavioural category failed to capture this variation. Consequently, behavioural scores were recorded for each horse at each encounter.Distance between the horses and game was intended to be recorded, however following the pilot study this was excluded due to logistical difficulties in recording accurate distances between the multiple individuals in the groups of horses and game.2.8. Data AnalysisAll data were recorded in Microsoft Excel (Microsoft Office, Microsoft Corporation, Redmond, WA, USA), excluding pilot study data which were not included in the final analysis.A descriptive account of the behavioural response of game and horses is reported as mean initial, final and extreme behavioural scores for each behaviour category across all encounters grouped by the seven plains game species observed.As both horses and game species were in groups at the time of the encounters, the individual level data were not truly independent. This confounding effect meant that statistical analysis was inappropriate for these data. Consequently, only descriptive results are reported.3. ResultsIn total, 17 group safari rides were observed over the data collection period. The rides lasted for a mean duration of two hours (range 1 h 14 min–2 h 22 min) and during these rides all 20 horses and 50 different riders were observed (the researcher, four full-time staff, 22 equestrian centre volunteer staff and 23 guests). Rider ability as scored by the researcher ranged from 1 (total beginner) to 7 (professional), with a median score of 4 corresponding to confident novice. Female riders made up 72% (n = 36) of the rider cohort. There were a median of 7 horses and riders per ride, with a range from 2–11.There were 72 encounters with plains game species across the 17 rides, with a mean of 4.24 encounters per ride (range 1–8) and a mean encounter duration of 2 min 43 s (range 38 s to 7 min 32 s), generating 196 min of footage of horseback safari horses–plains game species encounters.3.1. Response of Game to HorsesWildebeest were the most frequently encountered plains game species (26/72 encounters; Table 5) and were also encountered in the biggest groups, while red hartebeest were the least frequently encountered with only one individual encountered across the 17 safari rides (1/72 encounters; Table 5). Response to the approaching horses varied between species (Figure 1a), with zebra approaching the horses at a walk in 21% of encounters, and retreat behaviours seen when horses approached the game in 12% of wildebeest and 11% of impala encounters. Most frequently, game responded to the approaching horses by standing still, either grazing/browsing, standing at rest or standing alert. Kudu and impala more frequently retreated from the horses at higher speeds (Figure 1b; Table 5), thereby ending encounters rather than the horses moving away first.3.2. Response of Horses to GameThe response of horses to game varied between horses and with the game species they were encountering (Table 6; Figure 2). Horses most frequently approached game in walk or trot, although horses in 11% of giraffe encounters were observed to retreat from the encounter by walking forward. The most extreme ear position was ears held back during encounters with wildebeest, giraffe and impala. Two horses retreated by cantering from the same encounter with zebra, and one from an encounter with wildebeest. Fourteen horses, across nine encounters, retreated from wildebeest at a trot, nine horses across six encounters retreated from zebra at a trot and nine horses across seven encounters retreated from giraffe at a trot. All encounters with giraffe, waterbuck and red hartebeest ended when the horses moved away at a walk, as did the majority of encounters with impala and zebra.Across the 17 rides (72 encounters), there were 37 shies by twelve horses, with some individual horses exhibiting up to four instances of shying during one encounter. There were 8 shies during encounters with wildebeest (n = 6 horses), 14 during encounters with giraffe (n = 3 horses) 10 shies with zebra (n = 7 horses), 2 with impala (n = 2 horses), 2 with kudu (n = 1 horse), 1 with waterbuck (n = 1 horse). No horses shied during the sole encounter with Red Hartebeest. Of the twelve horses observed to shy, two were Safari horses and ten were Thoroughbreds.No horses were observed to rear or buck during any of the encounters with plains game species on any of the rides.4. DiscussionThis preliminary study provides some initial insights into the welfare implications of horseback safari rides for both the horses and the plains game species they meet, based on the behaviour of both species at the time of the encounter.The findings indicate that there were differences between game species in how they responded to their encounters with the horseback safari ride. Interestingly, zebra were typically calm and curious around the horses with 12% approaching the ride at the start of the encounter. The Burchell’s zebra (Equus quagga burchellii) encountered in this study are a subspecies of the plains zebra which are reported to be highly vigilant and reactive to human approach [36]. The findings from this study suggest that zebra may recognise horses as fellow equids and therefore not perceive them as a threat, despite the presence of human riders. Generally, the antelope species (wildebeest, impala, kudu, waterbuck and hartebeest) were highly alert and exhibited flight response behaviours, often retreating at speed. The response of kudu was the most extreme as 100% retreated at a gallop, although it should be noted that, due to the small sample sizes, conclusions about the behaviour of the kudu, waterbuck and hartebeest cannot reliably be made.The likelihood of encountering each species of game can in part be explained by their prevalence in the park, but also corresponds with their behaviour. Wildebeest were the most commonly encountered, which was expected due to their high abundance. They were also found to be less vigilant and prone to flight than the other antelope species, which may be due to habituation or simply their natural behaviour [40]. Although there are far fewer giraffe and zebra in the park, they were often encountered. This may be explained by their behaviour as they were found to be the least vigilant and most curious animals; they were the most likely to approach and least likely to retreat from the ride. Despite the large numbers of impala in the park, they were not often encountered, and this may be due to their high vigilance and flighty, easily alarmed nature [34]. The response behaviours observed by impala also support Schenkel’s [34] findings of their general behaviour. Waterbuck, hartebeest and kudu were rarely seen which is likely due to low numbers in the reserve, in addition to their high vigilance. Some degree of habituation to horseback safaris by the plains game species encountered was likely, as has been suggested in other human encounters [15] and in other species visited by tourists [41], and this may also have affected the behaviour observed.Equine response behaviour appeared to be influenced by the species of game encountered. The horses seemed more wary of giraffe than the other species, with a higher percentage of horses showing stationary and retreat behaviour at the start of giraffe encounters. They were also most likely to shy at the giraffe. Flight animals such as horses are more likely to flee from a persistent threat [34]. If the game retreats, it increases the distance between itself and the horses, thereby reducing the need for the horse to do so itself. However, the exhibited response behaviours towards zebra contradict this; the zebra showed low vigilance behaviour similar to the giraffe, and were observed to actively approach the horses, but this did not result in more flight responses from the horses. This may therefore support the earlier suggestion that zebra and horses recognise each other as equids and are consequently more at ease with one another. In addition to the influence of the game behaviour, it is possible that the differences in equine behavioural responses to different game species may also be affected by their size, appearance and smell.Overall, most horses displayed vigilant response behaviours during game encounters, with ears pricked forwards and standing alert. These behaviours do not, however, necessarily indicate fear or stress, but may rather show alertness, interest and curiosity. Flight behaviour, such as shying or retreating, is the most extreme response [36] and was rarely observed except for the occasional shy or backing up (4% of encounters). The horses were generally willing to calmly approach the game at a walk (71% of encounters). These results suggest that the horses did not usually exhibit behavioural indicators of severe stress during game encounters.Individual differences in how the horses responded to game encounters were evident but are impossible to draw out in this observational study. Personality differences may be involved [42] and are known to be associated with breed differences [43]. Within our sample of 20 horses, clear breed/type differences were observed. Thoroughbreds were more vigilant and less likely to approach the game than safari horses. Thoroughbred ex-racehorses are generally thought to be more highly strung [44] and are bred to be highly reactive for racing [43]. The Thoroughbreds in this study were generally younger and less experienced than their safari horse counterparts. This may have influenced the breed differences, as older horses with more experience of game encounters are more likely to be habituated to the situation and therefore be calmer and less reactive. Age is therefore a significant confounder that should be controlled for in further studies, alongside management. In this study the Thoroughbreds were stabled at night whereas the safari horses were grazed out in the game reserve. This meant that safari horses had greater exposure to game species. Furthermore, individual stabling has been shown to affect equine responsiveness to the environment [45]. It is important to note that behaviour may not necessarily reflect the internal stress state of the animal, particularly in prey species such as the horse, as it is adaptive for them to conceal their awareness and fear [36]. Additionally, horses are trained to suppress their natural reactions to perform behaviours desired by their riders [42]. The rider is likely to influence a horse’s behavioural response [46]; an experienced rider is more likely to give appropriate ridden cues (such as leg or rein aids) and ask for specific behaviours to control their horse, and the horse may be affected by the riders’ emotions and confidence [47]. Unfortunately, we were unable to explore this effect with data from the current study. The data do illustrate the potential value of employing gradual habituation protocols for new and young horses, to introduce them to all species of game they may encounter in a calm and positive manner.There were a number of limitations to this study which impair interpretation of the findings. Observations were conducted opportunistically at one horseback safari establishment where the researcher was unable to control any of the variables such as horse age, breed, management, rider ability and game species encountered. Furthermore, both the horses and game species were with at least one other conspecific during each encounter (with the exceptions of the waterbuck and red hartebeest), so the behaviour of each individual within the group was potentially influenced by the behaviour of other conspecifics, as well as by the other species (game or horses). This limited the ability to conduct any meaningful statistical analysis on the data as it was impossible to control for all confounders nor to treat the data as independent. Physiological measures, such as faecal glucocorticoid metabolites, heart rate and heart rate variability, were not taken to support the behavioural observations, although as previously noted in many instances behavioural and physiological indicators of stress do not always align [22,42,48]. Future studies should attempt to tease out these confounding variables to produce a more robust evaluation of the welfare implications of safari rides on the animals involved, including evaluating the influences of variables such as horse breed/type and rider ability.That said, our findings still have merit and suggest that the welfare implications of horseback safari encounters are generally not extreme for either party. In a recent review, Bateman and Fleming [49] conclude that there is often little empirical evidence for negative interpretations of wildlife–tourist encounters. Wildlife–tourist encounters may impact the animals involved in a myriad of ways both short- and long-term, directly and indirectly [49]. This study looked at the short-term behavioural impact of direct encounters for the horses and game species involved to provide an initial insight into the potential implications of this industry. Future studies would benefit from exploring the longer-term implications of these encounters using a range of behavioural, physiological and survival measures.5. Animal Welfare Implications and ConclusionsThis study aimed to provide an initial insight into the behavioural response of horses and plains game species during horseback safari encounters, an area which has not been previously studied. The behavioural responses exhibited by the horses and game animals suggest that game encounters can elicit a stress response in both animal groups, although it is not usually extreme, indicating that some degree of habituation has potentially occurred. These preliminary findings imply that the welfare implications of horseback safari rides are not entirely negative, although this varies with the game species encountered. Kudu, waterbuck and red hartebeest showed the most extreme flight response towards the ride, while giraffe and zebra appeared less affected, and even curious, with a small percentage approaching the horses. That said, the horses’ behaviour suggested that they were more wary of giraffe than the other game species. Balancing the welfare of both the horses and the plains game species along with the preferences of the tourists may be challenging in this context. Before firm conclusions can be drawn on the welfare implications of horseback safari rides, it is recommended that future studies utilise multiple welfare indicators, encompassing behavioural and physiological measures, and evaluate the long-term and indirect consequences of horseback safari rides. This information may then be used to inform best practice guidelines within the industry, such as maximum distances that different species can be approached by riders on horseback, to reduce stress, improve welfare, and ensure safety and enjoyment for riders.

animals : an open access journal from mdpi

10.3390/ani12070934

PMC8997108

Velvet antler is the only organ in mammals that can completely and circularly regenerate, which involves the co-development of a variety of tissues including cartilage. Thus, velvet antler can provide an ideal model for studying chondrogenesis, endochondral ossification and rapid tissue growth. However, the mechanism of rapid growth and regeneration of velvet antler is still unclear. In this study, we conducted integrated analysis of the transcriptome and proteome of antler cartilage tissues at different growth stages. The results showed that gene13546 and its coding protein rna13546 were annotated in Wnt signaling pathway. They may play roles in antler rapid growth and regeneration.

The velvet antler is a unique model for cancer and regeneration research due to its periodic regeneration and rapid growth. Antler growth is mainly triggered by the growth center located in its tip, which consists of velvet skin, mesenchyme and cartilage. Among them, cartilage accounts for most of the growth center. We performed an integrative analysis of the antler cartilage transcriptome and proteome at different antler growth stages. RNA-seq results revealed 24,778 unigenes, 19,243 known protein-coding genes, and 5535 new predicted genes. Of these, 2722 were detected with differential expression patterns among 30 d, 60 d, and 90 d libraries, and 488 differentially expressed genes (DEGs) were screened at 30 d vs. 60 d and 60 d vs. 90 d but not at 30 d vs. 90 d. Proteomic data identified 1361 known proteins and 179 predicted novel proteins. Comparative analyses showed 382 differentially expressed proteins (DEPs), of which 16 had differential expression levels at 30 d vs. 60 d and 60 d vs. 90 d but not at 30 d vs. 90 d. An integrated analysis conducted for DEGs and DEPs showed that gene13546 and its coding protein protein13546 annotated in the Wnt signaling pathway may possess important bio-logical functions in rapid antler growth. This study provides in-depth characterization of candidate genes and proteins, providing further insights into the molecular mechanisms controlling antler development.

1. IntroductionGansu red deer (Cervus elaphus kansuensis) is commonly known as the white rump deer or Qilian red deer. It is an endemic species of the deer family in China and is mainly distributed in the forest areas of the Qilian Mountains. Research on C. elaphus kansuensis is limited with the focus on ecology, production performance, active substances, and functional gene cloning; however, omics studies are rare [1].Deer antler is a unique cranial appendage derived from the forehead of male deer. It is attached to the skull by a tissue called the pedicle, from which the antler grows. Deer antler growth is relatively slow in the first month after the base shedding (30 d). Two months later (60 d), antler enters a rapid growth phase, which is the fastest growth rate of any mammal. At this stage, antler can grow up to 2 cm per day, and antler cells can reach more than 30 times faster than the proliferation of cancer cells, but no cancerous changes occur. At 90 days, the growth rate of velvet antler slows down and gradually ossifies, entering the ossification stage [2]. Histologically, deer antler is made up of several tissues such as velvet-like skin, cartilage, bone, nerves, and blood vessels [3]. Deer antleris the only mammalian organs that iscapable of annual regeneration and rapid growth [4]. Therefore, it is a premier model organ for cancer and regeneration research. The underlying molecular mechanisms involved in antler regeneration and rapid growth are the focus of attention of many biologists.Advances in high-throughput technologies, including genomics, transcriptomics, proteomics, and metabolomics, have provided powerful methods to study antler development [5,6,7]. However, few studies have used an integrated multi-omics approach to characterize antlers in greater detail and comprehensively [8,9,10,11]. It is difficult to explain the biological regulatory networks of complex traits using only a single omics approach [12]. Integrative transcriptomics and proteomics analysis are more conducive to studying models of the regulatory mechanisms underlying the biological processes [13].Transcriptomic and proteomic techniques have provided promising and powerful tools for mechanistic investigation as reflected in the increasing number of studies [13,14,15]. In addition, integrated transcriptomic and proteomic analyses comprehensively explain the influencing factors through experimentally observed differences in messenger ribonucleic acid (mRNA) and protein expression.In this study, we combined mRNA sequencing with isobaric tags for relative and absolute quantitation (iTRAQ) to generate datasets encompassing primary (30 d), rapid (60 d), and ossified (90 d) stages of antler development. Our results provide a deeper understanding of rapid antler growth and cartilage development. To the best of our knowledge, this is the first study comprising genetic research on C. elaphus kansuensis the results of which provide a molecular basis for the further study of this species.2. Materials and Methods2.1. Animals, Sample Collection, and PreparationIn this study, C. elaphus kansuensis were fed on a semi-wild Shandan horse farm. Velvet antlers at different growth stages (30 d, 60 d, and 90 d) were collected from three adult male deer at each time point. Before we collecting antlers, the male deers were were anaesthetized with Mian Naining (Lu Mian ning, Beijing, China). After the velvet antlers were sawed off from the antler pedicle, potassium permanganate was used to stop the bleeding immediately. Then Lu xingling (Beijing, China) was injected to wake up red deers and release them into the wild. Cartilage parts in the antler tip were separated for transcriptome and proteome analyses and data verification. All samples were stored in liquid nitrogen tanks, brought back to the laboratory, and stored at −80 °C. All experimental protocols were approved by the Institutional Animal Care and Use Committee of Qinghai University (Xining, China), and all methods were carried out in accordance with approved guidelines and regulations (Code: SL-2022024).2.2. RNA Extraction and SequencingTotal RNA from the antler cartilage tissues of three Gansu red deer was extracted using The TRIzol reagent (Thermo Fisher Scientific, Waltham, MA, USA), and the extraction procedure was performed according to the manufacturer’s instructions. The extracted total RNA was quality checked using Agilent Bioanalyzer 2100 (Agilent, Santa Clara, CA, USA) and quantified using NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). The RNA samples with RNA Integrity Number (RIN) ≥ 8 were selected for preparing libraries.In total, 1 μg of RNA per sample was used as the input material for the RNA sample preparations. The NEBNext9 UltraTM RNA Library Preparation Kit for Illumina (NEB, USA) was used to conduct sequencing libraries according to the manufacturer’s procedures, and index codes were added to attribute sequences to each sample. Sequencing was performed using an Illumina HiSeq 2500 system (Illumina).2.3. Protein Extraction, iTRAQ Labeling, and Liquid Chromatography-Electrospray Ionization-Tandem Mass Spectrometry AnalysisTotal proteins of each sample were extracted using lysis buffer (7 M urea, 2 M thiourea, 2% CHAPS, proteasome inhibitor). The Qubit fluorescent protein quantification kit (Invitrogen) was used to quantify the protein concentration according to the manufacturer’s instructions. Protein peptides (100 μg) from each sample were labeled using the 8plex iTRAQ reagents multiplex kit (ABI, Foster City, CA, USA).The iTRAQ-labeled samples were analyzed using a Q-Exactive mass spectrometer (Thermo Fisher Scientific) coupled with a nano high-performance liquid chromatography system (UltiMate 3000 LC Dionex; Thermo Fisher Scientific).Peak recognition was performed on the peptide obtained by mass spectrometry, and a reference database was established. Then, Mascot 2.3.02 and Proteome Discoverer 1.4 (Thermo Fisher Scientific) were used for library search identification and quantitative analysis. Finally, the obtained peptides and proteins were analyzed using Uniport and NCBI databases, and their functions and metabolic pathways were annotated by Gene ontology (GO) annotation (q < 0.05) and Kyoto Encyclopedia of Genes and Genomes (KEGG) annotation (q < 0.05). The p value was adjusted using q value.2.4. Transcriptome and Proteome Data AnalysisTranscriptome data were analyzed in accordance with RNA-Seq workflow (https://github.com/twbattaglia/RNAseq-workflow (accessed on 15 February 2017)). Raw data (raw reads, FASTQ files) from each library were preprocessed using in-house Perl scripts. Briefly, the adapters, sequences with unknown nucleotides larger than 10% (poly-N, N% > 10%) and low-quality reads (quality score < 20) were filtered from raw data. Concurrently, the sequences quality including Q20, Q30, GC content, were calculated. All downstream analyses were based on high-quality clean data. Hisat2 [16] was used to verify reads with a perfect match or one mismatch were further analyzed and annotated based on the reference genome of C. elaphus (https://www.ncbi.nlm.nih.gov/genome/?term=red+deer (accessed on 2 January 2018)). Then, clean reads were assembled and quantified using StringTie [17] to obtain contigs and unigenes. Differential expression analysis was performed using the EBSeq (default parameters, qtrm = 0.5) [18].Significant differentially expressed genes (DEGs) among three groups were screened with the threshold of fold change ≥ 2 and false discovery rate (FDR) < 0.01 [19,20]. FDR was adjusted p value [7]. Only DEGs were subjected to further analyses.For proteome analysis, the raw mass data were processed for peptide data analysis using Proteome Discoverer 1.4 (Thermo Fisher Scientific) with FDR = N(decoy) ∗ 2/(N(decoy)+ N(target) of < 1% and expected cutoff or ion score of < 0.05 with 95% confidence to search the Uniprot Human Complete Proteome database. Protein probabilities were assigned using the Protein Prophet algorithm [21], and proteins with at least two unique peptides were identified. Differential expression levels of proteins were calculated using the Mann–Whitney test and calibrated using the Benjamini–Hochberg correction. Only proteins with p value < 0.05 and fold-change ≥ 1.5 were defined as significant differentially expressed proteins (DEPs). The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE [22] partner repository.2.5. Gene Functional Annotation of DEGs and DEPsBased onClusters of Orthologous Genes (COG) [23], GO [24] and the KEGG database [25], the functions of DEGs and DEPs were annotated.The Database of Clusters of Orthologous Genes (COGs) [23] is a Database of homeologous protein information maintained by NCBI. A protein can be compared to all the proteins in COGs and grouped into the appropriate COG cluster.GOseq software was used for GO enrichment analysis of differential genes based on the Wallenius noncentral hypergeometric distribution mathematical model [26]. GO terms were considered to be significantly enriched with q < 0.05.KEGG [25] is a database that integrates genomic, chemical, and phylogenetic information. The core of them are KEGG PATHWAY and KEGG ORTHOLOGY databases. In the KEGG PATHWAY database, biological metabolic pathways are classified into six categories of processing: cellular, environmental information, genetic information, human diseases, metabolism, and organismal systems. We used KOBAS software [27] to test the statistical enrichment of DEGs and DEPs in the KEGG pathways. KEGG pathways were considered to be significantly enriched with q < 0.052.6. Integrated Analysis of Transcriptomic and Proteomic DataIntegrated analysis was conducted to explore the consistency between proteome and transcriptome levels of antler cartilage at 30 d, 60 d, and 90 d. Spearman’s correlation test [28] was used to assess the correlation between gene expression levels in the transcriptome and corresponding proteins in the proteome. The results were divided into three categories: DEGs and DEPs had the same expression trend, DEGs and DEPs had reverse expression trend, and DEGs and DEPs had no expression difference.3. Results3.1. Data AvailabilityRaw FASTQ files of the transcriptome were submitted to the National Center for Biotechnology Information Sequence Read Archive (NCBI SRA) database under the BioProject Accession Number PRJNA772802 [29](Transcriptome profiles of velvet antler in Gansu red deer. Available online: http://www.ncbi.nlm.nih.gov/bioproject/772802 (accessed on 19 October 2021)). Proteomic data have not been submitted. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium (http://www.proteomexchange.org (accessed on 19 October 2021)) with the dataset identifier PXD032668.3.2. RNA Sequencing and AssemblyWe performed RNA-seq to investigate the mRNA expression profiles of cartilage in antler tips of C. elaphus kansuensis at different growth stages. After sequencing and filtration, we generated 50,552,622, 69,330,832, and 44,833,400 clean reads of the antler growth center for 30 d, 60 d, and 90 d stages, respectively. We obtained 40,317,002, 56,820,843, and 35,010,141 mapped reads after alignment to the reference genome, accounting for 79.75%, 81.96%, and 78.09% of clean reads from the three libraries, respectively. After mapping the read assembly using StringTie software, we obtained 24,778 unigenes. The lengths of the assembled unigenes ranged from 90 bp to 82,366 bp, with an average of 2269 bp and N50 of 3894 bp. Of these 24,778 unigenes, 19,243 were mapped to known protein-coding genes and 5535 were mapped to new predicted genes. After applying the fragments per kilobase of transcript per million mapped reads (FPKM) filter (FPKM = 0), 18,707, 19,694, and 18,985 unigenes were detected in 30 d, 60 d, and 90 d libraries, respectively. Among these genes, 17,527, 18,307, and 18,004 were detected (FPKM ≥ 0.1), whereas the remaining 7251, 6471, and 6774 genes were considered to be very low in expression or not expressed (FPKM < 0.1) in the three libraries.Expression analysis showed that gene4406 and gene13363 were the most highly expressed in all three libraries (Table 1). The co-expressed gene7568, gene2851, and gene21360 also had higher expression levels. Among the most highly expressed genes, most were known genes; hence, only one and three predicted novel genes were selected in the 30 d and 90 d antler growth centers, respectively.3.3. Differentially Expressed GenesGene expression levels were calculated according to FPKM arithmetic; FDR < 0.01 and |log2 (foldchange)| ≥ 2 were set as the threshold for significantly differential expression. Finally, we generated 2722 differentially expressed genes (DEGs) among 30 d, 60 d, and 90 d libraries. Of these, 122 DEGs were co-expressed in the three libraries, 1504 DEGs in 30 d vs. 60 d (897 upregulated and 607 downregulated), 1432 DEGs in 30 d vs. 90 d (871 upregulated and 561 downregulated), and 1406 DEGs in 60 d vs. 90 d (680 upregulated and 726 downregulated) (Figure 1 and Figure 2).3.4. Functional Annotation of DEGsBasic Local Aalignment Search Tool (BLAST) [30] software was used to align DEGs to COG [23], GO [24], and KEGG [25] databases using an E value cut-off of 10−5. The results indicated that among 2722 DEGs, 2290 were annotated against the GO database, 1705 against the KEGG database, and 529 against the COG database. Annotation of DEGs showed that 529 DEGs were predicted and classified into 25 functional categories after COG annotation (Figure 3). Most DEGs were classified into “R: General function prediction only,” followed by “O: Posttranslational modification, protein turnover, chaperones” and “T: Signal transduction mechanisms”.In the GO annotation (Figure 4), “cellular process” (1525 DEGs), “single-organism process” (1425 DEGs), and “biological regulation” (1385 DEGs) were dominant in the biological process category; “cell part” (1722 DEGs), “cell” (1718 DEGs), “organelle” (1335 DEGs), and “membrane” (1139 DEGs) were dominant in the cellular component category, and “binding” (1418 DEGs) and “catalytic activity” (736 DEGs) were dominant in the molecular function category.According to the KEGG annotation results (Supplementary Figures S1), the most classifications include “Pathway in cancer” (82 DEGs), “P13K-Akt signaling pathway” (76 DEGs), “Focal adhesion” (54 DEGs), “Rap1 signaling pathway” (53 DEGs), “HTLV-I infection” (51 DEGs), “Protein digestion and absorption” (51 DEGs), “Axon guidance” (51 DEGs), and “Proteoglycans in cancer” (50 DEGs).The KEGG analysis showed that DEGs were mainly associated with the “Pathways in cancer” and “PI3K-Akt signaling pathway”, followed by pathways of “Proteoglycans in cancer”, “Rap 1 signaling pathway”, “Axon guidance” and “Protein digestion and absorption” (Figure 5).In addition, GO and KEGG annotations for DEGs in the comparisons of 30 d vs. 60 d, 30 d vs. 90 d, and 60 d vs. 90 d were also performed (Supplementary Figures S2 and S3).3.5. Antler Proteins Revealed by iTRAQ AnalysisProteome profiles of deer antlers were obtained by using the iTRAQ method. In total, 1540 proteins were identified, and all these proteins were expressed in 30 d, 60 d, and 90 d libraries (Table 2). Of these predicted proteins, 1361 were partially sequenced similar to known proteins, and 179 proteins were predicted as novel proteins.3.6. Differential Expression of ProteinsIn total, 382 DEPs were screened in the three comparative analyses, of which 53 DEPs were expressed in 30 d vs. 60 d (18 upregulated and 35 downregulated), 269 DEPs in 30 d vs. 90 d (132 upregulated and 137 downregulated), and 304 DEPs in 60 d vs. 90 d (136 upregulated and 168 downregulated) (Figure 6 and Figure 7).3.7. Annotation of DEPsBased on the COG annotation, DEPs were classified into 19 functional categories (Figure 8). “Translation, ribosomal structure, and biogenesis” was the most popular group, followed by “Posttranslational modification, protein turnover, chaperones” and “General function prediction only.”In the GO annotation, “biological regulation” and “cellular process” were dominant in biological process’s category. In cellular component category, “organelle,” “extracellular region part,” and “extracellular region” were dominant. Furthermore, “Binding” was dominant in molecular function category (Figure 9). The results were different from those of DEGs.According to the KEGG annotation results (Figure 10), DEPs were annotated and assigned to 46 KEGG pathways. The pathways of “Tight junction,” “Focal adhesion,” “Pertussis,” “Staphylococcus aureus infection,” “Systemic lupus erythematosus,” and “Complement and coagulation cascades” classified two DEPs. In addition, other pathways only classified one DEP.GO and KEGG annotations of DEPs at 30 d vs. 60 d, 30 d vs. 90 d, and 60 d vs. 90 d were also performed (Supplementary Figures S3 and S4).In addition, we found 34 DEGs and 5 DEPs classified into the “Wnt signaling pathway,” and 82 DEGs and 7 DEPs classified into “Pathway in cancer.” These DEGs and DEPs require further study.The rapid growth period (60 d) is a special physiological period in the growth process of the velvet antler, during which the growth rate of velvet antler cells was much higher than that of cancer cells. Moreover, the ordered tissue structure is still maintained without cancerization, and complete ossification eventually occurs, resulting in the death of velvet antler tissues and effectively preventing the growth of velvet without restriction. As velvet antler cell proliferation can be stopped eventually, the relationship between the growth and development mechanism of velvet antler and the malignant proliferation of cancer cells has been an attractive research topic. Furthermore, the velvet antler growth mechanism can be used to identify new ideas for cancer treatment. Thus, in the present study, we focused mainly on the DEGs and DEPs that had differential expression levels at 30 d vs. 60 d and 60 d vs. 90 d but had no differential expression at 30 d vs. 90 d. Finally, 488 DEGs and 16 DEPs with these characteristics were selected.To comprehensively understand the functions of the selected DEGs and DEPs, GO and KEGG functional enrichment analysis was conducted for the 488 DEGs and 16 DEPs. “cellular process” and “biological regulation” in biological process category, “cell” and “cell part” in cellular component category, and “binding” in molecular function category were the most enriched GO terms for the 488 DEGs. For the 16 DEPs, “organelle” classified most proteins (Figure 11). KEGG analysis showed that most of the selected DEGs were annotated in “metabolic pathway,” and then in “PI3K-Akt signaling pathway” and “pathways in cancer.” However, all KEGG pathways classified only one of the selected DEPs (Figure 12).3.8. Integrated Analysis of Transcriptome and Proteome Data and Integrated Analysis of DEPs and DEG during Antler GrowthIntegrated analysis of DEGs and DEPs during antler growth was performed. For integrated analysis, if a gene was detected to be expressed at the mRNA and protein levels simultaneously, there is a correlation between the mRNA and protein of the gene. The relationships between the number of proteins and genes were shown in Table 3.Compared datasets of proteomics and transcriptomics showed that in 30 d vs. 60 d comparison, 108 proteins conjoined with their corresponding mRNAs. Among them, 76 NDEPs (Non-differentially expressed proteins) conjoined with DEGs, 24 DEPs conjoined with NDEPs, while 8 DEPs conjoined with DEGs. The eight identified DEGs/DEPs had reversed expression trends, which meant that the eight genes collectively exhibited up- or down-regulated expression profiles at mRNA and protein levels. In the 30 d vs. 90 d comparison, 314 proteins or transcripts were identified. Among them, 76 NDEPs conjoined with DEGs, 213 DEPs conjoined with NDEPs, and 25 DEPs conjoined with DEGs, of which 9 DEPs and DEGs had the same expression trend and 16 DEPs and DEGs had the reverse expression trend. In addition, 382 proteins or transcripts were identified in the 60 d vs. 90 d comparison. Among them, 80 NDEPs conjoined with DEGs, 257 DEPs conjoined with NDEPs, and 45 DEPs conjoined with DEGs, of which 16 DEPs and DEGs had the same expression trend and 29 DEPs and DEGs had the reverse expression tend.In the three comparison groups, the number of conjoined DEPs/DEGs was lower than that of DEPs/NDEGs and NDEPs/DEGs. Therefore, integrative DEGs and DEPs were used for further analyses.We performed GO classification for DEPs and their corresponding DEGs with the same expression trends in the three libraries. In total, 22 DEPs and 12 DEGs were selected after filtering the repeat ID. Then, these DEPs and DEGs were classified into GO terms (Figure 13). The term “cellular process” classified the most DEGs but “organelle” classified the most DEPs. All terms annotated by DEPs were also annotated by DEGs, but some terms were annotated only by DEGs.Then, GO classification for DEPs and their corresponding DEGs with the reverse expression trend in the three libraries was conducted; we screened 40 DEPs and 29 DEGs. For GO classification (Figure 14), the term “binding” classified the most DEGs but “extracellular region part” and “organelle” classified the most DEPs. Similar to the DEGs and DEPs, DEGs also annotated all terms annotated by DEPs, but some terms were only annotated by DEGs.Among the 488 DEGs, 2 genes (gene13546 and gene6151) encode proteins (protein13546 and protein6151) in the 16 selected DEPs. Gene13546 (protein13546) was annotated in “Wnt signaling pathway” and gene6151 (protein6151) was annotated in “complement and coagulation cascades,” “Prion diseases, Pertussis,” “Chagas disease (American trypanosomiasis),” “Staphylococcus aureus infection,” and “Systemic lupus erythematosus” KEGG pathways. It has been reported that the Wnt signaling pathway participates in antler development [31]. Thus, gene13546 and its encoding protein (protein13546) may play important roles in rapid antler growth. Unfortunately, owing to the incomplete genome of red deer, detailed gene names were not annotated in the transcriptome data. However, BLAST results showed that gene13546 was similar to the SFRP4 gene in other mammals and humans, which could be predicted as the SFRP4 gene in Gansu red deer. A phylogenetic tree was constructed on SFRP4 amino acid sequence of C. elaphus kansuensis and 18 other species including Cervus elaphus and Bos tauus (Figure 15). The results showed that C. elaphus kansuensis was closest to Cervidae, followed by bovidae, and furthest to the fish. This was similar to the results of previous studies [32,33].4. DiscussionDeer antler cells are normal, non-cancerous cells but can proliferate and differentiate rapidly. This property makes the antler a valuable model for studying potent growth factors, unique signaling pathways, and novel regulatory systems [9]. The growth center of the antler tip determines the rapid growth rate of the antler and is precisely regulated so that the antler does not become cancerous [35]. The antler tip region is referred to as the proliferation zone [36]. The rapid growth of antlers is mainly achieved through the activity of cells residing in the proliferation zone [37]. Therefore, the apical tissue is often used as the research object to study the rapid growth and endochondral ossification mechanisms of deer antlers. Presently, the molecular mechanism of antler regeneration and rapid growth needs further study.4.1. General Features of the Transcriptomes and Proteomes of Antlers at Different Growth StagesIn this study, transcriptomic and proteomic analyses were performed on the antler cartilage tissue of Gansu red deer grown at 30 d, 60 d, and 90 d, and DEGs and DEPs were screened at different growth stages. For RNA-seq, clean reads as a percentage of total raw reads, Q30 values, and GC content are often used to assess the quality of transcriptome sequencing [4]. The Q30 values were 94.50%, 95.31%, and 94.15% at 30 d, 60 d, and 90 d antler cartilage tissues, respectively. The GC content was 56.66%, 55.32%, and 55.37% in the three samples, respectively. These data indicate that the transcriptome sequencing quality was qualified for subsequent analysis in this study. After assembly, we obtained a total of 24,778 unigenes, with an average of 2269 bp and an N50 of 3894 bp, which is longer than the previous transcriptome studies of deer antler [38,39]. Proteins expressed in antlers at different growth stages were also identified by the iTRAQ method in this study. There were previous studies have reported differentially expressed proteins involved in antler regeneration [8,11]. How our data differ from the reported findings has not been analyzed. Proteins that regulate rapid antler growth and chondrogenesis require further exploration.In this study, 3 male red deer were selected to collect antler samples at about 30 d, 60 d and 90 d growth stages, so that there were 3 samples from different individuals at each time point. In high-throughput sequencing, every sample pool was a mix of equal amount of RNAs from three individual male deer, as was the case in other studies [7,40,41,42], which indicated that this method was acceptable. However, studies with at least biological replicates may be more meaningful and acceptable compared with RNA pools, though each pool is collected with at least three samples. To explore the moleculars involved in rapid antler growth, integrative analysis of transcriptomic and proteomic data was meaningful, which helped us to screen a set of genes and proteins that may be related to antler rapid growth.4.2. Analysis of Expression of Key DEGs and DEPs Involved in Rapid Antler GrowthHigh neuralization and vascularization are important tissue characteristics of the velvet antler [43]. The velvet antler enters the rapid growth stage from the beginning to about 60 days [44]. At the rapid growth stage, the growth rate of the velvet antler reaches 2 cm/d. Many scholars call this phenomenon cancer-like growth [45,46]. The behavior of the velvet antler is very similar to the expansion of cancer cells, but the fascination is that it does not become cancerous. During the ossification period, the growth rate begins to slow down again. This unique growth process makes the rapid growth and ossification mechanism of the velvet antler a major focus of biological research. In our study, comparative transcriptome analysis showed that DEGs in comparisons of 30 d vs. 60 d, 30 d vs. 90 d, and 60 d vs. 90 d were mainly classified in “pathways in cancer” and “PI3K-Akt signaling pathway.” This was consistent with the enrichment of DEGs. As antler growth is a tumor-like development, the genes classified in pathways in cancer may be potential regulators in antler development. The PI3K-Akt signaling pathway can be activated by a variety of factors and has a wide range of biological functions, such as transcription, translation, cell cycle, proliferation, differentiation, survival, apoptosis, metabolism, angiogenesis, and migration [47,48]. It has been implicated in various cancers, such as gynecological tumors [49], prostate cancer [50], medulloblastoma [51], and gastric cancer [52]. In addition, the PI3K-Akt signaling pathway participates in chondrocyte proliferation, apoptosis, autophagy [53,54], cartilage protection [55,56], cartilage degeneration [57], cartilage cell apoptosis [58], and osteoarthritis cartilage regeneration [59]. Liu [60] found that the PI3K/Akt pathway affects the generation and regeneration of antler stem cells in vitro. Dong [61] predicted that the PI3K-Akt signaling pathway plays an important role in regulating the regeneration of antlers. Furthermore, proteomic analysis of velvet cartilage showed that DEPs were classified in the “PI3K-Akt signaling pathway” and “Wnt signaling pathway.” Thus, the genes and proteins involved in the development of cartilage tissue in the growth center of deer antlers at the fast growth stage require further study.Integrated analysis showed that among the 488 DEGs and 16 DEPs, 2 genes (gene13546 and gene6151) and proteins (protein13546 and protein6151) corresponded to each other. Gene13546 and its encoding protein protein13546 were both annotated in the “Wnt signaling pathway.” Wnt signaling plays a crucial role in embryogenesis [62]. In adults, Wnt is mainly involved in cell proliferation and differentiation [62]. Clinical findings suggest that Wnt signaling is critical for trabecular and cortical bone mass [63]. The Wnt signaling pathway has been reported to regulate antler regeneration [31,64]. Secreted frizzled-related proteins (SFRPs), a family of tumor suppressor candidate genes, act as Wnt antagonists in the Wnt signaling pathway [65]. SFRP4 is a member of the SFRP family of proteins. SFRP4-dependent Wnt signaling modulation has been reported to be essential for bone remodeling [66]. The sequence of gene13546 was analyzed with ORF Finder [67] and other online softwares [68,69]. Bioinformatics analysis showed that the ORF of C. elaphus kansuensis SFRP4 was 624 bp, encoding 207 amino acids. The molecular weight of C. elaphus kansuensis SFRP4 protein was 23.37 Kda. The theoretical isoelectric point (pI) was 6.74. The SFRP4 protein contains a conserved domain, CRD_FZ, belonging to the CRD_FZ superfamily.The higher expression of gene13546 and lower expression of protein13546 in the rapid growth stage indicate that certain factors inhibit the translation of gene13546, which in turn activates the Wnt signaling pathway and promotes the rapid growth of the velvet antler. In the ossification stage, this inhibitory effect is relieved, protein13546 protein accumulates and inhibits the activity of the Wnt signaling pathway, and the growth rate of velvet antler slows down and gradually ossifies. This may explain the timely cessation of deer antlers during proliferation without unlimited growth or cancerization.GO and KEGG annotations provide useful resources for the further identification of specific cellular structures, pathways, processes, and protein functions in antlers. Our data revealed that many DEGs and DEPs in the cartilage tissue of antlers at different growth stages. Thus, the genes involved in cancer, angiogenesis, and chondrogenesis may play key roles in antler development during the rapid growth stage.5. ConclusionsThe selected 488 DEGs and 16 DEPs that had differential expression levels at 30 d vs. 60 d and 60 d vs. 90 d but not at 30 d vs. 90 d may have research potential in regeneration of velvet antler. The gene13546 and its coding protein protein13546 annotated in the Wnt signaling pathway may possess important biological functions in rapid antler growth.

animals : an open access journal from mdpi

10.3390/ani11061775

PMC8232148

Nest boxes are considered a valid conservation tool in order to sustain wild populations of birds. The Cyprus Scops Owl was recently defined as a new species. However, the extant information on the species is sporadic and the population level is not confirmed. In order to evaluate the ability of the species to use nest boxes, and to understand its habitat preferences, we placed boxes in rural areas, at the forest edge, and in the Paphos Forest. We found that the Cyprus Scops Owl displayed a preference for the forest edge and rural areas; although we also had, several pairs occupy nest boxes in the forest. We show that the nest box strategy can be implemented if in the future the species is threatened.

As is well-known, endemic island bird species are especially vulnerable to extinction from anthropogenic environmental change and reduced fitness compared with mainland taxa. The Cyprus Scops Owl, Otus cyprius, is a recently recognized island endemic species whose ecology and breeding biology have not been studied. It nests mainly in holes in trees and buildings, so the felling of old trees, modern architectural practices, and the renovation of old houses in villages may reduce nest site availability. Its population trend is also unknown. Therefore, to better determine its ecological requirements and habitat preferences we placed nest boxes in rural areas adjacent to the forest, in the forest, and in the ecotone between them, and used breeding success as our indicator of habitat suitability. We found that breeding parameters like laying date, clutch size, length of the incubation period, hatching day, hatching success, and number of nestlings did not differ between the three habitats. Despite the low level of nest box occupancy rate (5–11%) the endemic Cyprus Scops Owl readily breeds in artificial nests. Therefore, although we are unaware of any current threats to the Cyprus Scops Owl, we recommend that its conservation be prioritized, including studies, monitoring, habitat conservation, and the provision of nest boxes.

1. IntroductionNatural cavity-nesting animals that roost or breed use holes in buildings or human-made constructions like nest boxes [1]. Nest boxes can either supplement existing natural cavities or replace them when destroyed and can be a technique in the toolbox of conservation in order to recover declining populations (e.g., Lesser Kestrel, Falco naumanni; [2]). Some countries, such as Germany [3] and Poland [4], have legislated rules that require homeowners to install nest boxes as compensation for destroyed nest sites.The use of nest boxes has allowed the study of life history traits and strategies of many cavity-nesting species by allowing easier access for monitoring and handling of the study organisms [5]. Nest boxes also help control stochastic events, thereby enhancing the chances of survival by artificial, conservation oriented intervention [6]. However, in sensitive species (declining populations, endemics, etc.) it is advisable to test the efficacy of this technique while viable populations still exist in the wild (e.g., [7]). Strigiformes is an avian order that readily occupy nest boxes (e.g., Tyto alba [8], Aegolius funereus [9,10], Athene noctua [6], Otus scops [11]).One of the smallest owls of the Strigidae family is the Scops Owl (genus Otus). A recently recognized species is the island-endemic Cyprus Scops Owl (O. cyprius; [12,13,14,15]).The endemic species is presently considered to be of ”Least Concern” by the International Union for Conservation of Nature (IUCN) Red List, with a breeding population of 10,000–24,000 individuals and its current population trend unknown [16]. The local status of this species is considered as common, mainly resident and breeding in villages, lightly wooded areas, and open pine forest up to 1900 m ([17,18,19,20] Cyprus Bird Reports). Hadjisterkotis [21] discovered that they also use nest boxes and recommended their widespread provision because of the frequent felling of the very old forest trees that develop natural cavities. This, combined with the fact that Cyprus has no woodpeckers to create holes in the forest trees, and that the most common forest tree, the Calabrian pine (Pinus brutia), rarely develops cavities until it is old, emphasizes the importance of leaving these old trees unfelled.Owls are affected by edge-effects of transition zones between two habitats [22]. In our case, these are the transitions from rural areas to the Paphos Forest. Therefore, we set up an experimental design and placed nest boxes in rural areas adjacent to the forest, on trees in the ecotone at the forest line, and in the forest to understand the ecological requirements of the endemic Cyprus Scops Owl. We used breeding success and density of occupied nest boxes as our indicators of habitat suitability and hoped to understand which of the three aforementioned habitats best suited the study species. We expected to find that rural areas would have the highest levels of nest loss owing to predation (e.g., domestic cats Felis catus, [23]), while the forest edge would have the lowest occupancy and nesting success (cf. [22,24]). We considered the forest to be the optimal habitat, which would allow for the safest, most successful colonization of the nest boxes. We reasoned that if the species can breed in artificial nests independent of habitat type, further anthropogenic-related changes of Cyprus would have less impact on the population of this endemic species.2. Material and MethodsOur study was conducted in the Paphos Forest Reserve, an area of 62,000 ha, which is situated within the Troodos massif in the west of the Republic of Cyprus (Figure 1), and ranges from sea level to >1300 m above sea level. It is a typical Mediterranean forest with coniferous (Calabrian pine, Cyprus cedar Cedrus brevifolia) and broadleaf trees (Golden oak Quercus alnifolia, Planetree Platanus orientalis). The Calabrian pine is the predominant forest tree in our study area; with a closed canopy and an understory with dense low maquis shrubs (e.g., Cistus creticus). In this area, between 2015 and 2018 we placed a total of 238 nest boxes (Figure S1). Of these, 91 were placed in rural areas/traditional villages on houses or walls (2.5 km2, density 36.4/km2), 34 on trees in the transition zone between the villages and the forest (the ecotone, 2 km2, density 34.0/km2), and 113 in the forest (12.2 km2, density 9.26/km2). The altitude above sea level ranged from 525 m in the lowest box to 1015 m in the highest. In the rural areas, nest boxes were placed at an average height of 3.5 m (±1.2), in the ecotone and the forest, most nest boxes were placed on Pinus brutia (56, 85%) at an average height of 5.8 m (±1.5) and depended on tree availability.We considered a nest box as occupied if we observed both adults entering it. All nests included in the study were observed periodically with the help of a camera mounted on a telescopic pole. We observed all breeding pairs weekly through the complete breeding cycle from early April to early June.We analyzed breeding parameters, such as laying date, incubation period (from first day of incubation to hatching date), hatching date, clutch size (number of eggs in nest), number of eggs hatched, and length of breeding attempt (from laying of the first egg to fledging of the last young). Laying and hatching dates were expressed as Julian Days. Owing to the loss of nestlings, we separated the number of hatched eggs from the number of nestlings that subsequently fledged. We considered that Scops Owl females lay one egg every 24 h, usually at dawn. Scops Owl is known to be an ultimate brooder [25,26]. Based on this we established the laying dates and brooding periods for clutches laid in between visits using the Mayfield method [27,28], wherein the median day between visits was considered to be the day of laying, hatching or fledging. The number of nestlings was calculated based on the inspection after hatching. A reproductive attempt was considered successful if at least one young fledged. The breeding success was defined as the proportion of successful nests to all inspected nests and allowed us to compare the breeding performance between the three kinds of artificial nest sites. Because these parameters could be influenced by year, we performed Factorial ANOVA where year (2015–2018) and habitat type for nest boxes (rural, forest, forest edge) were considered as factors [29]. Bonferroni corrections were applied to adjust the alpha values for the increased probability of obtaining statistical significance from multiple testing. Not all information was obtained for all nests studied and resulted in differences in sample sizes between the different analyses. The Kruskall-Wallis test with Dunn’s test [30] as a post-hoc was employed to analyze density differences between years. Mean values are presented with 95% confidence limits (CL) or standard deviation (SD).3. ResultsOf 238 nest boxes available during the four breeding seasons of 2015–2018 in three habitats, 91 (38%) in total were occupied, i.e., 9.5% per year, of the 91 nest boxes placed in the rural areas, an average of 6 (6.6%) were occupied per year (in total, 24 nest boxes were occupied i.e., 26.4%), of 34 placed at the forest-edge 5.25 (15.4%) per year were occupied (20, 62%), and of 113 boxes placed in the forest, an average of 11.75 (10.3%) were occupied per year (47, 42%). However, these differences were not significant (chi-square = 5.89, df = 2, p = 0.052). The mean (±SD) density of occupied nest boxes per year for rural, forest-edge, and forest were respectively: 2.4 (±0.56), 2.62 (±0.81), 0.96 (±0.33)/km2. Although, these differences were significant (Kruskal-Wallis test: H = 7.59, df = 2, p = 0.02), according to Dunn’s test, the difference was only between forest and forest-edge (Z = −2.55, p = 0.031, in other cases, p for Dunn’s test > 0.05).The occupancy rate of the artificial nest sites was 6.3% (CL: 2.9–9.8) in 2015, 8.4% (CL: 2.9–9.6) in 2016, 11.7% (CL: 2.9–16.1) in 2017, and 10.5% (2.8–16.4) in 2018, and did not differ between the years (Chi-square = 1.53, p = 0.64). The laying date, clutch size, incubation period, hatching day, number of hatchlings, and number of fledglings did not differ between the artificial nest sites located in three different places (Factorial ANOVA; laying dates: F3, 87 = 1.52, p = 0.211; clutch: F3, 87 = 1.42, p = 0.233; incubation: F3, 74 = 0.696, p = 0.556; hatching: F3, 74 = 1.41, p = 0.232; eggs hatched: F3, 87 =1.56, p = 0.203; fledglings: F3, 87 = 1.47, p = 0.227; Table 1). Furthermore, overall breeding success was 84.8% (N = 92); with 80.8% in the forest, 95.2% at the forest edge, and 83.3% in rural areas and did not differ between years (chi-square = 0.67, df = 2, p = 0.71).4. DiscussionWe found that the endemic Cyprus Scops Owl will breed in artificial breeding boxes and that the technique can be an alternative solution for the protection of endemic species in highly exploited habitats. This finding corresponds with other studies, which stress that nest boxes ensure the survival of the bird population where nesting habitat is a limiting factor (cf. [31]). This conservation tool has been successfully used in other species [24,32,33,34,35]. However, no previous studies have evaluated the viability of nest boxes for Cyprus Scops Owl. Hence, the importance of this study is in verifying the hypothesis that this endemic species will breed in nest boxes independently of habitats in a rapidly changing island landscape.We did not find any differences in the phenology and the basic reproductive parameters between the nest boxes located in three habitats and do not substantiate our hypothesis. We had predicted that we would find increased predation, and hence lower breeding success, in rural areas and at the forest edge, and expected the forest to be the optimal habitat with the highest density and breeding success. Although breeding success was indeed highest in the forest and lowest in the rural area, the differences are not significant. However, one must take the evaluation of breeding success with care because it could be similar in habitats with different “quality” if, for example, a lower owl density balances the lower availability of food in the poorer habitat. However, occupancy rate can also suggest preference by the studied organism. In the Cyprus Scops Owl, occupancy was significantly higher at the forest-edge than in the forest, taking intermediate values at the rural areas. We think that this preference is because of the foraging opportunities offered by the open spaces at the forest-edge and in rural areas, which facilitate prey detection and increased foraging success (cf. [36]).The reproductive output of the Cyprus Scops Owl (83.3%, N = 91) was similar to other studies, but we were surprised to find that relatively few studies report breeding success in the nominate Eurasian Scops Owl O. scops in Europe. Bavoux et al. [37] reported breeding success of 64% (N = 142) on Ile d’Oleron (Charente-Maritime, France) and claimed that the loss of wooded areas and decline in insect abundance because of the reduction in cultivated areas may have affected the species. Blanco et al. [38] found breeding success to be 69% (N = 32) in Spain, but did not study the environmental effects on breeding success. Toyama et al. [39] reported breeding success of 96% (N = 53) in the Japanese Scops Owl (O. semitorques) and 77% in the Ryuku Scops Owl (O. elegans; N = 150) on the island of Okinawa and considered predation to be the main factor affecting breeding success. Furthermore, on the island of Minami-Daito, in the northwest Pacific Ocean, breeding success of the Daito Scops Owl (O. e. interpositus) was 79% (N = 95; [40]), while only 25% (N = 8) in the Seychelles Scops-Owl (O. insularis; [41]), where alien predators were considered the main cause. The latter three are of interest because they are all island endemics, like our Cyprus Scops Owl, displaying low breeding success owing to either high predation levels or habitat change. However, the differences in occupancy could also reflect differences in nest-hole availability between different habitats.We speculate that at present the Cyprus Scops population is stable with high reproductive output when compared to other studies. Other factors influencing the evolution of the population may have been the absence of native mammalian predators [42] or Tawny Owls Strix aluco [43], the latter of which are significant predators of O. s. scops elsewhere [44], probably resulting in reduced predation and higher breeding densities compared to the mainland. However, one must take into account that all of the above mentioned studies, including ours, did not evaluate the rate of loss of holes due to building restoration, or logging, or hole-availability in their respective study areas. Also, in comparisons between habitats or study areas there is no data on hole availability or density, prey type and density, tree and hole preferences, other avian species densities, and other possible perturbations that could influence nest-site selection, and should be included in future studies.In Northern Italy, agricultural intensification in the form of vineyards and in the intensive use of pesticides was probably responsible for the decline of the Eurasian Scops Owl. Nevertheless, the opposite trend has happened in Cyprus, and could contribute to the success of the Cyprus Scops Owl and the stability of its population. Flint [45] stated that because of rural depopulation and agricultural abandonment, vineyard area has greatly decreased, from 414 km2 in 1929, to 288 km2 in 1960, to 190 km2 in 1999 [46] and to 66 km2 in 2015 [47]. For the same reason it is likely that pesticide use within these former vineyards, which all lie within the breeding range of Cyprus Scops Owl, has also decreased or largely ceased. Furthermore, since the middle of the last century there has been a long-term, ongoing, and very large increase in the quality and area of forest and woodland on the island, including in the Paphos Forest. This policy of re-afforestation and the extensive regrowth of forest and woodland on abandoned agricultural land following rural depopulation (e.g., [21,48,49,50,51,52]) has provided extensive suitable habitat for Cyprus Scops Owls, hopefully offsetting any losses in renovated villages.Denac et al. [36] concluded that the Eurasian Scops Owl faced threats throughout its range in continental Europe, as was also found by Marchesi and Sergio [11], Treggiari et al. [53], and Malle and Probst [54]. They found that agricultural landscapes that were converted to monocultures, and where perches, hedges, and vineyards were removed, adversely affected the foraging capabilities of the species. In Central Italy, parakeets (Psitaculla krameri) occupied the nest cavities earlier in the year while the migratory Scops Owls were absent, resulting in a reduction of the breeding density [55]. They recommended that natural cavities should not be filled with any materials in traditional homes and villages, and that nest boxes can be placed where there are no natural cavities, but where suitable foraging habitat exists. This recommendation was also forwarded for the Daito Scops Owl [40] because the species nest in invasive trees and their removal could affect the island population and nest boxes were proven to be a viable alternative.As is well known, endemic island bird species are especially vulnerable to extinction from anthropogenic environmental change and reduced fitness compared with mainland taxa (e.g., [45,56,57,58,59]). In this context it is noteworthy that one of the other two Cyprus endemic bird species, the widespread and common Cyprus Warbler Sylvia melanothorax, is now in serious decline following the recent colonization by the congeneric Sardinian Warbler S. melanocephala (e.g., [60,61,62]). Therefore, although we are unaware of any current significant threats to the Cyprus Scops Owl, we recommend that its conservation be prioritized, including further studies, regular monitoring, habitat conservation, and the widespread provision of nest boxes.5. ConclusionsOur study illustrated that the Cyprus Scops Owl did not show a preference for any of the three habitats–rural, forest edge, or forest. Also, we found no differences in the breeding parameters between habitats. However, habitat preference, expressed as nest-box occupancy, was highest at the forest-edge and lowest in the forest. We conclude that nest boxes are a viable alternative to nest cavities in the endemic Cyprus Scops Owl.

animals : an open access journal from mdpi

10.3390/ani11072027

PMC8300316

Necrotic enteritis (NE) is one of the most serious diseases in terms of economic losses. Aspects related to application of nanotechnology to control outbreaks of NE due to Clostridium perfringens (C. perfringens) are not completely understood. Hence, the purpose of this study was to evaluate the beneficial effects of garlic nano-hydrogel (G-NHG) on the performance, intestinal integrity, economic returns and alleviation of the intestinal C. perfringens levels using an NE challenge model in broiler chickens. Dietary inclusion of 400 mg/kg of G-NHG improved body weight gain (BWG) and feed conversion ratio (FCR). The digestive enzymes and tight junction and gut barrier-related genes expression were negatively impacted post C. perfringens challenge, resulting in a decrease in BWG with an increase in FCR. Meanwhile, G-NHG supplementation decreased C. perfringens levels, mortality rates and intestinal lesion score, and thereby improved intestinal permeability measurements, which consequently resulted in improved growth performance parameters. In conclusion, G-NHG markedly ameliorated the negative effects of C. perfringens challenge, which positively reflected on the growth performance of challenged birds, suggesting its beneficial effects in controlling C. perfringens infection in broiler chickens.

Necrotic enteritis (NE) caused by Clostridium perfringens (C. perfringens) results in impaired bird growth performance and increased production costs. Nanotechnology application in the poultry industry to control NE outbreaks is still not completely clarified. Therefore, the efficacy of dietary garlic nano-hydrogel (G-NHG) on broilers growth performance, intestinal integrity, economic returns and its potency to alleviate C. perfringens levels using NE challenge model were addressed. A total of 1200 male broiler chicks (Ross 308) were assigned into six groups; four supplemented with 100, 200, 300 or 400 mg of G-NHG/kg diet and co-challenged with C. perfringens at 21, 22 and 23 d of age and two control groups fed basal diet with or without C. perfringens challenge. Over the total growing period, the 400 mg/kg G-NHG group had the most improved body weight gain and feed conversion efficiency regardless of challenge. Parallel with these results, the mRNA expression of genes encoding digestive enzymes (alpha 2A amylase (AMY2A), pancreatic lipase (PNLIP) and cholecystokinin (CCK)) and intestinal barriers (junctional adhesion molecule-2 (JAM-2), occludin and mucin-2 (Muc-2)) were increased in groups fed G-NHG at higher levels to be nearly similar to those in the unchallenged group. At 14 d post challenge, real-time PCR results revealed that inclusion of G-NHG led to a dose-dependently decrease in the C. perfringens population, thereby decreasing the birds’ intestinal lesion score and mortality rates. Using 400 mg/kg of G-NHG remarkably ameliorated the adverse effects of NE caused by C. perfringens challenge, which contributed to better growth performance of challenged birds with rational economic benefits.

1. IntroductionGut health encompasses a variety of physiological and functional features essential for a cost-effective and higher growth rate, especially in intensive poultry production systems. The small intestine is not only the principal site for regulating an animal’s digestion and absorption, it also tightly regulates the passage of pro-inflammatory molecules, microorganisms and toxins to act as a barrier against pathogens and toxins. Bacterial infection is among the main causative agents [1,2] that adversely affects the intestinal health and impairs the productive performance and immune status of poultry. Necrotic enteritis (NE), especially necro-hemorrhagic enteritis, is one of the most infectious diseases that threatens the poultry industry [3], resulting in a great economic burden in terms of treatment costs and estimated productivity losses of 6 billion US dollars worldwide [4]. It is a result of over proliferation of Clostridium perfringens (C. perfringens), which represents the most common member of the normal intestinal microbiota of poultry. It is an anaerobic Gram-positive, spore-forming bacterium that is grouped into five types of toxins (A, B, C, D and E) based upon the production of four main toxinotypes (alpha, beta, epsilon and iota) with C. perfringens type A being the major causative agent of hemorrhagic NE [5]. Based on C. perfringens expansion of the toxin-based typing scheme, two new toxinotypes have been established (C. perfringens type F producing C. perfringens enterotoxin, but not beta, epsilon or iota toxins and C. perfringens type G producing NetB toxin) [6]. The principles are described, as is a mechanism by which new toxinotypes can be proposed and subsequently approved. these criteria consist of isolates that produce Type F strains will include strains responsible for C. perfringens-mediated human food poisoning and antibiotic associated diarrhea. Its growth rate is greatly affected by the temperature; it grows rapidly at elevated temperatures with a generation time of 8 to 10 min [7]. The pathogenesis of C. perfringens infection can be broken down into a number of stages including colonization of the host tissues, nutrients acquisition to permit further multiplication, evasion of the host immune defenses and transmission of toxins causing host damage [8]. C. perfringens α-toxin suppresses innate immunity via neutrophil differentiation inhibition, which explains how pathogenic C. perfringens can evade the host immune system [9]. Additionally, potent inflammatory reactions caused by C. perfringens infection may impair nutrient digestion and absorption, and thus, suppress the chicken’s growth rate [10,11,12,13]. Collectively, many studies have indicated that inhibition of pathogenic bacteria and reinforcement of the intestinal barrier integrity may be closely interlinked [14].Banning of antibiotic growth promoters (AGPs) used to control NE has forced the poultry industry to develop natural alternatives for improving the growth performance and maximizing the production efficiency [15]. Thus, essential oils (EOs) are generally recognized as safe and effective promising natural alternatives with growth promoting activities [16] and antibacterial properties against a wide range of pathogenic bacteria [17]. Furthermore, EOs have been described to strengthen the chicken’s mucosal barriers and intestinal integrity [18,19] and modulate the expression of immune related genes [20]. Garlic and garlic-related products, such as garlic oils, essential oils, aged garlic extracts and so forth, provide several beneficial effects for human and animal health. They have been reported to exert antioxidant, antimicrobial, antiviral, antifungal, hypercholesteremic and immuostimulating properties [21]. Moreover, they have positive effects on the digestion of broiler chickens due to its very rich aromatic essential contents [22,23]. Interestingly, the beneficial single and combined effects of garlic essential oil and oregano in reducing the ileal clostridia counts in broiler chickens were documented [24]. However, the instability and volatility of garlic bioactive compounds may limit its application in poultry farms. Therefore, searching for new techniques that can enhance garlic stability and preservation is a major concern for feed manufacturers to maximize its efficacy and application. Nanotechnology is one of the most innovative and promising strategies used to protect bioactive constituents against oxidation, heat or volatilization, maintain delivery, uniform size distribution and storage stability, mask undesirable flavors and increase the shelf life without altering their physical, chemical and functional properties [25]. The incorporation of plants extracts into nanocomposite hydrogels can enhance their activity at low doses when used in poultry production [26]. Nanotechnology application in poultry production system is still in its nascent stage. Despite this interest, there have been no reports to the best of our knowledge on the use of nanoparticles (NPs) from garlic essential oil in poultry farming. Therefore, the current study was undertaken to explore, for the first time, the efficacy of garlic nano-hydrogel (G-NHG) on broilers growth performance, intestinal integrity and net economic returns. Moreover, in this study, we adopted a C. perfringens challenge model to investigate the potency of G-NHG on the intestinal proliferation of C. perfringens.2. Materials and Methods2.1. Garlic Nano-Hydrogel PreparationSodium alginate (A-2033), garlic oil (W250320) and acrylic acid (147230) were purchased from Sigma-Aldrich (Saint Louis, MO, USA). Sodium alginate was dissolved in 1% distilled water using various ratios of acrylic acid. Network structure of sodium alginate was formed by gamma irradiation using hydrogels solutions, and then acrylic acid and sodium alginate solutions were blended until complete compatibility. The prepared mixture was then subjected for gamma irradiation (35 kGy) at the Atomic Energy Authority, Egypt. Characterization of the prepared G-NHG was done using a scanning electron microscope (Figure 1A) and Fourier-transform infrared spectroscopy (FTIR, Figure 1B).2.2. Birds, Diets and Experimental DesignApproval for the experimental protocols was obtained from the Local Animal Ethics Committee of Zagazig University, Egypt (Approval No. ZU-IACUC/F/141/2020). A total of 1200 one-day old male broiler chickens (Ross 308), purchased from a local commercial hatchery, were used for a 38-day experiment. Chicks were individually weighed and randomly assigned into six groups (200 birds/group) and each group involved 20 replicates with 10 birds each. Birds in the negative control (NC) group were unchallenged, but birds in the positive control (PC) group were challenged with C. perfringens. Both control groups were fed the basal diet without G-NHG supplementation. Correspondingly, birds in the four treated groups were fed the basal diet containing 100, 200, 300 or 400 mg/kg diet of G-NHG [27] and challenged with C. perfringens at 21, 22 and 23 d of age. Broilers were fed mash form diets for starter (d 1–10), grower (d 11–20) and finisher (d 21–38) periods, with free access to feed and drinking water. The antibiotic- and coccidiostat-free balanced commercial diets were formulated to meet the recommendations of Ross broiler nutrition specifications [28], as shown in Table 1. Chemical analysis of various diets was performed in line with the Association of Official Analytical Chemists, AOAC [29].2.3. Monitoring Chicken’s Growth PerformanceThe average daily feed intake (FI) and body weight (BW) were determined to calculate the body weight gain (BWG) and feed conversion ratio (FCR) at the end of each period [30]. The FI, BWG and FCR were calculated for the entire experimental period (d 1–38) as previously reported by Ibrahim et al. [31,32].2.4. Clostridium Perfringens Challenge ModelA coccidia-free C. perfringens challenge model was applied in this study as previously described by Yin et al. [33]. Clostridium perfringens type A strain was freshly isolated from a field avian case suffering from NE in a commercial broiler chicken flock at Sharkia Governorate, and it was able to induce the enteric disorders and severe lesions typical for NE. The strain was cultivated anaerobically on tryptose sulphite cycloserine agar (Oxoid, Basingstoke, UK) plates at 37 °C for 18 h. The black colonies of C. perfringens were confirmed by conventional and molecular methods. The strain was further confirmed to be of type A by PCR amplification of the alpha toxin gene. The actual C. perfringens concentration in the prepared challenge inoculum was approximately 1.0 × 108 colony forming units (CFU)/mL.On d 21, 22 and 23 of the experiment, all birds in PC and G-NHG treated groups were individually challenged twice a day with freshly prepared inoculum of C. perfringens utilizing a crop gavage, whereas birds in the NC group were treated orally gavaged with 1 mL of physiological saline solution. Establishment of C. perfringens infection in birds displaying NE typical gross lesions was confirmed by detection of C. perfringes and their alpha toxin gene by culture and PCR-based methods, respectively, as mentioned above. The birds were frequently observed for any clinical signs of NE and the mortality was registered daily. Birds that died or culled due to unhealthy conditions were necropsied and scored for the gross lesions of NE according to earlier established criteria [34]. Before necropsy, the birds were anesthetized via intraperitoneal injection of sodium pentobarbital (40 mg/kg) and euthanized by cervical dislocation according to the American Veterinary Medical Association guidelines for the euthanasia of animals [35].2.5. Expression Analysis by Reverse Transcription Quantitative Real-Time PCR (RT-qPCR)Pancreatic and duodenal mucosal samples were collected at the end of the experiment (38th day of age) for determining the expression levels of genes encoding the digestive enzymes (alpha 2A amylase (AMY2A), pancreatic lipase (PNLIP) and cholecystokinin (CCK)) and three molecules related to the intestinal barrier (junctional adhesion molecule-2 (JAM-2), occludin and mucin-2 (Muc-2)). Total RNA was then isolated using QIAamp RNeasy Mini kit (Qiagen, Hilden, Germany) as recommended by the manufacturer. The concentration of the eluted RNA was determined spectrophotometrically at 260 nm and the RNA purity was then assessed by calculating the ratio of absorbance values at 260 and 280 nm. One-step RT-qPCR assay was performed on the Stratagene MX3005P real-time PCR detection system using a commercial QuantiTect SYBR Green RT-PCR Kit (Qiagen, Hilden, Germany). All PCR measurements were applied in triplicate. The specificity of each PCR amplification assay was verified using a final melting curve analysis. The levels of different transcripts were then normalized to the expression of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as an endogenous control. All gene-specific primer sequences utilized in RT-qPCR assay are listed in Table 2. The results of relative mRNA expression of investigated genes were evaluated using the 2−ΔΔCt method [36].2.6. Biochemical IndicesBlood samples were aseptically obtained from the wing vein of birds (one bird/replicate) for immunological assays and biochemical analysis. Serum was separated by centrifuging the blood samples for 15 min at 2000 rpm and then it was stored at −20 °C for various analysis. At 30 and 38 d of age (7 and 14 d post challenge, respectively), estimation of lysozyme, nitric oxide (NO) and myeloperoxidase (MPO) activities were conducted using commercial kits (Jiancheng Biotechnology Institute, Nanjing, China). For determining the biochemical parameters at the end of experimental period, the concentrations of alanine transaminase (ALT), aspartate transaminase (AST), creatinine, uric acid, cholesterol, triglycerides total, very low-density lipoprotein (VLDL), low density lipoprotein (LDL) and high-density lipoprotein (HDL) were estimated using standard kits (Span Diagnostic Ltd., Sachin, India).2.7. Intestinal Lesion ScoreOn d 30 and 38, five birds/replicate were randomly picked out, slaughtered and sacrificed. The small intestine from each bird was aseptically removed and subjected to lesion scoring using a scale from 0 to 4 as follows: 0 = no apparent lesions (normal intestinal appearance); 0.5 = extremely congested serosa and mesentery distended with blood; 1 = friable and thin walled small intestine; 2 = focal necrotic lesions, ulceration and/or gas production; 3 = gas-filled and hemorrhagic intestines and patches of necrosis and 4 = severe and diffused mucosal necrosis typical of the field cases [34]. Birds that exhibited lesion scores of 2 or more were considered to be NE positive.2.8. Quantitative Detection of Ileal C. perfringens by Real-Time PCROn d 30 and 38, absolute quantitation of C. perfringens populations was carried out by quantitative real-time PCR (RT-PCR) assay. Genomic DNA was extracted from ileal digesta using the commercial QIAamp DNA stool kit (Qiagen, Hilden, Germany) according to the manufacturer’s recommendations. Eluted DNA concentration was assessed with a NanoDropTM 2000 spectrophotometer (Thermo Fisher Scientific Inc., Waltham, MA, USA). After that, purified DNA samples were stored at −80 °C until analysis. The ileal C. perfringens numbers were enumerated, in duplicate, by SYBR-Green-I based RT-PCR assay performed on the Stratagene MX3005P RT-PCR machine using SYBR Premix Ex Taq™ kit (TaKaRa, Maebashi, Japan) following the manufacturer’s protocol. The primer pair; CPerf165F: 5′-CGCATAACGTTGAAAGATGG-3′ and CPerf269R: 5′-CCTTGGTAGGCCGTTACCC-3′ (Invitrogen, Mulgrave, VIC, Australia) targeting the 16S rRNA gene of C. perfringens was used [37]. Ten-fold serial dilutions of DNA extracted from pure C. perfringens cultures were done on the 96-well plate to generate a standard curve for RT-PCR. The concentration of C. perfringens in each sample was measured in terms of log10 CFU per gram of the ileal digesta.2.9. Economic AnalysisCost return analysis was performed to evaluate the economic advantage of supplementing broilers diet with varying levels of G-NHG. According to Ibrahim et al. [31], the feed costs (average variable costs) were calculated by multiplying the actual FI for the whole feeding period with the prevailing prices. The total revenue (TR) = live body weight × price/kg. The net profit (NP) was calculated by subtracting total costs (TC) from TR. Feed costs/kg weight gain = feed conversion × cost of one kg diet. The economic efficiency was calculated from the input/output analysis as per the prevailing market price of the experimental diets at the time of the experiment. Economic efficiency (EE) = NP/feed costs. Profitability index (PI) = NP/TR. Moreover, the mortality losses were estimated as previously documented by Williams and Pant et al. [38,39]:Cost of mortality = no. of died bird × (average fixed cost + average cost of bird that reared until death)(1) Profit loss = (no. of dead birds x TR) − (no. of dead birds x total costs of bird that reared until death)(2)The sum of Equations (1) and (2) implies the mortality losses.2.10. Statistical AnalysisStatistical analysis was performed using the General linear method (GLM) method of SPSS. The homogeneity among treated groups was performed using the Levene test and normality was evaluated using the Shapiro–Wilk test. Tukey’s test was carried out to separate the mean values when the differences were significant. Data variation was expressed as standard error of the mean (SEM) and the statistical significance was set at a P value less than 0.05 (typically ≤ 0.05). The experimental unit was a pen of broilers. All graphs were made by GraphPad Prism software Version 8 (San Diego, CA, USA).3. Results3.1. Growth Performance Parameters The results concerning growth performance parameters of broiler chickens at starter, grower (preinfection) and finisher periods (postinfection) are presented in Table 3. During starter period, the BWG of broiler chickens was significantly (p < 0.05) increased in all groups supplemented with G-NHG, except for the group fed 100 mg/kg of G-NHG. Moreover, the FI during this period was significantly (p < 0.05) decreased in the 400 mg/kg G-NHG-supplemented group, while it was not affected by dietary G-NHG in other groups. Broilers fed 400 mg/kg of G-NHG showed improved FCR when compared with other experimental groups. During the grower period, all experimental birds fed G-NHG at different levels showed increased BWG. Additionally, groups fed 300 and 400 mg/kg of G-NHG exhibited the most improved FCR. During the finisher period, the BWG of broiler chickens fed 400 mg/kg of G-NHG was not suppressed by C. perfringens challenge. Moreover, other groups supplemented with G-NHG showed a significant (p < 0.05) increase in BWG when compared with the PC group. Furthermore, FCR was enhanced in groups supplemented with higher levels of G-NHG unlike the PC group. At the end of experimental period, C. perfringens challenge caused significant growth retardation in all experimental groups, except for the 400 mg/kg G-NHG-supplemented group, which displayed increased final BWG regardless of C. perfringens challenge. Moreover, groups supplemented with 200 and 300 mg/kg of G-NHG exhibited a significant (p < 0.05) increase in the final BWG when compared with the PC group. Overall, during the growing period, FCR was not affected by C. perfringens challenge in birds fed 400 mg/kg of G-NHG. Additionally, FCR of birds that received 200 and 300 mg/kg of G-NHG were affected by C. perfringens challenge, but it was not impaired as in the PC group.3.2. Gene Expression Analysis3.2.1. Digestive EnzymesAt the end of feeding trail, the mRNA expression of AMY2A, PNLIP and CCK genes were increased in response to dietary inclusion of G-NHG when compared to PC group (Figure 2). The highest upregulation of AMY2A was observed in groups received 300 and 400 mg/kg of G-NHG regardless to C. perfringens challenge (Figure 2A). Additionally, both PNLIP (Figure 2B) and CCK (Figure 2C) genes were upregulated in groups that received G-NHG in a dose-dependent manner. Regardless of C. perfringens challenge, dietary inclusion of G-NHG at higher levels maintained the upregulated levels of genes encoding digestive enzymes to be nearly the same as those in the NC group.3.2.2. Tight Junction Proteins and Mucin-2The transcriptional levels of genes encoding tight junction proteins, TJP (occludin and JAM-2), were significantly (p < 0.05) increased in groups received G-NHG regardless to challenge with C. perfringens. Meanwhile, all groups except that received 100 mg/kg of G-NHG showed a significant (p < 0.05) upregulation of Muc-2 gene (Figure 3). The expression of occludin was significantly (p < 0.05) increased with increasing levels of G-NHG (up to 2.06-fold) when compared with PC group (Figure 3A). Groups received 300 and 400 mg/kg of G-NHG showed the most significant (p < 0.05) upregulation of JAM-2 gene (1.7 and 1.8-fold, respectively, Figure 3B). The highest significant (p < 0.05) expression of Muc-2 gene was observed in birds received 400 mg/kg of G-NHG (1.5-fold, Figure 3C).3.3. Serum Activities of Lysozyme, Nitric Oxide and MyeloperoxidaseSerum activities of lysozyme, NO and MPO were significantly (p < 0.05) increased in all groups challenged with C. perfringens at 7 and 14 d post challenge, unlike the NC group (Table 4). At both intervals, serum activities of lysozyme and MPO were significantly decreased (p < 0.05) in all groups that received G-NHG compared to the PC group. Moreover, the NO contents were reduced in birds received 300 and 400 mg/kg of G-NHG when compared with the PC group. Notably, no significant differences were detected in serum activities of MPO and NO in the NC and 400 mg/kg G-NHG-supplemented groups.3.4. Serum Biochemical ParametersAt the end of the experiment, it was noticed that C. perfringens adversely affected the liver and kidney functions’ parameters (Table 5). Serum AST, ALT, uric acid and creatinine levels were reduced in all groups received G-NHG unlike PC group. Concerning lipid profile, serum cholesterol, triglycerides, LDL and VLDL were significantly (p < 0.05) decreased in all groups that received a higher dosage of G-NHG regardless of C. perfringens challenge; the highest level of HDL was detected in birds fed 400 mg/kg of G-NHG.3.5. Intestinal Lesion ScoringThe results revealed that unchallenged birds exhibited no intestinal lesions (Figure 4). Generally, intestinal lesion scores were decreased in all groups supplemented with G-NHG in comparison with the PC group. At 7 d post challenge, the decline in intestinal scoring was significant (p < 0.05) in all groups that received G-NHG, except the group that received 100 mg/kg of G-NHG. Compared with PC group, intestinal lesion severity in birds fed G-NHG was significantly (p < 0.05) reduced in a dose-dependent manner at 14 d post challenge.3.6. Quantitation of Ileal C. perfringensThe results of C. perfringens quantification in the ileal digesta are presented in Figure 5. The results demonstrated that G-NHG at various supplementation levels significantly (p < 0.05) reduced C. perfringens log10 numbers of copies with respect to the PC group at 7 and 14 d post challenge. The C. perfringens populations decreased with increased G-NHG levels at both intervals. At 7 d post challenge, the lowest C. perfringens counts were observed in the birds fed 300 and 400 mg/kg of G-NHG (2.16 and 2.30 log units decreases than the PC group), but with no detectable significant differences. At 14 d post challenge, the mean values of C. perfringens loads corresponding to the different supplementation levels were statistically significant (p < 0.05). Supplementing diets with 400 mg/kg of G-NHG was found to cause a 2.14 log CFU/g reduction of C. perfringens ileal counts than the PC group. The mean value for C. perfringens populations in this group was restored near to that in the NC group.3.7. Economic Evaluation ParametersThe morality rates throughout the entire rearing period ranged from 4–32% (Table 6). Use of dietary G-NHG reduced the mortality rates in birds, especially those fed 400 mg/kg of G-NHG. Herein, the highest mortality cost (p < 0.05) was observed in the PC group. Meanwhile, dietary G-NHG supplementation reduced mortality losses that resulted from C. perfringens challenge. Feed cost and total expenses increased significantly (p < 0.05) with increasing G-NHG levels. The group that received 400 mg/kg of G-NHG displayed the highest (p < 0.05) total expenses, total revenue, net profit, cost/benefit ratio, economic efficiency and profitability index irrespective of C. perfringens challenge. In contrast, the most negative impact on economic indicators was detected in the challenged group that did not receive G-NHG (Table 6).4. DiscussionClostridium perfringens infection has been evidenced to decrease feed efficiency and increase gut lesions and mortality rates, which account for higher productive losses in poultry. Using natural feed additives in the form of nanoformulation can provide great protection against C. perfringens infection. In the current study, we found that inclusion of G-NHG in the broiler’s diet preceding C. perfringens challenge could significantly improve growth performance and reduce both mortality and intestinal lesions. During the starter period, the most improved BWG and FCR were detected in the group that received 400 mg/kg of G-NHG; in the grower period, the highest BWG and superior FCR were observed in groups that received 300 and 400 mg/kg of G-NHG. During the finisher period, a similar improvement pattern was observed for BWG in 400 mg/kg G-NHG-supplemented group, with values similar to those observed in the NC group without infection. These results are in agreement with Brzóska et al. [23], who described that dietary supplementation of garlic extract at the levels of 1.50 and 2.25 mL/kg stimulated the appetite and significantly increased body gain compared to control group. Moreover, Kirubakaran et al. [40] described that garlic in broilers’ diet might increase the secretion of gastric juice, causing better digestibility and BWG. A previous study showed that supplementation of garlic to broilers’ diets improved their immune systems and digestion efficiency due to the abundance of bioactive compounds of garlic, namely, alliin, daily lsulphide and allicin [41]. Additionally, improvement in weight gain and better FCR of broiler chickens following the dietary supplementation of garlic can be attributed to allicin-active ingredients that promote the beneficial microflora in the gut, thereby improving the digestion efficiency and enhancing the energy utilization [42,43]. Peinado et al. [44] found that the compounds obtained from garlic had increased digestibility and activities of intestinal mucosal enzymes, confirming their application as alternatives to antibiotics in broiler nutrition. Moreover, higher growth rates of chickens are associated with the antimicrobial impact of garlic active ingredients on intestinal microorganisms, producing toxic metabolites and/or competing with the host for accessible nutrients.Injured mucosa caused by C. perfringens results in inferior growth rate and feed conversion efficiency in poultry [45,46]. Herein, C. perfringens caused a retardation of BWG with an inferior FCR and high mortality rates in broiler chickens that did not receive G-NHG over the entire experimental trial. In contrast, groups received G-NHG and infected with C. perfringens showed an improvement in BWG and FCR in a dose-dependent manner. Interestingly, BWG and FCR of birds received 400 mg/kg of G-NHG were similar to those observed in the NC group without infection. Similarly, a previous in vivo study evaluating the effect of the dietary supplementation of garlic metabolites revealed increased BWG and higher antibody response in NE challenged chickens when compared with challenged birds with no garlic supplementation [47]. The authors concluded that the aforementioned effects were due to phenolic compounds and allicin in garlic. Moreover, rapid growth and enhanced digestion and immunity of poultry can result from the reduced expanding range of pathogens in the digestive tract upon dietary garlic supplementation [48]. Decreased BWG losses and lesion scores upon A. Hookeri dietary supplementation in commercial broilers experimentally challenged with NE caused by C. perfringens infection were reported [49]. Additionally, garlic at different concentrations lowered the cecal counts of C. perfringens, which have been reported to account for better performance in broiler chickens [50]. Notably, integration of NPs as promising feed supplements for poultry is a way to further improve the overall poultry health and FCR. Until now, there were no data describing the efficacy of G-NHG on broilers performance and its protection against C. perfringens infection. The better performance of broiler chickens even after infection with C. perfringens could be attributed to the incorporation of garlic extract in the nanoform (nanohydrogel). This is considered a valuable approach used to protect its bioactive ingredients from oxidation controlling its delivery, distribution and storage stability and expanding its shelf life with no effects on the chemical, physical and functional characteristics [26]. The preparation of plant-derived bioactive components by nanotechnology can enhance their activity especially at low dosage [51].Previous studies described the enhanced effects of garlic active compounds on the digestion of broilers; however, describing this point at the molecular levels has not been investigated until now. Parallel with the increased growth rate and feed efficiency in G-NHG received groups, the expressions of genes encoding digestive enzymes (AMY2A, PNLIP and CCK) were also upregulated. Garlic can stimulate the digestive systems by controlling the digestive pH and the activity of digestive enzymes [52]. Similarly, substances derived from garlic increased the activity of pancreatic enzymes, and thereby, they provided an environment for better nutrient absorption and activated the digestive process [44,53].The intestinal mucosal barrier mainly consists of epithelial cells, tight junctions between adjacent enterocytes and critical components of the mucosal immune system. The TJP consist of diverse types of proteins, including occludin and JAM-2, which are essential for establishing continuous intact physical barriers between the intestinal epithelial cells [54]. They regulate major immune functions, increase the absorption rate of nutrients, maintain homeostasis and protect against invading pathogens [55]. In the pathogenesis of several inflammatory diseases, disturbances in the production and formation of TJP occur [56]. The disruption of TJP can lead to decreasing nutrient absorption, increasing permeability to the luminal antigens, sustained inflammation, bacteria translocation and tissue damage [57]. Dietary supplementation of essential oils has been documented to strengthen the mucosal barrier and maintain intestinal integrity [19]. In the current study, the expression levels of occludin and JAM-2 genes were significantly reduced in the infected group, which did not receive G-NHG. However, the expression levels of occludin and JAM-2 in birds given 400 mg/kg of G-NHG showed 2.06- and 1.8-fold increase when compared to NE-challenged birds fed a basal diet, respectively. This increased expression may be due to improved intestinal barrier functions, especially during the invasion of pathogenic microorganisms. In accordance, supplementation of Allium hookeri (AH) root by 3% upregulated the expression of TJP genes; JAM-2 and occludin in broiler chickens infected by C. perfringens [49]. Our results are consistent with previous findings on the active ingredients of other essential oils, where increased TJP gene expression and improved intestinal barrier functions were observed in thymol- and carvacrol-treated broilers challenged with C. perfringens [11]. Moreover, Muc-2 is one of the major secreted mucins expressed by intestinal goblet cells and it acts as a protective barrier for the intestine [58,59]. Several studies have demonstrated that necrotic pathogens can induce decreased the expression of Muc-2 in chickens [60,61]. In agreement with previous studies, decreased expression of Muc-2 was observed in the NE challenged group compared to the unchallenged group. However, dietary supplementation of higher levels of G-NHG led to upregulation of Muc-2 expression. Likewise, dietary supplementation of AH at the levels of 1 and 3% dramatically led to increased regulation of Muc-2 [49]. Moreover, higher mRNA relative expression of Muc-2 in ileum was found after dietary supplementation of encapsulated essential oils in laying hens [62]. In a recent study, Muc-2 gene expression was upregulated in broiler chickens challenged with Salmonella Typhimurium fed thymol nanoemulsion, indicating the maintenance of the intestinal barrier integrity [16]. Furthermore, nanoencapsulation of cumin essential oil increased Muc-2 gene expression in broiler chickens [63]. The boosting effects of G-NHG on genes expression of TJP and Muc-2 in the gastrointestinal tract can be attributed to its bioactive compounds, besides its incorporation into the nanoform, which protects and controls its delivery.Herein, the inclusion of higher levels of G-NHG reduced the serum lipid parameters of broiler chickens even after infection with C. perfringens. In accordance, lower triglycerides, total cholesterol, LDL and VLDL levels and higher HDL were detected upon dietary garlic inclusion for broiler chickens when compared with the control group [64]. This can be explained by the possible hypolipidemic and hypocholesterolemic activities of garlic products, which impairs the hepatic functions of cholesterogenic and lipogenic enzymes [65]. Moreover, this effect may be probably due to the inhibition of the Acetyl CoA synthetase enzyme that is necessary for the biosynthesis of fatty acids. In a previous study, garlic extract significantly reduced triglycerides and total cholesterol in diabetic rats [66]. This effect can be explained by the potential antiperoxide action of alliin; the isolated product from garlic or reduction in the hepatic synthesis of VLDL, which considers the precursor of LDL in blood circulation [67].Clostridium perfringens is an important anaerobic pathogen that inhabits the broiler intestine. The proliferation of C. perfringens in the poultry intestine results in NE, leading to increased mortality and productivity losses [45]. There are many concerns within the poultry industry to prevent or eliminate the resistance pathogenic bacteria causing gastrointestinal infections [68,69,70,71]. It is well established that garlic extract has been reported to exert an in vitro antimicrobial activity against the potentially pathogenic C. perfringens populations [72]. The beneficial effects of garlic supplementation in broiler diets have been previously reported. Nevertheless, there is currently a lack of information about its use in the nanoform as a valuable alternative in diets of broiler chickens to control the pathogenic bacteria. Herein, dietary G-NHG supplementation significantly reduced C. perfringens numbers in challenged broiler chickens at 7 and 14 d post challenge (up to 2.837 and 1.857 log10 CFU per gram of the ileal digesta, respectively), which in turn alleviated intestinal lesion scores in a dose-related manner. Our present in vivo findings are in accordance with those of early in vitro studies concerning the inhibitory potentials of garlic on C. perfringens. Accordingly, garlic reduced the C. perfringens cecal loads at different supplementation levels compared with the control group [50]. Moreover, Clostridium species counts were significantly decreased in birds supplemented with garlic oil compared with the control group [24]. The antibacterial activity of garlic is mainly attributed to allicin (allyl 2-propenethiosulfinate), which is generated via the enzymatic activity of alliinase on alliin [73]. Interestingly, EO delivery systems, such as nanoemulsions, microcapsules, NPs and liposomes, are models for the enclosure of the natural bioactive compounds to reduce their volatility and improve their effectiveness. Therefore, the tested G-NHG extended the antimicrobial effectiveness and inhibitory spectrum of garlic essential oil in controlling C. perfringens by improving the evaluated parameters in broiler chickens. Additionally, garlic essential oil nanoemulsions exhibited a better in vitro antimicrobial activity than the garlic essential oil itself [74].Indeed, dietary essential oil supplementation has the capability to enhance the broiler chicken’s immune response, which could be involved in affecting the intestinal microbiota and gut health and enhancing the poultry resistance against bacterial infections. To explore the effects of G-NHG on poultry immunity, MPO, NO and lysozyme levels were evaluated. The bactericidal properties of neutrophils and monocytes/macrophages have been attributed to the action of MPO, NO and lysozyme, which are beneficial in terms of the protective immune responses to eliminate the invading bacterial pathogens. Elevated serum MPO, NO and lysozyme activities could be considered to be a response to C. perfringens challenge and are vital indicators of inflammatory responses, suggesting activated neutrophils and monocytes in blood [75]. Interfering with MPO, NO and lysozyme production might be helpful for the health status of poultry. Herein, the reducing trend of lysozyme, NO and MPO levels observed in G-NHG-fed birds at 7 and 14 d post challenge demonstrated that dietary G-NHG exerted an important role in mitigating C. perfringens-induced stimulation to phagocytes, and subsequently conferring a health benefit to broiler chickens. The enhancing effects exerted on humoral immune response of broiler chickens have been already documented in previous studies post garlic [23,76] and nano garlic [76] supplementation. The positive responses of humoral non-specific defense mechanisms, such as lysozyme activity, and the key mediators of host defense and oxidative tissue injury, such as NO, were reported after garlic extract incorporation into fish feed in a recent study [77]. Moreover, there is very limited information regarding these effects in broilers. To date, the knowledge of the impact of garlic nano materials on the broiler’s immune system has yet to be elucidated. Therefore, more thorough investigations on this subject are still needed. The antibacterial effects of G-NHG on pathogenic C. perfringens in the gastrointestinal tract and the stimulatory effects on the immune system may have contributed to the lower mortality rates of broiler chickens in our study, as was previously illustrated [23].It is interesting to pay attention to the strategy that allows monitoring of the production costs and helps the producer to get the best financial return of the production system. From this point, efforts were made to research the use of phytogenic compounds in the broiler diet with satisfactory effects on the overall performance and economic efficiency [78,79,80]. Under the C. perfringens challenge imposed in our research, supplemental 400 mg/kg G-NHG presented a positive impact on the net profit, cost/benefit ratio and total revenue. The profitability ratio and economic efficiency were similar to the NC group, suggesting the use of G-NHG as a potential alternative for the AGPs. In accordance, garlic powder was proved to be the cost effective natural additive [81]. Moreover, supplementation of garlic extract had the highest net return and revenue offering the lowest cost/benefit ratio [82]. Incorporation of garlic extract in the form of nano-oil enhanced its stability and efficiency in improving the production parameters, with great economic benefits.5. ConclusionsBased on our data, it could be concluded that dietary supplementation with G-NHG at 400 mg/kg noticeably enhanced the broilers’ growth performance and maximized the economic returns. Additionally, supplemental G-NHG has a promising role in motivating the birds’ immune response and boosting the expression of genes encoding tight junction protein and mucin-2. This, consequently, counteracted the negative effects of C. perfringens challenge through decreasing C. perfringens loads, intestinal lesion score and mortality rates. Keeping in view the practical relevance of these findings, G-NHG can be used as a reliable alternative feed additive to the AGPs in the concurrent control or prevention of the economically important enteric diseases.

animals : an open access journal from mdpi

10.3390/ani13050947

PMC10000142

In order to further standardize and simplify the operation of the nylon bag method, rumen degradation kinetics of 25 feedstuffs were first determined using in situ nylon bag technique and then the differences of degradation parameters fitted with five or seven time points measuring data were evaluated. Additionally, the goodness of fit (R2) of degradation curves obtained at different time points was compared. In protein feeds, energy feeds and roughages, there were no significant differences in rumen degradation parameters except for several feedstuffs. By comparing the R2, it was found that the R2 of degradation curves which obtained five time points was better than that which obtained seven time points. These results indicate that it is feasible to determine the rumen degradation characteristics of feedstuffs by only setting five measuring time points, and based on the reasonable criteria, where the optimal selection of incubation time points for protein feeds and energy feeds was the set of ② 2, 16, 24, 36, 48 h, and that for roughages was ① 4, 8, 16, 48, 72 h.

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.

1. IntroductionThough 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 inter- and 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 [1,2].However, the incubation times recommended procedures which might remarkably influence the accuracy and efficiency of the evaluation. Ørskov [3] suggested that concentrates should be cultured in rumen for 2, 6, 12, 24 and 36 h; Lindberg [4] recommended concentrates for 2, 4, 8, 16 and 24 h, and roughages for 2, 4, 8, 16, 24, 36, 48 h; AFRC [1] 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 [5] and Vanzant [6] 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 [7], L. Tao [8], B. Ghorbani [9], Xiaogao Diao [10], and so on [11,12,13,14,15,16]. 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 MethodsAll 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 DietsEight 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 AnalysisA 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 °C 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 [17]. Nitrogen (N) content was measured by the Kjeldahl method [17] using a FOSS semi-automatic nitrogen analyzer, and crude protein (CP) content was calculated as N × 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. [18].2.3. In Situ Nylon Bag ExperimentThe in situ degradabilities of DM, CP, and OM in the 25 feeds were determined according to the procedure described by Mehrez and Ørskov [19]. 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 μm pore size, 6 cm × 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 [20]. Then, the bags with clean residue were dried to a constant weight at 65 °C for 48 h and weighed. The residues were further ground through a 1 mm sieve for nutrient analysis.2.4. Calculations of Degradation Kinetics ParametersThe 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 [21], DDGS 3.99%/h, silage feeds 2.53%/h, and oil-seed-meals 5%/h [22]. 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 SelectionThe 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 AnalysesThe 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. Results3.1. The Parameters a, b, c and ED of DM, CP and OM of the FeedstuffThe 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 PointsThe 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. DiscussionThe 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 [23], 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 [5] and Vanzant [6] suggested that the number of time points could be used to describe the curve. The British AFRC [1] 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. ConclusionsThe 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.

animals : an open access journal from mdpi

10.3390/ani11082153

PMC8388491

Mesenchymal stem cells (MSCs) are available in minuscule numbers in the body or placental tissues. These cells have mostly been harvested from bone marrow and adipose tissue. To broaden the currently available knowledge, the current study provides (a) information on the feasibility of isolation of MSCs at different ambient temperatures, (b) details of MSCs’ culture characteristics with respect to the physiological status of the donor, and (c) information on the viability of cryopreserved cells. Bone marrow harbors a higher mononuclear cell fraction than that of the adipose tissue, although percent adherent cells are comparably more in adipose tissue. MSCs from a pregnant donor show enhanced proliferation and differentiation potential, although further studies are desired. The cryopreserved cells have comparable characteristics to that of the fresh cells. In conclusion, donor animals’ tissue type and physiological status may affect MSCs’ characteristics and should be taken into consideration while applying in clinical settings.

The current study demonstrates the culture characteristics of adipose tissue and bone marrow-derived mesenchymal stem cells (MSC). The study evaluates the effect of ambient temperature, physiological status of the donor and the tissue source on sheep (Ovis aries) mesenchymal stem cells. The tissue samples were harvested from full term pregnant female sheep (n = 9) and male sheep (n = 10). Adipose tissue was harvested from n = 9 sheep and bone marrow from n = 10 sheep. The samples (adipose tissue, n = 2; bone marrow, n = 3) transported at cold ambient temperature (<10 °C) failed to yield MSCs while those (n = 14) at higher (>20 °C) ambient temperature successfully yielded MSCs. Bone marrow mononuclear cell (MNC) fraction was higher than the adipose tissue-derived stromal vascular fraction (SVF), but the percent adherent cells (PAC) was higher in the later cell fraction. Adipose tissue-derived MSCs from the full term female sheep had a significantly (p < 0.05) higher proliferation potential as compared to those of the male sheep-derived MSCs. Female sheep MSCs also had rapid differentiation potential. The cryopreserved MSCs had morphological features comparable to that of the fresh cells. In conclusion, the tissue type and physiological status of donor animal may affect MSCs’ characteristics and should be taken into consideration while applying in clinical settings.

1. IntroductionMesenchymal stem cells (MSCs) are increasingly being used in regenerative medicine due to their specialized properties of self-renewal, multiplication and differentiation, in addition to their ability to home-in and immune-modulate [1,2]. These cells are available in almost all the adult tissues and foetal membranes. Among various sources, mainly bone marrow (BM)- and adipose tissue (AD)-derived MSCs have been evaluated for in vivo applications, although foetal membrane-derived MSCs are being increasingly studied. There are various in vitro animal studies that have shown variability in the concentration, multiplication and differentiation properties of MSCs with respect to the tissue source [3,4,5,6,7,8,9,10], health status [11,12,13] and the physiological status of the donor animal [14,15], although with exceptions [16]. Some sources, such as bone chip, may harbour higher MSC concentration as compared to others, such as bone marrow [7]. The proliferation potential of MSCs also varies with respect to the tissue source as AD-MSCs tend to proliferate at a higher rate as compared to BM-MSCs and liver MSCs [5]. Even MSCs from a particular source may show differentiation potential more predisposed towards that particular lineage. MSCs from musculoskeletal sources such as bone marrow and synovial membrane have higher osteogenic differentiation potential as compared to those from adipose tissue [3,8]. This may further differ among the particular tissue sources as synovium MSCs may have higher chondrogenic potential than BM-MSCs [10]. Health status may also affect MSCs’ characteristics. In osteoporosis, goat BM-MSCs may have reduced proliferation and osteogenic differentiation potential [11]. Similarly, in equine metabolic syndrome (EMS), AD-MSCs are senescent and show lower proliferation potential [12]. In the case of bovine endometritis, endometrial MSCs show reduced proliferation and have little tendency towards adipogenic differentiation [13]. Anestral goat endometrial MSCs tend to show higher proliferation as compared to the cyclic endometrial MSCs [15]. MSCs’ proliferation potential reduces with the ageing, although a sheep study has failed to demonstrate such an observation [16]. All these studies show MSCs from particular sources at specific physiologies may have differential characteristics. Thus, such types of characteristics need detailed evaluation for effective utilization of MSCs in clinical settings. No literature on MSCs’ characteristics with respect to full term pregnancy in relation to the non-pregnant-derived tissue sources has been reported. Sheep are a suitable model animal for humans and previous studies using the species have been extrapolated for humans. The current study reports the effect of ambient temperature, tissue source and physiological status on characteristics of MSCs derived from sheep adipose tissue and bone marrow. 2. Materials and Methods2.1. Ethics StatementThe Institute Animal Ethics Committee of Sher-e-Kashmir University of Agricultural Sciences and Technology, Kashmir, India granted approval for this study vide order no. AU/FVSc/PS-57/15196. Sheep bone marrow and adipose tissue samples were harvested after obtaining written consent from the owners. All applicable national and/or institutional guidelines for the care and use of animals were followed.2.2. AnimalsThe current study reports the characteristic features of mesenchymal stem cells (MSCs) derived from sheep (Ovis aries) bone marrow (BM-MSCs) and adipose tissue (AD-MSCs). The samples were collected from 19 mixed breed sheep (aged between 10 months to 2 years) that underwent caesarean section (Gender: Female; n = 9) and tube cystostomy (Gender: Male; n = 10), being restrained under the lumbo-sacral anaesthetic block. Initially, 5 samples (bone marrow (n = 3); adipose tissue (n = 2)) were transported and maintained for 30–45 min in the ambient temperature of < 10 °C (peak winter) until processing. Thereafter, the samples were transported and kept in a slightly improved temperature (ambient temperature >20 °C) till the processing. The samples were harvested in the small ruminant operation theatre of the Veterinary Clinical Complex, FVSc and AH. In female sheep, n = 7 samples of adipose tissue and n = 2 samples of bone marrow were harvested. In male sheep, n = 2 samples of adipose tissue and n = 8 samples of bone marrow were harvested. The culture and characterization work was conducted in the stem cell laboratory, Division of Veterinary Clinical Complex, FVSc and AH. 2.3. Bone Marrow and Adipose Tssue CollectionBM was collected from iliac crest with a bone marrow biopsy needle (18 gauge) by an aseptic procedure. In short, the biopsy needle (stylet in place) was inserted into the bone and a syringe (10–20 mL), loaded with 2500 IU units of heparin (celparin, Celon Labs, Hyderabad, India) was attached to the needle. About 2.5 mL of bone marrow was aspirated into a hypodermic syringe and a similar procedure was followed on the other side to harvest equal volume. A total of 5.0 mL of BM collected was taken to the laboratory for processing. The adipose tissue (5.0 gm of omental fat) was collected from female sheep that underwent celiotomy (full-term caesarean section). The harvested tissue was maintained into Dulbecco’s phosphate buffer saline (DPBS) till the sample processing. 2.4. Stem Cell Isolation and Culture Expansion2.4.1. Bone MarrowBM samples were processed for isolation and culture expansion of MSCs as per the standard procedure [17]. BM samples were mixed with equal volume of Dulbecco’s phosphate buffered saline (DPBS) (Hyclone, GE Healthcare Life Sciences, Chicago, IL, USA) (1:1 v/v). The samples were passed through a 21-gauge intravenous catheter to disaggregate cell clumps and create a single cell suspension. Marrow samples with DPBS were loaded onto a half volume of Hisep LSM (HiMedia Laboratories, Mumbai, India) (1:0.5 v/v). The mono-nucleated cells collected from the interface were diluted with a double volume of DPBS (1:2 v/v) and collected by centrifugation at 300 gm for 30 min. After centrifugation, 3 mL of RBC lysis buffer (Himedia Laboratories) was added to the cell pellet for lysis of RBCs, if any, and again centrifuged at 200 gm for l0 min. The pellet obtained was washed with DPBS at the same speed of centrifugation. The cells were suspended in growth media (Dulbecco’s Modified Eagle’s Medium-low glucose (DMEM-LG) (Hyclone, GE Healthcare Life Sciences) +l0% foetal bovine serum (FBS) (VWR) and antibiotics (mixture of 100 units/mL of penicillin and 100 mg/mL of streptomycin (Himedia Laboratories). The cells were counted by Neubaeur’s counting chamber method (table), plated in T-25 culture flasks and maintained at 37 °C and 5% CO2 in a humidified atmosphere in a CO2 incubator (CellXpert, Eppendorf, Hamburg, Germany). The cells were plated at an average of 2.5 × 105 in T-25 flasks. The non-adherent cells were removed after 3 days followed by media change (Table 1). Subsequently, growth medium (GM) was changed every 3 days. Upon 70–80% confluency (assessed by visual inspection under inverted microscope), the cells were passaged at lower densities into new culture flasks. The confluent flasks were trypsinized (0.075%) for about 3–5 min, followed by addition of GM to stop trypsin activity. The contents were collected in a centrifuge tube and centrifuged at 300 gm for 10 min. The supernatant was discarded and the pellet was re-suspended in 10 mL of GM. The single cell suspension was created after passing the cells through a 20-gauge needle three times. The cells were replated at the density mentioned earlier. Cultures were maintained at 37 °C and 5% CO2 in humidified conditions. For further cell passaging (up to passage 4) a similar procedure was followed. 2.4.2. Adipose TissueThe isolation and culture expansion of AD-MSCs was performed as per the standard procedure [18] with slight modification. In brief, the collected fat was triturated and mixed in equal volume (1:1 w/v) of collagenase (1 mg/mL). The assembly was kept in a shaking incubator at 37 °C for 40 min. The enzymatic action was neutralized by adding equal volume of GM followed by centrifugation at 300 gm for 40 min. Half of the volume of the supernatant was removed followed by addition of GM, and the tubes were then centrifuged (200 gm for 20 min). The pellet obtained after discarding the supernatant was mixed with RBC lysis buffer (Himedia Laboratories, Mumbai, India) to lyse RBCs. The tubes were then again centrifuged at the same speed for 10 min. After complete RBC lysis, the clear white pellet was mixed in GM and implanted into the T-25 culture flasks at a density mentioned above and maintained at 37 °C and 5% CO2 in an incubator in a humidified environment. After obtaining the cell confluency of 70–80%, the cells were passaged as mentioned above up to passage 4.2.5. Percent Adherent Cells (PAC) PAC from mononuclear cell (MNC) and stromal vascular fraction (SVF) were calculated after day 6 from initial cell seeding as detailed below:PAC after initial seeding = C2/2h1/h0 × C1 × 100(1) where h1 = time in hours from initial seeding to calculation of total adherent cells (C2), and h0 = population doubling time of MSC.2.6. Characterization of MSCsThe cells of two sources were characterized based on the morphology, surface marker expression and differentiation potential as recommended by the International Society for Cellular Therapy (ISCT) [19]. MorphologyMorphological characteristics of the MSCs were observed at different magnifications using an inverted microscope (LMI, Leeds, England, UK) at regular intervals.2.7. Colony Forming Unit-f (CFU-f) The self-renewal property of the cells was observed by their colony forming capacity (CFU-f). The passage 3 cells were seeded at a low density and media were changed twice a week. To enumerate the colonies, PBS rinsed cells were fixed in 10% formalin at day 10. Aggregate of more than 50 cells stained with crystal violet (0.5%) (Himedia Laboratories, Mumbai, India) to lyse RBCs. The tubes were then again centrifuged at the same speed for 10 min. After) was considered as a colony, and total number of colonies was counted for the each cell line from two sources. 2.8. Growth Kinetics and Population Doubling Time (PDT)Growth kinetics of passage 3 MSCs was evaluated in culture media seeded in a 12-well plate at the rate of 10 × 103 cells per well for each cell source. GM was changed twice a week. At every 48 h, two wells were harvested for each cell type and the cell number was counted using a haemocytometer. The growth curve was plotted for each type.The cells (10 × 103) were cultured in two 12-well culture plates. Two wells at 24 h intervals for 3 consecutive days were harvested and the cells were counted in each culture well using a haemocytometer. The population doubling time was calculated by:PDT = Culture time (CT)/Cell doubling (CD)(2) where CD = log (C2/C1)/log 2, C2 is harvested cell number, and C1 is initial cell number. Culture time is in hours.2.9. Surface Marker Expression AnalysisThe passage 3 MSCs from female and male sheep adipose tissue and bone marrow were analysed for their phenotypic marker expression (CD73, CD90, CD34, and CD45 markers) by reverse transcription polymerase chain reaction (RT-PCR).Semi-Quantitative RT-PCRTotal RNA was extracted by RNeasy Micro Kit (Qiagen, Germantown, MD, USA) as per the manufacturer’s recommendations. Eluted total RNA samples were stored at −20 °C until use. Concentration and quality check were confirmed by Qubit® 2.0 Fluorometer (Life Technologies, Carlsbad, CA, USA). The synthesis of first strand cDNA from RNA templates was conducted by following the manufacturer’s protocol (RevertAid First Strand cDNA Synthesis Kit, Thermo Scientific™). The product of the first strand cDNA synthesis was used directly in qPCR. A total of 2 µL of the first strand cDNA synthesis reaction mixture was used as template for subsequent PCR in the 25 µL reaction volume. Amplification reaction was set in a gradient thermal cycler (BioRad, Hercules, CA, USA). The results were analysed based on the presence or absence of specific amplification. Cycling conditions for different primers and their sequences are given in Table 1.2.10. Tri-Lineage DifferentiationThe tri-lineage differentiation was conducted on the MSCs (AD and BM) harvested from female sheep.2.10.1. Adipogenic DifferentiationAdipogenic differentiation of MSCs (10 × 103 per 9.6 cm2) was evaluated using AdvanceSTEM Adipogenic differentiation kit (Hyclone, GE Healthcare Life Sciences, Bangalore, India). The cells from two sources were cultured for 3 weeks and evaluated through staining starting from day 10, as per the given instructions. Adipogenic differentiation was assessed by the presence of lipid droplets after staining with oil red O stain (Modified Promo Cell staining protocol). Expression analysis of PPARG with conventional RT-PCR (Table 1) was also made at day 15. As a negative control, an equal number of cells were maintained in the growth media for similar period.2.10.2. Chondrogenic DifferentiationChondrogenic differentiation was evaluated by AdvanceSTEM Chondrogenic differentiation kit (Hyclone, GE Healthcare Life Sciences, Sciences, Bangalore, India) following the given instructions. MSCs (10 × 103 per 9.6 cm2) from two cell types were cultured for 3 weeks and evaluated through staining starting from day 10. Differentiation was assessed by alcian blue staining. BGN expression with conventional RT-PCR (Table 1) was carried out on day 15. As a negative control, an equal number of cells were maintained in culture and supplemented with growth media for a similar period. 2.10.3. Osteogenic DifferentiationOsteogenic differentiation of each cell (10 × 103 per 9.6 cm2) type was evaluated using an AdvanceSTEM Osteogenic differentiation kit (Hyclone, GE Healthcare Life Sciences, Sciences, Bangalore, India). Calcium deposition was evaluated by alizarin red staining (Modified Promo Cell staining protocol). As a negative control, an equal number of cells were maintained in the growth medium. The cells were cultured for 3 weeks with weekly medium changes and evaluated through staining starting from day 10. At day 15, expression analysis of BGLAP was also carried out with conventional RT-PCR (Table 1).2.11. Cryopreservation and Post-Thaw ViabilityConfluent cultures with an approximate cell number of 2.5 million cells of both sources (female sheep samples) were cryopreserved. The cells after trysinization and DPBS washing were resuspended into the 1 mL cryopreservation media (Hyclone, GE Healthcare Life Sciences) and kept in the 2 mL cryovials (GWare, Genetix, Asia Biotech Pvt Ltd., Hyderabad, India). The cryovials were kept in a 1 °C cooler and kept at −20 °C for 3–4 hrs, followed by overnight transfer to −80 °C; finally, the vials were kept in liquid nitrogen (LN2) over a 2-month period. Cryopreserved MSCs were revived by thawing them in a water bath at 37 °C. The cells were later poured into a 15 mL centrifuge tube containing 10 mL of GM. 2.11.1. Cell Viability The viability was measured by trypan blue staining as per the standard protocol [20]. In short, 10 μL of cell suspension was stained with equal volume of 0.4% trypan blue (1:1 v/v) (Himedia Laboratories, Mumbai, India) to lyse RBCs. The tubes were then again centrifuged at the same speed for 10 min. The stained cell suspension was loaded onto the Neubauer’s haemocytometer chamber and unstained cells were counted as live cells. The live percentage of cells was calculated as:Post-thaw viability = live cell count/total cell count × 100(3)2.11.2. Cellular Expansion and CFU (f)The cells of each source were culture expanded as per the protocol mentioned above. The cells were implanted in T-25 culture flasks (SPL Life Sciences, Pocheon-si, Korea). The CFU (f) was evaluated as described for fresh cells.2.11.3. Differentiation of CellsThe post-thaw cells from each source were put to differentiation for adipogenic, chondrogenic and osteogenic and evaluated as per the protocol described for fresh cells.2.12. Statistical AnalysisThe statistical analysis was conducted with IBM SPSS version 16. The independent t-test was applied to compare the means of two groups while for among the groups the statistical analysis was confirmed by the one-way ANOVA (analysis of variance) with Duncan’s post-hoc test. The level of significance was set at p < 0.05. The data are presented as mean ± standard error (SE).3. Results3.1. Bone Marrow and Adipose Tissue CollectionMSCs were isolated from bone marrow and adipose tissue of the sheep. Interestingly, we failed to isolate MSCs from samples (both the tissue types) under lower ambient temperature (<10 °C). All the samples at higher ambient temperature (>20 °C), however, were successfully processed and yielded MSCs. The overall success rate of MSC isolation from adipose tissue and bone marrow was 78% and 70%, respectively. 3.2. Cell Population and Percent Cell Adherence (PAC)The initial cell count (stromal vascular fraction) for adipose tissue was lower as compared to bone marrow cell fraction (mononuclear cell fraction). The stromal vascular fraction (SVF) ranged from 0.97 million to 3.25 million with a mean ± SE of 2.53 × 106 ± 3.43 per 5 gm of the tissue. Mononuclear cell fraction (MNCs) ranged from 2.05 million to 6.5 million with a mean ± SE value of 4.57 × 106 ± 6.2 per 5 mL of bone marrow (Figure 1A). The initial cell count and average PAC were higher in MNCs and SVF, respectively, but statistically non-significant (p > 0.05) (Figure 1B).3.3. Growth Kinetics and PDTThe cells were maintained in GM up to passage 4. The growth characteristics of sheep BM-MSCs and AD-MSCs had shown a typical growth curve encompassing lag, log and plateau phases. Female sheep AD-MSCs had a lag phase of 24–36 h, while in female sheep BM-MSCs and male AD-MSCs and BM-MSCs, a lag phase of 1–2 days was seen. A log phase of 7–8 days followed by plateau up to day 12 was seen. Thereafter a declined growth rate was seen in all these cells (Figure 1C). The PDT for female sheep AD-MSCs and BM-MSCs ranged from 29.41 h to 51.61 h (mean 39.26 h) and 32.74 h to 59.70 h (mean: 44.35 h), respectively. The PDT for male sheep-derived BM-MSCs and AD-MSCs ranged from 39.05 h to 86.96 h (mean 60.51 h) and 38.30 h to 74.53 h (mean: 56.73 h) (Figure 1D). Female sheep AD-MSCs had a significantly (p < 0.05) lower PDT as compared to the male sheep AD-MSCs and BM-MSCs, while female sheep BM-MSCs had lower PDT but statistically non-significant (p > 0.05) as compared to male sheep tissue-derived cells. 3.4. Cell MorphologyThe adherent cells initially (P0–P1) were of mixed shapes ranging from polygonal, rounded to spindle shaped fibroblast-like cells. Subsequently, in advanced passage (P2–P4) the cells had typical uniform spindle shaped fibroblast-like morphology (Figure 2). 3.5. Colony Forming Unit (Fibroblasts) AssayFemale sheep AD-MSCs had a higher clonogenic potential (48 clones in all the fields) followed in decreasing order by female BM-MSCs (44 clones in all the fields), male sheep-derived AD-MSCs (36 clones in all the fields) and BM-MSCs (27 clones in all the fields) (Figure 3).3.6. Phenotypic Gene ExpressionMSCs from two cell sources evaluated for surface markers (CD73 and CD90) and haematopoietic markers (CD34 and CD45) revealed positive surface marker expression while expression for haematopoietic markers was lacking (Figure 4). 3.7. Tri-Lineage DifferentiationMSCs from two sources (AD and BM) were subjected to tri-lineage differentiation post-establishment of the monolayer. MSCs from these two cell sources were comparable in their ability to differentiate into the adipogenic, chondrogenic and osteogenic lineages, as demonstrated by the special staining techniques. MSCs from both the sources could differentiate into adipogenic and chondrogenic as early as 10 days and effective osteogenic differentiation was achieved by day 15. MSCs could maintain morphology in chondrogenic differentiation until 10 days, while in adipogenic differentiation cellular morphology was changed and cells started detaching as early as 6–8 days. Osteogenic characteristics started appearing post 10 days and better at day 15. The staining of chondrogenic, osteogenic and adipogenic-differentiated MSCs was characteristic for proteoglycans, calcium crystals and oil droplets, respectively (Figure 5). The semi-quantitative RT-PCR-based gene expression analysis showed higher expression of PPARG, BGN and BGLAP corresponding to the adipogenic, chondrogenic and osteogenic differentiation, respectively, in concerned differentiation cocktails (Figure 6). 3.8. MSCs Cryopreservation and Post-Thaw CharacteristicsThe cryopreserved female sheep-harvested AD-MSCs and BM-MSCs could successfully revive with a viability percentage of 90% and 89.6%, respectively. The cells could be successfully re-cultured. The cellular morphology appeared fibroblast-like. The cryopreserved cells successfully achieved >80% confluency within 8–10 days (Figure 2). The CFU (f) was comparable to that of the fresh cells although a bit lower (44 colonies in AD-MSCs and 42 colonies in BM-MSCs) (Figure 3). These cells also had successful and similar differentiation properties to those of the fresh cells. 4. DiscussionMSCs are being increasingly utilized in regenerative medicine with a positive in vivo healing response, although currently no definitive use in clinical settings has been made. The main limitation that restricts their definitive utilization is the lack of understanding of their cellular physiological processes [2,23]. MSCs lack uniform properties across donor tissues and also with respect to the physiological status of the donor. For reliable and effective clinical utilization of MSCs, it is imperative to study their properties with respect to the tissue source and physiological status of the donor. The current study demonstrates MSCs’ properties harvested from adipose tissue and bone marrow at full term pregnancy and from male donors suffering with urolithiasis.In the current study, unsuccessful attempts were made to harvest MSCs in peak winter. The transportation and maintenance of the samples at lower temperature for around 30–45 min might have affected the tissue samples. Some of the studies have been able to demonstrate isolation of MSCs from cryopreserved foetal membranes and umbilical cord blood, although with limited success [24,25,26]. The success rate of MSCs’ isolation from umbilical cord blood had been <60% and a delay in achieving the culture expansion has been demonstrated [24,25]. Furthermore, MSCs’ isolation from foetal membranes has been achieved through the explant method [26]. In the current study, the direct exposure to cold might had led to cold shock to the cells; moreover, the adipose tissue under cold environment undergoes hardening, which complicates its enzymatic digestion. The adipose tissue had been processed through an enzymatic method, which could have further stressed the cells. As the tissue samples contain a miniscule number of MSCs, it becomes imperative to culture expand and characterize them before in vivo application. There are variable cell concentrations in tissue sources, and they also tend to vary as per the physiological status of the donor. MNC fraction isolated from bone marrow samples had higher cell concentration as compared to the SVF derived from adipose tissue. The higher cell concentration in bone marrow as compared to that of adipose tissue in the current study is similar to that reported for equines. However, comparably less initial cell concentration in both bone marrow as well as adipose tissue was particular to our study as compared to that demonstrated for equine tissues [27,28]. The higher percent adherent cells in SVF as compared to MNC fraction in our study suggests that a higher MSCs fraction may be available in adipose tissue than bone marrow. The limited cell concentration, however, can be compensated for by harvesting more donor tissue for desired therapeutic concentrations. Although AD-MSCs had shorter PDT as compared to the BM-MSCs, considerable differences with respect to PDT were evident in the samples harvested from full term parturient female sheep as compared to those MSCs harvested from male sheep. One of the previous sheep studies had demonstrated lower proliferation potential of AD-MSCs as compared to the BM-MSCs [5]. The possible reasons behind such a significantly higher proliferation rate and higher cellular concentrations of MSCs from full term parturient female sheep may be due to the higher systemic cortisol and oestrogen concentrations at parturition [29,30,31]. The higher oestrogen concentration at term might favour epithelial-to-mesenchymal transition [31]. However, a delicate balance of such hormones is required as higher cortisol concentration can be detrimental to cells [30] and in absence of progesterone, estrogenic effect may not be obvious [31]. As urolithiasis (urinary obstruction) is an acute condition, the adverse effect on the cell count or their growth is remote. It is also worth mentioning that although the effect of the above-mentioned hormones remains transient, in current study the cells showed higher proliferation for advanced passages and, thus, further studies are desired. The cells from two sources effectively followed the standards of the ISCT [19]. Both these cell types were plastic adherent and appeared fibroblast-like, although at early passage the cells exhibited diverse morphology as has also been reported in other studies [32,33]. The two cell types positively expressed genes for some surface markers while simultaneously lacking expression for haematopoietic marker expression. The two cell types effectively underwent tri-lineage differentiation. These cells showed chondrogenic and osteogenic differentiation as early as 10 days, while most of the studies have reported a 2–3-week time period for cellular differentiation. The possible reason for early chondrogenic and adipogenic differentiation of MSCs may be due to the estrogenic effect [31]. However, contrary to the oestrogen effect reported for MSCs’ osteogenic differentiation [31], we could achieve osteogenic differentiation by day 15 and, thus, further insights are desired. On gene expression analysis, adipogenic (PPARG), chondrogenic (BGN) and osteogenic (BGLAP)-specific genes were upregulated in differentiated cells. These findings have also been reported by others [21,22]. However, one study failed to show higher BGN expression in the chondrogenic differentiation cocktail [21], contrary to another study [22]. This could possibly be due to the differences in the cells based on the source and the physiological status of the donor. Comparative phenotypic and differentiation marker expression potential of MSCs from parturient female and male sheep may be studied for better understanding.The cryopreserved cells from two sources had a comparable viability. MSCs’ viability from two sources was comparably better than that reported for goat foetal membrane-derived MSCs [20] but slightly lower than that reported for sheep dermis-derived MSCs [34]. The cellular viability is affected by the donor tissue type and cell freeze-thaw procedure and needs further advancements to enable the harvesting of an acceptable percentage of cells. The cellular features such as morphology, growth and differentiation were comparable to those of the fresh cells, in agreement with results reported for sheep BM-MSCs [16] and foetal membrane MSCs [20]. 5. ConclusionsTissue samples transported under cold ambient temperature may not be suitable to isolate MSCs, irrespective of the donor tissue type. The tissues harvested from full term pregnant female sheep may yield MSCs with improved proliferation and differentiation potential. The cryopreserved cells tend to show similar cellular features to those of fresh cells and may be a suitable source for their ready-to-use application(s).

animals : an open access journal from mdpi

10.3390/ani13091464

PMC10177495

The impacts of pollutants on the aquatic environment have become an increasingly important subject of study over the past few decades. Pollutants, including pharmaceuticals, can have direct and/or indirect effects on biota, affecting individual trophic levels in the food chain, the composition of populations, or even the degree of parasitism, a secondary stressor to the host. In this study, we assessed uptake of pharmaceutical compounds in tissues of common carp (Cyprinus carpio L.) and parasite community response to the change in environmental conditions six months after relocation from a control to a treatment pond loaded by organic pollution from a sewage treatment plant outlet using partial cross-over experimental design. By comparing fish from control and treatment ponds, we observed higher pollutant uptake and the concentration of pharmacological compounds in fish tissues restocked to the treatment pond, along with changes in fish biometric parameters and parasite load. Fish from polluted environment exhibited decreased parasite diversity and higher ectoparasite abundance; however, the major differences were observed between families within taxonomic groups. Our results, therefore, highlight the need for more detailed taxonomic analyses in studies using parasites as potential environmental bioindicators.

The response of parasite communities to aquatic contamination has been shown to vary with both type of pollutant and parasite lifestyle. In this semi-experimental study, we examined uptake of pharmaceutical compounds in common carp (Cyprinus carpio L.) restocked from a control pond to a treatment pond fed with organic pollution from a sewage treatment plant and assessed changes in parasite community composition and fish biometric parameters. The parasite community of restocked fish changed over the six-month exposure period, and the composition of pharmaceutical compounds in the liver and brain was almost the same as that in fish living in the treatment pond their whole life. While fish size and weight were significantly higher in both treatment groups compared to the control, condition indices, including condition factor, hepatosomatic index, and splenosomatic index, were significantly higher in control fish. Parasite diversity and species richness decreased at the polluted site, alongside a significant increase in the abundance of a single parasite species, Gyrodactylus sprostonae. Oviparous monogeneans of the Dactylogyridae and Diplozoidae families and parasitic crustaceans responded to pollution with a significant decrease in abundance, the reduction in numbers most likely related to the sensitivity of their free-living stages to pollution.

1. IntroductionFreshwater ecosystems have long been subjected to serious threats from human activities, with pollution among the most significant [1,2,3]. Over recent years, there has been increasing concern over the unintentional presence of pharmaceuticals and personal care products (PPCPs) in different compartments of the aquatic environment (e.g., water, sediments, and biota) at concentrations capable of causing detrimental effects to aquatic organisms [4]. Indeed, PPCPs have now been detected worldwide in treated sewage, rivers and streams, seawater, groundwater, and drinking water [5]. In part, this is because present conventional systems of wastewater treatment are not designed to fully remove these contaminants [6]. Though a wide range of pharmaceutical classes are used in human and veterinary medicine, only a few are considered of environmental importance due to their consumption volumes, toxicity, and/or persistence in the environment [7,8]. Despite this, little is known about the ecotoxicological effects of PPCPs on non-target aquatic organisms exposed to such wastewater residues over their life [7], or their natural interactions [8].The majority of fish populations are exposed to a wide variety of anthropogenically sourced chemical compounds, including PPCPs. Though these chemicals are often found at concentrations not directly toxic to fish, exposure at sublethal concentrations may still induce harmful effects [9]. Aquatic habitats under chronic exposure to pollutants often suffer reduced species richness and a loss of community integrity, which may subsequently impact the whole ecosystem [10]. Further, when subjected to many pollutants, other common natural stressors, such as parasites and pathogens, may have an additional effect on fish host physiology and population structure [8]. The combined effects of multiple stressors, i.e., parasites and pollution, may then subsequently reduce either fish host resistance or tolerance to infection [11]. On the other hand, aquatic contaminants may also affect the parasites. As the parasites are, for part of or their whole life, in direct contact with their environment, and thus the toxic substances, their vitality may decrease or mortality increase, with subsequent impacts on parasite community composition and diversity [12]. Despite an increasing number of recent field studies, our understanding of the synergistic effects of pollution and parasites on fish host populations remains limited.Fish parasite abundance, community composition, and structure may be affected by a range of factors, including host ecology and physiology, environmental factors, and anthropogenic stressors [13,14]. Pollution, and other anthropogenic disturbances to the aquatic environment, may affect a parasite community directly by acting on free-living parasite stages [15,16] or on ectoparasites [17], or indirectly by acting on intermediate or definitive host populations [13,18]. This wide variability in the effects of aquatic contaminants on parasites can cause alterations at both organism and population levels [19]. For example, increases in parasite levels in the affected environment may reflect stress-related immune suppression and reduced resistance in the host [20]. Alternatively, an increase in intermediate host abundance due to improved living conditions, e.g., following eutrophication, may favor parasite transmission [13], while decreases in parasitism may result from direct toxicity to either parasites [15] or their intermediate hosts, leading to a decrease in abundance [13].As early as the 1980s, Möller [18] postulated that the composition of ectoparasitic fauna in aquatic organisms might be a useful and quickly reacting indicator for the effects of certain pollution conditions in freshwater ecosystems. A later summary by Blanar et al. [21] confirmed this, with the authors showing that environmental pollution generally has a stronger, mostly negative, effect on directly exposed parasites, i.e., free-living stages and ectoparasites, when compared with endoparasites. Moreover, monoxenous parasites, i.e., parasites with direct life cycles, tend to show higher susceptibility to a larger variety of environmental stressors [21]. Most such studies, however, have been focused on eutrophication and heavy metal pollution [12,22,23], while the effect of pharmaceutical pollutants on host–parasite interactions remains less explored [8,24,25].In the present study, a partial cross-over experimental design was used to assess uptake of pharmaceutical compounds in tissues of common carp (Cyprinus carpio L.) six months after relocation from a control pond to a treatment pond, and to assess parasite community response to the change in environmental conditions. As Blanar et al. [14] pointed out, comparisons of localities in ecotoxicological studies usually consider polluted (treatment) localities without any deep specification of the pollutants involved. In our study, we determined pharmaceutical pollutants as high concentrations of PPCPs have recently been detected in both fish and water samples from the treatment pond [26,27,28], while heavy metal concentrations at the same locality are generally low [29]. Although the fish parasite infection was not evaluated prior the experiment, previous studies in the study area (i.e., pond system in Vodnany region) indicate that parasite communities of common carp share similar parasite species with dominance of monogenean and cestode parasites [25,30], but they differ in parasite prevalence and abundance, reflecting seasonal changes [25,30] and water quality [25]. By relocation of experimental fish in both directions, similar initial pool of parasites was achieved at both sites. The experimental design of our study therefore allowed us to assess how parasites relocated to environmental conditions affected by a high concentration of PPCPs cope with such a change. We hypothesized that parasites sensitive to pollution will limit their reproduction during the main growing season, leading to low abundance at the end of the experiment. Parasites that can take advantage of their host being weakened by other stressors were then expected to increase in their abundance, particularly in restocked fish, as original fish were predicted to be longer adapted to the polluted environment.2. Material and Methods2.1. Study Area and Experimental DesignFor this study, common carp were collected from two experimental ponds with different pollution levels. The treatment locality (Cezarka pond; 49.1466617 N, 14.1915231 E) is a treated wastewater pond (2.6 ha, average depth 1–2 m) located below the sewage treatment plant (STP) for the town of Vodnany (Czech Republic). The STP runs a standard treatment process, including mechanical filtration, sedimentation, biological treatment, sludge concentration, and secondary settlement in tanks. The treated effluent is then fed directly into Cezarka pond, which provides wastewater stabilization as a tertiary treatment process prior to introduction into a fish production pond. STP effluent (except for precipitation) is the sole source of water for the pond. In recent years, studies have recorded high concentrations of PPCPs in both fish and water samples from the pond [27,28,31]. The control pond (49.1577281 N, 14.1623011 E; 0.12 h, average depth 1 m), located near the University of South Bohemia’s Faculty of Fisheries and Protection of Waters in Vodnany, was selected as a locality with no significant pollution [32], being fed by water from the adjacent River Blanice, which has low background pollution concentrations typical of a regional river. As the two ponds are approximately 2 km apart, they are both subject to comparable climatic conditions.Both ponds were stocked with common carp of the same genetic origin that were allowed to feed on natural food only, i.e., there was no supplemental feeding with grain. For the first year of life, the fish were reared in the Cezarka treatment pond and control pond under standard rearing conditions. In April 2018, both ponds were harvested and one year-old fish of similar size were selected from each pond and group-marked. Two groups were then created from the fish from each pond, resulting in four groups for further monitoring, i.e., T-T (original fish from treatment Cezarka pond, returned to Cezarka); T-C (fish originally from Cezarka treatment, restocked to control pond), C-C (original fish from control pond, returned to control pond), and C-T (fish originally from control pond, restocked to Cezarka treatment pond). The ratio between stocked/restocked fish for the two groups in both ponds was approximately 1:1, with final density in both ponds being 0.14 fish × m−2 (see [32] for further details). The fish were kept in particular ponds for a period of six months, corresponding to the length of growing season in this type of habitat. After six months (October 2018), fish were collected from both sites using seine and gill nets until 20 fish from group T-T, 20 from group C-T (both from the Cezarka treatment pond), and 20 from group C-C (control pond) had been collected. Unfortunately, due to technical problems, fish from group T-C (originating from Cezarka, restocked to the control pond) could not be used for further analysis; thus, this study only represents a partial cross-experiment.2.2. Fish Processing and Chemical AnalysisThe fish from each treatment group (T-T, C-T, C-C; n = 20 per group) were humanely dispatched prior to dissection for subsequent parasitological and chemical analysis and the standard length (SL, mm), total length (TL, mm), and total weight (Wt, g) recorded. The fish were then eviscerated and the eviscerated body weight (WE, g) taken, after which the weight of the liver (W[liver], mg), spleen (W[spleen], mg) and gonads (W[gonads], mg) were recorded. Four fish body condition indices were then calculated for each fish, i.e., condition factor (K) = WE × 105/SL3; hepatosomatic index (HSI) = W[liver] × 102/ WE; splenosomatic index (SSI) = W[spleen] × 103 WE; and gonadosomatic index (GSI) = W[gonads] 102/WE. For some fish, it was not possible to retrieve the gonads, and thus confirm the sex; in which case, the fish was not included in any further analysis. WE was used rather than Wt for condition factor analysis to eliminate errors caused by different levels of stomach fullness [33].Liver and brain tissue samples (0.5 g) were taken from each fish during dissection, and the concentration of 69 pharmaceutical compounds was determined according to validated methodologies [34]. Briefly, isotopically labeled internal standards, an extraction solvent, and a homogenizing ball were added to the pre-weighed tissue, after which it was homogenized and centrifuged. The supernatant was then filtered (regenerated cellulose, 0.45 µm pores), frozen at −20 °C for 24 h, then defrosted and an aliquot analyzed via liquid chromatography using a high-resolution mass spectrometer (Thermo Fisher Scientific, Waltham, MA, USA). For further analysis, all pharmaceuticals detected were sorted into medicinal classes according to their clinical effects (Supplementary Tables S1 and S2).2.3. Parasite Collection and IdentificationAll fish were examined for the presence of parasites under a binocular microscope using standard protocols. Protozoan parasites living on gills and fins were examined under light microscopy. Metazoan parasites collected were preserved in glycerin ammonium-picrate mixture (monogeneans), 4% formaldehyde (cestodes, crustacean, hirudineans, glochidia), or 70% ethanol (nematodes). Cestodes were stained using iron acetic carmine, dehydrated in ethanol of increasing concentration and then mounted in Canada balsam as permanent slides [35], while nematodes were mounted in glycerol as temporary slides for light microscopy. Larval parasites, impossible to identify using standard morphological methods (e.g., larval trematodes), were preserved in 96% ethanol and identified using molecular methods following Georgieva et al. [36]. All parasites were identified to species level or to the lowest possible taxa following the respective keys [37,38,39] under an Olympus BX51 light microscope equipped with phase contrast and Stream Motion v.1.9.2 digital image analysis software (Olympus, Tokyo, Japan). Levels of parasite infection were expressed as prevalence, calculated as the proportion of parasitized fish from all fish in each group, mean abundance was expressed as the mean number of parasites in all hosts in the sample and intensity of infection was calculated as the mean number of parasites in infected hosts [40]. All infection parameters were calculated for individual parasite species. Parasite diversity was measured using the Species richness, Shannon–Wiener, Dominance, and Equitability indices for all fish in each group [41], with diversity index values calculated using PAST software [42]. The same software was also used to compare index values between parasite communities using permutation tests generating 1000 random matrices with two samples, the p-value being computed as the proportion of randomly permutated matrix combinations resulting in index difference higher or equal to the difference observed.2.4. Data AnalysisInter-group differences in composition of both parasite community and pharmaceuticals were visualized using non-metric multidimensional scaling (NMDS) and tested using permutational multiple analysis of variance (PERMANOVA). Parasite data were fourth-root transformed and Bray–Curtis distances were used to describe the sample distances. Pharmaceutical data were log (x + 1) transformed, scaled, and centered (to a mean of 0 and SD of 1) and Euclidean distances were used to describe sample distances.Inter-group differences in the four parasite assemblage characteristics (i.e., species richness, abundance, abundance of Gyrodactylus spp., and abundance of Dactylogyrus spp.) were tested using generalized linear models (GLM; Poisson distribution for richness, negative binomial for abundance), with fish SL as a covariate. Owing to their low abundance, other parasite species were not compared. The Tukey HSD approach was used to control for type II errors in multiple post-hoc pairwise comparisons in all models (using the glht and mcp functions from the multcomp package; [43]). Inter-group differences in pharmacological load and fish biometric parameters were tested using analysis of variance (ANOVA) on log-transformed data, with Tukey HSD post-hoc pairwise comparisons.Potential associations between parasites, pharmaceuticals, and fish biometric parameters were examined using co-inertia analysis (COIA, [44,45]), testing whether the variability in parasite assemblage structure, pharmacological load, and biometric parameters were pair-wise correlated to each other. For each fish group (C-C, C-T, T-T), separate COIA analyses were conducted. The first step of COIA involved separate principle component analyses (PCA) of three matrices: parasite abundance, concentration of pharmaceuticals (the same datasets that were used for inter-groups comparisons of parasite assemblage and composition of pharmaceuticals), and fish biometric parameters. In a second step, each pair of PCA ordinations was combined in COIA to explore the co-structure between them. The assessment of a possible link between the two tables was obtained by performing pairwise Monte Carlo permutation tests on the value of the RV coefficient (expressing the amount of correlation between matrices) using 999 random permutations. All statistical analyses were undertaken using R v.4.1.1 [46].3. Results3.1. Parasite CommunityA total of 18 parasite taxa were recorded in the three groups of common carp. Of these, 16 were identified in the C-C group, 10 in the T-T group, and 10 in the C-T group (Table 1), with significantly higher species richness exhibited in control C-C compared with the treatment T-T site (permutation test, p = 0.001; Table 2). The parasite community mainly consisted of ectoparasites, with the dominance of monogeneans (Gyrodactylidae, Dactylogyridae, Diplozoidae) and crustacean (Ergasilidae, Argulidae) species. Gyrodactylus sprostonae was the most abundant and prevalent parasite in both the T-T and C-T groups from the Cezarka treatment pond. Dactylogyridae (Dactylogyrus falciformis, D. achmerowi, D. extensus, and D. molnari), along with Eudiplozoon nipponicum, dominated in fish from the control C-C group. In addition, a high prevalence of parasitic crustaceans (Ergasilus sieboldi, Argulus coregoni, A. foliaceus, A. japonicus) was also recorded in fish from the C-C group. Endoparasitic species, represented by one larval trematode and three cestode species, occurred relatively rarely (Table 1).There were clear differences in parasite diversity between fish from the C-C group and those from the T-T and C-T treatment groups, with the C-C control having significantly higher Shannon–Wiener diversity (all p < 0.001) and Equitability indices (all p < 0.001) and a significantly lower Dominance index (all p < 0.001, Table 2).Fish from the C-C group also had a significantly different parasite community composition compared to both the C-T and T-T groups (PERMANOVA, both p < 0.001, Table 3), with no significant differences between C-T and T-T fish (PERMANOVA, p = 0.058, Figure 1A). There were also significant differences in all four univariate parasite assemblage characteristics (GLM, all df = 2,56, all p < 0.001). While there was no difference in parasite species richness between T-T and C-T (post-hoc comparison, p = 1.000), both groups were significantly less rich compared to C-C (post-hoc comparisons, both p < 0.001; Figure 2A). Parasite abundance in C-T was significantly higher than that in C-C (post-hoc comparison, p = 0.002), while there was no significant difference in abundance between T-T and both C-C and C-T (post-hoc comparisons, p = 0.109 and 0.143; Figure 2B). Abundance of G. sprostonae in C-C was almost negligible, and significantly lower than that in either T-T or T-C (post-hoc comparisons, both p < 0.001), with no significant difference between the latter two (post-hoc comparison, p = 0.238; Figure 2C). In contrast, Dactylogyrus spp. abundance in C-C was significantly higher than that in both C-T and T-T (post-hoc comparisons, both p < 0.001), which again, were not significantly different from each other (post-hoc comparison, p = 0.801; Figure 2D).3.2. Fish Biometric ParametersFish SL and WE were significantly lower in fish from the control C-C group compared with both the C-T and T-T treatment groups (ANOVA, F2,57 = 36.7, p < 0.001 and F2,57 = 11.9, p < 0.001, respectively; all Tukey post-hoc comparison tests p < 0.001). K, HSI, and SSI differed significantly between groups (ANOVA, F2,57 = 26.9, p < 0.001; F2,57 = 16.3, p < 0.001; F2,57 = 9.7, p < 0.001, respectively), with higher values observed in C-C compared to C-T and T-T (post-hoc comparisons, all p < 0.002; Figure 3). No significant differences were observed between C-T and T-T for all parameters mentioned above. For GSI, while no significant differences were found between groups for females (ANOVA, F2,27 = 1.3, p = 0.299), male fish from T-T showed higher GSI values than those from C-C (ANOVA, F2,25 = 6.4, p = 0.006, post-hoc comparison p = 0.004), with no difference between C-T and the other two groups (post-hoc comparisons, p = 0.237 and 0.133; Table 4).3.3. Presence of Pharmaceuticals in Fish TissuesOf the 69 pharmaceuticals and/or metabolites determined in liver and brain tissue, 15 compounds were detected in at least one tissue type. Antidepressants and beta-blockers were the most frequently registered (eight and three compounds, respectively), with analgesics, anti-inflammatory drugs, antiepileptics, and CNS stimulants represented by a single compound each. In brain tissue, six, ten, and nine pharmaceutical compounds were found in the C-C, C-T and T-T groups, respectively, while four, nine, and eleven compounds, respectively, were found in the liver (see Supplementary Table S1). Fish in the C-C group had a significantly different composition of pharmaceutical loading compared to C-T and T-T fish (PERMANOVA, all p < 0.001; Table 5), with no significant differences between C-T and T-T fish (PERMANOVA, p = 0.179; Figure 1B). Antidepressants were found in significantly higher concentrations in both brain and liver tissue, and analgesics in liver tissue, in fish from the C-T and T-T groups compared with the C-C control (ANOVA, F2,57 = 17.89, p < 0.001, F2,57= 9.31, p = 0.001, and F2,57 = 6.54, p = 0.002; post-hoc comparisons all p < 0.005; Figure 4), with no significant difference between the C-T and T-T groups (post-hoc comparisons, p = 0.884, 0.997, and 0.942). Beta-blocker concentrations were significantly higher in fish from the T-T group than the C-C and C-T groups (ANOVA, F2,57 = 8.59, p < 0.001; post-hoc comparisons p = 0.002 and p = 0.002), while anti-inflammatory drug concentrations were significantly higher in T-T than C-C (ANOVA, F2,57 = 3.19, p = 0.049; post-hoc comparison p = 0.042), with no difference between the C-T and T-T and C-C groups (post-hoc comparisons, p = 0.232 and 0.688, respectively).3.4. Association between Parasite Infection and Pharmaceutical LoadSignificant covariance between parasite assemblage structure and composition of pharmaceutical load in fish tissues were found in both T-T and C-T fish but not in C-C fish (Table 6). No such covariance was observed between fish biometric parameters and either parasite assemblage structure or pharmaceutical load in all fish groups (Table 6).4. DiscussionIn this study, we addressed the effects of aquatic pollution on common carp by assessing the uptake of pharmaceutical compounds and their effect on biometric parameters, parasite community composition, and parasite abundance, using a partial cross-over semi-experimental approach. While changes in parasite community composition, diversity, and species richness have previously been used as an indicator of environmental impact [47], Marcogliese et al. [48] suggested that parasite communities may not be sensitive enough to detect the effects of low to moderate pollution, or that the effects may be overshadowed by those of natural environmental variation. In our study, the less polluted control site was characterized by higher parasite diversity and equitability and lower dominance. This corresponds to findings reported from a range of other freshwater ecosystems, e.g., [47,49,50,51], and supports the indication value of parasite communities in partially controlled natural studies. The parasite community of carp restocked into the treatment pond (C-T) from the control pond (C-C) adapted quickly to the new environmental conditions and, after six months exposure, matched the composition of those carp in the treatment pond. This change was manifested by a dramatic decrease in dactylogyrid and diplozoid monogeneans and ergasilid crustaceans and the disappearance of Argulus spp., alongside an increase in the abundance of gyrodactylid parasites. Though a detailed analysis of endoparasites could not be performed due to the generally low levels of infection, our data clearly show differences in parasite community composition between the control and treatment sites (Figure 1). Thus, unsuitable environmental conditions in Cezarka pond resulted in a decrease in parasites species richness and a decrease in the abundance of viviparous monogeneans and parasitic crustaceans, possibly due to the direct effect of toxic substances on free-living parasitic stages [52]. On the other hand, massive gyrodactylid infection levels in the treatment pond resulted in a significant decrease in parasite diversity and equitability, alongside high dominance indices.Proliferation of viviparous gyrodactylid parasites tends to be attributed to a reduction in host resistance under certain pollution conditions [18]. Moreover, polluted environments are assumed to negatively affect fish gills and damage the skin’s protective barrier, facilitating access to infection [53]. As a result, weakened individuals are more likely to be infected by parasites, particularly on the host’s surface [54,55]. Alternatively, contaminant exposure has been shown to result in hosts producing excess mucous, which gyrodactylids feed on [56,57], possibly explaining the high gyrodactylid numbers at polluted sites. Thus, gyrodactylids may prosper in such polluted environments as their reproductive strategy allows them to reproduce rapidly, resulting in large-scale invasion of hosts [58]. These findings were supported by our own study, where hundreds of G. sprostonae were found on the fins and gills of a single common carp in both the C-T and T-T treatment groups, while only a few specimens infected control (C-C) fish. Overall gyrodactylid abundance was almost two-times higher in fish restocked to Cezarka pond (though the difference was not significant), indicating that stress associated with relocation to a polluted site may contribute to such high infection intensities. As summarized in Gilbert and Avenant-Oldewage [19], similarly high gyrodactylid abundance levels have been found in fish exposed to eutrophication [22] and sediments contaminated with polynuclear aromatic hydrocarbons (PAHs) and PCBs [59] or pharmaceutical compounds released from sewage treatment plant effluent [24]. Surprisingly, opposing results were observed in three-year-old carp in our previous study [25]; however, in this case, the absence of gyrodactylids in carp from the Cezarka treatment pond was explained by the extremely high condition status of the fish due to excess food availability [31], which helped the fish cope better with parasite infection [25]. In the present study, carp in the two treatment groups had much lower condition factors as a result of decreased natural food availability in autumn [31].Unlike gyrodactylids, oviparous monogeneans, including dactylogyrids and diplozoids, and parasitic crustaceans, which were all abundant at the control site (C-C), were significantly reduced in fish from both treatment groups (T-T and C-T). Carp from the control site, which were naturally infected with a rich and abundant dactylogyrid community, lost most of their parasites after being restocked to the Cezarka treatment pond, retaining just 4% of dactylogyrids originally found at the control site. While almost all control fish were infected with E. nipponicum, for example, only one parasite was found in each of the treatment groups, suggesting that the free-living larval stage, the oncomiracidium, which actively searches for hosts after hatching in water, may be highly sensitive to environmental stress [15], with a resultant drop in numbers in polluted environments. A similar explanation may also be applied for the absence of Argulus branchiuran and the decrease in abundance of the ergasilid copepod E. sieboldi at the Cezarka treatment pond. A similar reduction in parasite load has been observed for oviparous monogeneans (Cichlidogyrus spp.) infecting Mozambique tilapia (Oreochromis mossambicus) in African reservoirs, where parasite abundance and species richness decreased dramatically with increased levels of contamination [60]. Likewise, a decrease in the diversity and abundance of Dactylogyrus spp. was also found in chub (Squalius cephalus) at downstream sites along an increasing pollution gradient in the River Bilina, Czech Republic [50]. Moreover, Gilbert and Avenant-Oldewage [17] found that the diplozoid Paradiplozoon ichthyoxanthon had disappeared from a polluted site in South Africa within 14 years of the water quality decreasing, despite the plentiful presence of its fish host, while changes in parasite prevalence in the control lake over the same period clearly reflected the parasite’s natural seasonal variance. It should be noted, however, that the response of dactylogyrid parasites to pollution varies between studies, with data analyzed from 14 published studies showing an equal number of positive and negative responses of dactylogyrids to contaminants (see [19]). While eutrophication has been reported as promoting the abundance of dactylogyrid and diplozoid monogeneans [22,61], we observed a reduction in infection in our study, possibly related to the composition of pharmaceutical compounds in the Cezarka treatment pond. For example, while antibiotics and antihelminthics were not detected in fish at significant concentrations during the study, these compounds are regularly present in the water [25,31] and may possibly have had a negative effect on the survival of free-living parasite stages.Our data showed that fish restocked from the control (C-C) to the treatment pond (C-T) increased their growth rate, resulting in a significant increase in SL and Wt over six months that matched the values of fish living in the treatment pond their whole life (T-T group), while condition indices in the treatment group were significantly lower than those in control. However, no covariance was observed between fish biometric parameters and pharmaceutical load. In a previous study at the same site [31], extreme growth rates of juvenile carp in the Cezarka treatment pond led to a high total fish biomass within the first three months. The resultant high feeding pressure caused a significant reduction in the availability of natural food, leading to a dramatic decrease in K and his over the following three months due to high competition for food, though SL and Wt remained higher in the Cezarka pond [31], as in our own results. It is also likely that the massive gyrodactylid infection observed contributed to the decrease in carp condition parameters in both treatment groups.As pharmaceuticals usually form complex mixtures with additive, synergistic, or antagonistic impacts under natural conditions [62], separation of impacts from individual pharmaceuticals is at the least complicated, if not impossible [63]. Variability in pharmacological load at treatment site was significantly associated with that in parasite assemblage structure in both C-T and T-T treatment groups, while no such association was found in C-C group, supporting our prediction of parasites response to organic pollution. Nevertheless, no significant differences in parasite abundance and species composition, as well as pharmaceutical load between C-T and T-T groups suggest that relocated fish quickly adapted to the new conditions, showing similar vulnerability to parasites as T-T fish. High pharmacological load in fish tissues have previously been associated with overall parasite abundance of brown trout exposed to STP effluent in a small river [24], suggesting that exposure to pollutants impairs the host’s immune system, resulting in a higher susceptibility to parasite infection [13,20,64]. Over the six-month period of exposure in this study, carp restocked to the Cezarka pond mainly acquired antidepressants and their metabolites (clomipramine, sertraline and its metabolite norsertraline, citalopram and its metabolite N-desmetylcitalopram), anti-inflammatory drugs (diclofenac), and analgesics (tramadol), with the antidepressants and analgesics reaching almost the same concentrations as fish living in the pond their whole life (T-T group; Figure 3), reflecting their relatively rapid accumulation in fish tissue, particularly the brain [65]. In the study by Pravdová et al. [24], the high concentrations of antidepressants in brown trout resulted in a significant increase in gyrodactylid parasites. Accordingly, our results showed massive gyrodactylid infection in common carp exposed to pharmacological pollution where antidepressants were among the most concentrated compounds (Supplementary Table S1). Recent studies examining the effects of neuroactive pharmaceuticals on fish behavior [66,67] have suggested that ecological endpoints (e.g., behavior) are more sensitive to pharmaceuticals than the more commonly used toxicological endpoints [68]. This is because such compounds are specifically designed to affect mood or nerve function and reduce stress, with possible consequences for feeding, predator avoidance, or schooling behavior [6]. Transmission of gyrodactylids from one host to another occurs through direct contact [58]. Increased gyrodactylid infection at the treatment site may, therefore, be also associated with behavioral changes potentially induced by neuroactive compounds [69,70], in addition to the negative impacts of contaminants on the immune response of the host fish enhancing the infection rate [13,20,71].5. ConclusionsThis semi-experimental study increases our knowledge of how fish and their parasites respond to organic pollution in aquatic habitats. By comparing fish from control and treatment ponds, it was possible to observe differences in both pollutant uptake and the concentration of pharmacological compounds in fish tissues, along with changes in fish biometric parameters and parasite load. Though our data confirmed higher ectoparasite abundance and a slightly lower endoparasite abundance in polluted environments, which agrees with general trends elsewhere [21], such trends are not universal, and major differences were observed between families within taxonomic groups, especially in ectoparasites. Monogeneans are often considered useful environmental bioindicators [12,19,59]; our data, however, show that their bioindicative abilities can vary widely among families. Our results, therefore, highlight the need for more detailed taxonomic analyses, at least to the levels of family or genus, in the parasites evaluated for studies using parasites as pollution indicators.The experimental design of this study did not allow us to study consecutive development of parasite community in fish translocated to polluted environment, as all fish were dissected after six months growing season. In future studies, assessment of parasite communities in different phases of fish host exposure to organic pollution would help to clarify the differences between parasite groups in their response to inappropriate environmental conditions.

animals : an open access journal from mdpi

10.3390/ani11041171

PMC8074133

To elucidate the genetic basis of reproductive seasonality in Rasa Aragonesa sheep breed, we performed a genome-wide association study (GWAS) in order to detect single nucleotide polymorphisms (SNPs) or regions associated with traits related to ovarian function and behavioural signs of estrous. The GWAS included 205 ewes with genotypes for 583882 SNPs. Only one SNP overcame the genome-wide significance level. Nine potential SNPs overcame the chromosome-wise significance level (FDR 10%). Gene annotation demonstrated that CD226 molecule (CD226) and neuropeptide Y (NPY) genes that could be involved in reproductive seasonality were close to the significant SNPs. To validate the results, we sequenced the entire coding region of the NPY gene and four exons of the CD226 gene to search for polymorphisms that could be involved in the phenotypes studied. Two synonymous and two nonsynonymous SNPs in the NPY and CD226 genes, respectively, were genotyped in the whole population. We demonstrated that the AA genotype of the SNP rs404360094 located in exon 3 of the CD226 gene was associated with higher and lower total days of anoestrus and oestrous cycling months, respectively. Therefore, this SNP could be utilized as a genetic marker for assisted selection marker to reduce seasonality.

A genome-wide association study (GWAS) was used to identify genomic regions influencing seasonality reproduction traits in Rasa Aragonesa sheep. Three traits associated with either ovarian function based on blood progesterone levels (total days of anoestrus and progesterone cycling months) or behavioral signs of oestrous (oestrous cycling months) were studied. The GWAS included 205 ewes genotyped using the 50k and 680k Illumina Ovine Beadchips. Only one SNP associated with the progesterone cycling months overcame the genome-wide significance level (rs404991855). Nine SNPs exhibited significant associations at the chromosome level, being the SNPs rs404991855 and rs418191944, that are located in the CD226 molecule (CD226) gene, associated with the three traits. This gene is related to reproductive diseases. Two other SNPs were located close to the neuropeptide Y (NPY) gene, which is involved in circadian rhythms. To validate the GWAS, partial characterization of both genes by Sanger sequencing, and genotyping of two synonymous and two nonsynonymous SNPs in the NPY and CD226 genes, respectively, were performed. SNP association analysis showed that only SNP rs404360094 in the exon 3 of the CD226 gene, which produces an amino acid substitution from asparagine (uncharged polar) to aspartic acid (acidic), was associated with the three seasonality traits. Our results suggest that the CD226 gene may be involved in the reproductive seasonality in Rasa Aragonesa.

1. IntroductionSheep reproduction at temperate latitudes is widely known to exhibit marked seasonality [1]. The photoperiod represents a temporal signal to initiate changes in sheep reproductive status [2,3]. Lambing normally occurs at the end of winter-early spring, which causes seasonal variation in lamb production throughout the year. Accordingly, there is an imbalance between the availability of animal products and consumer demand. To overcome this disequilibrium, several alternatives have been developed, such as hormonal and/or photoperiod treatments. Nevertheless, at the initiative of the European Commission and to achieve “clean, green, and ethical” animal production [4], the use of hormones is being reviewed, primarily because it generates hormonal residues in animal carcasses. Therefore, breeders need other alternatives that minimize or completely avoid the use of hormonal treatments.Differences in the duration of the breeding season between breeds and between individuals within a breed raised in the same region have been reported [5,6]. Ewes exhibiting spontaneous out-of-season ovulatory activity (SOA) are of considerable interest for identifying genes and mutations involved in molecular pathways controlling reproductive seasonality in sheep. In Rasa Aragonesa, Folch and Alabart [7] reported that approximately 25% of ewes have spontaneous ovulations in spring and can be naturally mated throughout the year with good management and feeding conditions. Therefore, developing a genetic approach for improving the out-of-season breeding ability of animals may represent a useful way to address this challenge. Hanocq et al. [8] reported relatively high heritability and repeatability estimates (0.20 and 0.30, respectively) for SOA in the Merinos d’Arles breed. Similar heritability of SOAs was found in the Chios breed in Greece [9] and in the Latxa breed in Spain [10]. Hence, the estimation of genetic parameters for SOA indicated that this trait could be used in selection [11].Advances in genomic research and high-throughput genotyping techniques have enhanced the ability of researchers to search for mutations that underlie variations in complex traits [12,13]. Genome-wide association studies (GWAS) have become an important method for identifying genes and genomic regions associated with economically important traits in livestock. These studies are used to screen the whole genome for target genes that correlate with phenotypic traits using single nucleotide polymorphisms (SNPs) as genetic markers [14]. In sheep, progress has been made in the genetic basis of reproductive seasonality. In fact, three genes have been reported to be associated with out-of-season breeding: melatonin receptor subtype 1A (MTNR1A) [15,16,17,18,19,20,21,22,23,24,25], arylalkylamine N-acetyltransferase (AANAT) [26], and leptin receptor (LEPR) [27]. The first gene acts through high-affinity G-protein coupled receptors, one of which is melatonin receptor 1A encoded by the MTNR1A gene. Calvo et al. [28] and Lakhssassi et al. [27] showed that the SNPs rs403212791 in exon 2 of the MTNR1A gene and rs403578195 in exon 8 of the LEPR gene were associated with reproductive seasonality traits in the Rasa Aragonesa sheep breed. AANAT is involved in the biosynthesis of melatonin and controls daily changes in melatonin production. Additional candidate genes involved in seasonal breeding in sheep, such as the aryl hydrocarbon receptor nuclear translocator-like protein (ARNTL), casein kinase 1 epsilon (CSNK1E), clock circadian regulator (CLOCK), cryptochrome circadian regulator 1 (CRY1), period circadian regulator 1 (PER1), period circadian regulator 2 (PER2), and neuronal pas domain protein 4 (NPAS4), have been reported in functional genomic studies of genes associated with circadian and circannual rhythms [29,30,31], although association studies of these genes with seasonality in ovine species have not been published to date.As an initial attempt to identify genomic regions associated with reproductive seasonality in Rasa Aragonesa, our research group carried out a GWAS using 110 ewes genotyped by the Illumina OvineSNP50 Beadchip [32]. Several genes were identified near the significant SNPs, namely, neuropeptide s receptor 1 (NPSR1), heparan sulfate-glucosamine 3-sulfotransferase 5 (HS3ST5), regulatory associated protein of mtor complex 1 (RPTOR), and neuronal pentraxin 1 (NPTX1), which are related to circadian and circannual rhythms. The aims of this study were the following: first, to perform a GWAS with a larger animal sample (205 ewes) using medium (50k) and high-density (680k) arrays from an Illumina Ovine Beadchip to identify new SNPs underlying sheep reproductive seasonality traits. Second, we confirmed the results obtained in the GWAS analysis through partial characterization of the genes located close to significant SNPs with putative functions related to seasonal reproduction and subsequent genotyping of some SNPs identified in these genes for association studies.2. Materials and Methods2.1. Animal SamplesPhenotypic seasonality data were collected from an experimental flock of Rasa Aragonesa sheep, described by Martinez-Royo et al. [32]. Flock management was the same for all ewes. Briefly, 3 different flock age groups were considered for a total number of 265 ewes: 155 mature ewes (5.2–7.2 y; 5.5 ± 0.5; mean ± SD), 84 young ewes (1.9 ± 0.0 y), and 26 lambs (0.94 ± 0.0 y). From these 265 ewes, 205 were used for GWAS distributed as follows: 122 mature (5.4 ± 0.45 y), 66 young (1.9 ± 0.0 y) and 17 ewe lambs (0.94 ± 0.0 y). Every three weeks, individual live weight (LW) and body condition score (BCS) on a 1 to 5 scale [33] were assessed. The mean LW and BCS were similar in the mature and young ewe age groups. The pooled overall means and standard deviations for mature and young ewes for the entire experimental period were 52.5 ± 7.7 kg and 2.9 ± 0.3 for LW and BCS, respectively. Ewe lambs had LW and BCS values of 40.6 ± 3.8 kg and 2.8 ± 0.1, respectively. The experimental period lasted from January to August 2012.2.2. Measurement of Reproductive Seasonality TraitsIn Martinez-Royo et al. [32] are reported the three reproductive seasonality phenotypes analysed in this study in detail. The first two phenotypes were based on weekly individual plasma progesterone level measurements. The total days of anoestrous (TDA) were the sum of days in anoestrous. This period of ovarian inactivity was characterised by three or more consecutive progesterone concentrations lower than a threshold of 0.5 ng/mL. The second reproductive seasonality phenotype was the progesterone cycling months (P4CM), defined for each ewe as the rate of cycling months according to the progesterone level measurements. Progesterone was determined using a commercial ELISA kit designed for ovine plasma (Ridgeway Science, St. Briavels, Gloucestershire, UK). An ewe was considered cyclic in a month when the progesterone concentration was higher than or equal to the threshold of 0.5 ng/mL in at least one progesterone determination in that month. It is outstanding that ewes with progesterone levels below the 0.5 ng/mL threshold in all samples taken in January and with more than 4 consecutive samples higher than the threshold (potential pathological ewes) were discarded for the GWAS. In the same way, the ewes had to be cycling in the preceding breeding season, according to three progesterone determinations performed one week apart in October. Finally, the oestrous cycling months (OCM) was the third phenotype analysed. This trait was explained for each ewe as the rate of months cycling based on daily oestrous records, using eight vasectomised rams fitted with breeding harnesses with a replaceable marking crayon [34]. Consequently, oestrous was recorded daily as crayon marks on the rumps of the ewes.2.3. Sampling and Genotyping AnalysisThe GWAS included 110 ewes genotyped with the OvineSNP50 Infinium Beadchip (Illumina Inc., San Diego, CA, USA) designed by the International Sheep Genome Consortium [35], as employed in the study performed by Martinez-Royo et al. [32], and 97 ewes genotyped with the 680k (IlluminaAgResearchSheep HD). Genomic DNA was extracted from blood samples using the SpeedTools DNA Extraction kit (Biotools, Madrid, Spain). SNP genotyping services were provided by the Spanish centre “Centro Nacional de Genotipado (CEGEN-ISCIII)” (https://www.usc.es/cegen/; accessed on 19 April 2021) and “Xenetica Fontao” company (https://www.xeneticafontao.com; accessed on 19 April 2021).2.4. Data Quality Control and Genome-Wide Association AnalysisThe software Plink1.9 [36] was used for quality control (QC) of each genotyped data. Individuals with a low call rate (< 0.90) were excluded from additional analysis. The SNPs that met the following criteria were selected: call rate > 0.97 and minor allele frequency (MAF) > 0.01. SNPs that failed Hardy–Weinberg equilibrium (HWE) (p-Value < 0.001) were excluded. Next, the two datasets were merged with PLINK 1.9, and the Beagle4.0 program [37] was used to impute 50k to 680k genotypes. We performed clustering and multidimensional scaling (MDS) to check for population stratification using PLINK 1.9. SNPs that passed QC were pruned using the linkage disequilibrium (LD) pruning parameters of r2 < 0.2 over a window size of 50 SNPs, and a step of 10 SNPs. Genome-wide identity-by-state (IBS) pairwise distances were calculated using all SNPs that remained after pruning. A pairwise population concordance test constraint was applied to the clustering procedure (-ppc option). GWAS was performed using the GCTA (Genome-wide Complex Trait Analysis) program [38] running a mixed linear model association (MLMA) and excluding the chromosome on which the candidate SNP is located (leaving-one-chromosome-out, or LOCO). The model also considered age and clusters obtained by MDS as a fixed effect, and BCS and LW effects as quantitative covariates. The estimated genetic relationships matrix (GRM) was included in the mixed model analysis to correct the effect of population substructure. The significance of association was assessed using Bonferroni correction and the false discovery rate (FDR) multitest correction tests. Chromosome-wise significance association was also assessed using a false discovery rate (FDR = 0.1) multitest correction threshold [39]. The choice of a threshold of 10% can be explained by the fact that in this study, the objective is mainly exploratory in order to identify new SNPs underlying sheep reproductive seasonality traits, and these are low heritable female traits. Visualization of the association data in Manhattan plots and quantile-quantile plots was performed using SNPEVG software [40]. To control the number of false positives, genomic inflation factors were calculated in R v3.5.1 software for each reproductive seasonality trait. The genomic inflation factor was estimated as the observed median χ2 divided by the expected median χ2.2.5. Gene IdentificationGenes located within a 500 kb-long interval centered on the significant SNPs associated with the three seasonality traits were obtained according to the sheep genome assembly (Oar_v3.1) and based on Ensembl release 81.2.6. Validation of GWAS Results2.6.1. CD226 and NPY Gene CharacterizationGene annotation based on the results of GWAS demonstrated that CD226 and NPY genes could be involved in reproductive seasonality. The ovine CD226 gene is located on chromosome 23, covering approximately 100.7 kb with 6 exons (GenBank acc. Number NC_040274), whereas the NPY gene is located on chromosome OAR4 and covers approximately 6.8 kb with 4 exons (GenBank acc. Number NC_040255). All exons of the NPY gene were characterized, while four exons were chosen to characterize the CD226 gene. The significant SNPs were located in intron 2 of CD226, approximately 52 and 4 kb from exons 2 and 3, respectively. These exons were selected because they have nonsynonymous polymorphisms in the Ensembl Variation database (https://www.ensembl.org/info/genome/variation/index.html; accessed on 19 April 2021) of the Oar 3.1 version of the sheep genome. Primer3 software was used to design the primers (https://bioinfo.ut.ee/primer3–0.4.0/; accessed on 19 April 2021). Target-specific primers were designed in intron-flanking regions around the targeted exon. Table 1 shows the oligonucleotide sequences, the amplified exon, the annealing temperature, and expected product sizes. Genomic DNA was extracted from blood samples using standard protocols. The genomic DNA (25 ng) was amplified in a final polymerase chain reaction (PCR) volume of 25 µL containing 5 pmol of each primer, 200 nM dNTPs, 2.4 mM MgCl2, 50 mM KCl, 10 mM Tris-HCl, 0.1% Triton X-100, and 1 U Taq polymerase (Biotools, Madrid, Spain). The following cycling conditions were used for all amplification fragments: an initial denaturation step of 94 °C for 3 min followed by 35 cycles of PCR, with cycling conditions of 30 s at 94 °C, 30 s at annealing temperature, and 30 s at 72 °C, and a final extension step of 72 °C for 5 min.Direct Sanger sequencing of the PCR products from the 8 exons in 18 ewes with extreme and intermediate values for TDA (low TDA: 0 days, n = 6; intermediate TDA: 56 ± 19.8 days, n = 6; high TDA: 142.3 ± 15.7 days, n = 6) was utilized to look for polymorphisms in the experimental population. The PCR products were purified using the FavorPrep Gel/PCR purification mini kit (Favorgen, Ibian, Zaragoza, Spain), and sequenced in both directions by STAB Vida company (Caparica, Portugal) using an ABI 3730XL sequencer (Applied Biosystems, Foster City, CA, USA). Homology searches were performed using BLAST (National Centre for Biotechnology Information: https://blast.ncbi.nlm.nih.gov/Blast.cgi; accessed on 19 April 2021). To align the sequences, BioEdit [41] software and CLUSTAL Omega (http://www.ebi.ac.uk/Tools/msa/clustalo/; accessed on 19 April 2021) were used. The impact of amino acid substitution on the structure and function of the protein was predicted using the PolyPhen-2 (http://genetics.bwh.harvard.edu/pph2/; accessed on 19 April 2021) [42] and Variant Effect Predictor (VEP: http://www.ensembl.org/Ovisaries/Tools/VEP?db=core; accessed on 19 April 2021) softwares. The locations of SNPs were determined based on the genome version of Ovis aries Oar_v3.1.2.6.2. CD226 and NPY Polymorphism GenotypingGenomic DNA was extracted from blood samples of 265 ewes of the flock using standard protocols. Two nonsynonymous SNPs in the CD226 gene were selected for genotyping the whole population by Kompetitive allele-specific PCR (KASP) following the manufacturer’s instructions: one in exon 2 (rs588529642) and the second in exon 3 (rs404360094). The sequence surrounding the target polymorphism was submitted for assay design to the KASP genotyping provider (LGC Genomics, Biotools, Spain). Assays were performed in 96-well formats in a 10 μL volume containing 1 µL of DNA (20 ng final concentration of DNA), 5 µL of 2X KASP V4.0 master mix standard ROX (LGC Genomics, UK), 0.14 µL of assay mix (KASP by Design assay mix, LGC Genomics, UK) and 3.86 µL nuclease-free water. Reactions were performed in a CFX96 Bio-Rad thermocycler (Bio-Rad, Madrid, Spain) with conditions as follows: 94 °C for 15 min followed by 9 touchdown cycles of 94 °C for 20 s and 57 °C for 60 s (decreasing −0.6 °C per cycle) followed by 25 additional cycles of 20 s at 94 °C and 60 s at 55 °C. Following PCR, the plate was cooled to 30 °C, and fluorescence was read using a single quantification cycle for 1 s.The assay designed to genotype the NPY gene failed. Therefore, the two synonymous SNPs located in exon 2 of the NPY gene (OAR4: g.71593018G > T, and rs594346709) were genotyped by Sanger sequencing (as described above in Section 2.6.1).2.6.3. SNP Association StudiesHaploview software v4.2 [43] was used to calculate the Hardy–Weinberg equilibrium exact test, the observed and expected heterozygosities and the minor allele frequency (MAF) for each SNP. Statistical association studies between the SNPs and reproductive seasonality traits (TDA, P4CM, and OCM) were performed by fitting a linear model using the Rcmdr package of R software (http://socserv.socsci.mcmaster.ca/jfox/Misc/Rcmdr/; accessed on 19 April 2021) [44], including the genotype of the SNPs (S) and age (mature, young and ewe lambs) (A) as fixed effects. The LW and (CS were fitted as covariates. The least square means (LSMs) for each pairwise comparison were calculated to test differences between genotypes. The Bonferroni correction was used to consider multiple tests. The CD226 and NPY SNPs were independently analysed with the same statistical model.2.6.4. Haplotype Association StudiesHaploview software v4.2 was used to define blocks of LD based on the 4-gamete rule [43]. D’ and r2 within the CD226 and NPY were estimated. SNPs were phased using the expectation-maximization (E–M) algorithm to assign individual haplotypes with PLINK1.9 [45]. Diplotypes with a posterior probability lower than 0.7 were discarded. Haplotype association studies were performed using the Rcmdr package by fitting a similar lineal model to that used for the SNP association studies but included the haplotype (H) effect instead of the S effect. The number of copies of each haplotype were codified as 0, 1, or 2 copies. Haplotypes with <1% frequency were not included in the analysis. The LSMs for each pairwise comparison were calculated to test differences between haplotypes. The Bonferroni correction was used to consider multiple tests.3. Results3.1. GWAS ResultsAfter the QC was performed on the imputed genotypes, 205 ewes with genotypes for 583882 SNPs distributed on the 27 ovine chromosomes were retained for subsequent analyses. In the MDS analysis, 188633 autosomal SNPs were used to calculate the pairwise IBS distance after SNP pruning. MDS analysis revealed a substructure within the total dataset and identified 4 principal clusters in the analysed population (Figure S1). These clusters were taken into account for subsequent association analysis. The GWAS results obtained through MLMA and LOCO were very similar. The genomic inflation factors for each trait were less than 1 (TDA: 0.96; P4CM: 0.97; OCM: 0.95), and then no more adjustment was needed.Only one significant SNP (rs404991855) for P4CM was found at the genome-wise significance level after Bonferroni correction (Table S1). In the same way, a trend was observed for the same SNP and TDA trait (p = 0.07). Figure S2 presents the Manhattan and Q-Q plots for the three traits. Furthermore, 2, 7, and 4 SNPs were significantly associated at the chromosome-wise level (FDR p < 0.10) with TDA, P4CM, and OCM traits, respectively (Figure 1 and Table 2). Two SNPs (rs404991855 and rs418191944) located on chromosome 23 were found to be significantly associated with the three traits at the chromosome level (Figure 1 and Table 2). The seven SNPs associated with P4CM variability were located on chromosomes 4 (rs424340754, rs410373132), 6 (rs409834034), 7 (rs428238419 and rs405959180), and 23 (rs404991855 and rs418191944) (Figure 1c). Finally, four SNPs on chromosome 23 (rs405024177, rs404991855, rs418191944 and rs410842314) were associated with OCM (Figure 1b). The genes located 250 kb upstream and downstream of the most significant SNPs for each seasonality trait are shown in Table 2. Two genes were of interest because they may be related to reproductive seasonality trait variation. The NPY gene, and CD226 gene that is also known as DNAM-1 (DNAX Accessory Molecule-1). The significant SNPs for P4CM in chromosome 4 were located 35 (rs424340754) and 47 kb (rs410373132) from NPY and were completely linked. The SNPs rs404991855 and rs418191944 were located in intron 2 of CD226.3.2. Validation StudiesWe sequenced the entire coding region of the NPY gene and four exons of the CD226 gene to search for polymorphisms that could be involved in the phenotypes studied. No deleterious mutations were detected. For the NPY gene, we found two synonymous SNPs in exon 2. One of these mutations was not previously described (OAR4:g.71593018 G > T) (Table 3). These SNPs showed low MAFs, ranging between 0.025 (g.71593018 G > T) and 0.082 (rs594346709) (Table S2). Although these mutations were synonymous, they were genotyped in the whole population, since they might be in linkage disequilibrium with putative causative mutations.For the CD226 gene, three SNPs were detected in exon 2 (rs427511555, rs403900117 and rs588529642), and one SNP was detected in exon 3 (rs404360094). Two of these SNPs (rs588529642 and rs404360094) were nonsynonymous substitutions and were selected to be genotyped in the whole population. These SNPs were predicted in silico to be tolerated by VEP and benign by PolyPhen-2, showing that the SNP located in exon 2 (rs588529642) had a lower SIFT value (0.1) (Table 3). This SNP produces a change of methionine to valine (Met60Val), both of which belong to hydrophobic groups (nonpolar). Conversely, the SNP located in exon 3 (rs404360094) produces an amino acid substitution from asparagine (uncharged polar) to aspartic acid (acidic) at position 243.To determine the extent of LD among these markers, we estimated the parameters D’ and r2 between the pairwise combination of the SNPs in the same chromosome. Very low LD was found. LD indices for SNPs located in the CD226 gene were 0.06 and 0 for D’ and r2, respectively. For SNPs located in the NPY gene, 1 and 0.002 were observed for D’ and r2, respectively. One haplotype block was predicted for SNPs located in exon 2 of the NPY gene.3.3. SNP Association StudiesAssociation analyses included 265 ewes. Table 4 shows results after Bonferroni correction. Only the SNP located in exon 3 of the CD226 gene was significantly associated with the three reproductive seasonality traits (see Table S3 for further details). In fact, significant differences were observed among alternative genotypes. Ewes with the AA genotypes had higher TDA values than ewes with AG (50.80 ± 12.76; p < 0.01) or GG (42.52 ± 12.81; p < 0.05) genotypes. Similarly, ewes with the AA genotype showed fewer oestrous records than those with AG (−0.17 ± 0.05; p < 0.01) or GG (−0.13 ± 0.05). Similar results were observed for the P4CM trait.3.4. Haplotype Association StudiesHaplotype association studies were performed, considering block 1, which was predicted with Haploview (the two SNPs in exon 2 of the NPY gene), and a second block containing the two nonsynonymous SNPs of the CD226 gene (block 2). Three haplotypes were identified for each block (Table S4). Only diplotypes with a posterior probability higher than 0.7 and haplotype frequency > 1% were considered. The results of haplotype analysis are presented in detail in Tables S5 and S6 for blocks 1 and 2, respectively. After Bonferroni correction, only haplotypes H1 and H2 of block 2 were associated with the three reproductive seasonality traits, thereby confirming the previous association found in SNP analysis (Table 5). In this sense, ewes with one or 2 copies of haplotype H1 (AG) had lower TDA and more oestrous records than those without copies (in bold the SNP allele associated with the trait). This haplotype contains the G allele of the SNP located in exon 2 (rs418191944), which is associated with lower TDA and higher P4MC and OCM values. Conversely, ewes with 2 copies of haplotype H2 (AA) had more TDA and fewer oestrous records.4. DiscussionNine potential SNPs reaching the chromosome-wise level of significance were identified to be associated with the TDA, P4CM, and OCM reproductive seasonality traits. Only one SNP (rs404991855) associated with P4CM overcame the genome-wide significance level. The SNPs rs404991855 and rs418191944 on chromosome 23 appeared to be associated with the three traits and were located in intron 2 of the CD226 gene. This gene has been related to such reproductive diseases as cystic ovarian teratoma and mature teratoma of the ovary in humans [46,47]. On the other hand, SNPs (rs424340754 and rs410373132) in chromosome 4 were significantly associated with P4CM. These SNPs are located in an intergenic region approximately 34 kb from the NPY gene, which influences many physiological processes, including circadian rhythms, anorexia, and weight loss [48]. Three other SNPs (rs409834034, rs428238419, and rs405959180) in chromosomes 6 and 7 were also significantly associated with this trait. However, none of the genes annotated in the 250 kb region around the significant SNPs was related to reproductive traits. Finally, four SNPs located in chromosome 23 were associated with the OCM trait: two in intron 2 of the CD226 gene (rs404991855 and rs418191944), one in an intergenic region approximately 412 kb from the CD226 gene (rs405024177), and one in a different intergenic region in chromosome 23 (rs410842314).The NPY and CD226 genes have been reported in other studies to be potentially associated with reproductive processes. NPY plays a role in the regulation of the secretion of gonadotropin releasing hormone (GnRH) in rats [49], rabbits [50], monkeys [51], and sheep [52,53]. It has been shown that NPY-containing neurons in the hypothalamus hold oestrogen receptors in rats [54]. In the same way, Skinner and Herbison [55] indicated that changes in photoperiod may regulate oestrogen receptor expression within the preoptic area. These authors suggested that hypothalamic NPY and β-endorphin neurons are involved in the seasonal regulation of ewe reproductive activity. Barker-Gibb and Clarke [56] reported a reduction in the number of NPY cells detectable by immunohistochemistry in the nonbreeding season compared to the breeding season in ovariectomized ewes either treated or not treated with oestrogen. Williams et al. [57] noted that NPY and other unidentified neurons in the hypothalamus interact with leptin, which is an important mediator of energy homeostasis and reproductive status in sheep. In pigs, NPY modulates hypothalamic neuronal activity (LH secretion) and serves as a putative link between the metabolic state and the reproductive axis [58]. Clarke et al. [48] determined that NPY stimulates feed consumption and inhibits reproduction in sheep. However, Miner [59] showed that NPY is a potent orexigenic agent in sheep, as in other species, and a seasonal change in the expression of this peptide may be related to stimulation of feed intake, rather than seasonality. On the other hand, the CD226 gene encodes the glycoprotein CD226, also known as DNAM-1 (DNAX Accessory Molecule-1), which is expressed on the surface of natural killer (NK) cells and is involved in T cell-mediated cytotoxicity against certain tumours [60]. NK cells express a repertoire of activating and inhibitory receptors that control NK cell cytotoxicity and interferon-γ (IFNG) production to ensure self-tolerance while allowing efficacy against such insults as viral infection and tumour development [61,62]. NK cells are also known to play an important role in human reproduction. These cells can be categorized into two main populations based on the relative expression of the surface markers CD16 and CD56: CD56bright/CD16– functioning to primarily produce cytokines in the circulating blood, and CD56dim/CD16+ performing cytotoxicity in the tissues [63,64]. Lukassen et al. [65] have shown that the proportion of CD56dim/CD16+ NK cells in follicular fluid (FF) in women suffering from idiopathic infertility is significantly higher than that in FF from patients undergoing in vitro fertilization (IVF) for tubal or male factor infertility. Furthermore, Křížan et al. [66] reported that the FF of patients with successful IVF outcomes was enriched with CD56bright/CD16– NK cells. Additionally, Fainaru et al. [67] indicated that CD56bright CD16– NK cells are abundant in maturing ovarian follicles and that their presence correlates with the ovarian response to gonadotropins. These cells have been shown to be proangiogenic through the secretion of such cytokines as vascular endothelial growth factor and placental growth factor [68]. In patients with a good response to ovarian stimulation, CD56bright NK cells migrate into the ovarian follicle, supporting follicular angiogenesis and oocyte development [67]. Recent research conducted by Stannard et al. [46] described a new CD56dim NK cell subset characterized by a lack of expression of DNAM-1. These researchers reported that CD56dimDNAM-1neg NK cells displayed reduced motility, poor proliferation, lower production of interferon-γ, and limited killing capacities compared with CD56bright and CD56dimDNAM-1pos NK cell subsets.According to the aforementioned findings, it can be inferred that mutations in CD226 could lead to glycoprotein alteration and could therefore affect follicle development. Additionally, mutations in the NPY gene would explain one of the neuroendocrine and physiological mechanisms that link nutrition and reproduction. Accordingly, the NPY and CD226 genes were selected for validation studies for their possible involvement in seasonal reproduction in sheep. Using 18 ewes with extreme values of TDA, we detected 2 and 4 SNPs in NPY and CD226, respectively (Table 3). This design could increase the power to detect polymorphic SNPs associated with the trait but minimize the probability of detecting other polymorphic SNPs.In this study, no association was found with the NPY gene. These SNPs show a low MAF (0.025 and 0.08 for OAR4: g.71593018 G > T and rs594346709, respectively). Notably, we did not sequence regulatory regions, such as the promoter or 5’ and 3’ UTR regions. Therefore, we could have missed the responsible mutation, as it was not in LD with the detected mutations. Similarly, no association was found with the SNP located in exon 2 of the CD226 gene. Importantly, no homozygous animals were found for the G allele, and a low MAF was found for this SNP (0.042). However, the analysis demonstrated that the SNP located in exon 3 of CD226 (rs404360094) was associated with the three reproductive seasonality traits. Notably, this SNP is close and in LD with the significant SNPs found in intron 2 in the GWAS analysis (4 and 4.7 kb from SNPs rs418191944 and rs404991855, respectively). In this sense, the GWAS analysis could have detected the effect found in exon 3 of CD226. Heterozygous and homozygous ewes for the G allele had less TDA and showed more oestrous events than those with the AA genotype (Table 4), which were observed at low frequency (4.7%) in our sample (Table S2). These results were confirmed by haplotype analysis such that animals carrying 1 or 2 copies of the H1 haplotype (containing the G allele) showed less TDA and higher P4CM and OCM values. Similarly, animals with two copies of the H2 haplotype (containing the A allele) showed higher TDA and lower P4CM and OCM values. This SNP produces an amino acid substitution from asparagine (uncharged polar) to aspartic acid (acidic) at position 243 at the end of the second immunoglobulin V-like domain in the extracellular region of the protein and close to the transmembrane motif [69]. However, the real effect of this variant is not clear because it has been predicted in silico to be a tolerant nonsynonymous substitution. It should be noted that we did not sequence the complete coding or regulatory regions of the CD226 gene; thus, the observed relationship could indicate that the SNP should be in LD with the true causative mutation.In summary, GWAS results were confirmed using a candidate gene approach. First, two SNPs located in intron 2 of the CD226 gene reached the chromosome-wise significance threshold on chromosome 23 using the GWAS approach. As noted above, because this gene could be related to follicular angiogenesis and oocyte development in humans, we decided to confirm the GWAS results using a candidate gene approach, determining that the SNP rs404360094 located in exon 3 of the CD226 gene was associated with reproductive seasonality traits in Rasa Aragonesa ewes. Therefore, this SNP could be utilized as a genetic marker for assisted selection to reduce seasonality. These results should be confirmed in more animals and in other breeds.5. ConclusionsThis study employed a GWAS approach to identify genomic regions associated with traits involved in reproductive seasonality in sheep. We demonstrated that the G allele of the SNP rs404360094 located in exon 3 of the CD226 gene is associated with lower TDA and higher P4CM, which are both traits related to ovarian function based on blood progesterone levels. This allele was also associated with higher OCM scores, which is an indicator of oestrous behaviour. These findings enabled us to validate the GWAS results and demonstrated the involvement of the genomic region where the CD226 gene is located. This SNP could be utilized as a genetic marker along with other SNPs already characterized in Rasa Aragonesa as being associated with traits related to reproductive efficiency, such as prolificacy or reproductive seasonality.

animals : an open access journal from mdpi

10.3390/ani13101630

PMC10215893

Cats are popular pets in Australia, being present in around one-third of households. As pets, cats are managed in a wide variety of ways, from fully indoors in apartments to completely outdoor free roaming. Australian wildlife is uniquely vulnerable to cat predation. Roaming cats also create a nuisance and are at risk of accidents and injuries. Councils, veterinarians, animal welfare organisations and conservation groups all have an interest in encouraging cat owners to change their behaviour and prevent their cats from roaming. Understanding what influences cat owner decisions can help design effective programs. This study asked cat owners about their cats, living circumstances, current cat management behaviour and agreement with statements reflecting their ability to contain their cats and their social opportunity and motivation to do so. More than half of participating cat owners already fully contain their cats. The most important influence for cat owners to keep their cats contained was having the skills, knowledge and belief that they could do so successfully. Those who lived in apartments, were renting or were motivated by their cat’s safety, to protect wildlife or to care for their community were also more likely to contain their cats.

There are over 5 million pet cats in Australia managed on a spectrum from fully indoors to completely outdoor free roaming. Roaming cats threaten biodiversity, can create a nuisance and are at risk of accidents and injury. Hence, there is substantial interest in behaviour change interventions to increase cat containment. An online questionnaire collected information on cat owner demographics, the number of cats owned, current containment behaviours and an agreement with 15 capability, opportunity and motivation (COM) items. Responses were received from 4482 cat owners. More than half (65%) indicated that they currently keep their cat(s) fully contained. Another 24% practiced a night curfew. Owners’ psychological capability had the greatest influence on containment behaviour. Motivation (community- and cat welfare-framed), living in an apartment and renting were also associated with a greater likelihood of containment. Cat owners not currently containing their cats could be divided into six profiles who differed on agreement with COM themes, age, future intentions, current behaviour, location and gender. Understanding differences between cat owner segments can assist with designing behaviour change interventions. Increasing cat owners’ psychological capability to contain their cats and encouraging the adoption of a night curfew as a first step towards 24 h containment are recommended.

1. IntroductionCats are valued companions and family members to many people all over the world. Almost one-third of Australian households are home to one or more cat, with an estimated population of more than 5.3 million pet cats nationally [1]. However, pet cats are managed by their owners in vastly different ways, from living fully indoors in apartments to completely outdoor free roaming and everything in between.Australian wildlife is uniquely vulnerable to predation by cats, having evolved without exposure to felids. Cats were introduced to the island continent at the time of European colonisation and are thought to have since caused or contributed to the extinction of more than 30 mammalian species [2,3,4]. Cats mostly predate mammals but will also predate birds, reptiles and amphibians. The type and amount of prey will depend on prey abundance, and the cat’s individual preference [5,6,7]. Research suggests that most pet cats will hunt if given the opportunity, even if well fed, and that only a proportion of what is caught is brought home [5,8,9,10]. However, the composition of the food may play an important role in reducing hunting; increasing the meat content of food, along with providing opportunities to engage in predatory play, have been shown to reduce the predation of wild animals by pet cats [11]. Pet cats are estimated to predate substantially fewer animals compared with a wild free-living or ‘feral’ cat; however, the greater population density of pet cats has been estimated to result in wildlife impacts 18–50 times higher per square kilometre [8]. This represents an important threat to biodiversity, given that urban areas can provide valuable habitats for threatened species [12].Free roaming also poses risks to cat safety. Trauma is a leading reason for pet cats to present to veterinary practices, especially due to road traffic accidents [13], and mortality rates from road traffic accidents can be 60% or higher [14]. Roaming cats are frequently injured when attacked by other cats or dogs [15] and are at risk of contracting infectious diseases such as feline immunodeficiency virus [16]. Roaming cats can also be a nuisance to neighbours through noise, property damage, soiling with urine and faeces and disturbing other companion animals.A variety of stakeholders have an interest in encouraging a greater uptake of cat containment by cat owners, including wildlife and conservation organisations, local councils, animal welfare organisations, veterinarians and the non-cat owning public. A twenty-four-hour containment of cats to their owner’s property, either exclusively indoors or with controlled outdoor access, is recommended by RSPCA Australia to prevent the potential negative consequences of roaming [17]. In addition, mandatory containment or ‘cat curfews’ are increasingly being legislated across Australia; in a large 2020 survey, close to one-third of the 250 participating Australian local governments had either a cat curfew, 24 h containment or cat prohibition zones (or a combination of these) in place [18]. More recently, the Australian Capital Territory has required all cats born from 1 July 2022 onwards to be contained to owner premises 24 h a day [19].A range of factors have been identified that influence cat containment behaviour, including owner demographics (age, gender, education and area of residence), owner ability to successfully contain their cat, their beliefs and attitudes around a cat’s rights to roam and their predation of wildlife, their concerns for cat safety, social norms around cat containment, as well as the cat’s characteristics (such as sex, breed, health and behaviour) [20,21,22,23,24,25,26,27,28,29]. Understanding these factors is the first step in designing more effective and targeted human behaviour change interventions.Changing human behaviour can be difficult. Education and providing information are rarely enough [30]. Social psychology and behavioural economics have generated an array of models and frameworks designed to increase audience understanding, engagement and, ultimately, the adoption of desired behaviours. Two such frameworks that have been applied to domestic cat management include Community-based Social Marketing [31,32,33] and the Behaviour Change Wheel (BCW) [20,25,26,33,34]. This later framework, along with its associated integrative Capability-Opportunity-Motivation (COM) Behaviour model, was initially developed by Michie and her colleagues for application in the health field. It conceptualises influential factors into three categories:(1)Capability: An individual’s physical and psychological ability to perform a behaviour. For example, does the cat owner have the physical skills or knowledge and cognitive skills to contain their cat. Interventions that increase capability incorporate techniques that educate, train and provide personal support.(2)Opportunity: The physical and social factors external to an individual that prompt or enable a behaviour to occur. For example, does the cat owner have access to the relevant containment resources, and do they have support from family, neighbours and community to keep their cat contained. Interventions that increase opportunity incorporate techniques that: provide access, enable, facilitate, prompt or constrain.(3)Motivation: Factors internal to an individual that energise or direct behaviour. These factors can be either reflective (incorporating conscious deliberation and reasoning) or automatic (usually outside conscious control, e.g., impulse, habitual or emotional) [35]. For example, a cat owners’ decision to contain their cat may occur after careful cost-benefit deliberation, after their cat experienced a traumatic traffic incident or because that is what they have always done. Interventions that increase motivation incorporate techniques that: inform, persuade, discuss, demonstrate, incentivise or coerce.Another important aspect in improving the behavioural impact of an intervention is matching its content to a specific audience need. Not every cat owner views their cats’ needs and their management approaches in the same way; thus, the patterns of the drivers of and barriers to cat containment will vary across individuals. Interventions can be designed or targeted to best match the characteristics of segments with specific driver/barrier profiles, and messages can also be crafted for specific individuals, as opposed to larger segments [36,37].The objectives of this study were to: (1) document the containment behaviour of cat owners across New South Wales (NSW), (2) identify the main drivers and barriers to cat containment and organise them according to the COM Behavioural model, (3) determine the importance of these COM items, along with other demographic/situational variables in influencing current containment behaviour and future intentions of cat owners to restrict their cats roaming and (4) segment cat owners using these COM items and identify leverage points that may be useful for targeting interventions and informing policy and legislation.2. Materials and Methods2.1. QuestionnaireAn online questionnaire was developed and advertised throughout NSW, with links available through the RSPCA NSW website and social media and shared by other external stakeholders, including veterinary clinics and councils throughout NSW (Appendix A). NSW residents aged 18 years and over were eligible to participate in the study. Only responses from current cat owners were included in the analysis. The human ethics approval was obtained from the University of Sydney’s Human Research Ethics Committee (Project Number 2021/473).The questionnaire collected basic demographic information (age and gender) from all respondents. Cat owners were asked about the number of cats they owned, their current cat containment behaviours, their likelihood of future adoption of cat containment and an estimate of the time their cat currently spent roaming freely outside across specific parts of the day (which were combined to gain a total time spent outside freely roaming). They were asked about the characteristics of their home (location, type of dwelling, access to an outside space and home ownership) that had the potential to influence containment behaviour (physical opportunity). In addition, they were asked to rate their agreement (on a 5-point Likert scale) to 15 capability, social opportunity and motivation (COM) items relating to cat containment (Table 1). These COM items addressed four important themes that had been identified from a review of previous research: cat owners’ capability to contain their cat, social opportunity for cat containment, motivation for containment associated with improving cats’ welfare and motivation for containment associated with supporting the community [26,28,29,37].2.2. QuestionnaireAs COM items were worded as either drivers or barriers in the survey, all barrier items were reversed-scored for analysis. All data was tested for compliance with the assumptions for parametric statistical analyses: normality, outliers, multicollinearity, non-linearity, homoscedasticity and non-independence assumptions. COM agreement data from Likert scales were treated as interval data, following the common practice used in medical and psychological research [38]. Internal consistency of the COM variables containing multiple items was tested using the Cronbach’s Alpha Test [39].ANOVAs and Pearson Chi-Squared were used to compare the differences in demographic and situational variables between various groups of respondents. A multiple regression was performed to identify the variables associated with cat free-roaming behaviour (measured as time spent outside freely roaming). A Latent Profile analysis (LPA) was conducted to classify cat owners into homogenous segments based on their responses to the COM items. The relative model fit was assessed using the Bayesian information criteria (BIC) [40] relative entropy [41] and the Lo–Mendell–Rubin likelihood ratio test (LMR) [42]. A significant p value from the LMR test (p < 0.05) indicated that the given profile solution fit the data significantly better than the solution with one fewer profile groups. MANOVAs, ANOVAs and Pearson Chi-Squared were used to compare the differences in demographic and situational variables between the cat owner LPA profiles. All analyses were conducted using SPSS version 26.0 (IBM, Armonk, NY, USA, 2019) except for the LPA which was conducted in MPlus version 8.9 (Muthén and Muthén, Los Angeles, CA, USA, 2019).3. Results3.1. Participant CharacteristicsThe online questionnaire received 4482 complete responses from cat owners from 105 of the 128 NSW local government areas. Over two-thirds of respondents (71%: 3177) lived in major urban centres (Sydney, Newcastle, Lake Macquarie, Central Coast, Wollongong and Shoalhaven), with the remaining third coming from regional areas [43].The overall average age of respondents was 46.1 years (±13.7). Over three quarters of respondents were female (3684, 80%), with 601 males (13%) and 186 identifying as non-binary (4%). As a response to this question was not compulsory, 11 respondents chose not to offer a response.Just over half of the respondents (2325; 52%) owned one cat, a third (1453; 33%) owned two cats, and 665 (15%) owned 3 cats or more (Table 2). More than half of the cat owners (2896 of 4482; 65%) indicated that they currently keep their cat(s) fully contained on their property (24 h contain), either indoors all the time (1606, 36%) or with restricted outdoor access using a cat enclosure or on a lead (1290, 29%). A further 1088 cat owners (24%) practiced a night curfew, whereby they kept their cat(s) indoors during the night but allowed their cats to roam freely during the day. The remaining 492 cat owners (11%) allowed their cats to roam freely during the day and night (24 h roam).Comparisons between cat containment behaviour and demographic, situational and cat ownership variables demonstrate that:Cat owners who practiced a night curfew were significantly older (mean 48.9 years) than both owners who 24 h contained their cats (mean 45.2 years) and owners who allowed their cats to 24 h roam (mean 44.8 years) (F = 29.41, df = 2, p < 0.001, η2 = 0.01).Male cat owners were more likely to allow their cat(s) to 24 h roam than female owners (15% vs. 10%; Pearson Χ2 = 14.01, df = 4, p = 0.01, r = 0.04).There was no statistical difference in containment behaviours between urban and regional locations (Pearson Χ2 = 5.08, df = 2, p = 0.08, r = 0.03).Cat owners who rented were more likely to 24 h contain their cat than those who owned their home (74% vs. 62%; Pearson Χ2 = 46.42, df = 2, p < 0.001, r = 0.09).Owners living in an apartment or unit were more likely to 24 h contain their cat than owners living in free-standing houses (82% vs. 59%; Pearson Χ2 = 182.80, df = 2, p < 0.001, r = 0.18).Owners without access to outdoor spaces at their homes were more likely to 24 h contain their cat than owners with access (73% vs. 63%; Pearson Χ2 = 25.75, df = 2, p < 0.001, r = 0.07).Cat owners who owned five cats or more were more likely to 24 h contain their cats compared to cat owners who owned one cat (75% vs. 62% Pearson Χ2 = 23.19, df = 8, p = 0.003, r = 0.05).Those cat owners who allowed their cats to roam away from their property (i.e., night curfew and 24 h roam) were asked to estimate the time their cat currently spent outside. These cat owners were also asked about their future intentions of 24 h containment. Cats who were allowed to 24 h roam spent significantly more time outdoors than those under a night curfew (Figure 1; Pearson Χ2 = 158.02, df = 3, p < 0.001, r = 0.27). Cat owners currently practicing a night curfew were more likely to have future intentions of preventing their cat from roaming more often (F = 6.09, df = 1, p = 0.01, η2 = 0.01), or 24 h containing their cat (F = 26.1, df = 1, p < 0.001, η2 = 0.02) compared to cat owners who were currently allowing their cats to 24 h roam (Figure 2).3.2. COM Theme Reliability and ComparisonsAll items reflected an adequate internal consistency (Table 1) [39]. Scale scores for each of these themes were computed by averaging the items which were then used for subsequent analysis.The Capability, Social Opportunity, Cat Welfare Motivation and Community Motivation theme ratings of cat owners who were currently keeping their cat 24 h contained were significantly higher than those of cat owners who were currently practicing a night curfew. Likewise, the ratings of cat owners who were practicing a night curfew were significantly higher than those owners who currently let their cat 24 h roam (Table 3 and Figure 3).3.3. Variables Influencing Cat Free-Roaming BehaviourA multiple regression was conducted to identify which variables had a significant effect on the time companion cats spent roaming freely (Table 4). Variables added to the model included cat owner age and gender (dichotomous: 0 = not female, 1 = female), the number of cats owned, location (dichotomous: 0 = urban, 1 = regional), type of dwelling (dichotomous: 0 = apartments/units, 1 = standalone house), home ownership (dichotomous: 0 = rent, 1 = own), access to an outside space (dichotomous: 0 = no access, 1 = access available) and the COM themes (Capability, Social Opportunity, Cat Welfare Motivation and Community Motivation; 1 = low agreement, 5 = high agreement).Three of the COM themes (Capability, Cat Welfare Motivation and Community Motivation) along with the cat owners’ home ownership and type of dwelling predicted significant amounts of the unique variance of time spent by their cats outside, with no restriction on their movements (Table 4). Overall, the final regression model explained 47% of the variance. Cat owners’ capability to contain their cats explained 12% of the unique variance in the regression, while the other two COM items and type of dwelling explained 1% each, and home ownership explained less than 1%.These results indicate that an increase in cat owners’ capability and motivation (both cat welfare and community framed) to contain their cat will likely reduce the amount of time spent by cats roaming freely, along with living in an apartment or unit. The cats of renters also tended to spend less time roaming freely outside, although these effects were minimal.3.4. Reasons for Allowing Cats to RoamCat owners were given the opportunity in an open-ended format to list the factors they have considered when deciding to allow their cat to roam freely (Figure 4). The factor listed by most online respondents was that it was okay for their cat to roam during the day, just not at night. The next most popular factor was that their cat did not roam very far from their property.3.5. Cat Owner SegmentationTo develop the most effective policies and engagement interventions, we not only need to understand why cat owners are willing or not to adopt containment practices, but also if these reasons are similar across all cat owners. Latent profile analysis indicated that cat owners who currently allow their cat to roam away from their property (n = 1580) could be classified into six profiles. This solution produced the lowest BIC value, and highest entropy value, with the Lo–Mendell–Rubin test indicating that it fitted the data significantly better than the 7-profile solution (Table 5).The demographic and behavioural characteristics for each profile are described in Table 6. There were significant differences between the profiles for:All four COM themes (MANOVA F = 326.91, df = 20, p < 0.001, η2 = 0.49; Capability F = 84.81, df = 5, p < 0.001, η2 = 0.21; Social opportunity F = 892.98, df = 5, p < 0.001, η2 = 0.74; Cat Welfare Motivation F = 460.15, df = 5, p < 0.001, η2 = 0.59; Community motivation F = 242.16, df = 5, p < 0.001, η2 = 0.86);Age (F = 12.21, df = 5, p < 0.001, η2 = 0.04);Future intentions (MANOVA F = 70.04, df = 10, p < 0.001, η2 = 0.18; Prevent from roaming more often F = 99.54, df = 5, p < 0.001, η2 = 0.24; Keep 24 h contained F = 110.99, df = 5, p < 0.001, η2 = 0.26);Current containment behaviour (Pearson Χ2 = 39.62, df = 5, p < 0.001, r = 0.15);Location (Pearson Χ2 = 17.43, df = 5, p < 0.01, r = 0.10);Gender (Pearson Χ2 = 11.04, df = 5, p = 0.05, r = 0.07) (Figure 5).4. DiscussionFree-roaming cats are a global challenge, causing nuisance through noise pollution, property damage, urine and faecal soiling and disease transmission [44]. Roaming cats worldwide are also at risk of injury from motor vehicle accidents and animal attacks, as well as being at risk of becoming lost or stolen and contracting infectious diseases. In addition, some regions, including Australia, have wildlife populations that are especially vulnerable to cat predation, making cat containment an important target for human behaviour change interventions. We report a relatively high rate of cat containment compared to previous studies from a large sample of Australian cat owners. This is consistent with the literature over several decades that demonstrates increasing rates of cat containment in Australia, from less than 1 in 4 cats in the 2000s [45], to close to half of all cats in a 2019 study [46], which presumably reflects shifting social norms. Importantly, cat owners who do allow their cats to roam differ and can be divided into six segments, with major implications for the intervention design and delivery. These findings will be applicable to international readers, particularly those from Western countries where companion animal management norms are similar to those in Australia.A cat owner’s capability to contain their cat had the greatest influence on the amount of time cats spent contained. Cat owners who believed preventing their cat from roaming would be too difficult, were not confident they could prevent their cat from roaming or were not confident they could meet their cat’s needs if not roaming were significantly more likely to allow their cats to roam. Cat owners’ motivation and opportunity also significantly influenced their likelihood to prevent roaming. Cats living in apartments, and cats with younger and female owners were also more likely to be fully contained; however, the owner capability was by far the most important contributor.Variables reflecting a cat owner’s physical capability to contain their cats, such as location (urban vs. regional), home ownership status and access to outdoor space, did not influence whether cats were prevented from roaming, suggesting that a cat owner’s physical capability is less important to undertaking this behaviour than their psychological capability, i.e., their knowledge or psychological skills, strength or stamina to engage in the necessary mental process [34]. Indeed, we found that cat owners who owned their homes, who lived in free-standing houses (as opposed to apartments), who had access to outdoor space and who had only one cat were significantly more likely to allow their cats to roam; however, these are all scenarios that might be expected to improve a cat owner’s physical capability to contain their cat.Our findings suggest that audience segments already employing a night curfew for their cats (Concerned Protectors, Conscientious Caretakers and some Laissez-faire Landlords) will be most receptive to full 24 h containment. Two of these segments (Concerned Protectors; Conscientious Caretakers) reported an intention to increase their containment behaviour in the future and hence can be considered the ‘low-hanging fruit’. As quantified by McLeod et al. [33], 24 h containment more effectively reduces the number of free-roaming cats but has a lower likelihood of adoption than a night curfew. Concerned Protectors, while a small group of cat owners, are already highly motivated to keep their cats contained and have the physical capability and opportunity to do so. Hence, this group might be the most receptive to behaviour change techniques focused on education that might be ineffective on their own for other audience segments [27]. These include providing ‘information about social and environmental consequences’, ‘information about health consequences’ and ‘prompts and cues’, for example, to ‘close the door’, ‘bring your cat in’ or ‘check—where is your cat now?’ [34]. Prompts and cues (words or images related to the concept of keeping their cat at home) presented close to the decision point (when feeding their cat, or when they would usually let them in or out) might work well for this group, as they are already committed to the goal of containing their cats and already have the skills and knowledge to perform these behaviours effectively [47].Conscientious Caretakers were the largest segment and might be the most promising target of behaviour change campaigns. Cat owners in this segment are sensitive to the community impacts of cats roaming (wildlife predation; nuisance to neighbours), are somewhat motivated to contain their cat more in future and are mostly already employing a night curfew. In addition, Laissez-faire Landlords are a group with no firm opinions on cat management and as such might be more open to discussions about management than those with strong, existing viewpoints. However, because of their lack of prior interest they might be relatively difficult to engage initially [21]. This is a group that might be less likely to seek cat management advice or invest resources into cat management. Crowley et al. [21] suggest that this segment might be receptive to prominent, coherent messaging promoting simple, inexpensive and easy-to-implement strategies.Several audience segments (Tolerant Guardian, Laissez-fare Landlord and Freedom Defender) perceive a 24 h cat containment as having considerable barriers with limited benefits. Those allowing their cats to roam can be strongly motivated by their belief that cats need to roam, and also by a dislike of the smell of cat urine inside [48]. Twenty-four-hour containment is also a behaviour that is difficult to incentivise due to its complexity. Consistent with the findings of this study, previous research has reported that a proportion of Australian cat owners who oppose 24 h containment will agree with a night curfew [26,28,48]. As such, interventions for these audience segments could target a night curfew as a behaviour that is easier to adopt and might act as a catalyst to encourage the more difficult behaviour of 24 h containment in the future [33,48,49].Our findings suggest that interventions aiming to reduce the roaming of pet cats should target cat owners’ psychological capability to contain their cats. As such, behaviour change techniques focused on education, enablement and training are likely to be most effective [34]. However, interventions that only provide general education content often fail to produce a significant behaviour change [30], and training might be cost-prohibitive to provide at scale, hence, a focus on enablement might be the most practical. Relevant enablement behaviour change techniques include ‘demonstration of the behaviour’, ‘social support’, ‘goal setting’ and ‘action planning’ [34]. A final two enabling behaviour change techniques might be especially valuable in the context of cat containment: ‘adding objects to the environment’—for example, enhancing a cat’s at-home environment with vertical space, scratching surfaces, opportunities for scent marking and predatory play to ensure cat owners can meet all their cat’s behavioural needs at home [50] and a ‘restructuring of the physical environment’ through the construction of secure catios, or modifying fencing.Interventions for most audience segments (Conscientious Caretaker, Tolerant Guardian, Laissez-fare Landlord and Freedom Defender), whether with 24 h containment or a night curfew as their target behaviours, should aim to encourage cat owners to act while also improving their psychological capability. The behaviour change techniques that might most effectively encourage cat owners in this context, in addition to those discussed above for enablement, include ‘persuasion’, ‘incentivization’ and ‘modelling’, which could be effectively delivered using communications and marketing strategies [34]. Seeing a ‘similar other’ (in this instance, a cat owner like them) modelling the behaviour has been associated with increased self-efficacy and increased engagement in the target behaviour [51]. Incentivisation using competitions might be beneficial to increase engagement without excessive cost, especially as the behaviour change effects from competitions might be the strongest for those initially less motived [52]. Face-to-face exchanges between individuals are likely to be particularly effective [34], especially when using messengers that are most trusted by cat owners such as veterinarians, or RSPCA staff [1,37,46]. Motivational interviewing—a client centred, evidence-based counselling method aiming to strengthen a person’s motivation and commitment to behaviour change [53]—might be applicable in this context but has to-date been underutilised in veterinary practice [54].Consistent with Crowley et al. [21], our findings suggest that campaigns promoting benefits of containment for cat safety and for wildlife conservation—recommended by previous research [23,28,37]—might align with the values of Concerned Protectors and Conscientious Caretakers, but not with values of other important segments, especially Tolerant Guardians and Freedom Defenders. These segments often have ‘working cats’; they like that their cat hunts and feel strongly about their cat’s need to roam [21]. A different intervention approach is needed for these segments that aligns with their different values and priorities, potentially with messaging focused around protecting wildlife, being a good neighbour and caring for the community.Interventions focused on increasing the opportunity for cat containment might have the greatest promise for reducing numbers of cats roaming in the medium- and long-term. These interventions aim to decrease the barriers to containment and increase the benefits on a systemic level [34]. Interventions might include making housing more cat-friendly, for example by changing legislation to allow cats in more private rentals and removing regulatory barriers to cat owners modifying fences and constructing cat enclosures. Most importantly, interventions should directly address cat overpopulation. While rates of desexing are high amongst owned pet cats [46,55], close to half of the pet cats in Australia are passively acquired, i.e., acquired for free ‘to give them a home’ [1]. These passively acquired cats, along with cats acquired from animal pounds and shelters, largely originate from the semi-owned and unowned cat population, few of whom are desexed [17]. We did not ask how cats were acquired in this study (whether passively or actively); however, this is an important area for future research. Are those who passively acquire cats more or less likely to allow them to roam? How does the acquisition source relate to target audience segmentation?McLeod et al. [48] demonstrated that most adopters of cats from an animal shelter in Australia intend to keep their cats contained, which might suggest that owners who actively acquire their cats are more likely to adopt this behaviour. Reducing cat overpopulation, and consequently reducing the proportion of pet cats who are passively acquired, might be important to increase the opportunity for pet cat containment, while also directly reducing numbers of free-roaming cats. Desexing cats has been identified as the most effective intervention to reduce numbers of roaming cats, and is the intervention with the greatest likelihood of adoption [33]. There is likely to be strong support from cat owners and the public for humane strategies for reducing cat populations [21]. In addition, there is a growing body of research on humane and effective cat population management interventions [44].This online survey sample was not randomly selected and hence likely experienced some sampling bias in favour of people already containing their cats. Consequently, the study might have overestimated the proportion of people fully containing cats in the general population. Future surveys of cat owners might benefit from the use of random online panels to reduce sampling bias.5. Conclusions and RecommendationsIt is important to recognise cat owners as key partners in reducing the negative impacts of cats. Cat containment in Australia is increasingly being adopted by cat owners, with more than half the participants in our study already fully containing their cats. Understanding the differences in priorities, values and perspectives of cat owners who are not containing their cats can assist with designing behaviour change interventions. Increasing cat owners’ psychological capability to contain their cats, i.e., their knowledge or psychological skills, strength or stamina, as well as encouraging the adoption of a night-curfew as a first step towards 24 h containment, are recommended. Maintaining a constructive tone and focusing on actions cat owners can take to reduce their cat’s impacts rather than demonizing cat owners is likely to be met with the most success. Concurrent interventions that address cat overpopulation and reduce the number of cats who are passively acquired might be important for increasing cat owners’ opportunity to contain their cats.

animals : an open access journal from mdpi

10.3390/ani13050814

PMC10000197

The level of supplementation of low-quality forage diets before and in the early gestation period may influence the performance of different hair sheep breeds. Dorper, Katahdin, and St. Croix sheep consumed wheat straw ad libitum supplemented with 0.15% initial body weight (BW) of soybean meal or a 1:3 mixture of soybean meal and rolled corn at 1% BW for 162 days with 84 or 97 days pre-breeding. Supplement treatment by breed did not generally influence feed intake, BW, body condition and mass indexes, or reproductive performance. Although soybean meal given alone boosted straw intake, it resulted in lower total feed intake, litter size, and total litter birth weight compared with the higher level of supplementation. Body condition score and mass indexes and BW were affected by breed, but reproductive performance was not influenced. Therefore, hair sheep regardless of breed fed low-protein and high-fiber forage such as wheat straw should be supplemented at a higher level with consideration of the inclusion of a feedstuff(s) high in energy along with protein during these physiological periods.

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 × 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.

1. IntroductionThe 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 [1]. 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 [2].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 [3,4,5]. 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 [6]. Additionally, proper nutrition and body condition score (BCS) during gestation influence the performance, health, reproduction, and metabolic responses of progeny [7,8].The three most common breeds of hair sheep in the USA are Dorper (DOR), Katahdin (KAT), and St. Croix (STC) [9,10]. After a project in which the resilience of these breeds to stress factors was expected to increase in importance with anticipated future climate change [11,12,13], 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 [14]. 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 Methods2.1. AnimalsThe 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. [12,13] and Hussein et al. [11], 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 HousingBefore the experiment, sheep were vaccinated against clostridial organisms, and ones with a FAMACHA© 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 (Figure 1). Each group was housed in a pen with two sections, a 3.66 m × 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 × 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 [15], 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 [16], 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 (Figure 2). 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 moderate- to high-quality forage.2.3. BreedingOne 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-breed™ CIDR®, Pfizer Animal Health, Auckland, New Zealand) inserted on day 0, and 10 mg of PGF2α (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© 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 MeasuresBody condition score (BCS), as described by Ngwa et al. [17], 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. [18], the 4 most meaningful ones as recommended by Wang et al. [19] were calculated as noted below. BMI–WH = BW/(Wither × Hook) [g/cm2](1) BMI–WP = BW/(Wither × Pin) [g/cm2](2) BMI–GH = BW/(Heart × Hook) [g/cm2](3) BMI–GP = BW/(Heart × 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 3000× g and frozen at −20 °C. 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® 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 [20].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 [21,22,23]. Heart rate measurement was as described by Puchala et al. [24,25]. 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. [24,25]. 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 [26] equation without the consideration of urinary nitrogen.2.5. Statistical AnalysesMost data were analyzed with the MIXED procedure of SAS [27], 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 [27]. Breed × 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 [27].3. Results3.1. Feed Intake, BW, and ADGThe chemical composition of wheat straw and supplements (Table 1) was similar to that in the earlier experiment of Lourencon et al. [14]. 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 BMIBody 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 HEBreed 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 ConcentrationsAll 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 PerformanceThe 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 RelationshipsCorrelation 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. Discussion4.1. Feed IntakeFor low-quality forages, digestive capacity including fermentation, the breakdown of feed particles, and digesta passage rate are important determinants of feed intake [28]. Low-quality forages are fermented in the rumen relatively slowly which can result in limited feed intake because of high ruminal NDF fill [29,30]. 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 [31]. 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. [14], 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. [14], 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. [14] 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 [32,33]. 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 [29] and 2.1% and 1.76% BW for ewes of 45 and 90 kg BW, respectively [30]. 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 [29,34]. This latter finding in the present experiment was similar to one of Lourencon et al. [14] 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. [14] 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. [14] 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 ADGIn contrast to the findings of Lourencon et al. [14] and other studies [11,13,35,36], 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 [37,38]. 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 [37,39,40]. 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 [41]. 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 BMIThe 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 [42,43,44]. 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. [14], 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. [14] 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 [14,45,46]. 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 HEHeat 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 [47,48,49]. 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 ConcentrationsThere 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 [50,51]. 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 [52], 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 [53], 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 [54,55]. 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 PerformanceA high plane of nutrition before breeding has been shown to improve the reproductive performance of sheep in many studies [5,56] via improved reproductive hormonal balance and follicular development [3,57]. Supplement treatment had a much greater effect on reproductive performance measures than in the previous experiment of Lourencon et al. [14], 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 [5,58]. 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 [4]. However, differences in experimental conditions between the present study and that of Lourencon et al. [14] could have had an influence. For example, the trial of Lourencon et al. [14] 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 RelationshipsThe 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. [14] and Wang et al. [19], 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 ConclusionsThe 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.

animals : an open access journal from mdpi

10.3390/ani11123589

PMC8698017

Oral disease in cats is a significant concern in the small animal practice setting. The specific cause of this disease is inadequately understood; however, antibiotics are commonly used for the management, although many cats respond poorly to these treatments. Antibiotics have been overused and misused in the context of both human and veterinary medicine. Consequently, many antimicrobial drugs are becoming less effective in treating infections. This study aimed to evaluate the presence of antimicrobial resistance genes in swabs obtained from the mouth of cats. Moreover, the study looked at simultaneous occurrence between these genes and one type of oral fungi. We found that antimicrobial resistance genes are extremely common in both clinically healthy and sick cats. Furthermore, we established that Malassezia (a type of fungi) co-occurs with some resistance genes. The findings are important because antimicrobial resistance genes present in the mouth of cats have the potential to transfer to humans and thereby make certain antibiotics less effective.

Feline chronic gingivostomatitis (FCGS) is a severe immune-mediated inflammatory disease with concurrent oral dysbiosis (bacterial and fungal). Broad-spectrum antibiotics are used empirically in FCGS. Still, neither the occurrence of antimicrobial-resistant (AMR) bacteria nor potential patterns of co-occurrence between AMR genes and fungi have been documented in FCGS. This study explored the differential occurrence of AMR genes and the co-occurrence of AMR genes with oral fungal species. Briefly, 14 clinically healthy (CH) cats and 14 cats with FCGS were included. Using a sterile swab, oral tissue surfaces were sampled and submitted for 16S rRNA and ITS-2 next-generation DNA sequencing. Microbial DNA was analyzed using a proprietary curated database targeting AMR genes found in bacterial pathogens. The co-occurrence of AMR genes and fungi was tested using point biserial correlation. A total of 21 and 23 different AMR genes were detected in CH and FCGS cats, respectively. A comparison of AMR-gene frequencies between groups revealed statistically significant differences in the occurrence of genes conferring resistance to aminoglycosides (ant4Ib), beta-lactam (mecA), and macrolides (mphD and mphC). Two AMR genes (mecA and mphD) showed statistically significant co-occurrence with Malassezia restricta. In conclusion, resistance to clinically relevant antibiotics, such as beta-lactams and macrolides, is a significant cause for concern in the context of both feline and human medicine.

1. IntroductionFeline Chronic Gingivostomatitis (FCGS) is a severe immune-mediated inflammatory disease of the oral cavity in domestic cats [1]. Initial bacterial biofilm formation leading to plaque deposits results in activation of the immune system and gingivitis, which impacts individual teeth with gradual spread to surrounding tissues [2]. Despite substantial research efforts dedicated to the exploration of disease causation in FCGS, the etiology has yet to be established. The syndrome is likely multi-factorial, including the involvement of different pathogens, nutritional factors, environmental stressors, and more. Conventional treatment strategies to address FCGS involve medical management through the prescription of immunosuppressants and antibiotics or surgical management aimed at partial or complete dental extraction dependent on disease severity [3]. Given the variation in how patients with FCGS respond to immunosuppressants, efforts have increasingly focused on differentiating between the relative importance of bacterial pathogens, hereunder anaerobic and Gram-negative bacteria, such as Pasteurella multocida [1]. Broad-spectrum antibiotics are frequently prescribed empirically in cases with FCGS in the absence of culture and sensitivity analysis, which is a One Medicine (i.e., intersection between human, animal, and environmental health) issue when viewed in light of growing antimicrobial resistance concerns worldwide.While antibiotics are routinely administered in an attempt to combat feline oral pathogens, our team has demonstrated that the oral cavity of patients with FCGS also harbors fungal species, such as Malassezia restricta, that are virtually absent in clinically healthy cats [4]. The coexistence of bacterial and fungal communities might suggest that fungi play a role in FCGS. The existing literatures suggest Malassezia species are pathogenic in human gut inflammation and some cancers [5]. Conversely, Malassezia species have been documented to serve prominent roles as commensal microorganisms in the oral cavity of humans [6]. In companion animal medicine, Malassezia species have been implicated in dermatitis, but no association has been reported to oral disease in either cats or dogs. Culture-independent analyses confirm that the oral cavity in cats is polymicrobial [7]. Metagenomic analyses of the feline oral mycobiome still remain scarce, and reports have thus far focused on cats with allergies versus controls [8]. A single report exists on the oral mycobiome in FCGS patients [4]. This study presents patterns of antimicrobial resistance (AMR) genes in cats with FCGS versus controls, as well as the co-occurrence of AMR genes with one highly abundant oral fungal species.2. Materials and Methods2.1. Subjects Included and Study DesignA total of 28 samples were analyzed in this study, 14 from clinically healthy (CH) cats and 14 from cats with chronic gingivostomatitis (FCGS). The CH samples were all collected at Western University of Health Sciences (WUHS), Pet Health Center (PHC), Pomona, CA, USA (n = 14). The FCGS samples were obtained from WUHS-PHC (Pomona, CA, USA), Saddleback Animal Hospital (Tustin, CA, USA), Advanced Veterinary Specialty Group (Tustin, CA, USA), and Cat Care Clinic (Orange, CA, USA). Six different cat breeds were represented (Domestic Short Hair, 17; Domestic Long Hair, 8; Siamese, 1; Scottish Fold, 1; Main Coon, 1; Persian, 1) across a wide age range (2 to 13 years old) with an average of 4 ± 0.5 years in the CH group and 8 ± 1.1 years in the FCGS group. All cats from the FCGS group were diagnosed with chronic gingivostomatitis ≥6 months prior to the inclusion in our study. Co-existing viral infection status and previous medical/surgical therapy information (e.g., tooth extractions and antibiotic therapy) were not available at the time of the oral sample collection. The CH cats were recruited from the surgery section of the WUHS-PHC at the time of admission for elective procedures (e.g., neutering). All cats in the CH and FCGS groups underwent the same examination protocol and were classified based on a previously published FCGS scale with minor modifications [9]. Briefly, the oral cavity was examined thoroughly, and lesions were sorted according to severity, as previously described [4]: grade 0, absence of lesions; grade 1, mild gingivitis; grade 2, moderate gingivitis; grade 3, severe gingivitis; grade 4, gingivitis associated with proliferative and/or ulcerative lesions in the caudal oral cavity/palatoglossal fold and/or alveolar, labial, buccal, sublingual, and lingual mucosae (extra-gingival lesions). Only cats with grade 4 lesions were included in the FCGS group. Cats in the CH group presented no evidence of oral lesions or only mild gingivitis (grade 1 in six of 14 cats) at the time of examination.2.2. Sample Collection, DNA Extraction, Preparation, and SequencingOral samples from the FCGS cats were collected in mucosal transition areas (affected tissues and their contiguous normal areas) using a sterile DNA-free swab (HydraFlock®, Puritan® Cat. No. 25-3406-H, Guilford, ME, USA). For the CH cats, swab samples were collected from the gingival, hard palate, rostral dorsal tongue, and other oral mucosal surfaces. All samples were immediately immersed and preserved in DNA/RNA ShieldTM (Zymo Research Corp.; Cat. No. R1108, Irvine, CA, USA) until processing at the MiDOG LLC testing facility (Tustin, CA, USA). Genomic DNA was purified using the ZymoBIOMICSTM-96 DNA kit (Cat. No. D4304, Zymo Research Corp., Irvine, CA, USA). Sample library preparation and data analysis for bacterial and fungal profiling were performed by Zymo Research Corp. using the Quick-16S NGS Library Prep Kit (Cat. No. D6400, Zymo Research Corp., Irvine, CA, USA) with minor modifications. Primer sequences are proprietary to the MiDOG LLC service and target the 16S rDNA V1–V3 region for bacteria and the ITS-2 region for fungal analysis. Libraries were sequenced using an Illumina HiSeq 1500 sequencer, and reads were filtered through Dada2 (R package version 3.4) [10]. Phylotypes were computed as percent proportions based on the total number of sequences in each sample. The relative abundances of bacteria compared to fungi were determined, assuming an equivalency of one 16S rDNA copy to one fungal ITS copy. Species-level resolution of the sequencing approach used here has previously been demonstrated by shot-gun sequencing [11].2.3. Detection of AMR Genes, and Correlation Analysis with Malassezia restrictaThe presence of AMR genes in the oral cavity of the study subjects was evaluated by using a proprietary sequencing workflow that targets at least eighty AMR genes. An amplicon based sequencing approach was applied using proprietary PCR primers, which were designed based on AMR gene sequences retrieved from NCBI (National Center for Biotechnology Information). Sequencing reads were mapped back to the reference using a proprietary pipeline. To ensure specificity and reproducibility of the tests, sequencing reads were further confirmed using the Comprehensive Antibiotic Resistance Database (CARD) [12]. Patterns in the occurrence of AMR genes (proportions) between groups were compared using two-sample z-tests (p < 0.05). Point-biserial correlation tests (stats v3.6.1 R Core Team, R Foundation for Statistical Computing, Vienna, Austria, 2013) were applied to analyze any relationship between the presence of specific AMR gene (dichotomous variable) and relative abundance of Malassezia restricta (continuous variable), a species found solely in oral swabs from FCGS cats based on prior microbial core analysis [4].3. ResultsAMR genes were detected in all 28 samples regardless of feline health status (Figure 1). A total of twenty-four acquired AMR genes were detected with the potential to confer resistance to a wide range of antimicrobials, including clinically relevant and commonly used antibiotic classes such as beta-lactams, tetracyclines, aminoglycosides, phenicols, lincosamides, macrolides and sulfonamides. A total of 21 and 23 different AMR genes were detected in CH and FCGS cats, respectively (Table 1). Statistically significant differences were found in occurrence of genes conferring resistance to aminoglycosides (ant4Ib, p = 0.012), beta-lactam (mecA, p = 0.005), and macrolides (mphD, p = 0.014 and mphC, p = 0.024). These four AMR genes were found in 7, 8, 5, and 10 cats with FCGS, as compared to 1, 1, 0, and 4 CH controls, respectively (Table 1).Additionally, we established co-occurrence between Malassezia restricta, the most frequently found fungal species in the oral cavity of FCGS cats [4], with two of the twenty-four AMR genes. Specifically, we found a positive correlation between the abundance of M. restricta and the presence of mecA (r = 0.49, p = 0.0076) and mphD (r = 0.62, p = 0.0004) genes (Table 2).4. DiscussionThis is the first study to investigate patterns of AMR gene expression in cats with FCGS versus CH controls. Twenty-four acquired AMR genes were identified in the oral cavity of all 28 cats in this study, and at least one AMR gene was detected in each sample. Compared to CH controls, AMR genes conferring resistance to aminoglycosides, beta-lactams, and macrolide antibiotics were found more commonly in oral swabs from cats with FCGS.The mecA (beta-lactam) AMR gene is of considerable clinical relevance because it confers resistance to commonly prescribed antibiotics used in the management of FCGS, including amoxicillin/clavulanate and cephalexin [13]. The study includes a relatively small sample size, but the data give pause for thought when deciding on the empirical use of amoxicillin/clavulanate in the management of FCGS. The cautious use of amoxicillin/clavulanate (Clavamox) is especially important because this is one of the most common antibiotics used for the treatment of infections in feline medicine, such as FURC (feline upper respiratory complex) and feline eosinophilic plaques and lip ulcers [14]. Our findings moreover have One Medicine implications because beta-lactams, such as amoxicillin/clavulanate (Augmentin), and cephalexin (Keflex) are among the top five most commonly prescribed outpatient antibiotics in the USA [15]. Furthermore, the mecA gene is expressed by multidrug-resistant bacteria, such as methicillin-resistant Staphylococcus aureus (MRSA). MRSA is among the most important causes of nosocomial outbreaks and is responsible for significant morbidity and mortality around the world [16]. Further research is needed to explore the public health significance of mecA and other AMR genes in domestic cats, hereunder the transmission of AMR genes to humans (Figure 2).An additional two AMR genes were found more commonly in the oral cavity of cats with FCGS compared to CH controls, including mphD and mphC, which confer resistance to macrolide antibiotics such as azithromycin and clarithromycin. Azithromycin has been suggested as an alternative antibiotic in refractory cases of FCGS but has shown only limited success. The rationale behind the use of azithromycin is that some non-responsive FCGS cases present with positive Bartonella henselae titers. Interestingly, pradofloxacin (a third-generation quinolone antibiotic used in companion animals) has superior in vitro antimicrobial activity against B. henselae [17]. Azithromycin has been used for more than a decade in feline medicine as a treatment for Bartonellosis, empirical treatment of FURC in shelter cats [18], and acute cytauxzoonosis (infection with tick-borne haemoprotozoan parasite) [19]. Public health concerns about the presence of this AMR gene in client-owned cats has not been investigated, but it warrants serious consideration since azithromycin is the second most prescribed outpatient antibiotic for humans in the USA [20] and a critically important antibiotic for human medicine according to the WHO [21].The AMR tetW/N/W gene was ubiquitously present in the oral cavity of all cats enrolled in this study (Figure 1). This gene confers resistance to tetracycline and all its synthetic derivatives that inhibit bacterial aminotransferase-tRNA [12]. Doxycycline (a synthetic tetracycline derivative) has been one of the most commonly used antibiotics for FCGS but has shown limited success in changing clinical outcomes [13]. According to the Centers for Disease Control and Prevention [20], doxycycline was prescribed 19.5 million times in 2020, making it the fifth most used oral antimicrobial in outpatients in the USA. The presence of the tetW/N/W gene in all 28 cats in this study raises important public health questions about the potential of cat-to-human AMR gene transfer and the original source of this gene in the cat population.The presence of a single AMR gene that confers resistance to aminoglycosides (ant4Ib) in affected cats appears to have less veterinary medical relevance considering that cats with chronic gingivostomatitis rarely are treated with this class of antibiotics. The use of aminoglycosides in cats is typically avoided because of well-established adverse effects such as nephrotoxicity and ototoxicity [22].Our group recently documented the oral microbiome and mycobiome of CH and FCGS cats [4], which showed that the oral cavity of cats with FCGS harbors Malassezia restricta—a pathogen commonly associated with seborrheic dermatitis in humans [23]—at a significantly higher abundance and frequency as compared to CH cats. Notably, the composition of cell wall polysaccharides of M. restricta is distinctly different from all other fungal species analyzed to date [24], suggesting that M. restricta has evolved unique traits to adapt more effectively to the skin and mucosal host microenvironments. Moreover, M restricta expresses a metallo-beta-lactamase [25] capable of catalyzing the hydrolysis of all beta-lactam antibiotic classes, including carbapenems [26]. This constitutes a serious health concern because there is currently no clinically relevant inhibitor available for the fungal metallo-beta-lactamases [26]. In light of these observations, we decided to explore the co-occurrence of this fungal species and clinically relevant AMR genes in the oral cavity of FCGS cats and established a significant association of M. restricta with two highly relevant AMR genes (mecA and mphD) in the oral cavity of cats with FCGS. Although the co-occurrence between M. restricta and select AMR genes does not offer proof of causation based on this study alone, it opens the door for further investigating the role that M. restricta may play in a feline oral health context.In summary, the AMR genes documented in this descriptive study have the potential to confer antimicrobial resistance to the five most frequently prescribed outpatient oral antibiotics in 2020 in the USA, including amoxicillin, azithromycin, amoxicillin/clavulanic acid, cephalexin, and doxycycline [20].5. ConclusionsThe feline oral resistome, as described here, includes at least 24 acquired AMR genes with potential for horizontal gene transfer to the bacteriome of humans and other animals. We also found the occurrence of four AMR genes to be significantly different between FCGS and CH cats, where genes conferring resistance to critically important antibiotics, including aminoglycosides, beta-lactam, and macrolides, were more commonly found in cats with FCGS. The study furthermore showed the co-occurrence between M. restricta (a beta-lactamase producing yeast) and two AMR genes in FCGS cats, strongly suggesting that scientifically based manipulation of the oral microbiome (bacterial and fungal) may prove extremely helpful in designing novel and effective strategies to manage a feline syndrome that has been a medical conundrum for small animal clinicians for decades. Finally, our findings also have important One Medicine implications that warrant further investigation of the public health significance of resistomes in cats.

animals : an open access journal from mdpi

10.3390/ani11113261

PMC8614545

During the hot season, ruminants can easily suffer from heat stress. Heat stress can inevitably lead to loss of livestock production. Supplementing their diet with probiotics is an effective approach to improving livestock welfare. This study showed that dietary supplementation of heat-stressed goats with Clostridium butyricum, both in vitro and in vivo, can effectively alleviate heat stress by improving the rumen fermentation and growth performance of goats. This study provides a reference for the use of this probiotic in goat production when heat stress occurs.

This study aimed to evaluate the effects of Clostridium butyricum on rumen fermentation and the growth performance of heat-stressed goats. The in vitro fermentation was carried out using Clostridium butyricum supplement at 0% (CG), 0.025% (CB1), 0.05% (CB2), 0.10% (CB3), and 0.20% (CB4) of the dry matter (DM) weight of basal diet. Results showed that ruminal pH and the concentrations of ammonia nitrogen, total volatile fatty acids, acetic acid, propionic acid, as well as the acetic acid to propionic acid ratio were significantly increased (p < 0.05) in CB2 and CB3 compared with the CG group. Additionally, significant increases (p < 0.05) in the degradability of DM, neutral detergent fiber, and acid detergent fiber were observed in CB2 and CB3 compared with the CG group. For the in vivo study, 12 heat-stressed goats were divided equally into three groups: the control (HS1) was fed the basal diet, and groups HS2 and HS3 were fed with 0.05% and 0.10% Clostridium butyricum added to the basal diet, respectively. The experiment was designed as a 3 × 3 Latin square. Similar effects on rumen fermentation and digestibility parameters were obtained with 0.05% of Clostridium butyricum supplement compared to the in vitro study. Moreover, the dry matter intake and average daily gain were significantly increased (p < 0.05) in HS2 compared with other groups. These results indicated that an effective dose of Clostridium butyricum supplement (0.05%) could improve the rumen fermentation and growth performance of heat-stressed goats.

1. IntroductionIn recent years, intensive goat breeding has rapidly developed in the Jianghuai region of China, where the climate is characterized by high temperatures and humidity in summer [1]. The large amounts of heat produced by rumen fermentation contribute to the low tolerance that ruminants have against high environmental temperatures, hence goats in this region are prone to suffering from heat stress during the summer [2]. Heat stress causes various adverse impacts on ruminants, including lowered rumen pH, decreased production of rumen volatile fatty acid (TVFA), reduced digestibility of feed, and oxidative stress [2,3,4,5]. These effects eventually lead to a decline in goat production and economic loss [6].Probiotics are defined as live microbial feed additives that beneficially affect the host’s health when supplemented in adequate amounts [7]. They have been widely used in ruminants to enhance feed digestion, and improve performance and health status [8]. Clostridium butyricum is a promising candidate as a microbial feed additive [9]. Most previous studies of Clostridium butyricum focused on monogastric animals and poultry, with few studies carried out on ruminants [10,11,12]. For ruminants, Clostridium butyricum has the potential to improve rumen fermentation and nutrient degradability [13,14]; however, there are few such studies.Supplementing a diet with probiotics has been reported as being effective in reducing the negative effects of heat stress in livestock production [15,16]. A previous study claimed that active dry yeast fed to cows could reduce rectal temperature and prolong the period of peak milk production during heat stress [17]. Furthermore, Saccharomyces cerevisiae culture fed to mid-lactation dairy cows during the summer improved the feed efficiency, although no effect was found on the yield of energy-corrected milk and dry matter intake (DMI) [18]. Clostridium butyricum has the potential to enhance rumen fermentation and degradability, and thus may alleviate the adverse effects of heat stress on rumen fermentation and enhance the growth performance of ruminants. However, studies on heat-stressed goats are still rare. Hence, the objectives of this study were to evaluate the effects of Clostridium butyricum on rumen fermentation and the growth performance of heat-stressed goats, both in vitro and in vivo. This study could provide a scientific reference for the use of Clostridium butyricum in goats to alleviate the adverse effects of heat stress on rumen fermentation and growth performance.2. Materials and Methods2.1. Animals, Diet and TreatmentThis study was carried out from July to October and was approved by the Animal Care and Use Committee of Huazhong Agricultural University (Approval code HZAUGO-2015-008). Twelve female crossbred goats (Macheng Black × Boer) aged 6.0 ± 1.0 months with a bodyweight of 23.23 ± 3.10 kg were kept in a house equipped with slatted floors and manure scraper systems, with individual feeding pens (1.0 × 1.50 m). The goats were fed twice daily (8:00 h; 17:00 h) with a 1.31 kg/day maintenance diet and had free access to water. The ingredients and nutritional composition of the diet are provided in Table 1. No antibiotics and probiotics were offered to goats before this study.2.2. Modeling of Heat-Stressed GoatsThe modeling process was as described by Cai et al. [2]. Basically, all 12 goats were kept in a thermally controlled environment with the room temperature and relative humidity maintained at 33.2 ± 2.7 °C and 74.4 ± 2.3%, respectively. The temperature-humidity index (THI) was used as an indicator for the evaluation of heat stress in goats. THI was calculated as described by LPHSI [19]. In this environment, the THI was 87.0. When the THI is greater than 82, goats are considered to be heat stressed [20]. Goats were kept in this indoor environment for two weeks. On day 14, blood was collected for measurement of the gene expression of the heat shock protein 70 (HSP70) [21] and cortisol concentration [22] to determine the occurrence of heat stress in goats.2.3. Measurement of Physiological Indices of GoatsRectal temperature, skin temperature, pulse, and respiratory rate were all measured three times a day, at 8:00, 12:00, and 17:00, throughout 14 days before and after heat-stress modeling. Rectal temperature was measured using a mercury glass thermometer (Fangda Pharmceutical machinery Co., Ltd., Hefei, China). Skin temperature was measured with an OS543 infrared thermometer (Omega, Norcross, GA, USA). A stethoscope (Yuwell, Shanghai, China). was placed laterally in the thoracic area to monitor inhalation and exhalation, with the respiratory rate recorded. A stethoscope was placed ventrally to measure the pulse.2.4. Gene Expression Analysis Using Real-Time Quantitative PCRPeripheral blood lymphocytes from the blood of the goats were isolated using a peripheral blood lymphocyte isolation solution kit (Solarbio Science & Technology, Beijing, China). Total RNA was extracted from the peripheral blood lymphocytes using TRIzol® Reagent (Life Technologies, Carlsbad, CA, USA). A Revert Aid First Strand cDNA Synthesis kit (Thermo Fisher Scientific, Waltham, MA, USA) was then used for reverse transcription, following the manufacturer’s instructions. Primers were designed using Primer 5.0 software (Premier Biosoft, Palo Alto, CA, USA). and were synthesized by Sangon Biotech Co., Ltd. (Shanghai, China). The details of the gene-specific primer sequences are shown in Table 2. A SYBR RT-PCR Kit (Bio-Rad, Hercules, CA, USA) in conjunction with an ABI QuanStudio TM6 flex real-time fluorescent quantitative PCR system (Life Technologies, Carlsbad, CA, USA) were used for the RT-PCR conduction. Each sample was analyzed in triplicate to ensure the accuracy of the results. The levels of relative expression were quantified using the 2−ΔΔCt method [23].2.5. Cortisol Concentration MeasurementThe next day of the heat-stressed goat modeling, blood samples were taken from the jugular veins of 12 goats before the morning feeding by vein puncture and were placed in 10 mL vacuum blood collection tubes. The blood samples were centrifuged at 3000 rpm for 10 min to obtain the serum. The blood serum samples were immediately frozen at −20 °C and stored until analysis. The cortisol concentration was measured in serum samples using a cortisol assay kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China), following the manufacturer’s instructions.2.6. Rumen Fermentation Experiments In VitroThree heat-stressed goats were randomly selected as rumen fluid donors in this study. Rumen fluid was collected using a soft plastic stomach tube with a GM-0.33A vacuum pump (Jinteng, Tianjing, China), 4 h after the morning feed. Then, the rumen fluid was strained through four layers of gauze to remove the large feed pellets, and the filtrate was transferred to flasks prepared for the in vitro fermentation. The rest of the filtrate was immediately stored at −20 °C for further analysis. The commercial Clostridium butyricum live cell product (Huijia Biotechnology Co. Ltd., Huzhou, China) with a live cell number of 1.0 × 108 CFU/g was supplemented at the level of 0% (CG), 0.025% (CB1), 0.05% (CB2), 0.10% (CB3), and 0.20% (CB4) of the dry matter (DM) concentration in the basal diet for in vitro incubation. Feed substrates were ground through a 1.0 mm screen grinder (Hongguang Machinery Co., Ltd., Zhejiang, Jaxiing, China). Before incubation, 400 mg of dry substrates and Clostridium butyricum were added to a 100 mL flask, and all flasks were prewarmed using a water bath at 39 °C. In each flask, 32.0 mL of McDougall’s buffer [24] and 8.0 mL of rumen fluid were added and then flushed with CO2. The flasks were sealed with rubber stoppers and covered with aluminum foil. Finally, the flasks were shaken (125 rpm) using an Environ-Shaker (Guohua, Beijing, China) incubator at 39 °C for 24 h. Three flasks were prepared for each supplement level. The flasks were placed in an ice water bath for 15 min to stop the incubation before the rumen cultures were collected.2.7. Rumen Fermentation Experiments In VivoTwelve heat-stressed goats were randomly allocated into three groups and assigned to a 3 × 3 Latin square design; each experimental cycle lasted for 20 days. Clostridium butyricum was supplemented with 0% (HS1), 0.05% (HS2), and 0.10% (HS3) of the DM concentration in the basal diet. Five grams of Cr2O3 were added to the diet, as an exogenous indicator for the determination of nutrient digestibility, in the morning feeding on days 17 to 19 within each experimental cycle. Between experimental cycles, all goats were fed a basal diet for 20 days to eliminate the influence of the previous treatment. The collection, pre-treatment, and storage of rumen fluid were consistent with that of the in vitro experiment. Before the morning and afternoon feedings, fecal samples were collected from each goat during days 18 to 20 within each experimental cycle. Fecal samples were stored at −20 °C for further analysis.2.8. Sample AnalysisThe ruminal pH and oxidation-reduction potential (ORP) of rumen cultures were measured immediately at the end of the in vitro incubation using a digital pH meter and a digital ORP meter (Thermo Scientific, Waltham, MA, USA), respectively. In the in vivo experiment, these two parameters were measured immediately after the rumen fluid collection. The filtrate of rumen cultures or rumen fluid was centrifuged at 12,000× g at 4 °C for 15 min, and the supernatants were collected for ammonia nitrogen (NH3-N) and volatile fatty acids (VFAs) analysis. NH3-N was measured as described by Maitisaiyidi et al. [25] using spectrophotometry. The concentrations of VFAs were determined as described by Yang et al. [26] using gas chromatography. Briefly, 1.0 mL of 25% (w/v) metaphosphoric acid was added to 0.20 mL of supernatant and centrifuged at 10,000 r/min for 10 min. Then, the supernatant was injected into a Chrompack CP-Wax 52 fused silica column (30 m × 0.53 mm × 1.00 μm) in a gas chromatograph equipped with a Model 2010 flame ionization detector (Shimazu, Kyoto Japan).DMI was calculated by subtracting the weight of the remaining feed from the weight of feed provided per meal. The body weights of the goats were measured with an electronic scale (Salter Brecknell, Fairmont, MN, USA) in the morning before offering feed and water. The body weights were recorded at the start and end of each experimental cycle to allow average daily gain (ADG) calculations. The dry matter (DM), neutral detergent fiber (NDF), and acid detergent fiber (ADF) of feedstuff, fermentation substrate, and fecal samples were analyzed, as described by Zhang et al. [27].2.9. Statistical AnalysisRumen fermentation and growth performance parameters were analyzed using GrapgPad Prism (vv8.0.2) (GraphPad Software Inc., San Diego, CA, USA). for one-way analysis of variance (ANOVA) tests followed by post-hoc Dunn test for multiple pairwise-comparison. p values of less than 0.05 were considered statistically significant.3. Results3.1. Evaluation of the Model of Heat-Stressed GoatsThere were no significant differences in the body temperatures of goats across the whole time before (CG) and after heat-stressed modeling (HS). After heat stress was imposed, the goats exhibited significantly higher skin temperature, heart rate, and respiratory rate (p < 0.05) than they exhibited before. The physiological indices of the goats are shown in Table 3. To evaluate whether the goats suffered heat stress or not, the expression of the heat shock protein 70 (Hsp 70) family member gene, including HSPA 1, HSPA 6, and HSPA 8, in the blood lymphocytes was determined. Increased expression of HSPA 1 was observed in the blood lymphocytes of HS compared to CG (p < 0.01; Figure 1A). However, there were no differences in the expression of HSPA 6 and HSPA 8 in the blood lymphocytes between CG and HS (p > 0.05; Figure 1A). Moreover, increased cortisol concentrations were observed in the serum of HS goats compared to CG goats (p < 0.001; Figure 1B).3.2. Rumen Fermentation In Vitro with Clostridium butyricum SupplementAfter 24 h of incubation, the pH; the concentrations of NH3-N, TVFA, acetic acid, and propionic acid; and the acetic acid to propionic acid (A/P) ratio in rumen cultures were significantly increased (p < 0.05) in CB2 and CB3 compared with the CG, CB1, and CB4. However, there were no significant differences of these parameters among CG, CB1, and CB4. The ORP was significantly decreased (p < 0.05) in CB2 and CB3 compared with the CG, CB1, and CB4, whereas there were no significant differences among CG, CB1, and CB4. Moreover, there was significantly higher degradability of DM, NDF, and ADF (p < 0.05) in CB2 and CB3 than in the CG, CB1, and CB4, while there were no significant differences among CG, CB1, and CB4. The rumen fermentation parameters in rumen cultures with Clostridium butyricum incubation in vitro are shown in Table 4.3.3. Rumen Fermentation In Vivo with Clostridium butyricum SupplementThe rumen pH; the concentrations of NH3-N, TVFA, acetic acid, and propionic acid; and the A/P ratio were significantly increased (p < 0.05), while the ORP was significantly decreased (p < 0.05) in the HS2 group compared with those in the HS1 and HS3 group, respectively. The rumen fermentation parameters of the heat-stressed goats supplemented with Clostridium butyricum are shown in Table 5.3.4. Growth Performance of Heat-Stressed Goats with Clostridium butyricum SupplementCompared with the HS1 and HS3 group, the DMI, ADG, and the degradability of DM, NDF, and ADF were significantly increased (p < 0.05) in the HS2 group. The growth performance parameters of the heat-stressed goats supplemented with Clostridium butyricum are shown in Table 6.4. DiscussionProbiotics have been shown to be effective in improving the anaerobic environment, stabilizing pH and supplying nutrients for ruminants [28,29,30,31,32]. Clostridium butyricum, an excellent probiotic resource, could act as a regulator in balancing the gut microflora, providing nutrients and antioxidants, as well as improving immunity, and promoting the growth of livestock [33]. It has been widely used in pig and poultry production [10,34]. However, to our knowledge, few studies have evaluated the effects of it on rumen fermentation and growth performance in ruminants, especially in heat-stressed goats. In this study, both environmental THI and HSP 70 family member gene, cortisol concentration, and the physiological indices of the goats were determined to characterize the exact occurrence of heat stress in goats. Then, the in vitro and in vivo studies of heat-stressed goats supplemented with Clostridium butyricum were carried out. The pH of the rumen cultures and rumen fluid increased when the diet was supplemented with Clostridium butyricum. This result was consistent with a previous study in which ruminal pH significantly increased in calves whose diet had been supplemented with Clostridium butyricum. The result suggested that Clostridium butyricum was effective in alleviating the pH reduction [35]. The probiotic may prevent a decline in rumen pH by decreasing lactic acid production and increasing the utilization of lactic acid by ruminal microbiota [31,32,36]. Moreover, probiotics could also enhance the abundance of rumen protozoa, contributing to a reduction in the ruminal lactic acid concentration [37]. In contrast to a large number of studies on the effects of probiotics on rumen pH, few studies have focused on the effects of probiotics on the ruminal ORP. Ruminal ORP can reflect the fermentation processes in the rumen and the fluctuation of rumen pH. In this study, both incubation and feeding with Clostridium butyricum significantly decreased the ORP in the rumen cultures and fluid. The results are consistent with previous studies showing that live yeast reduced ruminal ORP by −20 and −34 mV [38,39]. The reduction of ruminal ORP can be attributed to the consumption of oxygen on the surface of feedstuff and in the rumen promoted by Clostridium butyricum [40]. In this study, the rumen NH3-N concentration was increased by Clostridium butyricum supplements, both in vitro and in vivo. Similar to our results, a previous study using yeast supplements observed an increase in the NH3-N concentration in the rumen [40,41]. This increase in NH3-N may result from enhanced microbial activity in the rumen that breaks down protein into carbon skeletons and ammonia after Clostridium butyricum supplements have been administered [42]. Since this study did not evaluate the effect of Clostridium butyricum on the composition and function of rumen microbiota, future studies are required to confirm this beneficial effect of Clostridium butyricum. Few studies have investigated the effects of Clostridium butyricum on ruminal VFAs production. A previous study reported that calves fed with Clostridium butyricum showed no effect on their ruminal VFAs concentrations [35]. In contrast, significant increases in the concentrations of TVFA, acetic acid, and propionic acid, and in the A/P ratio with Clostridium butyricum supplements were obtained both in vitro and in vivo in the present study. The simulation of the activities of rumen microbes (especially fibrolytic bacteria) by Clostridium butyricum perhaps contributes to the increase of the TVFA concentration [39,43]. Future studies are required to confirm these effects of Clostridium butyricum on the composition and function of the rumen microbiome.In this study, Clostridium butyricum was shown to have a positive effect on the DMI, ADG, and digestibility of DM, NDF, and ADF in heat-stressed goats, both in vitro and in vivo. This result is consistent with a previous study that found that supplements of Clostridium butyricum fed to calves significantly increased their DMI and ADG [14]. Similarly, it has been reported that supplements with Clostridium butyricum in the diet of weaning piglets and chickens improved weight gain and feed efficiency [44]. The effect of Clostridium butyricum on growth performance may be attributed to its ability to provide amino acids, short-chain fatty acids, and vitamin B to animals. Moreover, it can produce a variety of digestive enzymes including amylase, lipase, and protease, which could promote the digestibility of nutrients [14,33]. Future studies to detect metabolite production along with culture-based studies are required to confirm the function of Clostridium butyricum, which could provide a better understanding of the contribution of this probiotic to rumen fermentation and the growth parameters of heat-stressed goats.5. ConclusionsClostridium butyricum improves the rumen environment by increasing pH and decreasing ORP. Rumen fermentation can be improved by increasing NH3-N and VFA production, and enhancing the feed digestibility both in vitro and in vivo with Clostridium butyricum supplements, resulting in improved growth performance of heat-stressed goats. In conclusion, supplementing their diet with Clostridium butyricum can be effective in alleviating the negative effects of heat stress on goats. For stressed goats, the optimum addition amount of Clostridium butyricum is 0.05% of the DM concentration in the basal diet.

animals : an open access journal from mdpi

10.3390/ani12060690

PMC8944494

There is a demand to replace fishmeal with protein sources of plant origin in fish feeds. Biotechnology strategies, such as fermentation, can improve the bioavailability of plant proteins and decrease the presence of antinutrients, optimizing the results obtained. Fermented soybean meal has already been evaluated for different fish species as a replacement for fishmeal, and there is evidence that it can improve the intestinal health of animals. Lactobacillus acidophilus is a strain used as a probiotic in fish feeding but it remains to be evaluated as a potential fermentation bacterium for feed ingredients. This study aimed to evaluate the effect of diets containing different inclusion levels (0%, 7%, 14%, 21% and 28%) of soybean meal fermented by L. acidophilus (SMFL) on the zootechnical performance and intestinal health of South American catfish juveniles (Rhamdia quelen). The inclusion of SMFL up to 21% in replacement of fish meal did not affect the zootechnical performance of fish and also decreased the concentration of Vibrionaceae bacteria present in the intestine compared to the control group. The results demonstrate that fermentation with L. acidophilus enables greater inclusion of soybean protein in South American catfish diets and promotes the control of intestinal pathogenic bacteria.

The objective of this study was to evaluate the effect of diets containing different inclusion levels (0%, 7%, 14%, 21% and 28%) of soybean meal fermented by Lactobacillus acidophilus (SMFL) on the zootechnical performance and intestinal health of South American catfish juveniles (Rhamdia quelen). The experimental design was completely randomized with five treatments and four replications and lasted 56 days. Five isoproteic (39% crude protein) and isoenergetic (4300 kcal of gross energy kg−1) diets were formulated where SMFL was included in replacement of fish meal. Two hundred forty South American catfish juveniles (3.0 ± 0.5 g) were distributed in 20 tanks (70 L) connected in a recirculation aquaculture system. At the end of the experiment, the inclusion of SMFL up to 21% in replacement of fish meal did not affect the zootechnical performance and also decreased the concentration of Vibrionaceae bacteria present in the intestine compared to the control group. The amount of total lactic and heterotrophic bacteria, the enzymatic activity and the intestinal morphometry did not differ between dietary treatments. The results demonstrate that fermentation with Lactobacillus acidophilus enables greater inclusion of soybean protein in South American catfish diets and promotes the control of intestinal pathogenic bacteria.

1. IntroductionSoybean meal is an ingredient that is the most common vegetal protein source in animal nutrition due to its availability and balanced amino acid profile [1,2]. However, the palatability of soybean meal is low [3], and there are problems with antinutritional factors that limit its use in feeding aquatic organisms [4]. Some of the antinutritional factors, such as trypsin inhibitors and hemagglutinants, are inactivated by the toasting process [5]. However, other factors are heat-stable, such as saponins, non-starch polysaccharides, antigenic proteins, and some phenolic compounds. Furthermore, the excess of oligosaccharides can impair the digestive process [5], and the presence of antinutritional factors such as phytic acid can impair protein digestibility [1].Fermentation is a method that can enhance the quality of feed ingredients, inactivate antinutritional factors, improve nutritional bioavailability and increase soluble proteins and small peptides [6]. Fermented products are foods or beverages that serve as substrates for microorganisms, which through enzymes (amylases, proteases and lipases, among others), transform and reduce the size of organic compounds [7]. Fermented foods are part of the human diet due to their nutritional and functional qualities [8]. In recent years, the use of fermented ingredients as functional diets in aquaculture species has attracted the attention of academia and industry [9].Microbial fermentation is carried out using fungi or bacterial strains [8]. Lactobacillus spp. are widely used in the process as they are considered nonpathogenic and safe bacteria [10]. Like other lactic acid bacteria, Lactobacillus spp. can inhibit other competing microorganisms by combining rapid carbohydrate utilization and lactic acid production [11]. Several strains of Lactobacillus spp. have functional properties and can be used in fermentation processes. Lactobacillus acidophilus are important microorganisms in the composition of the human intestinal microbiota [12]. This strain is used as a probiotic in fish feeding [10] as it has therapeutic activities on intestinal health and produces a variety of antimicrobial compounds that are effective against pathogenic bacteria [13]. Positive results have already been obtained with Lactobacillus spp. mixed cultures in the fermentation of soybean meal [14,15], but the use of L. acidophilus remains to be evaluated as a potential fermentation bacterium for feed ingredients.Fermentation is an advantageous technique for nutritional improvement of soybean meal, through biodegradation and reduction of oligosaccharides and phytic acid, and for enhancement of the amino acid profile [16,17,18,19]. Fermentation also causes a release of bioactive peptides, which have functional properties in animal metabolism by modulating the immune system [20]. In studies with fish, the use of fermented soybean meal showed positive results on weight gain [21,22,23,24]. Enhancement in the intestinal health of fish fed with fermented soybean was observed, evidenced by the increase in beneficial bacteria in the intestinal microbiota [23,25,26], by the increase in the activity of gastrointestinal enzymes [27,28,29,30] and by the improvement of intestinal condition histological morphometric indexes [27,31,32,33,34].The South American catfish (Rhamdia quelen) is an omnivorous species with a tendency to exhibit carnivore behavior [35]. Regarding nutrition, the South American catfish is considered a demanding fish in terms of quality [36] and quantity of protein [37]. Diets for this species are generally formulated with high levels of animal protein sources, whereas when fishmeal has been replaced with alternative protein sources such as soybean meal, the zootechnical performance of this species has been compromised [37,38]. In order to overcome such a limitation, fermentation may be used as a strategy for enabling higher levels of soy protein in compound feeds and for benefiting the intestinal health of the South American catfish. Therefore, the objective of this work is to evaluate the effect of diets containing different levels of inclusion of soybean meal fermented by L. acidophilus (SMFL) on the zootechnical performance and intestinal health condition of South American catfish juveniles.2. Materials and Methods2.1. Experimental DesignThe experiment was carried out at the Santa Catarina State University (UDESC) Fish Farming Laboratory, in Lages, SC, Brazil. The study was approved by the Ethics Committee on Animal Use (CEUA) of UDESC (protocol number 4384210621). SMFL was evaluated at four levels (7%, 14%, 21% and 28%) of inclusion compared to a control diet (0%) during 56 days. The experimental design was completely randomized, with five treatments and four replications.2.2. Production and Characterization of Fermented Soybean MealSoybean meal was fermented using an adaptation of the methodology of Azarm and Lee [39]. In brief, soybean meal and bacteria were purchased from local suppliers. Initially, the soybean meal was autoclaved (AV-100, Tecnal, Piracicaba, Brazil) at 100 °C for 20 min. For this purpose, the soybean meal was autoclaved at a temperature of 100 °C for 20 min and inoculated with L. acidophilus (strain LA14-1.109 CFU g−1, Aché, São Paulo, Brazil) in the proportion of 44 g kg−1. Then, deionized water was added in the proportion of 50% of the soybean meal weight (model ML 600, Marte, Brazil), and the mixture was placed in trays previously sterilized with 70% ethanol. The mixture was distributed in trays in order to obtain an average thickness of two centimeters and then placed in an incubator (SSDcr-336L, SolidSteel, Piracicaba, Brazil) at 36 °C. Every 12 h, the material was weighed to quantify the evaporated water, replaced and homogenized to maintain moisture at 50%. Every 24 h, up to a maximum time of 96 h, samples were collected from the material that was dried in the incubator until reaching a constant weight, and later kept in the freezer. To determine the optimum fermentation time, three different production cycles were carried out to analyze lactic acid bacteria, enzyme activity and soluble protein. To count lactic acid bacteria, 1 g samples of SMFL were serially diluted (1:10) in 0.65% sterile saline solution and plated on MRS (Man Rogosa Sharpe) agar culture medium (Kasvi, São José dos Pinhais. Brazil). Fermented soybean samples plated in Petri dishes were placed in an incubator, and the temperature was maintained at 35 °C. Total counts of colony-forming units (CFU) were performed after 48 h of incubation on agar.Analyses of the enzymatic activity of α-amylase and total alkaline proteases were performed at different times of SMFL fermentation. Samples were placed in 50 mL tubes where they were diluted in chilled distilled water (1:6, w/v). The tubes were centrifuged (centrifuge K14-4000, Kasvi, São José dos Pinhais, Brazil) for 15 min at 4000 rpm, and the supernatants were used in the analyses. All assays were analyzed in duplicate in the three production runs described above. Alpha-amylase activity was measured (spectrophotometer Spectroquant Pharo300, Merck, Darmstadt, Germany) at λ = 580 nm using soluble starch (0.3%) as substrate in Na2HPO4 buffer solution (pH 7.4) [40]. A unit of α-amylase activity (U) was defined as a milligram of starch hydrolyzed in 30 min at 25 °C per milliliter of the supernatant.The activity of total alkaline proteases was determined after 30 min of incubation (dry block incubator Model K80-200, Kasvi, São José dos Pinhais, Brazil) at 25 °C, using 0.5% (w/v) casein as substrate in 50 mM Tris-HCl (pH 8.0). The reaction was stopped with trichloroacetic acid (20% w/v), the mixture was centrifuged (centrifuge K14-4000, Kasvi, São José dos Pinhais, Brazil) (5000 rpm, 20 min) and the absorbance of the supernatant was measured (spectrophotometer Spectroquant Pharo300, Merck, Darmstadt, Germany) at λ = 280 nm at room temperature. A unit of total alkaline protease activity (U) per milliliter was defined as 1 µmol of casein hydrolyzed per minute per milliliter of supernatant [41]. In order to measure the soluble protein, the fermented soybean meal samples were macerated until obtaining a fine powder. Then, 50 mM Tris-HCl buffer, pH 7.0, containing protease inhibitors (1 mM phenylmethylsulfonyl fluoride, 1 mM benzamidine, 2 mM thiourea and 10 mM EDTA) and 2% (m/v) polyvinylpolypyrrolidone, was added. Soluble protein was measured according to the Bradford method [42], using bovine serum albumin (Sigma-Aldrich, St. Louis, MO, USA) as a standard.Based on lactic acid bacteria counts, enzymatic activity and bromatological composition, the time of 48 h for SMFL fermentation was determined. Samples were collected before and after fermentation for bromatological analysis [43], pH and amino acid profile [44] (Table 1). The amino acid composition of SMFL was determined according to White, Harty and Fry [44]. The pH was measured in solution, according to the potentiometric method described by the Association of Official Analytical Chemists [45]. Ten grams of the samples were placed in a 250 mL Erlenmeyer flask, where 100 mL of distilled water at 25 °C was added. After adding water, the contents were mixed in an electronic shaker (model DT3110H, DiagTech, São Paulo, Brazil) for 30 min. The contents were placed in a 500 mL beaker and allowed to rest for 10 min, and then pH (model AT-355, Alfakit, Florianópolis, Brazil) was measured.2.3. Experimental DietsFive isoproteic (39% crude protein) and isoenergetic (about 4300 kcal of gross energy kg−1) diets were formulated, according to the nutritional requirement of the South American catfish [47], with five levels of inclusion (0%, 7%, 14%, 21% and 28%) of SMFL (Table 2). Dietary levels of crude lipids were 11.1%, 10.4%, 9.7%, 7.1% and 7.9%. The adjustment in the lipid levels was necessary to balance the energy levels of the diets. In addition to the fermented soybean meal, diets were formulated using corn and soybean oil as energy sources and fishmeal and soybean meal as protein sources. Marine fishmeal was purchased from Agroforte (Laguna, Santa Catarina, Brazil). The ingredients were crushed in a knife mill with a 2 mm diameter mesh sieve, mixed, pelleted (meat grinder MTU 08, Arbel, São José do Rio Preto, Brazil) with the addition of water (30%) and placed in an incubator (SSDcr-336L, SolidSteel, Piracicaba, Brazil) at 45 °C for 36 h. The proximal composition of experimental diets was analyzed using the methods described in AOAC [43]. The diets were stored in plastic containers and kept under refrigeration (4 °C) until the moment of use.2.4. Animals and FacilitiesFish were acquired from Piscicultura Nossa Senhora Aparecida, located in the city of Ijuí, Rio Grande do Sul (Brazil), and previously acclimated to the experimental conditions. A total of 240 South American catfish juveniles (3.0 ± 0.5 g) were used, which were distributed in 20 polyethylene tanks with a useful volume of 70 L. The tanks were connected to a water recirculation system equipped with a mechanical and biological filter and a heating system. Fish were fed until apparent satiety, once a day, at 9 a.m. This feeding protocol has already been determined as suitable for the South American catfish [48].Every day, organic waste was removed from the bottom of the tanks, and the presence of dead animals in the tanks was checked. Temperature (28.5 ± 0.3 °C), pH (7.12 ± 0.25) (HI98130, Hanna, Brazil) and dissolved oxygen (6.41 ± 0.54 mg L−1) (HI9147-10, Hanna, Barueri, Brazil) were monitored daily. Total ammonia was checked weekly (LabconTest, Alcon Pet, São Francisco de Assis, Brazil) (0.17 ± 0.2 mg NH3 L−1). Salinity was maintained around 4 g L−1 [49] in all polyethylene boxes throughout the experimental period. Water quality parameters remained within the recommended parameters for the cultivation of the South American catfish [50].2.5. Productive Performance and Sample CollectionAt the beginning and at the end of the experiment, biometrics were performed to assess the animals’ growth. All fish were anesthetized in a clove oil solution (1 g 10 L−1 of water) and individually weighed on a semianalytical digital scale with a precision of 0.01 g (model ML 600, Marte, São Paulo, Brazil). The following zootechnical performance indicators were evaluated: final weight, specific growth rate (SGR), apparent feed conversion (FC), feed ingestion (FI) and weight gain (WG). Mortality was recorded to assess the survival rate (S). (1) SGR%.day−1=In final weight−In initial weightexperimental period×100 (2) FC=feed consumptiontotal weight gain (3) FIg=total feed consumptiontank (4) WGg=final average weight FW−initial average weight IW (5) S%=total animals harvestedtotal animals stocked×100 At the end of the experiment, two fish per replicate (eight fish per dietary treatment) were collected, anesthetized and euthanized for analysis of intestinal histomorphometry, intestinal microbiology and gastrointestinal tract enzyme activity.2.6. Count of Total Heterotrophic, Lactic Acid and Vibrionaceae BacteriaThe intestinal tracts were homogenized, serially diluted (1:10) in sterile saline solution at 0.65% and seeded in the culture media with MRS (Man Rogosa Sharpe) agar (Kasvi, Brazil), TSA (tryptone soy agar) (Kasvi, São José dos Pinhais, Brazil) and TCBS (thiosulfate, citrate, bile and sucrose) agar (Acumedia, Indaiatuba, Brazil) to quantify lactic acid, total heterotrophic and Vibrionaceae bacteria, respectively. Intestinal samples were plated on Petri dishes and placed in incubators at 36 °C. Total counts of colony-forming units (CFU) were performed after 24 h of incubation in TSA and TCBS agar. In the MRS medium, the counts were performed after 48 h of incubation according to Jatobá et al. [51].2.7. Enzyme AnalysisStomachs and intestines were extracted through a longitudinal incision in the abdominal cavity and immediately frozen at −80 °C until the moment of analysis. The gastrointestinal tracts were weighed, ground and added in 50 mL tubes. The homogenates and total alkaline protease activity analysis were realized as described in Section 2.2. Lipase activity was measured at λ = 410 nm using p-nitrophenyl laurate (3 mM) in propanol as substrate [52]. The reaction was stopped by the addition of acetone [53]. A unit of lipase activity (U) was defined as the amount of enzyme required for the hydrolysis of 1 µmol of p-nitrophenyl laurate in 20 min at 25 °C per milliliter of enzyme extract.2.8. Intestinal MorphometrySections of 3 cm in length were collected from the midgut (4 cm after the junction of the stomach and the intestine) and dipped in a 10% buffered formalin fixative solution for 24 h, which was replaced by 70% alcohol, remaining preserved until the evaluation of the morphohistological characteristics of the mucosa. For this purpose, the intestinal sections were cut into slices of approximately 0.3 cm, dehydrated with graded series of ethanol and embedded in histological paraffin. Paraffin blocks were cut in a rotating microtome, with 5 µm thick cuts.Two slides were made from each sample, and the sections were stained according to the Harris eosin staining technique [54]. Slides were photographed using a digital camera (300 dpi) coupled to a microscope (10× objective). Fifteen villi of each fish were measured. Total height and height (corresponding to the distance from the apex of the villi to the beginning of the muscle layer and from the apex of the villi to the end of the serosa, respectively) and villous width values were measured using the ToupTek (Hangzhou, China) ToupView-x64 image analyzer software, version 2270/07/03.2.9. Statistical AnalysisAll data were subjected to tests to verify the normality of errors (Shapiro–Wilk test) and homoscedasticity of variances (Levene test). Broken-line model, linear, and polynomial regressions were performed using the SAS statistical program, version 9.0. The model with the best coefficient of determination was chosen to estimate the response.3. ResultsThe lactic bacteria count (Figure 1A; p = 0.0002; R2 = 0.86) and amylase activity (Figure 1B; p = 0.0106; R2 = 0.56) during soybean meal fermentation showed better fit to a quadratic equation. Values increased faster in the first 48 h. Protease activity (Figure 1C; p < 0.0001; R2 = 0.67) and soluble protein (Figure 1D; p < 0.0001; R2 = 0.85) showed a linear response with their values as fermentation time increased.No fish mortality was observed throughout the experiment. Regarding growth and feed performance variables, the broken-line analysis showed that dietary SMFL levels close to 21% may be included in the diets for South American catfish juveniles without any negative effect on somatic weight gain (Figure 2A; p = 0.0002), specific growth rate (Figure 2B; p = 0.0002) or apparent feed conversion (Figure 2C; p = 0.0178) of South American catfish juveniles. There was a trend (mean 31.21 ± 3.84, p = 0.0978) towards a reduction in feed ingestion in inclusions of SMFL higher than 14%.The count of Vibrionaceae in the intestine of South American catfish juveniles fed with SMFL showed a quadratic effect (Figure 3; p = 0.0316). Among the evaluated treatments, the lowest counts were observed in fish fed the diets containing 7%, 14% and 21% SMFL. There was no effect of SMFL inclusion on the count of heterotrophic bacteria (mean 3.32 ± 0.93 CFU g−1, p = 0.9827) or lactic acid bacteria (mean 3.01 ± 0.93 CFU g−1 p = 0.30)The inclusion of SMFL did not modify the specific activity of total alkaline proteases (mean 1.43 ± 0.87 U mg−1 protein, p = 0.5404) or lipase (mean 1.47 ± 079 U mg−1 protein, p = 0.4612) in the digestive tract of South American catfish juveniles. Intestinal histomorphometry was also not affected. The inclusion of SMFL did not change the total height (mean 616.23 ± 144.94 µm, p = 0.3482), height (mean 481 ± 121.73 µm, p = 0.7533) or width (mean 88.09 ± 14.33 µm, p = 0.3486) of the intestinal villi of South American catfish juveniles.4. DiscussionDuring fermentation, probiotic bacteria multiply and produce metabolites with bioactive properties [20]. Lactic acid bacteria increased in the SMFL from 0.00 to 5.80 CFU g−1 after 48 h of fermentation. These values are in agreement with the literature and demonstrate that the colonies grew as expected [18,55]. The colonies of lactic acid bacteria grew exponentially in the first 24 h of fermentation. After 48 h, growth stabilized, and this was considered the optimal fermentation time. As nutrients decrease and metabolites accumulate, the culture enters a stationary phase and reduces the production of organic compounds of interest [56]. As a result of fermentation, an increase in soluble protein and amino acids was observed. The increase in soluble protein is related to the presence of low-molecular-weight bioactive peptides [57] These peptides are also found in protein hydrolysates and can improve fish growth and intestinal health [58]. The synthesis of amino acids may be the result of lactic acid fermentation [8] and indicates an improvement in the nutritional value of SMFL. There was an increase in the activity of protease and amylase enzymes in SMFL. During fermentation, different types of enzymes are secreted by Lactobacillus [18], and SMFL can be a source of exogenous enzymes. Lactic acid is also secreted during fermentation, which leads to a drop in pH and can benefit the digestive process [59]. In the present study, the pH values remained relatively stable; the reduction in pH was from 6.64 to 6.47. It was not a significant decrease, but similar results were obtained using another bacterial species, Bifidobacterium animalis, in soybean fermentation [55].The dietary inclusion of up to 21% SMFL replacing fishmeal did not affect the zootechnical performance of the South American catfish juveniles. The zootechnical parameters are within what is expected for the species [58,60], and this result confirms the efficiency of fermentation as a strategy to include a higher percentage of soybean meal in carnivorous fish feeds. It has been shown that soybean meal fermentation reduced the amounts of antinutritional factors and allowed greater inclusion of soybean meal in the largemouth bass (Micropterus salmoides) diets [25]. Previous studies showed that an inclusion of above 35% soybean meal worsened the growth of the South American catfish [37]. In the present study, it was possible to include up to 46% soybean meal, considering the ingredients in the fermented and in the unfermented form. The higher inclusion of soybean meal may have been made possible by the improvement in the nutritional characteristics of the SMFL. Soybean meal fermentation increased soluble protein, indicating a higher proportion of low-molecular-weight peptides [57]. These peptides find specific absorption sites in the intestine, which results in more efficient absorption and thus compensates for the lower inclusion of fishmeal in diets [61].The highest level of SMFL inclusion (28%) reduced the weight gain of South American catfish juveniles. The result obtained in this experiment corroborated other studies that included levels of fermented soybean meal between 24% and 30% [3,62,63,64]. At the highest levels of inclusion, there was a trend towards a reduction in food intake, indicating that possibly the antinutritional factors were not completely removed. In addition, another possible explanation is the increase in the soluble protein present in the fermented meal. At high levels of inclusion, excessive peptides can compromise their absorption due to saturation of transport mechanisms and thus reduce animal performance [65,66,67,68]. Furthermore, there may be an increase in amino acid oxidation and a reduction in dietary protein retention due to the large number of free peptides present in the fermented meal used in this study, which may also compromise their absorption [69]. These results indicated that more studies are still needed to clarify how the use of fermented ingredients affects protein absorption and how this knowledge may be applied for guaranteeing higher levels of SMFL in aquafeeds.Increasing the inclusion of SMFL in the diets of South American catfish led to a reduction in dietary crude lipids. This adjustment was necessary to balance the energy levels of the diets. Although it is a protein source, SMFL does have moderate levels of starch and other carbohydrates that can be used as an energy source. South American catfish does not use carbohydrates as well as other omnivorous freshwater species, but it can adapt to different carbohydrate sources in diets [70]. Furthermore, if the energy intake had not been sufficient, an increase in the ingestion of these diets would be expected to compensate [71]. The result was the opposite: there was a tendency to reduce feed ingestion with the increase in the inclusion of SMFL in diets. Furthermore, we also find no differences in growth performance among fish fed diets differing in their crude lipid content (7.1–11%), which indicated that nutritional lipid requirements were already covered with the lowest crude lipid content tested (21% SMFL diet). These results are of relevance since they indicate that it is possible to reduce dietary lipid inclusion levels in diets for South American catfish juveniles when dietary crude protein (37.5–39.2%) and energy levels (4205–4300 kcal g−1) are covered.In terms of microbial load in the intestinal mucosa, the inclusion of 21% SMFL promoted a decrease in bacteria from the family Vibrionaceae in the intestine of the South American catfish when compared to the control group. Cheng et al. [72] obtained a similar result with shrimp (Litopenaeus vannamei) when using fermented soybean meal, causing the reduction of two species of Vibrionaceae bacteria. Other authors have demonstrated that fermented soybean meal can improve the composition of the intestinal microbiota in fish [23,25,26]. Most of the bacteria in the fermented products die during the drying process; however, the residues of dead bacteria and their metabolites produced may have contributed to these positive results [25]. The fermented meal would work as a paraprobiotic additive in which the microorganisms are not in their viable form but still bring benefits to intestinal health [73]. Another explanation for the decrease in Vibrionaceae bacteria would be the presence of bioactive peptides that have antimicrobial activities and that are abundantly present in soybean meal [74]. It should be noted that at the highest level of SMFL inclusion, the same response was not obtained in terms of controlling Vibrionaceae bacteria. As performance was compromised in this treatment, the bacterial count may also have been affected.Changes in the bacterial counts of Vibrionaceae were not related to changes in the abundance of heterotrophic and lactic acid bacteria in the intestine of South American catfish, which suggested that these changes in the Vibrionaceae were not due to competitive exclusion mechanisms with heterotrophic and lactic acid bacteria. Fermented ingredients are sources of probiotic bacteria and contain functional metabolites that can positively affect the intestinal microbiota of fish [73]. In the present study, due to technical limitations, only two fish were collected per replicate for each analysis. The low number of repetitions may have prevented the detection of differences between treatments. More studies are needed to understand the effects of ingesting fermented ingredients on intestinal bacteria counts. It is also necessary to evaluate, through molecular biology studies using massive sequencing approaches, the dietary effects of SMFL on autochthonous microbiota.The enzymatic activity and the intestinal morphometry of South American catfish juveniles were not affected by the inclusion of SMFL. The use of fermented ingredients can bring benefits to intestinal health, such as increased enzymatic activity [27,28,29] and improvement of intestinal villi [16,27,31,32,33,34]. However, these beneficial effects were not seen in this study. One of the potential reasons for explaining such results was the low number of replicates used, which may have hampered detecting statistical differences among dietary groups. On the other hand, there are several factors involved in the fermentation process that may affect the functional properties of the fermented product [8]. Ranjan et al. [75] and Azarm and Lee [39] also did not observe changes in the digestive enzymes of Labeo rohita and Acanthopagrus schlegeli fed with fermented soybean meal. Even with no changes, the obtained results can be considered positive, as high inclusions of soybean meal can compromise the enzymatic activity [76,77] and the intestinal morphometry of fish [6]. Previous studies have shown that soybean meal fermentation can protect the fish intestinal epithelium from possible damage [25,78].5. ConclusionsFermentation of soybean meal with L. acidophilus improved its nutritional profile and microbiological load. In particular, there was an increase in lactic acid bacteria count, enzymatic activity and soluble protein in the SMFL. The inclusion of SMFL up to 21% as a strategy for replacing fishmeal did not affect zootechnical performance in terms of growth and feed efficiency performances, nor did it affect intestinal morphology or enzymatic activity (total alkaline proteases and lipase) in the gastrointestinal tract of South American catfish juveniles. The amount of the Gram-negative bacteria from the Vibrionaceae family in the intestine decreased with the inclusion of up to 21% SMFL. These results demonstrate that soybean meal fermentation with L. acidophilus enabled a greater inclusion of soybean protein in diets and also promoted the control of intestinal pathogenic bacteria.

animals : an open access journal from mdpi

10.3390/ani13050778

PMC10000058

Peste des petits ruminants (PPR) is a highly contagious animal disease affecting small ruminants that causes high morbidity and mortality. To prevent outbreaks, Karnataka state, India, has implemented the PPR-Control programme (PPR-CP) with a ‘mass vaccination’ strategy since 2010–11, resulting in a significant reduction in the number of outbreaks. However, the state continues to report outbreaks every year due to various reasons. Presently, the state is planning to eradicate the disease by 2025–26 by employing a new mass vaccination programme in coordination with the government of India’s PPR eradication plan. In this study, we report on the current status of PPR, its economic cost, the financial viability of vaccination plans, and the perspectives of field veterinarians in controlling and eventually eradicating the disease in Karnataka state. The disease incidence in the state declined significantly due to the implementation of mass vaccination and the benefits of vaccination outweighed the cost many-fold. The majority of the veterinarians concurred with the various activities of PPR-CP but a few indicated disagreement with the plan per se, the coordination between the functionaries, the available funding and the acceptance of the programme by farmers.

In this study, we assessed the PPR disease status, its economic cost, the financial viability of vaccination, and the perspectives of field veterinarians on the PPR vaccination programme implemented in Karnataka state, India. In addition to secondary data, cross-sectional surveys undertaken during 2016–17 (survey I) and 2018–19 (survey II) from 673 sheep and goat flocks and data collected from 62 veterinarians were analysed. The economic costs and perceptions of veterinarians were analysed using deterministic models and the Likert scale, respectively, and the financial viability of vaccination programmes under the best (15%), base (20%), and worst-case (25%) PPR incidence scenarios, considering two different vaccination plans (plan I and plan II), was assessed. The disease incidence in sheep and goats was found to be 9.8% and 4.8% in survey I and survey II, respectively. In consonance with the increased vaccination coverage, the number of reported PPR outbreaks in the state declined significantly. The estimated farm-level loss of PPR varied between the surveyed years. Even under the best-incidence scenario, under vaccination plan-I and plan-II, the estimated benefit–cost ratio (18.4:1; 19.7:1), the net present value (USD 932 million; USD 936 million) and the internal rate of return (412%) implied that the vaccination programmes were financially viable and the benefits outweighed the cost. Though the majority of veterinarians perceived that the control programme was well planned and rolled out in the state, a few of them disagreed or were neutral towards the plan per se, towards the coordination between functionaries, the availability of funding, and the programme acceptance by farmers. Despite many years of vaccination, PPR still persists in the Karnataka state for various reasons and in order to eradicate the disease, a review of the existing control programme with strong facilitation from the federal government is needed.

1. IntroductionPeste des Petits Ruminants (PPR) is an acute febrile viral disease of sheep and goats and is generally referred to as ‘sheep and goat plague’. The disease is characterised by mucopurulent nasal and ocular discharges, necrotising and erosive stomatitis, enteritis, diarrhoea, and bronchopneumonia [1,2]. The causative agent, PPR virus (PPRV), belongs to the genus Morbillivirus under the family Paramyxoviridae. It is closely related to the Rinderpest virus that causes cattle plague, which was eradicated globally by 2011. The disease was first reported in sheep and goats in Côte d’Ivoire, West Africa in 1942 [3] and later spread to different countries in Asia and Africa. The global annual loss due to PPR is estimated to range from USD 1.45 billion to USD 2.1 billion [4]. Small ruminants are an important asset of landless, marginal and small farmers and disease outbreaks in flocks undermines the food and livelihood security of these poor farmers. Hence, considering the importance of PPR for small holder’s, the Food and Agriculture Organisation (FAO) and the World Organisation of Animal Health (WOAH) launched a global plan to eradicate PPR by 2030 [4].In India, the disease was first reported in 1987 in a small sheep flock in the village of Arasur in Tamil Nadu state, which was caused by a lineage III virus [5]. Subsequently, large outbreaks were reported in 1994–1995, which was caused by a lineage IV virus. Later, the disease spread to the entire country, and now PPR is enzootic in India and outbreaks are reported regularly among small ruminant population [1,2,6,7,8]. Very high rates of morbidity and mortality (> 50 percent) due to high fever, pneumonia, diarrhoea, and dehydration have been reported in the affected flocks [9]. Epidemics have enormous consequences on livestock productivity and affect the livelihoods of farmers and associated stakeholders in the sheep and goat value chain. Outbreaks have been reported regularly in the majority of the states of India since 2002. The losses due to PPR, as estimated by various researchers in India during different time periods, were INR 16116 million (USD 230.2 million) during 2015 [10], INR 88,951 million (USD 1270.7 million) based on reported outbreaks during 2008 to 2012 [11], and INR 45,710 to 46,830 million (USD 653–669 million) during 2016 [12]. Considering the devastating nature of the disease, an effective Vero-cell-line-based live attenuated indigenous freeze-dried vaccine (Sungri 96) with a shelf life of more than one year at 4 °C, which provides immunity for three to six years, was developed by the ICAR–Indian Veterinary Research Institute [13,14]. In India, since 2002, focused vaccinations of sheep and goats have been undertaken, and since 2010–2011, in southern states, the PPR-Control Programme (PPR-CP), with mass vaccination drives, has been implemented. This programme was implemented even before the global framework to control and eradicate PPR was developed. The Department of Animal Husbandry, Dairying and Fisheries (DADF), Government of India, sponsors the PPR-Control Programme (PPR-CP) in India and the participating states adopted a plan to carry out 100% vaccination of the risk population of small ruminants in the first year, followed by 30% bi-annual vaccination in the subsequent years to cover the naïve population [2,15]. Among the southern states, Karnataka and its contiguous state, undivided Andhra Pradesh, have implemented focused vaccination since 2002 and carried out mass vaccinations during 2004 and 2007–2008, respectively. Thereafter, they conducted annual vaccination programmes until 2010. After the first phase of the implementation of PPR-CP in these states during 2010–11, a significant reduction in the number of outbreaks in Karnataka state and a 99% reduction in PPR burden, with a flock immunity level of 81% to 95.6%, was observed in undivided Andhra Pradesh [2]. This was achieved due to the adoption of a systematic vaccination programme under PPR-CP. In central India, Chhattisgarh state has carried out annual mass vaccination campaigns (MVCs) since 2010 on the ‘pulse polio vaccination mode’ for a designated period of 11–12 days in a year, resulting in no PPR outbreaks having been reported in the state since 2013–14 [2,16]. In the second phase of PPR-CP, since 2015, although the rest of India’s states and Union Territories (UTs) have implemented PPR-CP [2], the continuation of the programme is largely elusive in these states.Karnataka accounts for 17.21 million sheep and goats.PPR is endemic in the state and is the single largest cause of mortality in small ruminants. Hence, to control the disease, ‘focused vaccination’ has been undertaken since 2003–2004, and the PPR-CP strategy, including ‘mass vaccination’, was implemented in 2010–2011 [10,15]. The main objectives of PPR-CP are 100% vaccination coverage, with the aim of bringing the outbreaks and epizootics to the level of zero and to make the state a PPR-free zone. The number of outbreaks has significantly decrease in the state due to the implementation of the vaccination programme. Now the state has planned to eradicate the disease by 2025–2026, implementing a further planned mass vaccination programme in consonance with the government of India’s recent PPR eradication plan. In general, the economic costs and financial effectiveness of PPR vaccination programmes are limited in PPR-endemic countries, including India. Hence, the present study was undertaken to understand the disease status after implementing the vaccination programme, vaccination coverage vis-à-vis outbreaks, the economic cost of the disease, the financial viability of the vaccination programme, and the perceptions of veterinarians in the PPR-endemic state of Karnataka, India.2. Materials and Methods2.1. Study AreaKarnataka is the 6th largest state, comprising 30 districts, which cover an area of 191,791 square kilometers or 5.83% of the total geographical area of India. Karnataka is the only southern state to have land borders with all of the other six southern Indian sister states. It is bordered by the Arabian Sea to the west, Goa to the northwest, Maharashtra to the north, Telangana to the northeast, Andhra Pradesh to the east, Tamil Nadu to the southeast, and Kerala to the south. As per the 20th livestock census, 2019, the total livestock population in Karnataka state was 29 million, of which 59.35% (17.21 million) comprised sheep and goat, of which, sheep constitute 64.21% (11.05 million). To understand the PPR incidence and the disease cost, the primary survey was undertaken during 2016–2017 and 2018–2019 and the geographical locations of the survey districts are presented in Figure 1.2.2. Sampling ProcedureCross-sectional surveys were undertaken in six districts of Karnataka state among sheep- and goat-rearing households during 2016–2017 (survey I) and 2018–2019 (survey II). Initially, the districts in the state were grouped into three risk groups (high, medium, and low) based on the cumulative number of outbreaks that occurred in the districts in the three years preceding 2016–2017, the total number of PPR attacks per 1000 heads of sheep and goats, and the frequency of outbreaks and livestock density per 100 km2, and one district was selected in each of the risk group for the primary survey. During survey I, three districts, Kolar (high PPR risk), Bangalore rural (medium PPR risk), and Bagalkote (Low PPR risk), and in survey II, three districts, Chikkaballapura (high PPR risk), Kalaburgi (medium PPR risk) and Bidar (low PPR risk), were surveyed. A multistage random sampling procedure was followed to conduct survey I and survey II. In the first stage, one district from each of the risk groups was selected randomly and in the second stage 2–4 blocks (a block is a smaller administrative unit in a district and each district may comprise 2 to 5 or more blocks, depending on the population of the district) were randomly selected in each of the selected districts. In the third stage, in each selected block, one veterinary dispensary/hospital was selected randomly. In the fourth stage, the sheep- and goat-rearing villages under the jurisdiction of the dispensary/hospital were enlisted, of which 5–10 villages were selected randomly. In the final stage, in each of the identified villages, the individual farmers/households were selected randomly for the primary survey. The number of flocks to be surveyed in the identified villages was determined based on the proportion of sheep- and goat-rearing households in the village.2.3. Sample SizeThe sample size for the primary survey was estimated as below [17], (1)SS=z2p1-pe2 where SS is the required sample size; Z is the z-value at a 95% confidence interval (1.96); p is the proportion of sheep- and goat-rearing households to the total number of households rearing livestock (0.224), and e is the acceptable sampling error (5%). The estimated total sample size for the survey was 267 sheep- and goat-rearing flocks. However, the primary data was collected from 350 flocks during 2016–2017 (survey I) and 323 flocks during 2018–2019 (survey II) using pre-tested schedules.2.4. Data Collection and Identification of PPR-Affected FlocksThe primary data on socio-economic parameters, sheep and goat inventories, production parameters, morbidity and mortality, PPR vaccination and treatment cost, etc. were collected from the selected sheep and goat farmers in the surveyed villages as per the sampling plan. Among the sampled farmers, photographs of various clinical signs of PPR [18] were shown to the farmers and based on their observations of clinical signs, the flocks were diagnosed and grouped into PPR-affected and non-affected groups. Furthermore, the PPR incidence reported by the farmers during the survey was triangulated with the data provided by jurisdictional veterinary doctors to confirm the occurrence of PPR in the surveyed flocks, as these veterinarians are the ‘gatekeepers’ for the animal diseases occurring in their jurisdiction.2.5. Estimation of the Flock-Level Economic Cost of PPRSheep and goat inventory, clinically diagnosed PPR cases, and deaths were considered in order to estimate the primary metrics of morbidity and mortality levels. The mortality loss was estimated based on the number of animals that died in each age and sex group multiplied by the market value of the apparently healthy animals and subtracting the recovered value of the animal, if any. To calculate the weight loss after PPR outbreaks, measurements of the pre- and post- disease weights of the animals across the breed, age, and sex were required. As this was a cross-sectional survey-based study, on the day of the survey, it was possible that diseased flocks may not have been encountered. Hence, to calculate the weight loss due to PPR, the actual weight of the animals of various breeds, ages, and sexes from the apparently healthy flocks were documented by physically weighing the animals during the survey. A conservative 10% reduction (although a 15% weight reduction for sheep and goat pox was reported earlier [19]) in the recorded weights for the healthy flocks was considered as the weight loss in the PPR-affected flocks, and accordingly, the weight loss was calculated across breed, age, and sex for the flocks reported to be affected by PPR. The distress sale loss was calculated based on the number of animals sold under distress and the difference in the actual price obtained during a healthy state and the distress sale value of the animals. The treatment cost was calculated based on the expenditure made for drugs/medicines and veterinarian charges during the PPR outbreak in the flock and converted to the per-animal cost. Similarly, the opportunity cost of labour engaged for nursing the sick animals was calculated based on the incremental labour hours spent by the farm family or hired members multiplied by the prevailing wage rate in the PPR-affected villages. After calculating the loss per flock, appropriate weights were considered based on the number of animals that died and that were infected and recovered among the sheep and goats reared by the sample farmers to calculate the per-animal loss. The market value/prices of animals of various age and sex groups and labour wage rate prevailing in the surveyed villages during the survey periods (2016–2017 and 2018–2019) were considered in order to estimate the various losses. The estimated disease cost per animal during survey I (2016–2017) was converted to the 2018–2019 constant price, based on the prevailing consumer price index during the respective years [20].2.6. Estimation of the Financial Viability of Vaccination2.6.1. Vaccination PeriodThe cash-flows were estimated in order to assess the benefit versus the cost of the vaccination programme from the initial year of PPR vaccination in Karnataka (2003–2004) up to the year selected for the potential control and eradication of PPR (2025–2026) as envisaged in the draft plan for PPR eradication in India. The vaccination period was divided into two stages—vaccination before PPR-CP implementation in the state (from 2005–2006 to 2010–2011) and vaccination after PPR-CP implementation (2011–2012 to 2025–2026).2.6.2. Vaccination Coverage and PlansThe data on the number of sheep and goats vaccinated in Karnataka state were collected from secondary sources from 2003–2004 to 2020–2021 in order to calculate the vaccine coverage (%). The benefits and costs of vaccination were estimated under two methods of vaccination plans. The first plan involved actual vaccination coverage from 2003–2004 to 2020–2021, covering 100% of the risk population from 2021–2022 to 2023–2024, followed by the need-based vaccination of 10% for the next two years (2024–2025 and 2025–2026) and the second plan was as per the PPR-CP plan, i.e., the actual vaccination coverage from 2003–2004 to 2020–2021 and the vaccination of 100% of the at-risk population during 2021–2022, followed by 30% bi-annual coverage for the two subsequent years (2022–2023 to 2023–2024) and 10% need-based vaccination coverage for next two years (2024–2025 and 2025–2026).2.6.3. Population ProjectionThe livestock population census in Karnataka was available only for quinquennial periods (i.e., 2003, 2007, 2012, and 2019) and hence to calculate the sheep and goat population in Karnataka for in-between years, the interpolation method was employed, using the compound annual growth rate (CAGR) of the sheep and goat population between respective livestock censuses. Furthermore, to predict the population for the period from 2020–2021 to 2025–2026, the average CAGR of the sheep and goat population between the 2003 and 2019 livestock censuses was considered.2.6.4. Incidence InterpolationThe PPR incidence levels with and without vaccination intervention were crucial for our financial evaluations. Due to various surveillance bottlenecks in developing countries, the reported disease incidence differs from the field-level incidence. For Karnataka state, the PPR incidence in the field based on surveys was available only for two years (2016–2017; 2018–2019). Hence, the 8% incidence level reported in the literature [21] during 2008–2009 for Madhya Pradesh state was considered as the initial incidence level in the study state (Karnataka) for the first year of financial assessment (i.e., 2003–2004). Furthermore, in determining the PPR incidence under a scenario with a vaccination intervention, we assumed that this 8% incidence level increased gradually and reached 12% during 2010–2011, as the progress of vaccination in the initial years was very slow due to the adoption of the ‘focussed vaccination’ policy in the state. Thereafter, mainly after PPR-CP implementation (since 2010–2011), the incidence level gradually decreased due to the adoption of the ‘mass vaccination’ drive. In the primary surveys undertaken in this study, we estimated an incidence of 9.8% and 4.8% in Karnataka during 2016–2017 and 2018–2019, respectively. Considering the initial incidence (2003–2004), the incidence under focussed vaccination (2010–2011), and the estimated incidence after PPR-CP implementation (2016–2017 and 2018–2019), the possible incidence levels in in-between years under the vaccination scenario were derived via interpolation through the linear method. At the end of 2023–24, as per the draft national strategic plan, 100% of the sheep and goat population [2] will be vaccinated, and as a result, the disease incidence will be very low during 2023–2025 to 2025–2026. Hence, for the financial analysis, a PPR incidence of zero was assumed during 2023–2025 to 2025–2026 (In India, the financial year lasts from April until March of the following year (for example, the year 2010–2011 year represents the period from April 2010 to March 2011). Similarly, the stream of incidences in different years between 2003–2004 and 2025–2026 under the scenario without vaccination intervention was derived via interpolation through the linear method. The three possible incidence scenarios (Supplementary Table S1) were as follows: an 8% incidence level during 2003–2004 that increased to 15% by 2010–2011 and remained at the same level until 2025–2026 (low incidence); an 8% incidence level during 2003–2004 that increased to 20% by 2025–2026 (medium incidence); and an 8% incidence level during 2003–2004 which increased to 25% by 2025–2026 (high incidence). These differences between incidence levels under scenarios with and without vaccination in different years were considered in order to estimate the avoided costs/benefits of morbidity and mortality reductions that occurred due to PPR vaccination. The details of interpolated incidence levels in scenarios with and without vaccination (with three possible incidences under without vaccination) are presented in Supplementary Table S1.2.6.5. Benefit StreamThe benefits of vaccination in different years were calculated based on the difference in the PPR incidence (the disease avoidance level) under scenarios with and without vaccination, the projected risk population in the respective year, the per-animal disease cost, the morbidity and mortality levels, and the effectiveness of vaccination (80%). The disease cost included mortality, body weight reductions, distress sales, treatment costs, and the opportunity cost of labour per animal due to PPR. The disease cost, incidence, morbidity, mortality, and distress sale proportions were calculated based on the data collected in the primary surveys undertaken in Karnataka state during 2016–17 and 2018–19. An annual 3% deflation rate (from 2003–2004 to 2017–2018) and inflation rate (from 2019–2020 to 2025–2026) were applied [22] to calculate disease cost per animal at current prices.The total benefit of vaccination against PPR was calculated as follows. (2)Bv=∑i=1n(ΔIi×Pi) Mpi×Lmi+Wpi×Lwi+Dpi×Dli+Tci+Oci×VEi where Bv = total benefits of vaccination/PPR avoidance cost (USD); (ΔI)i = difference in PPR incidence under scenarios with and without vaccination in the ith year (%), Pi = projected population in the ith year (No.), Mpi = proportion of mortality due to PPR in the ith year (%), Lmi = average mortality loss per animal in the ith year (USD), Wpi = proportion of morbidity due to PPR in the ith year (%), Lwi = average body weight reduction loss per animal in a PPR-recovered animal in the ith year (USD), Dpi = distress sale proportion due to PPR in the ith year (%), Dli = average distress sale loss per animal in the ith year (USD), Tci = average treatment cost per animal in the ith year (USD), Oci = average opportunity cost of labour per animal in the ith year (USD), VEi = vaccination effectiveness in the ith year (80%), and n = number of years (i = 1, 2, 3, …n).2.6.6. Vaccination Cost StreamThe total vaccination cost per annum was calculated based on the proportion of sheep and goats vaccinated, multiplied by the vaccination cost (vaccine cost per dose and the vaccination logistics and accessories cost per dose/animal). The vaccine cost and vaccination logistics and accessories cost varied depending on the vaccination strategy adopted, viz., focussed/routine vaccination or using the PPR-CP model. In Karnataka, the focussed/routine vaccination strategy was adopted from 2003–2004 to 2010–2011 and, thereafter, the PPR-CP vaccination strategy was implemented up to 2025–26. Hence, for the focussed/routine vaccination period (from 2003–2004 to 2009–2010), the vaccine cost per dose was assumed to be twice the vaccine cost for the control programme vaccination period (2010–2011 to 2025–2026) the vaccine cost was INR 1.8/dose and the vaccination logistics and accessories cost was INR18.2/dose [16]. The vaccination logistics and accessories cost included expenditure for accessories (needles, syringes, etc.); payment for hired vaccinators, vaccine storage, and transportation; the cost of sensitisation and technical workshops, extension awareness materials, programme dissemination, and publication; expenditure directed towards the strengthening of district diagnostic laboratories, staff salaries, etc. [16]. The total vaccination cost for the respective year was calculated as follows. (3)Cv=∑i=1nVpi× Vci where Cv = total cost of vaccination (USD million), VPi = the proportion of sheep and goats vaccinated in the ith year (%); Vci = cost of vaccination (including the vaccine cost and the vaccination logistics and accessories cost per dose) per animal in the ith year (USD million), and n = number of years (i = 1, 2, …, n).All the benefit and cost streams were estimated in INR and converted to millions of USD (One USD = INR 75).2.6.7. Financial Assessment of PPR VaccinationThe evaluation of the financial benefits of PPR vaccination was based on the benefit:cost ratio (BCR), net present value (NPV), and the internal rate of returns (IRR) as per standard procedure [16,23] under two vaccination plans, designated as plans I and II. Plan I refers to the actual vaccination coverage from 2003–2004 to 2020–2021 and vaccinating 100% risk population from 2021–2022 to 2023–2024 (for three years), followed by 10% need-based vaccination coverage from 2024–2025 to 2025–2026 (for two years). Plan II refers to the actual vaccination coverage from 2003–2004 to 2020–2021 and vaccinating 100% of the naive population during the year 2021–2022, followed by 30% bi-annual coverage from 2022–2023 to 2023–2024 (the two subsequent years) and 10% need based vaccination coverage from 2024–2025 to 2025–2026 (the next two years), as per the PPR-CP plan.2.7. Perspectives of Veterinary Officers on PPR-CPA survey was conducted among field veterinarians, who are the custodians of implementing the PPR vaccination programme for the sheep and goats in their jurisdictional veterinary hospitals/dispensaries through the support of the Animal Disease Surveillance Scheme (ADSS), Department of Animal Husbandry and Veterinary Services, Government of Karnataka, using the pre-tested schedules. In addition to implementing PPR-CP, field veterinarians also implement other disease prevention programmes and animal treatment and extension activities. The schedules were mailed to the 176 veterinarians working in veterinary hospitals/dispensaries in the state via e-mail and 62 responded to the survey. The survey schedule comprised statements about the planning and rollout of the PPR control programme in the state, the performance of the functional components of the programme, and the respondent’s opinions regarding its improved implementation. The statements were assessed through a two-point assessment (Yes/No) and a three-point Likert scale (Disagree (SD), Neutral (N), and Agree (A)) with positive and negative statements. The frequencies were calculated based on the responses of the field veterinarians to various statements about PPR control and eradication in Karnataka state.2.8. Statistical AnalysisDescriptive statistics and a two-sample z-test for proportions were used to test the significant differences in terms of diagnosed and dead cases in sheep and goats in the sample farms between survey I (2016–2017) and survey II (2018–2019). These were carried out using SPSS version 22.0 (SPSS Inc., Chicago, IL, USA).3. ResultsThe results obtained from the secondary data on the reported PPR outbreaks and vaccination coverage in Karnataka state and the results of the cross-sectional primary surveys undertaken in the sampled districts are presented below.3.1. Reported Outbreaks and PPR Vaccination CoverageKarnataka practiced focussed vaccination from 2003–2004 to 2010–2011. The vaccination coverage increased from 6% during 2003–2004 to 51% during 2009–2010, whereas after the implementation of PPR-CP in 2010–2011, the vaccination coverage increased consistently and reached 100% in the period from 2014–2015 to 2020–2021, except during 2017–2018 (Figure 2). In consonance with this increased vaccination coverage, the reported PPR outbreaks in the state declined significantly (Figure 2).3.2. Socio-Economic Characteristics of Sheep- and Goat-Rearing FarmersThe socio-economic characteristics of flocks surveyed in survey I and survey II are presented in Table 1. The median ages of farmers rearing sheep and goats during the two surveys were 49 and 46, respectively. The majority of the farmers were illiterate, and the majority of them were small farmers having less than 2.0 ha in land holdings. A considerable proportion of surveyed farmers were landless and they depended entirely on sheep and goats for their livelihoods. The observed average numbers of sheep and goats reared/flock sizes were 40 and 55 during survey I and survey II, respectively (Table 1). The majority (40%) of the sample farmers’ nominal incomes were less than USD 715 during 2016–2017, whereas the majority (52%) of the sample farmers’ nominal incomes were between USD 715 and USD 1429 during 2018–2019. The other socio-economic details of the surveyed flocks in different surveyed districts in Karnataka state during survey I and survey II are presented in Table 1.3.3. PPR Incidence in the Surveyed FlocksThe PPR-affected flocks and the disease incidence in sheep and goats among the sample flocks during surveys I and II are presented in Figure 3. The proportion of PPR-affected flocks among the surveyed sheep and goat flocks was 19.7% and 16.1%, respectively (Figure 3A), whereas the PPR incidence at the animal level was 9.8% and 4.8% in survey I and survey II, respectively (Figure 3B).3.4. Distribution of Incidence, Mortality and Case Fatality Rate (CFR)The age- and sex-wise distributions of PPR incidence, mortality, and CFR in sheep and goats in survey I and survey II are presented in Table 2. The PPR incidence in sheep and goats was 9.8% in survey I, whereas it was 4.8% in survey II. In sheep, age-wise and sex-wise comparisons of incidence showed a significant difference (p < 0.01) between the two surveys, whereas, in goats, we observed a significant difference in all the age groups (< 6 months (z = 5.65, p < 0.01), 6–12 months (z = 2.52, p < 0.1) and > 1 year (z = 5.43, p < 0.01)), whereas a non-significant difference was observed in the sex-wise comparison between the two surveys. In sheep, age-wise and sex-wise comparisons of mortality levels showed a significant difference between two surveys, whereas, in goats, a significant difference was observed in the group < 6 months old (z = 6.75, p < 0.01) and the 6–12 month old (z = 2.62, p < 0.01) group. Furthermore, the CFR was lower in survey I, compared to survey II (Table 2).3.5. Estimated Loss of PPRThe various components of the farm-level loss per animal in surveys I and II are presented in Table 3. The estimated mortality loss ranged between USD 30.1 and USD 128.6 per animal, depending on the severity of the disease in the respective years and the distress sale loss ranged between USD 19.6 and USD 101.8. The body-weight reduction, treatment cost, and opportunity cost of labour ranged between USD 1.7 and USD 7.4, USD 0.3 and USD 7.3, and USD 0.2 and USD 8.3 during the two surveys, respectively. The estimated loss components in sheep and goats independently during the two surveys are presented in Table 3.3.6. Financial Viability of PPR VaccinationThe results of the financial viability assessment of PPR vaccination in Karnataka under the best-, base-, and worst-case PPR incidence scenarios of 15%, 20%, and 25%, respectively, under two methods of vaccination plans are presented in Supplementary Tables S2 and S3. The estimated BCR under the best-, base-, and worst-case incidence scenarios were 18.36:1, 23.45:1, and 28.55:1, whereas the NPV was USD 931.97 million, USD 1205.75 million, and USD 1479.52 million, respectively, under vaccination plan I (Supplementary Table S2). Under vaccination plan II, the estimated BCR was 19.65:1, 25.11:1, and 30.57:1 and the NPV was USD 935.51 million, USD 1209.29 million, and USD 1483.07 million in the best-, base-, and worst-case incidence scenarios, respectively (Supplementary Table S3). Remarkably, an IRR of 412% was observed in all three incidence level scenarios with the two methods of vaccination plans (Supplementary Tables S2 and S3).3.7. Perspective of Field Veterinarians on PPR-CP3.7.1. Planning and Rollout of PPR-CPThe perceptions of veterinarians with regard to the planning and rollout of PPR-CP in Karnataka state are presented in Table 4. The majority (93.5%) of the surveyed field veterinarians perceived that the PPR-CP had been planned well in the state. For the statement regarding the ‘proper coordination of various programme functionaries’, although the majority agreed on the existence of co-ordination, around 19% remained neutral. Regarding the negative statement concerning the weak support from local Panchayat authorities, the 42% level of agreement indicates that there is still scope for fostering support from the local authorities. Regarding the statement that a ‘delay in the release of funds affected the PPR-CP implementation’, 26% agreed and 42% remained neutral, indicating that there were delays in the release of funds, which in turn affected the implementation of PPR-CP.3.7.2. Performance of Functional Components of PPR-CPThe majority (82%) of veterinarians perceived that the required quantity of PPR vaccine doses was supplied on time and 72% were able to cover the targeted population in the stipulated time. Regarding the statement on the ‘availability of cold-chain facilities’, 86% of the veterinarians perceived that good facilities were available (Table 5).3.7.3. Opinions of the Veterinarians on Improving the Implementation of PPR-CPThe majority (84%) of the veterinarians opined on the need for training regarding vaccine efficacy and effectiveness and 77% opined that provision of funds, mainly for mobility and contingency expenditure, were necessary for the success of the PPR-CP implementation in the state. The majority (92%) of the field veterinarians opined that intensive awareness meetings for farmers before the implementation of vaccination would benefit the programme considerably (Table 6).4. DiscussionThe socio-economic profile of the sampled farmers revealed that the majority of them were illiterate and small farmers (with landholdings < 2.0 ha) and their annual income was <1500 USD. An outbreak of disease in flocks affects the animal asset pattern, as well as financial and social security, and pushes these farmers to transitional poverty. Hence, to ensure the livelihoods of these farmers, it is important to control and prevent the incidence of PPR in their flocks. Karnataka state reported 150 to 200 outbreaks per year during 2004 to 2006 [2,15]. Due to the adoption of PPR vaccination in the state, the numbers of reported outbreaks have declined significantly since 2006–2007. The maximum vaccination coverage during the focussed vaccination period (2003–2004 to 2009–2010) was only 52%, whereas after the implementation of PPR-CP (2010–2011), a significant increase was observed. In consonance with the increased vaccination coverage, the number of reported PPR outbreaks in the state has declined considerably over the years. A similar pattern of a decline in outbreaks due to mass vaccination was observed in the undivided Andhra Pradesh area, which is a contiguous state of Karnataka.Though there were fewer reported outbreaks in Karnataka in recent years, the two primary surveys conducted here, covering 673 flocks, revealed incidences of 9.8% (survey I) and 4.8% (survey II), respectively. Similar results showing a higher incidence (17.5%) before mass vaccination (Singh et al., 2014) and a lower incidence (0.8%) after a mass vaccination campaign (MVC) were reported in Chhattisgarh state, India [16]. Age-wise and sex-wise comparisons of incidence and CFR revealed a significant difference between the two surveys. The observed PPR incidences during surveys I and II implied that a significant number of animals became infected with PPR across species, age, and sex groups and a considerable burden was being inflicted upon various stakeholders associated with small-ruminant rearing despite the state being a vaccine-adopted state since 2003–2004. This also implies that vaccination coverage in this particular state alone will not be sufficient to eradicate the disease and the implementation of parallel programmes in the neighboring and contiguous states are also equally important as the regular movement of sheep and goats between these states occurs for trade, transit, and for grazing. Though the number of reported outbreaks significantly decreased in Karnataka state after the implementation of the vaccination programme, in order to obtain a further definitive decline and disease eradication by 2025–2026 through ‘mass vaccination’ in the state, in consonance with the government of India’s PPR eradication plan, an appropriate strategy covering vaccination in all the contiguous regions or epi-systems—mainly in the regions where the maximum level of animal movement is observed between the states—is warranted.The observed variations in the estimated flock-level losses due to PPR in the surveyed years could be due to differences in the risk population, the number and severity of outbreaks, the age and sex compositions of the diseased flocks, the vaccination coverage in the preceding years, and the time and season of outbreaks. These findings are also in agreement with those of earlier reports [16]. Furthermore, the sensitivity analysis for the various disease incidence scenarios under the two vaccination strategies indicated high gains even under the low-incidence scenario (BCR 18.36 and 19.65; NPV 931.97 and 935.51; and IRR 412% under vaccination plan I and plan II, respectively). A previous study conducted in Chhattisgarh state, India, reported BCRs of 4.9:1, 12.4:1, and 13.5:1 under low, medium, and high PPR incidence levels, respectively, for a 100% yearly vaccination strategy and BCRs of 13.7:1, 34.7:1, and 37.8:1 for a five-year vaccination cycle (100% vaccination in the first year, followed by 30% coverage for three years and need-based coverage in the fifth year) [16]. A study on the economic feasibility of the PPR control programme using the PPR vaccine at the national level, which used an economic surplus model [24], revealed a significant NPV, IRR, and BCR, with values of USD 6988 million (at 1 USD= INR 70), 119%, and 123:1, respectively. Furthermore, a study using a dynamic herd model estimated a BCR of 12.0 for adopting a 5-year period vaccination cycle in Niger [25]. These results imply that PPR vaccination is economically viable and generates more outflows (benefits) than inflows (costs). However, if the Karnataka state intends to eradicate PPR in consonance with the government of India’s plan by 2025–2026, in addition to vaccination, other bio-security measures also need to be promoted in order to reduce the PPR incidence to zero.The perception of the veterinarians regarding the PPR-CP’s rollout revealed that the majority of the veterinarians concurred with the various activities implemented in Karnataka state, viz., the control programme was planned well, the vaccination programme was accepted by the farmers, proper coordination existed between various functionaries of PPR-CP, and they agreed upon the effectiveness of extension services. Furthermore, regarding the performance of the functional components of the control programme, the majority of the veterinarians perceived that vaccines were available on time in sufficient quantities, they could vaccinate the targeted animals on time, storage and cold-chain facilities were available, and they could manage the outbreaks on time. Though the majority of veterinarians expressed positive opinions on the existing PPR control plan, including its rollout and the performance of the various functional components implemented in the state, a few of the veterinarians disagreed and some remained neutral towards the plan per se, as well as towards the level of coordination between the functionaries, the availability of funding, and the programme’s acceptance by farmers. The veterinarians’ opinions regarding the improvement of the existing control programme included imparting training on vaccine efficacy and effectiveness, increasing the provision of funds for various activities, and increased coordination with various authorities. This implies that there is scope for improvement in the existing programme. Furthermore, since India is planning to eradicate PPR by 2025–2026, all facets of the programme need to be revisited, revised, implemented, and monitored on a regular basis and, more importantly, the perspectives of the important ‘gatekeepers’ in the PPR eradication programme, such as field veterinarians, need to be included in the current programme. Although the availability of effective vaccines, diagnostic tests, veterinary infrastructure, and technical manpower were what prompted the government to initiate PPR control and eradication efforts in small ruminants in India [26], it may be difficult to achieve the eradication target unless shortfalls in the existing programme are addressed. Previous studies also reported the need for strengthening of the veterinary infrastructure, vaccine production, disease diagnoses, and surveillance measures, as well as faster notification in order to achieve PPR disease eradication in Nigeria [27] and in Karnataka [28]. Furthermore, a study on the eradication and control of animal diseases [29] revealed that a combination of measures may be employed to avoid the spread of disease from infected animals to clean animals and their success is dependent on a variety of factors, including the strength and capacity of veterinary services, cross-border efforts for disease vaccination and disease surveillance, political will and support, diagnostic facilities, and financial support. Furthermore, another obstacle that will limit the success of the eradication efforts in India is the unabated movement of animals between and within states for trade and transit, as well as for migration in search of fodder.Vaccination is an important palliative method to prevent infectious diseases [30]. However, voluntary efforts by farmers to vaccinate their animals on a regular basis are limited in developing countries such as India and hence all the stakeholders in the livestock sector, including farmers, need to be involved for the success of the eradication programme that is planned in India. The results of this study provide evidences and necessary directions for the up-scaling of this strategy in similar socio-economic environments with the aim of eradicating PPR from India and the world by 2030 [16].At a broader level, the study provides evidence on the operational and financial feasibility of PPR-CP, as implemented in Karnataka state, India. However, the results of the present study need to be visualised with certain limitations, for instance, PPR was confirmed based on the clinical signs observed by farmers and triangulated with those of field veterinarians in the respective jurisdictions and was not based on laboratory confirmation; we also did not consider concomitant diseases; only major farm-level costs were considered in the financial assessments; and only 62 field veterinarians who responded to the survey were considered in the study.5. ConclusionsThough Karnataka state followed a ‘focussed vaccination’ strategy after 2003–2004 and a ‘mass vaccination’ strategy after 2010–2011 under the PPR-CP plan, the disease continues to persist in the state. If the state and the country plan to eradicate PPR, the revisiting of the existing programme is warranted. Furthermore, strong facilitation measures are needed from the federal government for the effective implementation of the eradication programme in these states and to ensure effective coordination between the states. Despite long years of vaccination against PPR, the benefits outweigh the costs, mainly because of the low cost of the vaccine (USD 2.4/100 doses). However, developing countries such as India cannot afford to extend the current control strategies and continue to observe incidences of PPR. Hence, a strong mass vaccination programme that does not exclude a single susceptible animal from vaccination for a considerable period is necessary. Furthermore, the migration routes, trade routes, state borders, and high-risk regions within the states need to be covered during vaccination drives to eradicate the disease. Furthermore, the syndromic surveillance and attending the outbreaks on time, as well as stamping out policy, if needed with compensation to farmers need to be implemented in the final stage of the eradication process.

animals : an open access journal from mdpi

10.3390/ani11082256

PMC8388513

The dead end (dnd) gene encodes an RNA-binding protein that plays a role for migration of primordial germ cells (PGCs) to the gonadal region during embryogenesis in vertebrates. Here, the starry flounder (Platichthys stellatus) dead end (psdnd) gene was characterized and its expression patterns were analyzed. Full-length psdnd mRNA was 1495 bp long, encoding 395 amino acids. psdnd was only expressed in gonadal tissues, we detected no psdnd expression in somatic cells. Furthermore, psdnd was strongly expressed during early embryogenesis. Our findings suggest that psdnd expression is gonad-specific and could therefore be used as a germ cell marker in starry flounder.

dnd is a germline-specific maternal RNA expressed in various vertebrate classes, which encodes an RNA-binding protein that is essential for PGC migration. The purpose of this study is fundamental research about starry flounder dnd gene for germ cell marker development. In this study, we cloned and analyzed the expression levels of Platichthys stellatus dead end (psdnd) in various tissues and embryonic stages. The psdnd gene was isolated from starry flounder ovaries, cloned into a pGEM-t vector, and sequenced. Full-length of psdnd cDNA was 1495 bp long, encoding 395 amino acids. psdnd expression levels were investigated by real-time polymerase chain reaction (qRT-PCR) in various tissues and embryo developmental stages. psdnd transcripts were detected in the testes and ovaries, but not in somatic tissues. Embryonic psdnd expression levels were higher during early embryo development stages than during late embryogenesis; psdnd expression was highest at the 1 cell stage, then gradually decreased throughout the subsequent developmental stages. The spatial expression pattern was analyzed by whole-mount in situ hybridization (WISH). The psdnd transcripts migration pattern was similar with zebrafish (Danio rerio). Our results suggest that psdnd may function as a germ cell-specific marker.

1. IntroductionPGCs are the only cells that can transmit genetic material to the next generation. PGCs move to the gonadal location during early embryogenesis where they develop into gonads [1]. The proliferation of PGCs become the germline stem cells which can develop to mature gonads. Furthermore, migration of PGCs impacts on the fertility [2]. The cell fate and development of PGCs are determined by germ plasm mRNA derived from the maternal line [2]. Maternal germplasm mRNAs encode evolutionarily conserved proteins, including vasa, nanos C2HC-type zinc finger 3 (nanos3), dead-end (dnd), tudor-domain-containing protein 7 (tdrd7), and deleted in azzospermia-like (dazl) [2,3,4,5,6].dnd is a germ plasm-specific marker in several vertebrate species that encodes an RNA-binding protein essential for PGC migration [7]. Uracil-rich sequences in the microRNA 430 bind to DND in zebrafish (Danio rerio). dnd prevents the degradation of germ plasm mRNAs targeted by mi430, thus maintaining germ cell development [8].Germ cell-specific expression of dnd was first discovered in zebrafish after which similar expression patterns were found in various vertebrate species including mice (Mus musculus), frogs (Xenopus tropicalis), chickens (Gallus gallus), and humans (Homo sapiens) [8,9,10,11]. Thus, dnd is an evolutionarily conserved gene. In zebrafish and medaka (Oryzias latipes), germ cells are essential for female gonadal differentiation and the absence of PGCs induces sterility in males [12,13]. In addition, the removal of PGCs from pond loach (Misgurnus anguillicaudatus) and goldfish (Carassius auratus) induces sterility both males and females [14,15]. Abnormal PGC localization was induced in zebrafish by knocking down dnd expression [12]. In frogs, PGCs were lost when dnd was knocked down using morpholino oligonucleotide [16]. These studies indicate that dnd is essential for germ cell development during embryogenesis.Most farmed fish reach gonadal maturation before they are released to the market, and gonadal maturation is an important factor that determines the marketability of fish [17]. However, infertile fish exhibit improved growth rates, meat quality, and disease resistance; these effects are achieved by minimizing the energy required for gonadal development [18,19,20,21,22]. In addition, sterilization is an effective method to reduce ecological genetic contamination by protecting natural species from escaped farmed fish, thus maintaining genetic diversity [17].The development of sterilization strategies is an important goal for researchers. To achieve this, genetic modification techniques such as chromosome manipulation, transgene expression, morpholino oligonucleotide treatment, and CRISPR/Cas9 are increasingly undergoing development and application in the aquaculture industry. dnd knockdowns can induce sterility in organisms by blocking PGC migration to the gonad developmental region. Genetic engineering techniques have been applied to knock-down dnd in channel catfish (Ictalurus punctatus) [23]. Furthermore, salmon were successfully sterilized by targeting dnd with CRISPR/Cas9 [24]. Infertility was induced in sterlet (Acipenser ruthenus) by knocking down dnd with an antisense morpholino oligonucleotide [25].To support novel technologies for producing infertile eggs in starry flounder, we conducted fundamental research including identification of psdnd and expression pattern during embryogenesis. Improving our understanding of the mechanisms driving gonad development, such as dnd expression, would enable the development of new sex maturation control technologies.2. Materials and Methods2.1. Animals and Sampling3 females and 3 males which fully matured starry flounders were used in this experiment from Marine seed fish farm (Yeosu, Korea). These fishes were raised in a 15 ton tank controlling the water temperature. The mean total length and weight of females was 33.8 ± 2.8 cm and 825.7 ± 186.5 g. The mean total length and weight of males was were 32.6 ± 0.9 cm and 504.6 ± 87.4 g. Starry flounders were kept on 16:8 light:dark cycle. Exogenous salmon gonadotropin-releasing hormone analog (sGnRHa) (Ovaplant, Syndel, Ferndale, WA, USA) pellets were inserted into the dorsal muscles at a concentration of 50 µg/kg. Various tissues including brain, gill, heart, kidney, eye, stomach, gut, spleen, liver, muscle, testis, and ovary were collected from fully matured female and male starry flounders. Fertilized eggs were cultured at 10 ± 1 °C after artificial insemination. Embryo samples including unfertilized egg, 1 cell, 2 cell, 4 cell, 8 cell, 16 cell, morula, blastula, early gastrula, late gastrula, somite, and hatching larva were collected for analysis. Tissues and embryos were stored in liquid nitrogen prior to RNA extraction.2.2. Total RNA Extraction and cDNA SynthesisTotal RNA was extracted followed by the manufacturer’s instructions. Total RNA was extracted from 100 mg of each tissue (brain, gill, heart, kidney, eye, stomach, gut, spleen, liver, muscle, testis, and ovary) using TRIzol® (Invitrogen, Waltham, MA, USA). In addition, total RNA was extracted from 100 mg of embryo tissue at 12 distinct developmental stages using TRIzol® reagent. 200 μL of chloroform were added and reacted at −20 °C for 5 min. Centrifugation at 12,000 rpm, 4 °C for 15 min. The supernatant was mixed with the same amount of isopropanol and reacted for 10 min. Centrifuge at 12,000 rpm, 4 °C for 10 min. The supernatant was removed and 1 mL of 70% ethanol was added for washing the pellet. The ethanol was removed completely by centrifuging at 12,000 rpm, 4 °C for 5 min. 100 μL of diethylpyrocarbonate (DEPC) water was added to the pellet and resuspended. DNase I (Qiagen, Hilden, Germany) was treated to prevent DNA contamination. cDNA was synthesized from purified total RNA using a Maxima First Strand cDNA Synthesis Kit (Thermo, Waltham, MA, USA). Two microliters of Maxima Enzyme Mix, 4 μL of 5 × reaction mix, and 14 μL of RNA were mixed for real-time PCR. The cDNA synthesis conditions were 25 °C for 10 min, 65 °C for 30 min, and 85 °C for 5 min. The synthesized cDNA was quantified using a NANODROP ONEC (Thermo, Waltham, MA, USA) and stored in a deep freezer (−80 °C).2.3. PCR Amplification of cDNA FragmentPrimers were designed based on the dnd mRNA sequence of olive flounder to detect and amplify psdnd. The olive flounder dnd sequence (Accession No. KP224455.1) was obtained from the National Center for Biotechnology database (Table 1).The PCR cycling conditions were as follows: pre-denaturation at 94 °C for 5 min; 30 cycles of 94 °C for 30 s, annealing at 55 °C for 30 s, and extension at 72 °C for 2 min; then a final extension at 72 °C for 10 min. The PCR products were analyzed by electrophoresis with a 1% agarose gel. The DNA was gel extracted and sequenced by Macrogen Inc. (Seoul, Korea).2.4. 5′ and 3′ Rapid Amplification of cDNA Ends (RACE) PCR5′ and 3′ gene-specific primers (GSPs) were designed based on the partial fragment of starry flounder dnd to obtain the full transcript (Table 1). 5′ RACE PCR was conducted using a 5′ RACE system for Rapid Amplification of cDNA Ends kit version 2.0 (Invitrogen, Waltham, MA, USA), in accordance with the manufacturer’s instructions.3’ cDNA was synthesized using a SMART cDNA synthesis kit (Clontech, MountainView, CA, USA). Total RNA was isolated from starry flounder ovarian tissues using TRIzol® reagent. Single-step PCR and semi-nested PCR were performed using the synthesized 3’ cDNA. One microliter of 3’ GSP1 primer, 1 µL of universal primer mix, and 2 μL of 3’ cDNA were added to the reaction mixture containing 0.25 μL of Advantage Taq polymerase (Takara, Nojihigashi, Japan), 1.5 μL of 10 × buffer, 1 μL of dNTPs, and sterile water to a total volume of 10 μL. The single-step PCR cycling conditions were as follows: pre-denaturation at 94 °C for 2 min 30 s; 35 cycles of 94 °C for 30 s, annealing at 58 °C for 30 s, and amplification at 72 °C for 2 min; then a final extension at 72 °C for 5 min. The semi-nested PCR was conducted with 1 μL of nested universal primer, 1 μL of 3’ GSP2, 1 μL of the single-step PCR product, 17 μL of 1.1 × master mix, and sterile water to a total volume of 20 μL. The semi-nested PCR was performed using thermocycling conditions identical to the conditions used for single-step PCR. PCR products were an-alyzed by means of electrophoresis and sequencing, performed by Macrogen Inc. (Seoul, Korea).2.5. CloningPrimers were designed based on the 1525 bp psdnd cDNA sequence obtained via 5’ and 3’ RACE PCR. One microliter of ovary cDNA was amplified with AccuPower® PCR premix (Bioneer, Deajeon, Korea) and 10 pM of each primer (sense and antisense). The PCR cycling parameters were as follows: initial denaturing at 94 °C for 5 min; 35 cycles of denaturing at 94 °C for 30 s, annealing at 53 °C for 30 s, and elongation at 72 °C for 2 min; then a final elongation at 72 °C for 10 min. PCR products were analyzed on a 1% agarose gel and DNA within a size range of 1400–1600 bp was extracted using a Gel extraction kit (Bioneer, Korea). cDNA (30 ng/μL) was ligated into the pGEM-T vector system I (Promega, Madison, WI, USA), in accordance with the manufacturer’s instructions. Ten microliters of ligation mixture were used to transform 50 μL of Escherichia coli DH5α Competent Cells (Takara, Nojihigashi, Japan), and the cells were incubated at 37 °C for recovery. The transformation culture was then incubated for 16 h at 37 °C on MacConkey agar supplemented with ampicillin for colony selection. Plasmid DNA was extracted using an Accuprep® plasmid Mini extraction kit (Bioneer, Korea). The extracted plasmid DNA was sequenced by Macrogen Inc. (Seoul, Korea).2.6. Phylogenetic Analysis of psdndMolecular phylogenetic analysis was performed to compare the dnd sequences of different vertebrate species. The full dnd gene sequences of various species were retrieved from the NCBI database. The GenBank accession numbers of the sequences examined were as follows: atlantic salmon (Salmo salar), JN712911.1; channel catfish (Ictalurus punctatus), XM_017484732.1; chicken (Gallus gallus), XM_015293536.2; common carp (Cyprinus carpio), XM_019103334.1; atlantic halibut (Hippoglossus hippoglossus), XM_034598806.1; human (Homo sapiens), NM_194249.3; pond loach (Misgurnus anguillicaudatus), MH283870.1; mouse (Mus musculus), NM_173383.2; olive flounder (Paralichthys olivaceus), KP224455.1; pacific bluefin tuna (Thunnus orientalis), KF128758.1; rainbow trout (Oncorhynchus mykiss), MK887177.1; turbot (Scophthalmus maximus), KC460339.1; western clawed frog (Xenopus tropicalis), NM_001044434.1 and zebrafish (Danio rerio), AY225448.1.A phylogenetic tree was constructed with the Mega X version 10.1 software using the maximum-likelihood method with a bootstrap analysis of 1000 replicates. The percentage identities between the starry flounder dnd sequence and the homologues genes from other species were analyzed with CLUSTALW.2.7. qRT-PCRqRT-PCR was performed using a Pikoreal 96 RealTime PCR System (Thermo, Waltham, MA, USA) to examine the psdnd expression profiles in various tissues and stages of embryo development. The concentrations of cDNA from eight distinct tissues and 12 developmental stages were adjusted to 50 ng/μL and 500 ng/μL respectively. The primers used for qRT-PCR are listed in Table 1. dnd transcripts were detected using SYBR Green PCR Mastermix (Thermo, Waltham, MA, USA), mixed with 1 μL of each primer (10 pM), 1 μL of cDNA, and 7 μL of sterile water. The qRT-PCR cycling conditions were as follows: pre-incubation at 50 °C for 2 min; pre-denaturation at 95 °C for 7 min; and then 40 cycles of denaturation at 95 °C for 15 s, annealing at 55 °C for 30 s, and extension at 72 °C for 20 s. Expression levels of the housekeeping gene glyceraldehyde3-phosphate dehydrogenase were used to calculate the relative expression levels of psdnd. psdnd expression levels in the spleen and fertilized eggs were used to calculate Ct values. Relative expression levels were determined by calculating 2−ΔΔCt using mean Ct values. The qRT-PCR primer sequences are shown in Table 1.2.8. Whole Mount In Situ Hybridization (WISH)Digoxigenin-labeled antisense probes were synthesized from linearized plasmids containing the dnd sequence using a DIG RNA Labeling kit (Roche, Basel, Switzerland). The primers used for WISH are shown in Table 1. Embryo samples were fixed in 4% paraformaldehyde with phosphate-buffered saline. The embryo samples were dehydrated using 50% and 30% methanol, and the pigments were removed from the embryo samples using 6% hydrogen peroxide and 0.5% potassium hydroxide. Then, proteinase K solution (10 μg/mL) was applied to permeabilize embryo membranes, and the embryo samples were fixed again in 4% paraformaldehyde. Samples were incubated in hybridization solution at 65 °C for 3 h; 1 μg/mL antisense RNA probe was then added to the hybridization solution and incubated at 85 °C for 3 minutes. A longer hybridization reaction was also conducted at 65 °C for 12 h. The dnd transcripts were visualized using purple alkaline phosphatase substrate.2.9. Statistical AnalysisThe qRT-PCR results were expressed as mean ± SD. Statistical analyses were performed using SPSS Statistic version 26.0 (SPSS Inc., Chicago, IL, USA). One-way analysis of variance and Tukey’s test were used to determine significance (p < 0.05).3. Results3.1. Identification of P. stellatus cDNA SequenceThe full-length psdnd mRNA was 1495 bp long, containing an 1185 bp coding sequence encoding 395 amino acids (Figure 1). psdnd cDNA had five exon regions; the length of exons 1 to 5 were 151 bp, 111 bp, 327 bp, 133 bp, and 536 bp respectively. The length of the 5′-untranslated region was 73 bp. The 3′-untranslated region was 239 bp long, including the complete 3′ end with a poly-(A)-tail. psdnd genomic DNA had 4 introns with lengths of 1240 bp, 1001 bp, 751 bp, and 1509 bp (Figure 2). 3.2. Multiple Sequence AlignmentsA multiple alignments comparing the amino acid sequence deduced from psdnd with the dnd sequences of other species is shown in Figure 3. The range of sequence identities of the dnd amino acid sequences was 31–85% (Table 2). Of the alignment, data suggested that several regions of the dnd protein were conserved between species. The amino acid sequence determined using the psdnd cDNA sequence possessed three conserved motifs including two RNA-recognition motifs (RRMs) and one double-stranded RNA-binding motif (DSRM). The RRMs and DSRM were shared among dnd homologues.3.3. Phylogenetic Tree and IdentityThe deduced psdnd amino acid sequence was aligned with related protein sequences from various vertebrate species (Table 2). According to the neighbor-joining tree shown in Figure 4, psdnd and atlantic halibut dnd formed a clade supported by a bootstrap value of 100%. psdnd exhibited identities of 85%, 77%, 62%, 61%, 50%, 49%, 39%, 39%, 37%, 35%, 33%, 32%, 32%, and 31% with dnd homologues in atlantic halibut, olive flounder, turbot, pacific bluefin tuna, rainbow trout, Atlantic salmon, pond loach, channel catfish, common carp, zebrafish, human, mouse, chicken, and western clawed frog respectively (Figure 4).The highest protein sequence identity is 85% of Atlantic halibut. As followed DND protein identity ranked high 77% of olive flounder and 62% of turbot. The lowest protein sequence identity is 31% of western clawed frog.3.4. qRT-PCRpsdnd transcripts were detected in both male and female gonads by means of qRT-PCR. However, psdnd expression was significantly greater in the ovaries than in the testes. No psdnd expression was detected in the other tissues examined (Figure 5a).psdnd mRNA was expressed during all stages of embryonic development and expression was maintained until hatching (Figure 5b). Strong expression was detected from the fertilized egg stage until the 16 cell stage. After fertilization, psdnd transcript expression increased and peaked at the 1 cell stage. psdnd expression decreased slightly until 16 cell stage, then decreased dramatically from the 16 cell stage to the hatching stage.3.5. Whole-Mount In Situ HybridizationTwo spots of dnd positive cells were detected at the edges of the first furrows during the 2 cell stage (Figure 6a). psdnd was expressed in four spots at the ends of the second furrows from the 4 cell to the morula stage (Figure 6b–d). At the blastula stage, multiple sister cells were generated close to the psdnd positive-cells (Figure 6e). During the early gastrula stage, psdnd expression was detected at the blastoderm margins (Figure 6f). At the late gastrula stage, few psdnd transcripts were dispersed however they were on move at the trunk mesoderm (Figure 6g). The psdnd positive signals then moved toward the dorsal mesentery, where the gonads would later develop; psdnd expression continued in this area until the hatching stage (Figure 6h,i).4. DiscussionIn this study, the psdnd protein sequence contains two RRM motif and one DSRM motif at its C-terminus, which are characteristic of DND protein [1]. The interspecific similarities in the DND amino acid sequences suggest that they have similar structures and functions, and that DND is well conserved. The first RRM motif, RRM1, is the main binding site for DND1 protein that directly binds to nanos1. The second RRM motif, RRM2, plays a role in promoting nanos1 translation and germline survival in xenopus [26]. DSRM is not essential for RNA binding, although it increases the target specificity of the RRM regions in DND1 in human [27]. Zebrafish dnd has a binding site to bind miR-430 for preventing degradation from miRNA [8]. However, the structure and function of which has not been evaluated in starry flounder. Further study is needed to understand the relationship between psdnd and miR-430 in starry flounder.The psdnd protein sequence exhibited strong homologues with DND proteins of other species, especially other flatfish species such as Atlantic halibut (85%), olive flounder (77%), and turbot (62%). The phylogenetic tree and identity results indicated that dnd in starry flounder and Atlantic halibut have the highest evolutionary closeness in our researched target species.The transcript expression of psdnd was analyzed in various tissues by means of qRT-PCR. psdnd expression was only detected in the ovaries and testes, suggesting that psdnd can be used as a germ cell marker in starry flounder. In some species, such as mouse and xenopus, dnd expression is sex-dependent [13,28]. However, in fish species, dnd is expressed in both sexes, as observed in this study [7,13,18,29,30]. The expression in both ovary and testis means psdnd has specific roles for oogenesis and spermatogenesis during sexual maturation in starry flounder.The expression levels of psdnd at various embryo developmental stages were also investigated by qRT-PCR. We found that psdnd was highly expressed during the early developmental stages, from the 1 cell to the early gastrula stage. Higher expression patterns during early embryogenesis also revealed in olive flounder and turbot [7,28]. This suggests that embryonic psdnd expression is of maternal origin, similar with the germplasm marker, vasa. psdnd was also detected at the zygote stage. After the gastrula stage, psdnd transcripts began to decrease more dramatically. Maternal genes are often highly expressed until the zygotic genome is activated. Depending on the genes or species, continuously sex-pressed gene can interact with the zygotic genome [31]. The pattern of psdnd expression pattern may be related to the expression of the microRNA 430. MicroRNAs are small molecules that regulate the expression of target mRNAs by degradation. In zebrafish, miR-430 targets nanos and tdrd7, which are essential for PGC development. DND1 prevents miR-430 from binding to its target mRNAs. After zygotic genome activation at the gastrula stage, miR-430 and DND1 expression levels decrease [8,32,33]. In this study, we found that psdnd was strongly expressed before the blastula stage, suggesting that psdnd was of maternal origin.The successful migration of PGCs to the gonads is essential for PGC development and survival. PGCs require maternally supplied germplasm and mRNAs for their specification and maintenance. Our WISH results revealed that the psdnd expression pattern was similar with the PGC localization pattern, suggesting that psdnd is a germplasm involved in PGC migration. Similar quantitative and spatial expression patterns occur for dnd in other vertebrate species, including olive flounder, turbot, and rare minnow [7,30,31].This finding of this study suggests that psdnd could be used as a germ cell marker in starry flounder; moreover, dnd could be a potential target for starry flounder sterilization.5. ConclusionsIn this study, we identified and cloned full-length psdnd, which was 1495 bp long and encoded 395 amino acids. psdnd was specifically expressed in starry flounder ovaries and testes. Moreover, psdnd was highly expressed during the early embryonic developmental stages, from fertilized egg to the 16 cell stage, after which expression significantly decreased. Our results suggest that psdnd is maternally derived and specifically expressed in germ cells, similar with dnd homologs in organisms.

animals : an open access journal from mdpi

10.3390/ani11113274

PMC8614568

Consumers are increasingly interested in the health and nutritional aspects of meat products, with the result that they are willing to pay more for meat products that have been produced naturally, taking into account high standards and animal welfare. Therefore, we decided to examine in a slightly wider perspective the muscles of Ross 308 chickens kept in an ecological system, taking into consideration an additional factor, i.e., sex. As sexual dimorphism is considered to be a factor in meat quality, we decided to examine this factor in our study. At the same time, we investigated the suitability of these fast-feathering broilers for ecological production. The aim of the study was to investigate the effect of sex on the growth performance, carcass traits, meat quality, fatty acid profile and histological traits of the pectoral muscles in organic Ross 308 broiler chickens. The suitability of these fast-growing broilers for organic production systems was also analyzed. As expected, the study confirmed the influence of sex on the analyzed parameters of the pectoral muscles in chickens reared in the organic system.

Given the growing interest of consumers in naturally produced meat, we decided to examine the muscles of Ross 308 broiler chickens kept in an ecological system, with the division into two research groups depending on sex. All the analyses were carried out using the appropriate methods recommended by the AOAC and in accordance with the Polish standards (PN), which are described in detail in the relevant section of the publication. The aim of the experiment was to investigate the effect of sex on the growth performance, carcass traits and meat quality, as well as the fatty acid profile and histological parameters, of the pectoral muscles from organic broiler chickens. A total of 60 one-day-old Ross 308 chickens (half males and half females) were divided into two groups, according to the sex, and reared under organic conditions (Org.) until 82 days of age (ten birds, i.e., five males and five females in each of three pens; replications for experimental groups). Compared with the female group, the male group had a higher final BW and carcass weight (p < 0.05). The males had a better growth and slaughter performance than the females. The meat quality traits and fatty acids content were also affected by sex. The meat from females showed a significantly higher (p < 0.05) protein, dry matter, fiber diameter and shear force and a significantly lower (p < 0.05) fat level than the male group. In this research, the suitability of these fast-growing broilers for natural and organic production systems has been researched with regard to the performance, meat quality and histological characteristics of the muscles.

1. IntroductionMeat products with high nutritional value and coming from organic systems with animal welfare in mind are becoming more and more popular among consumers. Therefore, in the USA, the EU and other regions of the world, this fact has contributed to a significant development of poultry meat production in less intensive systems [1]. Moreover, consumers in the European Union are showing an increased interest in poultry from alternative management systems, i.e., free-range and organic, which has resulted in a 14% increase in the production of poultry reared in these conditions [2]. However, organic animal production still remains limited compared to the total animal production in Europe and the European Union, ranging from 0.5 to 4% depending on the animal species [3]. The standards of organic poultry production are specified in Commission Regulation (EC) No. 889/2008 [4]. In this regulation, organic poultry production is defined as the production of slow-growing or fast-growing strains reared to a minimum age of 81 days. The housing conditions should provide a high level of animal welfare, and the animals should have access to pastures and receive feed coming from the local farm. The animals should also have free access to outdoor areas [4,5]. It is a production system in which synthetic and chemical compounds are not used as feed components or to maintain the health of the chickens [1], and, importantly, it is environmentally friendly [6]. The existence of all these guidelines, related to the aspects of ecological chicken breeding, means that consumers are able to pay a higher price for products that have been produced naturally and present high nutritional value and quality [7,8].Poultry meat production accounts for about 36% of the global meat production, and chicken meat accounts for about 89% of the total poultry production [5,9]. In 2020, the global production of poultry meat was over 136.5 million tons and increased by 2.4% compared to 2019 [9]. Poultry meat is characterized by its high quality, affordable price and short production cycles, and, most importantly, it is safe for consumers [10]. The quality of poultry products, including the content of nutrients and functional properties, is influenced by intensive selection, genotype, age, sex and type of production system [5,6,7,11,12]. In addition to the physicochemical and nutritional properties of meat, special attention has recently been paid to the histological traits of the pectoral muscles, also due to the increasing incidence of new myopathies in breast muscles, such as white striping, wooden breast and spaghetti meat [13,14,15,16,17,18,19]. Most of the available studies mainly investigated the effect of the genotype and management system on the quality of the chicken meat and fiber diameter without considering the difference between the sex of the birds. Sex, however, is a vital factor determining both the physicochemical parameters as well as the growth and slaughter performance of chickens [7]. In addition, the evident impact of sexual dimorphism on the size of carcasses directly affects other parameters of the slaughter performance of chickens and the quality of meat [6,7,20,21]. Usually, males have a higher final live body weight (BW), daily weight gain and carcass weight, which can affect and be related to other parameters [7,14]. Therefore, we decided to verify the hypothesis that sex has an influence on the growth performance, carcass traits and meat quality in organic Ross 308 broiler chickens and that this rapidly feathering breed will also prove useful in ecological production.The content of fatty acids and their ratios are important criteria related to the health-promoting properties of meat [22]. Poultry meat is a good source of PUFA, especially n-3 PUFA, including eicosapentaenoic acid C20:5 n-3 and docosahexaenoic acid C22:6 n-3, which have a positive effect on the function of the brain and the cardiovascular system [23]. Other important criteria are, for example, the n-6/n-3 ratio of fatty acids and the atherogenic index (AI) and thrombogenic index (TI), and their values indicate a lower or higher risk of coronary heart disease or cancer [22,24]. Lower AI and TI values are positively correlated with a lower risk of serious abnormalities in the coronary arteries. In the human diet, the recommended values should be lower than 1 for AI and 0.5 for TI [25]. The sex of birds, in addition to their diet and genotype, is also an important parameter influencing the ratio and concentration of individual fatty acids [24,26,27].The main aim of the experiment was to investigate the effect of sex on the growth performance, carcass traits and meat quality, as well as the fatty acid profile and histological parameters, of the pectoral muscles from organic broiler chickens.2. Materials and Methods2.1. Animals and Experimental DesignThe experiment was conducted during the spring season, from March to May, in a poultry farm located in the central region of Poland (Kujawsko-Pomorskie Voivodeship). A total of 60 one-d-old Ross 308 chickens (half males and half females) were divided into 2 groups, according to the sex, and reared under organic conditions (Org.) till 82 d of age (10 birds, i.e., 5 males and 5 females in each 3 pens; replications for experimental groups). The area of the poultry house for each group was 3 m2 (3.3 birds/m2) with an outdoor yard of 13.3 m2/bird outdoor run availability. For the Org. group, the outdoor access from the pens was provided after 4 weeks of age during daylight hours (from 8:00 a.m. to 3:00 p.m.), and those chickens were exposed to natural environment (the average temperature was 13–15 °C). Birds were confined to indoor pens at night. The experiment was performed according to the Polish Local Ethical Commission (No. 22/2012) and in accordance with the animal welfare recommendations of European Union directive 86/609/EEC. During the whole time of the experiment, the birds’ health was under constant supervision. The birds were fed from the beginning with organic feed, consisting of 54% grains (wheat, triticale and oats), 30% legumes (pea, yellow lupine) and the remaining part press cake and rape oil, vitamins and fodder chalk and salt. Feed was prepared in an organic farm (certificate No. PL-EKO-07-04187, Łabiszyn, Poland) where the birds were kept. Basic chemical composition of the feed is presented in Table 1. All birds had unlimited access to water.2.2. Slaughter SurveysOn the last day of rearing, all birds (males and females) were individually weighed (after a fasting period of 12 h) and transported (including careful catching and loading) to a commercial poultry slaughterhouse. After careful unloading and hanging in randomized order, all birds were electrically stunned and slaughtered. After evisceration, the hot carcass weight was recorded, and carcass yield was calculated. At slaughter, the pectoral muscle (PM) was removed from all carcasses and its percentage based on hot carcass weight was calculated. Afterward, it was vacuum packaged and stored frozen (−20 °C) until analyses.2.3. Physicochemical PropertiesThe PM pH was measured using a portable pH-meter (pH-star Matthäus GmbH, Pöttmes, Germany) at 15 min (pH15) and 24 h (pHu) post mortem (according to Polish Standard PN-77/A-82058) [28]. Color measurements were performed at 24 h post mortem using the CIE system (L*, lightness; a*, redness; b*, yellowness) according to the method given by Litwińczuk et al. [29] using a spectrophotometer Shimadzu UV-1800 (Shimadzu Corporation, Tokyo, Japan).The tenderness of PM was assessed using a multifunctional machine Instron 3342 (Instron Corporation, Norwood, MA, USA, 2005) with Bluehill Application for tensile tests with Warner–Bratzler shear device (Instron force transducer, Model 2519-104, Series No. 47452, Capacity 500N, S/N 47452), which allowed to register the maximum shear force at crosshead speed of 150 mm/min. Five cores (10 mm2 cross-sectional area and 50 mm length) were cut parallel to the muscle fibers, and each core was sheared 3 times. The average of 15 shears was expressed in N/cm.Water-holding capacity (WHC) was measured on the right PM 24 h after slaughter. The sample was minced and analyzed by a method of Grau and Hamm [30] modified by Pohja and Ninivaara [31]. The measurement was performed using Whatman No. 1 filter paper. The obtained value was expressed as % hygroscopicity.The protein content was calculated using Kjeldahl method, while the content of fat was determined by Soxhlet method [32].The amount of total collagen was determined based on the content of hydroxyproline (conversion factor 7.52) according to PN-ISO3496:2000 [33]. Soluble collagen was determined using the method described by Palka [34]. The collagen solubility was calculated as the percentage of soluble collagen in the total collagen.2.4. Histological EvaluationFrom the carcasses, approximately 1 cm3 muscle sample of each PM was removed and immediately frozen in liquid nitrogen (−196 °C). Each specimen was cut in a cryostat (Cryostat Microm HM 525, GmbH, Germany, Thermo SCIENTIFIC, Series No. 52827, Runcorn, UK) into sections of 10 μm thick, which were then used for histochemical staining based on the hematoxylin and eosin method [35]. The microscopic images of the specimens (at the magnification of 200×) were taken using Opta-Tech microscope equipped with an Opta-View™ camera (Opta-Tech microscope, Warsaw, Poland), Model: MN-800, Series No. 04783. Histomorphometric analysis, including the calculation of the shortest diameters of 300 muscle fibers in each individual according to Brooke [36], was conducted by means of Multiscan 18.03 software for computer analysis of microscopic pictures (Computer Scanning Systems II Ltd., Warsaw, Poland).2.5. Analysis of Fatty Acid ProfileThe samples were subjected to lyophilization in a freeze-dryer (Lyovac GT2, Finn-Aqua, Tuusula, Finnland) and subsequently to homogenization using homogenizator (MPW-309, Warsaw, Poland) in an extraction mixture composed of chloroform and methanol in a 2:1 ratio in accordance with the method described by Folch et al. [37]. Next, fatty acid methyl esters were prepared. For this purpose, methylation of fatty acids with a 0.5 M solution of sodium methoxide was conducted. The samples were kept in an incubator at 37 °C for 22 h. Next, in order to extract fatty acid methyl esters, isooctane was introduced to the samples.The analysis was performed using the 3800 GC type gas chromatograph with FID detector (Varian 3800 GC, Walnut Creek, CA, USA). The separation was conducted on the Supelcowax 10 GC column (dimensions 30 m × 0.25 mm × 0.25 µm) at a transfer temperature line of 230 °C, and, of the detector, 250 °C. The flow rate of the carrier gas (helium) was 1.5 (mL/min), and the volume of the injected sample was 1 µL. The fatty acid methyl esters were identified with Supelco PUFA-2 Animal Source and Supelco 37 Component Fame Mix standards (Supelco, Bellefonte, PA, USA). The composition of fatty acids was expressed as a percentage of the total fatty acids.2.6. Measurement of Oxidative Stability (TBARS)Prior to the analysis of oxidative stability, left breast muscle samples were kept in a freezer (−20 °C) for 3 mo. Lipid oxidation was determined by the thiobarbituric acid reactive substances (TBARS) method as described by Pikul et al. [38] on 10 g of raw meat after allowing it to thaw for 15 min. Briefly, 5.0 g of thawed meat was minced and homogenized using a 4% perchloric acid and alcohol solution of butylated hydroxytoluene (0.01%) at 9500 rev/min. After filtering with Whatman filter paper, the filtrates were diluted and washed with 4% perchloric acid and mixed. Next, a 0.02 M solution of TBA was added and the samples were heated (100 °C for 1 h). The absorbance at the wavelength of 532 nm was measured using a spectrophotometer UV-VIS (Shimadzu Corporation, Tokyo, Japan). The TBARS value was expressed as mg of malondialdehyde (MDA) per kilogram of raw meat using a standard curve prepared from 1,1,3,3-tetraethoxy-propane.2.7. Statistical AnalysesThe statistics meet the assumptions of normal distribution (which was verified using the Shapiro–Wilk test) and requirements for the homogeneity of variance, which are necessary for the use of parametric tests. The significance of differences between experimental groups were evaluated by the Student’s t-test. The values were given in terms of mean values and standard deviation (SD). The obtained data were processed using Statistica 13.1 software. Each bird formed the experimental unit.3. Results and Discussion3.1. Growth and Slaughter PerformancesThe growth and slaughter performances in the broiler chickens (males and females) are presented in Table 2.As expected, the males were heavier (+5.7%) and the differences were significant (p < 0.05) and showed a higher carcass weight (+12.3%; p < 0.05) and higher carcass yield (+5.6%; p < 0.05), while the weight and yield of the PM were similar between the sexes (p > 0.05). Moreover, the daily weight gain (DWG) in males was significantly higher at p > 0.05. A similar trend was observed by van der Sluis [39] in ISA broiler chickens fed with organic feed and Tůmová et al. [20] for Ross, JA and Dual chickens. In traditional rearing systems (intensive vs. semi-intensive), the sex of the birds was a factor determining slaughter performance, as confirmed in studies by Cygan-Szczegielniak et al. [7]. Moreover, in this case, the males were characterized by a statistically significantly higher final live body weight (final BW), DWG and carcass and pectoral muscle weight [7]. A study by Maiorano et al. [40] also confirmed significantly higher values of final BW, carcass weight, carcass yield and pectoral muscle weight in male Ross chickens slaughtered at the age of 42 days. A clear sexual dimorphism in relation to body weight was also noted in Milanino hens managed in the free-range system [41]. The inherent differences in the size of carcasses between the sexes directly influence other parameters of the slaughter performance in chickens [6,7,20,21].3.2. Physicochemical Traits and Fiber DiameterThe physicochemical traits and fiber diameter of the PM are reported in Table 3.Sex did not influence (p > 0.05) the pH measured at 15 min and 24 h post mortem, as well as the color and WHC. A high pH of muscles usually results in a shorter shelf life of the meat, and the post mortem drop in pH is one of the most important processes necessary for the transformation of muscles into meat, which, in turn, directly influences the meat’s tenderness, color and WHC [42]. In our study, the sex of the birds had no effect on the WHC, which corresponded to the results obtained for the parameter directly related to it, i.e., the pH. Similar findings were reported by Połtowicz and Doktor [43], who studied the effect of the free-range raising system using the fast-growing Ross 308 line. The pHu value (5.63) is within the pH range accepted for commercial meats. A relatively low and stable pH is characteristic of meat from organic production systems where chickens have constant access to outdoor areas, which ensures their welfare and reduces pre-slaughter stress, thus reducing the amount of glycogen released in the muscles [5]. Consistent with our study, Goo et al. [21] reported that sex had no effect on meat quality traits such as the pH, WHC or meat color. Color has been considered an important indicator of meat quality driving consumer choices [5,44]. One factor that may affect the meat color is the poultry production system. In our study, the relatively high values of a* for chicken meat could be attributed to the physical activity of the birds and their access to outdoor areas. This trend was also observed by Galvez et al. [5] in the case of organic chickens. A higher level of physical activity promotes an increase in the content of myoglobin, which is directly related to a greater value of redness [5]. The higher yellowness (compared to that usually noted in intensive systems) of the meat (b*) measured in our own study could also be related to the access of chickens to outdoor areas and, thus, to a diet rich in plants containing a high amount of carotenoid pigments [8,45]. Consistent with our study, Fanatico et al. [46] found no differences in the value of b* between male and female chickens.Statistically significant differences (p < 0.05) were found between the males and females reared under the organic system in regard to the dry matter (%) and protein (%) in the PM (Table 3). In both cases, the results were higher in the females, respectively, by 3.4% and 4.14%. In the meat of the females, statistically lower fat content was demonstrated (p < 0.05). Although the sex of the birds played a key role in this case, in general, the pectoral muscles of chickens, regardless of their sex, are characterized by a high content of protein and a low content of fat. The high level of physical activity in the chickens managed in the organic system, compared to the results reported by other authors for intensive production systems, could be a reason why myogenesis is favored instead of lipogenesis [5,8], and this explains the obtained results. All the parameters measured in this study for the physicochemical traits of the breast muscles did not differ from the values measured in the muscles from chickens managed in the same production system and reported by other authors [5,6]. Our study revealed that the sex of the birds was a factor determining the value of the analyzed traits, and, therefore, this aspect should be taken into consideration.The appearance and tenderness are two extremely important traits in poultry meat quality [47]. In particular, meat tenderness is the single most important sensory property affecting the final quality assessment [47], and it is an important attribute for consumers. Tenderness is affected by several factors, such as breed, sex, age, fiber resistance, sarcomere length, pH and collagen morphology [14,48,49,50]. In the present study, the meat from male chickens had a lower (p < 0.05) shear force value than that of female birds, indicating that the meat from the male birds is more tender. This could be due to the fiber diameter that was significantly smaller (p < 0.05) in the male chickens (Figure 1).The smaller thickness of the fibers beneficially affects the meat quality and might be considered an indicator of fibrillarity and a delicate structure of the meat [40]. Berri et al. [51] also confirmed the significant effect of sex on most of the analyzed characteristics, including the fiber diameter. The greater diameter of the muscle fibers in the female birds was associated with a higher plasma creatine kinase activity, i.e., a factor influencing muscle growth, which differentiated both sexes [51]. The content of total collagen and soluble collagen and other parameters were similar (p > 0.05) between the sexes. The collagen content was lower in the female birds, but the differences were not statistically significant. A similar trend was noted by Tavaniello et al. [52] in quails, whose collagen content was significantly lower in females, and this could be related to differences in the hormone metabolism between male and female birds [52]. Collagen content is an important parameter influencing the hardness and quality of meat [45,52]. In our study, the low collagen content certainly had an effect on the measured high tenderness of the meat from the chickens managed in the organic system. As has been demonstrated for the aforementioned system, sex is one of the factors that may affect the meat traits in question. A great deal of data that would confirm this tendency for the organic system were observed. The research revealed that the crucial factors affecting most of the parameters under investigation were undoubtedly the sex and feeding system, which has also been proved by other authors [7,53].3.3. Fatty Acid Profile and Oxidative Stability (TBARS)Table 4 presents the fatty acids composition in broiler meat depending on the sex of the birds.Considering saturated fatty acids (SFA), the highest concentration was found for C16:0, which was about 39% of the total SFA for both groups. With respect to monounsaturated acids (MUFA), the highest concentration was found for C18:1 n-9, and it ranged from 23.96 to 24.41% of the total MUFA in the females and males, respectively (Table 4). There were no statistically significant differences between the meat samples from male and female chickens in the content of this acid. The total concentration of PUFA was similar in the meat from female and male birds and accounted for approximately 19% of the total content of fatty acids. Considering polyunsaturated acids (PUFA), the highest concentration was measured for C18:2 n-6, and it was significantly higher (14.98%) in the meat from males, similar to the studies by Onk et al. [27] and Cerolini et al. for Milanino chickens [26]. An analysis of the meat from the broilers revealed the highest levels of n-3 fatty acids in the samples obtained from females. The levels of eicosatrienoic acid (C20:3 n-3), eicosapentaenoic acid (C20:5 n-3) and docosahexaenoic acid (C22:6 n-3) were significantly higher in females and accounted for 4.51, 0.336 and 0.683% of the total fatty acids, respectively, which is in line with the studies by Cerolini et al. [26]. Most of the identified PUFAs represented those from the n-3 group, which is characteristic of meat from organic production systems. These beneficial proportions can have a positive effect on the juiciness and tenderness of the meat [26,27]. The n-3/n-6 ratio is important due to the effect of these polyunsaturated fatty acids on health. Moreover, the values of AI and TI are crucial for the health of consumers. There were no statistically significant differences in the atherogenic and thrombogenic indices between the male and female birds. The AI values for the meat from both groups were within the recommended levels, but the TI was 3-fold higher than the recommended value. The analysis of the meat from the broilers revealed that the n-3/n-6 ratio of fatty acids was significantly higher in the samples obtained from females than in males (0.395 vs. 0.257%, respectively). On the other hand, the n-6/n-3 ratio was significantly higher in the males, and the values ranged between 3.23 for females and 4.76% for males, which was also consistent with the results of Cerolini et al. [26]. Because of the risk of coronary heart disease and cancer, the recommended values of the n-6/n-3 ratio should be lower than 4.0, and the ratio of PUFA to SFA higher than 0.4, which is very difficult to achieve in poultry and other meat products [22]. Considering the relationships identified in our study, i.e., a higher content of n-3 polyenoic acids, higher n-3/n-6 ratio and the n-6/n-3 ratio in females lower than 4.0, meat from female birds is more beneficial for consumer health.The TBARS value was measured to verify the oxidative stability of the muscle tissue, and the results are presented in Table 4. The TBARS represents the level of lipid oxidation and is a value describing the content of malondialdehyde, ketones and similar oxidation products. There were no significant differences in the TBARS levels between the male and female birds. The oxidative stability was similar in both experimental groups and ranged from 0.672 to 0.681 mg MDA/kg of muscle. The slightly higher TBARS values obtained in the present study can be explained by the higher physical activity of the animals reared in systems with access to the outdoors, which translates into an increased content of total and heme Fe catalyzing peroxidation, and this, in turn, promotes the oxidative metabolism in muscles and, thus, increased lipid peroxidation [45]. Moreover, Funaro et al. [45] obtained a higher TBARS concentration in the chicken meat from the free-range system than from the conventional one. Research by other authors confirmed that the TBARS level is mostly influenced by the rearing system, physical activity and cooking of the meat [24,45].Consumer preferences regarding poultry meat from the broilers managed in organic systems may concern not only factors related to the improved growth performance and meat quality but also those related strictly to animal welfare.4. ConclusionsIn conclusion, the results of the research confirm the hypothesis that sex is a factor influencing some of the characteristics related to the growth, carcass traits and quality of the meat of Ross 308 broiler chickens kept in an ecological system. They also prove the suitability of this breed for ecological production. Compared to the female birds, the male birds exhibited: (i) a lower fiber muscle diameter and shear force; (ii) a lower dry matter and protein; (iii) a meat with higher fat content. Sex had an effect on the performance and meat quality of the fast-growing chickens reared under the organic system: the males had a heavier carcass weight and yield, final BW and a higher DWG. Sex also had an effect on the fatty acid profile: the males had a lower content of n-3 polyenoic acids, lower n-3/n-6 and higher n-6/n-3 ratio. Moreover, the suitability of these fast-growing broilers for natural and organic production systems has been researched with regard to the performance, meat quality and the histological characteristics of the muscles. However, this part of the research should be supplemented with further analyses.

animals : an open access journal from mdpi

10.3390/ani11113047

PMC8614318

Staphylococcus aureus-associated human clinical infections are predominantly caused by the encapsulated strains, with non-typeable strains representing less than 25%. In contrast, 80% of the S. aureus from bovine mastitis cases are non-typeable as they do not possess the Capsular Types 1, 2, 5, and 8. In our previous studies, it was demonstrated that the extent of mammary tissue damage was associated with the strength of biofilms formed by encapsulated S. aureus strains. This study assesses the impact of biofilm formation, as a virulence factor of non-typeable Staphylococcus aureus, causing mammary tissue damage in a mouse mastitis model. The study demonstrates no association between the strength of biofilm production by non-typeable S. aureus and the mammary tissue damage. However, the mice infected with strong biofilm producing non-typeable S. aureus died 6h earlier than those infected with weak biofilm producing non-typeable S. aureus suggesting the role of biofilm in the advancement of the time of mice mortality.

Non-typeable (NT) Staphylococcus aureus strains are associated with chronic bovine mastitis. This study investigates the impact of biofilm formation by clinical NT S. aureus on cytokine production and mammary tissue damage by using a mouse mastitis model. Mice infected with two different NT S. aureus strains with strong and weak biofilm forming potential demonstrated identical clinical symptoms (moderate), minimal inflammatory infiltrates, and tissue damage (level 1 histopathological changes) in the mammary glands. However, the S. aureus load in the mammary glands of mice and the level of pro-inflammatory cytokines (IL-1β, IL-6, IL-12, IL-17 and IFN-γ) in serum were significantly higher (p ≤ 0.05) in those infected with the strong biofilm forming NT S. aureus strain. The level of IL-6 in sera samples of these mice was extremely high (15,479.9 ± 532 Pg/mL). Furthermore, these mice died in 24h of post infection compared to 30 h in the weak biofilm forming NT S. aureus infected group. The study demonstrates no association between the strength of PIA (polysaccharide intercellular adhesion)-dependent biofilm production by clinical NT S. aureus and mammary gland pathology in a mouse mastitis model. However, the role of biofilm in the virulence of S. aureus advancing the time of mortality in mice warrants further investigation.

1. IntroductionPresence of capsular polysaccharide (CP) and the biofilm forming ability of S. aureus are the major virulence determinants of the pathogen [1,2]. Capsular polysaccharide helps the organism to evade phagocytosis allowing the pathogen to persist in tissues and blood stream of the infected host [3]. The bacteria can invade and persist in both nonprofessional and professional phagocytic cells making the infection persistent through one lactation to the next [4,5,6,7]. The predominant capsular types in S. aureus are CP5 and CP8 and the prevalence of non-typeable (NT) S. aureus in human clinical isolates are less than 25% [3]. In contrast, S. aureus isolates from bovine mastitis cases are mostly non-typeable up to 86% [8], only 14–40% of the strains producing CP5 or CP8 [4,5,6,7]. While investigating the prevalence of S. aureus CP types associated with bovine mastitis cases in Australia and India, around 30% and 40% of the isolates were detected to be non-typeable, respectively [9]. NT S. aureus strains are associated with chronicity of infection as they can invade the mammary epithelial cells in higher numbers than the encapsulated ones leading to persistence of intramammary infection for longer duration [8].Biofilm forming ability of S. aureus is an important virulent factor associated with bovine mastitis [10,11]. After entering the mammary gland, S. aureus adheres into the mammary epithelial lining and start forming biofilm [12]. Biofilm helps the pathogen to resist phagocytosis and antimicrobial agents by aggregation of colonies and formation of exopolysaccharide matrix leading to persistent infections of the mammary gland [13] and antibiotic treatment failure [14,15,16]. Bacteria in biofilm are 10–1000 times more resistant to antimicrobial agents than its planktonic form [17]. In addition, there is evidence of stimulation of biofilm formation by certain antibiotics such as tetracycline and erythromycin if used in sub inhibitory concentrations [18,19,20,21,22,23]. In addition, sub-inhibitory concentration of antibiotics is one of the major reasons of mastitis treatment failure leading to relapses and reinfections of mastitis in dairy cows [24]. The development of biofilm occurs in three steps including initial adherence, subsequent maturation and final detachment or dispersal and these steps are physiologically different from one another. The wide variety of extracellular virulence antigens of S. aureus have been reported to be associated with biofilm formation including PNAG (poly-N-acetylglucosamine), surface associated MSCRAMMS (microbial surface components recognizing adhesive matrix molecules) such as FnBPA, FnBPB, clfA, clfB, cna, Bap, ProteinA, SasG, phenol soluble modulins and BBP, extracellular DNA and toxins (hla and hlb) [18,19,20,21,22,23]. However, the widely described method of development of biofilm is the PIA (polysaccharide intercellular adhesion)/PNAG production which is synthesized by icaADBC operon of S. aureus [25,26]. S. aureus in biofilm is entirely a different entity compared to its planktonic form regarding phenotypic characteristics [27], antibiotic resistance pattern [12] and development of innate immune response [28]. While studying the immunogenicity of S. aureus in biofilm versus planktonic cultures in an experimental mouse mastitis model, we demonstrated that S. aureus in biofilm induced stronger and differential immune responses from its planktonic counterpart [29]. In a pilot study using a non-invasive mouse mastitis model, we noted severe mammary tissue damage with significantly higher levels of TNF-α in mice infected with strong biofilm producing CP8 positive S. aureus compared to weak biofilm former CP8 positive S. aureus [30].Considering the predominance of NT S. aureus strains in bovine mastitis etiology, the ability of S. aureus to produce biofilm in the mammary gland and to induce distinct immune responses than its planktonic form, it is important to understand the extent of mammary tissue damage associated with intramammary infection with strong versus weak biofilm forming NT S. aureus. Few earlier studies focused on the virulence potential of S. aureus as slime versus non-slime producers [31], coagulase positive versus negative strains [32], and small colony variants of S. aureus [33]. However, there is paucity of knowledge about the impact of strength of in vitro biofilm formation by clinical NT S. aureus on the mammary gland pathology. To the best of our knowledge, no studies have been conducted to investigate the association between strength of biofilm formation by NT S. aureus and the mammary tissue damage due to production of inflammatory cytokines, the central mediators of inflammatory response during mastitis [34]. This study was undertaken to investigate the impact of biofilm formation by clinical NT S. aureus on cytokine production and mammary tissue damage. In our previous studies, it was discovered that damage caused to the mammary gland by infection with encapsulated S. aureus was associated with the strength of biofilm formation with an increase in TNF-α level [30]. The current study hypothesised that mice infected with NT S. aureus with strong biofilm forming potential will develop severe mammary tissue damage compared to the NT S. aureus with weak biofilm forming potential. The aim of this experiment, therefore, was to investigate association between PIA-dependent biofilm production by clinical NT S. aureus and mammary gland pathology by using a mouse mastitis model. The use of large animal models including cow, goat, and sheep, to study bovine mastitis has its associated problems such as cost and management, even using minimal number of animals. Besides, only limited number of hypotheses can be studied in large animals [35]. Mouse is still considered a suitable animal model for bovine mastitis research due to cost effectiveness, minimum management and similarity between mouse and cow’s mammary glands in respect to neutrophil infiltration and tissue damage [35]. Both the species have two pairs of anatomically and functionally independent mammary glands in inguinal region [36]. In addition to these two pairs, mouse has three additional pairs of mammary glands in the thoracic region which can be used to study additional parameters in mastitis research [36].2. Materials and Methods2.1. S. aureus PhenotypesTwo NT S. aureus strains isolated from mastitis cases of cows, with strong or weak biofilm forming potential, were used in this investigation (Table 1). Both the strains were selected from a collection of 154 strains from cows in dairy farms in Victoria and Queensland, Australia suffering from clinical and subclinical mastitis. The phenotypic characteristics that were used to select these 2 strains were capsule formation, biofilm forming potential, presence or absence of biofilm-associated genes including ica, spa, bbp, hla, and hlb. The reference S. aureus strains used in this study were strain M (CP1), Smith diffuse strain (CP2), USA 400 MW2 (CP8), Strain Newman and USA 100 NRS 648 (CP5), CP-negative isolates (USA 300 LAC and USA 300 NRS 648) and a strong biofilm-forming strain ATCC 29,213 as a positive control.2.2. Capsular Typing of S. aureus Capsular typing of the S. aureus strains used in this investigation was carried out using molecular as well as serological methods as described elsewhere [9]. Briefly, Extraction of DNA from the 2 strains of S. aureus was accomplished using the extraction kit (MO BIO laboratories, Inc, Carlsbad, CA, USA). The PCR cycling parameters for cap1, cap5 and cap8 have been described previously [16]. For serotyping CP 1, 2, 5, and 8 specific antisera were produced in Quackenbush Swiss line 5 mice after gaining approval from the Animal Ethics Committee of Curtin University (Approval number: AEC_2011_65). The preparation of the vaccines and production of CP-specific sera were carried out according to the methods previously described [9]. A slide agglutination test was performed to determine the serotype of the 4 strains of S. aureus. Each strain was grown on Mueller Hinton (MH) agar plates at 37 °C overnight and a single colony was picked and suspended in a drop of 0.9% normal saline on a clean glass slide. A drop of serum was added to the suspension and checked for formation of agglutination within 20 s. The strains, which did not show agglutination against CP1, 2, 5 and 8—specific antisera were considered as non-typeable (Table 1). 2.3. Determination of Biofilm Forming Potential of S. aureus Biofilm forming potential of the two NT S. aureus strains were determined by Congo red agar (CRA) method and Tissue Culture plate (TCP) method as described previously [16] (Table 1).2.4. Detection of PIA-Dependent Biofilm Production Related Genes of S. aureus The ica typing of the S. aureus isolates were accomplished by using conventional PCR described elsewhere [37] (Table 1).2.5. Detection of Virulence Genes of S. aureus Conventional PCR was carried out to detect biofilm related MSCRAMM and toxin genes of the two NT S. aureus strains. The primers, Tm, for all the MSCRAMM-encoding (cna, clfA, clfB, spa, fnbpA, fnbpB, bbp, isdA, isdB, sdrD, sdrE and bap) and toxin genes (hla, hlb, eta, etb, pvl and tsst-1) of the S. aureus strains used in this study have been described elsewhere [38] (Table 1).2.6. Infection of Mammary Gland Using NT S. aureus Strains2.6.1. Animal Ethics ApprovalAll animal work described in this investigation was approved by the Animal Ethics Committee of Curtin University (Approval number: AEC_2012_14) prior to commencement of the experiment. The mice were used for the study ensuring compliance with the Western Australian Animal Welfare Act 2002.2.6.2. Preparation of Bacterial InoculaThe two NT S. aureus strains, S. aureus 83 and 87 were harvested on MH agar plates at 37 °C for 18 h. The colonies were washed from the plates using 20 mL of isotonic saline and suspended in isotonic saline to give a final viable bacterial count of 4 × 1011 mL−1 [39].2.6.3. MiceA total of 12 Balb/c first-pregnancy mice, in three groups (such as strain 83, strain 87, control groups) comprising 4 mice in each group were used for the experiment. The 5–15 days old pups were removed from the lactating mice approximately 1 h prior to the experiment and euthanized.2.6.4. Method of Infection of the Mammary GlandInfection of mammary glands using S. aureus 83 and 87 was carried out using a slightly modified procedure (Protocol S1) [40] described elsewhere [30]. Briefly, mice were anaesthetised using 100 mg kg−1 ketamine and 10 mg kg−1 xylazine administered by the intraperitoneal route and surrounding area of the fifth pair of mammary glands (L5 and R5) was disinfected with 70% ethanol. The duct orifice of the teat was located using a binocular dissecting microscope and 0.05 mL of bacterial suspension equivalent to 2 × 1010 CFU (Colony Forming Unit) S. aureus was injected using a blunt smooth 31-gauge hypodermic needle to a depth of not more than 4 mm. The mammary glands were harvested for 48 h and the mice were observed at six-hour intervals to assess development of macroscopic clinical signs of infection. The control group of mice was injected with normal saline following the same procedure.2.6.5. Post Inoculation ExaminationMacroscopic ExaminationThe mice were monitored at an interval of 6 h for the clinical symptoms or any mortality. The 48 h post-infection was chosen for euthanasia as per the experimental mastitis model standerdised by Anderson and Chandler to study histological and bacteriological changes caused by S. aureus [39]. However, in our study none of the mice survived until 48 h. The level of clinical signs was graded as 0 (no macroscopic changes), + (low) grade, ++ (medium grade) and +++ (severe grade) based on the observed clinical features including redness, swelling, and discolouration of mammary gland, exudate, morbidity, and mortality (Table S1).Bacteriological ProcedureMammary GlandAfter 48 h of infection, L5 mammary glands from both control and test mice were collected aseptically and processed for bacteriological load study [39]. The mammary glands were ground individually in sterile Griffith’s tubes containing 2 mL of sterile normal saline. The homogenates from the mammary glands were subjected to serial tenfold dilutions and inoculated on Baird Parker (BP) agar plates (Pathwest, Laboratory Medicine, WA) by the spread plate method and incubated at 37 °C for 48 h, followed by determination of colony counts of S. aureus per mammary gland. Blood, Liver, Lung, and SpleenBlood samples obtained by cardiac puncture and organs including liver, lung and spleen homogenates were inoculated on BP agar plates and incubated at 37 °C for 48 h. Histological and Cytological ProcedureMammary GlandAfter 48 h of infection, R5 mammary glands were collected aseptically for histological examination [39]. Prior to embedding in paraffin wax, glands were fixed using 10% neutral buffered formalin for 24 h and processed on an automatic tissue processor. Sections were cut at 4 µm thickness at three levels and stained by the Haematoxylin and Eosin stain [41]. An additional section was stained for bacteria using the Gram Twort Method [42].BloodBlood smears were prepared following standard procedure and stained by the Diff Quik method [43].Grading of Histological Changes Observed in Mammary GlandsThe histopathological changes observed in mammary glands of mice, infected with S. aureus 83 and 87 were graded as follows:Level 0: No reaction.Level 1: Organisms identified with minimal inflammatory response in mammary tissue.Level 2: Moderate inflammation in peri-mammary and intramammary tissue with intra luminal organisms observed.Level 3: Marked inflammatory cell infiltration into mammary tissue in the presence of organisms with evidence of tissue degeneration including necrosis.2.6.6. Quantification of Inflammatory Cytokines BD cytometric Bead Array (CBA) Mouse/Rat soluble protein Master Buffer Kit (BD Biosciences), USA was used to quantify inflammatory cytokines, IL-1β, IL-6, IL-10, IL-12, IL-17A, IFN-γ and TNF-α in serum samples of mice. Standard protocol provided with the kit was used to prepare Mouse/Rat soluble protein flex set standards, capture beads and detection reagents. Briefly, 50 µL of Mouse/Rat soluble protein flex set standard dilutions ranging from 1:2 to 1:256 and one negative control containing only assay diluent was prepared. To 10 µL of each unknown serum sample, 10 µL of each capture bead and mixed PE (phycoerythrin) detection reagent was added. After adding capture beads and PE detection reagent tubes were incubated at 4 °C for 1 h each after in dark. Immediately after incubation, 200 µL of wash buffer was added to each tube and centrifuged at 200× g for 5 min. The supernatant was aspirated, discarded and the remaining pellet was reconstituted using 200 µL of wash buffer. This reconstituted pellet was used for acquiring on an Attune Acoustic Focusing Flow Cytometer (Thermofisher Scientific, Waltham, MA, USA). Samples were analysed using the FlowJo software.2.6.7. Statistical AnalysisStatistical analysis was carried out using Student’s t-test to compare total viable counts of S. aureus recovered from mammary glands injected with non-typeable strong biofilm forming S. aureus and weak biofilm forming S. aureus. The Student’s t-test was also performed to compare the IL-1β, IL-6, IL10, IL-12, IL-17, TNF-α and IFN-γ levels between groups of mice injected with S. aureus phenotypes. Statistical significance was set at p < 0.05. 3. Results3.1. Detection of Capsular Types of S. aureusBoth S. aureus 83 and 87 were found to be non-capsulated as these strains carried none of the three loci (cap5, cap8 or cap1) and were not agglutinated by any of the CP-specific sera (CP 1, CP 2, CP 5 and CP 8) subjected to slide agglutination test. 3.2. Determination of Biofilm Forming Potential of S. aureus Isolates 3.2.1. CRA and TCP MethodIn both CRA and TCP methods, S. aureus strain 83 (OD value 0.775) was detected to be strong biofilm former in vitro whereas S. aureus strain 87 (OD value 0.367) was a weak biofilm former in vitro.3.2.2. ica Typing of S. aureus IsolatesS. aureus strain 83 confirmed PIA dependant biofilm formations by harbouring the icaA and icaD genes whereas these genes were not detectable in S. aureus strain 87. 3.3. Detection of Different MSCRAMM-Encoding Genes of S. aureus Using Conventional PCRBoth S. aureus strains harboured genes encoding alpha and beta toxin. S. aureus 83 carried clfA, clfB, spa, bbp, isdA, isdB, sdrD and sdrE MSCRAMM genes. S. aureus 87 was found to carry clfA, clfB, isdA, isdB, sdrD and sdrE genes (Table 1).3.4. Macroscopic Examination of Mammary Glands for Clinical Symptoms The control group of mice injected with normal saline did not show any clinical symptoms and the mammary glands appeared normal. Both the test groups of mice infected with S. aureus 83 and 87 strains showed medium grades of clinical symptoms. However, mice injected with S. aureus 83 died 24 h of post inoculation and the group injected with S. aureus 87 died 30 h post inoculation (Table 2).3.5. Bacterial Load and Histopathological Changes of Mammary GlandThe log average number of bacteria (CFU) isolated from the mammary glands of all the 3 groups of mice including control group and the associated histopathological changes in the mammary glands are presented in Table 3. The bacterial load of mammary glands of mice infected with S. aureus 83 was significantly higher (p < 0.05) with 8.23 ± 0.001 CFU compared to those infected with S. aureus 87 which was 7.91 ± 0.003 CFU.3.5.1. Bacteriology of Blood and Histopathology of Liver, Lung, and SpleenThe culture of blood and organs (liver, lung and spleen) in BP agar plates was negative for S. aureus indicating no evidence of systemic infection. There was no evidence of inflammation in tissue sections of lung, liver and spleen from any of the mice.3.5.2. Histopathology of Mammary Glands Post-infection with Biofilm Forming S. aureusNo evidence of inflammatory response was recorded in mammary tissue of control mice which were inoculated with sterile normal saline. The mammary tissue of all the mice infected with S. aureus 83 and 87 demonstrated identical Level 1 inflammation.3.6. Quantification of Inflammatory Cytokines in SerumQuantification study of inflammatory cytokines, IL-1β, IL-6, TNF-α and other cytokines including IL-10, IL-12, IL-17A and IFN-γ showed that the levels of IL-1β, IL-6, IL-12, IL-17 and IFN-γ were significantly higher (p < 0.05) in the sera of mice inoculated with non-typeable strong biofilm forming S. aureus 83 than those inoculated with weak biofilm forming non typeable S. aureus 87 (Table 4).4. DiscussionGlobally, S. aureus remains one of the predominant causes of clinical and subclinical mastitis in dairy ruminants. Non-typeable strains of S. aureus can survive in the mammary gland for longer duration than the encapsulated strains [2,8]. Higher degree of inflammation appeared to have been induced by encapsulated S. aureus than the non-typeable S. aureus strains, which leads to quick clearance of these cells by the host immune system. The non-typeable cells are quickly internalized by the mammary epithelial cells due to absence of capsule and thus they are protected from the action of phagocytic cells [8] and allows bacteria to persist leading to chronic infection [44]. Biofilm producing S. aureus can attach more effectively to the epithelial lining of mammary glands to develop intra-mammary infection [31]. In fact, S. aureus isolated from mammary glands are more likely to form biofilm than S. aureus isolated from external sources including milking machines [45]. Bacteria growing in biofilm demonstrate increased resistance to antimicrobial therapy [14] due to delayed penetration of antimicrobial agents crossing the barrier of slimy biofilm matrix, modification in the growth rate of pathogens residing in biofilm and certain physiological and genotypic changes in pathogen residing in biofilm [46]. Furthermore, the ability of the macrophages to invade into biofilm is limited and the pathogen in biofilm is able to polarise the macrophages from proinflammatory microbicidal M1 phenotype to an M2 phenotype which exert anti-inflammatory properties and restrict phagocytosis [28,47]. To make the situation worse, in a biofilm environment, S. aureus initiate a favourable interaction with the Myeloid derived suppressor cells (MDSCs) which exert immunosuppressive properties [48] and this phenomenon is partly aggravated by cytokines such as IL10 [49] and IL12 [50]. As the NT S. aureus can remain longer in the mammary epithelial cells well protected from host phagocytosis and additionally the biofilm formation is one of the important survival strategies of the pathogen [51] in intramammary infection, it is important to understand the magnitude of mammary tissue damage post infection by NT S. aureus with different strength of biofilm forming potential for future therapeutic interventions. In the current study, both the NT strains of S. aureus with different strength of biofilm formation produced identical clinical symptoms and mammary tissue damage. Both the strains produced moderate level of clinical symptoms of mastitis and level 1 histopathological lesion in the mammary gland. However, the bacterial load in the mammary glands of mice injected with the non-typeable S. aureus 83 strain with strong biofilm forming ability was significantly higher (p ≤ 0.05) than the weak biofilm forming S. aureus 87. The period of observation in the study was 48 h. Both the test groups of mice died before 48 h. The mice injected with S. aureus 83 (strong biofilm former) survived only 24 h followed by the mice injected with S. aureus 87 (weak biofilm former). Mice in the latter died at 30 h post inoculation. The analysis of sera samples collected from mice immediately before death showed higher levels of IL-1β, IL-6, IL-10, IL-17A, IFN-γ and TNF-α (Table 4). In one study, it was suggested that quick internalization of non-typeable S. aureus cells by the mammary epithelial cells may have prevented the clearance of S. aureus from the mammary gland providing scope for production of high levels of cytokines of different types [52]. The highly elevated level of various cytokines can lead to cytokine storm, a fatal immune response which may result in sudden death [53,54]. It has been demonstrated that patients who died due to cytokine storm had higher levels of anti-inflammatory cytokine, IL-10 and pro inflammatory cytokines, IL-1β, IL-6 and TNF-α in serum samples [55]. In the present study, between both the groups of test mice, the mice injected with S. aureus 83 produced significantly higher levels of IL-1β, IL-6, IL-12, IL-17 and IFN-γ (p < 0.05) than the mice injected with S. aureus 87. The level of IL-6 in sera samples of mice injected with S. aureus 83 was extremely high (15479.9 ± 532 Pg/mL) which could have been responsible for the death of the mice within mere 24 h post inoculation. In addition, IL-12 is a cytokine with both pro and anti-inflammatory effects, can assist in the recruitment of MSDCs. IL-10, an anti-inflammatory cytokine mainly produced by the MSDCs during S. aureus biofilm infection is capable of promoting the growth of biofilm [49]. In the current study, IL-10, IL-12 and MSDCs may be the contributing factors for the development of anti-inflammatory environment in the mammary gland resulting only Grade 1 mammary tissue damage. However, this warrants further investigation.The role of biofilm in complication of bovine mastitis has been established previously [30,46,56]. In this study, the only phenotypic difference between both the strains was the biofilm forming ability. Though not conclusively, it can be suggested that biofilm forming ability of non-typeable S. aureus may play role in the virulence of S. aureus as increased higher levels of PIA/PNAG-associated biofilm could be helpful in dispersion of biofilm facilitated by bacterial cell to cell interaction in the mammary gland [57] leading to higher colonisation of the pathogen in mammary tissue [31] and possibly systemic dissemination of infection [56]. This might have resulted in higher bacterial load and significantly higher levels of cytokines in mice infected with strong biofilm producing S. aureus which might have contributed to the mortality of the mice. In the mammary glands, inflammation has been associated with neutrophil chemo-attractants and the cytokines (IL-1β, IL-6, TNF-α and IL8) [58,59]. Local induction of cytokines including IL-1β and IL- 6 post infection of the mouse mammary gland with S. aureus was reported by Breyne and co-workers [32]. IL-6 in quarter milk has been proven as a prediction marker of bovine mastitis [60]. In the current study, the extraordinarily high amount of IL-6 in sera samples of mice infected with biofilm forming NT S. aureus suggests an important role of IL-6 in virulence of strong biofilm forming S. aureus in mouse mastitis model. It will be interesting to investigate the neutralisation effect of IL-6 by anti-IL-6R antibody in preventing mortality in mice in the future investigations. Due to non-availability of commercial mutant NT S. aureus strains of different biofilm forming potential, we have emphasized on using clinical isolates and included selected biofilm related phenotypic characteristics to be compared in the selected two S. aureus strains used in this study. This is one of the limitations of our study. Further investigation will be important to compare the virulence and pathogenicity of mutant S. aureus strains that are genotypically and phenotypically similar but with different biofilm forming abilities. Additionally, the expression of S. aureus biofilm in vitro may not correlate with the expression that occurs in vivo in the mammary gland. The future research will focus on comparing the in vitro attachment potential of NT S. aureus strains (used in this study) to bovine mammary epithelial cells (MAC-T) to in vivo response in either a mouse mastitis model or in dairy cows. It will be interesting to replicate this study using S. aureus with different biofilm forming potential from different species to understand if there are any species-specific differences to the host response. The potential information generated from this study will contribute new knowledge in mastitis pathology associated with NT S. aureus and may aid in future research to strategize different treatment options with advanced pharmacological interventions to reduce tissue damage aiming to control mastitis.5. ConclusionsFrom this study it was concluded that phenotypic PIA-dependent biofilm production by clinical non-typeable S. aureus is not associated with the intensity of inflammation in mammary gland. Clinical non-typeable S. aureus strains isolated from bovine mastitis cases developed identical moderate clinical symptoms, Grade-1 mammary tissue damage and generated minimal inflammatory infiltrates in the mammary tissue when tested in a mouse mastitis model.

animals : an open access journal from mdpi

10.3390/ani11061700

PMC8228742

Aquaculture effluents with high levels of phosphorus (P) and nitrogen (N) contribute to eutrophication in the aquatic ecosystem. The environmental impact of phosphorus and N aquaculture waste may be diminished by modifying diet ingredients that improve phosphorous (P) digestibility, and therefore, reduce the P in metabolic waste. The content of P in fishmeal is high (30 g/kg), but the inclusion of fishmeal in the diet is reducing due to its high costs and limited accessibility; therefore, the addition of an inorganic P source is necessary to ensure a satisfactory level of available P in fish diets. Consequently, the present study aimed to evaluate the effect of four different inorganic P sources on P digestibility and excretion in rainbow trout (Oncorhynchus mykiss), as one of the most relevant aquaculture species. Monosodium/monocalcium phosphate with 2% of sodium source presented a P digestibility similar to monoammonium phosphate, but with lower nitrogen and phosphorus excretion into the environment, which is advantageous from a nutritional, environmental and industrial point of view (biofilters and recirculation systems in fish farms).

This study was conducted to evaluate the apparent availability and P and N excretion in rainbow trout (Oncorhynchus mykiss) using different inorganic phosphorus sources. With this goal, fish (153 ± 14.1 g) fed four inorganic P sources were assayed: monoammonium phosphate (MAP, NH4H2PO4), monosodium/monocalcium phosphate (SCP-2%, AQphos+, NaH2PO4/Ca(H2PO4)2·H2O in proportion 12/88), monosodium/monocalcium phosphate (SCP-5%, NaH2PO4/Ca(H2PO4)2·H2O in proportion 30/70) and monocalcium phosphate (MCP, Ca(H2PO4)2·H2O). Phosphorus (P) digestibility, in diets that included MAP and SCP-2% as inorganic phosphorus sources, were significantly higher than for SCP-5% and MCP sources. In relation to the P excretion pattern, independent of the diet, a peak at 6 h after feeding was registered, but at different levels depending on inorganic P sources. Fish fed an MAP diet excreted a higher amount of dissolved P in comparison with the rest of the inorganic P sources, although the total P losses were lower in MAP and SCP-2% (33.02% and 28.13, respectively) than in SCP-5% and MCP sources (43.35% and 47.83, respectively). Nitrogen (N) excretion was also studied, and the fish fed an SCP-5% diet provided lower values (15.8%) than MAP (28.0%). When N total wastes were calculated, SCP-2% and SCP-5% showed the lowest values (31.54 and 28.25%, respectively). In conclusion, based on P and N digestibility and excretion, the SCP-2% diet showed the best results from a nutritional and environmental point of view.

1. IntroductionAquaculture is still a sector with a great growth potential, with improvements in fish feed formulations being essential to achieve a greater expansion. On the other hand, it is also necessary to promote a more sustainable production, reducing waste and environmental pollution [1], without affecting the nutritional attributes, quality and cost-effectiveness. Therefore, environmental management of intensive aquaculture is essential for the achievement of sustainable aquaculture in the coming years.With this aim, a decrease in phosphorus (P) and nitrogenous (N) discharge from aquaculture effluents would reduce water eutrophication, and therefore, its environmental impact. The main strategy to achieve this reduction is through the optimization of diet formulation. P is a macromineral component of nucleic acids and cell membranes in living beings, also participating in energy processes [2,3]. Additionally, it plays an essential role in the formation and maintenance of bone structures, as well as the synthesis of other tissues such as muscle mass [4]. Despite the fact that fish can absorb minerals from water through their gills, fish require an additional source of P in their diet [5], since P is usually a limiting mineral in most natural waters, and its absorption rate from water is low.P in fishmeal, mainly in the form of hydroxyapatite or bone phosphate as well as inorganic supplements, is relatively available to rainbow trout (Oncorhynchus mykiss). In contrast, approximately two-thirds of the P in plant sources is bound to phytate, being only partially available to fish [6], presumably due to low levels of intestinal phytase. On the other hand, the scarcity of fishmeal production and its high price is limiting its inclusion in the feed, it being substituted by plant protein sources [7,8,9,10]. Therefore, due to the greater inclusion in the diet of plant protein sources and their low P digestibility, the inclusion of an inorganic P source in feed is required. However, previous studies in common carp (Cyprinus carpio) production [11] reported reduced environmental effects in diets with high plant protein levels when the P dietary level is optimum. Therefore, the selection of the inorganic P source is an important issue and it will be based on its solubility and digestibility [12], which may be affected by the calcium level in the diet, as well as changes in pH under gastrointestinal conditions [13]. In general, monobasic phosphates from monovalent cations are more digestible and soluble, followed closely by monocalcium phosphate (MCP) and, beyond this, by tricalcium phosphates or bone apatite. This is generally applicable to marine and freshwater fish with a stomach, such as rainbow trout [14]. The combined use of phytase with a highly digestible inorganic P source, such as monosodium phosphate (MSP) and monoammonium phosphate (MAP), to meet a species’ P requirement might be an accurate choice to optimize P retention efficiency, and thereby, to reduce P excretion into the environment [15]. Moreover, the dietary supplementation of an organic acid mixture enhanced the growth and nutrient efficiency of dicalcium phosphates [16].A low absorption and retention of nutrients will mean a higher discharge into water, affecting the sustainability of the production. Aquaculture effluents with high levels of P and N contribute to the pollution of the aquatic ecosystem through the eutrophication of natural fresh water. Consequently, aquaculture faces a dilemma: feed must meet P levels, but at the same time, feeding practices must comply with environmental guidelines to minimize the P load in the aquatic environment [17]. Inorganic commercial phosphates, under European Union Regulation [18], can be easily found on the market, being categorized according to the Ca/P ratio (Figure 1). This is due to commercial phosphates coexisting without a defined chemical composition, in the case of mixtures of monocalcium phosphate (MCP), dicalcium phosphate (DCP), phosphoric acid, carbonate and impurities. This undefined chemical composition is explained by the appearance of unwanted collateral reactions, events difficult to avoid at the industrial scale. This compositional variability causes different degrees of solubility in both water and citric acid, as well as variations in pH and the presence of other minerals. The mixture of chemical products ends up directly influencing the degree of bioavailability of the phosphate species in each species, which is defined as the degree to which a nutrient ingested from a particular source is absorbed and remains available for the animal’s metabolism [19].Studies on rainbow trout [20] have stated that both MAP and MSP have higher levels of apparent P digestibility (≈90%) compared to other inorganic phosphates, such as MCP. Likewise, a recent study [21] reported an improvement in nutrient digestibility, mineral bioavailability and immune functions of MAP, monopotassium phosphate (MKP) and MSP when compared with MCP in high plant ingredient-based diets. On the other hand, from an environmental point of view, it should be considered that MAP can release a greater amount of non-protein nitrogen (N) into the water, probably as undigested ammonia through the feces released by fish [22].In conclusion, the aim of the present study was to evaluate in vivo the P availability and excretion level of diets including four different inorganic P sources in rainbow trout. As a novelty, new formulations of inorganic phosphates were assessed from nutrition and environmental point of view for its inclusion in fish diets.2. Materials and Methods2.1. Fish, Facilities and Rearing ConditionsAround 200 rainbow trout with an average weight of 130 g were moved by an authorized service from the fish farm “El Zarzalejo” located in Zamora (Spain) to the Fish Nutrition Laboratory belonging to ICTA-UPV (Valencia, Spain). Before starting the feeding assay, all fish were acclimated to indoor rearing conditions for two weeks and fed a standard diet for rainbow trout (45% crude protein, CP; 22% crude lipid, CL; 6.8% ash; 0.81% Ca and 1% phosphate, Biomar Iberia S.A., Dueñas, Spain).After 15 days of acclimation, 16 trout (153 ± 14.1 g) were redistributed into cylindric-conical tanks of 200 L with sedimentation columns based on the Guelph system [23] (four replicates per treatment). Afterwards, the fish were acclimated to new tanks for two weeks being fed once per day with a control diet (10:00 a.m.). The rest of the rainbow trout were housed in 4000 L tanks with an open water circuit, as a preventive measure.In order to ensure the correct functionality of the facility and animal welfare, physic-chemical water parameters and mortality were checked daily. The oxygen concentration in the water and the temperature were monitored using a portable oximeter (Oxyguard Handy Polaris, Farum, Denmark). pH, ammonium and nitrites concentrations were analyzed with a pH meter and colorimetric test (MERK, Darmstadt, Germany), respectively.The environmental conditions were as follows: salinity: 0 g/L, temperature: 16.10 ± 1.06 °C, oxygen: 8.04 ± 1.41 mg/L, pH: 7.9 ± 0.32, ammonium and nitrites: 0.0 mg/L and nitrates: 15 ± 2.3 mg/L. The photoperiod was natural (May to July). The duration of the experiment was 120 days. All tanks had aeration supply. The water temperature was kept constant thanks to the fact that the freshwater was taken from a freshwater well.2.2. Experimental DietsFour supplemental inorganic P sources were chosen for determining the apparent P availability and non-fecal P excretion: monoammonium phosphate (MAP, NH4H2PO4, Yara S.L., Helsingborg, Finland), monosodium/monocalcium phosphate (SCP-2%, AQphos+, Global Feed, S.L.U., Huelva, Spain), monosodium/monocalcium phosphate (SCP-5%, Global Feed, S.L.U., Huelva, Spain) and monocalcium phosphate (MCP, Ca(H2PO4)2·H2O, Global Feed, S.L.U., Huelva, Spain). Chemically, SCP-2% and SCP-5% were mixtures of monosodium and monocalcium phosphates, being NaH2PO4/Ca(H2PO4)2·H2O at a proportion of 12/88 and 30/70 for SCP-2% and SCP-5%, respectively. All experimental groups were isonitrogenous (45% crude protein) and isolipidic (18% crude lipid), with a P content of 0.8% (Table 1). Additionally, the diets contained denatured egg albumin (Vicens i Batllori S.L., Girona, Spain) as the sole protein source. The basal diet contained negligible amounts of P (0.02%).Before formulating the diets, each ingredient was individually weighed and analyzed in triplicate, then mixed. Subsequently, the diets were prepared using a cooking-extrusion process with a semi-industrial twin screw extruder (CLEXTRAL BC-45, St. Etienne, France) at the UPV facilities. The processing conditions were as follows: a pressure of 4–5 MPa, a temperature of 110 °C and a screw speed of 100 rpm. Finally, the composition of all experimental diets was analyzed, also in triplicate, and are reported in Table 1.2.3. Digestibility TrialTo obtain the feces, a Latin square experimental design of 4 treatments × 4 tanks × 4 trials was followed (consisting of four trials or periods, and in each of them, four tanks were fed with one of the feeds, so that the fish in all the tanks were fed with the two experimental feeds) [25,26,27]. Each collection period or trial lasted 14 days, 7 days of adaptation to the new diet without feces collection and 7 days collecting feces. The feces collection was performed in a settling column (water flow rate: 8.5 L/min, average density: 12 g/L) supplied with filtered freshwater.For each experimental group, this procedure was repeated four times (n = 4). To reduce the tank effect, the tank location was changed in each repetition. Every day, fish were fed by hand until apparent satiation. The feeding rate was once per day at 10.00 a.m. from Monday to Saturday, with a starvation day on Sunday. Pellets were distributed slowly, allowing all fish to eat. Before feeding, the remaining uneaten feed was collated and feces were collected from trays, pooled by tank and frozen at −20 °C. At the end of the experimental period, feces collected from each tank were freeze-dried, prior to analysis. Before changing diets, the fish were starved for 24 h to help the ejection of the previous diet.2.4. N and P Excretion EstimationThe excretion of ammonia and P was estimated as a function of the difference between the concentration of ammonia/P dissolved in a given time. The tanks were isolated for 20 min every two hours, closing the water and aeration of the tanks, where ammonia/P could only be accumulated by fish excretion.Feeding was carried out at a restricted rate, according to the average weight of the fish and at water temperature, and the ration per tank was calculated according to the biomass of each fish. The same experimental design was followed as that described in the digestibility test.Total ammonia N (N-TAN) and P excretions were calculated at each given time using the following equation:Excretion (mg nutrienth)=((Final nutrient (mg)t0−Initial nutrient (mg)t1)time (h)t1)The results of ammonia and P excretions were used to calculate the average hourly and/or daily excretion of ammonia/P and kg of fish. The initial and final amount of nutrient (N-TAN or P) was calculated by multiplying the volume of water in the tank by the concentration of N-TAN or P measured in mg/L.2.5. Chemical Analyses and CalculationsFish diets, feed ingredients and feces were analyzed based on the Association of Official Analytical Chemists (AOAC) [28] procedures: dry matter, official method 934.01 (105 °C to constant weight); crude protein, official method 990.03 (analyzed by the direct combustion method DUMAS using LECO CN628, Geleen, the Netherlands); crude lipid, official method 920.39 (extracted with methyl-ether using ANKOMXT10 Extractor, Macedon, NY, USA); and ash, official method 942.05 (incinerated at 550 °C for 5 h). All analyses were performed in triplicate.An atomic absorption spectrophotometer (Perkin Elmer 3300, Perkin Elmer, Boston, MA, USA) was used for the analysis of Cr in feces and feed after acid digestion of the sample with HNO3 1.5 N + KCl 0.38% [29].The concentration of N-TAN in water was measured by the indophenol method after the addition of phenol, nitroprusside, sodium citrate and alkaline sodium dichloroisocyanurate (NaDTT). After 6 h, the absorbance of the compound was measured at a wavelength of 640 nm using a T60V UV-VIS spectrophotometer (PG Instruments, Leicester, UK).Apparent digestibility coefficients (ADCdiet, %) of N and P (%) were calculated using the following formulae described previously by [30]:ADCdiet(%)=100−(Markerdiet×NutrientfaecesMarkerfaeces×Nutrientdiet)In this equation, the terms Markerdiet (%) and Markerfeces (%) represent the marker content (Cr) of the diet and the feces, respectively, and Nutrientdiet (%) and Nutrientfeces (%) are the nutritional parameters of concern (e.g., N or P) in the diet and the faces, respectively.2.6. Predicted DigestibilityDue to the critical importance of being able to distinguish between the different phosphates with higher bioavailability in each species, a predictive program has been developed considering the different chemical species that compound each phosphate, by means of different chemical parameters. Each of these chemical species has been assigned a digestibility weighting value based on previous in vivo studies [31,32,33,34,35]. The combination of the chemical balance with the weightings leads to Predictive Equations for Digestibility Comparison (EPCD) of commercial phosphates. In vivo digestibility estimation requires specialized settings, expensive operating costs and a high number of fish, and it is difficult to perform and achieve the desired experimental working conditions [36]. In this regard, EPCD can help to successfully evaluate the effect of diets on nutrient digestibility providing very reliable information.From a chemical point of view, the EPCD predictive digestibility technology developed by Global Feed, S.L.U. (Tervalis group, Huelva, Spain) is based on the analysis of a multitude of chemical parameters, which are directly correlated with the presence of different chemical species that make up the phosphates on the market. These analyses are:▪Percentage of P, Ca and Na: determination of the composition of the product.▪Solubility in water: above 80% correlates with MCP, while solubility around 50% is due to the presence of MDCP.▪Solubility in alkaline ammonium citrate: almost completely dissolves DCP but not TCP, which remains insoluble.▪Solubility in 2% citric acid: difference in TCP (> 95%).▪Solubility in neutral ammonium citrate: good correlation with bioavailability.▪Percentage of CaCO3: presence of impurities and type of phosphate.▪Humidity: allows for quantification of free water.▪Loss 200–250 °C: estimate of hydration water.▪Ca/P ratio: estimation of the presence of different phosphates, as well as their composition.Collecting the information described in previous studies [12,13,20,37,38,39,40,41,42], the parameters of the predictive equation have been modified and it has been estimated that, in the current test carried out on rainbow trout, the expected values will be in the proportion shown in Table 2. The predictive digestibility has been calculated by establishing the different chemical species in each inorganic phosphate and assigning a digestibility value based on bibliographic data such as those referred to above.2.7. Statistical AnalysesPrior to analysis, the normal distribution was checked through a Kolmogorov–Smirnov test, while the homogeneity of variances using a Levene test. N-TAN and P values were measured as dissolved N concentration (mg N-TAN or P/L) and were transformed into mass units (mg N-TAN or P) to calculate ammonia excretion. The data on ammonia excretion are presented as produced ammonia N (mg N-TAN or P) divided by fish biomass (kg) and time (h). Two series of data are presented according to the approach of the trials. Firstly, the different values of N-TAN and P excretion that occur according to the time of day are graphed, where the daily variation between treatments can be observed. Secondly, all measured N-TAN and P consumption and daily excretion values were combined for statistical analysis.Univariate ANOVAs were performed to determine the significant differences between treatments, using a Newman–Keuls test for comparison between means for a confidence interval greater than 95%. All statistical analyses were performed using Statgraphics® Centurion v.XVII.II for Windows®.3. Results3.1. Effect of P Inorganic Sources on DigestibilityThe digestibility data from diets and feces are shown in Table 3. ADC values of P were markedly influenced by inorganic P sources, with MAP and SCP-2% registering higher digestibility (92.26 and 90.08%, respectively) than SCP-5% and MCP sources (75.21 and 71.11%, respectively).Fish fed an SCP-2% diet presented the highest values (92.80% and 93.07%) of protein and energy digestibility, while fish fed the SCP-5% and MCP diets registered statistically lower levels.In Figure 2, the relationship between the predicted and observed values is shown. Values predicted for MAP and SCP-2% are very similar to those obtained in vivo, although in the case of inorganic phosphates which obtained lower P digestibility, the predicted values differ slightly.3.2. Effect of Inorganic Phosphates on N and P ExcretionThe N excretion patterns are shown in Figure 3. In spite of dietary treatment, the peak N excretion occurred at 6 h post meal ingestion, decreasing afterwards until basal levels at 18 h after feeding. If the dietary treatment is considered, at 6 h, N excretion was lower in the SCP-5% and SCP-2% diets (8.57 and 9.87 mg N-NH4+/kg fish × h, respectively); however, at 8 h after feeding, SCP-2% maintained higher levels than the rest (9.09 mg N-NH4+/kg fish and h) (Figure 3).As N excretion, regardless of inorganic P sources, the peak P excretion occurred at 6 h post meal ingestion, followed by a decrease down to basal levels at 12 h after feeding (Figure 4). Fish fed MAP as P inorganic sources significantly presented the highest P excretion at 6 h post feeding (2.19 mg P/kg fish and h).3.3. Total N and P LossesConsidering both soluble and solid N and P discharged fractions, N and P wastes for each inorganic P source expressed per percentage of feed intake have been evaluated as shown in Figure 5.Fish fed a diet supplemented with MAP released a higher amount of P in its dissolved form to the water in comparison with the rest of inorganic P sources (Figure 5), although the total losses were lower in MAP and MCP (33.02 and 28.13%, respectively) than in SCP-5% and MCP sources (43.35 and 47.83%, respectively).In the case of N, SCP-2% showed the lowest N discharge by fecal losses (7.2%), but in the case of N excretion, SCP-5% provided lower values (15.8%) than MAP (28.0%). Therefore, SCP-2% and SCP-5% showed the lowest total waste of N (31.54 and 28.25%, respectively) and MAP showed the highest values (39.01%) of total N intake.4. DiscussionThe N and P digestibility as well as the excretion of commercial inorganic phosphates has been evaluated assessing its nutritional and also its environmental value for fish diets. Both treatments, MAP and SCP-2%, showed higher values than SCP-5% and MCP in terms of P digestibility (92 and 90%, respectively). If it is considered that the only P source comes from diet, we can state that the results obtained are also very similar to those shown in the study carried out by Morales with rainbow trout [20], who also reported P digestibility coefficients above 90%. In Japanese amberjack (Seriola quinqueradiata), with a similar feed, formulation greater than 90% of P digestibility was obtained [37].Compared with the digestibility results in other species, the values of the digestibility of different inorganic phosphates were similar to those obtained in the present experiments. The MSP and DCP values were in concordance with those obtained for shrimp [43] but considerably lower than the values of 90–98% and 46–71%, respectively, reported for fish [40]. The 73% value for the MCP is higher than the 49% reported for shrimp [43]. These differences in digestibility are mainly due to the different degrees of solubility between phosphates, where, for example, MCP has a much higher solubility to water (>85%) than DCP (<25%). This difference in solubility is very relevant, since it reflects the ability of P to solubilize at neutral pH, simulating the intestine (absorption of P). This fact is even more important in fish, since they lack a true stomach in which a first stage of solubilization begins prior to digestion.The needs of P in the diet depend on several factors [39], including the bioavailability of the element, food intake, the requirement for new tissue synthesis and the amount of endogenous loss, among others; these factors being dependent on the life cycle and size, and even environmental factors such as salinity and temperature. Commercial feed usually contains from 10 to 15 g P/kg with varying P digestibility content, which depends on ingredient quality and P sources used in aquafeeds. If both the level of dietary P and P ADC are taken into account, the levels of digestible P would be 0.923 and 0.90%, respectively, for MAP and SCP-2% treatments, which would cover the needs of digestible P since around 0.8% is recommended in salmonids [5,44]. Nevertheless, it is remarkable that the SCP-2% diet registered higher ADCs in terms of N and energy. The improved digestibility of SCP-2% requires further investigation, since the sources of protein and lipids have been at the same proportions in both treatments. It is difficult to explain the differences observed in the present experiment regarding N digestibility, because the protein source was the same in all treatments and only the inorganic phosphate source differed between diets. One possible explanation could be the differential buffering capacity (BC) of inorganic phosphates, which could affect the pH, and consequently, modulate digestive enzyme activities, and therefore, raising the protein and mineral content [45]. In previous studies, BC was tested in ingredients used for animal and fish diets. For example, cereals reported lower buffering capacity than plant protein meals [46]. On the other hand, plant protein registered lower capacity than fish meal and gluten [46]. Thus, the ingredients used in diets can modify feed BC, as has been reported in sea bass (Dicentrarchus labrax) [47], Atlantic salmon (Salmo salar) [48] and sea bream (Sparus aurata) [49,50]. In the current experiment, the buffering capacity of SCP-2% was significantly lower (866 meq HCl/kg) than the rest of the inorganic phosphates (1966 meq HCl/kg), which might imply an advantage of including this additive in fish diets, as has been reported [51].A high correlation (>0.9) was obtained between the results of the in vivo P digestibility assay and the estimated results using the EPCD index. This index could become a useful tool to formulate practical diets. Nevertheless, it is noteworthy that the only factor not taken into account in the EPCD index is the variability stemming from the different particle sizes of phosphate; therefore, an inclusion of this factor might be needed for products with a different granulometry or the comparison only of products with similar textural properties.The P excretion into the water system provided similar values independent of the diet, with a maximum at 6 h after feeding, similar to previous studies [16]. Additionally, the daytime pattern of soluble P is in accordance with other studies in trout fed semi-purified diets [52]. On the other hand, the MAP diet provided higher P excretion at almost all sampling points. Some researchers have applied the classical concept of balance study to determine the effect of diet on the fecal excretion of soluble P. Using this concept, the authors indicate that when fish consume excess available P, the excess P is mainly eliminated through the gills and kidneys as non-fecal soluble P [52,53,54]. Excretion of soluble non-fecal P also depends primarily on P sources, i.e., when fish ingest an excess of highly available inorganic P, excretion of soluble non-fecal P should increase compared to digestion from sources of low P availability [52]. Therefore, differences in P excretion observed in the present study might be due to the available P contained in the diets. Here, the excretion of soluble P per fish is generally independent of dietary P up to a certain level of dietary P, but then increases considerably above this level. This suggests that excretion of soluble P occurs when the available P intake is above levels sufficient for retention and/or when the mechanisms of intestinal absorption and renal reabsorption are saturated. In the rainbow trout intestine, there is a sodium-dependent inorganic P carrier that is closely regulated by dietary P [16]. Intestinal absorption rates of inorganic P decrease as dietary P levels increase, suggesting that the effectiveness of phosphate transport systems in retaining dietary P may decrease when dietary P levels are very high [55]. Vitamin D3, which improves intestinal absorption of inorganic P and renal reabsorption in birds and mammals, has no effect on the intestinal absorption of inorganic P in diets fed for trout containing sufficient amounts of P [56]. A collection of data from other studies confirms that the production of soluble P per kg of fish is a linear function of dietary P and presumably independent of P source [52]. Soluble P is the most flexible component of effluent P. There is no excretion of soluble P at low levels of dietary P, but soluble P becomes the main route of excretion as the available P concentration increases above hypothetical levels of P requirements for the species. In fact, in diets containing less than 0.22 ± 0.03% available P, there is no production of soluble P [52] and excretion increases at a rate of 6.6 ± 0.67 mg/day/kg for every 0.1% increase in available dietary P [54], which is roughly in line with the data obtained.Analyzing the overall data on daily excretion rate, the results corroborate that fish fed MAP feed had significantly higher N excretion, probably due to the ammonium content of MAP, as has been reported in previous studies [20]. These excretion data have been calculated by way of the difference of N-TAN concentration in the water; thus, based on the results obtained, it can be stated that there is a higher concentration of N-TAN in the water system in the MAP experimental group. This excess excretion must be considered when sizing biofilters in fish recirculation systems, or in the case of open systems, since it will mean a greater release of ammoniacal N into the natural environment, which will lead to greater eutrophication of the water. In the case of soluble P, there were no significant differences in the MAP treatment with respect to SCP-2% and MCP.Few studies have registered an increase of N residues from inorganic P dietary supplement containing indigestible N, such as MAP, compared to other phosphate sources. Morales [20] reported that higher discharge of ammonium ions directly through the feces, when MAP is used as a dietary supplement, could lead to an overload of undesirable reduced N fraction dissolved in aquaculture recirculation systems or other high intensity systems. Therefore, in addition to optimizing dietary P, the use of diets with an optimal level of digestible N will become another key to sustainable aquaculture.Considering fecal and soluble P losses, even though data on P retention are not available, it was possible to carry out an approximation of P and N losses in each of the treatments. As can be seen, in general, fish fed with MAP show a higher percentage of N losses than those fed with SCP-2% and SCP-5%, under the experimental conditions tested. Sugiura et al. [6] found that 38% of the total P fed was not assimilable and the P excretion represented up to 20% of total P ingested by trout, reporting P losses [57] similar to those obtained only with MCP.Considering N and P as the most relevant nutrients for inducing water eutrophication [57], it would be relevant to minimize these components into the column water. Currently, there are a wide range of commercial inorganic phosphate available; however, the most used in aquafeed are the monoammonium, monocalcium and monosodium phosphates due to their good availability in fish, although the new formulation (SCP-2%) can improve the wastes generated in the aquaculture production, which is crucial to improve the environment.5. ConclusionsThe SCP-2% source (AQphos+) presents a phosphorus digestibility comparable to MAP (without significant difference at a statistical level), but with lower P and N excretion, and thus, it is more environmentally friendly. Therefore, SCP-2% as a phosphorus source is more advantageous from a nutritional, environmental and industrial point of view (biofilters and recirculation systems in fish farms).

animals : an open access journal from mdpi

10.3390/ani13091541

PMC10177385

Invasive tunicates have become a global threat to shellfish aquaculture sites in recent decades, particularly mussel farms. In our study, the effectiveness of five eradication treatments (air exposure, freshwater immersion, sodium hypochlorite, hypersaline solution and acetic acid) was tested for the solitary tunicate Styela plicata. The effects on blue mussel Mytilus edulis survival and growth were also evaluated. The acetic acid treatment was the most effective in eliminating tunicates, although further studies are needed to achieve total survival in mussels. We suggest that the treatments are also likely to produce more effective results as prophylactic measures, applied in controlled environment in mussel seed.

In 2017, aquaculture producers of the Albufeira lagoon, Portugal, reported an invasion of tunicates that was disrupting mussel production, particularly the tunicate Styela plicata (Lesueur, 1823). A totally effective eradication method still does not exist, particularly for S. plicata, and the effects of the eradication treatments on bivalves’ performance are also poorly understood. Our study examined the effectiveness of eradication treatments using three laboratory trials and five treatments (air exposure, freshwater immersion, sodium hypochlorite, hypersaline solution and acetic acid) for S. plicata, as well as their effects on survival and growth of blue mussel Mytilus edulis Linnaeus, 1758. While air exposure and freshwater immersion caused a 27% mortality rate in S. plicata, the acetic acid treatment was the most effective in eliminating this species (>90% mortality). However, a 33–40% mortality rate was registered in mussels. Both species were not affected by the hypersaline treatment in the last trial, but the sodium hypochlorite treatment led to a 57% mortality rate in mussels. Differences in mussels’ growth rates were not detected. These trials represent a step forward in responding to the needs of aquaculture producers. However, further studies are needed to investigate the susceptibility of tunicates to treatments according to sexual maturation, as well as to ensure minimum mussel mortality in the most effective treatments, and to better understand the effects on mussel physiological performance in the long-term.

1. IntroductionThe growth of the human population leading into the middle of the 21st century poses significant challenges to the supply of high-quality, nutrient-rich food, such as edible marine bivalve mollusks, which are mainly supplied by the aquaculture industry [1]. Mussel aquaculture production has increased globally over recent decades as a result of a decline in wild captures since the early 1990s. In particular, cultured sea mussels (Mytilidae) represent 6% of total mollusk production, reaching 1108 thousand tons in 2020 [1]. Blue mussels (genus Mytilus) are cultured globally, particularly in China, the EU and Chile [1,2]. However, despite a 4% increase in value, production has generally declined 26% over the last two decades [1,2,3,4]. Mussel farming is still a slow-growing sector, with low product value and innovation, mainly performed in small-scale semiculture systems, with basic production technology, using longline, raft, bottom or ‘bouchot’ culture techniques [2,5]. In this context, several factors have contributed to the decline in mussel production, including algal blooms, predation, diseases and biofouling [2,6].Biofouling is the settlement and development of sessile species of microorganisms, plants, algae or animals, known as epibionts, on natural and artificial surfaces [7,8]. The equipment used in mussel farms (e.g., ropes, floats and other infrastructures), in combination with the cultivated mussel shells themselves, represent a favorable habitat that provides substrate, refuge and food, ideal for biofouling settlement [8,9]. Biofouling may result in negative impacts on bivalve cultivation: through increased weight on culture equipment, hampering the harvest process, damage, erosion and altered functioning of the shell, seed losses, competition for space, oxygen and food, and reduced survival and growth. Ultimately, all these factors contribute to a lower bivalve marketability, biosecurity and yield [6,7,10,11].Fouling communities of shellfish aquacultures are commonly dominated by tunicate species, which may reach high densities in aquaculture sites [12,13,14]. The solitary hermaphroditic tunicate Styela plicata (Lesueur, 1823), native to the NW Pacific Ocean, is commonly found in shallow and protected habitats including estuarine areas, most frequently attached to artificial structures, in warm and temperate waters [12,15,16]. With a cosmopolitan distribution, it was introduced into several coastal regions worldwide [12,17,18] and its biological and ecological traits made its expansion successful, including resilience to stressful abiotic conditions [19]. As a pioneer fouling species, S. plicata has become a global threat to shellfish aquaculture, over recent decades, particularly mussel farms [12,20,21,22,23,24]. In the Albufeira lagoon, Portugal, mussel farmers have struggled in recent years with an increasing invasion of tunicates, particularly S. plicata. Besides prevention strategies for management of tunicates invasions, the development of effective eradication methods is mandatory for introduced populations, despite the associated financial and technical constraints [25,26]. In both research and commercial contexts, physical and chemical eradication techniques have been tested for adult individuals of the genus Styela: these include hand removal [27], air exposure [28,29,30], freshwater immersion [30,31], heated seawater [32], acetic acid [26,29,31,32], citric acid [32], sodium hypochlorite [31] and hydrated lime [26]. However, while most of these reports specifically address Styela clava (Herdman, 1881), the industry still lacks a totally effective and practical method to effectively address biofouling [7]. Furthermore, current eradication treatments are often detrimental to the bivalves (e.g., induce significant mortality rates); thus, there is a need for further study [7,33]. Our study examined the effectiveness of five eradication treatments on the tunicate S. plicata, as well as their effects on survival and growth of the blue mussel Mytilus edulis Linnaeus, 1758. We also evaluated gametogenic development of the tunicates to assess a possible association between sexual maturation and susceptibility to treatments.2. Materials and Methods2.1. Animal Collection and MaintenanceAt three periods (June, September and November 2021), we manually collected individuals of the tunicate S. plicata and the mussel M. edulis from the ropes of a mussel raft culture located in the Albufeira lagoon (Sesimbra, Portugal). Animals were transported to the MARE—the Marine and Environmental Sciences Centre (Polytechnic of Leiria)—in isothermal boxes. In the laboratory, individuals of both species were carefully selected and cleaned before an acclimation period of three days in recirculating aquaculture systems (RAS). No mortality was registered during this period in the three trials. Tunicates and mussels were kept in 50 L conical tanks, suspended in fishing nets to simulate the aquaculture environment and supplied with constant aeration. Individuals were exposed to a simulated natural photoperiod. During the trials, temperature, pH, salinity and dissolved oxygen (DO) were measured every two days with a YSI Professional Plus multiparameter probe (YSI Inc., Yellow Springs, OH, USA). Ammonia, nitrite and nitrate were monitored every two days with API® Test Kits (Mars Fishcare, Inc., Chalfont, Pennsylvania, United States of America). The natural seawater was previously filtered through a sand filter and treated with UV light, and a total water exchange was performed daily in all trials. Animals were fed daily with a mixture of live (Dunaliella tertiolecta and Chaetoceros calcitrans) and frozen (Tetraselmis chuii) microalgae, at a concentration of ~100,000 cells/day/animal [34].Before the application of each treatment and in the end of the trials, tunicates and mussels were measured with a Vernier Calliper (Insize, code 1205-150S, INSIZE Co., Ltd., Zamudio, Spain; ±0.05 mm accuracy) and weighed using an electronic precision balance (Kern PCB 2500-2, Kern & Sohn GmbH, Balingen, Germany; ±0.01 g accuracy). Survival of both species was monitored daily and was assessed as described by Sievers et al. [32] and Cahill et al. [33], for tunicates (siphons closure and response to touch) and mussels (valves closure), respectively. Dead individuals were immediately removed.2.2. Experimental Treatments2.2.1. Trial 1A total of 90 S. plicata individuals (4.10 ± 0.91 cm length; 2.11 ± 0.46 cm width; 10.27 ± 5.59 g total weight) and 135 mussels (6.79 ± 0.67 cm length; 3.47 ± 0.34 cm width; 29.61 ± 8.92 g total weight) were selected for trial 1. Two experimental treatments for tunicate eradication were tested (air exposure and freshwater immersion) (Table 1). This trial was run in triplicate and included a control group. After the initial exposures, animals were reared for 30 days. For the air exposure treatment, tunicates and mussels were submitted to 6 h of air exposure at the beginning of the trial (T0). The same process was repeated once after 15 days of rearing. For the freshwater immersion, animals were submerged in freshwater for 30 min (T0) and 15 days later, the treatment was repeated for 1 h. Animals were uniformly distributed in 9 tanks, with 10 tunicates and 15 mussels per tank, in a total of 30 and 45 individuals per treatment, respectively. During the trial, the parameters were the following: 20.1 ± 0.6 °C, 8.1 ± 0.3 (pH), 32.6 ± 0.4 (salinity) and 91 ± 1% (DO). The room temperature was 20 ± 1 °C. The biometric parameters of both species are represented in Table 2. Mussels’ specific growth rate (SGR) was calculated as follows:SGR (% day−1) = [(ln (Mfinal) − ln (Minitial))/t] × 100(1) in which Mfinal and Minitial represent the final and initial average mass (g) of the individuals, respectively, in each replicate tank and t represents the number of days.2.2.2. Trial 2A total of 180 tunicates (5.62 ± 1.09 cm length; 3.23 ± 0.55 cm width; 29.22 ± 12.05 g total weight) and 180 mussels (5.42 ± 0.59 cm length; 2.91 ± 0.29 cm width; 15.48 ± 4.59 g total weight) were selected for trial 2. Three experimental treatments for tunicate eradication were tested (acetic acid, sodium hypochlorite, hypersaline solution) (Table 1). This trial was run in triplicate and included a control group. For the acetic acid (AcOH) treatment, organisms were submerged in a 4% AcOH solution for 1 min using glacial acetic acid (ACS grade) (Carlo Erba, Val de Reuil, France) (adapted from Sievers et al. [32]). In the sodium hypochlorite (NaClO) treatment, animals were submerged in a 0.5% NaClO solution, prepared with commercial grade bleach (7.5%) for 1.5 min (adapted from McCann et al. [35] and Denny [36]). For the hypersaline group, individuals were submerged in a hypersaline solution (60-salinity) for 20 sec (adapted from Carman et al. [37]), using commercial sea salt (Aquaforest, Poland) to adjust the salinity of the natural seawater. Animals were then uniformly distributed in 12 tanks, with 15 individuals per tank, for a total of 45 individuals per treatment for each species, and maintained for 15 days. The parameters were the following during the trial: 19.6 ± 0.8 °C, 8.0 ± 0.5 (pH), 32.4 ± 0.5 (salinity) and 91 ± 2% (DO). The biometric parameters of both species are represented in Table 3. 2.2.3. Trial 3A total of 270 tunicates (4.72 ± 0.75 cm length; 2.66 ± 0.37 cm width; 16.85 ± 5.59 g total weight) and 270 mussels (5.01 ± 0.85 cm length; 2.70 ± 0.36 cm width; 12.73 ± 5.89 g total weight) were selected for trial 3. Based on the results of trial 2, two additional experimental treatments were tested using acetic acid and the hypersaline solution (Table 1). This trial was run in triplicate and included a control group. Two distinct size classes of mussels were selected (lower class: 4.23 ± 0.35 cm length; higher class: 5.79 ± 0.33 cm length) for a total of 6 treatment groups. Animals were uniformly distributed in 18 tanks, with 15 individuals per tank, for a total of 45 individuals per treatment for each species, and maintained for 15 days. The parameters were the following during the trial: 19.1 ± 0.5 °C, 8.2 ± 0.4 (pH), 32.1 ± 0.8 (salinity) and 92 ± 1% (DO). The biometric parameters of both species are represented in Table 4. Tunicates were immediately fixed in a 4% buffered formaldehyde solution for 48 h and stored in 70% ethanol for histological analysis.The tunicates’ gonadosomatic index (GI) was calculated as follows:GI (%) = (gonads weight/total weight) × 100(2)2.3. Tunicates’ Gametogenic DevelopmentThe gonads of tunicates were processed in a Leica® TP1020 Automatic Tissue Processor (Leica Microsystems GmbH, Wetzlar, Germany) with sequential submersions in graded ethanol for dehydration followed by xylene for clarification and impregnation with paraffin wax at 60 °C. After the gonad samples were embedded in 100% (v/v) paraffin, they were cut with a thickness of 7 µm using an Accu-Cut® SRM™ 200 Rotary Microtome (Sakura Finetek Europe BV, Alphen aan den Rijn, The Netherlands) and stained with Harris’ haematoxylin solution (Scharlab S.L., Sentmenat, Barcelona, Spain) and eosin Y (yellowish) (VWR International, Leuven, Belgium). Gonad tissues were then analyzed using a Leica® DM 2000 LED light optical microscope equipped with a Leica® MC170 5MP HD Microscope Camera and the combined LAS v4.4.0 software (Leica Application Suite) for monitor display (Leica Microsystems GmbH, Wetzlar, Germany). Determined by size and histological characteristics, the oocytes were classified according to their developmental stage into three classes (Sciscioli et al. [38] and Pineda et al. [39]). This allowed us to identify the stage of maturation of each individual as follows:Stage I (pre-vitellogenic)—oocytes smaller than 50 μm, strongly basophilic, with a large nucleus occupying most of the cytoplasm.Stage II (vitellogenic)—oocytes between 50 and 150 μm; the first follicular cells become visible, and the primary follicle begins the process that will give rise to two layers of cells, an outer layer composed of flattened cells and an inner layer accompanying some test cells.Stage III (mature)—oocytes larger than 150 μm; the cytoplasm continues to accumulate nutrient material and increase in volume, accompanying the test cells and the two layers of follicular cells (inner and outer).For male follicles, a categorical maturity index was established, according to the same authors:Stage I (immature)—male follicles filled only by spermatogonia.Stage II (mature)—male follicles filled with several mature sperm in the lumen.Stage III (spawning)—male follicles with empty spaces in the lumen.2.4. Statistical AnalysisResults were expressed as mean ± standard deviation (SD) and a significance level of α = 0.05 was used for statistical tests. These were performed using IBM SPSSTM Statistics for Windows, version 28 (IBM Corporation, Armonk, NY, USA). The Pearson’s chi-square test was applied to assess a possible association between treatments and organisms’ mortality. Mortality was expressed as mean ± SD of the three replicate tanks by treatment group. To assess differences between treatments, for each biometric parameter (length, width, total weight, mussel SGR), results were analyzed through one-way ANOVA. A Kruskal–Wallis test was used when the data did not meet the assumptions of ANOVA. Data were tested for normal distribution with the Shapiro–Wilk normality test and for homogeneity of variances with the Levene test.3. Results3.1. Trial 1Both experimental treatments led to a total tunicate mortality of 26.7 ± 11.6%, and 10.0 ± 10.0% in the control (Figure 1). No significant association between treatments and mortality was detected [χ2 (2) = 3.34; p = 0.19]. A mortality rate of 8.9 ± 3.8% was registered in the mussels control group (Figure 1), showing a significant difference between treatments [χ2 (2) = 8.24; p = 0.02]. No significant differences were detected for biometric parameters (Table 2) between T0 and T1, for both species, including mussel SGR (Table A1 in Appendix A).animals-13-01541-t002_Table 2Table 2Biometric parameters of the tunicate Styela plicata and the mussel Mytilus edulis at the beginning (T0) and end (T1) of a 30-day rearing period in which both species were submitted to experimental treatments (air exposure and freshwater immersion). Experiments were run in triplicate with a control group. ControlAir ExposureFreshwater ImmersionT0 Tunicate length (cm)4.15 ± 1.014.11 ± 0.854.06 ± 0.89Tunicate width (cm)2.06 ± 0.422.13 ± 0.532.15 ± 0.44Tunicate total weight (g)10.12 ± 5.6110.44 ± 6.1610.24 ± 5.13Mussel length (cm)6.81 ± 0.706.79 ± 0.526.76 ± 0.78Mussel width (cm)3.46 ± 0.363.48 ± 0.363.48 ± 0.31Mussel total weight (g)29.87 ± 9.9529.40 ± 8.0129.56 ± 8.90T1 Tunicate length (cm)3.14 ± 0.693.21 ± 0.893.13 ± 0.40Tunicate width (cm)1.91 ± 0.362.02 ± 0.442.09 ± 0.38Tunicate total weight (g)5.90 ± 2.786.35 ± 3.996.98 ± 2.89Mussel length (cm)6.88 ± 0.706.82 ± 0.516.90 ± 0.62Mussel width (cm)3.53 ± 0.403.49 ± 0.343.51 ± 0.36Mussel total weight (g)30.24 ± 10.4129.69 ± 8.0729.84 ± 8.83Mussel SGR (% day−1)0.04 ± 0.070.03 ± 0.000.03 ± 0.013.2. Trial 2The acetic acid treatment caused the highest tunicate mortality (91.1 ± 7.7%), associated with a 33.3 ± 6.7% mussel mortality (Figure 2). The hypersaline solution promoted a mortality rate of 73.3 ± 6.7% in tunicates, while all mussels survived in this treatment. In the sodium hypochlorite immersion, 66.4 ± 3.9% of the tunicates died, but the highest mussel mortality was also registered (55.6 ± 20.4%). In the control group, a mortality of 57.8 ± 10.2% and 2.2 ± 3.9% was registered in the tunicates and mussels, respectively. A significant association between treatments and mortality, both in tunicates [χ2 (3) = 13.87; p = 0.00] and mussels [χ2 (3) = 54.42; p < 0.001], was registered. No significant differences in biometric parameters (Table 3) were found between T0 and T1 for both species, including mussel SGR (Table A2).animals-13-01541-t003_Table 3Table 3Biometric parameters of the tunicate Styela plicata and the mussel Mytilus edulis at the beginning (T0) and end (T1) of a 15-day rearing period in which both species were previously submitted to experimental treatments (acetic acid, sodium hypochlorite, hypersaline solution). Experiments were run in triplicate with a control group. ControlAcetic AcidSodium HypochloriteHypersaline SolutionT0 Tunicate length (cm)5.72 ± 1.135.55 ± 1.125.56 ± 1.095.60 ± 1.06Tunicate width (cm)3.24 ± 0.503.19 ± 0.663.24 ± 0.533.26 ± 0.51Tunicate total weight (g)29.91 ± 11.8228.56 ± 12.2329.05 ± 11.6529.34 ± 12.84Mussel length (cm)5.44 ± 0.595.44 ± 0.525.34 ± 0.655.45 ± 0.61Mussel width (cm)2.93 ± 0.332.93 ± 0.272.87 ± 0.262.93 ± 0.32Mussel total weight (g)15.69 ± 4.7915.45 ± 4.6015.35 ± 3.4415.43 ± 5.45T1 Tunicate length (cm)5.48 ± 1.304.80 ± 1.265.07 ± 1.285.19 ± 1.12Tunicate width (cm)3.49 ± 0.523.03 ± 0.703.28 ± 0.753.29 ± 0.62Tunicate total weight (g)26.65 ± 11.2921.39 ± 12.1724.32 ± 10.9425.91 ± 12.50Mussel length (cm)5.48 ± 0.595.53 ± 0.575.51 ± 0.475.45 ± 0.58Mussel width (cm)2.88 ± 0.322.98 ± 0.292.94 ± 0.232.92 ± 0.31Mussel total weight (g)16.11 ± 5.0016.43 ± 5.2816.31 ± 3.6415.91 ± 5.79Mussel SGR (% day−1)0.18 ± 0.120.42 ± 0.180.37 ± 0.160.21 ± 0.163.3. Trial 3As shown in Figure 3, the mortality rate of tunicates in the lower size class of the mussel trial was the following: 4.4 ± 7.7% (control); 97.8 ± 3.8% (acetic acid); 2.2 ± 3.8% (hypersaline). Within the mussel higher size class groups, the mortality of tunicates was the following: 0.0 ± 0.0% (control); 97.8 ± 3.8% (acetic acid); 2.2 ± 3.8% (hypersaline). A significant association was detected between treatments and the mortality of tunicates in both cases [χ2 (2) = 117.96; p < 0.001] [χ2 (2) = 126.20; p < 0.001], respectively.The mortality of mussels in the lower size class was as follows: 0.0 ± 0.0% (control); 40.0 ± 6.7% (acetic acid); 2.2 ± 3.8% (hypersaline) (Figure 3). A similar result was also obtained in the larger mussel groups: 0.0 ± 0.0% (control); 35.6 ± 10.2% (acetic acid); 2.2 ± 3.8% (hypersaline). A significant association was detected between treatments and mortality in both cases [χ2 (2) = 37.61; p < 0.001] [χ2 (2) = 32.44; p < 0.001], respectively. However, mortality was similar between size classes, either in the acetic acid [χ2 (1) = 0.19; p = 0.66], or in the hypersaline treatment [χ2 (1) = 0.00; p = 1.00]. No significant differences in biometric parameters (Table 4) were detected in T0 or T1 for both species (Table A3), including mussel SGR (Figure 4). The maturity index of S. plicata is detailed in Table 5.animals-13-01541-t004_Table 4Table 4Biometric parameters of tunicate Styela plicata and two size classes of mussel Mytilus edulis at the beginning (T0) and end (T1) of a 15-day rearing period in which both species were previously submitted to experimental treatments (acetic acid, hypersaline solution). Experiments were run in triplicate with a control group. Mussel Lower Size Class GroupMussel Higher Size Class Group ControlAcetic AcidHypersaline SolutionControlAcetic AcidHypersaline Solution T0 Tunicate length (cm)4.70 ± 0.704.81 ± 0.764.66 ± 0.824.63 ± 0.724.85 ± 0.804.66 ± 0.72Tunicate width (cm)2.69 ± 0.432.66 ± 0.392.71 ± 0.402.65 ± 0.352.59 ± 0.282.63 ± 0.37Tunicate total weight (g)16.83 ± 6.0416.82 ± 4.9916.93 ± 5.7716.87 ± 5.7916.72 ± 5.4116.92 ± 5.82Mussel length (cm)4.22 ± 0.314.23 ± 0.334.24 ± 0.405.77 ± 0.355.80 ± 0.335.81 ± 0.31Mussel width (cm)2.40 ± 0.162.37 ± 0.212.40 ± 0.183.02 ± 0.193.04 ± 0.202.99 ± 0.15Mussel total weight (g)7.18 ± 1.467.23 ± 1.537.22 ± 1.6118.19 ± 2.8518.20 ± 2.0218.35 ± 2.47Tunicate GI (%)4.23 ± 1.00Tunicate oocyte diameter (µm)141.31 ± 50.85 T1 Tunicate length (cm)3.50 ± 0.653.203.55 ± 0.903.61 ± 0.574.003.92 ± 0.71Tunicate width (cm)2.63 ± 0.452.502.57 ± 0.552.69 ± 0.362.802.46 ± 0.44Tunicate total weight (g)12.43 ± 4.489.3112.97 ± 5.8813.14 ± 4.7215.4814.20 ± 5.73Mussel length (cm)4.32 ± 0.364.34 ± 0.284.37 ± 0.305.83 ± 0.345.80 ± 0.305.88 ± 0.33Mussel width (cm)2.46 ± 0.202.45 ± 0.192.47 ± 0.173.06 ± 0.233.10 ± 0.243.01 ± 0.16Mussel total weight (g)8.03 ± 1.467.80 ± 1.437.96 ± 1.6218.35 ± 2.7218.30 ± 2.4118.60 ± 2.33Tunicate GI (%)1.41 ± 0.781.421.82 ± 0.611.74 ± 0.850.911.64 ± 0.96Tunicate oocyte diameter (µm)86.99 ± 39.9349.7893.55 ± 42.32101.96 ± 44.6967.63 ± 39.8995.82 ± 41.51Mussel SGR (% day−1)0.75 ± 0.120.50 ± 0.050.65 ± 0.160.06 ± 0.070.05 ± 0.250.09 ± 0.114. Discussion4.1. Air Exposure and Freshwater TreatmentsThe eradication treatments for the tunicate S. plicata showed distinct levels of effectiveness for tunicate mortality and survival of the mussel M. edulis. Air exposure and freshwater immersion only promoted a mean tunicate mortality of 27% at the end of the 30-day rearing, observed mainly after the second exposure to the treatments. Hillock and Costello [28] obtained total mortality of S. clava submitted to air exposure for 24 h in full sun ambient (15–29 °C) and 48 h in shade ambient (15–27 °C). Therefore, the efficiency of the 6 h air exposure treatment (~20 °C) that we applied would likely be higher in field conditions but may compromise mussel survival. In mussels, air exposure in increased temperatures promote higher oxidative stress, with a negative impact in physiological performance [40]. A shorter exposure would likely be more effective, and particularly more feasible, if applied to mussel seeds, in a controlled environment. Darbyson et al. [41] showed that a 48 h exposure was not effective against S. clava, possibly demonstrating that the probability of survival is dependent on size/age [28] and on the level of aggregation of the organisms. Because a 6 h period is the average period of emersion of mussels during low tide [42], a longer exposure would likely compromise mussel productivity, as demonstrated by LeBlanc et al. [29]. Those authors obtained a 40% biomass reduction of M. edulis after 7 months in the field, following a 40 h air exposure at 21 °C.The relatively low mortality of S. plicata in the freshwater treatment may be explained in part by its capacity to adapt to low salinity conditions [43]. Rolheiser et al. [44] reported that a freshwater immersion for 0.5–10 min did not reduce the colonial ascidian Didemnum vexillum biofouling, as indeed much longer exposures are needed [35], but induced a slight mortality in oysters. Carver et al. [45] reported a 10% mortality in solitary tunicate Ciona intestinalis exposed to freshwater for 1 min at 15 °C. However, they reported 66% mortality at 40 °C. At this temperature, some mussel mortality was registered, perhaps due to the synergistic effects of osmotic and heat stress [46]. A 5 min freshwater spray appeared to be effective in eliminating various species of tunicates attached to oysters grown in commercial operations, but detailed information is missing [20]. Although relatively low salinities promote physiological stress in M. edulis, the species show highly efficient acclimatization mechanisms in long- and short-term exposures [46,47]. Furthermore, a pronounced osmotic shock promotes a lasting valve closure in M. edulis, which likely served as the main mechanism for resisting hyposalinity in the present trial [47]. Therefore, given the absence of mussel mortality and the high tolerance of solitary ascidians to freshwater immersions, increasing exposure periods to freshwater at higher temperatures (30–40 °C) should be tested in S. plicata and M. edulis [31]. However, the applicability of the treatment in field conditions should be further investigated. A prophylactic approach with mussel seeds translocated from other locations to the aquaculture facilities would also allow the preventing of the introduction of fouling species, using simpler and shorter freshwater exposures.4.2. Sodium Hypochlorite TreatmentThe collection of organisms for trial 2 took place in September 2021, when the lagoon water showed relatively higher levels of eutrophication, due to the closure of the tidal inlet by the end of the month. The potentially stressful abiotic conditions, particularly higher temperatures, as well as the transportation and laboratory acclimation periods, may have affected fitness of S. plicata, which was reflected in the mortality registered in the control group. Therefore, the results of tunicate mortality in our study should be analyzed with caution. Sodium hypochlorite (bleach) only eliminated 9% more of S. plicata comparatively to the control. Similarly, Carver et al. [45] obtained no mortality of C. intestinalis exposed to sodium hypochlorite for a longer period of 20 min. On the contrary, Piola et al. [48] reported a 75–100% removal of fouling biota, including the tunicate C. intestinalis, sprayed with 20% bleach (0.5–12 h), but only 0–50% removal with 5 and 10% bleach. In the work of Coutts and Forrest [31], S. clava was successfully eliminated in a 6 h exposure to sodium hypochlorite; however, in field trials performed on marina pontoons, treatments were not so effective, mainly due to the rapid decline in free available chlorine in the water. Given the short exposure time applied in our study (1.5 min), this issue probably did not represent a significant factor. Once again, the application of the treatment in earlier stages of mussel production, in more controlled conditions, would result in enhanced efficiency. Nonetheless, those results indicate that the concentration and exposure time applied in our study should have been higher to obtain total tunicate mortality. However, some results that have been reported in the literature are contradictory. McCann et al. [35] obtained total mortality of D. vexillum immersed in 1% bleach for 10 min, while immersions of 2 and 5 min only produced an initial decline in the surface area of the colony. However, Denny [36] indicates that bleach concentrations as low as 0.1%, applied for just 2 min, are effective in the same species. Nonetheless, the pronounced morphological differences between the colonial D. vexillum and the solitary S. plicata should be noted. Furthermore, Denny [36] also only obtained a maximum mortality of 6% in the mussel Perna canaliculus exposed to 0.5% bleach for 2 min, while in the present study, 55.6% of M. edulis died in the bleach treatment. Hypochlorite potentially causes a toxicological response in mussels, mostly in gills, promoting an oxidation process, but it also can affect the mussels’ byssus gland [36,49]. However, the resilience to sodium hypochlorite varies significantly according to different factors, including mussel size, sexual maturation, acclimatation temperature and species [50]. Therefore, despite the apparent resiliency of S. plicata to chlorine [31], further studies are needed to ensure M. edulis survival in more efficient treatments. Moreover, although the chemicals used in this study are considered to have a relatively low toxicity, with a high biodegradability [31,48], their application in the field must include a consideration of the safety of the operator, as well as environmental mitigation techniques [45].4.3. Hypersaline TreatmentThe hypersaline (brine) treatment was not successful in eliminating S. plicata which, according to Sims [43], exhibits some level of hyperosmotic regulation. The treatment only promoted a 16% higher mortality in the tunicates of trial 2, comparatively to the control. Similarly, Carver et al. [45] only obtained 25% mortality in C. intestinalis exposed to a more intense treatment of saturated brine for 8 min. Carman et al. [37] eliminated various species of tunicates from socks of juvenile M. edulis exposed to 10 or 20 s brine baths (70 salinity), but direct comparisons would also be biased due to the different target species. For D. vexillum eradication, brine treatments require at least 4 h immersions [35,44]. Vickerson et al. [51] and Rolheiser et al. [44] reported no mortality in mussels (Mytilus spp.) and oysters, respectively, while Carman et al. [37] reported 8–30% M. edulis mortality in a 20 s brine bath. In comparison with our study, this higher mortality may be due to the use of juvenile mussels, as well as a higher bath salinity. Therefore, increasing the exposure time to a 60-salinity bath may result in mussel mortality, or at least in higher metabolic activity and altered immune system, which would likely affect production [52]. According to Carman et al. [20], 10 min immersions in brine solution followed by a 2 h air exposure are effective in removing tunicates from oyster aquaculture operations. This procedure could potentially increase tunicate mortality while being safe for mussels. However, the logistical or financial viability of the treatment in field conditions should be further investigated [44].4.4. Acetic Acid TreatmentThe acetic acid treatment was the most successful in eliminating S. plicata. In trial 3, 98% of tunicates exposed to the treatment died, as well as 36–40% of mussels. Similarly, Coutts and Forrest [31] also achieved total mortality of S. clava with an identical procedure. In the work of Sievers et al. [32], 1 min immersions in 2 or 5% acetic acid, at ambient temperature, killed 50% of S. clava specimens. Only the 2% acetic acid treatment at 40 °C for 1 min led to total mortality. Compared to our study, the different results may derive from distinct physiological conditions and size of the organisms, experimental environments, as well as physiological differences between species. Furthermore, Sievers et al. [32] only assessed mortality in the first 48 h. Although in the present study most mortality was also detected in the first days of exposure, further mortality was registered during the trial period. Carver et al. [45] reported a 95% mortality of C. intestinalis exposed to 5% acetic acid for 15 to 30 s, either by spraying or immersion. Forrest et al. [53] also achieved an 84–100% biomass reduction of fouling organisms, mostly dominated by C. intestinalis, using 2 and 4% acetic acid in 1–4 min immersions. Nonetheless, Styela species may present a higher resistance to some eradication treatments given their thicker tunic, when compared to the soft-bodied C. intestinalis [32]. For D. vexillum, a 5% concentration was effective in significantly reducing tunicate coverage using 0.5–10 min exposures [37,44,48]. Tunicate mortality can be further enhanced by an air exposure period after immersions, but it would also enhance the probability of mussel mortality, particularly if the acid residue is not rinsed [33,53]. Moreover, as sexually mature mussels are likely more sensitive to chemical treatments [50], the use of mature S. plicata individuals in trial 3 may also have favored higher efficiency of the acetic acid treatment. However, further studies are needed to confirm this possibility and also the long-term effect of treatments on the reproductive performance of surviving individuals. Cahill et al. [33] registered a relatively higher sensitivity of the mussel P. canaliculus to acetic acid, compared to the oyster C. gigas. The authors recommended a concentration limit of 4% to avoid pronounced mussel mortality, which is in accordance with the present results. The authors tested in the laboratory a provisional treatment of 2% acetic acid for 1 min, which resulted in a higher mussel productivity in the field, as a result of biofouling reduction. Other works confirm these results. LeBlanc et al. [29] reported 67 and 74% reductions in M. edulis socks’ weight after exposure to 30 s and 2 min acetic acid immersions, respectively, as a 5% concentration was applied in mussel seed. However, loss of attachment may have also contributed to weight reduction, making it difficult to directly compare results. Carman et al. [37] also reported total juvenile M. edulis mortality in the acetic acid treatments (15–25 mm shell length). Although the authors suggested immersion times of less than 5 min in future works, larger mussels exposed to 1 min in the present study still experienced relatively high mortality. In fact, acetic acid concentration is likely more relevant than exposure time [33], although 5–10 s may be insufficient to eliminate tunicates [45]. Sievers et al. [32] also reported mussel (Mytilus galloprovincialis) and oyster mortality in the abovementioned treatments for S. clava, but it was only significant with the acetic acid at 50 °C. In the study of Rolheiser et al. [44], high mortality occurred in oysters exposed to a concentration of 5%. Apart from mortality, acetic acid may also affect mussel attachment, which represents an equally important issue for aquaculture operations, and further studies are needed [51,53]. Furthermore, one of the main factors influencing the survival of mussels exposed to various chemical treatments, besides size class, is valve-gaping, which determines the level of soft tissue exposure [29,33,45]. Valves are opened most of the time in raft-cultivated mussels, following mainly a circadian rhythmicity rather than a tidal one [54]. Therefore, it is crucial to ensure valve closure in most individuals prior to treatment exposure, using methods like shaking or freshwater immersion. In our study, all treatments were applied to individuals that were previously emerged for at least 2–3 min, thus promoting immediate valve closure [29,33,47]. However, the mortality obtained in the sodium hypochlorite and acetic acid groups likely indicates some level of contact between chemicals and soft tissues, as a result of gaping mussels. In addition, not only do declustered mussels present increased gaping and byssus production, but some individuals may have imperfect valve sealings as a result of variability in shell shape, enhancing the chemical exposure [29,53]. Interestingly, gapping may be more preponderant in higher size classes of mussels, which may represent another added advantage for applying these methods in earlier life stages, including prophylactic treatments [33]. In mussel seed, invasive tunicates are also likely to be in earlier development stages, with lower adhesion surface, and thus are more vulnerable to treatments, particularly mechanical removal. The length of the present trial was probably too short to detect possible differences in the mussels’ SGR. Andrade et al. [40] also did not obtain differences in condition index of M. galloprovincialis submitted to air exposure at increased temperatures. However, some treatments may impair physiological performance in the long term, as reported by Thompson et al. [55], who obtained lower growth rates in M. edulis exposed to chlorination, and further studies are needed.5. ConclusionsOur results highlight the efficacy of the acetic acid treatment for eradicating the ascidian S. plicata. However, the method still needs further research to minimize mussel mortality. On the other hand, exposure to air, freshwater and hypersaline solution did not induce significant mortality in tunicates. These treatments may need longer exposures to achieve better results. Furthermore, the duration of the present trial was too short to detect differences in mussel and tunicate growth. Therefore, further studies are needed to investigate the long-term effect of the different treatments on mussel growth, as well as the susceptibility of tunicates to treatments according to sexual maturation. Our results should also be interpreted in the context of laboratory conditions, namely the sample size. The application of treatments to declustered mussels may have favored a higher mortality; thus, the same procedures applied in the field are expected to produce different results. Other factors such as the species cultured, culture technique and the fouling community may also affect treatments success [7]. The treatments are likely to produce more effective results than prophylactic measures, applied in a controlled environment in mussel seed, thereby further inhibiting tunicate proliferation. Moreover, because S. plicata is a food product in Korea and the Mediterranean [56], this invasive species can represent a commercial opportunity.

animals : an open access journal from mdpi

10.3390/ani12030378

PMC8833600

Coronaviruses are a broad group of viruses that may infect a wide range of animals, including humans. Despite the fact that each coronavirus has a limited host range, frequent interspecies transmission of coronaviruses across diverse hosts has resulted in a complex ecology. The recently discovered SARS-CoV-2 virus is the clearest evidence of the danger of a global pandemic spreading. Natural infection with SARS-CoV-2 has been reported in a variety of domestic and wild animals, which may complicate the virus’s epidemiology and influence its development. In this review, we discussed the potential determinants of SARS-CoV-2 interspecies transmission. Additionally, despite the efforts that have been made to control this pandemic and to implement the One Health policy, several problems, such as the role of animals in SARS-CoV-2 evolution and the dynamics of interspecies transmission, are still unanswered.

In December 2019, the outbreak of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was reported in China with serious impacts on global health and economy that is still ongoing. Although interspecies transmission of coronaviruses is common and well documented, each coronavirus has a narrowly restricted host range. Coronaviruses utilize different receptors to mediate membrane fusion and replication in the cell cytoplasm. The interplay between the receptor-binding domain (RBD) of coronaviruses and their coevolution are determinants for host susceptibility. The recently emerged SARS-CoV-2 caused the coronavirus disease 2019 (COVID-19) pandemic and has also been reported in domestic and wild animals, raising the question about the responsibility of animals in virus evolution. Additionally, the COVID-19 pandemic might also substantially have an impact on animal production for a long time. In the present review, we discussed the diversity of coronaviruses in animals and thus the diversity of their receptors. Moreover, the determinants of the susceptibility of SARS-CoV-2 in several animals, with special reference to the current evidence of SARS-CoV-2 in animals, were highlighted. Finally, we shed light on the urgent demand for the implementation of the One Health concept as a collaborative global approach to mitigate the threat for both humans and animals.

1. IntroductionSince the first report of infectious bronchitis virus (IBV) in 1937 [1], numerous coronaviruses have been isolated and/or identified in various animal species as well as humans. The newly emerged severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the best example of a pandemic that had a global impact on the health, economic, and social aspects of communities [2,3]. At the time of writing this review, 350,292,303 SARS-CoV-2 cases and 5,611,459 deaths, according to Worldometer, had been reported worldwide. Although scientists have tried to control the coronavirus disease-19 (COVID-19) pandemic, the situation is still critical due to several global control strategy challenges. Like other coronaviruses, SARS-CoV-2 has acquired new mutations as a part of its evolution to evade host responses and transmit more effectively. Some of these mutations increased transmissibility through an increase in receptor binding or the ability to evade the host immune, in addition to the emergence of new variants, such as variant of interest (VOI) and variant of concern (VOC). The recently emerged “Omicron” variant (B.1.1.529) raises serious concern since it may significantly limit the antibody-mediated neutralization and increase the risk of reinfections due to the presence of numerous mutations in spike protein (30 nonsynonymous substitutions, 3 small deletions, and an insertion) [3].Cross-species transmission events that force viruses to adapt to new host settings result in species-specific adaptations [4]. These evolutionary modifications may influence the virus’s virulence and transmissibility in new host species [5]. It is believed that the initial spillover of SARS-CoV-2 has a zoonotic transfer from bats to humans [6], possibly by an unidentified intermediate host(s); for instance, snakes, turtles, and pangolins have been proposed as the intermediate hosts [5,7]. Human-to-human transmission, the driving force behind the pandemic, has been confirmed even from asymptomatic carriers and presymptomatic infected persons [8,9]. Additionally, there is an incalculable number of daily human–animal interactions potentially leading to unreported zoonotic and anthroponotic transmission of SARS-CoV-2. The repeated interspecies virus transmission has the potential to speed up viral evolution and provide a source of new strain emergence. This review sheds light on the diversity of coronaviruses in domestic animals and the potential determinants of interspecies transmission of SARS-CoV-2.2. Diversity of Coronaviruses in Domestic AnimalsCoronaviruses belong to the family Coronaviridae, order Nidovirales. This family is classified into Orthocoronavirinae and Letovirinae subfamilies. According to the phylogenetic clustering, the subfamily Letovirinae includes only one genus (Alphaletovirus), while the Orthocoronaviridae subfamily consists of four genera: Alphacoronavirus (α-CoV), Betacoronavirus (β-CoV), Gammacoronavirus (γ-CoV), and Deltacoronavirus (δ-CoV). To date, a total of 17, 12, 2, and 7 species belonging to α-CoV, β-CoV, γ-CoV, and δ-CoV, respectively, are known and classified by the International Committee on Taxonomy of Viruses [10]. The diversity of coronaviruses in different animal species and humans is summarized in Table 1.2.1. Avian CoronavirusesAccording to the ICTV 2018, all γ-CoVs from any bird species are considered avian coronaviruses (ACoVs) regardless of the host, tropism, antigenicity, cross protection, and genome identity. The ACoVs includes several subspecies, such as IBV, guinea fowl coronavirus (GfCoV), turkey coronavirus (TCoV), duck coronavirus (DCoV), and pheasant coronavirus (PhCoV) [34,35]. However, this classification failed to establish a standard classification scheme that may collect all TCoVs into one genotypic relationship [36]. IBV, the first isolated coronavirus in the 1930s, is characterized by respiratory, intestinal, and urogenital problems in chickens [1,11]. In addition to respiratory complications, secondary infections with other microorganisms lead to a high mortality rate and a reduction of animal performance. Some strains can cause kidney failure, leading to fatal course. IBV can also duplicate in the oviduct, causing permanent impairment in young hens. The primary host of IBV is chicken (Gallus gallus), but the virus has also been reported in pheasant and peafowl [37]. It was proposed that TCoV is emerged because of a recombination that occurred between IBVs and an unknown coronavirus. These recombinations and mutations lead to interspecies transmission from chickens to turkeys and change the virus tropism from respiratory to enteric [37,38]. TCoV is intended to be involved in the poult enteritis and mortality syndrome (PEMS) in turkeys [36]. The emergence of IBV variant strain β-CoVs, caused by the high mutation rate, makes the control difficult.2.2. Coronaviruses in PigsTo date, six coronaviruses are known in pigs, belonging to α-CoVs, β-CoVs, and δ-CoVs. Among the α-CoVs that can infect pigs are transmissible gastroenteritis virus (TGEV), porcine epidemic diarrhea virus (PEDV), porcine respiratory coronavirus (PRCV), and swine acute diarrhea syndrome coronavirus (SADS-CoV). The porcine hemagglutinating encephalomyelitis virus (PHEV) belongs to β-CoVs, and porcine deltacoronavirus (PDCoV) belongs to δ-CoVs, which are also known to infect pigs [16,17,18,19,20,21].2.3. Coronaviruses in DogsTwo canine coronaviruses in dogs are known: canine coronavirus (CCoV) and canine respiratory coronavirus (CRCoV). CCoV belongs to α-CoV and causes acute enteritis in young dogs (1- to 12-week-olds), where sometimes the infection is subclinical. The CCoV-β-CoV infection is sometimes accompanied with respiratory manifestation. Based on the spike gene, two distinct serotypes of CCoV are known: CCoV-I and CCoV-II. CCoV-II is subdivided into CCoV-IIa and CCoV-IIb, in which CCoV-IIb is a recombinant virus between CCoV-II and TGE [38,39]. On the other hand, CRCoV belongs to β-CoV, causes respiratory manifestations in dogs older than 2 years, and is the key player of canine infectious respiratory disease development (CRID), or “kennel cough” syndrome [22,23,40,41,42].2.4. Coronaviruses in CatsFeline coronavirus (FCoV) belongs to α-CoVs and causes disease in domestic and nondomestic felid species. The infection is associated with mild to severe immune-mediated disease (feline infectious peritonitis or FIP) [24,43].2.5. Coronaviruses in CattleIn cattle (Bos taurus), bovine coronavirus (BCoV) belongs to β-CoV and causes economic losses due to respiratory and gut problems [44]. In calves, BCoV causes severe bloody diarrhea due to the destruction of intestinal villi, resulting in high mortality rates [45]. In adult cattle, it also induces severe or fatal infection, especially in case of coinfections with other secondary respiratory pathogens [45,46,47].2.6. Coronaviruses in EquinesEquine coronavirus (ECoV) belongs to β-CoV and was first isolated from the feces of a diarrheic foal in 1999 in North Carolina, USA [26]. In the last decade, it has been associated with outbreaks of enteric disease in adult horses in the USA, Europe, and Japan [48,49,50,51,52,53]. The virus was detected also in other several countries, including Saudi Arabia and Oman [54]. Recently, ECoV antibodies were detected in horses in Israel [55].2.7. Coronaviruses in HumansTo date, seven coronaviruses are known to be able to infect humans, explicitly, 229E, NL63 (α-CoVs), OC43, HKU1, Middle East respiratory syndrome coronavirus (MERS), severe acute respiratory syndrome (SARS), and SARS-CoV-2 (β-CoVs) (Figure 1). NL63, 229E, OC43, and HKU1 cause only mild respiratory diseases and enteritis, similar to the common cold [56]. However, MERS-CoV and SARS-CoV-2 cause acute fatal pneumonia [33,57,58]. Three coronavirus pandemics/epidemics were reported in humans [47]: (i) SARS-CoV epidemic reported in Asia in 2002 (8422 cases and 11% mortality rate), (ii) MERS-CoV-2 reported in Saudi Arabia in 2012 (2468 cases, 34% mortality rate), (iii) the newly emerged SARS-CoV-2 in 2019 (~2% mortality rate).3. Diversity of Coronavirus ReceptorsCoronaviruses utilize different receptors to mediate membrane fusion and to enter the host cell and replicate in the cytoplasm. Therefore, interspecies transmission could be ascribed to the large S-glycoprotein. The S-protein homodimers consist of two subunits: S1 subunit (accountable for binding with a receptor) and S2 subunit (mediated membrane fusion). The S1 subunit is subdivided into the N-terminal domain (NTD) and the C-terminal domain (CTD). On the other hand, the S2 subunit is subdivided into a fusion protein (FP) that plays a role in membrane fusion, heptad repeat 1 (HR1) and HR2. Either NTD or CTD domains can bind to distinct receptors and serve as the receptor-binding domain (RBD) [58]. However, the RBD of coronaviruses is located within the S1 subdomain of the spike protein, which might allow binding to different receptors [59]. For example, although the receptor-binding domain (RBD) of most coronaviruses is located in the CT, the RBD of murine hepatitis virus (MHV-A59) is located within the NTD of the S-protein [60,61,62,63,64,65]. Additionally, although the RBD is relatively well conserved, the receptor-binding motif (RBM) tends to mutate frequently and determines receptor specificity [64]. In this section, we will shed light on the different receptors of coronaviruses. The receptors that are utilized by coronaviruses are shown in Table 2.3.1. Amino Peptidase ReceptorsAminopeptidase N receptors (APNs, CD13), a type II transmembrane zinc aminopeptidase and protein, are utilized by several species of the α-CoV genus, such as HCoV-229E, TGEV, PRCV, FCoV, and CCoV [66,67,69,80]. The APN is found in the gut, nervous system, dendritic cells, and monocytes of various hosts [81,82]. Coronaviruses that bind to the APN use their S1 CTD domain. However, the mode of interaction between the RBD and APN is distinct in different α-CoVs [83]. In [84], it was found that SARS-CoV-2 is unable to bind to the APN receptors.3.2. Carcinoembryonic Antigen-Related Cell Adhesion Molecule 1Carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) belongs to the immunoglobulin superfamily. As a cellular adhesion molecule, CEACAM1 is utilized by the mouse hepatitis virus (MHV) and belongs to β-CoV [85].3.3. Dipeptidyl Peptidase 4 (DPP4)The dipeptidyl peptidase 4 (DPP4) is a serine exopeptidase type II transmembrane protein. It is utilized by several coronaviruses such as MERS-CoV, camel-derived MERS-CoV, and BatCoV HKU4 [73]. The DPP4 receptors are expressed ubiquitously in the lungs, liver, intestine, immune cells, and kidneys [86]. The DPP4 receptor might be involved in SARS-CoV-2 infection [84]; however, their exact role in SARS-CoV-2 pathogenesis needs further investigation.3.4. Sialic Acids or Sialosides, Acidic CarbohydratesLike other viruses, such as parainfluenza, rotaviruses, adenoviruses, polyomaviruses, and influenza viruses [87,88], some coronaviruses can also bind to sialic acids or sialosides and acidic carbohydrates [73,75,78,79] (Table 2). Both HCoV-OC43 and BCoV bind to the N-acetyl-9-O-acetylneuraminic acid to mediate cellular entry [89,90,91,92]. Additionally, in [92] it was also suggested that the S-protein of SARS-CoV-2 might binds to α, N-acetyl neuraminic acid. This interaction needs to be investigated further because it might have a role in enteric tract infections.3.5. Angiotensin-Converting Enzyme 2 (ACE2)ACE2 is used by several coronaviruses, namely, SARS-CoV [68], NL63 [71], and SARS-CoV-2 [72]. The high sequence similarities between the receptor-binding domains (RBDs) of these viruses [92] might explain why these viruses interfere with the same receptor. The ACE2 receptor was discovered in 2000s. It is widely expressed in different organs and tissues, including pulmonary and extrapulmonary [93,94,95], and has specific functions, much more than just a receptor for SARS-COV-2: (i) catalytic function of the renin–angiotensin system (RAS); hence, it regulates and maintains the relative stability of the body homeostasis and the balance of blood pressure and water and electrolytes [96,97,98]. ACE2 has a catalytic function in RAS, which is summarized in Figure 2. (ii) ACE2 has a significant role in the transport of amino acids. (iii) ACE2 is abused by several viruses (i.e., SARS-CoV, SARS-CoV-2, and NL63) as a receptor to promote virus entry. The RBD of SARS-CoV-2 binds to the peptidase domain (PD) of ACE2, which is critical for viral entry [95,98].4. Interaction between SARS-CoV-2 and ACE2 ReceptorThe entry of SARS-CoV-2 into the cells correlates with the binding of the RBD of the S1 subunit to ACE2 and the priming by host cell proteases, which cleave the S-protein at the S/S2 and subsequently allow cellular membrane fusion and cell entry [72,84,92,99,100]. The general organization of the coronavirus S-protein is shown in Figure 3. In [101,102], it was found that transmembrane serine protease 2 (TMPRSS2) can cleave the S-glycoprotein at the S2′ site and furin proteases at the S1/S2 site. There are four sequential steps for the cell entry of coronaviruses: (i) cleavage of S1/S2 by the proprotein convertase furin, which cleaves at R-X-R/K-R↓. Furin is a ubiquitous protease expressed in eukaryotic cells. It has physiological functions in transport secretory pathways via the cleavage of several proteins, such as cell surface receptors, hormones, adhesion molecules, and growth factors [103]. Unfortunately, this enzyme is abused by several viruses, such as avian influenza, Ebola, yellow fever, human immunodeficiency virus, measles, and bacterial toxins, to facilitate cell entry [104]. The cleavage of the S1/S2 site might induce conformational changes that might be required for receptor binding [103,105,106]. The R-R-A-R motif of SARS-CoV-2 at the junction between the S1 and S2 subdomains is sensitive to furin cleavage [72]. (ii) SARS-CoV-2 binds with ACE2 receptors. (iii) The cleavage of the S-protein of SARS-CoV-2 by TMPRSS2 at single arginine or lysine residues (R/K↓), which, subsequently, allow internalization. (iv) Membrane fusion and virus entry are achieved by direct fusion with the plasma membrane or use of endocytic mechanisms.5. Interspecies Transmission of CoronavirusesBats (Rhinolophus affinis) are a reservoir for several human coronaviruses, such as NL63, 229E, MERS-CoV, and SARS-CoV [107,108,109,110,111,112]. Rodents are the natural host of both human CoVs OC43 and HKU1 [113]. Although the intermediate hosts of both HKU1 and NL63 are unknown, bovines, camelids, dromedary camels (Camelus dromedarius), and palm civets (Paradoxurus hermaphroditus) are the intermediate hosts to OC43, 229E, MERS-CoV, and SARS-CoV, respectively [57,107,108,109,110]. Recently, a novel canine–feline recombinant virus was found in a human pneumonia patient [111]. Generally, coronaviruses are observed to cross “species barriers” easily for several reasons, including: (i) The high mutation rates [112], site-directed mutations targeting the S-gene of the MHV to replace the ectodomain of its S-protein with the highly divergent ectodomain FIPV, resulting in the ability to infect feline cells and simultaneously missing the capacity to infect murine cells [113]. Moreover, the manipulation of the FIPV genome leads to switching species tropism [114]. Additionally, interspecies switching of IBV, cell tropism of FECoV, and FIPV highlight that the S-glycoprotein is a determinant of cell tropism. (ii) The large RNA (~29,903 nucleotides) genome of the coronaviruses raises the commonness of recombination and mutation events, resulting in the development of new strains/variants [115]; (iii) Coevolution between coronaviruses and receptors was cited by Bolles and others [116]. Although coronaviruses utilize specific cell receptors for cell entry, the S-protein binds with receptors and subsequently activates fusion; coevolution between virus (S-protein) and receptors is common, and hence, it can extend or alter the host range and jump the “species barrier”. For example, the RBD of SARS-CoV isolated in 2002–2003 demonstrated a greater binding affinity to ACE2 than that isolated in 2003–2004. Indeed, both receptors and coronaviruses are flexible and undergo mutations that lead to conformational changes required for host adaptation. Accumulation of the mutations of the S-protein is necessary for host adaptation [117,118]. The following section discusses the susceptibility of various animal species to the newly emerged SARS-CoV-2 and the determinants of different susceptibilities. Figure 4 illustrates the exposure of several animals to SARS-CoV-2.5.1. SARS-CoV-2 in Dogs and CatsSeveral studies have reported the detection of SARS-CoV-2 in pet animals, such as dogs (Canis lupus familiaris) and cats (Felis catus). In Hong Kong, SARS-CoV-2 was detected in asymptomatic Pomeranian and German shepherd dogs in which the owners also tested COVID-19 positive. The sequence of these viruses exhibited high identities with the respective human cases, highlighting the potential human–animal transmission [119,120]. Additionally, neutralizing antibodies against SARS-CoV-2 were also identified using plaque reduction neutralization assays. In Italy, extensive research was conducted to assess SARS-CoV-2 infection in companion animals (n = 999), sampled at a time of frequent human disease. Although all samples were negative using PCR, neutralizing antibodies against SARS-CoV-2 were recorded in dogs (3.3%) and cats (5.8%). The incidence of positive cases was significantly higher in dogs maintained in COVID-19-positive households than those maintained in COVID-19-negative households [120]. In Brazil, SARS-CoV-2 was also identified in dogs and cats that kept close to COVID-19-positive human cases [121]. In France, Fritz and others also found that 58.8% (20/34) of tested cats and 38.5% (5/13) of tested dogs were seropositive for SARS-CoV-2 [122]; positive samples were greater among animals kept close to COVID-19-positive households. In Lima, Peru, SARS-CoV-2 antibodies were also observed in domestic cats [123]. In Germany, during the first-wave COVID-19 pandemic, antibodies to SARS-CoV-2 were detected in 0.65% (6/920) of serum samples collected from cats [124]. Later on, during the second wave (between September 2020 and February 2021), the prevalence of positive samples was significantly higher. Out of 1173, a total of 16 samples were positive (1.36%) [124]. In the Netherlands, SARS-CoV-2 was detected in pets kept in an infected mink farm using PCR and serological examination. Although no cat-to-cat infection of SARS-CoV-2 was observed in this study, mink-to-cat transmission was documented, highlighting the role of mink as a potential threat for disseminating the virus [125]. The UK B.1.1.7 variant of SARS-CoV-2 was detected for the first time in a cat and a dog in the US. After their homeowners were diagnosed with COVID-19, the animals tested positive 2 days later [126]. Both animals exhibited respiratory manifestations several weeks after virus detection. In the United Kingdom, the B.1.1.7 variant was also detected in a dog and 2 cats whose owners were also COVID-19 positive [127]. These animals exhibited no respiratory signs but showed cardiac abnormalities (myocarditis). Additionally, SARS-CoV-2 was investigated in feral cats near mink farms diagnosed to be COVID-19 positive in the Netherlands. Seven out of 24 tested animals had antibodies to SARS-CoV-2, and 1 cat was confirmed positive for virus RNA [128].Based on the documented report, we can conclude the following: (i) Dogs that tested positive for SARS-CoV-2 did not exhibit any clinical symptoms [121], with the exception of transient respiratory distress and lethargy found in only one case [129]. (ii) Cats are more sensitive to SARS-CoV-2. Most SARS-CoV-2-positive cats exhibited diarrhea, vomiting, and respiratory manifestations [129]. These clinical findings are in accordance with the predicting susceptibility studies of Alexander et al. [130]. Additionally, although both dogs and cats have ACE2 receptors in the respiratory tract, dogs harbor low levels of ACE2 compared with cats [131,132]. This might explain why cats are more susceptible than dogs [133]. (iii) Cross-reactivity between coronavirus antibodies is possible, which must be considered in serological assays [134,135,136]. For example, SARS-CoV-2 exhibited a homology of 45% with CCoV. However, β-CoV, HCoV-OC43, and CRCoV exhibited a homology of 97% [137]. (iv) Although anthropozoonotic transmission (human to animal) of SARS-CoV-2 is possible, to date, there is a low risk of zoonotic transmission (animal to human) [137,138,139,140,141,142]. (v) The available data are not sufficient to rule out cat-to-cat transmission. Further investigations are required.5.2. SARS-CoV-2 in MinkTwo different minks, the family Mustelidae, are known, namely, European (Mustela lutreola) and American (Neovison vison) mink. Minks are aggressive animals and thus not suitable as pets. However, American minks are used worldwide for production [143]; Poland, the Netherlands, Denmark, and China are the main producing countries. Furthermore, in several countries in Europe and the Americas, SARS-CoV-2 has been found among minks (Neovison vison), suggesting that mink might be a reservoir of SARS-CoV-2 [128,144].In May 2020, SARS-CoV-2 was reported in mink in Denmark for the first time [145]; then the virus was also documented in the Netherlands in mid-June 2020. Due to the possible zoonotic transmission of the virus, a culling strategy of mink farms was implemented [146]. In the US, the virus was reported in farmed mink on 17 August 2020. The virus was also detected in Italy, France, Sweden, Spain, Poland, Canada, Greece, and Lithuania. However, Russia and China have not seen SARS-CoV-2 in their minks [143].Generally, SARS-CoV-2 multiplies effectively in the respiratory system of mink, leading to respiratory lesions like COVID-19 in humans. It was also cited that an S-protein-based vaccine in mink farms prevented the SARS-CoV-2 replication and lesions induced by SARS-CoV-2. This highlights the potential of mink as a valuable animal model for studying the pathogenesis of SARS-CoV-2 prior to the evaluation of an antiviral drug and vaccines [147].Considering the epidemiology and clinical picture of SARS-CoV-2 in farmed minks, the following points can be concluded: (i) The detection of SARS-CoV-2 in farmed mink demonstrated that mink might be an intermediate host and played a prospective role in the early phases of the pandemic transmission [143]. (ii) It also remains to be investigated whether mink has an impact on the virus evolution due to its rapid spread as a new host, which might lead to the emergence of mutations. It was found that SARS-CoV-2 evolved slower in humans than in minks [148] due to adaptation of the virus under selective pressure. Thus, continuous tracing of the molecular changes of SARS-CoV-2 in mink is essential to explore virus evolution.5.3. SARS-CoV-2 in RabbitsRabbits (Oryctolagus cuniculus) are vulnerable to SARS-CoV-2 [149]. After tentative infection of rabbits with SARS-CoV-2, the virus was detected in nasal swabs for 7 days postinoculation with a peak titer of 103 TCID50. However, the disease was asymptomatic [150]. Therefore, further surveys are needed to identify the prospective role of rabbits in SARS-CoV-2 evolution.5.4. SARS-CoV-2 in Other AnimalsThe first records of natural SARS-CoV-2 infection in lions (Panthera leo) and tigers (Panthera tigris) were reported in the US by McAloose and others (Table 3) [151]. The virus was noticed in feces and respiratory secretions. Most of the symptoms were related to the respiratory system, particularly cough. In Croatia, jackals (Canis aureus), free-living wild boars (Sus scrofa), and red foxes (Vulpes vulpes) showed positive antibody response to SARS-CoV-2 using ELISA. However, the virus neutralization test’s positive conclusions were not verified [152]. However, chickens, geese, and pigs were found to be nonsusceptible hosts for SARS-CoV-2 [153,154,155]. On the other hand, 8-week vintage crossbred pigs (Sus scrofa domesticus) were observed to be vulnerable to SARS-CoV-2 infection subsequent to oronasal infection. Although RNA from SARS-CoV-2 has been detected in nasal and oral fluids from pigs, the live virus has only been detected in the tissues of one animal. Moreover, SARS-CoV-2 neutralizing antibodies were found postinfection in oral fluid and serum in [156]. Experimental studies produced contradictory results since the infected animals were of different ages and breeds. In addition, they used different viral isolates and infectious doses [153,154,155,156]. Pickering and coworkers [156] applied an increasing viral level (10-fold) for investigational inoculation compared with previous experiments. In vitro, SARS-CoV-2 can replicate and induce cytopathic effects in the porcine kidney (PK-15) and swine testicle (ST) cell lines. Nevertheless, experimental inoculation of 5-week-old pigs did not show clinical signs in [153]. Additional investigations are needed to validate the exposure of pigs to disease.Although an ACE2 receptor is present in almost all animals, there are different susceptibilities to SARS-CoV-2. Therefore, considering the predicting susceptibility studies, animals can be categorized into four groups [84,119,130,155,157,158,159,160]. (i) Susceptible animals such as cats (Felis catus), tigers (Panthera tigris altaica), lions (Panthera leo), rhesus macaques (Macaca mulatta), and golden Syrian hamsters (Mesocricetus auratus). (ii) Intermediate susceptible animals such as pigs (Sus scrofa), ferrets (Mustela putorius furo), and dogs (Canis lupus familiaris). (iii) Nonsusceptible animals including chickens (Gallus gallus), ducks (Aythya fuligula), geese (Anser cygnoides), Japanese quails (Coturnix japonica), and mice (Mus musculus). (iv) Unknown susceptible animals were recorded so far, such as camels (Camelus bactrianus and Arabian camel), horses (Equus caballus), Malayan pangolins (Manis javanica), and sheep (Ovis aries).Generally, the binding of the SARS-CoV-2 S-glycoprotein to ACE2 is essential for virus duplication. However, the overall homology of ACE2 among animals is not the determinant for SARS-CoV-2 infection for the two following reasons: (i) ACE2 of mouse (not susceptible) is more similar to that of golden Syrian hamster (Mesocricetus auratus, susceptible animal) than ducks and chickens (nonsusceptible) [84,161]. (ii) Considering the phylogenetic analysis, mice are more similar to humans and rhesus macaques (susceptible species) than ducks and chickens (nonsusceptible species) [84,161].Indeed, there are some potential determinants for the host specificity and pathogenicity of SARS-CoV-2: (i) It is suggested that ACE2 amino acid residues, namely, 30, 83, 90, 322, and 354, can be used to differentiate between susceptible and nonsusceptible hosts. Alexander and coworkers [130] established a susceptibility score for SARS-CoV-2 infection in several animals, in which the lower cut-off for susceptibility is 23 and the upper cut-off for nonsusceptibility is 11. (ii) Coevolution between ACE2 receptors and the RBD of SARS-CoV-2 can lead to potential adaptation. Both ACE2 and SARS-CoV-2 are flexible and prone to mutations that affect the binding affinity to an ACE2 receptor, which defines the pathogenicity of the virus [61] and may lead to its adaptation in a new host. Several amino acid residues of the ACE2 receptor interface with certain residues of the SARS-CoV-2 RBD, namely, Y453F, F486L, and N501T residues, differ between humans and minks [162]. It is suggested that these differences may underlie the selective pressure for SARS-CoV-2 to adapt to a mink ACE2 receptor. It was found that the amino acid mutations Y453F, F486L, and N501T provide a better interaction between ACE2 and SARS-CoV-2 [98].animals-12-00378-t003_Table 3Table 3Susceptibility of animals to SARS-CoV-2 virus under experimental and natural infections.Risk LevelAnimalsExperimentalNaturalRemarksReferencesLowDog (Canis lupus familiaris)++No symptoms[129]Cattle (Bos taurus)+-No symptoms[163]Common marmosets (Callithrix jacchus)++No symptoms[164]Tree shrew (Tupaia belangeri)++No symptoms[95]HighCat (Felis catus) ++mild symptoms [155,165]Malayan tiger (Panthera tigris subsp. jacksoni)++Symptoms[63]Lion (Panthera leo)-+Symptoms[151]Puma (Puma concolor)-+Symptoms[165]American mink (Neovison vison)-+Symptoms[128,148]Egyptian fruit bats (Rousettus aegyptiacus)+-No symptoms[154]Ferret (Mustela putorius furo)+-Very mild [155,157,166]DetectedRabbits (Oryctolagus cuniculus)+-No symptoms [149]Raccoon dogs (Nyctereutes procyonoides)+-No symptoms [167]North American raccoons (Procyon lotor)+-No symptoms [168]Striped skunks (Mephitis mephitis)+-No symptoms [168]White Chinese geese (Anser cygnoides)+-No symptoms [169]Nonsusceptible Japanese quail (Coturnix japonica)+-No symptoms[170]White Chinese geese (Anser cygnoides)+-No symptoms[170]Turkeys (Meleagris gallopavo)+-No symptoms[170]Pekin duck (Anas platyrhinchos domesticus)+-No symptoms[170]Duck (Anatidae)+-No symptoms[155]Equine (Equus caballus) +-No symptoms [171]6. Control Measures and Implementation of One Health StrategyThe control of the SARS-CoV-2 pandemic still demands the One Health concept as a collaborative global approach to mitigate risk for both humans and animals (domestic and wildlife) and unravel and prevent the seriousness of complex a human–animal environmental health problem. To implement the One Health concept, all relevant stakeholders, including physicians and public health experts, veterinarians, epidemiologists, diagnosticians, pharmaceutical companies, vaccinologists, governments, and economists, must be engaged to identify clinical cases, perform laboratory diagnosis, trace the virus epidemiology, control the disease, enhance the isolation, quarantine, cure, vaccinate humans, and initially raise public awareness. In this regard, the following measures are described by several authors [172,173,174]: (i) developing strategies and funding needed for the application of preventative and control measures in the frame of One Health, (ii) engagement of well-trained and professional staff, (iii) fast and precise diagnostic tools and treatment of affected individuals, (iv) development and provision of efficient and safe vaccines for humans, (v) biosurveillance of live animal markets and humans in contact with animals to identity the possible reservoirs and to assess the risk factors, (vi) application of biosecurity in animal farms and implementation of good hygienic measures, (vii) assessment of the economic and social impacts of COVID-19 on people, and (viii) provision of efficient drugs and vaccines against SARS-CoV-2 and diagnostics. To control the spread of zoonotic diseases, vaccinating animals is more cost-effective, as well as (ix) cooperation among different agencies and taking advantage of veterinarians and human medicine experiences in the virus isolation programs and in the disinfection and collection of premises and clinics under the supervision of health authorities in order to prevent and/or reduce human outbreaks, (x) raising public awareness of SARS-CoV-2 transmission, and (xi) providing safe work practices in healthcare and nonhealthcare workplaces, and it is recommended to reassess the risk of COVID-19 infection from time to time. (xii) Protecting forests and changing agricultural practices are essential and cost-effective actions to prevent pandemics. Thus, destruction of the ecosystem by anthropogenic actions (such as urbanization, agricultural expansion, deforestation, and globalization) could be the reason for the emergence of pandemics. Unfortunately, it is widely highlighted that people became infected with SARS-CoV-2 through interaction with wild animals at the Huanan seafood wholesale market [175]. (xiii) As animal-to-human transmission of SARS-CoV-2 is not ruled out, the concept of One Health is urgently needed to control this pandemic virus. (xiv) Cleanliness and environmental hygiene are also important. Interaction with animals and improper use of animal products during COVID-19 outbreak must be avoided.7. Conclusions and RecommendationsCoronaviruses are diverse and can infect a wide range of animal species as well as humans. Currently, coronaviruses are categorized into four genera, specifically, αCoV, βCoV, γ-CoV, and δ-CoV. Although each coronavirus has a narrow-restricted host range, the frequent interspecies transmission of coronaviruses between different hosts leads to a complex ecosystem. The newly emerged SARS-CoV-2 is the clearest example of the risk of the spread of a disastrous pandemic worldwide. Natural infection with SARS-CoV-2 has been stated in several domestic and wild animals—dogs, cats, mink, ferrets, lions, tigers, pumas, snow leopards, and gorillas—which might contribute to complicating the epidemiology of the virus and have an impact on virus evolution. Experimentally, dogs, ferrets, cats, rhesus macaques, cynomolgus macaques, rabbits, white-tailed deer, and Syrian hamsters are susceptible to SARS-CoV-2 infections. Both humans and some animals have encountered unfavorable healthy problems due to SARS-CoV-2 infection. Therefore, while certain animals are resistant to SARS-CoV-2, adequate management, and strict hygienic processing of animal products should be considered during marketing and handling as a general rule for all animal supplies. On the other hand, the determinants of host susceptibility need further investigations to point out the factors that contribute to interspecies transmission. Although authorities, scientists, and industries have made several efforts to control this pandemic and have implemented the One Health strategy, several unresolved questions still pose significant challenges to researchers, such as: (i) What is the intermediate host of SARS-CoV-2? (ii) What is the role of animals in the early animal-to-human infection and in virus evolution? (iii) What are the dynamics and determinants of the interspecies transmission of this virus?A future pandemic could be worse than the ongoing COVID-19 because we are pushing nature to its limits by destroying and degrading amazing diverse ecosystems and ultimately removing natural buffers and expanding the interface between wildlife and people where pandemics emerge. Therefore, a multidisciplinary One Health task must be implemented to avoid the emergence of new pandemics. This is a call for scientists to explore in more detail the role of animals in this pandemic, maybe through experimental studies, expanding the ongoing surveillance and molecular epidemiology of SARS-CoV-2 to relevant animal populations. In addition, there is an urgent demand to implement the One Health concept to combat the COVID-19 pandemic with a holistic approach.

animals : an open access journal from mdpi

10.3390/ani11082170

PMC8388473

In recent years, the production and distribution of ZnO NPs have gradually increased. As the number of ZnO NPs containing products grows, and the release of these products into the environment—particularly to the aquatic environment—has increased, several questions about their toxic effects on aquatic organisms have arisen. In this study, we explore the embryotoxicity of ZnO NPs by using the newly introduced model organism Oryzias javanicus (Javanese medaka). We found that the 96 h LC50 of ZnO NPs on the embryo of Javanese medaka were 0.643 mg/L, 1.333 mg/L, and 2.370 mg/L in ultra-pure, deionized, and dechlorinated tap water. The toxicity of ZnO NPs increased as both the concentration and time of exposure increased. The results of this study demonstrate that ZnO NPs are extremely toxic for the early life stage of Javanese medaka.

(1) Background: Zinc oxide nanoparticles (ZnO NPs) are widely applied in various human products. However, they can be extremely toxic for aquatic organisms, particularly fish. This research was conducted to determine the LC50 of ZnO NPs on the embryos of Javanese medaka (Oryzias javanicus) in ultra-pure, deionized, and dechlorinated tap water; (2) Methods: The experiments were conducted in a completely randomized design (CRD) with three replicates for six treatments for acute (0.100, 0.250, 0.500, 1.00, 5.00, and 10.00 mg/L) exposures for each type of water; (3) Results: The LC50 of ZnO NPs at 96 h was determined as 0.643 mg/L in ultra-pure water, 1.333 mg/L in deionized water, and 2.370 in dechlorinated tap water. In addition to concentration-dependent toxicity, we also observed time-dependent toxicity for ZnO NPs. In addition, the sizes of ZnO NPs increased immediately after dispersion and were 1079 nm, 3209 nm, and 3652 nm in ultra-pure, deionized, and dechlorinated tap water. The highest concentration of measured Zn2+ in exposure concentrations was found in ultra-pure water, followed by deionized and dechlorinated tap water suspensions. Furthermore, Javanese medaka showed high sensitivity to acute exposure of ZnO NPs in all types of water.

1. IntroductionCurrently, several dangerous chemicals are considered a global threat to humans, other organisms and also the environment itself. Nevertheless, from time to time, the world is constantly generating and introducing large amounts of chemical substances into the environment. At the same time, the effects on organisms and the environment of these substances are not well known. Among evolving chemicals, nanoparticles (NPs) are one of those which are described as a particle with at least one dimension between 1 and 100 nm with different characteristics from bulk materials [1], and nanotechnology is known as the use of these materials [2]. Nanotechnology has recently developed as a rapidly growing market with efficient effects on major economic sectors with novel and unique properties that have been used in a diverse group of consumer goods such as agriculture, cosmetics, electronics, textiles, and pharmaceuticals [3,4,5]. Based on their composition, NPs can be classified into carbon-based NPs (carbon nanotubes and carbon black), inorganic NPs (generally this type of NPs contain metals (Al, Bi, Co, etc.) and metal oxides (ZnO, CuO Al2O3, etc.)), organic-based NPs (this type of NPs involve dendrimers, micelles, liposomes, and polymer NPs which are produced mainly through organic material, excluding carbon-based NPs and inorganic NPs), and composite-based NPs which are produced from the combination of NPs with other NPs or NPs with bulk materials (such as hybrid nanofibers) [5,6]. Among several NPs, ZnO NPs are known as one of the most efficiently used in the nano-scale range with a wide bandgap and large excitonic binding energy [7], high stability, anticorrosion and photo-catalytic properties [8], non-migratory, fluorescent, piezoelectric, absorptive, and scatters ultraviolet light [9], diverse nanostructures [10], and antimicrobial activity [11]. Zinc oxide NPs are already extensively implemented in consumer goods such as paints, UV filters, biosensors, paper, plastics, ceramics, building materials, rubber, power electronics, coatings, feed, photocatalytic, degradation of textiles, and printed matter [12,13]. ZnO NPs, which are the third most widely applied metal-based NPs with an approximate world-wide total production of 550 to 33,400 tons [5], can reach the environment, particularly the aquatic environment by (1) wastewater which contains the highest amount of ZnO NPs (0.3–0.4 μg/L), (2) direct use and (3) deposition from the air compartment [14,15].Once ZnO NPs are released to the aquatic environment, changes in their physico-chemistry occur, which modifies their environmental fate and toxicity to aquatic species. These changes mostly lead to a decrease in bioavailability and toxicity, although increases in bioaccumulation and toxicity were reported in some cases. One of the most important impacts on the fate of MeO-NPs in an aquatic ecosystem is the formation of a cluster of NPs called an aggregation/agglomeration. These alterations caused changes in the wide range of size distributions of ZnO NPs by forming aggregates, and some studies have reported that aggregations change the size of ZnO NPs 10-fold bigger than the primary ZnO NPs size [5,8]. The formation of aggregates of ZnO NPs are correlated to various parameters such as the presence of dissolved organic matter (DOM) in the surrounding media [12], dispersion method [8], pH, ionic strength [9], physico-chemical properties of ZnO NPs including particle size, shape [16], and surface properties such as surfaces modification [17]. Aggregation of ZnO NPs in the aquatic ecosystem will facilitate settling from suspension onto the bottom surface of the water body. Due to increased aggregation and sedimentation, estuarine and aquatic sediments have been proposed to be the endpoint for several NPs [5]. Most of the sedimentation of ZnO NPs started once they reached the water and occurred within 24 h, and then the sedimentation processes decreased over time [18]. Furthermore, Poynton et al. [15] showed that 97% of ZnO NPs dispersed in water settled out, and about 2% of Zn2+ was dispersed in the water. Another important transformation of ZnO NPs is their dissolution in the aquatic environment, which strongly affects their behavior in the environment. Surprisingly, data on the solubility of ZnO NPs are limited, and because of different laboratory conditions, such as the suspension medium, pH, salinity, dispersion methods, DOM, ionic strength and particle surface area, the reports appeared contradictory [1,3,10,15].Although ZnO NPs are widely produced and released in various industries, due to their toxic impacts on different aquatic organisms, they have been listed as extremely toxic [19]. Meanwhile, studies related to ZnO NPs toxicity on aquatic vertebrate organisms have concentrated largely on fish, in particular zebrafish. Several reports have shown that ZnO NPs can be highly toxic to zebrafish, particularly in the early developmental stages [20,21]. Furthermore, serious threats and higher toxicity compared to other NPs in aquatic environments have been reported in recent studies for ZnO NPs. For instance, Zhu et al. [22] reported that ZnO NPs showed higher toxicity compared to TiO2 NPs and Al2O3 NPs on the early life stage of zebrafish. Another study that has shown ZnO NPs are more toxic than TiO2 NPs is the study of Bhuvaneshwari et al. [23], who reported 27.62 and 71.63 mg/L for ZnO NPs and 117 and 120.9 mg/L for TiO2 NPs as 48 h LC50 on Artemia salina under pre-UV-A and visible light conditions. Meanwhile, the idea that water chemistry can affect the fate and behavior of chemicals, and their subsequent bioavailability to fish, is well established but, to date, there have been no systematic studies of the toxicity of ZnO NPs to the same species of organism in different types of water.Oryzias javanicus (Javanese medaka) belongs to the Adrianichthyidae family [24]. This species is widely distributed in Asian countries and highly adaptable to fresh, brackish, and saltwater [25]. The sensitivity of the species belonging to this family makes it an ideal test organism for toxicology and ecotoxicology studies. Recent studies have indeed used Javanese medaka as the test organism because of their high adaptability to both freshwater and saltwater, broad geographical range and availability throughout the year [26,27,28], short life span and life cycle, fast development [29,30], hardy, easy to identify and cultivate, short spawning period <1 min, and their transparent eggs [31]. These properties make it a suitable choice for studies, especially studies that involve early life stages. Hence, this study was conducted to determine the median lethal concentration (LC50) of ZnO NPs in ultra-pure, deionized, and dechlorinated tap water on the embryo of Javanese medaka.2. Materials and Methods2.1. Source of ZnO NPs and Test OrganismsZnO NPs (#677450 zinc oxide nanopowder, <50 nm particle size (BET), purity >97%) were purchased from Sigma-Aldrich, Missouri, United States. Adult Javanese medaka were collected from the estuary area in Sepang, Selangor, Malaysia. The fish were caught using scoop nets and immediately brought back to the laboratory and acclimatized in dechlorinated tap water with a 14 h/10 h light/dark cycle for at least 21 days. After acclimatization, the sexing process was carried out under a dissecting microscope. The sex of the fish was differentiated based on the characteristics described by Imai et al. [32]. Two males and four females were put in 3 L tanks and were maintained through the circulating system for breeding purposes. Consequently, to make them spawn daily, the photoperiod was maintained at 14 h light and 10 h dark, temperature (28–30 °C), dissolved oxygen (5.5–7.5 mg/L), pH (5.5–6.5), salinity (0 ppt). Fish were fed with newly hatched Artemia nauplii (Brine shrimp) larvae, since most recent studies reported that feeding Javanese medaka with newly hatched Brine shrimp resulted in active spawning with a high number of eggs [30,33,34].2.2. Physicochemical Characterization of ZnO NPsFor size verification, X-ray diffraction (XRD) was performed. The shape of ZnO NPs was visualized by transmission electron microscopy (TEM JEM-2100F, JEOL Ltd., Tokyo, Japan). Samples for TEM analysis were prepared from 10 mg/L suspensions of ZnO NPs on carbon-coated copper grids. Measurement of zeta potential and size distribution was carried out on solutions of ZnO NPs (1 mg/L) prepared in ultra-pure, deionized, and dechlorinated tap water, with samples taken rapidly after preparation. The size distribution and zeta potential were measured by dynamic light scattering (DLS) using the Malvern Zetasizer Nano-ZS instrument (Malvern Panalytical, Worcestershire, UK).2.3. Measured Exposure Concentrations and Zinc Ion ReleaseThe stock solutions of ZnO NPs (100 mg/L) were prepared in ultra-pure (18.2 MΩ cm), deionized, and dechlorinated tap water and then diluted to the exposure concentrations. To obtain a homogeneous suspension, the stock solutions were stirred with a magnetic stirrer for 30 min prior to use. In order to stimulate an environmentally relevant situation, the stirring method was used instead of utilizing surfactants or sonication. Range finding tests were conducted to determine the range for the acute toxicity tests. The nominal exposure concentrations of ZnO NPs in this study were (0.100, 0.250, 0.500, 1.00, 5.00, and 10.00 mg/L) in ultra-pure, deionized, and dechlorinated tap water. The concentrations of ionic Zn in ZnO NPs exposure concentrations were measured by ICP-MS. Samples for ICP-MS were taken rapidly after preparation. The ion release was shown as the measured zinc concentration (mg/L) of the total nominal exposure concentration of zinc.2.4. Toxicity TestsExperiments were conducted according to the Organization for Economic Cooperation and Development testing guidelines (OCED 2013) [35]. Newly spawned Javanese medaka egg clusters were carefully collected from the female’s body by hand, clusters of eggs were then separated with forceps and washed three times with saltwater, and the embryos at the blastula stage < 3 hpf were selected for further procedures. To start the 96 h exposure, the fertilized embryos were immediately transferred to 6-well plates, each well containing 10 embryos and 10 mL of exposure solution. Each well was one replicate and each concentration contained three replicates. Static toxicity tests were conducted after 24 h embryos were checked under a stereomicroscope (Olympus CX31 2D, Tokyo, Japan) and dead embryos were removed and recorded. Meanwhile, exposure solutions were removed and new stock and diluted solutions were prepared freshly. The light/dark ratio was 14 h/10 h and was maintained during the exposure. The water parameters were recorded throughout the experiments as follows: temperature at 26 ± 1 °C, salinity at 0 ppt, pH at 6.90 ± 0.4, and dissolved oxygen at 5.53 ± 0.6 mg/L.2.5. Statistical AnalysisTo determine the LC50, probit analyses were performed using log concentration in GraphPad prism version 8.0.2 for Windows (GraphPad Software, La Jolla, CA, USA).3. Results3.1. Characterization of ZnO NPSX-ray diffraction result confirmed the ZnO NPs with a crystal structure of 26 nm in primary size (Figure 1A). The TEM images (Figure 1B) revealed that most of the ZnO NPs in suspension had a hexagonal shape. The average hydrodynamic diameters of the ZnO NPs were 1079 nm, 3209 nm, and 3652 nm and the observed zeta potentials were −6.43 mV, 3.04, and 2.01 mV in ultra-pure, deionized, and dechlorinated tap water (Table 1).3.2. Dissolution of ZnO NPsWith increasing nominal concentration of ZnO NPs, the measured Zn2+ concentration also increased; however, different dissolution rates were observed for ZnO NPs in ultra-pure, deionized, and dechlorinated tap water. For instance, higher concentrations of Zn2+ were observed for ZnO NPs in ultra-pure water, which were 0.355 ± 0.064, 0.911 ± 0.010, 1.794 ± 0.136, 2.809 ± 0.154, 21.805 ± 0.417 and 42.235 ± 2.878 mg/L in the nominal concentrations (0.100, 0250, 0.500, 1.00, 5.00, and 10.00 mg/L, respectively) of ZnO NPs. However, at the same nominal concentrations, the measured Zn2+ concentrations were 0.917 ± 0.062, 1.054 ± 0.046, 1.454 ± 0.283, 3.073 ± 0.086, 20.295 ± 5.409 and 30.862 ± 0.860 mg/L, respectively, in deionized water and 0.711 ± 0.028, 1.211 ± 0.069, 2.457 ± 0.063, 3.485 ± 0.120, 7.935 ± 0.049 and 15.391 ± 0.268 mg/L, respectively, in dechlorinated tap water (Figure 2).3.3. Embryotoxicity of ZnO NPsThe mortality of Javanese medaka embryos increased as the concentration of ZnO NPs increased. For instance, mortality rates of Javanese medaka embryos exposed to ZnO NPs were: 3.33, 20.00 30.00, 83.33, 90.00 and 100.00%; 3.33, 6.67, 10.00, 33.33, 93.33 and 100.00%; and 6.67, 10.00, 13.33, 23.33, 83.33 and 96.67% in ultra-pure, deionized, and dechlorinated tap water at 0.100, 0.250, 0.500, 1.00, 5.00 and 10.00 mg/L of ZnO NPs, respectively. This indicates that ZnO NPs had a concentration-dependent toxicity on the embryos of Javanese medaka in all types of water.The lowest mortality was observed in 0.100 mg/L of ZnO NPs and did not increase throughout the experiment. Mortalities of Javanese medaka embryos exposed to ZnO NPs were less than 15% in all treatment groups at 24 and 48 h post-exposure (hpe). However, mortality increased sharply for 0.250, 0.500, 1.00, 5.00, and 10.00 mg/L of ZnO NPs at 72 and 96 hpe, which indicates that as well as concentration-dependent toxicity, toxicity increased with time for certain concentrations as shown in Figure 3.The lowest 96 h LC50 value determined by probit analysis for ZnO NPs on Javanese medaka embryos was in ultra-pure water (0.6438 mg/L) followed by deionized water (1.333 mg/L), and dechlorinated tap water (2.370 mg/L) (Figure 4).4. DiscussionIn the present study, the embryotoxicity of ZnO NPs on Javanese medaka was tested. Among several NPs, ZnO NPs are known as one of the most efficiently used in the nano-scale range with their unique characteristics ideally found in various applications in consumer goods such as cosmetics, textiles, rubber and electronic industries [9,10]. However, studies have shown that ZnO NPs are extremely toxic to fish, particularly at the early life stage, which causes several morphological deformities [19,36].The measured hydrodynamic diameters for ZnO NPs demonstrates that the hydrodynamic diameter of ZnO NPs in all types of water crossed the nanometer-scale immediately after dispersing, indicating that Javanese medaka embryos during these studies were exposed to both aggregates and well dispersed ZnO NPs. A similar finding was reported by Cong et al. [19], that the hydrodynamic diameter of ZnO NPs crossed the nanometer scale upon dispersion in filtered artificial saltwater. Furthermore, the observed zeta potential for ZnO NPs suspensions were between +30 mV and −30 mV, thereby suggesting that ZnO NPs suspensions were not stable based on the DLVO hypothesis [37], which revealed that the sedimentation of ZnO NPs aggregates had been unavoidable throughout the experiments.Differential dissolution of ZnO NPs in different types of water might be due to the presence of different amounts of ionic strength. High dissolution rates at low ionic strength were reported for ZnO NPs in previous studies. For instance, Li et al. [3] studied the effects of water chemistry on the dissolution of ZnO NPs and their toxicity on Escherichia coli and demonstrated that ZnO NPs had a concentration-dependent dissolution, and dissolution of ZnO NPs was reduced in the existence of ionic strength. A similar result was reported in the studies of Keller et al. [38] and Fairbairn et al. [39] who reported that the concentration of Zn2+ decreased as a result of increasing ionic strength in suspension. Zinc ions and ZnO NPs are two types of elemental zinc that can exist in water, soil, and organisms. In terms of water persistence, though, they have entirely different properties. Since Zn2+ is intrinsically persistent, it can be transformed into other compositions and can form zinc complexes with ions found in water, such as zinc chloro complexes or zinc hydroxide. Zinc oxide NPs, on the other hand, are not always persistent. Since the different types of water contain different types of ions, such as chloride, sulfate, and organic matter, an increase in ionic strength typically raises the degree of metal complication. Li et al. [9] compared the solubility of ZnO NPs in freshwater and saltwater and reported that ZnO NPs showed higher solubility in saltwater compared to freshwater, but due to an increase in ionic strength, the concentration of Zn2+ was lower in saltwater than in freshwater at the same concentration. Therefore, the solubility of ZnO NPs in different types of water was different.Concentration-dependent toxicity effects of ZnO NPs were reported in several recent studies. For instance, Zhu et al. [22] showed that during the 96 h exposure, the hatching of zebrafish embryos was not affected by 0.5 mg/L of ZnO NPs; however, the toxicity increased as the concentration of ZnO NPs increased, indicating that the toxicity of ZnO NPs had a concentration-dependent property. Bai et al. [10] on the other hand, reported that 50 mg/L and 100 mg/L of ZnO NPs caused mortality, and 1–25 ZnO NPs affected the hatching rate of zebrafish embryos in the E3 medium which ultimately caused abnormality during the 96 h exposure. Time-dependent toxicity of ZnO NPs was reported by Li et al. [9] who showed that the mortality rate of Mugilogobius chalae (yellow stripe goby) embryos in all ZnO NPs treatments was <20% on days 0–4, but increased sharply on day 5, at which time they increased to ≤50% for the 25 mg/L and 50 mg/L ZnO NPs concentration. Time-dependent toxicity for ZnO NPs was also reported on zebrafish embryos [10].The 96 h LC50 value of ZnO NPs on Javanese medaka embryos differed in different types of water. The lowest 96 h LC50 (0.6438 mg/L) value was observed in ultrapure water followed by deionized water (1.333 mg/L) and dechlorinated tap water (2.370 mg/L). This decrease in toxicity might possibly be due to an increase in aggregate size in the presence of high ionic strength for ZnO NPs. The DLS results showed that the size distribution of ZnO NPs in dechlorinated tap water was three-fold and ~one-fold higher compared to in ultra-pure water and deionized water. This demonstrates that the aggregate size of ZnO NPs increased with increasing ionic strength, which eventually decreased the toxicity of ZnO NPs. Many previous studies reported similar results for ZnO NPs. For instance, Young et al. [40] reported that due to the increase in the ionic strength of the suspension by increasing salinity, the size of aggregates of ZnO NPs increased and eventually decreased their toxicity to the marine diatom (Thalassiosira pseudonana). Another study that reported the size of aggregates of ZnO NPs increased due to higher ionic strength by increasing salinity of suspension is the study of Park et al. [41] who demonstrated that increasing salinity of the suspension affected the aggregation and dissolution of ZnO NPs and CuO NPs, and decreased their acute toxicity on Tigriopus japonicus. This correlation between the size of aggregates of ZnO NPs and ionic strength might be due to the attraction of van der Waals force, which takes place as a result of the combination of the electric double layers of particles, leading the particles to form larger aggregates [42]. The stability of the ZnO NPs suspension is determined by the combined effects of van der Waals attraction and electrostatic repulsion caused by an electric double layer of cations surrounding ZnO NPs in an aqueous medium. The electrical charge carried by ZnO NPs causes mutual electrostatic repulsion between nearest particles, and ZnO NPs can stay separate and stable in suspension if this electrical charge overcomes the van der Waals attraction force. However, a suspension with a high ionic strength induces compression of the electrical double layer, which decreases the electrostatic repulsion forces between the NPs and, as a result, encourages the NPs to aggregate and settle out of suspension [43].Dissolution of ZnO NPs is another phenomenon that might affect its toxicity to the embryos of Javanese medaka in different types of water. The dissolution of metallic oxide NPs serves a vital role in the determination of their toxicity. For instance, Auffan et al. [44] demonstrated that releasing metal ions from NPs can partially lead to their toxicity. Similar findings were reported in the study of Bai et al. [10]. Although the toxicity of ZnO NPs is predominantly related to the free Zn2+ in the aqueous solution, an increase in the ionic strength of the suspension could decrease the concentration of free Zn2+ and thus lower their toxicity. Similar findings were reported by Li et al. [3] who revealed that the concentration of Zn2+ in a ZnO NPs suspension decreased in the existence of ionic strength, which later on reduced their toxicity on Escherichia coli. Furthermore, the author also mentioned that during adsorption of Zn2+ by cells, the presence of cations in water such as (H+, Ca2+, Mg2+, Na+, and K+) may compete and thus lower the toxicity of ZnO NPs. According to the study of Hogstrand et al. [45] Zn2+ absorbs through a Ca2+ uptake mechanism and/or a Zn-specific absorption mechanism, where Ca2+ will inhibit Zn2+ uptake competitively in rainbow trout. Furthermore, Califford and McGeer [46] also demonstrate that the presence of Ca2+ and Mg2+ reduced the toxicity of Zn2+ on Daphnia pulex. A decrease in the toxicity of Zn2+ to the algae (Pseudokirchneriella subcapitata) in the presence of Na+, Mg2+, and Ca2+ was also reported by Heijerick et al. [47].We have observed lower LC50 values for the embryos of Javanese medaka compared to the LC50 values reported in previous studies for ZnO NPs. For instance, Zhu et al. [22] reported the value of 96 h LC50 as 1.793 mg/L after exposing the embryos of zebrafish to ZnO NPs in Milli-Q® water. Another study, which evaluated the acute toxicity of ZnO NPs at the early life stage of yellow stripe goby, by Li et al. [9], reported 45.40 mg/L as the 96 h LC50 value of ZnO NPs, which is higher than the LC50 value of this study. Furthermore, Saddick et al. [48] also reported that ZnO NPs were toxic to Nile tilapia and Red belly tilapia in deionized water, and calculated 5.5 mg/L and 5.6 mg/L as 96 h LC50. Other species of fish with different 96 h LC50 values have been summarized in Table 2.Similar to our results, different 96 h LC50 values were reported for the same species of fish in different types of medium in different studies. Zhu et al. [22] reported 1.793 mg/L as 96 h LC50 for ZnO NPs on the embryo of zebrafish in Milli-Q® water; however, Xiong et al. [20] reported 4.92 mg/L as a 96 h LC50 of ZnO NPs on the embryo of zebrafish in distilled water. However, Du et al. [49] studied the acute toxicity of ZnO NPs on zebrafish embryos in zebrafish culturing medium (E3 medium) and reported 60 mg/L as a 96 h LC50. Another study that reported that ZnO NPs were toxic toward zebrafish in early life stages (96-hpf) in pure water is the study of Wehmas et al. [50] who reported 2.20 mg/L as a 24-h LC50. However, in the same study, zebrafish embryos (8-hpf) were also exposed to the same ZnO NPs for 24 h and reported +50 mg/L as a 24 h LC50. Therefore, they concluded that as well as a medium dependent risk category, distinct life stages of the same species showed different sensitivities to the same ZnO NPs.5. ConclusionsFor the first time, we explored the toxicity of ZnO NPs and found that they are extremely toxic to Javanese medaka embryos. The 96 h of median lethal concentration (LC50) of the acute exposure of ZnO NPs were 0.6438 mg/L, 1.333 mg/L, and 2.251 mg/L in ultra-pure, deionized, and dechlorinated tap water. The mortality rate of Javanese medaka embryos increased as the concentration of ZnO NPs increased in all types of water, and there was a strong correlation between time of exposure and mortality of embryos. The toxicity of ZnO NPs was influenced by different types of water by affecting their size distribution, dissolution, and concentration of Zn2+ on the embryos of Javanese medaka. The LC50 value is a valuable criterion for nanoecotoxicity; it is not a representative concentration of contaminants in aquatic environments, but it is essential for indicating the toxicity of certain pollutants. Rather than using existing test organisms, Javanese medaka was used in this study as a new model organism of nanoecotoxicological exposure so that we could have existing indigenous organisms that live in tropical regions. As Javanese medaka show a higher sensitivity to ZnO NPs compared to other well-known model organisms, particularly zebrafish, the findings of this study can support the creation of the Javanese medaka as a novel model organism for tropical areas in aquatic nanoecotoxicological studies. In comparison to previous research conducted in saltwater, we observed high sensitivity for ZnO NPs in freshwater, which raises the importance of the physicochemical parameters of water on the toxicity of ZnO NPs. This indicates the need for a comparative study on the toxicity of ZnO NPs in freshwater versus saltwater. Moreover, further investigation needs to been carried out on the transcriptomic effects of ZnO NPs on this species.

animals : an open access journal from mdpi

10.3390/ani11061713

PMC8227745

A study was carried out to evaluate the effect of single cell protein (SCP) supplement as a protein source on nutrient intake, digestibility, nitrogen balance and in situ digestion kinetics in four Nili Ravi buffalo bulls. Four iso-caloric and iso-nitrogenous concentrates containing 3, 6, 9 and 12% of Saccharomyces cerevisiae-fermented citrus pulp were formulated and provided for 12 weeks. Chemical composition of fermented citrus pulp appeared as an excellent source of protein as no significant difference was observed on dry matter intake, digestibility of nutrients, SCP, ruminal pH and ammonia nitrogen. It is concluded that SCP could be used in the concentrate diet of ruminant up to 12%. Furthermore, the SCP has the potential of an alternative protein source in animal diet formulation.

A study was carried out to evaluate the effect of single cell protein (SCP) supplement as a protein source on nutrient intake, digestibility, nitrogen balance and in situ digestion kinetics in four Nili Ravi buffalo bulls. Four iso-caloric and iso-nitrogenous concentrates containing 3, 6, 9 and 12% of Saccharomyces cerevisiae-fermented citrus pulp were formulated. All animals were fed a ration with a concentrate/forage ratio of 50:50. Diets were provided ad libitum twice a day as a total mixed ration in a 4 × 4 Latin Square Design. Each experimental period lasted 3 weeks while the overall study 12 weeks. The first 2 weeks of each experimental period were used as adaptation period while the third week as collection period. Chemical composition of fermented citrus pulp appeared as an excellent source of protein. No significant difference was observed on dry matter intake, digestibility of nutrients and SCP among all the treatments. Moreover, no significant effect was observed on ruminal pH and ammonia nitrogen at different times. Rate of disappearance and lag time of in situ dry matter digestion kinetics remained nonsignificant regardless of SCP percentage. Based on results of similar nutrients intake, nutrient digestibility, and ruminal parameters it is concluded that SCP could be used in the concentrate diet of ruminant up to 12%. Furthermore, the SCP has the potential of an alternative protein source in animal diet formulation.

1. IntroductionSingle cell protein supplementation in the diet of small animals and ruminants has been widely acknowledged during the last decade [1,2,3]. As a single cell the Saccharomyces cerevisiae, a unicellular fungus belonging to the fungi kingdom, was selected. Although several yeast species are available on the market, Saccharomyces cerevisiae is considered as one of the best for culture production due to its growth and metabolic features [4]. Saccharomyces cerevisiae is a rich source of enzymes, vitamins and other unknown cofactors that increase the activity of microbes in the rumen [5,6]. It also has a good amino acids profile and is endowed with prebiotic activity [7,8,9]. It also has the ability to compensate vitamin and amino acid deficiencies [10]. Live culture of Saccharomyces cerevisiae chemically consists of 93% dry matter, 44.5% crude protein, 1.10% ether extract, 3.50% ash, 2.75% crude fiber and 1990 Kcal/kg metabolizable energy [11]. Moreover, it has a high biological value of protein that in turn improves the nutritional value of feed and makes it a valuable alternative to conventional protein sources [12].Saccharomyces cerevisiae has been added in ruminant diet to increase the number of ruminal bacteria and improve the dry matter intake along with digestibility of fiber and crude protein [13]. Supplementation of yeast culture was also shown to stimulate the growth of beneficial microorganisms in the rumen and reduce urinary nitrogen excretion [14,15,16,17]. Yeast supplementation also positively affects the feed intake and digestion process in the rumen [18]. Several studies indicate that yeast-fermented products can replace the conventional protein sources e.g., soybean meal, up to 75% in concentrate ration, which improves rumen fermentation and dry matter intake [19,20,21].Yeast culture reduces the accumulation of lactic acid and oxygen in rumen to improve fiber digestion and starch utilization [15], thus resulting beneficial for rumen fermentation and nutrient digestion [22,23,24].Yeast-fermented products have the ability to fully replace soybean meal in concentrated mixtures used for ruminants [25], increase the ruminal pH and ruminal fiber digestion rate extent, which ultimately improves the animal performance [26].Therefore, yeast-fermented products can be used as a nonconventional protein source in a concentrate ration without any negative impact on nutrients intake, digestibility, nitrogen balance and in situ digestion kinetics in ruminants. The present study aimed to investigate the effect of yeast-fermented citrus pulp as a protein source on nutrients intake, digestibility, nitrogen balance and in situ digestion kinetics in cannulated buffalo bulls.2. Materials and MethodsThe research study was conducted on four cannulated Nili Ravi buffalo bulls at Raja Muhammad Akram Research Center, University of Agriculture, Faisalabad. Operative procedures and animal care were performed in compliance with the national and international regulations. The protocol was examined and approved prior to the beginning of the study by the Veterinary Ethical Review Committee. The recommendations of the ARRIVE guidelines in animal research were also consulted and considered [27].Four iso-caloric and iso-nitrogenous concentrates containing 3, 6, 9 and 12% of yeast-fermented citrus pulp were formulated and represented as A, B, C, and D following the National Research Council guidelines [28] (Table 1). The yeast-fermented citrus pulp was prepared according the procedure of Sadh et al. [29].All animals were fed a ration of concentrate/forage ratio of 50:50 and the chemical composition of forage was 95.4 DM, 6.8% Ash, 93.2% OM, 6.64% CP, and 1.4% EE. The concentrate crude protein ratio was 18% while the crude protein of total mix ration was adjusted to 14%. Bulls were fed diets ad libitum twice a day as a total mixed ration in a 4 × 4 Latin Square Design. Each experimental period lasted 3 weeks. The first 2 weeks were used for adaptation while the third one as collection. Overall study lasted 12 weeks.Feed and feces were recorded daily and nutrient intake was calculated from the collected samples. Digestibility of nutrients was calculated by total collection method. During each collection period, complete urine and feces were collected on each day for nitrogen balance determination. For the first 2 days of each collection period, ruminal samples were collected from four different locations in the rumen at 3, 6, 9 and 12 h post feeding and pH values were determined. Portable pH meter (Orion portable Hanna HI 8314, Hanna industries, Romania model 230A, pH triode electrode; Orion Research, Inc., Boston, MA, USA) was used for immediate ruminal pH determination. Ruminal samples were squeezed through four layers of cheesecloth and 50 mL of the liquid were acidified with 3 mL of 6 N HCl to terminate fermentation. Samples were then used to determine ruminal ammonia by Kjeldhal’s method [30].In situ experiment was conducted to determine the digestion kinetics of yeast-fermented product using ruminally cannulated buffalo bulls. During this experiment, 10 × 23 cm nylon bags, with an average pore size of 50 μm, were used to determine dry matter (DM) and neutral detergent fiber (NDF) disappearance rate and extent. For each time point, 5 g of yeast-fermented product sample were weighed into bags, in triplicate. Two bags were used to determine DM and NDF disappearance while one bag served as blank. The bags were closed and tied with braided nylon fishing line. To remove soluble or 50-μm filterable materials, the bags were dipped into a specific amount of tap water for 15 min just before ruminal incubation. Weight loss due to dipping was expressed as pre ruminal dry matter disappearance. Three bags for each diet were incubated in the rumen of buffalo bulls for 0, 1, 2, 4, 6, 10, 16, 24, 36, 48 and 96 h intervals in reverse order and removed all at the same time. After rumen removal, bags were washed with running tap water until the rinse was clear. The bags were dried and residues were transferred to 100 mL cups and stored until the analysis. In situ digestion kinetics parameters, e.g., rate, lag and extent of DM and neutral detergent fiber NDF disappearance, were calculated for each period individually.The proximate composition of yeast-fermented citrus pulp was determined according to AOAC [30]. The collected feed samples and rumen residues were analyzed for DM and NDF. For DM and ash determination, hot air oven was used at 105 °C for 24 h and at 600 °C for 3 h, respectively. The nitrogen content was determined by Kjeldhal’s method [30] and CP was calculated as N × 6.25. The NDF was determined by the procedure described by Van Soest et al. with sodium sulphite [31]. Statistical AnalysisData were subjected to one-way analysis of variance using Latin Square Design and treatment means were compared by using Tukey’s multiple comparisons test. A p < 0.05 was considered significant.3. Results3.1. Nutritional CompositionNutritional values for fermented citrus pulp are given in Table 2 on dry matter basis. Results revealed that fermented citrus pulp had an excellent nutritional profile, resulting in a good source of protein, either extract and energy. 3.2. Nutrient Intake and Nutrient DigestibilityResults indicated no significant difference on dry matter intake among all the treatments. However, the highest numerical values for dry matter intake (DMI) were observed for diet D followed by diets C, B and A, respectively (Table 3). Use of yeast-fermented citrus pulp as a protein source in Nili Ravi buffalo bull’s diets did not affect crude protein intake. Neutral detergent fiber and acid detergent fiber (ADF) intakes remained unaltered among all dietary treatments. Dry matter digestibility showed no significant difference due to different levels of yeast-fermented citrus pulp as a protein source. Digestibility of CP, NDF and ADF also remained nonsignificant among all the treatments.3.3. Nitrogen BalanceInclusion of SCP in buffalo bull’s diets showed no significant difference among all dietary treatments (Table 4). Similarly, nitrogen in feces and urine remained unaffected among different treatments. Further, no significant effect was observed on nitrogen retention at different levels of yeast-fermented citrus pulp, despite the higher values observed in the diets.3.4. Ruminal pH and Ammonia NitrogenNo significant effect was observed on ruminal pH and ammonia nitrogen at 3 h postprandial and at all levels of yeast-fermented citrus pulp in buffalo bulls (Table 5). A similar trend was also observed on ruminal characteristics at 6 and 9 h post feeding among all dietary treatments.3.5. In Situ Digestion KineticsRate of disappearance and lag time of in situ dry matter digestion kinetics remained nonsignificant in buffalo bulls fed different levels of SCP supplement. In situ dry matter digestion kinetics extent also remained unaffected among the dietary treatments. Similarly, rate of disappearance and lag time of in situ NDF digestion kinetics remained nonsignificant among all dietary treatments. As for dry matter, no significant effect was observed on digestion extent of in situ NDF digestibility (Table 6).4. DiscussionCitrus pulp is a poor source of crude protein; however, fermentation can improve its value [32,33,34]. The increase in protein content after fermentation was presumably due to extracellular protein secretion, constituents metabolization or multiplication in the form of SCP by Saccharomyces cerevisiae [35]. Furthermore, the increase in growth and proliferation of the microorganisms in the fermenting substrates might possibly account for the apparent increase in the protein content of the fermented peels [36]. These results correlate with the findings of Oboh and Akindahunsi who observed increase in protein level in cassava products [37]. This could be due to possible secretion of some extracellular enzymes (proteins) such as amylase and cellulase into the substrates, which break the starch and other polysaccharides into simpler sugars that are easily metabolized by yeast as a carbon source. As far as concerns the nutrient intake, results of the current study are in accordance with the findings of Wanapat et al. who observed nonsignificant effects on dry matter intake after replacement of soybean meal with yeast-fermented cassava chips concluding that soybean meal could be fully replaced by yeast-fermented cassava chips without any adverse effect [38]. Similarly, Boonnop et al. reported that yeast-fermented cassava pulp could fully replace soybean meal without any negative effect on nutrient intake [25]. Additionally, Gobindram et al. investigated the effect of dried citrus pulp on the diet of lambs and concluded that dried citrus pulp had no significant effect on dry matter intake [39]. However, Williams et al. found that supplementation of yeast (10 g/d) in the diet of dairy cows increased dry matter intake [6]. Similarly, in dairy cows, Putnam et al. reported that supplementation of yeast culture (10 g/d) increased dry matter intake as compared to control group [40]. Pinos-Rodriguez et al. observed that supplementation of Saccharomyces cerevisiae increased DMI in ruminants [41]. Crosswhite et al. found that DM intake was higher in animals fed a diet supplemented with dried citrus pulp [42]. The reasons for increased intake might be likeness of animals for yeast-fermented citrus pulp due to its specific smell and taste as well as the better palatability of citrus pulp [43].Results concerning nutrient digestibility are in close resemblance with Khampa et al. who observed that animals fed yeast-fermented cassava chips had no significant effect on nutrient digestibility [20]. Similarly, Wanapat et al. found that addition of yeast-fermented cassava chips in the diet of animals had no significant effect on DM and NDF digestibility [38]. Studies on yeast-fermented cassava pulp substitution for soybean meal in the diet of ruminants also indicated no significant effect on DM, NDF and ADF digestibility [25]. Animals fed dried citrus pulp had no significant effect on nutrient digestibility [44]. De Lima et al. found that supplementation of dry yeast had no significant effect on DM, NDF and ADF digestibility [45]. These studies indicated that microorganism and substrate alone also have no adverse effect on animals’ performance. Conversely, Ghazanfar et al. reported that addition of Saccharomyces cerevisiae alone in the ration improved the digestibility of DM, CP, CF, NDF and ADF as compared to control group [46]. Ullah et al. also reported positive influence of Saccharomyces cerevisiae on nutrient digestibility [47]. The increased digestibility could be attributed to the increased nitrogen content of the rumen, which improved the growth of microbial population and led to increase in digestibility. In addition, Haddad and Goussous (2005) observed that supplementation of yeast culture improved CP and NDF digestibility compared to control group [48]. This can be ascribed to the increased concentration (5–40 times) of cellulolytic microorganisms in the rumen of yeast-supplemented animals rather than the nonsupplemented ones, resulting in a higher nutrient digestibility [14]. Our results regarding rumen characteristics in buffalo calves are in agreement with Wanapat et al. who reported that the addition of yeast-fermented cassava chips in the diet of animals had no significant effect on rumen pH [38]. Similarly, Khampa et al. also found that animals fed yeast-fermented cassava chips had no significant effect on ruminal pH and ammonia when compared with other conventional expensive protein sources (rice straw and rice bran) [19]. Other studies on crossbred native cattle, also observed that supplementation of yeast-malate-fermented cassava pulp and cassava as well as yeast-fermented lemon pulp did not induce any significant difference in the rumen pH regardless of treatments [20,49]. Conversely, Dolezal et al. found higher ruminal pH when yeast culture was added in the diet of dairy cows [5]. Boonnop et al. reported that yeast-fermented cassava pulp substitution for soybean meal in the diet of ruminants significantly increased ruminal ammonia regardless of treatments [25]. Dealing with nitrogen balance, our results are in line with other authors who observed that yeast itself and yeast-fermented product had no significant effect on nitrogen balance [50,51]. This might be due to the nonsignificant effect of yeast on nutrient intake and digestibility. However, Sawsan et al. found that addition of yeast culture in the lambs’ ration had more nitrogen balance as compared to control group [52]. Lambs had higher nitrogen balances raised on ration supplemented with yeast culture [53]. The higher nitrogen balance may be due to higher production of microbial protein synthesis as a result of yeast culture [54]. In situ digestion kinetics results revealed that SCP supplement had no significant effect on rate of disappearance, lag time and extent of digestion. Results are in the line with the findings of Lehloenya et al. who reported that supplementation of yeast culture in the diet of steers had no significant effect on ruminal digestion kinetics [51]. Doreau and Jounay found that addition of yeast in the diet increased ruminal dry matter content [55]. However, DM and NDF degradability was not significantly improved. Similarly, Olson et al. observed that supplementation of yeast culture did not affect rate or lag time of NDF disappearance [56]. Corona et al. recorded that animals fed yeast culture with basal diet (sorghum grain and corn stovers) had no significant on DM and NDF degradability [57].5. ConclusionsBased on our results about similar nutrients intake, nutrient digestibility, and ruminal parameters it is concluded that yeast-fermented citrus pulp could be used in the concentrate diet of ruminants. Our results indicated that yeast-fermented citrus pulp can be used successfully up to 12% of the concentrate in the diet of bulls without any adverse effect on growth performance and feed intake. Thus, the yeast-fermented citrus pulp holds the potential of an alternative protein source and economic ingredient in animals’ diet.

animals : an open access journal from mdpi

10.3390/ani12020216

PMC8773261

Minimising stress in intensive pig farms is paramount to raising immunocompetent pigs. This entails providing the pigs with living conditions (from birth to the point of slaughter) free of pain, stress, and suffering and simultaneously providing conditions that generate positive affective states. Our review aims to study the relationship between chronic stress, illnesses, their impact on antibiotic use (AMU), and potential housing and management improvements to tackle stress and AMU. According to the literature, pigs kept in crowded, barren conditions, with poor microclimatic conditions, and subject to painful and stressful weaning practices present redirected behaviours, poor immune-competence, and weaker bodies. In turn, pigs are more vulnerable to circulating pathogens and severe secondary infections, which is conducive to high AMU for the sake of the animals’ health. Simultaneously, we compiled a list of possible solutions for the current poor environment and practices, including a call for the pig industry to broaden its concept of animal welfare beyond the current biological/productivist scope. We propose that advocating for an industry with enhanced animal welfare is a crucial response to the international call to combat antimicrobial resistance and the social demand for ethically sustainable animal production.

Preventative measures, such as biosecurity and vaccinations, are essential but not sufficient to ensure high standards of health in pig production systems. Restrictive, barren housing and many widely used management practices that cause pain and stress predispose high-performance pigs reared in intensive systems to disease. In this context, antibiotics are used as part of the infrastructure that sustains health and high levels of production in pig farms. Antimicrobial resistance (AMR) is a global emergency affecting human and animal health, and the use of antibiotics (AMU) in intensive livestock farming is considered an important risk factor for the emergence and spread of resistant bacteria from animals to humans. Tackling the issue of AMR demands profound changes in AMU, e.g., reducing their use for prophylaxis and ending it for growth promotion. In support of such recommendations, we revise the link between animal welfare and AMU and argue that it is crucial to sustainably reduce AMU while ensuring that pigs can live happy lives. In support of such recommendations, we aimed to revise the link between animal welfare and AMU in pigs by analysing stress factors related to housing and management and their impact on pig welfare. In particular, we reviewed critical management practices that increase stress and, therefore, pigs’ susceptibility to disease and reduce the quality of life of pigs. We also reviewed some alternatives that can be adopted in pig farms to improve animal welfare and that go beyond the reduction in stress. By minimising environmental and management stressors, pigs can become more immunocompetent and prepared to overcome pathogenic challenges. This outcome can contribute to reducing AMU and the risk of AMR while simultaneously improving the quality of life of pigs and, ultimately, maintaining the pig industry’s social license.

1. IntroductionAnimal welfare, together with environmental issues, is one of the biggest challenges of agriculture in the 21st century [1]. Significant advances in the scientific and legal recognition of animal sentience and welfare have been achieved in the past few decades that guide this discussion. The OIE encompasses the basic elements that constitute animal welfare in its Terrestrial Animal Health Code, stating that “an animal experiences good welfare if the animal is healthy, comfortable, well-nourished, safe, is not suffering from unpleasant states, such as pain, fear, and distress, and is able to express behaviours that are important for its physical and mental state” [2]. However, intensive pig production systems, in general, fail many of these goals and offer few conditions for animals to experience positive affective states. As a result, many pigs spend their entire lives in conditions that do not ensure “a life worth living” [3]. Confinement housing limits free movement, the expression of highly motivated natural behaviours, such as nesting and rooting, and socialisation; additionally, pigs are exposed to painful management practices from moments after birth [4,5]. As a result, the pigs are stressed and more vulnerable immunologically and predisposed to contract environmental pathogens [6]. In addition, high stocking densities contribute to the spread of respiratory and enteric diseases. Altogether these factors contribute to the use of antibiotics (AMU) to control and prevent infectious outbreaks on farms [7,8]. The global consumption of veterinary antimicrobials is projected to increase 11.5% by 2030, over the estimated 93,000 tonnes used in 2017, when 10 countries used 75% of all veterinary antibiotics used in animal production (China = 45%; Brazil = 7.9%; the United States, Thailand, India, Iran, Spain, Russia, Mexico, and Argentina) [9]. Several countries have banned the use of antibiotic growth promoters and have limited active principles for human use only [10]. However, given the high use of antibiotics for disease prevention, this ban may be insufficient to reduce AMU [11]. Legislation restricting AMU in livestock production has changed attitudes and practices towards non-therapeutic AMU, most notably in the EU and particularly in some countries (see for example [8]), but the use of veterinary antibiotics in the world is still quite expressive. For example, in a survey of pig herds in Germany, the authors identified that pigs with an expectation of 200 days of life received antimicrobials for 48.5 days [12]. A Brazilian study identified that pigs received an average of 7 different antibiotic active principles during 73.7% of their life [13].The contribution of AMU in intensive pig farming to the emergence of antibiotic-resistant bacteria in humans raises important ethical, social, and public health concerns [14,15]. Resistance is a spontaneous process of bacteria that can be accelerated by the inappropriate use of antibiotics [16]. Resistance genes can be spread by vertical transmission, where the original bacterial cell transmits the resistance determinant to the offspring, or via horizontal, inter and intra-species transmission [17]. The potential for dissemination is greatest through horizontal transmission due to the mobile genetic elements that can spread in the environment and be incorporated by other bacteria, making animals highly relevant antibiotic resistance (AMR) agents [16,17]. AMR can be spread via direct or indirect contact between humans and farm animals or biological substances or via the consumption of contaminated food products; additionally, veterinarians, farmers, abattoir workers, and food handlers and their families may be an entry route of resistant bacteria into the community and health care settings [18]. The transfer of multi-resistant bacteria between animals and humans is a critical concern, considered by health agencies as a worldwide public health emergency [19]. For this reason, international leaders have called for an urgent reduction in AMU in livestock and the development of sustainable food systems [20].To promote strategies for prudent AMU and minimise AMR, the FAO, WHO, and OIE have created a three-pronged approach—One Health—that links aspects of human, animal, and environmental health into transdisciplinary public policies. One Health is a holistic health concept that proposes viewing human and animal health as interdependent and simultaneously connected with each other and with the ecosystems in which they coexist in a balanced relationship [21]. The One Welfare concept is based on the understanding that good animal welfare can reflect upon humans and the environment by ensuring food safety, improving human health, environmental sustainability, worker safety, rural development, gender equality, and social justice [22]. The aims of this review are to uncover the relationship between animal stress and health; to identify the main stressors in specific periods of the life of pigs reared in intensive systems and discuss how they affect pig welfare and health; and, finally, to point out some interventions that can improve the welfare and health of pigs and, potentially, contribute to the goal of reducing AMU in pig farming.2. Stress as a Trigger for DiseaseUnderstanding the physiological processes underlying the stress response and how they influence the pig’s immune system and health can shed light on the relationship between pig welfare and AMU. Therefore, we will define some terms that are relevant for this review.The stability of life-sustaining physiological parameters (pH, oxygenation, temperature, blood glucose) is referred to as homeostasis [23,24]. When an individual’s homeostasis is threatened in some way, e.g., by environmentally adverse situations, the homeostasis imbalances are compensated through a process called allostasis, which mobilises resources, such as energy, for the short-term adaptation/acclimatisation of the organism [23]. Evolutionary mechanisms of allostasis trigger the stress response through the autonomic nervous system and the hypothalamus–pituitary–adrenal axis (HPA). This prepares an alarm response, which is activated by the release of adrenocorticotropic hormone (ACTH), which, in turn, stimulates cortisol secretion, with metabolic, cardiovascular, and immunological effects. The excessive activation of allostatic mechanisms interferes with the basic energy mechanisms that maintain growth, reproduction, and the immune system, resulting in harmful stress (herein referred to as distress) that causes suffering and reduces animal welfare [6,25]. According to Moberg and Mench [6], the stress response can be divided into three stages: the recognition of a stressor, the biological protection against that stressor, and the consequences of the stress response. The consequences of the stress response will determine whether the animal is undergoing a temporary negative experience (i.e., acute stress) or suffering from chronic stress, the latter having the most damaging effects on the animal’s health and welfare [6].Psychological stressors (e.g., distress, frustration, boredom) may be as damaging to the immune system as pathogenic aggressors. The perception of a threat stimulus by the central nervous system triggers a biological defence that activates four types of responses: a behavioural response, an autonomic nervous system response, a neuroendocrine response, and an immune response [6]. Abnormal behaviours in pigs (e.g., belly-nosing, tail and ear biting, aggressiveness) are examples of behavioural responses to chronic stress [25].Fear plays an important role in helping animals cope with environmental stressors by motivating them to avoid potentially dangerous situations [26]. Fear influences animal performance and welfare via a classical stress response that involves physiological responses that aim to provide energy for immediate use by the body in preparation to flee or face aggression [26]. The reduction in staff/animals derived from intensification and the restriction of contact with humans with the advent of automated systems have contributed to more aversive reactions in routine management, poor handling, and negative interactions [27]. The intensification of livestock production also added problems, such as unskilled, often unmotivated workers due to high turnover, low pay, and lack of sufficient training [28,29]. Therefore, negative human–animal interactions at the farm may contribute to increased susceptibility to disease by activating energy-costly stress responses, especially when these interactions are persistent or frequent.According to the resource allocation theory, the animal metabolism will always spend the least amount of resources, selecting which metabolic function to benefit through a partition of metabolic resources; when certain resources are consumed by a given metabolic function, they are not available for other functions [30]. Pigs that suffer from chronic stress have a reduced natural ability to mount a successful response to an immune challenge [31]. Additionally, pig strains genetically selected for high production performance are more susceptible to disease because they allocate metabolic resources to meet physiological demands at the expense of the immune system [32]. For example, pigs that need to spend more metabolic resources for growth will have fewer nutrients available for the immune system, increasing their vulnerability to disease. Likewise, when the protein synthesis necessary for rapid growth, reproduction, and immune processes is depressed by chronic stress, energy reserves are mobilised [6]. In normal situations, this response favours the survival of the individual; however, chronic stress can be harmful to the organism due to its continuing nature.3. Sources of Stress in Pig Farm ManagementThe concepts and physiological mechanisms that we have reviewed shed light on the mechanisms that make pigs exposed to multiple stressors more vulnerable to disease. In this section, we will address some of the main stressors that challenge pigs reared in intensive conditions, dividing them into two types: those derived from the housing environment and those generated by management practices typically adopted in intensive pig farming (Figure 1). The first type is related to the physical limitations imposed by intensive housing, such as restriction to movement, socialisation and expression of natural behaviours, and unfavourable climatic conditions, which can cause discomfort, injuries, lameness, and abnormal and stereotyped behaviours. The second covers sow feeding management, prenatal stress, neonate management, weaning, early transport, mixing unfamiliar animals, mutilations, and human–animal interactions. When relevant, we point to the relationship between these stressors, disease, and AMU.3.1. Housing Stressors3.1.1. Housing That Limits the Ability of Pigs to Move and Express Natural BehavioursIntensive pig production models have been designed to raise as many animals as possible in small spaces and with short production cycles. In these systems, high stocking density often goes hand in hand with barren environments. Pigs are reared in barren housing environments in the breeding, weaning and fattening phases. Monotonous environments generate boredom, a negative emotional state [33]. High stocking densities exacerbate the problems of barren environments, resulting in aggression and redirected behaviours performed on conspecifics [34].The frustration associated with lack of environmental stimuli and the inability to root can manifest in behaviours such as tail and ear biting of pen mates [35], a major welfare problem in growing and fattening pigs and an important source of severe infections and abscesses [36,37]. Damage from tail biting can range from a bite mark to a serious injury and, in more severe cases, it can cause the bitten pig to die [37] and is a reason for AMU [36,38]. Partial tail docking of piglets at birth is the most commonly used practice to prevent tail biting [37]. Nevertheless, it has been shown that tail docking is not sufficient to eliminate biting behaviour when pigs are challenged by a stressful environment [37], and in some cases, when piglets have the tail docked, the behaviours may be redirected to other parts of the body [39].Individual confinement is used to house breeding sows, in many countries for their entire productive life. Gestating and lactating sows and boars are housed in barren crates where they cannot walk or turn around and their ability to perform highly motivated behaviours is restricted [40,41]. This housing facilitates feeding and management and optimises space use, but given that it limits the foraging behaviour, it causes chronic hunger and stress in gestating sows, which is expressed in abnormal and stereotypic behaviours [41]. Gestation crates have been increasingly banned in some countries but continue to be used in many parts of the world [42]. Farrowing crates, designed to facilitate management and to minimise piglet crushing, are the most common housing for farrowing and lactating sows in commercial farms in most countries [43]. Farrowing crates deprive sows of nesting, a highly motivated natural behaviour that has not been changed in modern pig lines genetically selected for productive traits compared to their predecessors [44]. Sows confined in farrowing crates show restlessness, frequent changes in body position, intermittent grunts, grinding teeth, and bar biting and biting other parts of the crates [45]; this housing can also generate frustration and aggressiveness in pre-parturient sows [46].The lack of movement resulting from permanent confinement has several health consequences. It can lead to poor cardiovascular function and bone and muscle weakness; in heavy pigs, it can also predispose to locomotor disorders, such as lameness [47]. In sows, lameness is an important predisposing factor for urinary tract infections. As pregnancy progresses, the sows become heavier and may have difficulty moving because of the pain, which predisposes them to remain in the sitting dog position for longer periods and reduce water consumption; this often leads to infrequent urination [48,49], which, together with faecal contamination of the perineal region, predispose sows to bacterial urinary infections [48], reported as the main cause of prophylactic AMU in pregnant sows [7,50]. These infections predispose animals to reproductive disorders, such as reduced litter size, return to oestrus, abortion, anoestrus, and postpartum dysgalactia syndrome [48], which also lead to increased AMU [51].3.1.2. Housing That Causes Thermal StressThe thermal comfort zone, i.e., the ambient temperature range in which the thermoregulatory effort is minimal, varies among individual pigs according to the amount of endogenous heat produced and the environmental conditions. Selection for lean tissue changed the proportion between body protein and fat, with protein accumulation generating more body heat, increasing pigs’ susceptibility to variations in temperature [52]. The general physiological response to thermal stress is similar to the chronic stress response discussed earlier in this review [6]. Two harmful physiological mechanisms result from thermal stress: the first mechanism is associated with a hormonal imbalance; the second mechanism involves an increase in pro-inflammatory cytokines that compromise intestinal integrity, alter immune function, and predispose pigs to infections [53,54]. Heat-stressed pigs are more prone to enteritis. To dissipate body heat, blood flow from the viscera to the periphery deviates, causing intestinal hypoxia, ATP depletion, and oxidative stress of enterocytes [53]; this also destabilises the intestinal barrier, making it permeable to Gram-negative bacteria and other disease-causing antigens [55,56]. Under low temperatures, ammonia concentrations within the housing facilities increase, which causes irritation and changes in the respiratory mucosa and leads to respiratory infections [57], one of the main causes of AMU [38]. Exposure to both cold and heat is an important stressor associated with the transport of weaned piglets. While high temperatures during travel may increase the risk of dehydration [58,59], cold-exposed piglets enter hypothermia and take time to recover when they reach their destination [58]. Moreover, thermal variations during travel can increase the stress level of piglets and affect their postweaning recovery [59].3.2. Common Management Practices as Stressors3.2.1. Feeding Strategies as a Source of StressFeed restriction in pigs is a management practice widely used to limit weight gain in breeding sows or for compensatory gain purposes in fattening pigs. Sows’ diets are designed to limit caloric intake and excessive weight gain during gestation [60]. Because they are usually fed twice a day and consume the low-fibre concentrated food quickly, sows remain hungry and highly motivated to seek food [61]. Chronic hunger stress can induce the expression of redirected and stereotyped oral behaviours [60]. In many cases, sows are housed in gestation crates, which adds to the stress caused by food deprivation; unable to forage, stressed sows bite cage parts, exhibit sham-chewing, and smell and lick the floor and other parts of their cages excessively [61]. In group housing, deficiencies in feed management can lead to social stress and an increase in competition for feed, resulting in agonistic interactions, injuries to the vulva and tail biting [60,61].Compensatory growth is a physiological phenomenon that occurs when an animal that has undergone a period of nutritional stress is fed ad libitum. Feed restriction during the growing and fattening periods is conducted by reducing the amount of feed or specific nutrients in the diet, as a way to decrease fat in pig carcasses at slaughter, or to stimulate compensatory weight gain in low-birth-weight piglets, improving feed conversion and reducing feed costs [62]. Providing low amounts of food or nutrients exposes pigs to unnecessary stress. The impact of these practices on pigs’ welfare needs to be investigated, e.g., the potential for hunger to increase activity, aggression, and tail biting.Lastly, long fasting periods and chronic hunger, together with environmental thermal variations and disease outbreaks, may lead to gastric ulcerations [63,64]. The prevalence of gastric ulcers at slaughterhouses can vary between 32% and 65% [64], but this figure may be underestimated, as ulcers are often subclinical. Other risk factors for gastric ulcerations include feed particle size, gastric microbiota composition, hormonal changes, Helicobacer suis infections, and low birth weight [64,65,66].3.2.2. Early Life ManagementPrenatal StressThe gestation environment may have repercussions on piglet welfare and health, given that prenatal stress in sows can impair growth and modify immune function, stress reactivity, and the behaviour of the offspring [67,68]. Piglets born to sows stressed during gestation have reduced immune capacity and are more susceptible to infections during the lactation and pre-weaning periods [67]. Stress in gestating sows results in high levels of glucocorticoids that cross the placental barrier and may affect the foetal HPA axis maturation through hippocampal cell death and loss of cognitive functions [69]. Prenatal stress can also alter the offspring phenotype, e.g., the daughters of stressed sows are more anxious, restless during farrowing, more reactive, and bite their piglets more [69].Neonatal ManagementNursing is a critical period for the survival of piglets when colostrum is the main source of antibodies, heat, and energy for the newborn [70]. The supply of colostrum in the first hours of life is essential for intestinal protection and passive immunity to piglets [71]. Piglets of different weights, typical of large litters, do not receive the same amount of colostrum. Weak or small piglets are especially susceptible to neonatal diarrhoea [72,73]. The main forms of diarrhoea control are vaccines and antibiotics, in particular penicillin and macrolides [12]. In Brazil, the use of injectable antibiotics in newborn piglets to prevent neonatal diarrhoea and other infections is commonly reported [13,50]. However, this practice may have deleterious effects on the intestinal microflora and the immune system of piglets, as it has been shown that early AMU can have a programming effect on the immune system. Gut dysbiosis in early life can promote an exacerbated inflammatory reaction, increasing the risk of colitis and impairing the immune response throughout life [74,75].Cross-Fostering and Artificial RearingCross-fostering and artificial rearing are two practices used to help small and slow-growing piglets gain weight. Cross-fostering consists of separating the newborn piglets by weight and distributing them in nursing sows according to piglet size and sow’s milk production, i.e., to equalise litters [76,77]. Artificial rearing is conducted with automatic systems that provide heat and feed to 2 to 14-day-old piglets [78]. Additionally, in some farms, piglets may be weaned 7 to 21 days after littermates and maintained with a nursing dam or nursed artificially, mixed with younger piglets of a similar weight [71], posing a disease risk to the younger pigs they get mixed with [79]. Hyperprolific sows, increasingly used in intensive farms, generate more viable eggs; however, the limitations of intrauterine space increase foetal competition, which is reflected in a greater number and proportion of piglets with low birth weight [80], which exacerbates the use of cross-fostering and artificial rearing practices [81]. However, cross-fostering and artificial rearing may have negative impacts on the health and welfare of piglets [78,79].During lactation, the sow transfers microorganisms to piglets, which is essential to the establishment of permanent intestinal and respiratory microbiomes that will assist in the maturation of the piglets’ immune system [82,83]. Piglets deprived of maternal contact and reared artificially present reduced pulmonary development, respiratory immune response and microbiome of the lungs [82,84].The recommendation is that cross-fostering is performed between 14 and 24 h postpartum to allow colostrum ingestion by piglets and the establishment of the teat order. Piglets transferred between 2 and 7 days postpartum or those transferred into groups with older piglets have difficulty integrating and exhibit more ambulation and vocalisations, taking a longer time to suckle compared to piglets transferred to younger foster litters [77]. Newborn piglets have not yet received maternal antibodies, and by mixing them with older piglets, they are exposed to pathogens to which they are not protected, increasing the risk of infection [85]. Early cross-fostering may be also harmful to piglets because colostrum production and the concentration of colostral immunoglobulins reach their peak within 14 h postpartum; thus, piglets that are fostered before this time will not have received sufficient colostrum, which makes them more susceptible to environmental pathogens [86]. However, in practice, cross-fostering is conducted after 7 days postpartum, or even later, and sometimes multiple times (e.g., [76,87]). The potential for transmission of pathogens in late cross-fostering is highly relevant to this discussion. Additionally, with each group change, the teat order needs to be re-established, which can be stressful and have detrimental effects on piglet survival, growth, and behaviour [71]. Growth may be impaired in adopted piglets due to fights over teats and shorter feeding bouts [88].Weaning StressWeaning is one of the greatest stressors in a piglet’s life [89] and one of the main risk factors for diarrhoea in the postweaning period [73], which contributes to a large proportion of AMU in pig farms (e.g., [50,90,91,92,93]). The time the piglets stay with their mother has important physiological and psychological effects on the piglets’ development. Under natural conditions, the piglets are gradually weaned, completely separating from their mother between 17 and 20 weeks of age [94]. Under typical commercial conditions, this process is conducted abruptly, between 3 and 5 weeks of age. Such a strategy, widely practised in intensive farms, eliminates or shortens important stages of the physiological and emotional development of piglets [89]. Early weaned piglets are subjected to the simultaneous social and psychological stress caused by losing milk, being socially separated from the mother and the siblings, and moved to a new environment, where they are usually mixed with unfamiliar animals and often transported between farms [87,89]. Biting, nosing, and abnormal behaviours are common among weanling piglets and constitute redirected sucking behaviours associated with early weaning, barren environments and hunger, and possibly have a genetic component; these behaviours can result in skin lesions on the recipient’s belly and flank, causing skin injuries, pain, and difficulty resting [95].From an emotional point of view, the early negative experience of maternal separation has effects on the hippocampus, so early weaned piglets have behavioural and cognitive impairment [96]. Sick and feverish piglets, in an attempt to conserve body energy, reduce their activity and remain lying down without feeding; however, weaning management prevents piglets from adopting such energy conservation strategies [89], making them more vulnerable to infections. Psychological and physical stressors associated with weaning imply energy expenditure and reduced food consumption, resulting in body weight loss in the first week after weaning [73,97]. All these factors, added to the lack of familiarity with solid feed, can lead to transient anorexia, intestinal inflammation, gut microbiota disorders, and behavioural disorders that result in a high occurrence of diarrhoea [73].Transportation of Young PigsWith the growing trend of pig production being conducted at specialised breeding sites, piglets may be transported for many hours after weaning to their destination at fattening units [87]. Every year millions of weaned piglets as young as 17 days of age are transported over long distances. The way this transport is carried out can have a significant impact on the welfare of these piglets [98]. There is a knowledge gap regarding this topic, as the scientific literature refers more often to the transport of pigs at slaughter age. However, it is understood that the stress factors during transport are the same in young and adult pigs, with the aggravation of the frailty of younger animals and the concurrent weaning stress. Transport stress is acute and may be followed by dehydration and protein catabolism [99]. Some stressors associated with transportation are temperature variation, the mixture of unknown animals’ hunger and thirst, loading and unloading, vibration, and noise [59]. Travel time affects the welfare of piglets during and after transportation [100]. For example, piglets transported for long journeys between 12 and 24 h are more prone to dehydration [98]. Transport speed can also be detrimental to the piglets; with fast motion, the piglets often lie down and stand up, indicating imbalance and vulnerability to falls [99]. Fasting associated with transport also causes deleterious effects on the health of weaning piglets. Pigs can lose about 4% of their body weight fasting for 18 to 24 h, causing catabolism of body reserves over 24 h [99,100,101]. Importantly, transport can be a pathogen carrier. Reducing vertical disease transfer, increasing productivity and overall efficiency of the farm are some justifications for the practice of site segregation of piglets [59]. However, the mixture of piglets from various origins is a source of pathogen transfer. Additionally, another epidemiological aspect to be considered is the emission of contaminating particles and the spread of resistant bacteria during transport [99]. The transmission of disease through transport and mixture of piglets of different origins and the impact on AMU and AMR are important knowledge gaps.3.2.3. Painful Procedures and Parturition as Sources of Pain in PigsMost piglets undergo several painful management practices during the first days after birth, mostly without any use of pharmacological tools to avoid or reduce the pain [5]. The main painful practices are teeth clipping or resection, tail docking, ear notching for identification, iron injection, and castration of male pigs [102]. These procedures, which are usually conducted simultaneously, are regarded within the industry as necessary to minimise problems caused by intensive production systems, such as large litters, high stocking density, successive social mixtures, together with lack of contact with soil and barren environments.Teeth clipping is the removal of canine teeth using pliers or other sharp objects. This practice, which is conducted to minimise biting injuries to the sow’s udder or possible fights over teat disputes between littermates [103], is extremely painful [5]. Teeth clipping causes oral lesions in piglets due to tooth fragments and the exposure of the dental pulp, predisposing to gingivitis [103]. An alternative to this practice is the abrasive grinding of the sharp end of the teeth using an electric whetstone grinder. Teeth clipping and wearing increase the piglets’ cortisol levels [102] and can be a gateway to neonatal infections [103]. Although the bites caused by piglets’ teeth are harmful, some studies suggest that both practices are also harmful and stressful to the piglets and can result in lower weight gain in early lactation [71].Tail docking, which is intended to control tail biting, can be conducted with sharp objects or cauterizers and is usually performed without the aid of analgesia or anaesthesia. Tail docking itself is painful [104], and, additionally, pigs with an amputated tail may experience pain that resembles neuropathic pain reported in humans, i.e., pigs experience persistent pain on the incision site long after the tail tissue has healed [105]. It has also been shown that besides acute pain and stress, tail docking may have adverse consequences on human–animal relationships via a fear response [104].Male piglets are usually castrated in the first week of life to eliminate boar taint in the meat of slaughtered pigs, caused by the volatile substances androstenone and skatole that accumulate in male pig fat [106]. Surgical castration is routinely performed without pain relief. Pigs’ castration is conducted cutting the skin, exposing and breaking the spermatic cord [4]. Circulating cortisol levels increase immediately after the procedure, possibly as a result of intense pain, added to the stressful handling of containment [4,5]. In addition, castration is a risk factor for infections and for AMU in weaned piglets [72].Sows feel pain during parturition, which can persist for up to 24 h after the birth of the last piglet [107] but awareness towards the issue is only recent and therefore interventions are rare. Providing analgesic medication for dystocic sows can reduce sows’ suffering and improve piglet immune competence. For example, oral meloxicam administered orally to sows at the beginning of farrowing increased the concentration of IgG in piglets’ serum [108] and the concentration of immunoglobulins and cytokines in the colostrum of medicated sows [109].3.2.4. Mixing Unfamiliar AnimalsIn intensive farms, pigs often are repeatedly mixed, starting during lactation when cross-fostering may be used, at weaning, during fattening, and after each cycle in group gestation systems. At weaning, piglets are typically separated by weight, a routine farm practice that aims to reduce fights over food that could negatively impact growth. Fattening pigs are mixed when they are moved to new housing for fattening or when they are transported to new farms. Gilts are mixed when moved to the breeding group and sows after weaning and insemination and, in some cases, after the first few weeks of gestation in crates [46]. Mixing unfamiliar animals generate fights related to the establishment of a social hierarchy, which is exacerbated by the stress associated with the novelty [97]. Management factors, such as sex, the weight of the animals, and the type of social mixtures, interfere in the occurrence of fights [110].Aggressive interactions are already observed in nursing piglets, but after the teat order is established, fights and bites tend to decrease. Throughout the growing and fattening period, the fights are frequent, especially involving males [111]. Aggressive behaviours may also be frequent in group-housed gestating sows, often related to the management of dynamic groups, resource availability and access, and disputes over resting areas; fighting occurs when a new group of pregnant sows is housed, and the frequency declines as the social hierarchy are established [46,112]. Increased fighting and aggressiveness within a group of sows may be observed when food is not supplied simultaneously to all the sows [61].Agonistic interactions resulting from social mixtures can impair the immune response of post-vaccination piglets, especially in castrated males, possibly because the stress of fights is associated with the suffering of castration [31]. Additionally, the HPA axis is activated in response to aggressive agonistic interactions, resulting in increased cortisol and impaired immune response [111]. Finally, skin lesions resulting from aggressive interaction predispose pigs to infections and AMU [46].3.2.5. Human–Animal Interactions and FearThe quality of human–animal interactions should be included as one of the predictors of quality of life for pigs. Although it is difficult to argue for a direct relationship between negative human–animal interactions and diseases or AMU, the relationship with fear and physiological stress is well documented in pigs and many other species [29]. Pigs and humans communicate through acoustic, visual, tactile, and chemical sensory cues [113], and it has been shown that animals have the ability to recognise and remember aversive handlers and respond accordingly [29]. Although many routine interactions may appear innocuous, aversive human contacts that trigger a fear response in animals occur in routine pig management [29]. Additionally, humans are present throughout stressful and painful procedures applied in intensive pig farming; therefore, the quality of the interactions may either exacerbate or minimise the physiological responses reviewed earlier, which challenge homeostasis and can be a risk to the health and welfare of pigs [113].4. So, Is There a Relationship between Animal Welfare and Use of Antibiotics in Pig Farming?We have discussed several housing and management factors that are a source of distress in pigs and that ultimately facilitate the occurrence of diseases, which are commonly prevented or treated with antibiotics. Unfortunately, to date, there is a scarcity of scientific literature reporting a direct causal relationship between distress (i.e., poor welfare) in pigs and AMU. However, the examples evidenced in this review are a relevant starting point for the discussion of the relationship between animal welfare, disease, and AMU in pig production. Additionally, growing evidence is provided by studies showing that when management is focused on adopting biosecurity practices, it is possible to reduce AMU in pig farms (e.g., [114,115,116,117].An important reason for AMU in pig farms is the prevention or treatment of respiratory, enteric, and reproductive diseases [50,93] that are facilitated by several housing and management stressors reviewed here. We have also summarised evidence that pigs’ early life concentrates a great deal on highly stressful management practices, while others have shown that this period coincides with high AMU [50,92,118]. For example, even in Germany, where antibiotics are not used for prophylactic treatments, piglets were found to be by far the most commonly treated age group [93]. Other studies have reported that farmers often use strategic oral treatment of all pigs with antibiotics around stressful managements highlighted in this review, such as castration, weaning, and at the start of the finishing period when pigs are exposed to novel physical and social environments [50,90,91].Additionally, we reviewed the evidence of an association between AMU and specific housing and management stressors or animal factors on pig farms. For example, cross-fostering and piglet low body weight or poor weight gain were associated with AMU [72]. Poor air quality and poor cleanliness combined with poor conditions of facilities were associated with increased AMU for respiratory diseases; inadequate drinking equipment, lack of enrichment, and a poor condition of pens combined with high stocking density were associated with AMU for joint infections; and inadequate stocking density and poor enrichment were associated with AMU for tail biting [38]. Supporting the role of many housing and management factors identified in this review as important stressors for pigs, a study showed that farms with lower AMU levels were those using access to outdoors and to roughage, natural ventilation, bedding, lower stocking densities, and later weaning [119].Lastly, it is important to bear in mind that although biosecurity and the quality of management are major risk factors for AMU [38,92,120] and AMR [121] on pig farms, they are a part of the puzzle of the current AMU challenges in the pig industry. However, good health through disease prevention is just one aspect of animal welfare. Our review has highlighted that to reduce AMU, we need to strive for happier pigs, i.e., pigs who are not stressed, bored, or fighting for a place in the group; pigs that are not left behind due to lack of space or resources. The question is how can we achieve these happier pigs? Below, we summarise evidence of how this could be achieved to some extent and thus improve pig welfare in commercial farms.5. Improving Housing Environment and Management Practices to Reduce Stress in Pig FarmingDuring the past decades, many changes in housing and management that may minimise or eliminate the environmental stressors described above have been proposed, tested, and even implemented with success in commercial farms [41]. Such housing and management changes, together with the elimination of painful procedures or their replacement with less invasive alternatives, have proven to allow the expression of species-specific behaviours, reduce stress, boost the immunological system, and even promote positive emotional states [41,122]. In this section, we present an overview of known strategies focusing on the gains attained through environmental enrichment (including better feeding practices and on-farm resources organisation), reduction in stocking density, group-housing and family rearing, increased age at weaning, neonatal socialisation, the prevention and treatment of lameness, automatic feeding, and supply of fibre for sows.5.1. Improved Housing and Environmental EnrichmentA key overarching solution to the distress and boredom suffered by pigs is good housing design and environmental enrichment of their living space. Good housing is a design that enables pigs to move, explore, and feel protected from threats and that ensures thermal comfort [41,123]. The provision of hiding opportunities is an area often forgotten in commercial farms; however, it can protect newly housed sows from aggressive interactions with resident sows [124], and building barriers and hiding places can also be an alternative to reduce agonistic interactions and stress [125]. On the other hand, the provision of space for locomotion is beneficial for the health of the locomotor system. Thus, increasing the space per sow in indoor gestation group housing can reduce the frequency of lameness [126]. Moreover, loose farrowing housing provides the additional space for sows to move and also improves maternal behaviour in sows and social behaviour in piglets; positive impacts are observed even when the sows’ movement is restricted for a few days after farrowing with the aim to reduce piglet mortality [127].Environmental enrichment consists of actions that make the living space attractive, giving pigs the opportunity to express highly motivated innate behaviours [128]. Substantial evidence exists about the positive behavioural value enriched environments can have in pigs, including the promotion of positive affective states at all stages of rearing. Examples of enrichment include the provision of straw, toys, and manipulable materials for weaned pigs that reduce the incidence of redirected oral behaviours [129,130]. In growing pigs, the supply of manipulable materials can aid in reducing the incidence of tail biting [37,131,132]. Recent work showed that environmental enriched housing could have positive effects on the development of the immune system and the establishment of gut microbiota in early life [133]. Enabling sows to build a nest and offering straw during farrowing and lactation can prevent skin and claw lesions in piglets [134], increase activity, and reduce abnormal behaviour in the sows [135]. Furthermore, there is a knock-on effect of reducing sows’ stress at the end of gestation in that it may positively affect the offspring, influencing the activity of the HPA axis and reducing the incidence of aggressiveness and belly-nosing [136]. Additionally, fibre (i.e., provision of straw in sow diet) increases the volume and absorbs water, stimulating the mechanical receptors of the stomach and decreasing gastric emptying, necessary for satiety [60]. Thus, straw inclusion will reduce levels of ghrelin and chronic hunger sensation, as well as the incidence of gastric ulcerations and agonistic interactions between sows [60,137]. Enrichment of the maternity housing with chewable materials can stimulate the exploratory behaviour of the piglets and increase the frequency of non-painful contact of the piglets with the udder, reducing the stress and severity of skin lesions in lactating sows [138].5.2. Group Housing-Increasing Space, Reducing Stocking Density or BothGood housing should also encompass the social dynamics and stocking density for it to fulfil the goal of improving welfare. Group housing can be beneficial for pregnant and lactating sows; if well managed, it may reduce serum cortisol concentration and reduce the frequency of vacuum chewing and sitting behaviour [139,140]. Improving the housing conditions of sows can have positive effects on the health of piglets; piglets of sows reared in groups showed greater resistance and resilience when challenged with LPS compared to piglets of individually housed sows [140]. Moreover, group housing enables early socialisation among piglets reducing agonistic interactions during weaning [35]. However, group housing can fall into a success in research/unsuccessful in practice paradox if provided with insufficient space (i.e., high stocking density), poor environmental enrichment, inadequate resource allocation, or poor feeding management. Such situations create failed living spaces that increase agonistic interactions, injuries, and overall reduction in welfare [41].5.3. Reducing Pre-Natal, Neonatal and Weaning Stress and Promoting Positive Human–Animal InteractionsWeaning piglets at later ages in the absence of early painful procedures (i.e., castration, tail clipping, teeth clipping) has positive impacts on piglet behaviour and GIT structure and function [73]. Additionally, later-weaned piglets with optimal feeding strategies (i.e., creep feeding) have greater diversity and abundance of bacterial microflora in the gastrointestinal tract, which may help reduce the incidence of diarrhoea and the use of antibiotics in this critical phase [73,89]. Delayed weaning by itself is not enough; maintaining the social group and the physical environment at weaning is equally important to reduce weaning stress and improve post-weaning feed intake [97]. Piglets with permanent social structures are better socialised, which improves their overall adaptability to the post-weaning environment and reduces cortisol levels, agonistic interactions, and the resulting lesions, whilst increasing play behaviour [89,141].Eliminating painful procedures or replacing them with known alternatives (e.g., immunocastration, environmental enrichment, reduced stocking density, family systems) facilitates caregivers’ attentiveness and empathy towards animals, central for pig production systems that advocate for high animal welfare [142]. In addition, calm, gentle, and pleasurable human–animal interactions (i.e., pleasant management routines) can decrease stress, reduce behaviours that denote fear and anxiety, and keep working environments peaceful [29]. Positive human–animal interactions can include friendly human presence, contact, and tactile stimuli (scratching or caressing), providing food and objects for interaction (i.e., environmental enrichment), and engaging in peaceful and pleasant routines with the animals [143]. Prolonged gentle handling has proven effective at reducing stress and anxiety in pigs [144], as well as in pregnant and lactating sows [145] and piglets that also gained more weight [146]. Finally, positive human–animal interactions are essential to ensure the welfare and quality of life of pigs on farms and maintain the social license of the pig industry and reduce the use of antibiotics in farms.5.4. Making a 180 Degree Turn into Genetic Selection to Improve Animal WelfareHistorical genetic selection has contributed to the success of intensive pig production through the development of genetic strains with highly productive characteristics, such as improved weight gain, feed conversion, and hyperprolificity. The use of this knowledge to develop animals more resilient to environmental stressors or more resistant to pathogens may contribute to the aim of reducing AMU in intensive pig production. For example, some promising studies have identified genotypes resistant to diseases such as circovirus [147] and Mycoplasma hyopneumoniae [148,149]. As noted in the discussion above, many stressors that affect pigs’ wellbeing and health are exacerbated in large litters, such as increased duration of farrowing, low birthweight piglets, and a lack of teats for all piglets. For example, a retrospective study of Swedish pig herds showed a correlation between the increase in litter size and an increased need for treatments with antibiotics in sows due to puerperal infections [150]. Thus, limiting litter size should be considered if striving for sustainability in the pig industry [151]. Genetic selection for lower aggressiveness, in the case of females, in addition to the reduction in agonistic interactions, can lead to increased maternal ability since these traits are correlated [152]. Another example is the selection for low levels of skatole, which would eliminate the need for castration, reducing fights between pigs during the fattening period without lowering the quality of meat by the presence of boar taint [153]. Gene editing technologies offer promising opportunities to introduce beneficial characteristics to commercial pig strains without competing with production traits, provided ethical and technical limitations are addressed [154].5.5. The Intrinsic Value of Pigs—Re-Centring Pig Industry ValuesWe have carefully selected evidence that current management systems, including the production system and the quality of practices within the system, are failing to keep pigs healthy and “happy”. In parallel, such evidence highlights an industry whose values and goals are guided by high productivity and financial returns. This fragile balance is sustained through reactive management practices (e.g., cross-fostering created as a response to hyperprolific breeds) [76] or reliance on the effectiveness of prophylactic and therapeutic AMU [50]. A recent scoping review of studies evaluating alternatives to antibiotics to prevent or control disease and reduce the need for antibiotics in nursery pigs identified a majority of studies covering feed additives and vaccines and relatively few studies evaluating housing or management practices [10], revealing a lower scientific effort/interest in systemic changes and a preference for “silver bullets” to replace antibiotics. On-farm changes are necessary to push for optimal pig welfare and reduced AMU, which demands an organisational re-centring of the pig industry values. Changes must recognise the intrinsic value of the pig within the system, the connections with its welfare, human well-being, and the environment [22].6. Implications and Closing RemarksA holistic reduction in stressors and boredom in pig herds (i.e., happier pigs) is crucial to the multi-actor and multi-action puzzle of reducing current AMU in intensive pig production farms. This will simultaneously address two demands from society: improving the welfare of pigs (ethical demand) and reducing the use of antibiotics (public health demand). We reviewed many husbandry practices used in intensive systems that challenge homeostasis, increasing the animals’ susceptibility to diseases. Examples centred around factors inherent to the housing environment (and its management), animal management, and the animal selection or genetic makeup. Likewise, we pointed out alternative actions to reduce and prevent stress. Although in some cases more applied research is needed, we have enough collective knowledge to act and seize the moment. Thus, it begs the question, why has the pig industry not done so? Post-war industrialisation of pig production was driven by increased productivity and cost reductions [155]. Although, arguably, initial industry changes developed holistically and sided with AMU [155,156,157], this review has highlighted current painful/stressful practices that are a result of the pig industry forgetting its holistic principles of production and that the transaction point is the animal, to the point of surpassing the balance of ethical and biological demands of these animals. Thus, today’s pig production is not sided with AMU; instead, it relies on using precious molecules (antibiotics) as palliative treatments to support such unsustainable production systems [50,157].Welfare is a complex balance of different aspects of the life of animals and how they perceive/feel them. We have argued that there is indirect evidence that happier and less stressed pigs are more immune-competent, more capable of naturally defending themselves from environmental pathogens, and therefore less dependent on preventive antibiotics. The adoption of good management practices that consider the characteristics and needs of the pigs holistically is essential to meet the international call for prudent use of antibiotics. Additionally, public opinion must be recognised as a vital force for change in production systems. There is a growing literature showing that restrictive, barren housing systems and painful and stressful managements have no societal support. In contrast, alternatives focused on free systems and naturalness are not just preferred but expected by consumers [158,159,160].A common understanding of the meaning of farm animal welfare is a necessary step to change to a production system that attends to these expectations. Yet, industry stakeholders define animal welfare in terms of biological function concerning production [87], thus minimising the importance of naturalness and affective states [3,42,142]. Thus, “good welfare” is seen as animals not falling sick or not dying on-farm before they reach their weight to slaughter and for sows that wean as many piglets as biologically possible. In turn, the current use of antibiotics is justified on the grounds that it protects the “welfare” of the animals. We agree that an animal’s health is an essential dimension that defines its welfare. Yet, there is a danger in reducing the welfare of an animal into not being sick. This reductionist approach fails to question how we got there and if there is another way to allow an animal to live happily, not just survive its environment. Thus, the lack of recognition of animal welfare in all its dimensions makes it more difficult to believe that many interventions suggested in Section 5 may improve pigs’ quality of life and make the industry less dependent on antibiotics.Farmers are seen as the ultimate people responsible for changing the current AMU. A simplistic and polarised portrait of why pig farmers use antibiotics is that of a neglectful or a protective individual of the welfare of their animals. Today, many farmers report feeling/being powerless to think differently, and if they do, act differently, quoting that economic constraints, production standards or technical advice do not leave room for change [87,161]. Just as the pigs in their farms, many farmers are surviving the system. Thus, investments needed for any changes must be supported by the industry, consumers, and governments. A call for pig farmers to rationally reduce AMU will succeed or fail pending such external support and structural changes in the network that currently uses antibiotics as a structural material for production, at local, national, and international levels. This means that individual behaviour changes are not enough nor sustainable in the long run. Ultimately, we urgently call for a re-centring of the industry objectives (inclusive of all stakeholders) into the intrinsic values of life (a life worth living) and nature (a place worth living) for all living creatures. The solutions demand leaving behind the conception that feeding the world means intensifying animal production, towards a genuinely sustainable approach where keeping our world means slowing down production.

animals : an open access journal from mdpi

10.3390/ani13101710

PMC10215676

Although the transcriptome of dairy cow mammary tissue has been reported, the transcriptome of yak mammary tissue remains unknown. The objective of this study was to assess the transcriptome of the mammary tissue of four yaks during the whole lactation cycle. The statistical analysis identified >6000 differentially expressed genes (DEGs) throughout lactation, with a large number of DEGs observed at the onset (1 d vs. −15 d) and at the end of lactation (240 d vs. 180 d). Bioinformatics analysis revealed a major role of lactation-associated genes on BTA3, BTA4, BTA6, BTA9, BTA14, and BTA28. Functions affected by the transcriptomic adaptation to lactation in mammary tissue of yak were very similar to those observed in dairy cows. Our study provides fundamental resources on yak mammary tissue transcriptome for the research community.

The objective of this study was to assess the transcriptome of the mammary tissue of four yaks during the whole lactation cycle. For this purpose, biopsies of the mammary gland were performed at −30, −15, 1, 15, 30, 60, 120, 180, and 240 days relative to parturition (d). The transcriptome analysis was performed using a commercial bovine microarray platform and the results were analyzed using several bioinformatic tools. The statistical analysis using an overall false discovery rate ≤ 0.05 for the effect of whole lactation and p < 0.05 for each comparison identified >6000 differentially expressed genes (DEGs) throughout lactation, with a large number of DEGs observed at the onset (1 d vs. −15 d) and at the end of lactation (240 d vs. 180 d). Bioinformatics analysis revealed a major role of genes associated with BTA3, BTA4, BTA6, BTA9, BTA14, and BTA28 in lactation. Functional analysis of DEG underlined an overall induction of lipid metabolism, suggesting an increase in triglycerides synthesis, likely regulated by PPAR signaling. The same analysis revealed an induction of amino acid metabolism and secretion of protein, with a concomitant decrease in proteasome, indicating a major role of amino acid handling and reduced protein degradation in the synthesis and secretion of milk proteins. Glycan biosynthesis was induced for both N-glycan and O-glycan, suggesting increased glycan content in the milk. The cell cycle and immune response, especially antigen processing and presentation, were strongly inhibited during lactation, suggesting that morphological changes are minimized during lactation, while the mammary gland prevents immune hyper-response. Transcripts associated with response to radiation and low oxygen were enriched in the down-regulated DEG affected by the stage of lactation. Except for this last finding, the functions affected by the transcriptomic adaptation to lactation in mammary tissue of yak are very similar to those observed in dairy cows.

1. IntroductionThe yak (Bos grunniens) lives throughout the Qinghai-Tibetan plateau of China. It is at home at high altitudes where a harsh climate exists, including low temperature, humidity, and oxygen levels, as well as strong winds, high ultraviolet radiation, and other hazards. The yak is crucial in this area, as it is the main source of meat and milk to the 6.5 million Tibetan people living in the Qinghai-Tibetan plateau area [1]. Unlike dairy cows, which can produce more than 3000 kg milk in a single lactation, the milk yield of the yak is extremely low (<500 kg in 180 d of lactation cycle). However, despite the low milk yield, yak milk contains higher concentrations of milk solids, protein, fat, organic calcium, and conjugated linoleic acid than Holstein cow milk [2].The mammary gland is specialized in the synthesis and secretion of milk, and undergoes drastic structural and metabolic changes during the transition from pregnancy into lactation and from lactation to the dry period [3,4,5]. As observed in the cow, mouse, and human, these events appear closely driven by changes in the transcriptome. Although basic functions of the mammary gland have been previously described [6,7], understanding the complexity of the adaptations to lactation is an active area of research [8]. High-throughput tools, including microarray and RNA-seq, have made it possible to uncover changes in the transcriptome of the mammary tissue in several species [3,4,5,9,10]. Although RNA-seq has gained tremendous popularity [11], researchers continue to use microarrays as well, especially in studies with large sample sizes [12]. Moreover, microarrays are more popular in clinical settings due to easier data processing [13].The biology of the mammary gland of the yak has received relatively little attention, although epithelial cells isolated from the yak mammary gland have been characterized [14]. These cells have been used to identify the proteins involved in mammary epithelial differentiation in this species [15]. However, transcriptome analysis, especially if performed through the whole lactation cycle, can provide further insights into the biology of the mammary gland of the yak. Prior work from our group using RTqPCR revealed a role of the transcription factor MYC on the adaptation of the mammary gland of the yak to lactation [16], and characterized the expression of protein synthesis-related transcripts during the whole lactation [17]. A recent study underlined the importance of non-coding RNA in the mammary gland of the yak during lactation and the dry period [18]. Our group performed metabolomics profiling of yak mammary tissue during the whole lactation, revealing changes in glucose and amino acid metabolism [14,19].All of the above studies have provided important insights into the adaptation of the yak mammary gland to lactation; however, they do not provide a comprehensive picture. Due to the important insights provided by high-throughput analysis of the transcriptome associated with bioinformatic tools in the adaptation to lactation of the mammary gland in other species [4,5,20,21], it is important to study the whole transcriptome of protein-coding transcripts in yak mammary tissue using similar approaches. Therefore, in the present study we performed a transcriptome profile of yak mammary tissue during the whole lactation cycle and compared it with prior data obtained in dairy cows.2. Material and Methods2.1. Milk Yield MeasurementAs previously reported [17], milk yield was measured just after calving and at 20, 40, 60, 80, 100,120,140, 160, 180, and 200 d relative to parturition. The milk yield values were the amount of milk collected twice a day (morning and night milking), and were adjusted for the milk consumed by the calves. It is estimated that yak calves consume approximately half the milk produced. We measured milk production in an additional ten yaks at the end of June. During the entire lactating period, at least 18 individual measurements were recorded for each yak.2.2. Animals, Experimental Design, and DietAll experiments performed in the present study were approved by Southwest Minzu University Institutional Animal Care Committee (permit number: 2012-3-1). Four multiparous MaiWa yaks from Southwest Minzu University Tibetan Research Center weighing 220–260 kg were selected. As is typical of the Tibet plateau, the yaks were grazed without any supplementation. All selected yaks calved at the end of June.2.3. Mammary Gland Collection and PreparationThe mammary tissue was collected via biopsy, alternating between the right or left rear quarter of the mammary gland at −30, −15, 1, 15, 30, 60, 120, 180, and 240 d relative to parturition [3,22]. Biopsy of the mammary gland was performed by a veterinarian. Briefly, to obtain mammary parenchyma, the mammary capsule was separated via blunt dissection and the mammary parenchyma was excised; the incision depth was 3.5–4 cm to ensure that the lactating tissue was obtained. The mammary parenchyma was subsequently washed with diethyl pyro carbonate-treated water. Approximately 1 g of tissue was cut into small pieces and immediately stored in liquid nitrogen for total RNA extraction.2.4. Microarray2.4.1. Hybridization of DNA Microarray and RNA ExtractionThe Agilent Bovine (V2) Gene Expression Microarray (Catalog Code: G2519F-023647; 4 × 44 K format) was used, with each array containing probes interrogating about 17,764 Entrez Gene ID in the array. A total of 36 microarrays were used for hybridization. Total RNA was extracted from each yak mammary tissue (50–100 mg) using Trizol reagent (Invitrogen, Karlsruhe, Germany) and purified according to the manufacturer’s protocol. The purity and concentration of RNA were determined by OD260/280 using a spectrophotometer (NanoDrop ND-1000). RNA integrity was determined by capillary electrophoresis using the RNA 6000 Nano Lab-on-a-Chip kit and the Bioanalyzer 2100 (Agilent Technologies, Santa Clara, CA, USA). RNA integrity number was >6 for all samples.2.4.2. RNA AmplificationEberwine’s linear RNA amplification was performed by double-stranded cDNA containing the T7 RNA polymerase promoter sequence synthesis from 100 ng total RNA using the CbcScript reverse transcriptase with cDNA synthesis system according to the manufacturer’s protocol (Capitalbio) with T7 Oligo (dT). After completion of double-stranded cDNA (dsDNA) synthesis using DNA polymerase and RNase H, the dsDNA products were purified using a PCR NucleoSpin Extract II Kit (Macherey-Nagel, Düren, Germany) and eluted with 30 μL elution buffer. The eluted dsDNA products were vacuum-evaporated to 16 μL and subjected to 40 μL in vitro transcription reactions at 37 °C for 14 h using a T7 Enzyme Mix. The amplified cRNA was purified using the RNA Clean-up Kit (Macherey-Nagel, Düren, Germany).2.4.3. Labelling and HybridizationcDNA was labeled with a fluorescent dye (Cy3-dCTP) produced by a Klenow enzyme labeling strategy after reverse transcription (Liu Zhihao et al., 2020). Briefly, 2 μg cRNA was mixed with 4 μg random nanomers, denatured at 65 °C for 5 min, and cooled on ice. Then, 5 μL of 4×first-strand buffer, 2 μL of 0.1 M DTT, and 1.5 μL CbcScript Ⅱ reverse transcriptase were added. The mixture was incubated at 25 °C for 10 min, then at 37 °C for 90 min. The cDNA product was purified using a PCR NucleoSpin Extract II Kit (Macherey-Nagel, Düren, Germany) and vacuum-evaporated to 14 μL. The cDNA was mixed with 4 μg random nanomer, heated to 95 °C for 3 min, and snap-cooled on ice for 5 min. Then, 5 μL Klenow buffer, dNTP, and Cy3-dCTP (GE Healthcare) were added to a final concentration of 240 μM dATP, 240 μM dGTP, 240 μM dTTP, 120 μM dCTP, and 40 μM Cy-dCTP, followed by addition of 1.2 μL of the Klenow enzyme. The reaction was performed at 37 °C for 90 min. Labeled cDNA was purified with a PCR NucleoSpin Extract II Kit (Macherey-Nagel, Düren, Germany) and resuspended in elution buffer. Labeled controls and test samples labeled with Cy3-dCTP were dissolved in 80 μL hybridization solution containing 3 × SSC, 0.2% SDS, 5 × Denhardt’s solution, and 25% formamide. DNA in hybridization solution was denatured at 95 °C for 3 min prior to loading onto a microarray. Array hybridization was performed in an Agilent hybridization oven overnight at a rotation speed of 20 rpm at 42 °C and washed with two consecutive solutions (0.2% SDS, 2 × SSC at 42 °C for 5 min, and 0.2 × SSC for 5 min at room temperature). The array data were analyzed for data summarization, normalization, and quality control using GeneSpring software V12 (Agilent). Transcriptome data were submitted to the NCBI public repository (accession number: GSE201438).2.5. Validation of Microarray Data with RTqPCRValidation of the microarray data was performed by comparing microarray data with the RTqPCR data of twelve transcripts, including RPS15, RPS23, UXT, TP53, FARP1, SLC1A5, LPL, FABP3, SCD1, AGPAT6, CSN3, and BDH1 from the same yak mammary tissues that were previously analyzed [17,23,24].2.6. Bioinformatics AnalysesThe Dynamic Impact Approach (DIA) [25] was used for bioinformatics analysis. The DIA was specifically developed for analysis of dynamic transcriptomic changes, and was validated using bovine mammary tissue data throughout the whole lactation [3,25], a very similar experimental design to the present study. The DIA provides two outputs: the “Impact”, which calculates the impact of a condition (e.g., phase of lactation) on specific biological terms (e.g., the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway or Gene Ontology (GO)) using the proportion of DEG, the log2 expression ratio, and the −log10 p-value of the comparison. By calculating the difference of the Impact between genes that induce the term and genes that inhibit the term, the tool provides the “Direction of Impact”. Specific details of the tool are available elsewhere [25]. The DIA was used to analyze KEGG pathways and chromosomes.Enrichment analysis of DEG was performed using Database for Annotation, Visualization, and Integrated Discovery (DAVID) v6.8 [26] and Ingenuity Pathway Analysis (IPA). Data were run in both tools using the default settings. In DAVID, three analyses were performed for each comparison: all DEG, up-regulated DEG, and down-regulated DEG. The last two analyses were used to determine whether the enriched KEGG and GO process terms were induced or inhibited. Results were downloaded using Chart Analysis. For IPA, the p-value and Z-score of each canonical pathway were downloaded. The Z-score in IPA infers activation states of each pathway (positive if increased and negative if decreased). For both tools, the annotated array was used as background.2.7. Statistical AnalysesData were analyzed by JMP genomics (version 7.0; SAS) using the default ANOVA model with time as the main effect and yak (n = 4) as a random effect. The overall effect of time was corrected by Benjamini–Hochberg False Discovery Rate (FDR) [27]. Differentially expressed genes (DEG) were determined when the overall time effect was with the FDR being ≤0.05 and the p-value between each comparison was ≤0.05.3. Results3.1. Overall Transcriptome Expression Profile in Yak Mammary TissueThe complete transcriptome data after statistical analysis are available in Supplementary File S1. Microarray data for a few selected genes through the lactation in yak were validated using RTqPCR. The data indicated a good concurrence between the data obtained from the two approaches (Supplementary Figure S1).The curve of lactation is reported in Figure 1A, with peak milk yield at 120 d and a rapid decline until 240 d. The statistical analysis of the mammary tissue transcriptome in yaks during the whole lactation identified 6256 DEG (Figure 1B). The number of down-regulated DEG was larger than that of up-regulated DEG throughout the entire lactation, although the up-regulated DEG had overall greater fold change (Figure 1B). Compared to −30 d, the number of DEG at each time point increased steadily until 30 d and remained high until 180 d, when a sharp drop in the number of DEG was detected, with almost no DEG in the 240 vs. −30 d comparison (Figure 1C). When compared with the previous time point, the largest increase in the number of DEG was detected between 240 d vs. 180 d, followed by the comparison between 1 d and −15 d (Figure 1D). The number of up- and down-regulated DEG vs. −30 d was similar among each time point vs. −30 d, with a relatively lower number of up- vs. down-regulated DEG between 30 d and 60 d. When considering the comparison between each consecutive time point, a lower and higher number of up- vs. down-regulated was observed in DEG between 30 d and 15 d and between 120 d and 60 d, respectively (Figure 1D).3.2. Impact of Stage of Lactation on ChromosomesThe results of Bos taurus autosome (BTA) analysis by DIA are available in Supplementary File S2. The top five most impacted chromosomes in mammary tissue during lactation in yaks were BTA6, BTA29, BTA23, BTA28, and BTA8 (Figure 2). Gene expression from most of the chromosomes was inhibited (p < 0.05) during lactation, except BTA6, BTA9, and BTA28, which were obviously activated (p < 0.05) during the period from day 1 to 60. In contrast, gene expressions from BTA3, BTA4, BTA14, and BTA20 were the most inhibited (p < 0.05) during lactation (Figure 2, Information and location in each chromosome of genes, is available in Supplementary File S2).3.3. Functional Analysis of DEG Revealed by DIAThe Impact and Direction of Impact of the categories of the KEGG pathways as analyzed using DIA are available in Figure 3. The same metrics for each pathway are available in Supplementary File S3.Metabolic-related pathways were activated overall during maximal milk yield increase, i.e., between 1 d and 60 d compared to −30 d, as well as at the onset of lactation, i.e., 1 d vs. −15 d. Among the most impacted sub-categories of pathways in the ‘Metabolism’ category were the sub-categories ‘Lipid Metabolism’, ‘Amino Acid Metabolism’, and ‘Metabolism of Cofactors and Vitamins’. The former two pathways were inhibited from 120 d on vs. −30 d, while the latter remained activated until the end of lactation. Among ‘Lipid Metabolism’ pathways, the most impacted and activated were ‘Synthesis and degradation of ketone bodies’, ‘Steroid biosynthesis’, ‘Glycerolipid metabolism’, ‘Glycerophospholipid metabolism’, ‘Arachidonic acid metabolism’, and ‘Linoleic acid metabolism’. The first two pathways were among the most impacted (see the ‘Pathway sort’ tab in Supplementary File S3).Among ‘Amino Acid Metabolism’, pathways associated with biosynthesis of Arg, Ile, Leu, Phe, Pro, Trp, Tyr, and Val were overall induced during first 60 days of lactation, while pathways associated with metabolism (i.e., degradation) of His, Lys, Trp, Tyr, Val, and taurine were overall inhibited during lactation. The ‘Phenylalanine, tyrosine and tryptophan biosynthesis’ pathway was the second most-impacted and induced pathway (see the ‘Pathway sort’ tab in Supplementary File S3), while ‘Cysteine and methionine metabolism’ was induced only at the onset of lactation and between 180 d and 240 d, and was inhibited from 15 d to 180 d vs. −30 d. All pathways related to amino acid metabolism were induced at onset of lactation and further induced from 180 d to 240 d vs. −30 d except for taurine, which was inhibited at the onset of lactation. The ‘Metabolism of Cofactors and Vitamins’ category of pathway was highly impacted and activated during early and mid-lactation (i.e., in all time points vs. −30 d up to 180 d) and at the onset of lactation, and was inhibited between 180 d and 240 d vs. −30 d. Particularly affected in this category of pathways were ‘Riboflavin metabolism’, ‘Nicotinate and nicotinamide metabolism’, ‘Pantothenate and CoA biosynthesis‘, ‘Folate biosynthesis’, and ‘Retinol metabolism’.‘Glycan Biosynthesis and Metabolism’ was impacted, and was slightly induced overall (Figure 3). The most impacted pathways in this sub-category of KEGG pathways were ‘O-Glycan biosynthesis’ and ‘N-Glycan biosynthesis’, which were strongly induced during lactation while being inhibited from 180 d to 240 d vs. −30 d, and ‘Glycosylphosphatidylinositol(GPI)-anchor biosynthesis’, which was overall induced during lactation, while ‘Glycosaminoglycan biosynthesis—heparan sulfate’ was strongly inhibited during the same time frame.All the pathways in the ‘Replication and Repair’ sub-category of pathways were highly inhibited starting two weeks prior parturition and during lactation, then induced between 240 d and 180 d. Similarly, the sub-category of pathways ‘Growth and Death’ was highly inhibited during lactation and induced at the end of lactation (i.e., 240 d vs. 180 d). ‘Cell cycle’ was the most impacted and inhibited among the pathways in the ‘Growth and Death’ sub-category of pathways.The sub-category of ‘Immune System’ and ‘Endocrine System’ pathways were highly impacted, with the former being mostly inhibited during and while the latter activated in the early stages of lactation and inhibited during the rest of lactation, with activation between 180 d and 240 d. The most impacted pathways in this sub-category of pathway were ‘Immune System’, ‘Chemokine signaling pathway’, ‘Toll-like receptor signaling pathway’, and ‘Complement and coagulation cascades’, which were overall activated, especially during early lactation; on the other hand, ‘Antigen processing and presentation’ was inhibited during the whole lactation. Among the ‘Endocrine System’ sub-category of pathways, the ‘PPAR signaling pathway’ was the most impacted, being induced during the whole lactation and inhibited between 180 d and 240 d. In the same sub-category of pathways, the ‘Renin-angiotensin system’ and ‘Progesterone-mediated oocyte maturation’ were highly impacted and inhibited during lactation, while being induced between 180 d and 240 d.3.4. Enrichment AnalysisComplete results from DAVID are available in Supplementary File S4. The KEGG pathways and Gene Ontology Biological Process terms enriched with a p-value < 0.01 are shown in Figure 4. The most enriched GO terms were associated with the cell cycle and proliferation, and were all enriched in down-regulated DEG in each time point during lactation vs. −30 d while being enriched in up-regulated DEG in the comparison of 240 d vs. 180 d. Enriched in down-regulated DEG during lactation were the GO terms associated with response to radiation (UV and gamma) and immune response. Among KEGG pathways, the most enriched among down-regulated DEG during lactation, though up-regulated between 240 d and 180 d, were pathways associated with the cell cycle. Others pathways similarly enriched were those associated with metabolism, with Cys and Met metabolism and pyrimidine metabolism enriched in up-regulated DEG at the onset of lactation and at the end of lactation; additionally, several time points during early and late lactation were enriched in down-regulated DEG. Steroid biosynthesis was enriched in up-regulated DEG between 1 and 60 day in lactation vs. −30 d. Protein processing in ER was increased at the onset of lactation and at 30 d compared to −30 d.The complete results of the IPA analysis are available in Supplementary File S5. The 25 most enriched (p < 0.05) canonical pathways are shown in Figure 5. Pathways that were estimated to be down-regulated by DEG during lactation (though up-regulated between 240 d and 180 d) were mostly involved with the cell cycle (including ATM Signaling, Estrogen-mediated S-phase Entry, and Role of CHK Proteins in Cell Cycle Checkpoint Control). Pathways estimated to be up-regulated were ‘Aryl Hydrocarbon Receptor Signaling’, ‘iNOS signaling’, and ‘OX40 Signaling Pathway’. Other pathways associated with immune response and the cell cycle (e.g., ‘Allograft Rejection Signaling’, ‘Antigen Presentation Pathway’, and ‘Cell Cycle Control of Chromosomal Replication’) were enriched.4. DiscussionIn the present study, a transcriptome profile of yak mammary tissue during the whole lactation was performed and compared with the same analysis performed in dairy cow [3].4.1. Impact of Lactation on Yak ChromosomeAnalysis of DEG affected by lactation and associated with chromosomes is critical in order to confirm chromosomal regions undergoing transcription changes during lactation. This can help to provide information about quantitative loci (QTL), which can benefit genetic selection to increase milk production and quality in yaks. For example, in the present study BTA20 was remarkably impacted during lactation, and harbors GHR and PRLR, both of which influence protein and milk yield [28,29,30]. As for dairy cows [25], in the present work on yaks the analysis of the transcriptome during lactation in mammary tissue revealed BTA6 as one of the most impacted and activated chromosomes, confirming it to be highly associated with QTL for milk production [31,32]. In dairy cows, casein coding genes are present in the BTA6 clustered in a small region together with statherin, all of which had a strong induction of transcription during the lactation cycle in yak mammary tissue (Supplementary File S1) as well as in bovine mammary tissue [25]. In addition to caseins, BTA6 has a plethora of other genes that were up-regulated during lactation (Supplementary File S1; e.g., ABCG2 and SPP1), with a potential strong effect on milk yield. For instance, ABCG2 plays a potential role in affecting the amount of water drawn into milk in vivo, thereby influencing milk volume [33,34].Although most of the genes in BTA14 were downregulated from 1 d to 180 d during lactation in our study, several QTL exist in BTA14 that are critical for milk fat yield [28,35,36]. For instance, protein coded by DGAT1 influences milk fat synthesis [37]. The DGAT1 gene was up-regulated two-fold during lactation (from 1–180 d) in our study (Supplementary File S1), which is consistent observations in bovines [38].4.2. The Onset and End of Lactation in Yak Mammary GlandIn mammals, the transition from pregnancy to lactation is characterized by metabolic adaptation of major organs (e.g., mammary, liver), enabling animals to adjust to the need to synthesize milk for the newborns [39]. In our study, the number of DEG indicates that transcriptional regulation in yak mammary gland is more intense at the onset and end of lactation. These data are very similar to the bovine mammary transcriptomic data [3], supporting a strong role of the transcriptome in the adaptation of the mammary gland to lactation. Similar to prior data in bovine [3], the onset of lactation is characterized by pathways associated with metabolism, especially lipid and amino acid metabolism. Pathways related to lipid metabolism revealed an increase in synthesis of triglycerides, which is very similar to observations in the bovine mammary transcriptome [3].The DEG in the comparison between 180 and 240 d in yak mammary tissue were highly associated with induction of ‘cell growth and death’, ‘immune system response’, ‘replication and repair’, and ‘amino acid metabolism’. The comparison between 240 d vs. 180 d corresponded to the time between end of lactation and dry period for yak. Thus, the comparison would be associated with mammary gland involution, in which more genes associated with ‘cell death’ and ‘immune response’ were induced. These data are consistent with the transcriptome of the mammary tissue in dairy cows [3], as well as with the typical large increase in mammary remodeling during involution [40,41].The large impact and activation of the ‘Metabolism of Cofactors and Vitamins’ sub-category of pathways in the mammary tissue of yaks differed from the inhibition of the same pathway observed in bovine mammary tissue [3]. High impact and induction were observed in pathways associated with mitochondrial respiration in yak mammary tissue during lactation (e.g., riboflavin, Pantothenate and CoA) [42]. Changes in mitochondrial abundance and function have been observed in people living in high vs. low altitude [43], and the same difference is present between yak and cattle [44]. GO Cellular Component terms related to mitochondrial structure and function were enriched in up-regulated DEG from 1 to 30 d, and enriched in down-regulated DEG afterwards, supporting a role of mitochondria during lactation. However, the ‘Oxidative phosphorylation’ pathway was barely impacted in the DIA and was not enriched in DAVID or IPA.The high impact on the ‘Folate biosynthesis’ pathway might indicate a role of this pathway in controlling the transcriptome. In folate metabolism, the 5,10-methylenetetrahydrofolate reductase (MTHFR) plays a key role in irreversibly converting 5,10-methylenetetrahydrofolate to 5-methylenetetra-hydrofolate, which is the main circulating form of folate. Mutations in the MTHFR gene, which is part of the ‘Metabolism of Cofactors and Vitamins’ pathway, is associated with changes in milk production in dairy goats [45]. The MTHFR transcript was upregulated during lactation in the present study (Supplementary File S1). Interestingly, this gene is associated with a genetic adaptation to high radiation in Tibetan people [46].In yak mammary tissue, ‘Cell growth and death’ was drastically induced at day 180 d compared with 240 d, and the fact that the lactation period of yak is around 180 days suggested an association with mammary gland involution, i.e., more genes associated with cell death were induced.4.3. Lipid Metabolism Is Induced in Mammary Tissue of Yak during the Lactation CycleMilk solids are composed of lactose, fat, and protein. Lipid metabolism was induced in mammary tissue during lactation in our study, similar to bovines [3], humans [4], and rodents [47]. Among the most induced lipid metabolism pathway in the yak mammary tissue during lactation was the ‘Synthesis and degradation of ketone bodies’. The expression of 3-hydroxybutyrate dehydrogenase type I (BDH1) was up-regulated between five and ninety-fold during the whole lactation compared to −30 d in our study (Supplementary File S1). This gene is the first step in the utilization of ketone bodies for the synthesis of milk fat [48,49], suggesting an important contribution of ketone bodies to milk fat synthesis in yaks. Recent data from buffalo suggests a similar role in that species [50] as well as in rats [51].Interestingly, as observed in dairy cows [3], ‘Steroid biosynthesis’ was revealed to be induced during lactation by DIA, IPA, and DAVID, indicating an important role of the mammary tissue in cholesterol synthesis. Milk contains cholesterol, and in bovines it is thought to be mostly derived from the liver [52], although evidence exists of cholesterol synthesis in the mammary gland [53]. While the importance of cholesterol synthesis by the mammary gland is unclear, the consistent increase in expression of cholesterol synthesis-related genes in mammary tissue of yak and cow, as observed in humans [4], strongly suggests an important role of cholesterol synthesis in milk production.Increases in milk fat synthesis shown by the transcriptome data were supported by the induction of several pathways involved in triglyceride synthesis; very similar pathways are induced in the mammary tissue of dairy cows during lactation [3]. These data confirm the important role of the transcriptome in controlling milk fat synthesis [38]. Additional pathways that support increased lipid synthesis in yak mammary tissue during lactation include the induction of ‘Riboflavin metabolism’, which was largely caused by the 11-to-32-fold increase in transcription of ENPP3 (ectonucleotide pyrophosphatase/phosphodiesterase 3) during lactation vs. −30 d (Supplementary File S1). The function of the enzyme encoded by this gene is to synthesize the cofactors FAD and FMN using riboflavin [54], which is essential for the proper function of the pentose phosphate pathway (not induced in our data). This is the main pathway for the provision of NADPH for the synthesis of lipids [55].Another important pathway associated with lipid metabolism was the ‘Pantothenate and CoA biosynthesis’. This pathway provides Coenzyme A for the activation of fatty acids prior to their utilization in the milk fat synthesis [56]. ‘Linoleic acid metabolism’ was strongly induced in yak mammary tissue, though it is not affected in dairy cows during lactation [3]. Yak milk is characterized by high concentration of conjugated linoleic acid, which is derived from the biohydrogenation of dietary linoleic acid in the rumen [57,58].The strong induction of the ‘PPAR signaling pathway’ during lactation in yak mammary tissue reflects the same induction observed in dairy cows [3,25,59]. These data confirm the importance of Peroxisome Proliferator-activated Receptors in regulation of milk synthesis; the role of PPARγ appears to be especially important in controlling milk fat synthesis, as has been previously argued [7,38,60]. A study of long non-coding RNA in yak mammary tissue during the dry period and lactation additionally underscores an important role of PPAR in lactation [18].4.4. Amino Acid MetabolismPathways related to amino acids indicate a strong role of the mammary gland in synthesis and utilization of amino acids, similar to bovine transcriptome data [3,20]. The importance of amino acid synthesis during lactation has been revealed by a recent study on long non-coding RNA in yak mammary tissue [18]. The pattern of ‘Valine, leucine and isoleucine degradation’ and the ‘Cysteine and methionine metabolism’ pathway were very similar between yak and bovine. However, the pattern of other amino acid-related pathways differed in yak compared to bovine. While in bovine these pathways were consistently activated during the whole lactation, in yak the pathways were activated early on during lactation and inhibited afterwards.As for bovine, these data indicate an important role of amino acid metabolism in the mammary gland, including the sparing of Met for the synthesis of milk proteins, as previously argued [3]. Transcriptome data, both microarray (as in the present manuscript) and RTqPCR [17], support an increase in expression of genes related to the regulation of milk protein synthesis, similar to the data for dairy cows [3]. RTqPCR data [17] suggest an important role of amino acid transport for milk protein synthesis. Rather than an increase in milk protein synthesis machinery, the present data suggest an increase in the metabolism of AA, providing additional support for an important role for the mammary tissue in handling amino acids for milk protein synthesis.In bovines as well as in other species, the expression of ribosomes mostly decreases during lactation, while transcription of genes coding for caseins and other typical milk proteins strongly increases, suggesting specialization of the mammary gland to synthesize milk proteins and not proteins for ancillary functions in the mammary gland [20]. In yak, the transcription of ribosomes was not strongly impacted; however, for other species [20], RNA degradation and protein degradation (i.e., proteasome) are inhibited and ER processing of proteins is induced during lactation, supporting an increase in synthesis of proteins that are secreted.4.5. Glycan Biosynthesis and MetabolismBovine milk N-glycome during early lactation is highly affected, with the alteration being largely due to the changes in N-glycosylation of IgG in colostrum [61]. Nearly forty oligosaccharides were detected in dairy cow milk, most of which stimulate the growth of beneficial bacteria and inhibit pathogen binding to epithelial cell surfaces in the intestine [62]. Among these, glycosphingolipids have attracted the most attention because of their probiotic roles [62]. In mammary tissue of dairy cows, ‘Glycosylphosphatidylinositol(GPI)-anchor biosynthesis’ is among the most impacted and induced pathways [3]. Although the role of this pathway in lactating mammary tissue remains unknown, it is of interest that in yaks this pathway appears to be associated with milk synthesis, prompting further research to understand its role.N-Linked glycans are attached in the endoplasmic reticulum, and O-linked glycans are assembled in the Golgi apparatus to produce modifications to proteins, as in glycoproteins and proteoglycans [63,64]. The strong induction of O-glycan and N-glycan biosynthesis in mammary tissue during lactation in yak strongly suggests enrichment of glycoproteins and proteoglycans in yak milk.4.6. Cell CycleCell cycle-associated pathways and GO terms were strongly inhibited according to DIA, and were the most enriched according to DAVID and IPA during lactation, though activation between 180 d and 240 d strongly suggested the cell cycle to be a major transcriptomic adaptation in lactating mammary tissue in yaks. Cell cycle and apoptosis were found to be important in mammary tissue of dairy cows [3]. As for dairy cows, the data clearly indicate that the mammary gland of the yak does not change its morphology/structure or experiences minimal changes in terms of cell profile during lactation (or even two weeks prior lactation). In contrast, strong morphological changes take place during the end of lactation (i.e., 240 d vs. 180 d).The microarray data indicated an important role of the ‘p53 signaling pathway’ in controlling the cell cycle in the yak mammary gland. A previous study with the human mammary MCF10A cell line showed that elevated p53 protein levels can arrest the cell cycle and initiate apoptosis [65,66], which further supports the stasis of cell proliferation suggested above. The increase in apoptosis after peak milk yield supports a likely gradual decrease in number of mammary epithelial cells after peak lactation, which might account for the decline in milk production with advancing lactation, as observed in dairy cows [67].4.7. Immune Response SystemImpact on functions related to the immune system in mammary tissue would be expected due to the evolutionary origin of the mammary gland and its known roles in the immune system [35]. The pattern of pathways associated with immune system in yak mammary tissue observed in the present work were very similar to that observed in dairy cows [3].The ‘Complement and coagulation cascades’ pathway belongs to the innate immune system. The activation of this pathway during lactation could be taken to mean an increase in the ability of ‘complement’ to kill pathogens by antibodies [68]. The induction of the ‘Toll-like receptor signaling pathway’ during lactation implies an increasing ability for mammary tissue to recognize invading microbes, especially gram-negative bacteria that present lipopolysaccharide [69].Similar to dairy cows, the overall induction of pathways associated with innate immune response, such as ‘Complement and coagulation cascades’ and ‘Toll-like receptor signaling’, along with strong inhibition of ‘Antigen processing and presentation’, appears to support previous conclusions [3] that lactating mammary tissue experiences an increase of amount/activity of immune cells with a concomitant decrease in the hyper-activation of the immune system. Considering the impact of mastitis in the dairy industry, this consistent finding between the two Bos species is important.4.8. Adaptation to Low Oxygen and High Radiation EnvironmentThe metabolism of the yak is adapted to the low oxygen content on the Tibetan plateau [70]. Among pathways potentially associated with the response to low levels of oxygen, the ‘VEGF signaling pathway’ was induced in DIA in the present study, and was not enriched in DAVID or IPA. Vascular endothelial growth factor (VEGF) acts as a signal protein to stimulate vasculogenesis and angiogenesis, restoring oxygen supply to tissues when blood circulation is inadequate. Transcription regulation of VEGF and the VEGF receptor plays a role in rodent mammary gland function during pregnancy, lactation, and involution [71], while its role in the dairy cow mammary gland during lactation appears to be minor [3]. Inactivation of VEGF in the mammary gland epithelium severely compromises mammary gland development and function [72]. Unlike dairy cattle, yaks must adapt to an environment with a low oxygen concentration. Therefore, induction of the ‘VEGF signaling pathway’ could be a potential adaptive mechanism in the yak mammary gland.As there is high solar radiation at the high altitude of the Tibetan plateau [73], the body has to adapt to this amount of radiation, as observed in humans [46] and, although not studied yet, likely in yak as well [74]. Because yaks must lactate under high solar radiation, the enrichment in down-regulated DEG of pathways associated with response to radiation is somewhat puzzling [75]. It is possible that the response to radiation is an important function in all yak tissues; the functional adaptation of the mammary gland to produce milk might involve the decrease of other biological functions of mammary tissue that are not teleologically ordered towards milk synthesis.4.9. LimitationsThis study presents several limitations. The use of microarray instead of RNAseq limits our ability to detect more transcripts, and microarray has lower sensitivity and reliability compared to RNAseq. Another potential limitation in our approach is that in mammary tissue the change in transcription of few highly expressed genes, such as caseins and lactalbumin, can “dilute” the level of other transcripts, generating an artificial down-regulation of transcripts, as previously demonstrated for RTqPCR (Bionaz and Loor, 2007). This would of course affect the interpretation of our findings. The dilution effect mentioned above can be very problematic when using quantitative techniques such as RTqPCR or RNAseq, though it might be not as problematic when using microarray. This is somewhat demonstrated by work done on pig mammary tissue during the lactation cycle of several transcripts, which was not affected by the day relative to parturition as potential internal control genes (Tramontana et al., 2008). Those genes were all “down-regulated” in the RTqPCR data prior to normalization. In order to evaluate whether a dilution effect was present in our microarray data, we evaluated the pattern of several transcripts previously used as references genes in the two above-cited papers. Fifty percent of the transcripts considered had a significant down-regulation that could be due to a dilution effect. We have also evaluated the three best reference genes for RTqPCR analysis of the same samples used in the present experiment (Jiang et al., 2016), and found that two transcripts were significantly affected through lactation, with MRPS15 being down-regulated (i.e., an apparent dilution effect), UXT having an increase through lactation, and RPS23 not being significantly affected (Supplementary Figure S2). Overall, we cannot exclude the possibility that certain down-regulated pathways may be a consequence of an artificial “dilution” of their transcripts; however, our data do not provide evidence that any dilution was apparent in our microarray data. In any case, this is an issue with normalization of RTqPCR vs. RNAseq or microarray that prevents full comparison between these techniques, which is an issue that lies outside the purpose of the present manuscript.5. Summary and ConclusionsThe transcriptome profile of yak mammary tissue during lactation was displayed by sampling nine time points. DIA was used to identify lactation-related genes on yak chromosomes, laying a foundation for QTL mapping of the chromosomes. KEGG analysis indicated that transcriptional regulation in the yak mammary gland is more intense at the onset and end of lactation. Functional analysis underscored the importance of induction of lipid and amino acid metabolism, glycan biosynthesis, and PPAR signaling along with inhibition of the cell cycle, response to radiation, and immune response during lactation. The transcriptome adaptation of mammary tissue to lactation in yaks appears to be very similar to that observed in dairy cows, though with several unique characteristics.

animals : an open access journal from mdpi

10.3390/ani11082272

PMC8388443

Doppler ultrasonography is frequently used to measure blood flow. The Ovsynch program is applied to synchronize the timing of ovulation in dairy cows. Heat stress can negatively affect the hormonal balance, ovarian activity, and blood flow. In this study, the effect of heat stress on corpus luteum blood flow, progesterone, and insulin-like growth factor parameters was investigated during and after Ovsynch synchronization. Our results showed that synchronization initiated with high progesterone values caused significantly higher blood flow and greater corpus luteum area in the comfort period when compared with the hot period. In addition, insulin-like growth factor values were found significantly higher during the comfort period compared to heat stress. Under heat stress circumstances, the Ovsynch synchronization provided better results when the progesterone levels were high. We suggest that it may be better to apply the modified Ovsynch program to increase progesterone levels in cows with low progesterone values when the protocol is initiated during the heat stress period.

The values of luteal blood flow (LBF), total corpus luteum (CL) area (TAR), and progesterone (P4), during and after OvSynch (OvS) protocol in comfort (CP; n = 40) and hot periods (HP; n = 40) were compared. We investigated how low and high P4 values obtained before the application affected the parameters above during CP and HP periods. Blood samples were collected before the OvS application on day 0 (OVSd0), day 9 (OeG), and day 18 (9th day after OeG: OvSd9). The P4 (ng/mL) values of the animals exhibiting dominant follicles were between 0.12–0.82 in HC and 0.1–0.88 in CP (P4-2: 4.36–4.38 and P4-3: ≥7.36 ng/mL). The LBF values were measured on days 7 (OvSd7) and 9 (OvSd9) after the OeG. The P4 mean values at day 0 (OvSd0) were classified as low (P4-1), medium (P4-2), and high (P4-3). The LBF and the TAR values in the P4-2 and P4-3 on OeG day 9 were higher than in HP (p < 0.05; 0.001), but there was no significant difference in the P4-1. In conclusion, when the OvS program was initiated with low P4 values, no difference was observed between HP and CP in terms of LBF values; however, when the program was started with high P4 values, there were significant increases in LBF and TAR values in the CP compared to the HP.

1. IntroductionHeat stress negatively affects fertility as temperatures rise [1]. When comfort and hot periods are compared, the pregnancy rate during the first insemination was 44% and 27%, and the anestrus rate was 1.2% and 12.9%, respectively, with results favouring the cooler period [2].Summer heat stress lowers the fertility in cattle in hot environments by influencing oocyte quality, follicular activity, and blood plasma progesterone (P4) levels [1,3]. However, the mechanisms by which elevated temperatures influence the corpus luteum (CL) functioning remain unclear. The upper limit of ambient temperature at which lactating dairy cows can maintain a stable body temperature (upper critical temperature) is as low as 25–27 °C. Therefore, heat stress is not confined to tropical regions of the world and imposes a considerable cost on milk and beef production [3].It has been revealed that the comfort zone for cows is between 5 and 25 °C, and the general condition and productivity of animals is not negatively affected at temperatures in this range [4,5]. Programs that stimulate ovulation by coordinating the development of the follicle and CL have been successfully implemented in lactating cows. Timed insemination programs, which eliminate the difficulties in estrus detection, enable the cow to be inseminated in estrus at a specified time and induce pregnancy [6]. Silent estrus is a severe problem in large herds during periods of heat stress [7]; previous research suggests that this program or its modified version is also successful during periods of heat stress, leading to increased pregnancy rates [8,9]. Although the OvSynch (OvS) program increases fertility performance during heat stress, it cannot prevent embryonic deaths [8].Color Doppler Ultrasonography (CDU) has long been used to measure the physiological changes and functions of the ovary and uterus [7]. For this purpose, with this technique, measurements of real-time changes in luteal blood flow (LBF) during induction [7,10] or spontaneous [11] luteolysis are made in cows. CDU has also been applied in cows to investigate the relationship between LBF, luteal size (LS), and P4 [7].In this study, the effect of heat stress in comfort and hot periods was investigated in dairy cows, one of the most significant breeding animals for human health and national economies. In the light of data from previous studies, the aims of the present study were i) to measure LBF, TAR, and the blood P4 values after OvS protocol in comfort and the hot periods and ii) to assess differences of these parameters exploring new approaches in the management of heat stress, revealing how especially low and high progesterone values affect the OVS protocols in comfort and hot periods.2. Materials and MethodsThe local animal ethics committee of Near East University approved the study (Decision No: 7/24.10.2016), and the ethical guidelines mentioned in the 1964 Declaration of Helsinki and its later amendments were followed. The study was carried out on a commercial dairy farm located in Ercan, Nicosia, North Cyprus, following the approval of the Veterinary Department of the Ministry of Agriculture and Forestry, North Cyprus.2.1. AnimalsThe study was performed during the following two periods; Comfort period (CP, winter (from December 2018 to February 2019), average temperature, precipitation, and humidity for 3 months: respectively 11.5 °C (min 2.5–max 19.7 °C), 142 mm, 74.0%; n = 40), and Hot period (HP, summer (from June 2018 to August 2018), average temperature, precipitation, and humidity for 3 months: respectively 35.5 °C (min 28.2–max 40.1 °C), 3.4 mm, 64.0%; n = 40). Additionally, there are no cooling or ventilation systems present on the farm. A total of 80 Holstein Friesian cows were assigned to this study (body condition score 2.75 and 3.00). The cows were kept at the same farm and provided with similar housing conditions and feeding regimes to exclude the influence of confounding factors such as feeding and environment. The average age of the study animals was 5 ± 1.5 years (3–8); all were multiparous, with an average milk yield of 27.0 ± 3.7 (min 22.0–max 35.0) litres. The animals were housed in a free-stall system and had ad libitum access to feed and water. The diet consisted of a base ration given ad libitum as a daily total mixed ration (TMR). The feeding frequency of TMR was twice per day. During the dry period, the ration was based on hay, green grass, TMR, an admixture of minerals and vitamins, and it was adjusted to contain 10–13% crude protein, 2–3.5% crude fat, and 35–40% neutral detergent fiber on a dry matter (DM) basis. The post-partum diet contained corn silage, hay, green grass, TMR, an admixture of minerals and vitamins, adjusted to contain 17–20% crude protein, 3–5% crude fat, and 27–34% neutral detergent fibre on a dry matter (DM) basis. Animals were screened before starting the applications (OvSd0). General (BCS 2.75–3.0; fat/protein ratio 1.16–1.18) and gynecological controls were performed (uterus symmetrical/close to symmetrical, no thickening, active ovaries, active CL/follicles, no vaginal discharge) of the animals. The status obtained from the control of animals is given in Table 1. It was determined that there was no difference between the parameters such as lactation number, days in milk, milk yield, and body condition score between the HP and CP groups. 2.2. Experimental Design The OvS protocol [12] was applied to start from the 35th day post-partum. LS, LBF, and P4 values were measured in an OvS protocol. The blood samples were collected (Vena jugularis) into serum separator tubes (SST) from each cow on day 0 (before the start of the application: OvSd0), day 9 (estrus: OeG), and day 18 (9th day after OeG: OvSd9). The first GnRH application was made after the blood was collected on day 0 (OvSd0) (Table 2). Sera were obtained following centrifugation at 1500 g for 10 min at +4 °C and stored at −80 °C until the analysis. The classification of P4 values was done retrospectively as low, medium, and high after P4 analyses were performed in the laboratory at the end of the study. The P4 (ng/mL) mean values at day 0 (OvSd0) were classified as follows: HP: 0.64 (0.10–1.42; n = 19), CP: 0.85 (0.16–1.97; n = 18) = (P4-1); HP: 4.36 (2.18–5.62; n = 12), CP:4.38 (2.56–5.80; n = 10), (P4-2); and HP: 7.36 (6.23–8.59; n = 9), CP: 10.33 (6.79–14.30; n = 12), (P4-3). The animals were not inseminated during estrus after the last GnRH administration (OeG). CL development on the 9th day postestrus was examined, and the LBF values were measured on days 7 (OvSd7) before PGF2-α administration (D16) and 9 (OvSd9) after the estrus (D18) (Table 2). Estrus (OeG) was determined according to known estrus criteria in follicle measurements (1.4–2.23 cm) and uterus examination, which was performed 24 h after the last GnRH administration [13,14]. 2.3. Measurement of Luteal Blood Flow (LBF) and Total CL Area (TAR)LBF of the CL was measured using a Color Doppler (portable LOGIQ Book XP ultrasound device-General Electrics Healthcare, Solingen, Germany; 10 MHz linear probe- Model 1739RS, Tokyo, Japan). Ultrasonographic controls and pictures taken were processed as described previously [14,15]. Pictures were frozen at the maximum cross-section and saved for measurements. The pictures were then examined using the same instrument equipped with a linear probe for imaging BF of CL in power Doppler mode (Doppler frequency: 5 MHz; gain: 19.5; pulse repetition frequency: 0.5 kHz). Five pictures without flash artefacts and a maximum number of colored areas were stored in system memory in the DICOM (Digital Imaging and Communications in Medicine) format. The stored Doppler images (5 images of blood flow regions in the LC) were analyzed using a specialised software program (Pixel Flux, version 1.0, Chameleon Software, Leipzig, Germany). For this, the entire CL structure and blood flow region was selected as the regions of interest and the colored areas were calculated (Figure 1). An average of five images was used to evaluate LBF further. 2.4. Progesterone (P4) and Insulin-Like Growth Factor-1 (IGF-1) ELISA AnalysesP4 and IGF-1 concentrations were measured using enzyme immunoassay test kits (Progesteron, DE1561, Lot. 23K039-2, Demeditec, Kiel, Germany; RayBio®® Bovine IGF-1 ELISA, ELB-IGF-1, Lot 120117 0659, Raybiotech, Inc., Peachtree Corners, GA 30092, USA). The tests were carried out following the manufacturer’s directions. The washing steps of the ELISA test were performed using an automated microtiter washer (MW-12A Microplate washer, Mindray, Shenzhen, China), and the results were obtained using a microtiter plate reader at 450 nm (MR-96A Microplate reader, Mindray, Shenzhen, China). The intra- and inter-assay coefficients of variation (CV) were 2.9% and 4.0% for P4, and 5.8% and 8.5% for IGF-1, respectively. 2.5. Statistical Analyses In the statistical evaluation, all analyses were carried out using the IBM SPSS software (version 21). The mean value and the standard deviations were obtained by applying descriptive statistics (X ± SD). The Shapiro–Wilk test was used to determine the homogeneity distribution. The Mann–Whitney U test was used as the nonparametric test in the case of non-homogeneous distribution of the values. In the case of homogeneous distribution, the Independent Samples T-test was used. A Chi-square test was used in the proportional comparisons. General Linear Models-Repeated Measures-Test was used for the independent values. Bivariate analysis was applied as correlation, and the Pearson test was selected as the Correlation Coefficient. A p-value of <0.05 was considered statistically significant.3. ResultsThere was no difference (p > 0.05) between the LBF-HP and the LBF-CP values on the 7th and 9th days after the OvS protocol (Table 3).On the 9th day after OvS, there was no statistically significant difference between the HP and the CP periods in terms of both P4-Serum and IGF1-Serum values (p > 0.05; Table 4).The TAR measurements showed a significant difference at the level of p < 0.0001 between the CP and the HP values both on the seventh and the ninth days after OvS. It was found that the CP values on both days were greater than the HP values (Table 5).No statistical difference was found in the follicle diameters between the HP and the CP. On the other hand, the IGF-1 values in the comfort period were significantly higher than those from the summer period (Table 6).As a result of the examination of blood samples obtained 24 h after the last GnRH administration (OeG) in the OvS protocol, it was determined that the IGF-1 values in the summer period were significantly lower than in the CP (p < 0.01). However, there was no significant difference in follicle size between the two periods. According to the classification of the initial P4 values at the beginning of the OvS protocol (P4-1, P4-2, and P4-3), when the LBF values corresponding to these values were examined on the 9th day after OVS, it was determined that the LBF values that belonged to the P4-2 and P4-3 categories were significantly higher in the CP period than the HP period. In addition, it was determined that the CL size corresponding to the initial P4-1, P4-2, and P4-3 values 9 days after OvS were significantly higher in the CP period than the HP period (p < 0.05; 0.001 and 0.0001; Table 7).While there was no significant correlation between the P4 grouping, based on samples measurements at the start of the OvS protocol, and LBF and TAR values corresponding to that grouping in the HP group, a significant positive correlation was determined between the P4 categories (P4-1, P4-2, P4-3), LBF, and TAR (0.999; p < 0.05 and p < 0.01) in the CP period, and a high level of correlation between LBF and P4, again in the CP period (1.0; p < 0.001), was detected (Table 8).4. DiscussionThe negative effect of heat stress on both the animal and its reproductive system has long been known [16,17,18]. In studies on the effect of heat stress on steroid hormones, while it has been determined that plasma estrogen concentrations decrease due to heat stress [1,19,20,21], studies on blood progesterone levels are contentious. While some studies suggest no change as a result of heat stress [21], other studies indicate both decreases [22,23,24] or increases [25,26]. Various synchronization programs are applied to create timed artificial insemination. Among these programs, the OvSynch (OvS) protocol has been used successfully in synchronizing ovulation in timed insemination of cows during the first service [6,13]. Since this study aimed to measure the LBF values and TAR in the luteal stage, the animals were not inseminated. In our study, the data obtained as a result of OvSynch synchronization were evaluated in two ways: During the OvS protocol and in the luteal periods after completion of the application, as the calculation of the differences between HP and CP without considering the P4 values at the beginning of the application, and as considering the P4 values in categories as low, medium, and high (P4-1, P4-2, P4-3) at the beginning of the application. Some research suggests that the OvS protocol increases pregnancy rates when applied during the summer [9,27]; however, other research indicates better results are obtained in winter as opposed to summer [28]. Based on these reasons, we investigated the differences obtained in terms of LBF and TAR in OvS applications based on P4 values in HP and CP periods. In our study, when the OvS protocol was initiated, there was no statistically significant difference between the HP and CP in terms of P4 values. No statistically significant difference was found between the HP and CP in terms of serum progesterone measurements performed on the 9th day after the end of the OvS protocol. This result supports previous publications that stated that heat stress has no effect on P4 values [29,30]. It has been reported that conditions such as hepatic metabolism, other stress conditions, and dry matter intake have a greater effect on progesterone levels [1].Data obtained as a result of Color Doppler Ultrasonography LBF measurements applied during the CL formation found no statistically significant difference between HP and CP in terms of the LBF and the P4 values. It has been demonstrated that heat stress causes a 20–30% reduction in blood flow in the ovaries of rabbits and poultry [31,32]. Conversely, there are publications related to cows that have found heat stress does not affect the reaction of the CL and P4 circulation [33] and that rising temperatures do not negatively affect the luteal function and luteal cells [3] Previous research has revealed that P4 values are lower in the summer during the luteal period of the estrus cycle [23], or P4 values are higher in winter on the 9th day of in vitro luteinization [34]. In our study, when the results obtained in the luteal period after OvS protocol were calculated (regardless of the difference between P4 concentrations at the beginning of the application), it was concluded that the LBF and the P4 values were not affected by heat stress. To our knowledge, there are no publications to date in which the differences between the LBF or the CL sizes according to the season were examined with color Doppler data. Only one publication has shown less dominant follicular blood flow in cows with heat stress than in cows under cold management [35].Although LBF and P4 were not affected, it was revealed that the TAR was higher during the CP period, both on the 7th and the 9th days after the OvS protocol. No significant relationship was determined between the CL and the P4 values in the early and mid-luteal periods. A significant association could be determined 5 days before ovulation [36]. The maximum number of colored pixels in the cross-sectional area of the CL revealed a correlation between progesterone and CL change during the cycle process, but it was also revealed that the CL size did not reflect the blood flow and peripheral blood P4 alteration [37]. In our study, the size of CL did not increase between the 8th and the 16th days of the cycle; however, there was an increase in the progesterone concentration between these days. There is a decrease in blood flow shortly after ovulation, but with angiogenesis on the 2nd–5th days of the cycle, progesterone also increases with an increase in CL volume [10]. However, in this study, the CL measurements were made on the 7th and 9th days after the OvS protocol. Many factors support the function of CL. Luteinizing Hormone (LH), and Growth Hormone (GH) are necessary for the development and function of CL. In addition, angiogenic factors such as vascular endothelial growth factor (VEGF) and fibroblast growth factor (FGF) are also effective [38]. In our study, no statistically significant difference in follicle diameter was found between CP (1.67 cm) and HP (1.52 cm). However, IGF-1 measurements made on the same day showed that the CP values were significantly higher than the HP measurements during this period (p < 0.01). Various studies have found no relationship between the follicle diameter and IGF-1 [39,40]. It has been determined that heat stress negatively affects mostly the follicular fluid, the oocyte, and the granulosa cells [41]. Heat stress affects follicle selection and extends the follicle wave duration, resulting in a decrease in the quality of oocytes [42]. These results show that follicle content and structure are affected rather than the follicle size, which supports our findings.IGF-1 values are lower in the summer than in the thermoneutral period [43,44]. IGF-1 plays a role in developing follicles in synergy with follicle-stimulating hormone (FSH) and LH and supports steroidogenesis by enhancing the LH receptor induction and inhibin synthesis [45]. IGF-1 also increases the sensitivity of follicular cells to FSH and LH [46]. This is important in follicle development and improvement of oocyte quality [47]. In our findings, IGF-1 values in the CP are significantly higher than in the HP in blood measurements made during the follicle period 24 h after the last GnRH administration.However, in this study, cows with high P4 values (P4-2 and P4-3 values) revealed that the average blood flow in CL on the 9th day post-application was significantly higher in the CP period. When the P4 value is high at the beginning of OvS (no difference between CP and HP P4 values during start treatment), which develops 9 days after the last application, there is a different response in the LBF between the two periods with an increase during the CP period.As P4 values increased, there was a significant increase in both LBF (r = 0.999) and TAR (r = 0.999) values during the CP period (p < 0.05), while this increase did not occur during the HP period. An interesting result from the study is that the TAR values were significantly higher in the CP than HP according to the P4 categories. In some publications, there is a correlation in the measurements made between P4, LBF, and TAR [14,15]. The detection of a correlation in this study during the CP period, but the absence of such a correlation during the HP period indicates that this interaction occurs during the appropriate environmental conditions (CP). It seems that when OvS protocol is started with high progesterone values, high BF and larger CL sizes can be obtained in CP. The OvS protocol can always be started, but when started in the early diestrus period (days 5–12), it has been shown that the ovulation rate is 70% higher than during other periods (53%) [48]. It has been shown that when the OvS protocol is initiated on days 5–12, the rates of conception also increase [49,50,51]. In another study [52], in animals without CL, after waiting for 7 days, the pregnancy rate was found to be 40.9% in the OvS protocol, which was started after the CL was determined, while the pregnancy rate in animals without CL was found to be 25.6%. In our findings, as a result of OvS protocol performed in two categories (P4-2 and P4-3) where progesterone was high, significant increases in LBF and TAR values during CP also support these findings. In addition, studies demonstrate that the data obtained from the OvSynch protocol are better when performed during the winter period compared to those performed in the summer period [53]. While the conception rate of the first artificial insemination (AI) was 17.5% in heat stress (HS) from the OvS protocol, this rate was 23.3% in the winter season (WS). Likewise, while days open were 179.6 days in HS conditions, it was found as 104.02 days (p < 0.05) in WS (52). One study showed the conception rate obtained from the OvS protocol in the HS period was found to be significantly (p < 0.05) lower (25% vs. 35%) compared to the WS period [28]. When the OvSynch protocol was administered, the rate of pregnant cows obtained in winter was higher than that obtained in the summer [54]. In our study, when the OvS protocol was initiated with high P4 values, the increases in LBF and TAR values during the CP period suggests that the low conception values obtained in the aforementioned studies during the hot period are primarily due to the initiation of the OvS program with low progesterone values.When the OvS protocol is started, corpora lutea can be found in approximately 52% of animals to be under 15 mm (D0). In this case, the subluteal function cannot sufficiently generate progesterone priming in the endometrium during the application of Ovsynch [55]. It has been determined that low P4 values due to subluteal function in the estrus cycle cause low fertility after insemination [56]. In this study, mean P4 values were determined as 0.642 ng/mL (0.10–1.42 ng/mL) in the HP and 0.85 ng/mL (0.16–1.97 ng/mL) in the CP. These low P4 values are due to the fact that when starting the OvS program, the CL is very small (<15 mm) and has a dominant follicle on the ovary with a size of approximately 2 cm. No difference was detected between the two periods (HP/CP) regarding LBF values at this P4 value concentration. However, the increase in both LBF and TAR values in the CP period as the progesterone value increased shows that when the OvS protocol was started with high P4 values, a better response was obtained than the HP. In support of the findings of the aforementioned authors, our findings also suggest that the ovaries with small corpora lutea did not receive sufficient response. Lopez-Gatius et al. [55] selectively applied OvS program (without normal selection procedures) showed significantly decreased rates of pregnancy during first AI (43% vs. 27%) and pregnancy during the second AI (36% vs. 53%) in the presence of CL. In the case of follicles, when OvS application is initiated, progesterone supplementation increases the P4 value above 2.0 ng/mL, and this value is necessary to increase the Pregnancy/AI ratio [57].Corpus luteum blood flow was measured by Herzog et al. [7] with color Doppler during the estrus cycle, and a significant correlation was revealed between LBF and P4 concentration. The same study revealed that high luteal blood flow was vital for P4 secretion due to the close relationship between LBF and P4. However, it has also been shown that blood flow does not differ between low or high progesterone values [58]. On the other hand, in our study, the P4-2 and P4-3 group values are lower in the CP compared to HP. Honig et al. [35], using colour Doppler, showed that the dominant follicle circulation and the cycle length were affected by heat stress.As a result, when OvS protocol is initiated with high P4 concentration, a significant difference is obtained favouring CP in terms of LBF and CL size in the comfort period (CP) compared to the summer period (HP). LBF of developing CL is higher when starting OvS with high P4 values in cows. It is thought that it may be better to apply the modified OvS programs that increase the progesterone value in cows with low progesterone values when the protocol is started. 5. ConclusionsIn conclusion, the degree to which luteal blood flow and size differed between cows in the CP and HP was determined by the P4 concentration at D0. However, without grouping the P4 values, there was no CP/HP difference in the LBF and the P4 values in the luteal period developing after OvS. On the 9th day after estrus, the P4 and the LBF values in the seasons CP and HP were not significantly different from each other despite the area of the CL being different. However, in cows with high P4 values (P4-2 and P4-3), higher LBF and TAR values are obtained from OvS applications in the CP compared to HP. This shows that the high P4 values were more affected by heat stress.

animals : an open access journal from mdpi

10.3390/ani13071233

PMC10093590

The chest bone, or sternum, protects the heart and lungs and aids in breathing motion. It is included on chest radiographs of dogs and cats, but little information is available for veterinarians on what abnormalities or diseases affect the chest bone in companion animals. We reassessed chest radiographs of dogs and cats taken in our hospital over a 2 year period to describe these changes. We found that abnormalities of the chest bone were visible in 24% of dogs and 29% of cats, with the most common abnormality being age-related degeneration. Most of the abnormalities noted were of minor clinical importance, but in some animals, conditions that could be painful or otherwise affect well-being were seen.

Evaluation of the sternum is part of the routine examination of small animal thoracic radiographs. However, descriptions on frequency and type of abnormalities are lacking. This retrospective observational study aimed to describe abnormal radiographic findings of the sternum in a cross-section of client-owned dogs and cats undergoing thoracic radiography between 1 January 2019 and 1 January 2021 for reasons unrelated to diseases of the sternum. The study population consisted of 777 dogs (mean age, 7.3 ± 3.9 years) and 183 cats (mean age, 7.3 ± 5.1 years). Sternal abnormalities were observed in 189/777 (24%) dogs and 53/183 (29%) cats, mostly around the intersternebral cartilages, accounting for 32/80 (40%) dogs and 20/35 (57%) cats. This was followed by an abnormal number of sternal segments (8% dogs, range 3–9 sternebrae; 15% cats, range 7–9 sternebra). Pectus excavatum was observed in 6/777 (0.8%) dogs and 6/183 (3%) cats, and pectus carinatum in 18/777 (2%) dogs and 2/183 (1%) cats. Post-traumatic changes, such as subluxation, were observed in nine dogs (1.1%) and three cats (1.6%). Presumed prostatic carcinoma metastasis and malignant lymphoma were observed in two dogs (0.2%). Incidental radiographic sternal abnormalities are common in cats and dogs but mostly of unknown clinical relevance.

1. IntroductionAn evaluation of the sternum is part of the routine examination of small animal thoracic radiographs, as it forms the ventral contour of the thoracic cavity. The sternum contributes to the bony protection of the intra-thoracic cardiopulmonary structures, as well as to the stability and breathing motion of the thorax.The small animal sternum has been defined as an unpaired segmented series of typically eight bones, called sternebrae [1,2,3,4]. The cranial first sternebra, or manubrium of the sternum (manubrium sterni), is wider and longer than the other sternal segments. The manubrium of the sternum is the point of insertion of the sternocephalicus muscle and has lateral shelves of bone accommodating the attachment of the costal cartilages of the first pair of ribs. The body of the sternum (corpus sterni) consists of six rectangular to cylindrical-shaped sternebrae. The most caudal last sternebra, or xiphoid process (processus xiphoideus), is flat and long, occasionally with a foramen in its caudal half. The xiphoid process is prolonged caudally by the xiphoid cartilage (cartilago xiphoidea), which supports the cranial part of the ventral abdominal wall and from which the linea alba extends caudally to the symphysis pelvis. The individual sternebrae are connected by intersternebral cartilages, forming cartilaginous joints (synchondroses sternales), and the sternum is covered on the ventral and dorsal surface by thickened periosteum, forming the sternal membrane (membrana sterni). Whilst the costal cartilages of the first pair of ribs articulate directly with the manubrium of the sternum, the costal cartilages of the second to seventh ribs articulate with the consecutive intersternebral cartilages between the individual sternal segments, and both the costal cartilages of the eighth and ninth rib pairs articulate with the intersternebral cartilage between the seventh sternebra and the xiphoid process. The first eight paired sternocostal joints are synovial joints, but no synovial joint was found at the ninth sternocostal articulation The costal cartilages of the last four pairs of ribs do not directly articulate with the sternum but connect via the costal arch or are floating ribs, and these ribs are therefore considered asternal ribs [1,2,3,4].Species differences in the shape of the sternum exist. In dogs the sternum is curved and the sternebrae of the body of the sternum are rectangular in form, with the height exceeding the width. In cats, the shape of the sternum is described as a straight and uniform cylinder [4,5]. On thoracic radiographs, only the sternebrae are visible as individual structures because their high mineral content results in increased x-ray beam attenuation, and therefore higher opacity, on the digital image. The soft tissue structures of the sternum, such as the intersternebral cartilages, are generally not individually identifiable on a radiograph because of border effacement with the neighboring soft tissue of the thoracic wall [6].Congenital malformation of the sternum can consist of dorsal or ventral deviations (pectus excavatum and pectus carinatum, respectively), as well as numeric or shape changes, such as cleft sternum. Sternal malformations sometimes form a part of a larger congenital defect with expansion to the adjacent thoracic and abdominal structures as well as diaphragmatic cupula [7,8,9,10,11,12,13]. Acquired sternal abnormalities include neoplasia [14], traumatic or pathological fracture and luxation [15,16,17], infection [18,19,20], and degenerative disease of the intersternebral cartilage [21].No reports on occurrence of sternal abnormalities, either congenital or acquired, as a part of routine radiographic thoracic evaluation are available in dogs and cats. Although not all congenital variations in sternal conformation might be clinically relevant, some of these malformations and abnormalities are causing clinical signs. Being familiar with these abnormalities and the normal variation present, is therefore considered essential in image interpretation. The aim of this study was to describe the occurrence of sternal abnormalities on routine thoracic radiographs of dogs and cats.2. Materials and MethodsThis observational study had a cross-sectional and retrospective design. Thoracic radiographic examinations of dogs and cats performed between 1 January 2019 and 1 January 2021 were retrieved from the Picture Archiving and Communication System (Agfa Healthcare Enterprise Imaging, Agfa Healthcare N.V., Belgium) of the Division of Diagnostic Imaging of the Faculty of Veterinary Medicine, Utrecht University, The Netherlands. Images from client-owned animals that had thoracic radiography performed for reasons unrelated to diseases of the sternum were used with anonymity preserved. Examinations were excluded if the thoracic radiographs did not include the entire sternum in the field of view. For animals with repeat examinations performed within the study period, only the first performed radiographic study of the thorax was included for analysis.The thoracic radiographic studies were obtained with a direct radiography system (Optimus NZR136, Philips, Best, The Netherlands) using a radiographic technique optimized for examination of the thorax (i.e., exposure during maximum inspiration and high kV, low mAs technique) with exposure factors based on patient body size, typically 105–110 kV; 1.2–1.4 mAs for dogs and 50 kV; 5 mAs for cats. A grid was used if object thickness was >10 cm. Animals were not routinely sedated and were positioned using manual restraint by trained personnel wearing protective clothing and personal dosimeters.Assessment of the sternum was performed on left lateral and dorsoventral thoracic radiographs displayed on medical-grade greyscale monitors (Coronis Fusion 6MP DL, Barco, Kortrijk, Belgium). Images were independently reviewed by two European College of Veterinary Diagnostic Imaging resident (DHNB and SCV) and two European College of Veterinary Diagnostic Imaging diplomates (MTR and SV). For each animal, the sternum was subjectively assessed on alignment, number, shape, and opacity of sternebrae, and width and opacity of the intersternebral cartilages on orthogonal radiographs. A total number of eight sternebrae, including the manubrium of the sternum and xiphoid process, was considered the normal number of sternal segments in both cats and dogs [1,3], and any animal with a higher or lower number of sternal segments was considered abnormal for the purpose of this study.The vertebral index was calculated as a measure of severity of pectus excavatum using the vertebra overlying the deviation at the most depressed point of the thoracic wall [11,12,22]. The severity of pectus excavatum was characterized as mild if the vertebral index was >9, moderate if the vertebral index was 6–8.99, and severe if the vertebral index was <6, as previously described [11,23]. The type of pectus excavatum was determined based on the anatomical location of the deformity of the sternum and rib cartilage. Pectus excavatum was characterized as the typical form when the condition affected the caudal sternum from the 5th to 8th sternebrae and as an atypical form when the dorsal deviation was noted in the cranial to mid-region of the sternum [10,22].Numerical data are reported as mean (standard deviation [SD]) if normally distributed or as median (range) if the distribution was skewed. Data were analyzed using IBM SPSS Statistics (Version 28). Independent samples t-test was used to assess differences in age between groups of animals with and without degenerative changes affecting the sternum. Statistical significance was set at p < 0.05.3. Results3.1. DogsThe study population consisted of 777 dogs. The group of dogs had a mean age of 7.3 (SD, 3.9) years and consisted of 400 males (188 neutered) and 377 females (252 neutered) of various breeds, the most common being mixed breed (n = 145), Labrador Retriever (n = 62), Chihuahua (n = 28), Bernese Mountain Dog (n = 26), Golden Retriever (n = 23), French Bulldog (n = 23), Labradoodle (n = 22), and German Shepherd (n = 21). One hundred and fourteen dogs were of brachycephalic breed. The three most common indications for thoracic radiography in dogs were metastasis screening (n = 252), cough (n = 94), and dysphagia (n = 57). An example of a thoracic radiographs of a dog on which the sternum was considered unremarkable is provided in Figure 1.Sternal abnormalities were observed in 189/777 (24%) dogs (Table 1). Nineteen dogs had more than one sternal abnormality, being degenerative changes in combination with abnormal number of sternebrae (n = 8), post-traumatic changes (n = 5) or shape deformity of the sternum (n = 1), and an abnormal number of sternebrae in combination with shape deformity (n = 4) or post-traumatic changes (n = 1).Abnormalities were most frequently observed in the area of the intersternebral cartilages in dogs (n = 108; 14%), most of which were considered degenerative type changes such as mineralisation of the sternal cartilages or formation of bony spurs (n = 98; 13%). Marked degenerative changes, such as narrowing of the intersternebral cartilage space (n = 13), vacuum phenomenon (n = 13), and subluxation (n = 5), were mainly observed in the mid-section of the sternum, between the 3rd and 4th and 4th and 5th sternebrae (Figure 1 and Figure 2). Predominantly medium to large breed dogs were affected, with Labrador Retriever as the most common breed (n = 16). The mean age of dogs with degenerative changes (mean, 9.8; SD, 2.5 years) was significantly higher than the mean age of dogs without these changes (mean, 6.9; SD, 3.9 years; p <0.001).An abnormal number of sternal segments was the second most seen abnormality in dogs (n = 62; 8%). Less than eight sternal segments were observed 46 dogs (6%): seven sternebrae were visible in 40 dogs, six sternebrae in four dogs, and three and four sternebrae in one dog each. The latter resulted in morphologically abnormal short sternum (Figure 2). The lower number of sternebrae predominantly affected small breed dogs, including four out of seven Pugs (57%), five out of fourteen Pomeranians (36%), and six of twenty-eight Chihuahuas (21%) in the study population. In eight dogs, the lower number was due to fusion of two (n = 5) or four (n = 2) sternebrae. Three dogs with lower number of sternebrae showed border effacement of the ventral thoracic diaphragmatic surface and the caudal ventral cardiac silhouette due to cranial excursion of the diaphragm, but none of these animals had evidence of peritoneopericardial herniation. Supernumerary sternal segments (9 sternebrae in all) were observed in 16 dogs (2%), affecting predominantly medium and large breed dogs. The most common breed affected was Labrador Retriever (n = 7).Pectus excavatum was observed in six dogs (0.8%), and the severity was considered mild in five dogs and moderate in one dog based on vertebral indices (median vertebral index, 10.5; range, 8.6–11.2). Pectus excavatum involved the caudal sternum in five cases and the mid-sternum in one mixed breed dog. One case was considered likely to have a traumatic instead of congenital origin based on clinical history. Four of the five dogs with suspect congenital pectus excavatum were brachycephalic breeds, including two out of seven Pugs (Figure 2). Mild pectus carinatum was observed in 18 dogs (2%) and affected the caudal part of the sternum in all cases. French Bulldog (n = 8) and Chihuahua (n = 5) were overrepresented.Presumed post-traumatic changes were observed in 12 dogs (1.5%), although none of these dogs were presented for thoracic radiography because of a history of trauma or suspect traumatic lesions of the sternum; the indication in eight of these dogs was metastasis screening. The presumed post-traumatic changes mainly consisted of dislocation (n = 9, of which five were in combination with other degenerative changes) (Figure 2). These changes affected predominantly the mid-section of the sternum. Changes compatible with chronic fracture of sternebrae were observed in two dogs.One Chihuahua with malignant lymphoma had multiple pathological fractures associated with osteopenia (Figure 2). An aggressive bone lesion, suspect prostatic carcinoma metastasis affecting the second and third sternebrae, was observed in another dog.3.2. CatsThe study population consisted of 183 cats. The group of cats had a mean age of 7.3 (SD, 5.1) years and consisted of 105 males (89 neutered) and 78 females (66 neutered) of various breeds, most commonly Domestic Shorthair (n = 112), Maine Coon (n = 16), mixed breed (n = 11), and British Shorthair (n = 10). The most common indications for thoracic radiography in cats were dyspnea (n = 40), metastasis screening (n = 37), and post-accident trauma (n = 18). An example of a thoracic radiographs of a cat on which the sternum was considered unremarkable is provided in Figure 3. Sternal abnormalities were observed in 53/183 (29%) cats (Table 2). Six cats had more than one sternal abnormality, being abnormal number of sternebrae in combination with pectus excavatum (n = 3) or degenerative changes (n = 3).Degenerative changes, consistent of mineralization of the sternal cartilages or formation of bony spurs, were observed in 15 cats (8%). Cats with degenerative changes were significantly older than those without (mean, 11.2; SD, 5.6 years versus mean, 6.9; SD, 4.9 years; p = 0.002). Concurrent gas opacity in the intersternebral cartilage space or associated subluxation of sternebrae was not observed in any of the cats with signs of degenerative changes of the intersternebral cartilages.An abnormal number of sternebrae was observed in 27 cats (15%), of which seven cats had nine sternebrae. Nineteen cats had seven individual sternal segments, which, in ten cats was caused by complete fusion of the first and second sternebrae (n = 5) or sixth and seventh sternebrae (n = 5). The other nine cats with seven sternebrae had an absence of one sternal segment. One Domestic Shorthair had six individual sternebrae, caused by complete fusion of the fourth and fifth sternebrae and the absence of one sternal segment (Figure 3).Pectus excavatum was observed in six cats (3%), and the severity was considered mild in four cats and moderate and severe in one cat each (median vertebral index, 9.8; range, 5.8–11.4) (Figure 3). The typical form of pectus excavatum, affecting the caudal sternum, was observed in 2/6 cats and the atypical form, affecting the cranial or mid-region of the sternum, was observed in 4/6 cats. Mild forms of pectus carinatum were observed in two cats (1%).Signs of traumatic luxation were observed in three cats (1%). One cat had undergone recent cardiopulmonary resuscitation, and the other two cases showed more chronic changes with malformed fusion of the dislocated sternebrae. One of these cats had a history of sternal fracture 10 years prior and the other cat had no known history of trauma (Figure 3). Other abnormalities observed in cats were a kinked manubrium of the sternum (n = 2), partial fusion of sternebrae (n = 3), and narrowing of the intersternebral cartilage space without signs of degeneration (n = 1).4. DiscussionThe sternum develops from the fusion of bilateral mesodermal bars which unite from day 25 of gestation in dogs and day 28 of gestation in cats, starting cranially at the manubrium of the sternum and ending caudally at the xiphoid process [1,24]. Ossification starts at day 40 in dogs but is inhibited at rib attachment sites, resulting in the individual sternebrae with interposed intersternebral cartilages in those areas where the ribs attach. However, this is not the case for the first rib pair, which connects to the ossified manubrium of the sternum but attaches at a later time than the other costal cartilages during development. Nonunion of the caudal part of the sternal bars, anomalous ribs, and uneven apposition of ribs can lead to various morphological abnormalities of the body of the sternum and its individual sternebrae [1]. Sternal defects have been reported with concurrent congenital midline anomalies, such as peritoneopericardial diaphragmatic hernia and cranioventral abdominal wall hernia in dogs and cats [7,8,9,13,25,26]. Abnormal number, fusion, and shape alterations of sternebrae were observed in both cats and dogs in our study population, but in none of these animals was clear evidence of bifid sternum or other ventral abdominal wall anomalies seen. This could in part be explained by the limited visibility of the sternum on straight dorsoventral radiographic projections, caused by superimposition of the vertebral column on the sternum. In addition, mineralized rib cartilage was often superimposed on the most caudal aspect of the sternum on left lateral views. Oblique dorsoventral radiographic views and computed tomography would be more sensitive techniques to identify cleft sternum and concurrent ventral abdominal wall defects [5].As in previous reports, pectus excavatum mostly affected the caudal sternum in dogs in our study population, which is the typical form of pectus excavatum [12]. Komsta and colleagues (2019) observed pectus excavatum in a large percentage of brachycephalic dogs (44.4%) [10], whilst we observed this condition in only 3.5% of brachycephalic dogs and 0.8% of dogs of all breeds. In that study, Maltese and English Bulldog breed dogs were overrepresented, but in our study population none of the three dogs for each of these two breeds showed evidence of pectus excavatum. A large proportion (67%) of affected cats in our study population had an atypical location of pectus excavatum, in which the dorsal deviation affects the cranial and mid-section of the sternum. This was also reported in a recent publication on computed tomographic features of pectus excavatum in cats, in which half the cats had the atypical form of the disorder [11]. That study found mild pectus excavatum in only 21.5% of cats, whilst most of the cats included had moderate or severe pectus excavatum. In contrast, a larger proportion of pectus excavatum was considered mild in cats in the present study (67%). In part, this could be explained by the fact that none of the cats included in our study were presented for radiographic examination because of complaints related to their thoracic malformation. However, although a strong positive correlation has been reported in brachycephalic dogs with the atypical form of pectus excavatum, no clear association has been found between the radiographic classification and the severity of clinical signs in brachycephalic dogs with the typical form of pectus excavatum [12] or in a group of kittens with pectus excavatum [27]. Moreover, it should be noted that our results are based on thoracic radiographs made on a single time point for each patient, whilst motion of the sternum in severely dyspneic animals could mimic conditions such as pectus excavatum [28]. Therefore, the number of animals with abnormal alignment of the sternum could be overestimated in our study population.In agreement with a previous publication by Hassan and colleagues (2018), we observed pectus carinatum predominantly in small breed dogs, except for in one English Springer Spaniel [12]. In that previous report, the Pug and French Bulldog were at increased risk of this disorder, with 41% and 18% of the dogs of these breeds affected, respectively. In our study population French Bulldog and Chihuahua were the most common breeds with pectus carinatum, with 8/23 (35%) and 5/28 (18%) animals affected, respectively. Only 1/7 (14%) Pugs showed radiographic evidence of pectus carinatum in our study.Although neoplastic disease was the most common indication for thoracic radiography in dogs, only two cases presented with radiographic aggressive bone lesions affecting the sternum. One dog diagnosed with malignant lymphoma presented with pathological fractures of multiple sternebrae, the other dog was diagnosed with prostatic carcinoma and had an aggressive bone lesion affecting the second and third sternebrae. However, the sternal lesions were not biopsied in these dogs and therefore it remains unknown if these aggressive bone lesions indeed were caused by neoplastic disease. Skeletal involvement affecting the spine has been described in canine multicentric lymphoma [29,30], but to the author’s knowledge it was not specifically affecting the sternum. Skeletal metastases are common in canine prostatic carcinoma, often accompanied by extraskeletal metastases, and the lumbar vertebra, pelvis, and proximal femur are mostly affected [31]. Metastases to the sternum from prostatic carcinoma appear uncommon, but have been documented in a dog [32]. In a necropsy study, metastatic lesions were observed in the sternum in nine out of twenty-one dogs with metastatic or multicentric tumors with bone involvement, and mammary carcinoma and lung carcinoma were the most common primary tumors [14]. Only one dog with prostatic carcinoma was included in that study, which did not have evidence of bone metastasis. Sternal osteomyelitis, caused by foreign body, systemic mycosis, following sternotomy or hematogenous spread of bacteria, is the main differential diagnosis to neoplasia for sternal aggressive bone lesions [18,19,20,33,34]. The dogs with aggressive bone lesions affecting the sternum included in this study had no other clinical signs or history indicative of infectious disease, but sternal osteomyelitis cannot be completely ruled out because the specific lesions were not biopsied.Age-related degeneration of the synchondroses sternales, mostly consistent of mineralization of the sternal cartilages or formation of bony spurs, was the most common finding in our study, with a prevalence of 14% in dogs and 8% in cats. In 13 of the dogs affected, gas opacity was observed in between the sternebrae on the left lateral view radiograph. Gas accumulation within synovial joints and intervertebral disc spaces is termed vacuum phenomenon. This gas consists of 90–92% nitrogen and its presence has been associated with intervertebral disc degeneration in dogs [35,36,37]. The sternal vacuum phenomenon has previously been described in three dogs [21] and, in agreement with our findings, this was reported in large breed dogs, frequently surrounding the fourth sternebra, and was associated with intersternebral joint space narrowing. Those authors proposed laxity of the intersternebral fibrocartilage and tractile forces caused by radiographic positioning as potential causes for vacuum phenomenon in the intersternebral cartilage space or superimposed sternocostal joints [21]. In our population, vacuum phenomenon was only observed in the intersternebral cartilage space in conjunction with other degenerative changes, such as narrowing of the intersternebral cartilage space, new bone formation or even dislocation of sternebrae. Gas opacity in the intersternebral cartilage space could therefore be considered a sign of degeneration of the synchondrosis sternalis in dogs, similar to the above-mentioned association between vacuum phenomenon in the intervertebral disc space and intervertebral disc degeneration in dogs [36,37].Sternal dislocation is infrequently reported in veterinary literature and is most often considered to be of traumatic origin [15,16,17]. Based on history and other findings, a traumatic origin was suspected in all three cats with sternal dislocation included in our study population. In five dogs, marked degenerative changes of the intersternebral joints coincided with sternal subluxation, which might suggest spontaneous dislocation following instability or arthropathy of these joints. However, due to the cross-sectional study design, it cannot be determined if dislocation preceded or followed intersternebral joint degeneration signs in these dogs. Future research is warranted to examine if sternal degeneration, now considered an incidental finding, affects animal well-being.Our study had several limitations. First, the retrospective design prohibits adequate correlation between radiographic findings and clinical signs. For example, it cannot be reliably determined if sternal dislocation in dogs with severe intersternebral cartilage degeneration was associated with pain or other clinical signs, because no specific history and physical examination addressing these findings were performed in these cases. Secondly, the large variety in breeds in combination with the relatively low number of cases for each disorder impedes drawing firm conclusions on any breed predispositions. Thirdly, the use of radiography instead of a cross-sectional technique such as computed tomography results in a lower sensitivity for detection of abnormalities because of the inferior contrast resolution and problems of superimposition inherent to radiography. Moreover, computed tomography would allow a better representation of the anatomy. For instance, computed tomography was reported to be useful for evaluation of thoracic asymmetry and sternal torsion in cats with pectus excavatum, which cannot be done with radiography [11]. In addition, the use of intravenous iodinated contrast medium in computed tomography would allow for better characterization of potential soft tissue abnormalities, such as inflammation [18].5. ConclusionsSternal abnormalities are common incidental findings on thoracic radiographs of companion animals and were observed on thoracic radiographs of 24% of dogs and of 29% of cats in our study population. Clinically relevant abnormalities, such as severe degeneration of the intersternebral cartilage space, dislocation, or aggressive bone lesions, were only recognized in few cases, whilst most animals showed abnormalities that were unlikely to cause clinical complaints.

animals : an open access journal from mdpi

10.3390/ani13071184

PMC10093577

Thermal imaging is gaining popularity in poultry, swine, and dairy animal husbandry for detecting disease and distress. In this study, we present a depthwise separable inception subnetwork (DISubNet) for classifying pig treatments, offering two versions: DISubNetV1 and DISubNetV2. These lightweight models are compared to other deep learning models used for image classification. A forward-looking infrared (FLIR) camera captures thermal data for model training. Experimental results show the proposed models outperform others in classifying pig treatments using thermal images, achieving 99.96–99.98% accuracy with fewer parameters, potentially improving animal welfare and promoting sustainable production.

Thermal imaging is increasingly used in poultry, swine, and dairy animal husbandry to detect disease and distress. In intensive pig production systems, early detection of health and welfare issues is crucial for timely intervention. Using thermal imaging for pig treatment classification can improve animal welfare and promote sustainable pig production. In this paper, we present a depthwise separable inception subnetwork (DISubNet), a lightweight model for classifying four pig treatments. Based on the modified model architecture, we propose two DISubNet versions: DISubNetV1 and DISubNetV2. Our proposed models are compared to other deep learning models commonly employed for image classification. The thermal dataset captured by a forward-looking infrared (FLIR) camera is used to train these models. The experimental results demonstrate that the proposed models for thermal images of various pig treatments outperform other models. In addition, both proposed models achieve approximately 99.96–99.98% classification accuracy with fewer parameters.

1. IntroductionOver the past few years, the number of applications for image classification has significantly increased. The goal of image classification is to determine the class to which a target object belongs. Classification is required whenever an object is assigned to a specific group or class based on the characteristics associated with that object. Image classification has many applications, including medical image analysis, human and animal face recognition, and monitoring and classifying animal behaviour [1]. It can be difficult to distinguish an object in an image if it is obscured by background clutter, noise, poor image quality, or other factors. Furthermore, the visible spectrum has limitations, such as lighting conditions and shadows, that could be overcome by thermal imaging. Thermal imaging is a non-destructive testing method that can be utilized to determine the surface temperature of objects. Thermal imaging is increasingly utilized in animal welfare to increase farm production efficiency. Calves [2], poultry [3], and pig production [4] have been evaluated for animal welfare using thermal imaging. In addition, it is used to identify the temperature increase in pigs to predict their health [5].In computer vision, animal classification using thermal images has been a crucial field of study. Continuous automatic systems for animal welfare typically provide information by collecting raw data and identifying key features through deep learning techniques. Farmers were better able to understand specific animal needs, such as welfare [6,7,8], and reproductive efficiency [9,10], with the aid of this method. The problem with automatic systems is that they use all nearby natural objects to represent animals in a scene, rather than just the animals themselves. In addition, animals can be viewed from various perspectives, scales, and shapes, as well as under different lighting conditions. However, this could be resolved using thermal images for animal classification. Thermal images capture heat emitted by animals, and these data can be used to identify patterns and detect abnormalities that are not visible to the naked eye. The use of thermal imaging in livestock applications has the potential to improve animal welfare, increase productivity, and reduce the environmental impact of livestock production. Ongoing research and development in this field will likely result in even more advanced applications in the near future. Thermal imaging can be used in livestock to detect indications of illness or injury. Changes in body temperature, for example, can suggest the existence of a fever, which is a typical sign of many disorders [11]. Thermal imaging can be used to detect estrus, which indicate when a female animal is in heat [12]. Furthermore, this information can be used to improve breeding programs and increase reproductive efficiency. Stress detection in livestock using elevated body temperature or changes in respiratory patterns for animal welfare [13]. Thermal imaging can be used to monitor individual animal growth as well as environmental factors such as temperature and humidity in livestock facilities [14]. Since there are many different animal classes, each with a complex intra-class variability and inter-class similarity, methods for classifying human faces have been developed with high accuracy, but those methods are incredibly inaccurate for classifying animal faces [6]. With each approach having advantages and disadvantages, researchers have tried various approaches to address these issues.Convolutional neural network (CNN)-based classification techniques have, however, drawn a lot of attention in recent years. Deep learning methods involve representation learning and have multiple levels of representation [1]. Each of the modules that make up these algorithms transforms the representation at one level into a representation at a higher, more fundamental level while still being relatively simple but non-linear. As a result, a combination of these transformations can be used to learn quite complex functions. The higher-level representation enlarges aspects of data input that are important for distinguishing and suppressing irrelevant changes in animal classification tasks. The benefits of deep learning techniques have been successfully demonstrated in numerous applications where the input values are characterized by high dimensionality, enormous quantities, and highly structured data [15]. Deep learning techniques have a good performance and are, therefore, widely used in animal classification. Additionally, they have been widely used with thermal images [16]. Deep learning tools are incredibly helpful in image classification because the structure of the image is made up of millions of pixels that can be aligned into distinct objects [17]. The development of deep learning models has practical implications for pig farm management, allowing farmers to make data-driven decisions that improve pig health, welfare, and productivity. The advancement of neural models has greatly enhanced our ability to predict and manage various aspects of pig farming. Deep learning can help farmers optimize feeding programs and predict growth rates by analyzing large datasets of pig growth and feed intake [18]. Deep learning techniques can help predict disease outbreaks early by finding patterns in pig behavior and health data. These data can be utilized to develop early warning systems and guide disease management strategies [19]. They may be used to predict temperature and humidity levels in pig barns, which can assist farmers in maintaining optimal environmental conditions for pig growth and health [20]. Deep learning models can enable farmers to identify breeding pairs that are likely to generate high-quality offspring with desirable traits by examining large datasets of genetic and phenotypic data [21].To achieve greater accuracy, the general trend has been to create deeper and more complex networks [22]. These improvements in accuracy are necessarily making networks less effective in terms of size and speed. The recognition tasks in many real-world applications, including robotics, self-driving cars, and augmented reality, must be completed promptly on a platform with constrained computational resources [23]. To solve this issue, scaling CNN can improve accuracy while keeping the model lightweight and efficient. We propose a lightweight model that employs depthwise convolution layers and inception modules to reduce computational load while increasing accuracy with fewer parameters. We use thermal images instead of standard RGB images to overcome varying lighting and background conditions.The main contributions of the paper are as follows:1.We propose a depthwise separable inception subnetwork (DISubNet), a lightweight model for pig treatment classifications that consist of depthwise separable layers and an inception module.2.We propose two versions of DISubNet: DISubNetV1 and DISubNetV2. The models are modified based on the concatenation of depthwise layers and inception modules.3.Experiments are carried out on the pig image thermal dataset collected from the FLIR camera. The collected dataset consists of four pig treatment categories, such as isolation after feeding (IAF), isolation before feeding (IBF), paired after feeding (PAF), and paired before feeding (PBF).4.Detailed experiments are conducted on both versions of DISubNet models with other image classification models using various evaluation metrics.The rest of the paper is organized as follows: Section 2 provides the related works on image classification. The proposed models are explained in detail in Section 3. Section 4 provides details about the experiment. Section 5 contains the results of the experiments and their discussion. Finally, we conclude in Section 6.2. Related Work2.1. Image Classification MethodsDeep learning methods are commonly used in image classification tasks. The image classification process begins with the input image and ends with a classified result based on the class. The same principle applies to animal classification. The CNN-based animal classification system can be divided into three phases: pre-processing, feature learning, and classification. Firstly, to maximize the impact of factors that influence the animal classification algorithm, the input image undergoes a rescaling and image augmentation process in the pre-processing stage [24]. Second, in the feature learning step, the convolution algorithm is used to calculate the features of the input image. Finally, in the classification step, a predictive model is constructed using the features from the training data [25]. These predictive models estimate their class labels by comparing learned features from training data with test data or validation data [26]. The output classes are specific, and the user can discover the precise name of the class based on the prediction ratio. Animal image classification has previously been carried out using a variety of conventional classifiers, including support vector machine (SVM) [27,28] random forest (RF) [29,30], and decision tree (DT) [31,32,33]. In various settings, the use of an ensemble has grown in popularity. An ensemble is a supervised learning strategy that uses multiple models to boost the performance of a single model [7]. Recent research has mainly used deep learning techniques due to the promising results it has demonstrated in challenging computer vision tasks. In their work on animal species identification, Villa et al. [34] used the AlexNet [35], VGGNet [36], GoogLeNet [37], and ResNets [23] to analyze images of animals taken with a digital camera and an infrared sensor. The wildlife detector [38] was provided as a CNN model that trains a multi-class classifier while also learning a binary classification with two classes: animal and non-animal. There are a few popular methods to divide and categorize animals in camera-trap images [39]. Animal recognition methods such as robust layer principal component analysis for segmentation, CNN for feature extraction, the least absolute shrinkage and selection operator (LASSO) for characteristics, and the SVM for classification of mammalian genera have been used in the Colombian forest [40]. As a classification model, ResNet50, ResNet101, ResNet152, GoogLeNet, and MixtureNet, which are all frequently used CNN models, were utilized [40]. CNNs have great potential in agriculture and livestock contexts for improving animal health and welfare, as well as for increasing efficiency and productivity on farms. As machine learning and computer vision technologies continue to advance, we can expect to see more innovative applications of CNN in the agricultural industry. CNNs can be trained to recognize individual animals, such as pigs or cows, based on their facial features or body markings [41]. This can be useful for tracking animal health and growth over time. CNNs can also be used to analyze animal behavior, such as monitoring pig or cow facial expressions to detect signs of pain or distress [42]. Tools such as ChickTrack use CNNs to track chicken activity levels, which can help farmers to monitor the health and welfare of their birds [43]. CNNs can help to automatically record and manage animals using different sensor technologies [44].2.2. Model Design and EfficiencyFor the past few years, researchers have been working on fine-tuning deep neural architectures to achieve the best possible balance between accuracy and performance. Small and effective neural networks are becoming increasingly popular in animal welfare [45,46]. Both compressing pre-trained networks and training small networks directly fall under the broad categories of many different approaches. There have been significant advancements over early designs such as AlexNet, VGGNet, GoogLeNet, and ResNet thanks to both manual architecture search and training algorithm improvements. In recent years, there has been significant progress in algorithmic architecture exploration, including hyperparameter optimization [47] network pruning [48] and connectivity learning [49]. As seen in ShuffleNet [50] or the addition of sparsity, much work has also gone into changing the connectivity structure of the internal convolutional blocks. Another advantage of deep learning is creating distributed representations that generalise newly learned characteristics and those observed during training. As a result, each of these representations can help model similar representations in other domains [49]. However, it is important to note that deep learning models are frequently complex models that involve the use of a large number of computational resources. Therefore, the goal of this paper is to design the model structure for the classification of pig treatments using thermal images with a focus on the need for smaller and more effective models.3. Materials and MethodsIn this section, we describe the various models used in the experiments, including LeNet5 [51], AlexNet, VGGNet, Xception [52], CNN-LeakyReLU [53], CNN-inception, and the proposed DISubNet model. These models are compared for the classification of the pig treatments.3.1. Image Classifcation ModelsOne of the first pre-trained models is LeNet5, which recognises handwritten and machine-printed characters. The main reason that the model is popular is due to its straightforward structure. It is an image classification multi-layer convolution neural network which is made up of five layers that have learnable parameters. This network comprises three sets of convolutional layers, followed by a combination of average pooling layers and two fully connected hidden layers [51]. The images are classified using a softmax classifier. AlexNet won the Imagenet large-scale visual recognition challenge in 2012. The network depth in this model was increased when compared to the LeNet5 network. It has eight layers with learnable parameters. The model has five layers, the first of which is a max-pooling combination, followed by three fully connected layers [35]. The layers use rectified linear unit activation (ReLU) as their activation function, which speeds up the training process. Dropout layers are also used in the model to avoid overfitting. The final layer employs softmax as its activation function. So, as we progress deeper into the architecture, the number of filters grows. As a result, it extracts more features as we progress deeper into the architecture. Furthermore, the filter size is decreasing, indicating that the initial filter was larger and that as we progress, the filter size is decreasing, resulting in a decrease in the feature map shape. The University of Oxford’s visual geometry group (VGGNet) [36] created a deep convolutional neural network, which is widely used in computer vision fields. It comprises VGG-16 or VGG-19, which refer to the 16 and 19 convolutional layers, respectively. Xception employs depthwise separable convolutions [52]. It was developed by researchers at Google. They interpreted inception modules in CNN as an intermediate step between conventional convolution and the depthwise separable convolution in which a depthwise convolution is followed by a pointwise convolution.3.2. Modified CNN ModelsThe CNN model with LeakyReLU [53] is a straightforward sequential model consisting of several convolutional layers and a batch normalization layer. Following the convolutional layers is LeakyReLU, which is based on ReLU but has a small slope for negative values rather than a flat slope. To reduce the spatial dimension of the feature map, max pooling is applied after each even convolution layer. The convolution layer has a filter size of 3×3 and a pooling size of 2×2 across all layers. Figure 1 depicts the CNN-LeakyReLU model structure.Similar to the CNN-leakyReLU model structure, the model consists of convolutional layers and batch normalization layers. The max pooling is followed after every even convolution layer. Convolutional layers are made up of 3×3 filters in each layer. After every two convolution layers, max pooling with a 2×2 filter is applied to reduce the spatial dimension of the feature map. Figure 2 shows a representation of CNN-inception. To extract features, the model is further modified with a tunable inception module [37] consisting of filters such as 1×1, 3×3, and dilation filters. Dilated filters increase the area covered by the input image without pooling. The goal is to extract more information from each convolution operation’s output. The different feature extraction from filters aids in focusing on different parts of images to detect complex patterns. In addition, the inception module includes a skip connection for identity mapping. The class scores will be processed by the fully connected layer, resulting in a volume in size, where each of the four numbers corresponds to a class score. The filters used in the inception module are more specifically shown in Figure 3.3.3. Proposed Model for Pig Treatment ClassificationIn comparison to large convolutional neural networks such as LeNet5, AlexNet, and VGGNet, DISubNet aims to make all of these networks smaller with fewer parameters while maintaining the same level of accuracy or even improving model generalization using fewer parameters. Larger networks are more prone to overfitting and raise the computation complexity. CNNs can also benefit from the extraction of features at different scales. Therefore, we propose DISubNet comprising of two subnetworks with alternating depthwise separable convolution layers [54] and an inception module. Additionally, we propose two DISubNet versions, DISubNetV1 and DISubNetV2. Figure 4 and Figure 5 provide detailed information about the DISubNetV1 and DISubNetV2 models.The depthwise separable convolution layers from both subnetworks are concatenated in the DISubNetV1. The concatenated output from both subnetworks is fed into the inception module. In the DISubNetV2 model, we concatenate inception modules from both subnetworks and feed them as input to the depthwise layers.Depthwise separable convolutions, also known as separable convolutions, are one approach. It separates the channel and spatial convolutions normally combined in convolutional layers. The number of output channels equals the number of input channels because we apply one convolutional filter to each output channel. We then apply a pointwise convolutional layer after the depthwise convolutional layer. A pointwise convolutional layer is a convolutional layer with a 1×1 kernel. A 1×1 kernel is to use non-linearity. A ReLU activation function is applied after each layer of a neural network. The inception module follows the same structure as the CNN-inception model. The inception modules from both subnetworks are concatenated and become inputs to the subsequent layers. Figure 6 illustrates a comparison of depthwise convolution layers and standard convolution layers.The DISubNet models can regularize our model by reducing the number of parameters and the number of computations required during training or inference. Additionally, the model takes advantage of the inception module’s capacity to extract features from input data at different scales by employing different convolutional filter sizes. DISubNet models use computing resources efficiently with minimal increase in computation load.4. Experimental Setup4.1. DatasetThe data were collected by Wageningen University and Research using a FLIR camera. The FLIR T1020 with a standard 28-degree lens and FLIR Thermal Studio was used to acquire the thermal videos. Thermal videos of different pig treatments are included in the dataset. For simplicity, we extract the images from the video and convert them to grayscale with 62,800 images in total. The pigs were filmed in pairs and separated before and after feeding as shown in Figure 7, resulting in four treatment groups: isolation after feeding (IAF), isolation before feeding (IBF), paired after feeding (PAF), and paired before feeding (PBF).The pigs were classified into four treatment groups to assess animal welfare during physical separation and transport using a thermal camera. These labels represent the four different pig treatments as well as the experiment’s required classified output. The thermal images of the IAF and IBF contained single pigs. The images in the PAF and PBF contain multiple pigs. Arousal in pigs is manipulated by delayed feeding due to short-term food restriction. Delaying feeding often increases the rate of eating, indicating higher arousal. Restrictive feeding tends to enhance aggression in pigs, which may result in adversarial social behavior when dealing with other pigs in the pen. To be able to build solutions and animal welfare monitoring systems for overcoming aggression and tail biting, it is crucial to analyze the impact of feeding intervals and pen mate manipulation behavior. The abnormal behavior of the pigs may be related to the redirection of the pig’s exploratory behavior, such as the ability to engage with the pen mate whether maintained in groups or in isolation. Hence these four treatments namely IAF, IBF, PAF, and PBF were chosen to understand the effect of feeding intervals and access to socializing conditions on the behaviour of pigs. The entire dataset is divided into 60, 20, and 20 ratios for train, test, and validation data, respectively. As a result, the training data have 37,680 images, and the test data have 25,120 thermal images.4.2. Implementation DetailsThe experiment uses images resized to 112×112 resolution. The models were trained using the Keras framework with a batch size of 32 and epochs of 100. All models have been trained on the Nvidia GeForce RTX 2070 SUPER GPU. For network training, the Adam optimization [55] method is used, which is an effective stochastic optimization that only requires first-order gradients and needs less memory. It combines the benefits of two common methods: AdaGrad [56], which works well with sparse gradients, and RMSProp [57], which works well in non-stationary and online settings. Instead of stochastic gradient descent, Adam is used to iteratively update network weights based on training data. The Adam technique is used to optimize the model at various learning rates, such as 10−2, 10−3, and 10−4.4.3. Loss FunctionThe categorical cross-entropy loss is also called softmax loss. It is closely related to the softmax function because categorical cross-entropy loss almost exclusively affects networks with a softmax layer at the output. The categorical cross-entropy loss is only employed in multi-class classification tasks where each sample precisely belongs to one of the C classes. Each sample is given a ground truth label, an integer value between 0 and C−1. A one-hot encoded vector of size C with a value for the correct class and zeroes everywhere can represent the label. The cross-entropy algorithm takes two discrete probability distributions as input and produces a single real-valued number indicating the correlation of both probability distributions. The categorical cross-entropy loss function is represented as, (1)Eloss(y,s)=−∑i=1Cyilog(si) where C denotes the number of distinct classes and i denotes the i-th element of the vector. The one-hot encoded label is fed into y, and the probabilities generated by the softmax layer are placed in s. The lower the cross-entropy, the closer the two probability distributions are to one another.4.4. Activation FunctionReLU is a non-linear activation function with output zero if the input x is less than zero and output equivalent to the input if the input is greater than zero. Hence, the ReLU function takes the maximum value of x. It has more advantages than the sigmoid function, which has more backpropagation errors. ReLU could be represented as (2)f(x)=max(x,0)However, there are a few drawbacks to ReLU, including the fact that it is not zero-centred and is not differentiable at zero. Another issue that the ReLU faces is the dying ReLU problem in which some ReLU neurons essentially die for all inputs and remain inactive regardless of input, resulting in no gradient flow and affecting performance. As a result, we use LeakyReLU in experiments where there is a small negative slope so that instead of not firing at all for large gradients, the neurons do output some value, making the layer much more optimized. LeakyReLU is represented as (3)f(x)=max(0.1x,x)4.5. Evaluation MetricsThe accuracy, loss, F1 score, precision, recall, and number of parameters are used to compare the various models. The accuracy of the validation data measures how often the classifier predicts correctly. The precision metric explains how many of the correctly predicted cases were positive. It is useful in situations where false positives are more serious than false negatives. Recall describes how many of the actual positive cases the model correctly predicted. It is useful when false negatives are more concerning than false positives. The F1 score is derived from precision and recall metrics. It is also used to balance precision and recall when dealing with uneven dataset distribution. The evaluation metrics for the model are described as (4)Accuracy=TP+TNTP+TN+FP+FN (5)Precision=TPTP+FP (6)Recall=TPTP+FN (7)F1score=2×Recall×PrecisionRecall+Precision where TP, TN, FP, and FN represent true positive, true negative, false positive, and false negative, respectively. The confusion matrix is a popular performance metric for classification problems with two or more classes as output.5. Results and Discussion5.1. Model ComparisonWe evaluated and visualized our results using an accuracy, loss, and confusion matrix. For our experiment, we have modified the LeNet5 for input data of 112×112. The network consists of two sets of convolution layers followed by max pooling. The filter size for the convolution layer is 5×5 with stride 1, and the pooling size is 2×2. There are 500 neurons in the hidden layers. The activation function used in this model is the ReLU activation function. With 19.6 M parameters, LeNet5 has an accuracy of 99.9%. After a certain epoch, the model converges but slightly overfits the model. With a learning rate of 10−3, the LeNet5 was able to close the generalization gap with a 0.006 error. LeNet5 is limited by the availability of computing resources because processing higher-resolution images require larger and more convolutional layers, which are difficult to implement. Figure 8a,b show the accuracy and loss plot of the LeNet5 with slight overfitting at the beginning of the training.The AlexNet model is slightly modified to use 4 convolutional layers instead of 5 for a 112×112 input size. The convolutional layers employ 11×11, 5×5, and 3×3 filter sizes. As a result of the varying convolution filter sizes, the network can learn various spatial patterns at different scales. The max pooling is applied with the size of 3×3 with stride 2. Despite having an accuracy of 90.22% with many parameters, AlexNet has several misclassified images. In comparison to LeNet5, AlexNet has 23.3 M parameters because of the addition of layers. As a result, AlexNet is not only a large model but also highly prone to overfitting. With a 0.27 error value, AlexNet has more errors than LeNet5. Figure 9a,b shows that AlexNet shows an accuracy plot and a loss plot.In this paper, we compare the 16-layer VGG-16 model with other models. VGG-19 was excluded from the experiment because it has a 55 M number of parameters. The convolutional layers are followed by single max pool layers. The layers use a 3×3 kernel size for a minimal receptive field. These are followed by the ReLU unit, which reduces training time compared to AlexNet. The number of depth layers has increased, and the hyperparameter tuning process has been simplified using only 3×3 filters. Consequently, increasing the depth of the model structure could enhance generalizability. Additionally, a larger receptive field might be offered. The number of parameters might be decreased by using a smaller filter size. Due to a large convergence gap between train and test data, VGGNet performed worse than other models. Figure 10a,b show that the VGGNet has a smoother learning curve than AlexNet. The model had an accuracy of 85.43% with 17 M parameters. Since the data are not evenly distributed, the VGGNet overfits similarly to AlexNet. With a 0.416 error, the VGGNet has a higher loss value than the AlexNet.The Xception model emphasizes the inception hypothesis. Hence, this model is known as the Xception model. Xception provides an architecture that consists of depthwise separable convolution blocks and maxpooling, all of which are connected using shortcuts similar to ResNet implementations. The distinguishing characteristic of Xception is that the depthwise Convolution is not followed by the pointwise convolution; instead, the sequence is inverted. The 1×1 convolutions capture the correlations between channels. Regular 3×3 or 5×5 convolutions capture the spatial correlations within each channel. Hence, 1×1 is applied to each channel, followed by 3×3 to each output. It is similar to substituting depthwise separable convolutions for the inception module. Xception model has the accuracy of 99.95% with 20 M parameters. According to the accuracy and loss plots of the Xception model presented in Figure 11a,b, depthwise separable convolutions reduce overfitting compared to AlexNet and VGGNet. The Xception model has classification accuracy similar to DISubNet V1 and V2 but requires more parameters and a larger model size.The confusion matrix in Figure 12 shows that the LeNet5 model classifies the paired before feeding treatment class more accurately than the other classes. When compared to other classes, the AlexNet model performs best at classifying isolation before feeding, followed by the class paired before feeding. Among the image classification methods, the VGGNet model illustrates the highly misclassified pig treatments. Furthermore, Xception performs a more accurate classification of pig treatments than LeNet5.In comparison to Lenet5, which uses 19.6 M parameters, the CNN-leakyReLU achieves an accuracy of 99.14% with 7.2 M parameters. Figure 13a,b demonstrate CNN-leakyReLU with more fluctuations in the learning curve at the beginning of the training. The model fluctuated during training due to the uneven data distribution, but it converged successfully after a certain number of epochs. With a 0.097 error, it displays a higher loss value than LeNet5. An L2 regularizer is used to lessen the overfitting of the proposed model. The confusion matrix shown in Figure 14a indicates that most pig treatment classes were also categorized with higher performance.The CNN-inception model makes use of the ability of the inception module to focus on different parts of images to find patterns that can be associated with classification labels. Working with different filters to capture the level of abstraction is possible with the inception. As a result, they are not limited to using a single filter size in a single image block, which is then concatenated and passed onto the next layer. After each max pooling, the inception module is added. When the dataset is trained with the CNN-Inception model, it captures better patterns. It thus achieves 99.97% accuracy with a slightly higher number of parameters (i.e., 7.4 M) than CNN-LeakyReLU. Figure 15a,b demonstrate that the CNN-inception model has a better learning and convergence curve than the other models.In the model, the filters are slid over the entire image, and the dot product of the image and filter values are calculated. The number of filters produces the same number of feature maps as the number of filters, which becomes the parameter for the model to be learned. Deep neural networks that are highly efficient must be large. A neural network had to have several more layers and units within these layers to be considered large. Multi-scale convolutional layers may also be able to learn more. However, large networks are prone to overfitting, and chaining multiple convolutional operations together raises the computational cost of the network [51]. In this case, the inception module is more advantageous. When compared to CNN-LeakyReLU, the model achieves a lower loss of 0.017. As a result, for use in any application, a trade-off between the number of parameters and accuracy could be considered. The CNN-Inception model correctly classifies three treatment categories, as shown by the confusion matrix in Figure 14b.The DISubNet model, which employs depthwise separable convolution layers, has significantly fewer parameters and a slightly lower train time per epoch. A normal convolutional layer differs from a depthwise convolution where the depthwise convolution applies the convolution along only one spatial dimension (i.e., channel), whereas a normal convolution applies the convolution across all spatial dimensions or channels at each step. Depthwise separable convolutions are more likely to perform more effectively on deeper models that may have an overfitting problem and on layers with larger kernels because there is a greater decrease in parameters and computations that would offset the high computation cost of performing two convolutions instead of one. Non-linear layers broaden the model’s possibilities, making a deep network superior to a wide network. We use a 1×1 kernel and add an activation layer after it to increase the number of non-linear layers without significantly increasing the number of parameters and computations. This adds a layer of depth to the network. Based on the model structure, our proposed model has two versions: DISubNetV1 and DISubNetV2. Depthwise convolution layers from both subnetworks are concatenated to form the DISubNetV1. Because the depthwise layers are close to the input, it extracts low-level features and concatenates features from both subnetworks to provide more information to the inception module. This version of the model achieves 99.96% accuracy, which is higher than all other models except CNN-Inception. In Figure 16a,b, the accuracy and loss plots of DISubNetV1 exhibit better convergence and fewer fluctuations. The DISubNetV2 concatenates inception modules rather than depthwise layers. At the beginning of the model, the input from different subnetworks goes through different levels of abstraction with different filters. As a result, it enables in obtaining more features when concatenated and provides better classification output. Regarding accuracy, the DISubNetV2 outperformed all other models with a score of 99.98% on thermal data. Although there are a few more fluctuations in the accuracy and loss of DISubNetV2 in Figure 17a,b, there is a better learning curve over the course of training. Even though DISubNetV2 has 0.002 more errors than DISubNetV1, it can still be used as a straightforward model with 4.5 M parameters.In comparison with other models, the confusion matrix of both proposed versions in Figure 18a,b shows correctly classified pig treatment classes. As a result, the model outperforms other models trained on thermal data from pig treatments.Table 1 summarizes our results for learning rate = 0.001. The proposed model DISubNet models, DISubNetV1 and DISubNetV2, provides increased accuracy compared to all the models for pig treatment classifcation.5.2. Comparison with Different Learning RatesOur proposed models were trained at various learning rates, including 10−2, 10−3, and 10−4. Table 2 summarizes the experiment and includes evaluation metrics such as accuracy, precision, recall, and F1 score.All models perform better with lower learning rates, such as 10−3 and 10−4. Furthermore, for the learning rate of 10−4, our proposed models outperformed all other models with improved accuracy in the range of 99.96–99.99%. It also clearly shows that at higher learning rates, all models have an accuracy of less than 40% excluding the Xception model. With a learning rate of 10−2, Xception outperforms all other models with an accuracy of 99.96%. However, the proposed model is smaller in compared to number of parameters. Though VGGNet has a similar accuracy of 99.98% to DISubNetV2, it is a relatively large model with 17.7 M parameters, particularly in comparison to DISubNetv2 which has 4.5 M parameters. The models are unable to converge well when the learning rate is 10−2, which may be caused by a smaller validation data sample or an uneven distribution of data. Since the dataset for paired before feeding data contains few samples, all models exhibit high learning fluctuations without increasing the accuracy. On the other hand, performance improves when the learning rate is reduced. Therefore, it is obvious that lowering the learning rate when training these models will result in better performance. In a few instances, the unbalanced dataset makes it challenging to learn the model for each batch, producing a high loss value.5.3. Comparison with Number of Parameters and Model SizeIn comparison to other models, our proposed models, DISubNetV1 and DISubNetV2, provide few parameters. The number of parameters typically rises when CNN models are expanded, potentially leading to a deeper model. However, this might impact the accuracy gain caused by the vanishing gradient. The depthwise convolution layer model requires fewer parameters and is more accurate. Table 3 compares all models in terms of parameter count and model size (in MB). With 4.5 M parameters, our suggested model yields a size of 53.7 MB.It is advantageous to have lightweight models in applications that run on mobile devices. Mobile-based deep learning applications have the potential to revolutionize pig farming by providing farmers with real-time data and insights that can help them optimize their operations and improve animal welfare. With the use of mobile-based deep learning applications, farmers can identify each pig in their herd and track their growth and health. This information can be used to monitor individual pig performance and to identify and address any health issues early on. Deep learning models can be trained to analyze pig behavior, such as eating and drinking patterns, activity levels, and social interactions. This information can be used to identify any abnormal behavior, which could be a sign of stress, illness, or other problems. With the use of mobile-based deep learning applications, farmers can use predictive analytics to forecast the growth rate of their pigs, identify potential health problems early, and optimize their feeding and breeding strategies. By monitoring the individual behavior and performance of pigs, farmers can optimize their resource allocation, such as feed and water, and minimize waste. The use of mobile-based deep learning applications can help farmers save time and money by automating data collection and analysis, reducing the need for manual labor, and increasing efficiency.5.4. Importance of Pig Treatment Classification in Animal WelfarePig treatment classification can be applied to many aspects of farming and animal care. The goal of the model is to create a framework for a decision support system for predictive analytics that can be used to identify changes in pig behaviour in response to environmental perturbations such as shifts in playtime, feeding interval time, and rest time. Isolated pigs develop behavioral stress reactions. Pigs that are completely isolated continue to display behavioural signs of stress, whereas pigs that are partially isolated (contact through a fence) eventually display fewer behavioral signs of stress [58]. Researchers working with animals can use these data to advocate for better treatment of animals. Future monitoring and treatment could benefit from using a non-invasive thermal camera to record the skin’s surface temperature. In veterinary medicine, thermal imaging is used to help diagnose diseases and to detect (early) signs of pain or stress in animals. Thermal imaging can also detect postoperative inflammation and changes in blood flow to the surgical site. Therefore, thermal images are a useful tool for identifying issues that may impact animal welfare.6. ConclusionsThis paper proposed the DISubNetV1 and DISubNetV2 models, which are made up of depthwise convolution layers and inception modules for classifying pig treatments. Various evaluation metrics are used to compare the proposed model to LeNet5, AlexNet, VGGNet, Xception, CNN-LeakyReLU, and CNN-inception models. The versions differ in terms of the concatenation of the layers in the subnetworks. Based on thermal data, the models classify four pig treatment categories. The proposed model outperforms all other models with fewer parameters and higher accuracy. Although the model improves accuracy, it misclassifies one of the paired before-feeding classes. It also shows fluctuations in learning due to the uneven distribution of the data. In the future, we plan to use this research for other applications such as emotion recognition to provide better information based on the features learned in the pig treatment classification. Since only thermal images were used, we intend to use videos instead. In addition, the conversion of thermal scores to grayscale may have resulted in the loss of some features. Therefore, future work on the model must target the feature loss to improve its accuracy.

animals : an open access journal from mdpi

10.3390/ani13101633

PMC10215438

One of the greatest challenges to achieving a sustainable aquaculture is finding alternatives to fishmeal as a primary protein source in aquafeeds. Insects represent one of the most promising alternatives being explored and produced as replacements for this ingredient. This review addresses the use of two insect species (black soldier fly, Hermetia illucens, and yellow mealworm, Tenebrio molitor) in freshwater and marine fish diet formulations and the effect of insect meal on fish gut microbiota. Furthermore, the effects of a probiotic, namely, Lactococcus lactis subsp. lactis, are considered. The study of fish gut microbiota is very important for aquaculture practice as gut microbiota plays a significant role in nutrition metabolism, also affecting a number of other physiological functions, including fish growth and development, immune response, and pathogen resistance. Along with recent and promising results in this field, new insights and future directions on fish gut microbiota research are highlighted.

Aquaculture is the fastest-growing agricultural industry in the world. Fishmeal is an essential component of commercial fish diets, but its long-term sustainability is a concern. Therefore, it is important to find alternatives to fishmeal that have a similar nutritional value and, at the same time, are affordable and readily available. The search for high-quality alternatives to fishmeal and fish oil has interested researchers worldwide. Over the past 20 years, different insect meals have been studied as a potential alternate source of fishmeal in aquafeeds. On the other hand, probiotics—live microbial strains—are being used as dietary supplements and showing beneficial effects on fish growth and health status. Fish gut microbiota plays a significant role in nutrition metabolism, which affects a number of other physiological functions, including fish growth and development, immune regulation, and pathogen resistance. One of the key reasons for studying fish gut microbiota is the possibility to modify microbial communities that inhabit the intestine to benefit host growth and health. The development of DNA sequencing technologies and advanced bioinformatics tools has made metagenomic analysis a feasible method for researching gut microbes. In this review, we analyze and summarize the current knowledge provided by studies of our research group on using insect meal and probiotic supplements in aquafeed formulations and their effects on different fish gut microbiota. We also highlight future research directions to make insect meals a key source of proteins for sustainable aquaculture and explore the challenges associated with the use of probiotics. Insect meals and probiotics will undoubtedly have a positive effect on the long-term sustainability and profitability of aquaculture.

1. IntroductionAquaculture is one of the fastest-growing food production sectors in the world, supplying more than half of the global fish supply. By 2050, it is expected that global aquaculture consumption will double. To guarantee long-term food security, efficient and sustainable animal production methods are urgently required. Aquafeeds are mainly based on fishmeal (FM) and fish oil (FO), the most abundant dietary protein source. However, the global increase in aquaculture production has required alternative feedstuff, which often has a detrimental effect on the growth, intestinal health, and immune response of farmed marine fish [1,2,3].One problem the aquaculture feed industry needs to solve is that of replacing FM with other protein sources. If this cannot be done, serious concerns exist about the industry’s capacity to remain economically and environmentally stable. Sardine, anchovy, herring, capelin, mackerel, and other forage and small pelagic marine fish species are sources of FM and FO. However, owing to the gradual decline in wild marine fish stocks [4,5,6,7,8,9,10,11,12,13,14], it will soon no longer be viable to use these aquafeed raw materials. Plant-based proteins and oils comprise the primary substitutes for FM and FO due to their greater availability and lower cost [4]. Indeed, soybean and other protein- and lipid-rich plants have replaced FM and FO in farmed fish diets [5,6,7,8,9,10]. Soybean meal is a top-rated source of protein in plant-based diets. However, plant-based diets may decrease fish growth and disease resistance due to anti-nutritional substances in plant meals that affect fish feed intake, digestion, and nutrient utilization, causing inflammation in many fish species’ intestines [4,5,6,7,8,9,10,11,12,13,14,15,16,17,18]. Furthermore, plant-based feed ingredients are deficient in protein, lack a balanced amino acid profile, are unpalatable, and compete with other food industrial sectors [16,19,20].As a result of all of these factors, the need to find better ways of getting protein from other valuable alternatives to FM, for example, animal feed ingredients such as by-products from slaughterhouses or insect meals (IM), has increased [18,21,22]. From this perspective, insects have the potential to open a new world of sustainable, protein-rich ingredients for aquafeeds. Furthermore, single-cell proteins (SCP) deriving from microalgae, bacteria, and yeast are also being used as fish feed ingredients. Insect, micro- and macroalgae, and microbial meals are becoming more popular as aquafeed components [23,24,25]. Among the scientific publications currently available in indexed databases, just 19% were focused on IM, whereas 16% discussed the use of microalgae: IM and microalgae are both components that offer much promise in the aquafeed sector. Animal health and metabolism are influenced by a complex relationship between the host, the gut microbiota, and their feed. A balanced microbiota is important for the overall health and well-being of the host. The fish gut microbiota significantly affects fish health and physiology [8]. It helps develop the immune system and promotes nutrient utilization [26,27]. The structure of the gut microbial communities, including microbial diversity, is highly influenced by the ingredients of the diet because the microbiota reacts quickly to dietary changes [28]. It is well established that replacing FM with plants, yeast, IM, or animal by-products influences the biodiversity and number of gut bacteria [22,29,30,31,32,33,34]. Furthermore, the bioactive compounds present in insects can alter the complex communities of intestinal microbiota. Consequently, the variety and richness of fish gut bacteria have changed as a result of replacing FM in their diets with IM, either from Hermetia illucens (HI) or Tenebrio molitor (TM) [35,36,37].The gut microbiota is usually called an “extra organ” because of its significant role in many physiological processes of the host, including digestion, metabolism, reproduction, development, and immunological response. In recent years, new alternative components have been investigated and used in aquafeeds. Since gut microbes are important for digestion and health, a number of studies have been carried out to determine how diet affects the gut microbiota of aquatic species. Therefore, this review highlights current developments and future perspectives of alternatives to conventional protein sources, that is, how IMs used as aquafeed ingredients affect the gut microbiota of marine fish. Furthermore, we review the effects of probiotics on fish intestinal microbiota.2. Insects for Sustainable AquacultureInsects are an environmentally sustainable protein-rich feed ingredient for farmed fish. IM is being considered a potential alternative to FM as a protein source in aquaculture feeds. Interest in using IM as an alternative to FM has increased since the European Union (EU) authorized the use of IM from seven distinct insect species in aquaculture feeds [38,39]. According to the circular economy concept, insects are worthy candidates for aquafeed ingredients. Many country’s aquaculture industries increasingly depend on IM instead of FM. Insects are nutritionally valuable due to their high protein (60–80%), fat (31–43%), essential amino acids, and mineral and vitamin content [24]. Due to their high protein content and balanced amino acid profile, IM has emerged as a popular alternative to FM and a new source of protein in terrestrial and aquatic animal diets [40,41]. Therefore, insects constitute an excellent alternative to conventionally produced animal-based protein sources for use as feed [42,43]. Many studies have investigated the effects of FM/IM substitution in various fish species diets. The European Commission has withdrawn the ban on using processed animal proteins generated from insects in aquafeed for farm fish under regulation EU-2017/893. As a result, IM can now be used in aquafeeds. The regulation lists the seven types of insects that are allowed: black soldier fly, Hermetia illucens; common housefly, Musca domestica; yellow mealworm Tenebrio molitor; lesser mealworm, Alphitobius diaperinus; house cricket, Acheta domesticus; banded cricket, Gryllodes sigillatus; and field cricket, Gryllus assimilis. Of these, flies in particular have been the focus of aquafeed industry research in recent years owing to their many advantages over other animal protein sources [44]. HI and TM are the main species presently receiving considerable attention for aquaculture feed formulations [45]. Most studies have shown that replacing FM with IM is a good approach to increase aquaculture sustainability; however, the results vary based on the fish and insect species used. We recently obtained promising findings in marine and freshwater carnivorous fish species with the dietary use of different inclusion rates of black soldier fly and yellow mealworm meals [36,37,46,47,48,49]. Table 1 represents the research that our group has done on the effects of IM on fish gut microbiota.2.1. Black Soldier Fly (Hermetia illucens, HI)When producing IM, the black soldier fly HI is an excellent potential species because its amino acid profile is similar to that of FM, making it a suitable alternative protein source [24]. HI is the most widely studied and used insect species, representing our research group’s primary alternative to FM raw material. Indeed, HI can be raised quickly, have a high fertility rate, and turn waste into high-quality protein [50]. An increasing number of feeding trials have been conducted, demonstrating that HI meals can be a suitable FM replacement in aquaculture diets [41,44,51]. During the last few decades, approximately 130 research publications with the terms “Black soldier fly,” “Larvae meal,” and “Aquaculture” have been indexed in PubMed, Scopus, Web of Science, and other databases. Prepupae of HI comprise an intriguing choice for producing IM since mass-rearing procedures for high-quality output currently exist [24]. Using HI in fish feed provides a way to solve problems in the aquaculture industry related to managing a sustainable aquatic environment. According to several studies, HI can replace conventional FM and totally replace SBM in aquaculture feeds without negatively influencing fish growth, feed efficiency, digestion, or fillet quality [37,52,53,54]. Our experiments have shown that rainbow trout (Oncorhynchus mykiss) can tolerate up to 50% HI meal in their diet with no negative effects on fish growth and survival [36,37,46,48,55] and with positive effects on the gut microbiota of fish.Effects of FM/HI Meal Replacement on Fish Gut MicrobiotaHI meals are becoming more popular in aquaculture feeds, but ideal inclusion levels still must be determined to ensure fish growth and health. An increasing number of studies have examined the effects of substituting HI meals for FM in the diets of different species of fish. Most research recommended partial replacements of FM with HI meals. However, some recent studies revealed 100% replacement without affecting fish growth, especially for carnivorous fish [52].Regarding fish growth, health, and gut microbiome, our group’s work has shown that partial or up to 50% inclusion of HI meal in the diet is well tolerated and has no negative effects on fish growth or survival. Diet has a significant role in shaping the gut microbiota, but the surrounding environment and environmental factors can also significantly impact microbiota composition. Our research group previously evaluated the effects of different HI inclusion levels in high-FM diets on fish gut microbiota using high-throughput sequencing technologies [36,37,46]. In all the experiments, we applied high-throughput sequencing of the 16S rRNA gene to assess the dynamics of major gut bacterial taxa in response to diet. PICRUSt1 bioinformatics software was used to determine gut microorganisms’ key active biological pathways. We reported that the partial substitution of dietary FM with 10%, 20%, or 30% of a defatted HI meal had an important effect in modulating the intestinal transient (allochthonous) and resident (autochthonous) bacterial communities in trout [36,37,46].HI diet increased butyrate-producing bacteria in the fish gut [36,37,49] and led to diversification and other alterations in the intestinal bacterial makeup of rainbow trout [37,53,56]. In addition, dietary IM increased the colonization of beneficial bacteria, such as lactic acid bacteria (LAB), which are often used as probiotics in animal nutrition [36,37]. This was a good result as it is known that beneficial bacteria species compete with gut detrimental bacteria for niche space and produce and secrete antimicrobial peptides, thus protecting the host from colonization and proliferation of environmental pathogens [57].Based on the metabarcoding results, three phyla, Firmicutes, Proteobacteria, and Tenericutes, were found to be the most abundant in the digestive tract of rainbow trout [37], and in fish fed with 10–30% HI meal; diversity was higher in allochthonous, but not in autochthonous gut microbiota [36,37]. Instead, another study [53] observed that trout fed a diet containing 20% HI meal had a higher species richness in their gut microbiota. Furthermore, the autochthonous bacterial community significantly influenced host metabolism and health status more than the allochthonous intestinal bacteria.Fish gut microbiota studies vary in many ways, including the techniques used to analyze the microbiome. The dietary HI meal’s effects on autochthonous microbiota of trout were first explored using the gradient gel electrophoresis (DGGE) method [53], which identified a lower number of bacterial species than the Illumina MiSeq method, which we used in all our studies [36]. We analyzed the inclusion of 10%, 20%, and 30% HI meals on the autochthonous intestinal microbiota of rainbow trout (O. mykiss) and found a reduced abundance of Proteobacteria and an increased abundance of Mycoplasma, which produce lactic and acetic acid as final products of its fermentation [36,37]. These differences in the composition of the autochthonous intestinal microbiota are due to the prebiotic characteristics of fermentable chitin. In one of our previous studies [37], Proteobacteria, Firmicutes, and Actinobacteria dominated trout’s allochthonous gut microbial community. Interestingly, also other studies reported that LAB (Firmicutes phylum) were only found in large numbers in the gut contents of trout that had been fed IM but they were absent in gut mucosa [53]. In contrast, trout intestinal mucosa in our study contained many Proteobacteria (Gammaproteobacteria) bacteria, which was in line with previous work on rainbow trout [53,58]. The most common phyla are not the only ones for which differences between these findings and our previously published data were observed. Fish mucosa samples contained considerably fewer operational taxonomic units (OTUs) (74 vs. 450, respectively) than fish gut digesta samples [37]. These results agree with another study [59], which found that microbial diversity was lower in the gut mucosa than in the luminal part. This indicates that certain species of bacteria colonize the gut mucosal layer poorly and that the number of bacteria and the diversification of the autochthonous bacterial community may be different from the allochthonous microbiota [60]. In our studies, 20% IM increased biodiversity (Shannon and Simpson evenness indices) but not bacterial richness [36,37]. In line with previous research, we found that HI meal inclusion in the trout diet had positive effects on gut bacterial biodiversity [37,53,56]. Furthermore, since dietary effects may in part be biased by taxa from the feed microbiome [61], we included the feed as control and did not use digesta as a proxy for the intestinal microbiome [36,37]. Indeed, to fully unveil the response of gut microbiota to dietary changes, we performed concurrent profiling of feed microbiota, and digesta- and mucosa-associated gut microbiota.In addition to their protein and fat content, insects contain a large amount of chitin, which is the building material that gives strength to the exoskeletons of insects. Studies have shown that the gut microbiota of fish may be altered by chitin [62]. In Atlantic salmon, a chitin-rich diet altered gut microbiota, revealing over 100 autochthonous bacterial species [63]. Dietary chitin or chitosan modulates fish gut microbiotas due to its prebiotic, antibacterial, and immunomodulatory properties [37,46,49,64,65]. Many fish cannot digest chitin, so it is possible to consider it as an insoluble fiber with possible prebiotic qualities. These properties may help maintain a well-balanced and healthy gut microbiota. The gut microbiota helps digest otherwise indigestible feed ingredients, generating short-chain fatty acids (SCFAs), which are the main energy source for intestinal epithelial cells [66]. Furthermore, our latest research [47] on the effects of chitin-rich shrimp head meal (SHM) and HI pupal exuviae on the gut microbiota of rainbow trout demonstrated that HI exuviae exert a modulatory influence on the fish gut microbiota by increasing the number of Firmicutes and Actinobacteria. Pupal exuviae thus represent a promising prebiotic for fish gut microbiota, increasing gut bacterial richness and the amount of beneficial chitin-degrading bacteria, such as Bacillus species, which promotes SCFA synthesis, especially butyrate. Similarly, adding krill or chitin into salmonid diets increased bacterial alpha diversity [62]. Therefore, our findings should not be unexpected when considering the chitin level of the IM.Chitin is a prebiotic that increases the diversity of the bacteria in the gut. A healthy gut is typically characterized by a diverse bacterial population. In contrast, decreased diversity is typically associated with dysbiosis and illness risk, due to low bacterial competition for space and resources and enteric pathogen colonization [67,68]. The addition of HI meal to the trout diet significantly decreased indigenous Proteobacteria in the intestinal digesta [36,37]. The same finding was obtained in a study on the digesta and mucosa-associated trout microbiota [56]. Chitin, an insoluble fiber, may reduce Proteobacteria in IM-fed groups. According to several investigations, chitin and deacetylated chitin derivatives are antibacterial and bacteriostatic against Gram-negative pathogens [47,69]. We reported that trout-fed HI meal showed decreased Gammaproteobacteria, including the genera Shewanella, Aeromonas, Citrobacter, and Kluyera, which are considered responsible for some diseases in fish [36]. Therefore, including IM meal in trout diets has a positive effect that inhibits potential pathogen growth. Fish fed 20% and 30% HI diets had more Mycoplasma-genus bacteria in their intestines, and these may be beneficial [36,37,47]. Many studies have identified Mycoplasma as the predominant genus in the distal intestines of rainbow trout and other farmed salmonids [33,70,71]. Bacilli and Clostridium, which are also included in the phylum Firmicutes, are closely related to Mycoplasma. They are generally obligate symbiotic microbes of the gastrointestinal ecosystem because their small genome size makes it unlikely that complex metabolic functions take place in the fish gut [36]. Lactic and acetic acids are the main metabolites of Mycoplasma bacteria [71,72]. Mycoplasma maintains intestinal homeostasis in trout by using fermentable substrates and releasing end products from bacterial fermentations [73]. Recent research on trout revealed that a lower level of Mycoplasma in the gastrointestinal tract makes the fish more susceptible to disease [74]. These findings suggest that Mycoplasma produces antimicrobial chemicals, such as lactic and acetic acids, which are the main metabolites that benefit host health.2.2. Yellow Mealworm (Tenebrio molitor, TM)Yellow mealworms are becoming more popular as an alternative source of protein in aquaculture diets due to their high efficiency in converting organic waste, being considered an ideal circular economy insect. Defatted TM provides up to 63.84% crude protein and an amino acid composition similar to that of FM [75]. Furthermore, TM contain anti-tumoral, antibacterial, antioxidant, and immunomodulatory, physiologically active compounds [76,77]. TM has been evaluated as a potential alternative to FM as a protein source in the diets of various fish species. The nutritional value of TM varies with its substrate composition and rearing settings. Although most studies have indicated that 25% to 30% of TM be included in the diet [78], rainbow trout fed different FM/TM meal replacement levels showed better performance [49]. Significant growth improvement was seen in red seabream (Pagrus major) fed diets containing 65% defatted TM larval meal, completely replacing FM [79]. TM showed the highest apparent digestibility coefficient of the four IMs tested in Nile tilapia [80]. This proves that TM larvae can replace FM as a protein source in fish diets. One of our studies examined the impact of replacing FM with TM meal in rainbow trout diets on fish weight gain and gut and skin microbiome [49]. Dietary FM substitution with TM has been explored extensively on fish development performance but less on host symbiotic microbial population [49]. Like HI, TM contains bioactive chemicals that are abundant in chitin and lauric acid and affect the gut microbiota [35,36,37]. Most current research on fish microbiota has focused on the bacterial diversity that may be discovered in the fish’s gut; however, fish also have distinct microbial diversity in other important body sites. Particularly, the skin microbiota of fish and most farm animals has not been thoroughly studied but would require careful consideration. Fish skin constitutes one of their vital mucosal barriers to the outer world. Thus, skin microbiota plays a very important role in preventing fish diseases. In one of our studies, therefore, we investigated how the gut and skin microbiota of trout changed when FM was replaced with TM larvae meal [49].Effects of FM/TM Meal Replacement on Fish Gut MicrobiotaA considerable amount of research has been conducted on mealworm meals in aquafeeds. TM is an excellent alternative to FM, positively influencing fish growth rates and gut microbiota. The appropriate TM meal inclusion rates in feeds for different fish species depend on the nutritional requirements of a given fish species and the nutritional quality of the TM, which in turn depends on the diet and culture conditions of the larvae. Insect meal manufacturers have increased defatted insect meal production in recent years. Defatting insect meal increases crude protein and degradation resistance [24]. In rainbow trout diets, 25% or 50% TM meal did not affect fish weight but significantly improved feed conversion and the protein efficiency ratio [81,82]. The amount of protein, amino acids, micronutrients, lipids, and fatty acids in TM meal makes it a suitable replacement for FM in aquafeeds based on its effects on fish growth performance. In contrast, 50% of full-fat TM diets in European seabass reduced fish growth compared to FM diets [83]. In marine carnivorous fish species, high TM levels in aquafeed have led to reduced growth [84]. Our study found no statistically significant differences in growth performance features in rainbow trout after 90 days of feeding with either a 100% substitution of FM with a partially defatted TM diet or a diet without TM [49].Many studies have focused on the impact of substituting TM meal for FM on growth and development without attempting to understand the processes that underlie these effects. It is important to use molecular genetics and genome sequencing to determine how TM meal works, how it is metabolized, and how it is absorbed by the digestive systems of different cultured fish species [49]. The gut microbiota plays an important role in enhancing feed digestion, which benefits the general health of fish [85]. As TM is being used in fish diets as a raw material, it is important to understand how gut microbes respond to adding TM to the diet. Several fish species, including rainbow trout, have been investigated to determine how dietary TM affects the composition and diversity of gut microbiota [35,49,86]. Our research group demonstrated how 100% of TM influences rainbow trout gut microbial populations [49]. Substituting FM with TM meal did not influence the species richness and variety of gut mucosal bacteria [50], a finding similar to that obtained from our previous studies [36,37]. Consistent with our findings, feeding rainbow trout (O. mykiss) or sea trout (Salmo trutta m. trutta) a hydrolyzed TM meal diet did not affect digesta-associated bacteria [86,87]. According to the results of our metagenomic analysis, the phylum Tenericutes was most represented in trout intestine irrespective of diet, followed by Proteobacteria and Firmicutes in descending order [49]; all these bacteria taxa play a key role in the host’s nutrition and metabolism. Furthermore, the abundance of Lactobacillus and Enterococcus bacteria increased in the intestines of juvenile rainbow trout fed a diet containing with 20% TM meal [86]. The prebiotic characteristics of chitin in dietary IM may be responsible for the increase in lactic acid bacteria. However, in our study on 100% FM substitution with a partially defatted TM diet, intestinal LAB did not increase [49]. This was a surprising result, especially compared to what we had seen in the intestines of trout-fed diets with HI meal [36,37]. Indeed, substituting FM with IM from HI larvae positively modulated rainbow trout gut microbiota by raising the levels of LAB, which are helpful bacteria commonly used as probiotics in the diet of fish and other vertebrates [36,37,53]. There is no doubt that LAB is crucial for degrading dietary fiber. In addition, they actively participate in host defense against pathogenic organisms by generating bactericidal chemicals, such as lactic acid, hydrogen peroxide, bacteriocins, and biosurfactants, which inhibit pathogen colonization of the intestinal epithelium [88,89]. The relative abundance of Actinobacteria increased in the digestive tracts of trout when TM larvae meal was added to their diet, but this effect was not evident in European sea bass or gilthead sea bream [35]. Indeed, the gut microbiota is usually changed towards Firmicutes and/or Actinobacteria when dietary fiber such as chitin is included [46,49,53,90]. Taken together, our data revealed that there were no negative effects on rainbow trout intestinal microbiota populations when FM was completely replaced with TM. No noticeable dysbiosis symptoms were found, but only slight microbial changes were seen [49]. The research revealed that TM larvae meal is a valid substitute for FM as an animal protein in aquafeeds.3. Probiotics for Sustainable AquacultureProbiotics are living microorganisms that, when administered correctly, positively regulate an organism’s health [91]. They are regarded as important modulators of many biological processes such as digestion, immunological activation, restoring microbial balance, and modulating the microbiota composition and have potent antioxidant qualities due to their effects on the gut microbiota [92]. Probiotics can inhibit pathogens in various ways, including by directly competing for nutrients and cell attachment space and generating inhibitory molecules, such as lactoferrin, lysozyme, bacteriocins, siderophores, and enzymes. Probiotics secrete proteases, amylases, and lipases that degrade those feed ingredients the fish gut cannot digest, leading to enhanced growth and nutrient conversion efficiency [93,94,95]. In aquaculture, many probiotic microbial strains are now used [96]. LAB, such as Lactobacillus sp., Bacillus sp., Enterococcus sp., and yeast, Saccharomyces cerevisiae, are the most common probiotics used in aquaculture [97,98]. These microorganisms are widely distributed in nature in the digestive tracts of farmed fish and regulate the fish microbiota as permanent or transitory inhabitants [99]. Probiotics in aquaculture are generally also used to reduce antibiotic use and promote aquaculture industry sustainability. The misuse of antibiotics has a negative effect on the aquatic environment, particularly in aquatic ecosystems where antimicrobials can persist for a long time and help bacteria become resistant to multiple antibiotics [100]. Antibiotics also help fish grow but their use as growth promoters has reduced the variety and abundance of indigenous gut microbiota, negatively affecting fish immune systems [101]. For these reasons, antibiotics have been restricted in farmed animals in the EU since 2006 [102,103]; therefore, several research projects have attempted to substitute antibiotics with probiotics to help the growth and development of farmed animals [104].Probiotics boost feed digestibility and nutrient absorption in cultured fish, leading to better fish growth and conversion rates [105]. They also maintain gut microbiota balance, especially at larval stages, when vaccination is challenging [101]. Probiotics are also being used more in aquaculture, and studies have confirmed the advantages for commercially important farmed fish [106,107,108]. In our recent work, gilthead sea bream fed low and high dosages of probiotic Lc. lactis subspecies lactis showed higher weight gain than control fish fed a diet without probiotics [109]. High-throughput sequencing was used in our study to analyze the alterations in sea bream gut microbial populations after Lc. lactis subsp. lactis feeding. The findings here indicate that digestion and nutrient utilization had improved in gilthead sea bream fed probiotics. The same results were seen when Lactobacillus spp. and Shewanella putrefaciens Pdp11 were administered to gilthead sea bream [110]. Many other farmed fish species showed improved growth performance when L. lactis was used as a probiotic [111,112,113]. Concerning the microbiota analysis, we also analyzed the microbiota populations associated with feeds at the end of our feeding trial to determine how stable the probiotics were in the fish diets. Firmicutes and Proteobacteria represented the most numerous bacterial phyla, followed by the Bacteriodetes and Fusobacteria in descending order. Compared to the most representative genera of Firmicutes phylum, the relative abundance of the probiotic L. lactis was higher.The proportion of L. lactis included in the control diet was 0%, while it was 64% and 71% in the treatment diets, respectively, which corresponds with administering a low and high dose of probiotics to fish [109]. Gilthead sea bream fed high dosages of Lc. lactis had an increase in Spirochete bacteria phylum in their gut, which were almost absent in fish fed low doses of Lc. lactis and in the control fish. Around 200 different genera in the Firmicutes phylum, including Lactobacillus, help maintain fish intestine health [37,109,114]. Commensal Firmicutes and Bacteroidetes produce butyrate, acetate, and propionate SCFAs by dietary fiber fermentation. The gut microbiota of sea bream on high-probiotic diets had a Proteobacteria/Firmicutes ratio five times higher than in the other groups. This result is not surprising since Lc. lactis subsp. lactis produces the antibiotic nisin, displaying strong activity against Gram-positive bacteria, and a vast majority of Firmicutes are Gram-positive [109,115].In our study, the analysis of gut-adherent (autochthonous) microbiota showed a lack of colonization of the probiotic Lc. lactis in the host’s intestinal mucosa [109]. This result was expected because it is well-recognized that the underlying mechanisms of establishing probiotics in the host intestinal mucosa are challenging and influenced by complex molecular interactions. Our probiotic modified the fish gut microbiota without colonizing the host’s intestinal mucosa, proving that colonization is not always required to trigger host modification [109]. In terms of diversity indices, the analyses of intestinal microbiota also found significant and controversial differences between groups of fish. There was a significant difference in the variety and diversification characterized by alpha diversity parameters in fish fed a low-probiotic diet compared to the control or high-probiotic diet fish [109].Consistent with our findings, the bacterial diversity in the intestinal mucosa of Atlantic salmon supplemented with LAB was higher [116]. On the other hand, in the probiotic-rich diet group, gut bacteria diversity was lowest despite reaching the highest growth rates. A functionally unbalanced ecosystem may reduce competition for opportunistic or invading bacteria if bacterial diversity decreases and is generally regarded as a negative outcome [110,117,118]. While it has been documented that administering prebiotics (specialized plant fibers that stimulate the growth of healthy bacteria) increases the microbial richness of the gut, evidence of the benefits of probiotics on fish remains less clear. According to findings in the literature, the dietary probiotic Bacillus subtilis, alone or in combination with prebiotics or microalgae, decreased gilthead sea bream species richness and diversity indexes [110,119]. Moreover, probiotics such as LAB produce antimicrobial substances that limit the growth of other microbes, which can change the gut microbiota’s composition and biodiversity [120].The correlations found in the aforementioned studies between diet and fish gut microbiota suggest that well-designed probiotics could provide a potential way to improve fish growth performance and digestive ability. However, traditional probiotics, such as lactic acid bacteria and yeasts are not the dominant indigenous microbes in the digestive tract of fish, and their use in fish may risk causing microbial dysbiosis in some cases [121]. Therefore, identifying commensal beneficial bacteria in fish is of great value for the development of novel probiotics for aquaculture [121].4. Metagenomic Analysis for the Identification of Gut MicrobiotaDifferent culture-dependent methods followed by identification based on biochemical and phenotypic characteristics of bacteria were used to identify and characterize fish microbiota in previous times. Unfortunately, culture-dependent techniques give a limited picture of intestinal microbiota because only a low fraction, down to about 1% of the bacteria from fish intestine, can be cultivated [22]. Therefore, culture-independent molecular technologies, such as next-generation sequencing (NGS) technologies, targeted amplicon sequencing of the 16S rRNA gene [122,123,124], polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) [125,126,127], or 16S rDNA PCR-DGGE and RNA polymerase β-subunit gene quantitative PCR [128], have been used more recently to evaluate the intestinal microbiota. New research approaches have boosted our understanding of the interplay between microbes and their hosts. In particular, NGS enabled the identification and quantification of fish gut bacteria at unprecedented resolution, providing novel insights into the role of the microbiota in fish growth and health [66,129].Metagenomics has thoroughly changed the study of the fish gut microbiota. With these methods, it is possible to directly look at the genome of microorganisms from samples taken from the environment [130,131]. It can provide a deeper understanding of the information that the retrieved DNA reveals about the host or environment-specific host species and help researchers understand microbial diversity in aquaculture. The use of 16S rRNA sequencing as the gold standard for identifying variability of the 16S rRNA to assess the composition of whole bacterial communities through culture-independent methods is being used by many researchers [59,132,133]. Additionally, metagenomics methods have been successfully used to find novel genes and microbial pathways as well as to discover functional dysbiosis [134].To investigate the effect of aquafeed ingredients on gut microbiota composition, we used the Illumina MiSeq platform (Illumina, Italy) for high-throughput sequencing of the 16S rRNA gene (Figure 1) to analyze and characterize the complete gut microbiome of different fish species [22,36,37,46,47,48,49]. Using the bioinformatics application PICRUSt (Phylogenetic Investigation of Communities by Reconstruction of Unobserved States), the active biological pathways of gut bacteria could be identified. This method is being used more frequently in fish studies. It can quickly and cost-effectively capture detailed sequencing data that provides additional information on even minute amounts of bacteria [66]. The Illumina, Roche 454, and Ion Torrent PGM (Personal Genome Machine) are the three leading platforms used to study fish gut microbiota [135]. Many studies have been performed using these platforms on the microbiota that live in different parts of a fish’s body, such as the gastrointestinal tract, gills, and skin that evolve to permit colonization on the mucosal surfaces by complex commensal microorganisms [136]. The skin, gills, and digestive tract of fish are the main entry routes for pathogens [136]. Thus, most research on microbial communities has focused on these body regions. Owing to the development and continuous growth of aquaculture, microbiota study has recently increased. More focus is now being directed to the microorganisms that inhabit the gastrointestinal systems of different finfish and crustaceans, such as prawns and crabs [136]. Furthermore, there is an increasing interest in the impact of aquafeed on the fish gut microbiome, as some recent findings [61] are consistent with a model wherein gut microbial profiles are to a different degree influenced by bacterial DNA present in the feed itself through a “feed microbiome” carry-over effect.5. Conclusion and Future Prospects for Applied ResearchThis review summarizes the results of studies from our research group on the effects of diets formulated to contain IM from either T. molitor or H. illucens to replace dietary FM on freshwater fish gut microbiota. It also emphasizes the connection between diet and fish gut microbiota, suggesting that “tuning” the microbiota composition through the use of new raw materials could offer a promising strategy towards a sustainable aquaculture. The effects of a probiotic (Lactococcus lactis subs lactis) used as a feed supplement on marine fish gut microbiota are also reviewed. According to metagenomic data, IMs from H. illucens, or T. molitor constitute valid alternative protein sources that can affect gut microbiota composition and overall fish health. With regard to Lc lactis, although this probiotic did not colonize the host’s intestinal mucosa, it positively influenced the fish gut microbiota by changing the abundance of different beneficial bacterial taxa and by impacting many metabolic pathways associated with protein absorption and digestion. Therefore, insects and probiotics used in fish diets have a positive effect on the composition of gut microbiota and on fish nutritional physiology.We believe this information will be helpful to researchers and aquaculture experts in fish nutrition, particularly when developing novel feed formulations or experimenting with various feed components and additives.

animals : an open access journal from mdpi

10.3390/ani11092636

PMC8467780

A article proved that, in rats with PTU-induced hypothyroidism, the E2 level as well as the expression of the uterine-receptivity factors homeobox A10 and osteopontin was decreased. Additionally, we observed changes in the expression of PGE2, PGF2α, and PGI2 signaling pathway elements, and changes in the concentrations of those prostaglandins in uterine tissue. The results suggest that hypothyroidism may interfere with the prostaglandin signaling pathway, which may further result in a reduction in uterine receptivity.

Thyroid hormones control the functions of almost all body systems. Reproductive dysfunctions, such as abnormal sexual development, infertility, or irregularities in the reproductive cycle, might be associated with thyroid disorders. Uterine receptivity is the period when the uterus is receptive to the implantation of an embryo. During the receptivity period (implantation window), a newly formed blastocyst is incorporated into the uterine epithelium. Prostaglandins are well-known primary mediators of pathological conditions such as inflammation and cancer but are also essential for the physiology of female reproduction. The aim of this study was to evaluate the possible relationship between hypothyroidism and changes in the prostaglandin signaling pathways in the uterus and in the process of uterine receptivity in a rat model. The results show that hypothyroidism impaired uterine receptivity by decreasing the level of E2 as well as decreasing the expression of the uterine-receptivity factors homeobox A10 and osteopontin. Moreover, hypothyroidism caused changes in the expression of elements of the prostaglandin E2, F2α, and I2 signaling pathways and changed the levels of those prostaglandins in the uterine tissue. The results suggest that the mechanisms by which hypothyroidism affects female reproductive abnormalities might involve the prostaglandin signaling pathway, resulting in a subsequent reduction in uterine receptivity.

1. IntroductionThyroid hormones control the functions of almost all the body’s systems. They stimulate growth and development, affect metabolism, and are essential for the proper function of the central nervous system, cardiovascular system, and immune system, as well as influencing the reproductive system [1,2,3,4,5]. Thyroid hormones regulate the secretion of the main reproductive hormones—estradiol (E2) and progesterone (P4). These hormones are necessary for the maturation and development of the oocytes, prepare the endometrium for embryo implantation, and are important in the establishment and maintenance of early pregnancy. It has been shown that, in hypothyroidism, the levels of both E2 and P4 are decreased [5]. Reproductive dysfunction, including abnormal sexual development, infertility, or irregularities in the reproductive cycle, is associated with thyroid disorders [6,7]. It is known that induced hypothyroidism in rats causes a reduction in the absolute volume of the endometrium and a decrease in its muscle layer [7,8]. In humans, hypothyroidism can disrupt the menstrual cycle and ovulation [5,9]. It can also cause problems with fertilization and implantation, miscarriage, and late-pregnancy complications [10,11].Uterine receptivity is the period when the uterus is receptive to an implanting embryo [12,13]. In rats, the receptivity period occurs between Days 4 and 5 of the estrous cycle [13,14], when the newly formed blastocysts incorporate into the uterine epithelium [13,15]. Estrogen and progesterone are hormones essential for implantation, which is mediated by an increase in the expression of the receptors for those hormones in the endometrium [13,16]. Several molecules are reported to be expressed in the endometrium exclusively during the uterine receptivity period and, therefore, could serve as markers of uterine receptivity [13,17]. Homeobox A10 (HOXA10) is expressed at high levels in adult human and mouse uteruses. Additionally, the significant increase in HOXA10 during the estrous cycle and at the time of implantation [18,19,20] suggests it plays an important role in cyclic endometrial development and uterine receptivity [20]. Other molecules considered as uterine-receptivity markers are osteopontin (OPN) and its receptor, β3 integrin (ITG3B). Both have been found to be coordinately expressed in the human endometrium across the menstrual cycle in fertile women. These glycoproteins are maximally expressed during the implantation window [21,22].Prostaglandins (PGs) are biologically active lipids. They are well-known primary mediators of pathological conditions such as inflammation and cancer, but are also essential for the physiology of female reproduction [23]. PGs belong to the group of prostanoids that are generated from arachidonic acid (AA). This acid is converted to PGH2 with the participation of prostaglandin endoperoxide synthases (PTGSs). There are two main isoforms of prostaglandin endoperoxide synthases, PTGS1 and PTGS2 [24,25]. PGE2, PGF2α, and PGI2 are synthesized from PGH2 by PGE synthases (PTGES-1, PTGES-2, and PTGES-3), PGF synthase (PGFS), and PGI synthase (PGIS) [25,26]. Prostaglandins act by interacting with specific G-protein-coupled receptors [27,28,29]. PGE2 transduces signals through four types of receptors—PTGER1, 2, 3, and 4 (EP1, 2, 3, and 4)—while PGF2α acts through PTGFR (FP) [29,30,31]. PGI2 acts through PTGIR (IP) [25,32], but it can also act via the peroxisome-proliferator-activated receptors PPARα, PPARγ, and PPARδ, which are members of the nuclear-hormone-receptor superfamily [25,33].PGE2 and PGF2α are very important factors in female reproduction. They are involved in blastocyst spacing, implantation, and decidualization, as well as in uterine contraction [29,31,34]. EP1, EP3, and FP affect smooth muscle contraction, while EP2 and EP4 affect the relaxation of smooth muscles [29,32,35]. The expression of EPs and FP in the human uterus varies during the menstrual cycle. EP1 dominates in the early-secretory phase, while EP2, EP3, and EP4 dominate in the mid-secretory phase, and FP, in the proliferative phase [29,36]. In pigs, the inhibition of the synthesis of PGs by blocking the activity of PTGS2 causes pregnancy loss [25,37]. An appropriate ratio between the luteoprotective PGE2 and the luteolytic PGF2α is very important for the successful establishment of pregnancy in pigs [25,38]. PGI2 is the most abundant prostanoid produced by the endometrium of mice and cattle. In mice, prostacyclin is critical for endometrial decidualization and embryo implantation. In rodents, cattle, and sheep, it has been demonstrated that signaling involving PGI2 and its receptor is an important component of the embryo–uterus interactions that are essential for successful implantation. PGI2 and PTGIR signaling are very important components of embryo–uterus interactions that are essential for successful implantation [25,39,40,41,42,43]. Furthermore, PGI2 increases embryonic cell proliferation and reduces apoptosis [25,44,45]. It also enhances the embryo hatching and live birth potential of mouse embryos [25,46,47].The role of prostaglandins in embryo implantation is indisputably essential [48,49]. Poor endometrial receptivity during embryo implantation has been linked to reduced prostaglandin synthesis in the human endometrium [50]. On the other hand, it is also known that hypothyroidism can cause problems with fertilization and implantation, miscarriages, and late-pregnancy complications [10,11]. The aim of this study was to evaluate the possible relationship between PTU-induced hypothyroidism and changes in prostaglandin signaling pathways in the uterus and in the process of uterine receptivity in a rat model.2. Materials and Methods2.1. AnimalsAll the experimental procedures were approved by the Local Animal Care and Use Committee in Olsztyn, Poland (Agreement No. 40/2015/DTN).Female Wistar rats aged 8–10 weeks were kept in the Animal Laboratory of the Institute of Animal Reproduction and Food Research of the Polish Academy of Sciences in Olsztyn. The rats were conventionally housed. They were divided into two groups: the control group (n = 20) and the experimental group with induced hypothyroidism (n = 20). The control group was fed ad libitum. Over the 90 days, the study group, besides the normal diet, also received 0.05% 6-propyl-2-thiouracil solution (PTU) (#46698-250MG, Sigma-Aldrich, Munich, Germany) by oral administration to induce hypothyroidism. The duration of the PTU treatment was selected based on a study by Jena and Bhanja [51]. Then, the animals were sacrificed. The blood serum and uterus were collected and immediately frozen. The tissues were stored at −80 °C until mRNA and protein extraction.2.2. Confirmation of HypothyroidismTo confirm hypothyroidism, the thyroid hormone index (T4, T3, and thyrotropin (TSH)) for the blood serum samples was obtained. Serum T3 (#EKU04275), T4 (#EKU04274), and TSH (#EKC39776) measurements were performed using ELISA kits according to the manufacturer’s instructions (Biomatik, ON, Canada).2.3. mRNA Isolation and Real-Time PCRmRNA was isolated using the Total RNA Mini Plus Kit (#036-100; A&A Biotechnology; Gdansk, Poland). The quality and quantity of the mRNA were measured using the NanoDrop 100 (Thermo Fisher Scientific; Waltham, MA, USA). Reverse transcription was performed using the Maxima First Strand cDNA Synthesis Kit for RT-qPCR (#K1642; Thermo Fisher Scientific; Waltham, MA, USA). Real-time PCR was performed with the ABI Prism 7900 (Applied Biosystems, Life Technologies, Waltham, MA, USA) sequence detection system using the Maxima SYBR Green/ROX qPCR Master Mix (#K0223; Thermo Fisher Scientific, Waltham, MA, USA). PCRs were performed in 384-well plates. The results for the mRNA transcription were normalized to β-actin (ACTB, internal control). The mRNA levels are shown in arbitrary units. The primers were designed using Primer3web version 4.0.0 (http://primer3.ut.ee; accessed on 23 April 2019), and their sequences are shown in Table 1. For the relative quantification of the mRNA levels, the Miner software was used (http://www.miner.ewindup.info; accessed on 23 April 2019).2.4. Protein Isolation and Western BlottingTotal protein (n = 7) was isolated using the Radio Immuno Precipitation Assay Buffer (150 mM NaCl, 50 mM Tris, 0.1% SDS, 1% Triton × 100, 0.5% sodium deoxycholate, and 5 mM EDTA; pH = 7.2). The protein concentration was measured using the Micro BCA method. The levels of the proteins involved in prostaglandin signaling pathways—PTGS2, PTGES-2, PTGES-3, PTGER1, PTGER2, PTGER3, PTGER4, PGFS, PTGFR, PTGIS, and PTGIR—and the uterine-receptivity proteins HOXA10 and OPN were measured by Western blotting using the semi-dry method of transfer (Trans-Blot SD Cell, Bio-Rad; California, USA) onto polyvinylidene difluoride membranes (Immobilon-P Transfer Membrane; #IPVH00010, Millipore; Burlington, MA, USA). The primary antibodies used are shown in Table 2. β-actin antibody (#A2228-100UL, Sigma-Aldrich; Munich, Germany), diluted 1:4000, was used as the internal control for protein loading. The secondary antibodies used were goat anti-mouse IgG (whole molecule)–alkaline phosphatase (#A3562, Sigma-Aldrich; Munich, Germany), diluted 1:30,000, and goat anti-rabbit IgG–AP (#sc-2007, Santa Cruz, TX, USA), diluted 1:5000. The immune complexes were visualized using the alkaline phosphatase visualization procedure. The blots were scanned for densitometric analyses (Versa Doc Imagine System), and the specific bands were quantified using the Image Lab Software Version 5.2 (Bio-Rad Laboratories, CA, USA).2.5. The Extraction of Prostaglandins and the Measurement of Their Concentrations in the Uterine TissuePGE2, PGF2α, and PGI2 were extracted from the uterine tissue using diethyl ether (#384210114, POCH, Gliwice, Poland). The concentrations of PGE2, PGF2α, and PGI2 were measured using the PGE2 high-sensitivity ELISA kit (#ADI-931-069; ENZO Life Sciences Inc., New York, NY, USA), the PGF2α high-sensitivity ELISA kit ((#ADI-931-069; ENZO Life Sciences Inc., New York, NY, USA), and the urinary prostacyclin ELISA kit (#ADI-901-025, ENZO Life Sciences Inc., New York, NY, USA), respectively.2.6. Statistical AnalysesStatistical analyses were conducted using GraphPad Prism 7 (GraphPad Software, Inc., CA, USA). All the data are shown as the mean ± SEM, and differences were considered to be significantly different at a 95% confidence level (p < 0.05). Analyses were performed using Student’s t-tests.3. Results3.1. The Levels of Thyroid Hormones in the Blood Serum SamplesFigure 1 shows the T3, T4, and TSH levels in the blood serum samples in the control group and the experimental group with PTU-induced hypothyroidism. The levels of T3 and T4 were significantly decreased (p < 0.05), whereas the level of TSH was significantly increased (p < 0.05) in the blood serum samples from the experimental group in comparison to the control group.3.2. Uterine ReceptivityFigure 2 shows the E2 and P4 levels in the blood serum samples in the control group and the experimental group with PTU-induced hypothyroidism. The level of E2 was significantly decreased (p < 0.05), whereas that of P4 was not changed (p > 0.05) in the blood serum samples from the experimental group in comparison to the control group.Figure 3 shows mRNA and protein expression patterns of the uterine-receptivity factors osteopontin (OPN/OPN) and homeobox A10 (HOXA10/HOXA10). In both cases, the mRNA and protein expression levels were significantly lower in the group of rats with hypothyroidism than the control group (p < 0.05).3.3. Prostaglandin Signaling in the Uterine TissueFigure 4 shows the mRNA and protein expression patterns of prostaglandin endoperoxide synthase 2 PTGS2/PTGS2 (COX-2). The mRNA level of PTGS2 was significantly higher in the group with hypothyroidism than the control group, while the protein level of PTGS2 was significantly lower in the group with hypothyroidism. Figure 5 shows the mRNA and protein expression patterns of PGE2 synthases. The mRNA and protein levels of PTGES-2/PTGES-2 were significantly higher in the experimental group with induced hypothyroidism than the control group (p < 0.05). There was significantly higher PTGES-3 mRNA expression in the group of animals with induced hypothyroidism (p < 0.05), while its protein expression was maintained at the same level in both groups. Figure 6 shows the mRNA and protein expression patterns of the PGE2 receptors. There was no significant difference in the mRNA expression of PTGER1 between the groups, whereas the expression of the PTGER1 protein was significantly lower in the experimental group with induced hypothyroidism than the control group (p < 0.05). The mRNA and protein expression levels of PTGER2/PTGER2 and PTGER3/PTGER3 show similar patterns. The expression levels were significantly lower in the group of animals with hypothyroidism than the control group of rats (p < 0.05). The mRNA and protein expression levels of PTGER4/PTGER4 were significantly higher in the experimental group in comparison to the control group of rats (p < 0.05). Figure 7 shows the mRNA and protein expression patterns of PGF2α synthase PGFS/PGFS and the receptor for PGF2α (PTGFR/PTGFR). The PGFS/PGFS expression pattern was similar for both mRNA and protein. It was significantly lower in the group of animals with hypothyroidism than the control group (p < 0.05). The mRNA and protein expression patterns of the PGF2α receptor PGFR/PGFR were opposite. The mRNA level was significantly higher, while the protein level was significantly lower, in the group of animals with hypothyroidism than the control group (p < 0.05). Figure 8 shows the mRNA and protein expression patterns of prostacyclin synthase PTGIS/PTGIS and the prostacyclin receptor PTGIR/PTGIR. The mRNA level of PTGIS was significantly higher in the group of rats with induced hypothyroidism than the control group of animals, while there was no difference in the protein level of PTGIS between the groups. In the case of the prostacyclin receptor, the mRNA level of PTGIR was significantly higher in the experimental group of animals than the control group, whereas the protein level of PTGIR was significantly lower in the group of rats with hypothyroidism than the control group (p < 0.05).3.4. The Concentrations of Prostaglandins in the Uterine TissueFigure 9 shows the concentrations of PGs in the uterine tissue. The concentrations of PGE2, PGF2α, and PGI2 in the uterine tissue were significantly lower in the experimental group of animals with induced hypothyroidism than the control group of animals (p < 0.05).4. DiscussionOur present study provides a new understanding of the possible relationship between thyroid hormones and uterine receptivity, as well as prostaglandin signaling pathways in the rat uterus. To the best of our knowledge, this is the first report to investigate the effect of PTU-induced hypothyroidism on uterine receptivity and the uterine synthesis of prostaglandins in rats.Hypothyroidism is a disease characterized by a defect in the production of thyroid hormones due to the insufficient stimulation by TSH of normal thyroid gland function. This condition is the consequence of an anatomic or functional disorder of the pituitary gland or the hypothalamus, resulting in variable alterations of TSH secretion [52]. Other causes include a congenital absence of the thyroid, radioiodine therapy for hyperthyroidism, surgical thyroidectomy, various drugs that affect thyroid function or cause thyroid inflammation, and a variety of other rarer causes [53]. Congenital hypothyroidism occurs in ~1/4000 infants [54]. The lifetime risk of overt hypothyroidism is around 5%, and this disease is usually preceded by subclinical hypothyroidism, which has an even higher prevalence (estimated to be up to 9%) [52].It is known from the literature that thyroid hormones affect reproductive capacity and, in particular, play an important role during implantation and the early stages of embryo development. Successful implantation is the result of reciprocal interactions between the implantation-competent blastocyst and the receptive uterus [55]. Thus, thyroid hormones may also influence angiogenesis and immune function in the uterus during implantation. Angiogenesis is crucial for successful implantation, decidualization, and placentation. In the case of immune function, natural killer (NK) cells have emerged as crucial modulators of implantation and placental angiogenesis. It is known that NK cell concentrations are higher in patients with thyroid autoimmunity and recurrent spontaneous abortion or unexplained infertility. This suggests their involvement in the decrease in uterine receptivity and the increase in pregnancy loss. Thyroid hormones have been reported to significantly influence the concentrations of the female reproductive hormones estradiol (E2) and progesterone (P4) [5,11,56]. In the current study, we observed that the administration of PTU in the drinking water decreased the circulating E2 level compared to control. We did not observe any effect on the P4 level. Our results are partially consistent with the results of Kong et al. [5] and Tohei [57], who found that, in rats with hypothyroidism, the levels of both E2 and P4 were decreased. However, Hapon et al. [58] found significantly higher luteal P4 content on Day 21 of pregnancy in rats with hypothyroidism compared to control, suggesting that hypothyroidism impairs CL function during gestation, delaying the onset of parturition in the rat. As P4 and E2 are necessary for the maturation and development of oocytes, preparing the endometrium for embryo implantation, and are important in the establishment and maintenance of early pregnancy, changes in the physiological levels of these hormones caused by TH hormones may contribute to disorders of or disrupt the reproductive system. Furthermore, in our experiment, in the groups of animals with PTU-induced hypothyroidism, the levels of homeobox A10 (HOXA10/HOXA10) and osteopontin (OPN/OPN), which are considered to be uterine receptivity markers [13], were significantly lower in comparison to those in the control group. Hoxa10/HOXA10 has been found to be directly involved in embryogenesis and embryo implantation via the regulation of downstream genes. The cyclical endometrial expression of this factor, with a peak of expression occurring during the window of implantation, is observed in response to E2 and P4 [59]. Osteopontin, a member of the extracellular matrix (ECM) protein family, is involved in many physiological and pathological processes, including cell adhesion, cell proliferation and differentiation, angiogenesis, and tumor metastasis. Weintraub et al. [60] showed that OPN-deficient mice manifested a decreased pregnancy rate during mid-gestation and that the knockdown of OPN in mouse endometrial stromal cells restrained trophoblast invasion in vitro. Additionally, OPN was proved to be activated in mouse endometrial stromal cells (mESCs) and human endometrial stromal cells (hESCs) by E2 and P4 [61,62]. These results suggest that OPN plays an important role in regulating blastocyst implantation and decidualization. Our data confirm that, in hypothyroidism, uterine receptivity is reduced. A disturbed E2 level and decreased expression of HOXA10 and OPN may result in difficulties with the implantation of blastocysts and the maintenance of pregnancy.Prostaglandin endoperoxide synthase 2 (PTGS2) is involved in inflammation and in essential reproductive processes, including ovulation, fertilization, implantation, and decidualization. PTGS2-deficient females are infertile, with abnormalities in ovulation, fertilization, implantation, and decidualization [63,64]. PTGS2 is the key enzyme involved in prostaglandin synthesis [65,66]. Gillio-Meina et al. [42] showed that the PTGS2 protein is gradually expressed in the rat endometrium in response to deciduogenic stimuli. In our study, we showed that the PTGS2 protein level was significantly lower in the group of rats with induced hypothyroidism in comparison to the control group of animals. Combining that information with the knowledge that induced hypothyroidism in rats caused a reduction in the absolute volume of the endometrium [7,8], it may be speculated that the decreased level of PTGS2 in the uterus in the group of rats with induced hypothyroidism was caused by the significantly reduced volume of endometrium.PTGES-2 and PTGES-3 are responsible for the conversion of PGH2 to PGE2, while PGFS converts PGH2 to PGF2α. PGIS is responsible for the conversion of PGH2 to PGI2. These enzymes are involved in the reproductive processes [25,26]. Their expression at implantation sites in rats and other species has been reported [48,49]. In our study, we showed higher expression levels of PTGES-2 in the experimental group of animals, whereas the expression of PTGES-3 and PGIS was at the same level in the experimental and control groups. The expression of PGFS was significantly lower in the group of rats with hypothyroidism. This suggests that hypothyroidism may stimulate the synthesis of PTGES2 and inhibit the synthesis of PGFS but does not affect the synthesis of PTGES-3 or PGIS. In the present study, the mRNA and protein expression patterns of PGFR as well as PGIR were opposite. The mRNA level was significantly higher, while the protein level was significantly lower in the group of animals with hypothyroidism in comparison to the control group. This could be a result of post-translational changes in proteins. These changes regulate the functional activities of proteins in cells and their interactions with other cellular molecules. It is considered that this modification plays a key role in modulating protein function.It has been reported that the prostaglandin receptors EP1, EP3, and FP promote uterine smooth muscle contraction, while EP2 and EP4 affect uterine smooth muscle relaxation [29,34,35]. Blesson et al. [29] also indicated that the expression levels of the receptors of PGs are regulated by steroid hormones. In the human endometrium, it has been found that the levels of PG receptors vary in a phase-specific manner. EP2, EP3, and EP4 dominate in the mid-secretory phase, while EP1 dominates in the early-secretory phase, and FP dominates in the proliferative phase. This suggests that the expression of those receptors may be regulated by E2 and P4 [29,36]. Our study showed that induced hypothyroidism caused lower expression levels of EP1, EP3, and FP and higher expression levels of EP2 and EP4. It suggests that hypothyroidism leads to the impairment of the uterus’ function and disturbances of the estrous cycle by inducing changes in the expression levels of PG receptors in the uterine tissue. Furthermore, EP2, EP3, and EP4 might participate in the regulation of stromal edema, endometrial blood flow, and blood vessel permeability [29,36]. This, in turn, indicates that hypothyroidism can cause abnormalities in the cyclic changes in the uterine blood supply via changes in the expression of those receptors in the rat uterus. It is known that, in rodents, PGI2′s signaling and the expression of its receptor (PTGIR) are very important components of embryo–uterus interactions and are essential for successful implantation [25,39,41,42]. In mice, PGI2 is also critical for endometrial decidualization and embryo implantation [25,39]. In addition, PGI2 increases embryonic cell proliferation and reduces apoptosis [25,44,45]. It also enhances embryo hatching and the live birth potential of mouse embryos [25,46,47]. In our study, we also demonstrated a lower level of PTGIR in the group of animals with induced hypothyroidism. This implies that hypothyroidism may cause changes in endometrial decidualization, leading to difficulties in embryo implantation and the maintenance of pregnancy.5. ConclusionsIn summary, this study is the first to characterize the influence of hypothyroidism on uterine receptivity and PG signaling in uterine tissue in a rat model. The results show that hypothyroidism impairs uterine receptivity by decreasing the level of E2 as well as the expression of the uterine-receptivity factors HOXA10 and OPN. Hypothyroidism also causes changes in the expression of elements of the PGE2, PGF2α, and PGI2 signaling pathways, and changes in the concentrations of those prostaglandins in uterine tissue. In general, it decreases the expression of those factors in the rat uterus. It is known that prostaglandins are strongly involved in uterine functions, such as decidualization and blastocyst implantation, and therefore, hypothyroidism causes abnormalities in the female reproductive system. Our results suggest that hypothyroidism may interfere with the prostaglandin signaling pathway, which may further result in a reduction in uterine receptivity. However, this hypothesis requires further investigation.

animals : an open access journal from mdpi

10.3390/ani11102761

PMC8532838

Anthelmintic resistance (AR) is a serious threat to animal health and has a major economic impact worldwide due to production and financial losses. Currently, there are three classes of anthelmintics most commonly used in small ruminants: the benzimidazoles (BZs), macrocyclic lactones (MLs) and cholinergic agonists (especially levamisole; LEV). The widespread use of those products has led to the emergence of drug-resistant parasite strains. In the present study, we describe for the first time a case of resistance to anthelmintics in goats in Romania. Resistance was detected and confirmed for two chemical groups of anthelmintics (MLs and BZs) by in vivo faecal egg count reduction test (FECRT) and in vitro methods: the egg hatch test (EHT) and larval development test (LDT). Considering the increasing prevalence of AR in goat herds in Europe and around the globe, we believe that the findings of our study on AR in goats in Romania do not represent a singular event and could hence be just the noticeable part of a much wider issue.

Currently, there are three classes of anthelmintics most commonly used in small ruminants: the benzimidazoles (BZs), macrocyclic lactones (MLs) and cholinergic agonists (especially levamisole; LEV). The widespread use of those products has led to the emergence of drug-resistant parasite strains which represents a serious threat to the livestock industry. In the present study, we describe for the first time a case of resistance to anthelmintics in goats in Romania. The study was carried out in 2021 in a dairy goat herd from the Transylvania region. Two types of diagnostic methods were used to confirm anthelmintic resistance (AR). First, the faecal egg count reduction test (FECRT), an in vivo AR diagnostic method, was used to evaluate the efficacy of eprinomectin (EPM). The results of this test were analysed applying two different calculative methods that are used only in treated animals (without the control group). Furthermore, two in vitro methods were used: the egg hatch test (EHT) for the detection of resistance to BZs, and the larval development test (LDT) for detection of resistance to all three classes of anthelmintics. The results of FECRT indicate the resistance of gastrointestinal nematodes (GINs) to EPM in both calculative methods (FECR1 = −88% and FECR2 = −202%). In addition, the results obtained for ivermectin aglycone (IVM-AG) in LDT also indicate resistance to drugs from MLs group, especially avermectins. Similarly, the results of in vitro methods (EHT and LDT) indicate resistance to BZs in this herd. LEV was the only drug that stopped the development of L3 larvae 100% (LDT). H. contortus was the only nematode species found in coproculture after EPM treatment. Furthermore, H. contotus L3 larvae was the only species found in the wells with the highest concentrations of thiabendazole (TBZ) and IVM-AG in LDT. This suggests that resistance to both BZs and MLs was present for that species.

1. IntroductionParasitic infections, especially those caused by gastrointestinal nematodes (GINs), are one of the main causes of economic losses in goat production worldwide [1]. Therefore, the infection with GINs, such as Haemonchus contortus, Trichostrongylus spp. and Teladorsagia spp. threatens the profitability and sustainability of goat production [1,2]. Their control is mainly based on the use of anthelmintics [3]. Currently, there are three classes of anthelmintics most commonly used in small ruminants: the benzimidazoles (BZs), macrocyclic lactones (MLs) and cholinergic agonists (especially levamisole; LEV) [4,5]. The widespread use of those products has led to the emergence of drug-resistant parasite strains [1]. Resistance may arise to one anthelmintic class, or even to several or all classes of anthelmintics, referred to as multidrug resistance (MDR). The distribution of anthelmintic resistance seems to correspond to the popularity of anthelmintic classes used in veterinary practice [6,7]. Therefore, the increase in MDR among GINs and the lack of sufficiently effective alternative methods of control and prophylaxis of parasitic infections are an increasing threat to small ruminant health and production around the globe [8,9]. For example, resistance of GINs to one or more anthelmintic classes in goats has so far been described in many European countries including Great Britain [10], the Netherlands [11], Spain [12], Italy [13], Lithuania [14], France [15,16], Switzerland [17], Denmark [18], Norway [19], Germany [20], Slovakia [21], Czech Republic [22], and Poland [7,23,24,25]. The situation is similar in goat herds outside of Europe as described in USA [26], South Africa [27], Kenya [28], Cuba [29], Malaysia [30], India [31], Australia [32], and New Zealand [33].Currently, the detection methods used for anthelmintic resistance (AR) are grouped into three categories: a) in vivo methods, mainly represented by the faecal egg count reduction test (FECRT); b) in vitro methods, particularly the egg hatch test (EHT) and the larval development test (LDT), and c) molecular biology techniques. The recommended standard AR detection method by the World Association for Advance in Veterinary Parasitology (WAAVP), is faecal egg count reduction test (FECRT) [4,5]. However, this method is time-consuming, variation between animals is high, and the pharmacokinetics of the anthelmintic in the host may negatively influence the results [21]. Therefore, interest was directed towards in vitro tests for detecting AR. These provide comparable and reliable results and can detect low proportions of resistant worms in a population, with the sensitivity that has potential in determining resistance with field tests [34,35,36,37].In Romania, AR has been so far documented only in horses [38]. In the present study, we aim to describe for the first time a case of AR in goats in Romania.2. Materials and Methods2.1. AnimalsThe study was carried out in a dairy goat herd from Transylvania region, Romania in 2021. The herd consisted of 3 adult males, 38 adult females, and 20 kids. The goats were housed in a 100 m2 wooden barn. The herd was established in 2016 by purchasing a few young adult females (<6 months) of Carpathian breed from the same village where the herd is located. In 2019, the owner bought 20 French Alpine adult goats (males and females) from Alba County, Transylvania, the same County where the herd is located. Then, the herd relied on their own replacement goats. The animals were grazed from May to November for 8–10 h per day on a large pasture (4 ha) fenced with electric wire. Additionally, they were fed on alfalfa hay and during milking; the goats received concentrates (oat and corn). Mineral supplements consisted of mineral blocks with selenium. The medical records of the herd attest that all adult goats had been regularly dewormed (starting from 2017) twice per year as follows: using ivermectin 0.2 mg/kg by subcutaneous injection (Evomec 10 mg/mL, Pasteur, Romania) in the autumn, and using albendazole 10 mg/kg per os (Gardal 10%, Intervet, Romania) in the spring. Despite regular deworming, in the spring of 2021, several adult goats presented weight loss, diarrhoea, and submandibular oedema. No parasitological examinations were previously performed, except a simple flotation exam which revealed infestation with GINs (strongyle type eggs).2.2. Sampling and Laboratory Analysis2.2.1. Faecal Egg Count Reduction Test (FECRT)In the spring of 2021, ten adult goats (>6 months) were randomly selected for this study. The selected animals had not been treated with any anthelmintics for at least 8 weeks prior to the study. They were kept indoors during the study. No control group was used in the present study. The FECRT was performed at the Department of Parasitology and Parasitic Diseases, Faculty of Veterinary Medicine, University of Agricultural Sciences and Veterinary Medicine of Cluj-Napoca, according to the guidelines of the World Association for the Advancement of Veterinary Parasitology (W.A.A.V.P.) [4,5]. Before treatment, each goat was weighed on an electronic scale. The goats received the recommended doses of eprinomectin (EPM) 1 mg/kg pour on [39] (Eprinex multi 5 mg/mL, Boehringer Ingelheim, France). The drug was administered by local veterinarians in the presence of the owner. Faecal samples (about 10 g) were taken straight from the rectum on the day of the treatment (day 0) and 14 days after the treatment (day 14). The samples were brought to the lab at 4 °C and examined within 24 h after collection by the modified McMaster procedure with an analytical sensitivity of 50 epg [4,5]. Coproculture was prepared by mixing 5 g of faeces collected from each animal before and after the treatment and pooled in one sample. After incubation, third stage larvae (L3) from each pool were identified at the genus level following the method described by van Wyk and Mayhew [40]. Differentiation between Trichostrongylus spp. and Teladorsagia spp. was performed by comparing specific morphological features of GIN species using 400x light microscope magnification after exsheathement of the L3 larvae in 3.5% sodium hypochlorite solution.2.2.2. Egg Hatch Test (EHT)The EHT was performed 10 weeks after FECRT at the Division of Veterinary Epidemiology and Economics, Institute of Veterinary Medicine, Warsaw University of Life Sciences, Warsaw, Poland. The test was carried out according to Coles et al. [4,5]. Fresh faecal samples from 10 goats were collected by the farmer, pooled, homogenised and then tap water was added to fill at least 100 mL bottles in order to provide anaerobic conditions [41]. The pooled sample was sent to the laboratory and performed within 72 h after collection. Helminth eggs were extracted from the pooled sample by sieving through sieves of 250, 100 and 25 μm, and centrifuged (3000 rpm, 10′) in Sheather’s sugar solution. The suspension of eggs thus obtained was suspended in deionised water. The thiabendazole stock solution (TBZ; Sigma-Aldrick, Merck, Germany) was prepared by dissolving in pure dimethyl sulfoxide (DMSO; Sigma-Aldrick, Merck, Germany) as described by von Samson-Himmelstjerna et al. [42]. Subsequently, five concentrations of TBZ solution (0.05, 0.1, 0.3, 0.5 and 1.0 μg/mL) were prepared and added to the 24-well plate. The final concentration was set by adding 10 μL of TBZ solution into 1.99 mL of the eggs suspension (100 eggs/mL). Each concentration of TBZ was tested in duplicate. DMSO without any anthelmintic served as the control. The 24-well plate was sealed to prevent drying and incubated at 25 °C for 48 h, and then stained with 10 μL of Lugol’s iodine per each well. The wells were subsequently examined under the inverted microscope (Olympus, CKX53, Poland) at 100× magnification and the number of unhatched eggs and first-stage larvae (L1) in each well were counted [5].2.2.3. Larval Development Test (LDT)LDT was performed simultaneously to the EHT at the Division of Veterinary Epidemiology and Economics, Institute of Veterinary Medicine, Warsaw University of Life Sciences, Warsaw, Poland. The test was performed according to Hubert and Kerboeuf [43], with further modifications made by Várady et al. [44]. The collection, storage, and extraction of eggs were the same as described above for EHT. Stock solutions of thiabendazole (TBZ; Sigma-Aldrich, Merck, Germany) and ivermectin aglycone (IVM-AG, Tebu-bio, France) were prepared by dissolving in pure DMSO (Sigma-Aldrich, Merck, Germany). Stock solution of levamisole (LEV; Sigma-Aldrich, Merck, Germany) was prepared by dissolving in deionised water. Subsequently, 12 concentrations for each drug were prepared by serially diluting (1:2) in DMSO (TBZ and IVM-AG) and deionised water (LEV). These concentrations ranged from 0.0006 to 1.28 μg/mL for TBZ, from 0.084 to 173.6 ng/mL for IVM-AG, and from 0.02 to 32 μg/mL for LEV. The test was performed in a 96-well cell culture plate (Sarstedt, Germany) with 150 μL culture medium containing (all in one test plate) 10 μL of TBZ, IVM-AG, LEV or DMSO solution and deionised water (control wells), 110 μL of deionised water, 20 μL of nutritive medium (yeast extract with Earle’s Balanced Salt Solution) [41], and another 10 μL of egg suspension (roughly 100 eggs) containing Amphotericin B (Sigma-Aldrich, Merck, Germany) at a concentration of 5 μg/mL. Each concentration of TBZ, IVM-AG and LEV was tested in duplicate. Next, the plate was incubated for 7 days at 25 °C and then stained with 10 μL of Lugol’s iodine per each well to stop larval development. The unhatched eggs and L1-L3 larvae from all wells were examined using an inverted microscope (Olympus, CKX53, Poland). Infectious L3 in the wells were examined for morphological features and identified at the genus/species level as described by van Wyk and Mayhew [40].2.3. Data Analysis2.3.1. FECRTFaecal egg count results were presented as the arithmetic mean (± standard deviation, SD), median (interquartile range, IQR), and range. The 95% confidence intervals (CI 95%) for percentages were calculated using the Wilson score method.The percent reduction in faecal egg count (FECR%) was calculated using two different formulae. (a)FECR1 = 100 × (1 − [T1/T0]) [45], where T0 and T1 represent the arithmetic mean of eggs per gram (EPGs) of the treated group before (day 0) and after treatment (day 14).(b)FECR2 = (1/n)Σ(100 × (1 − [Ti1/Ti0]) [46], where Ti0 and Ti1 represent the EPGs (before and after treatment) in host i from a total n hosts. In this case, each host serves as its own control.Anthelmintic resistance was considered in both methods if FECR% was less than 95% [45,46] and only for the first method also when simultaneously the lower limit of 95% confidence interval (CI 95%) was lower than 90% [5,45].2.3.2. EHTResults of the EHT were determined as the TBZ concentration required to inhibit 50% or 99% of the eggs from hatching (inhibition concentration, IC50) and the number of hatched eggs at a concentration of 0.1 μg/mL of TBZ, since the results of Coles et al. [5] suggest that this discrimination concentration (DC) prevents hatching of 99% of susceptible eggs. Thus, the percentage of hatched eggs is a direct measure of the percentage of BZ-resistant eggs in the sample [37]. The number of hatched eggs was corrected for natural mortality from control wells (corrected percentage inhibition, cPI%) [5]. The concentrations of TBZ were log transformed, and an S-shaped dose–response curve was fitted by converting cPI% to their probits, defined as normal equivalent deviates increased by 5 to avoid calculating with negative numbers [46]. Benzimidazole resistance was considered if the IC50 value was above the DC of 0.1 μg/mL and hatching of larvae was observed [5].2.3.3. LDTThe arithmetic mean percentage of developing larvae in the tested wells (PDT) at each anthelmintic concentration was corrected by the percentage of developing larvae in the control wells (PDC) using the formula below:cPD% = PDT/PDC, where cPD% stood for the corrected percentage of larvae developing in the tested wells.The four-parameter logistic curve was used to estimate the concentration of each anthelmintic that inhibited development of 50% (median lethal concentration, LC50), and 99% (LC99) of larvae [47]. The results of LDT were interpreted with respect to the DC of the anthelmintic agent which was defined as the concentration of anthelmintic at which the development of at least 99% of susceptible larvae (adjusted by the PDC) would have been inhibited [5,47]. In this study, the following DC were applied: 0.08 μg/mL for TBZ [21], 21.6 ng/mL [14,48] for IVM-AG, and 2.0 μg/mL for LEV [49]. Statistical analysis was performed in TIBCO Statistica 13.3.0 (TIBCO Software Inc., Palo Alto, CA, USA).3. Results3.1. FECRTThe results of the individual faecal egg count (FEC) are presented in detail in Table 1. The FECR1 was −88% (CI 95%: −248%, −1%) and the FECR2 was −202%, both indicating the resistance of GINs to EPM. The examination of the coproculture prepared from the pooled sample before treatment revealed the presence of T. circumcincta, H. contortus, and Oesophagostomum spp., whereas in the following coproculture treatment, only H.contortus was found (Figure 1).3.2. EHTHatching of eggs was observed in all the wells regardless of TBZ concentration. Percentage of eggs hatching at the DC (0.1 μg/mL) was 96.2% (CI 95%: 92.6%, 98.1%). The IC50 value was 221.1 μg/mL (CI 95%: 214.2, 227.9 μg/mL), which is significantly above the DC threshold value which indicates resistance to BZs (Table 2).3.3. LDTLarval development was observed in all wells regardless of the anthelmintics’ concentrations in the case of TBZ and IVM-AG, whereas LEV proved to be effective and stopped larval development at the threshold concentration. It was considered that the herd harboured resistant GINs if L3 larvae development was observed at DC threshold (TBZ—0.08 μg/mL; IVM-AG—21.6 ng/mL; LEV—2 μg/mL). Infectious L3 development at DC were of 94.9% (CI 95%: 90.0%, 97.4%) for TBZ, 94.8% (CI 95%: 90.5%, 97.2%) for IVM-AG, and 0% (CI 95%: 0%, 10.4%) for LEV. These results indicate high resistance to TBZ and IVM-AG. Detailed results of LDT are presented in Table 3. H. contortus L3 larvae was the only species found in the wells with the highest concentrations of TBZ and IVM-AG, respectively.4. DiscussionOur study is the first report of AR in GINs in the goat population in Romania. Resistance was detected to two classes of anthelmintics: MLs and BZs. We employed two types of diagnostic methods to confirm AR [46]. The FECRT, an in vivo AR diagnostic method, was used to evaluate the efficacy of EPM. The results of this test were analysed using two different calculative methods that are applied only in treated animals (without the control group) [50]. One method evaluated the FECR of the treated herd (group) based on average values, and the other evaluated the FECR using individual evaluations in treated goats (before and after treatment). Furthermore, two in vitro methods were used: EHT for the detection of resistance to BZs, and LDT for detection of resistance to all three classes of anthelmintics. The interpretation of the results of these in vitro methods depends on the DC threshold values selected [7]. We decided to use the DC rather than the LC50 criterion for differentiation between resistant and susceptible GINs. Results of previous studies suggested that application of the LC50 may sometimes underestimate the resistance and possibly other herds might have been incorrectly classified by the criterion as susceptible [51]. It has been suggested that using the IC99/LC99 or the DC values in the in vitro tests such as EHT or LDT can substantially increase the sensitivity and identify resistance when only a small proportion of the GINs population is resistant [35,37,51,52,53]. A similar resistance classification criterion was successfully implemented in AR field studies [7,14,21,22,24,25,48]. For the present study, we adopted threshold values accepted in the scientific literature.The results of FECRT carried out in this study indicate the resistance of GINs to EPM with both calculative methods (FECR1 = −88% and FECR2 = −202%). In addition, the results obtained for IVM-AG in LDT also indicate resistance to drugs from MLs group, especially avermectins. Similarly, the results of in vitro methods (EHT and LDT) indicate resistance to BZs in this herd. In both tests, IC50 and LC50 values were much higher than the DC threshold values and L3 larvae development or hatching of eggs in the threshold concentrations were also on a very high level. LEV was the only drug that stopped the development of L3 larvae 100% (CI 95%: 89.6%, 100%) at the DC threshold in LDT. Based on the in vivo and in vitro tests results and deworming history of the herd, we assume that only LEV remained the only effective anthelmintic against GINs in the examined herd, H. contortus was the only nematode species found in coproculture after EPM treatment. Furthermore, H. contotus L3 larvae was the only species found in the wells with the highest concentrations of TBZ and IVM-AG, respectively. This suggests that resistance to both BZs and MLs was present for the aforementioned nematode species.The BZs have been most widely used worldwide since the successful production of thiabendazole in the 1960s. Nowadays, the resistance of BZs has been reported worldwide [54]. In our study, we chose the EHT and LDT for the detection of BZs resistance because these methods have been suggested to have a higher sensitivity than FECRT in detecting BZ resistance [35]. FECRT is considered reliable only if more than 25% of the population of nematodes are resistant [55]. There are several studies indicating that FECRT could underestimate low levels of BZ resistance. Crook et al. [36] showed the disagreement between the results of FECRT and LDT on two goat farms, where susceptibility to albendazole were indicated by FECRT and LDT indicated resistance to BZs. In the survey of Diez-Banos et al. [56] performed on sheep flocks, the BZs resistance using FECRT was found in 18% (13) of flocks, whereas 29% (21) of flocks were found to be resistant to BZs when EHT was used. A similar study performed by Babják et al. [21] pointed to a moderate correlation between in vivo and in vitro tests for detecting BZs resistance among the 30 goat farms. Moreover, the EHT and LDT have the greater potential to detect low levels of AR by using DC to reduce the number of drug concentrations required and to increase the sensitivity of the tests [5]. It allows the reliable detection of a frequency of resistance alleles below 10% [57] and is fairly reliable for the detection of BZ resistance under field conditions [35,37,53].Thus far, the resistance to MLs has been reported in Europe in goat herds from Denmark [58], Czech Republic [22], Scotland [59], Slovakia [34,60], Switzerland [17,61,62,63], Germany [63], and Poland [7,24,25] on a timespan of almost 30 years. This implies that the ML resistance might be spread throughout Europe, but its prevalence is still at a lower level than in case of BZs [2,6].In Romania, albendazole/fenbendazole (BZs) and ivermectin/eprinomectin (MLs) are the most frequently used anthelmintics in small ruminants, while levamisole is used only sporadically (inference from personal experience). BZs are relatively cheap and the withdrawal period is short, which is of great importance for the farmers as they are mainly interested in obtaining dairy products from their herds. As for MLs, ivermectin is the most commonly used anthelmintic in small ruminants, despite the extended withdrawal time for milk. EPM has more recently been approved for use in goats and rapidly gained in popularity thanks to its easy administration (pour-on), being the only anthelmintic with no milk-withdrawal period authorised in goats. However, for many Romanian farmers, the financial aspect is the one that prevails. Thus far, no data are available in Romania regarding the resistance to any anthelmintics in goats or sheep. The only study about AR in Romania was performed on horses in 2015 [38].The resistance to EPM found in this herd was unexpected since the drug has never been used in the herd before. However, this could be explained by the fact that eprinomectin and ivermectin, as avermectins (MLs), share the same mechanism of action [64], the latter being one of the most widely used drugs for controlling GINs in goats from Romania. Another explanation of the resistance to EPM in this herd could be attributed to the cross-resistance between BZs and ML. The same nucleotide changes in IVM resistant H. contortus have been observed in the gene coding for B-tubulin isotype 1, which is responsible for resistance in BZs, even though these chemical groups have different mechanisms of action [65]. Both explanations seem possible since the goats from the studied herd were only treated with ivermectin and albendazole in the last few years. Levamisole is very rarely used in goats in Romania. This most likely coincides with the fact that, to date, no commercial product based on levamisole has been approved for usage in goats. The herd from this study had never been treated with LEV before and the results of LDT showed susceptibility of GINs to this drug.There are several factors limiting the effective use of anthelmintics. One of the most important factors contributing to the rapid development of AR is underdosing [66]. In Romania, the anthelmintics commonly applied by farmers are not licensed for goats so they frequently extrapolate the dosage from other species. The goats metabolise anthelmintic medications more rapidly than other species, so they require higher doses of drugs [67]. However, our experience shows that the farmers either do not have knowledge about these requirements or choose to neglect them due to financial reasons. The situation was similar in the herd under study, where the owner dosed the drug as indicated for sheep. Cringoli et al. [68] observed big differences in FECR% between a 0.5 mg/kg/bw single dose (dosage recommended for sheep) and 1 mg/kg/bw double dose of EPM. Other factors that may contribute to the AR are the following: constant and repeated use of an anthelmintic belonging to one chemical group, too frequent deworming, and a lack of quarantine of newly purchased animals [69].From our field experience, we know that very few goat farmers ask for faecal examination needed for the diagnosis of GINs. The veterinary practitioners and farmers should be educated about all the aspects of AR and advised on how they can implement efficient control strategies against GINs in goats.5. ConclusionsThis study provides the first report of AR to two classes of anthelmintics: MLs and BZs, in goats in Romania. Considering the increasing prevalence of AR in goat herds in Europe and around the globe, we believe that the findings of our study on AR in goats in Romania do not represent a singular event and could hence be just the evidence of a much larger problem.

animals : an open access journal from mdpi

10.3390/ani13071244

PMC10093053

The subterranean fauna is an important component of global biodiversity. However, research on the subterranean fauna of the Yunnan–Guizhou Plateau in Southwest China, one of three important karst landforms in the world, is limited. In this study, we performed a population genetic analysis and reconstructed the phylogenetic tree of six populations of Trogloneta yunnanensis in South China Karst. The results showed that there was high genetic divergence among six populations, and the divergence of these six populations can be traced back to the late Pleistocene. Our results suggested that isolation was a pivotal factor affecting the biodiversity of cave faunas, and the biodiversity of cave-dwelling faunas needs to be studied as soon as possible.

Subterranean karst caves can contain unexpected biodiversity, but few studies related to spider population genetics have been conducted in the karst area of Southern China. In this study, we investigated the population genetic structure of Trogloneta yunnanensis (Song & Zhu, 1994) based on 73 spider samples from six underground populations in South China Karst. Population genetic structure analysis showed a clear divergence (FST > 0.9 and Nm < 0.05) among populations according to mitochondrial genes. The phylogenetic gene tree constructed by BI and ML methods recovered six geographic clades. Divergence time estimation indicated that the divergence of these six populations can be traced back to the late Pleistocene. We supposed that the geographic isolation led to the extreme population structure. According to this study and previous studies about troglobites living in this region, the subterranean habitats of the Yunnan-Guizhou Plateau may contain many organisms with similar genetic structures. The subterranean biodiversity in the karst area of Southern China needs to be re-evaluated and protected.

1. IntroductionBiodiversity loss is one of the most serious environmental crises worldwide; therefore, it is important and urgent to study it [1]. Compared with surface species, subterranean fauna is less studied due to sampling difficulty, low population density, and the rarity of encountering some species [2]. Caves provide a unique habitat for organisms, where there is no or less sunlight, no or less plant growth, high CO2 concentration, constant temperature close to the mean annual region temperature, and scarcity of food [3]. Culver and Holsinger [4] estimated a global total of 50,000 to 100,000 obligate subterranean species. Because of the extreme environment, cave-adapted species tend to have a relatively simple structure of a community and are isolated from each other in time and space [5]. Thus, the cave faunas can help us understand the evolution and biogeography of species and speciation under geographic isolation [6,7,8]. The contribution of endemic and relict taxa to overall biodiversity are increasingly being reappraised for many habitats and living groups from the conservation perspective [9].Many cave species have extremely small ranges, with a large part of troglobitic species, and subspecies limited to a single county, and many species are even known from a single cave [10]. Isolation barriers between cave systems limit gene flow between populations of cave organisms, promoting differentiation among populations and effectively dividing parts of the cave systems into subterranean islands [11]. Many studies in Europe and North America have found strong genetic differentiation among populations of cave faunas, particularly cave terrestrial invertebrates [5,11,12,13,14,15]. However, some studies indicated that cave species also have moderate to high rates of gene flow [12,16]. The gene flow may attribute to the existence of subterranean interconnecting passages or recent secondary contact between subterranean and surface forms [16]. The diversity of cave-dwelling species was influenced by many factors, such as the intrinsic characteristics of each species, the degree of cave dependence of species, and the distribution of limestone in karst areas [6,16,17,18]. The role of caves as natural laboratories has not been fully explored, especially when macroecological and biogeographic patterns at a continental or global scale are considered [19].The Mountains of Southwest China are one of the 34 global biodiversity hotspots [20]. The Yunnan-Guizhou Plateau in Southwest China is one of three important karst landforms in the world [21], where thousands of caves exist. There are some studies related to cave fish in this area [22,23,24], but terrestrial cave invertebrates which are regarded to make up the majority of subterranean faunas are still understudied [25]. The biodiversity of cave-dwelling faunas needs to be studied as soon as possible, which are threatened by a range of climate change and human activity [26].In this study, we investigated the population genetics of a tiny cave spider, Trogloneta yunnanensis [27], which belonged to the family Mysmenidae Petrunkevitch, 1928. To date, this species was only found in underground caves of the Yunnan-Guizhou Plateau, usually living under humid rocks or in rock gaps [28]. Trogloneta yunnanensis has been showing some cave-adapting characteristics, such as lighter color skin with little pigmentation. The main aims of this study were: (1) to explore the genetic structure of Trogloeta yunnanensis populations; (2) to reconstruct phylogenetic relationships and estimate the divergence time of different populations.2. Materials and Methods2.1. SamplingThe samples of spiders were collected from six isolated caves in Southwestern China (Figure 1). The sampling locality information is provided in Table 1. Sampled individuals were preserved in 95% ethanol in the field and then stored at −20 °C in the key Laboratory of Bio-resources and Eco-environment after being taken back. We identified spider species by the morphology of copulatory organs in both sexes. In this study, we found no surface populations of T. yunnanensis outside the cave, and there were no surface records of T. yunnanensis individuals in previous studies [28,29].2.2. DNA Extraction, Sample Preparation, and Gene SequencingDepending on the abundance of specimens, ten to fourteen individuals per cave were selected to extract DNA. Trogloneta yuensis [30] Yamaneta kehen (MK895531, MK908789, MK908805, MK908797, MK895538) and Yamaneta paquini (MK895536, MK908794, MK908810, MK908802, MK895544) were selected as outgroups. Trogloneta yuensis is the most closely related species to T. yunnanensis, and two Yamaneta species are also cave-dwelling mysmenid spiders from the Mountains of Southwest China [31]. We used DNeasy Blood and Tissue Kit (Qiagen, Hilden, Germany; P/N: 69506) to extract genomic DNA from the prosomal tissue of 73 individuals according to the instructions (the abdomens and male palps were kept as vouchers). We sequenced two partial mitochondrial genes and three partial nuclear genes: cytochrome c oxidase subunit 1 (cox1), 16S ribosomal RNA (rRNA), 28S rRNA, H3, and ITS-2. The primers were provided in Table S1. Two × M5 HiPer plus Taq HiFi PCR mix with blue dye was used as the polymerase enzyme. PCR reactions were 30 s at 94 °C, 30 s at 45 °C to 55 °C, and 30 s at 72 °C (×35 times). PCR products were sent to the Chengdu Branch of Qingke Biotechnology Co., LTD for sequencing. The sequencing data were checked and edited using Bioedit 7.2.5 [32]. MEGA X [33] was used to translate and align the protein-coding sequences. Other sequences were aligned in Clustal X [34].2.3. Population Genetic AnalysisWe used five genes to analyze the genetic structure of six populations of T. yunnanesis. DNAsp v6.0 [35] was used to determine haplotypes, DNA sequence polymorphisms, the number of haplotypes (h), haplotype diversity (Hd), and nucleotide diversity (π). The TCS (Templeton-Crandall-Sing) Networks [36] were constructed in PopArt 1.7 [37]. The F-statistics and AMOVA were calculated among the populations by Arlequin 3.5 [38], and we calculated the Nm values based on the F-statistics. The uncorrected ‘p’ distances between populations were calculated by MEGA X [33].2.4. Phylogenetic AnalysisTo examine the monophyly and allow us to compare diversity between populations, we used the Bayesian Inference (BI) method to reconstruct the phylogenetic tree of T. yunnanensis based on the concatenated genes (cox1 + 16S + H3 + 28S + ITS-2). We used PartitionFinder2 [39] to identify the best-fit models of molecular evolution and partitioning schemes for the dataset (Table S3). The BI phylogenetic tree was constructed in Mrbayes [40], and four Markov Chain Monte Carlo (MCMCs) with default heating parameters were performed for 10,000,000 generations until the average standard deviation of split frequencies was less than 0.01. The Markov chains were sampled every 1000 generations, and the first 25% of sampled trees were burn-in.The maximum-likelihood phylogenetic analysis was conducted in IQ-TREE v1.6.12 [41]. We used ultrafast bootstrapping with 5000 replicates [42] and the Shimodaira-Hasegawa approximate likelihood-ratio test (SH-aLRT) with 1000 replicates [43] to estimate the node support. The best-fit models were selected using ModelFinder [44]. The results are shown in Table S3.2.5. Divergence Time EstimationTo estimate the divergence time among six populations of T. yunnanensis, the species tree was constructed in BEAST v1.10.4 [45] under the Yule process tree model. MCMC chains were run for 10 million generations, sampling every 1000 generations. All other parameters were default settings. We assessed convergence, posterior trace plots, and effective sample sizes (ESS > 200) in Tracer v.1.7.1 [46]. TreeAnnotator was used to generate a maximum clade credibility (mcc) tree with the first 25% as burn-in. The best-fit model was found in PartitionFinder2 (Table S3).Due to the lack of suitable calibration points, we used prior information on substitution rates of genes to estimate population divergence time based on available information for spiders [13,47,48,49]. To reduce errors, we used only the cox1 gene for divergence time estimation. Preliminary analyses using a lognormal relaxed clock for the cox1 gene showed that the posterior distribution of the ucld.mean parameter accreted to zero, and hence a strict clock was preferred. The prior rate parameter was set to normal distribution with mean ± SD = 0.0168 ± 0.0018 for cox1.3. Results3.1. Genetic Diversity and Structure of Trogloneta yunnanensisA total of 632 sequences were obtained from 5 gene segments (630 bp cox1, 420 bp 16S, 781 bp 28S, 312 bp H3, and 373 bp ITS-2) in 73 individuals of T. yunnanensis. However, several cox1, 28S, and ITS-2 sequences could not be recovered because of their high AT content. The details are provided in Table S2. Since these sequences were similar among populations of T. yunnanensis, we believe that these missing data did not affect our analyses. The cox1, 16S, H3, and ITS-2 datasets had 30, 10, 4, and 2 polymorphic sites, respectively. The genetic diversity parameters of these six populations are summarized in Table 2. Among these six populations, QX, SLD, and YLD populations showed genetic differences in mitochondrial genes and nuclear genes, and the BY population showed high genetic diversity in mitochondrial genes (Table 2). XR and GN populations had relatively lower genetic diversity, and they only showed genetic differences in 16S and H3 genes, respectively (Table 2). There were no genetic differences in the 28S gene dataset of all samples, so it was not analyzed nor discussed below.Based on the cox1 gene, the genetic differentiation among six populations was high, the pairwise FST values among populations were above 0.90, and the Nm < 0.05 (Table 3 and Table 4). Based on FST values, the lowest level of divergence was observed between the SLD and BY populations, and the GN and XR populations showed the highest level of divergence. The divergence between the geographically close population YLD from the QX was higher than those from the geographically much higher populations QX, BY, and SLD (Figure 1). The mean pairwise uncorrected p-distances of T. yunnanensis based on the cox1 gene ranged from 0.49% to 2.53% (Table 5). XR and SLD were the two populations with the largest genetic distance (2.53%), while YLD and GN exhibited the smallest genetic distance (0.49%). The overall mean distance was 1.59% ± 0.31%. We also calculated the overall mean distance of 16S, H3, and ITS-2 to be 0.73% ± 0.24%, 0.29% ± 0.18%, and 0.14% ± 0.13%, respectively.The results of AMOVA suggested a high degree of genetic divergence among populations. A lower proportion of the variance (2.6%) was attributable to interpopulation (within populations), and nearly 97.4% of the cox1 gene diversity was explained by variance among the different cave populations (Table 6).The analysis of haplotype networks displayed clear genetic structure in populations of T. yunnanensis. The analysis of the cox1 gene showed the distinct geographic structure in T. yunnanensis. There were 12 haplotypes in 72 individuals from six populations, and six populations had no shared haplotypes (Figure 2). The 16S haplotype network showed several shared haplotypes among different populations: XR and QX shared Hap_1, SLD and BY shared Hap_4, and XR, QX, and GN shared Hap_2 (Figure 2). The haplotype network based on nuclear genes was divided into two parts. The H3 haplotype network showed that XR, YLD, GN, and BY shared Hap_1, and QX and SLD shared Hap_3 (Figure 2). The ITS-2 haplotype network illustrated that XR, QX, SLD, and BY shared Hap_1, and YLD and GN shared Hap_2 (Figure 2).3.2. Phylogenetic RelationshipsPhylogenetic analyses based on concatenated genes recovered six cave spider clades, but the topologies of the ML tree and the BI tree are inconsistent, and only given a moderate level of support values for some nodes (Figure 3 and Figure S1). The BI tree showed higher level of support values for some nodes (Figure 3). In the BI tree, T. yunnanensis was split into two lineages: A including XR, YLD, and GN, and B including BY, SLD, and QX. In the ML tree, YLD and GN were clustered into one lineage, and other populations (XR, SLD, QX, BY) were clustered into the second one (Figure S1), respectively. There are some “comb-like” lineages at shallow divergence levels (Figure 3), because of the same haplotype present across multiple samples of each cave.3.3. Divergence Time EstimationThe time tree was similar to the BI concatenated gene tree (Figure 4). Divergence time analysis revealed that two main clades diverged approximately 0.415 million years ago (Ma, 95% HPD = 0.230–0.641 Ma). The earliest divergent population was QX, which can be traced back to 0.349 Ma (95% HPD = 0.201–0.555 Ma), and the XR population occurred approximately 0.242 Ma (95% HPD = 0.128–0.401 Ma). The split time between BY and SLD was 0.226 Ma (95% HPD = 0.112–0.382 Ma). YLD and GN diverged into two lineages 0.136 million years ago (95% HPD = 0.067–0.243 Ma).4. Discussion4.1. Variation and AdmixtureIn this study, we did not find the existence of T. yunnanense outside the cave. There are no records of T. yunnanense epigean individuals. According to the morphological characteristics of T. yunnanense, we suppose that it is an obligate cave species. According to the analysis results of the cox1 gene, the population genetic structure of T. yunnanensis indicated a pattern of low intra-population diversity and high inter-population diversity. The genetic variation within each population of T. yunnanensis was extremely small, and the number of polymorphic (segregating) sites ranged from 0 to 3. The genetic diversity varied significantly among geographical distinct populations of T. yunnanensis. The lack of shared haplotypes among these six populations (Figure 2) and the high FST values (>0.4, Table 3) indicated that there was currently little to no migration of spiders between caves. The results were similar to previous studies associated with cave spiders [11,12,13]. The pattern of genetic structure of the T. yunnanensis population was consistent with the predicted results of the population model in a fragmented habitat, which was mainly caused by geographical isolation and habitat. Species trapped in caves are unable to exchange genes with the outside individuals and develop further genetic differentiation [11]. Because of the special environment in caves and small population size, cave faunas may form extreme genetic structures. The genetic distance among different populations of T. yunnanensis ranged from 0.49% to 2.5%, and there was no significant correlation in geographical distance. The closest genetic distance is between YLD and GN of populations, but their FST was 0.96, which revealed a clear divergence between them.Such large genetic differences among populations have led us to consider the possibility of the existence of cryptic species. Cryptic species have been described in many taxa, such as birds [50], reptiles [51], amphibians [52], crustaceans [53], and others. Previous studies of cave arthropods in karst areas of southern China have found that the underground fauna contained a surprising diversity. Zhang and Li [54] have found that the Nesticella cave spiders inhabit Yunnan–Guizhou Plateau invaded caves only recently, and cryptic species probably exist within them because of the deep divergences within the species. A study by Zhang and Li [25] on the cave telemid found that Telema cucurbitina was a species complex with a genetic distance of cox1 among different cave populations ranging from 5.4% to 17.3%. They concluded that multiple cryptic species existed in the population of Telema cucurbitina. The genetic distance among populations of T. yunnanensis was much smaller than that of Telema cucurbitina, so we excluded the possibility that there were cryptic species in T. yunnanensis. Analysis of nuclear genes revealed a more conserved genetic structure, dividing the six populations into two clusters. It was possibly that maternal inheritance of mitochondrial genes reduced the effective gene flow by a factor of four compared with diploid nuclear systems [12].4.2. Impact of Geographical IsolationWe reconstructed the phylogenetic tree of T. yunnanensis based on concatenated genes, showing the high levels of support values for main clades. The populations of T. yunnanensis were genetically isolated, and each population was supported as a monophyletic group. Highly different lineages (YLD and QX) were observed occupying geographically adjacent areas, but there was no mixing, suggesting that migration was limited (Figure 1 and Figure 3). The phylogenetic tree showed that BY and SLD were sister groups, which indicated that they have the closest and most stable relationship among these six populations. The topological structure of the BI tree and the ML genes tree was different, maybe because of the recent differentiation of the six populations. We believe that the dispersal ability of T. yunnanensis is limited, and cave isolation has a significant impact on its genetic structure.4.3. Evolutionary HistoryThe colonization and speciation of cave animals is generally explained by two hypotheses, one is the “climate-relict”, and the other is the “adaptive-shift” [3,5]. The first model was proposed for continental temperate ecosystems, where the surface species colonize to the cave environment. The isolation of cave populations occurs as surface populations become extinct (due to climatic changes). Under the second model, with the active colonization of surface populations in cave environments, adaptive differentiation occurred between populations on the surface and in the cave, and gene flow decreased. In Asia, a general cooling has gradually replaced the warm and humid climates of the early Miocene [55]. Climate change has a greater impact on vegetation cover, especially in the mid-latitudes [55]. This includes the slow but steady decline of the once widely distributed warm-temperate evergreen forests, which have gradually moved to coastal and low-latitude regions and been replaced by boreal forests, grasslands, and savannas [56]. The climate change strongly affected the vegetation cover, particularly in middle latitudes. During the middle Miocene to late Pliocene, the topography of China forms a three-step staircase in which the Yunnan–Guizhou Plateau constitutes the southern part of the second step. Since the Middle Pleistocene, the continuous uplift of the Qinghai-Tibet Plateau has promoted the formation of the Yunnan-Guizhou Plateau, which greatly affected the tectonic of the Yunnan–Guizhou Plateau, forming mountains and deep valleys and rearranging major river drainages. These events in East Asia during the second half of the Miocene may have gradually created new surface conditions that were unfavorable to species adapted to tropical habitats [51].The divergence time in six populations of T. yunnanensis occurred in the middle-to-late Pleistocene. The results were similar to previous studies on Nesticella spiders in this region. Zhang and Li [54] found that the cave groups of Nesticella in the Yunnan-Guizhou Plateau originated in the Miocene, and most populations of different species formed in the Pleistocene. Further research by Ballarin and Li [8] found that climate change in the Miocene caused Nesticella to take refuge in caves and to begin rapid differentiation in the 5.5 Ma. We suggest that T. yunnanensis, which is similar to the Nesticell in body size and habitat, may have experienced a similar evolutionary history. Because of the absence of surface populations and closest sister species in Yunnan-Guizhou Plateau, we could not determine the origin of T. yunnanensis. Ideally, if we can collect related species of T. yunnanensis on the surface, the origin could be inferred, and how the geological change impacted the organisms in the Yunnan-Guizhou Plateau can be further explored.5. ConclusionsThis study systematically explored the population genetics of a cave spider in Yunnan-Guizhou Plateau. Our results suggested that the isolation was a pivotal factor increasing biodiversity of cave faunas. We believe that cave faunas with similar body sizes and habitats also have similar genetic structures in the Yunnan-Guizhou Plateau. A correct understanding of biodiversity is fundamental to conservation, and karst areas need to be further studied, because there are thousands of caves which are unexplored. Our study provides new insights into the diversity of subsurface life in karst areas. Further research could use larger datasets, such as NGS data, and new analytical tools to explore genetic structures between and even within populations. More research on the underground fauna in karst areas will shed light on the formation pattern of biodiversity in this region.

animals : an open access journal from mdpi

10.3390/ani11123348

PMC8698084

In ovine species, transcervical artificial insemination is not easy to apply, due to the tortuous lumen of the cervix that does not allow the passage of routinely used catheters. Moreover, the quality of frozen–thawed semen in small ruminants is poor and these factors negatively affect the wide spreading of superior genotypes. The aim of this study was therefore to preliminarily test three newly designed insemination catheters, with bent tips of different lengths, in terms of reproductive performances in pluriparous ewes inseminated with frozen–thawed semen. Afterwards, the outcomes of insemination with the best performing catheter were compared to those obtained in ewes previously submitted to surgical incision of cervical folds, a technique that allows transcervical intrauterine deposition of semen. The results obtained indicated that a catheter with a bent tip of 5.0 mm allowed deep and fast intrauterine insemination, leading to pregnancy rates similar to those obtained following surgical incision of the folds. Further tests on the efficiency of the catheter are needed in field conditions and on a larger number of animals to assess the feasibility of the method in wide commercial insemination campaigns.

In ovine species, transcervical artificial insemination (TCAI) is limited by the poor quality of frozen–thawed semen and by the convoluted cervical lumen hampering the passage of inseminating devices. The aim of the study was to test the efficiency of three newly designed catheters with bent tips of 3.5 mm, 5.0 mm or 8.0 mm in terms of reproductive performances (experiment 1) and to compare the results of TCAI with the best performing catheter of experiment 1 to those obtained in ewes submitted to surgical incision of cervical folds (SICF) prior to insemination (experiment 2). The following parameters were assessed: time to pass the cervix; depth of cervical penetration; site of deposition of semen; pregnancy (PR); and lambing rates (LR). The results of experiment 1 indicated that the 5.0 mm tip catheter resulted in deeper and faster TCAI and higher PR and LR compared to 3.5 mm and 8.0 mm tip catheters (p < 0.05). In experiment 2, TCAI with the 5.0 mm catheter did not differ from TCAI after SICF in terms of depth of semen deposition, time to pass the cervix, PR and LR (p < 0.05). In conclusion, the use of a catheter that allowed transcervical uterine deposition of semen without excessive manipulation led to satisfactory pregnancy rates.

1. IntroductionThe ovine cervix is characterized by a very narrow and misaligned lumen delimited by funnel-shaped folds that protrude caudally and are often in an eccentric position [1,2]. This peculiar anatomy makes the passage of an artificial insemination catheter difficult, often almost impossible, and contributes to the limited diffusion of transcervical artificial insemination (TCAI) in the ovine breeding system. Moreover, it needs to be pointed out that, apart from the cervical barrier, another significant restricting factor is represented by the low resistance of ram semen to cryopreservation. In ovine species, a relationship between the depth of penetration of the cervical lumen and the fertility rates achieved was reported in 1994 by Eppleston et al. [3]. The site of deposition of frozen–thawed sperm deep in the cervix (beyond the 3rd–4th fold) leads to consistently higher fertility rates compared to deposition at 1 cm depth in the lumen [3]. These observations are supported by the poor pregnancy rates obtained when frozen–thawed semen is deposited in the external os of the cervix, ranging from less than 5% [4,5] to 36% [6]. The problems related to the cervical barrier and the poor fertilizing ability of frozen–thawed semen in ovine artificial insemination have so far been overcome by laparoscopic AI (LAI). This technique has been widely used in recent decades since it allows direct uterine deposition of frozen–thawed semen via laparoscopy. The fertility rates range from 60% to 70% [5,7,8,9]. Nevertheless, it needs trained veterinarians, anesthesia and expensive equipment to be performed and it should be considered as provisional until an efficient TCAI technique is developed (as reviewed by [10]).Basically, an optimal TCAI should: (i) allow deep cervical or uterine deposition of semen; (ii) be easy and fast to perform; (iii) be respectful of animal welfare; (iv) require inexpensive instruments; and (v) be applicable in field conditions.In the past, different approaches have been attempted to improve the performances of TCAI but, although some of them appear convincing and successful in terms of fertility rates, none has had a wide in-field application. Recently, our research group presented a technique for surgical incision of cervical folds (SICF) that allowed us to achieve fertility rates comparable to those obtained with LAI [9,11] using frozen–thawed semen. Although it is a promising technique, to be performed once in the lifetime of the animal, it is a surgery that requires veterinarian skills and, as suggested by the authors, it should be devoted to small nuclei of selected high-value ewes. Among other methods, the use of drugs (hormones [12,13,14,15,16,17,18], chemokines [19], myorelaxants [20], hyaluronan [21], beta-adrenergic blocking agents [22]) and the design of inseminating catheters [23,24,25] associated with manipulation of the cervical canal (i.e., Guelph System [26,27]) have been investigated. Since hormones are in general expensive and, according to consumer demand, animal production should slowly move towards drug-free alternatives [28], the focus on the design of devices that could trespass the cervical lumen with no traumas on surrounding tissues could be the key to overcome the limitations of TCAI. Therefore, the primary objectives of the present study were to evaluate the reproductive performances of ewes inseminated with frozen–thawed semen in two sets of experiments: (1) preliminary testing of three different devices for transcervical insemination and (2) comparing two different TCAI techniques (i.e., the newly designed device and transcervical insemination following SICF).2. Materials and Methods2.1. Experimental DesignAll experimental procedures were carried out under European regulations on the care and welfare of animals in research and were ethically approved by the Organization in Charge of Animal Welfare and Animal Testing (Organismo Preposto al Benessere Animale ed alla Sperimentazione sugli Animali—OPBSA) of the University of Sassari (protocol number: 26064). All animals were fed hay and concentrates, and water was provided ad libitum. The experimental design is represented in Figure 1. A total number of 95 pluriparous Sarda ewes (3–4 years old and about 45 kg live weight) were randomly assigned to two different experiments. In experiment 1 (n = 36), three different catheters for TCAI were tested, and in experiment 2 (n = 59), two different TCAI methods, insemination with the best performing catheter of experiment 1 and insemination following surgical incision of the cervical folds (SICF), were compared.2.2. Estrous SynchronizationIn all experimental animals, synchronization of estrous cycles was achieved by inserting intravaginal progestagen sponges (Crono-gest 20 mg, Intervet Italia S.r.l, Segrate, Italy) pre-treated with antibiotic powder (Izoaspersorio, Izo, Brescia, Italy) for 12 days and injecting IM 300 IU of PMSG (Folligon, Intervet Italia S.r.l, Italy) at the time of sponge removal.2.3. Semen Collection and PreparationEjaculates of three adult Sarda rams of proven fertility were collected by artificial vagina. Only samples having a concentration of at least 3 × 109 spz/mL and mass motility ≥3 (score from 0 (no waves) to 5 (vigorous swirling waves)) were further processed. Semen was pooled and extended at 30 °C with Tris-EY (egg yolk, 20%) supplemented with 6% glycerol to reach a final concentration of 400 × 106 spz/mL. Extended semen was gradually cooled from 30 °C to 4 °C in 5 h and loaded into 0.25 mL straws (insemination dose: 100 × 106 spz/straw). Straws were then exposed for 7 min to LN2 (liquid nitrogen) vapors at 5 cm above the surface, plunged in LN2 and then stored until use. Post-thawing motility was assessed by CASA (Computer Assisted Sperm Analysis, IVOS, Hamilton Thorne, Biosciences) in a straw from each batch. Semen with ≥45% progressive motility was considered suitable for insemination. At the time of insemination, thawing was performed at 37 °C for 30 s.2.4. Experiment 1The experiment was carried out on 36 pluriparous Sarda ewes randomly allocated to three different groups (n = 12). Following synchronization of ovulation, 58 h after removal of the sponges, ewes were submitted to TCAI using three different insemination catheters. All the inseminations were performed by the same professional. Catheter DesignCatheters were composed of a stainless-steel tube 28.5 cm long with an inner diameter of 3.5 mm, containing a Cassou insemination gun for small ruminants (IMV Technologies, L’Aigle, France) fitting a sanitary sheath (Figure 2A). One end of the catheter had a screw system that kept the Cassou gun secured (Figure 2B), while the other end was composed of a modified epidural needle (Ø 14 G) with a 45° bent rounded atraumatic tip of different lengths: 3.5 mm, 5.0 mm or 8.0 mm (Figure 2C).2.5. Experiment 2The experiment was carried out on 59 ewes allocated to two different groups: n = 30 were inseminated using the catheter that in experiment 1 yielded the best performances; n = 29 were previously submitted to two incisions of cervical folds [9,11] and transcervically inseminated. These ewes were the same subjects used for the study published by Pau et al. (2019) [11]. Briefly, surgery was performed within 24 h from lambing and after confirming expulsion of fetal membranes. Following mild sedation and epidural anesthesia, ewes were placed in dorsal recumbency with the hindquarters slightly elevated, and the external os of the cervix was localized by the aid of a speculum inserted in the vagina. The most external fold of the os was grasped with a Duval forceps and the whole cervix, fold by fold, was gently retracted caudally up to the vaginal vestibulum until complete exteriorization. Afterwards, each fold was incised dorsally and ventrally. Local antibiotic treatment was provided, and the extruded folds were gently repositioned. Ewes were kept under post-operatory observation for 24 h.2.6. Ewe Restraint and Cervical Manipulation for TCAIFor transcervical insemination, animals were placed in a cradle in dorsal recumbence and the vulva and perineal area were carefully rinsed. The cervix was located by the insertion of a vaginal speculum fitted with a light source and the external fold of the os was grasped with the aid of Bozeman forceps. The cervix was then gently retracted caudally and positioned to allow proper manipulation with the modified tip fitted on the stainless- steel tube. During cervical manipulation, semen was thawed and loaded on the Cassou gun. Once the site of deposition of semen was reached, the gun was inserted into the stainless-steel tube and secured, and insemination was carried out. Ewes previously submitted to SICF were inseminated using a commercial AI catheter for small ruminants (Cassou mini-pistolet for ovine-caprine, IMV Technologies, France).2.7. Data CollectionIn experiment 1, the depth of penetration through the cervical lumen was assessed by reporting the number of cervical folds overcome by the three catheters (1st–2nd folds; 3rd–4th folds; 5th–6th folds; in utero; Figure 3A–E). For both experiments, the time taken to trespass the cervical lumen was recorded and data about site of deposition of semen (in cervix vs. in utero) were collected. Pregnancy rates (pregnant ewes/inseminated ewes) were assessed by transrectal ultrasound (MyLab One, Esaote, Genova, Italy) on day 30 after insemination and lambing rates were also recorded.2.8. Statistical AnalysesCollected data were statistically analyzed using STATA 11.2/IC (StataCorp LP, College Station, TX, USA) by the chi-square test or Fisher’s exact test to determine the effect of the three catheters (experiment 1) and of the two insemination techniques (experiment 2) on depth of cervical penetration, site of deposition of semen and pregnancy and lambing rates. The significance level was defined for p < 0.05.3. Results3.1. Experiment 1The results of experiment 1 are summarized in Table 1, Table 2 and Table 3. The device used for TCAI had a significant effect on all the measured parameters (i.e., time to reach the uterus, depth of semen deposition, pregnancy and lambing rates; p < 0.05). In general, TCAI with the 5.0 mm tip catheter resulted in higher pregnancy and lambing rates compared to the 8.0 mm tip catheter (p < 0.05), while there was no significant difference compared to the 3.5 mm tip catheter (Table 1; p > 0.05). However, the 5.0 mm tip catheter allowed deeper and faster deposition of semen compared to the other two devices (Table 2 and Table 3; p < 0.05).The site of deposition of semen, measured by the number of overcome cervical folds, had a significant effect on pregnancy outcomes (p < 0.05). Most of the ewes inseminated with the 5.0 mm tip catheter directly in the uterine lumen were pregnant (77.7%), while none of those that had semen deposition in cervical lumen were pregnant. Using the 8.0 mm tip catheter, the only pregnant ewes had semen deposited beyond the 3rd–4th cervical fold. Deposition of semen with the 3.5 mm tip catheter led to 16.6% pregnant ewes, depositing semen beyond the 5th–6th cervical fold (n = 1) and in utero (n = 1) (Table 2).The time taken to reach the cervical lumen affected pregnancy and lambing rates (p < 0.05). Overall, when insemination took more than 60 s, pregnancy rates were significantly lower (p < 0.05). In most of the ewes inseminated with the 8.0 mm (91.6%) or the 3.5 mm (75%) tip catheter, the procedure lasted more than 60 s and led to low pregnancy rates (9% and 11%, respectively). Conversely, insemination with the 5.0 mm tip catheter allowed faster semen deposition: 33.3% of ewes were inseminated in less than 10 s and most of them (3/4 ewes) were pregnant and lambed regularly (Table 3).3.2. Experiment 2Results of experiment 2 are summarized in Table 4, Table 5 and Table 6. In general, no significant differences in pregnancy and lambing rates were found between ewes inseminated with the 5.0 mm tip catheter and those inseminated after SICF (Table 4; p > 0.05).Time taken to reach the cervical lumen was not affected by the method of insemination (Table 5; p > 0.05). Reaching the cervical lumen with both insemination methods took less than 30 s in most of the animals. In four ewes submitted to SICF, semen was deposited in the cervix as it was impossible for the catheter to reach the uterine lumen in <60 s, resulting in one pregnancy (Table 6). Using the 5.0 mm tip catheter, in four ewes, semen was deposited cervically in >60 s, resulting in no pregnancies.4. DiscussionThe results presented in this report indicate that the design of an insemination catheter with a bent tip of 5.0 mm was successful in allowing deeper and faster transcervical insemination with frozen–thawed semen in Sarda ewes. The pregnancy and lambing rates obtained using this catheter were comparable to those obtained by insemination following SICF and, in most of the ewes, semen deposition occurred in the uterus. In ewes submitted to SICF, the overlapping of cervical folds that hampers the passage of routinely used inseminating devices is avoided by dorsal and ventral incisions of each fold. This procedure allows a smooth and atraumatic penetration of the cervical lumen and intrauterine insemination, obtaining pregnancy rates that are easily comparable to those deriving from laparoscopic AI [9]. We have sufficient evidence in the literature demonstrating that in ovine species, the closer the semen is deposited to the site of fertilization, the higher the pregnancy rates [3]. This is supported by the satisfactory pregnancy and lambing rates obtained by laparoscopic AI [3] or by TCAI following SICF [9,11]. With both techniques, the cervical barrier is bypassed, and the deposition of frozen–thawed semen is intrauterine. However, these techniques are invasive and require anesthesia, proper training of the operator is mandatory and, in our opinion, they should be gradually destined to limited nuclei of animals for genetic selection. Nevertheless, the influence of the depth of semen deposition by TCAI on pregnancy rates has been questioned by several authors. Among others, Kumar et al. [29] reported no correlation among depth of insemination with frozen–thawed semen on pregnancy and lambing rates.In the literature, several research groups designed catheters for enhancing fertility rates following TCAI and, among them, many authors reported that using catheters with bent tips allowed deeper penetration compared to straight ones [2,23,26]. Alvarez et al. [23] used a catheter with an eccentric bent tip of 6.0 mm, similar to the one used in the present experiment, and reported pregnancy rates of around 45% using chilled semen. In contrast, our results showed that inseminating ewes using the catheter with the 5.0 mm bent tip led to higher pregnancy rates (around 63%), although frozen–thawed semen was used. Besides small differences in the design of the catheters, the discrepancies in the results could be attributable primarily to the different techniques used to perform the insemination: the gentle cervical retraction exerted in the present experiments allowed deep penetration and semen deposition. Alvarez et al. [23] did not use this technique and reported semen deposition as deep as possible in the cervical lumen. Moreover, breed differences in the weight and size of the animals chosen for the experiments may have influenced the morphometry of the cervical lumen, significantly affecting the depth of cervical penetration [2,12,30,31]. Another crucial factor affecting the outcomes of TCAI in ewes is the age of the inseminated animals, more precisely intended as number of parturitions. Changes in the morphology and the complexity of the cervical lumen in mature pluriparous ewes have been previously described by several authors [1,2,31]. Prior to the present trials, the ewes lambed 3 to 4 times and we can therefore hypothesize that cervical folds underwent mechanical stretching, making the lumen less convoluted and allowing deeper passage of the inseminating catheter. A further advantage in the use of the newly designed catheter could be that cervical manipulation was carried out with the tip of the catheter fitted in the outer stainless-steel tube, and that the Cassou gun was inserted at a later stage, when the site of semen deposition (cervical lumen or uterus) was reached. In this way, semen was thawed and loaded in the Cassou gun during cervical manipulation. This procedure may have limited the decay of the quality of thawed semen, easily damaged by cryopreservation, shortening the time between thawing and deposition in the female genital tract.The time taken to reach and possibly pass the cervical lumen is the indirect measure of the amount of manipulation of the cervix that, if prolonged, could potentially affect the success of the insemination method. Excessive cervical manipulation induced tissue damage, with release of local prostaglandins and recall of neutrophiles, altered the composition of cervical mucus and the uterine micro-environment, leading to pregnancy failure [23,32]. Therefore, the less the manipulation, the higher the chance of having pregnant animals. In both experiments, we observed that when cervical manipulation took more than 30 s, pregnancy rates were low, ranging from 8.7% to 29.0%. This was in agreement with what has been previously published. Alvarez et al. [23] reported successful pregnancy rates when TCAI was performed in less than 10 s and conversely, when penetration took more than two minutes, pregnancy rates dropped, and the technique becomes practically unfeasible [33,34,35]. 5. ConclusionsThe site of semen deposition and time taken to pass through the cervical lumen are essential parameters for the assessment of the feasibility of an insemination technique. In light of these observations, the design of a bent catheter with a 5.0 mm blunt tip allowed fast and deep transcervical insemination in pluriparous Sarda ewes, achieving satisfactory pregnancy rates. The results obtained are comparable to those obtained by techniques such as SICF or LAI that ensure uterine deposition of semen. However, further tests on the efficiency of the catheter are needed in field conditions and on a larger number of animals.

animals : an open access journal from mdpi

10.3390/ani11113293

PMC8614360

Probiotics, prebiotics and synbiotics are biologically active substances that are commonly used in poultry feeding as an alternative to antibiotic growth promoters. It was found that they could improve the intestinal microstructure as well as the health status and productivity of animals. The aim of this study was to determine the effect of probiotics, prebiotics and synbiotics administrated in ovo on the 12th day of embryonic development on selected morphological parameters of the small intestine in broiler and native chickens. After hatching, the chicks were placed in pens and housed for 42 days. On the last day of the experiment, all birds were individually weighed and slaughtered, and samples for histological analysis were taken from the duodenum, jejunum and ileum. The following parameters were determined: the height, width and surface area of the villi, the thickness of the muscular layer and the depth of the crypts, as well as the ratio of the villi height to the crypt depth. Based on the obtained data, it can be concluded that the substances used have an impact on the production parameters and intestinal morphology in various utility types of poultry. In addition, the obtained results indicate that chickens with different genotypes react differently to a given substance; therefore, the substances should be chosen in relation to the genotype.

The aim of the study was to determine the effect of probiotics, prebiotics and synbiotics administered in ovo on selected morphological parameters of the small intestine (duodenum, jejunum, ileum) in broiler chickens (Ross 308) and native chickens (Green-legged Partridge, GP). On the 12th day of embryonic development (the incubation period), an aqueous solution of a suitable bioactive substance was supplied in ovo to the egg’s air cell: probiotic—Lactococcus lactis subsp. cremoris (PRO), prebiotic—GOS, galacto-oligosaccharides (PRE) or symbiotic—GOS + Lactococcus lactis subsp. cremoris (SYN). Sterile saline was injected into control (CON) eggs. After hatching, the chicks were placed in pens (8 birds/pen, 4 replicates/group) and housed for 42 days. On the last day of the experiment, all birds were individually weighed and slaughtered. Samples for histological analysis were taken directly after slaughter from three sections of the small intestine. In samples from the duodenum, jejunum and ileum, the height and width of the intestinal villi (VH) were measured and their area (VA) was calculated, the depth of the intestinal crypts (CD) was determined, the thickness of the muscularis was measured and the ratio of the villus height to the crypt depth (V/C) was calculated. On the basis of the obtained data, it can be concluded that the applied substances administered in ovo affect the production parameters and intestinal morphology in broiler chickens and GP. The experiment showed a beneficial effect of in ovo stimulation with a prebiotic on the final body weight of Ross 308 compared to CON, while the effect of the administered substances on the intestinal microstructure is not unequivocal. In GP, the best effect in terms of villi height and V/C ratio was found in the in ovo synbiotic group. Taking into account the obtained results, it can be concluded that chickens of different genotypes react differently to a given substance; therefore, the substances should be adapted to the genotype.

1. IntroductionThe development, structure and functions of the digestive tract in animals largely depend on the composition and type of diet [1,2,3,4,5,6,7,8,9]. Nutritional factors can both positively and negatively influence the composition of the gut microflora, leading to changes in the end products of the bacterial fermentation of carbohydrates and proteins in the gut [2,3,10,11,12,13,14,15]. The intestinal epithelium, involved in the absorption of nutrients, is also a barrier between the external and internal environment of the organism. Unfavorable changes in the intestinal mucosa, occurring under the influence of the pathogenic bacteria and toxic substances present in the digesta, negatively affect the performance of farm animals [16,17,18].Pro-, pre- and synbiotics are biologically active substances, which are commonly used as feeding supplements in poultry. Their use increased after the European Union banned antibiotic growth promoters (AGP). Probiotics are preparations that contain live microorganisms, mainly bacteria and yeasts, which, by influencing the composition of the intestinal microbiota, can affect the health of the host [19,20]. Prebiotics are selectively fermented components that positively affect the welfare and health of the host by selectively stimulating the growth and/or activity of the intestinal microflora [12,21,22]. Products containing both probiotics and prebiotics are called synbiotics. Bioactive substances reportedly improve the intestinal microstructure and have a positive effect on the health status and production performance of animals by influencing intestinal microbiota composition and short-chain fatty acids (SCFA) profiles [12,20,21,22,23,24,25], the digestibility of nutrients and the body’s resistance [26,27,28,29,30]. One of the main products of bacterial fermentation, apart from acetic and propionic acid, is butyric acid, which has a beneficial effect on both the digestive tract and peripheral tissues. The activity of butyrate in the organism is related to its regulatory influence on gene expression and limitation of the multiplication of pathogenic bacteria [31,32,33], which may have a beneficial effect on the structure of the intestinal mucosa. Studies conducted in recent years indicated the antibacterial, immunostimulatory and antidiarrheal effects of bioactive substances [34,35,36,37]. Such a pro-health effect, also found in poultry, results from their beneficial effects on the microflora of the gastrointestinal tract and the microstructure of the intestinal mucosa [38,39,40]. According to de Vrese and Schrezenmeir [41] and Yang et al. [42], the effectiveness of pro-, pre- and synbiotics operation depends both on the composition of the preparation as well as the time and method of their administration. In poultry production, biologically active substances are usually added to feed and water. The effectiveness of this type of delivery was documented in numerous studies. For example, it was found that the probiotics, prebiotics and synbiotics administered in-feed had a positive effect on the development and broiler performance, mediated by stimulated intestinal microflora, immune system and small intestine mucosa [43,44,45,46,47]. An alternative to the oral administration of bioactive substances is the in ovo method. Numerous studies show that the delivery of pro, pre- and/or synbiotics to the egg during the embryonic development of the chick has a positive effect on the development of the digestive tract [7] and the immune status of birds [48,49,50,51,52,53]. The in ovo-delivered bioactive substances primarily influence the composition of the intestinal microflora [54,55], but their effect depends on the time point of delivery and the composition of the injected compounds/probiotic strains [56].As a result of intensive breeding work, two utility lines were obtained: high-laying chickens (laying hens) and chickens of the meat type (broilers). The growth rates of the two production types were different, presumably due to the characteristic development of the digestive system. The studies by Uni et al. [57] showed that the intestinal morphological parameters, such as the height and width of the intestinal villi, as well as the depth of the crypts and the size of the absorption area, could be related to the greater body weight of broilers compared to laying hens. Additionally, Simon et al. [58] showed that the genetic background could influence the ileal IgA, IgM and IgY expressions, which were higher in broiler chickens compared to laying hens. These differences were most likely attributed to the differences in the gut microbiota composition between distinct chicken genotypes [59]. There are few reports in the available literature comparing the physiological parameters of commercial chickens with native breeds. It is known, however, that native breeds are characterized by a greater resistance and better ability to adapt to environmental conditions [60]. The chicken breed native to Poland is the Green-legged Partridge (GP), which is treated as general-purpose poultry, characterized by a high tolerance to very low temperatures and extensive rearing. GP are phenotypically and genetically distinct compared to highly selected broiler chickens [61]. In this study, we hypothesized that there is a relation between the bioactive substances administered in ovo and chicken genotype expressed in the microstructure of the small intestine in broiler chickens and native chickens. This study aimed to determine the effect of probiotics, prebiotics and synbiotics administered in ovo on selected morphological parameters of the small intestine in broiler chickens and GP chickens.2. Materials and Methods2.1. Animal ProceduresThe research was carried out on broiler chickens (Ross 308) and native chickens (Green-legged Partridge, GP). The experiment began with egg incubation (600 eggs/genotype) in a commercial hatchery using an automated incubator at 37.8 °C and a relative humidity of 61–63%. The broiler eggs were obtained from a commercial breeding flock, while the GP eggs came from the conservation flock managed by the University of Life Sciences in Lublin, Poland. On day 12 of egg incubation, aquatic solutions of the respective bioactive substance was delivered in ovo into the eggs’ air cells: probiotic (Lactococcus lactis subsp. cremoris, 105 CFU/egg), prebiotic (GOS, galactooligosaccharides, 3.5 mg/egg), or synbiotic (GOS, 3.5 mg /egg + Lactococcus lactis subsp. cremoris, 105 CFU/egg). The selection of the bioactive compounds and their doses was based on previous research [55,62]. GOS was derived from Clasado Biosciences Ltd. (Jersey, UK) and is known under trade name: Bi2tos. Lactococcus lactis subsp. cremoris. IBB477 was obtained from the collection of the Institute of Biochemistry and Biophysics Polish Academy of Sciences (Warsaw, Poland). Sterile physiological saline was injected into the control eggs. The injection volume was 0.2 mL for each egg. After in ovo injection, the puncture hole was sealed, and incubation continued. Detailed information on the in ovo procedure is presented in Sławińska et al. [63]. The experiments were conducted at an experimental farm of Wroclaw University of Environmental and Life Sciences (Wroclaw, Poland) with the consent of the Local Ethics Committee for Animal Experiments (Bydgoszcz, Poland, no. 16/2014). After hatching, the chicks were placed in deep litter pens with a surface area of 3.75 m2, and with a stocking rate of 17.33 birds/m2 (32 birds per group: 8 birds/pen, 4 replicates/group) where they were kept for 42 days. All pens had the same environmental conditions. During the course of the experiment, both Ross 308 and GP were fed standard diets (Table 1), and both feed and fresh water were available ad libitum. The environmental conditions were adjusted to the age of the birds. At the end of the experiment (42 d of age), all birds were individually weighed, stunned and slaughtered. Samples for histological analysis (ca. 3–4 cm) were taken immediately after slaughter from three segments of the small intestine: from the midpoint of the duodenum, the midpoint between the point of entry of the bile duct and Meckel’s diverticulum (jejunum), and midway between Meckel’s diverticulum and the ileocecal junction (ileum).2.2. Analytical MethodsIndividual segments of the intestine were rinsed with 0.9% physiological saline, and then fixed with 4% CaCO3 buffered formalin for 24 h. Then, they were dehydrated in graded ethanol series, cleaned in xylene and infiltrated with paraffin in a tissue processor (Thermo Shandon, Chadwick Road, Astmoor, Runcorn, Cheshire, UK), and then embedded in paraffin wax (Medite, Burgdorf, Germany). Samples were cut on scraps at 10-μm thickness using a microtome (Thermo Shandon, Chadwick Road, Astmoor, Runcorn, Cheshire, UK) and mounted on microscope slides coated with an egg albumin and glycerin mixture. Sections were stained with periodic acid-Schiff (PAS) for morphometric evaluation.The measurements of the height and width of the villi, crypt depth and muscle thickness were performed using a Nikon Ci-L microscope equipped with a Nikon DS-Fi3 camera with a resolution of 5.9 MPix and NIS ELEMENTS software. Next, the villi area (VA) was calculated using the formula cited by Sakamoto et al. [64]:VA = 2π × (VW/2) × VH, where VW = villus width, and VH = villus height. The villus height/crypt depth ratio (V/C) was also calculated. Linear measurements of the thickness of the muscular layer of small intestine were conducted on five consecutive slices.2.3. Statistical AnalysisThe data are presented as means and standard deviation (SD). The results were statistically analyzed by one-way ANOVA using STATISTICA 13.1 software (StatSoft®, Tulsa, OK, USA). For data that corresponded with the normal distribution, the post hoc Dunkan’s multiple range test was applied. All differences were considered significant at p < 0.05.3. Results3.1. Performance Indices of Ross 308 and GP Chickens Stimulated In Ovo with Pro-, Pre- and SynbioticsTable 2 presents the performance indices of 42-day-old Ross 308 and GP stimulated in ovo with pro-, pre- and synbiotics. In the conducted experiment, a significant effect of in ovo stimulation with prebiotics and synbiotics on the final body weight of Ross 308 was found. In PRE, the body weight of Ross 308 was the highest (p < 0.05), and in SYN it was the lowest (p < 0.05), compared to CON. Similar relationships were found in GP, but without the significant effects of in ovo stimulation on the final body weight (p > 0.05). There was also no effect of the tested factor on feed intake (FI) and feed conversion rate (FCR) in both Ross 308 and GP chickens.3.2. Histological Parameters of Small Intestine of Ross 308 and GP Chickens Stimulated In Ovo with Pro-, Pre- and SynbioticsThe histological parameters of three segments of the small intestine (duodenum, jejunum and ileum) in 42-day-old Ross 308 and GP chickens are presented in Table 3 and Table 4. In the conduced experiment, the effects of the additives used in ovo on the villus height and width, villus area, crypt depth, muscle thickness and V/C ratio in the duodenum and jejunum of Ross 308, and in the duodenum of GP chickens, were not determined (p > 0.05). There were significant effects of the stimulation in ovo with pro-, pre- and synbiotics on the morphometric parameters in the ileum of Ross 308 and in jejunum and ileum of GP chickens (p < 0.05). 3.3. Effect of Chickens Genotype on Histological Parameters of Small IntestineBy analyzing the influence of the genotype on the histological parameters of the small intestine (Table 5), we found that the height of the villi, the area of the villi and the muscle thickness were greater in Ross 308 than in GP chickens (p < 0.05), regardless of the group (Figure 1). The intestinal villi width was greater in the duodenum in Ross 308 from the CON and PRO groups, and in the jejunum from PRE and SYN groups compared to GP chickens (p < 0.05). The V/C ratio was more favorable in the duodenum of Ross 308 from CON, PRE and SYN groups, and in jejunum of Ross 308 from CON and PRE, compared to the GP (p < 0.05). In the ileum, apart from the control group, no evidence was found for the influence of the genotype on V/C ratio in the experimental birds (p > 0.05).4. DiscussionWe conducted a histological study in two distinct genotypes of chickens stimulated in ovo with prebiotics, probiotics and synbiotics. The obtained results clearly indicated that the development of the intestinal morphology depended on the substance applied in ovo as well as on the chicken genotype. 4.1. Performance Indices of Ross 308 and GP Chickens Stimulated In Ovo with Pro-, Pre- and SynbioticsIn the conducted study, the in ovo stimulation with prebiotics significantly increased the FBW of 42-day-old Ross 308, compared to CON and SYN groups. These results correlated with the results of studies by Bogucka et al. [7] conducted on broiler chickens injected in ovo with bioactive substances. The authors found a beneficial effect of in ovo administered transgalacto-oligosaccharides on the final body weight of 35-day-old Ross 308 chickens compared to the control, inulin and synbiotic groups. On the other hand, Maiorano et al. [65] and Berrocoso et al. [66] did not find effects of raffinose family oligosaccharides (RFO) administered in ovo on the BW of 42-day-old Ross 308 and 21-day-old Cobb 500. Additionally, Miśta et al. [67] found that the in ovo injections of prebiotics did not affect broiler body weight. Other studies carried out on meat-type chickens confirmed the beneficial effect of adding prebiotics to feed on the performance indicators of broilers. Nabizadeh [14] showed that the inclusion of 1% inulin, but not 0.5%, into the diet of broilers significantly increased body weight after 42 days of rearing. Rebole et al. [68] demonstrated that a diet supplemented with inulin (at a level of 1%) positively affected the BWG of broilers. Moreover, Mookiah et al. [46] noted improvements in body weight gains in broiler chickens that were fed a diet supplemented with isomalto-oligosaccharides, compared to control broiler chickens. Xu et al. [16] observed improvements in the body weight gain of broiler chickens fed a diet with 4% of fructooligosaccharides (FOS). The higher body weight obtained in the experiment in chickens stimulated in ovo with the prebiotics can be explained by its beneficial effect on the organism. Numerous studies show that the positive effect of prebiotic substances administered in ovo or in feed is associated with the growth of beneficial bacteria, as well as a reduction in the number of potential pathogens in the intestines and the improvement of immune functions. Substances with prebiotic effects also affect the condition and functions of the digestive tract by increasing the secretion of digestive enzymes [69], thus affecting the digestibility and absorption of nutrients. In addition, volatile fatty acids, resulting from the fermentation of prebiotics, have a positive effect on the structure and functions of the small intestines [7,66,70]. 4.2. Histological Parameters of Small Intestine of Ross 308 and GP Chickens Stimulated In Ovo with Pro-, Pre- and SynbioticThe small intestine is a barrier that separates the internal environment of the body from the external environment. It is also an organ highly specialized in the digestion and absorption of nutrients. In poultry, the small intestine is relatively short, and the absorption area mainly depends on the surface area of the intestinal villi [71]. The structure of the small intestine provides important information on the health status of the digestive tract. Due to the proximity of the mucosa surface and the intestinal contents, dietary toxic substances and pathogenic bacteria may affect the condition of the intestinal mucosa, and their effect may be manifested in changes of the structure of the intestinal villi and the depth of the crypts [16]. In this study, a statistically significant increase in villi width in the jejunum in GP after the in ovo administration of probiotics resulted in the largest intestinal villi area. This may indicate a beneficial effect of probiotics on the absorption surface in the jejunum of GP chickens. It is well known that probiotics and other bioactive substances improve the morphological parameters of the intestinal mucosa and may have a beneficial effect on the absorption surface. This may be due to the competition for resources and ecological niche between lactic acid bacteria (LAB) and pathogenic microorganisms. This competition is mainly based on the ability of LAB to produce volatile fatty acids and bacteriocins that inhibit the growth of pathogenic bacteria [72]. Yang et al. [24] found a linear relationship between the amount of Bacillus and Salmonella bacteria in the cecum of chickens. Awad et al. [27] indicated that probiotics had beneficial effects on the morphology of the intestine and protected against pathogenic bacteria, but also improved the electrophysiology of the small intestine. The effect of the bioactive substances administered in ovo in the ileum of GP chickens was not clear and difficult to explain. The elongation of the intestinal villi in the SYN group did not increase their surface area. This was likely due to the fact that the in ovo administration of synbiotic did not influence the width of the villi in the third segment of the small intestine. The largest villi area was recorded in native chickens injected with prebiotics, which were also characterized by the highest body weight. Prebiotics are selectively fermentable substances that modify the composition and activities of the gastrointestinal microbiota [21]. The most common prebiotics include inulin, galacto-, fructo- and mannan-oligosaccharides, as well as raffinose-oligosaccharides [12,22,55]. These substances are fermented by commensal bacteria, and the volatile fatty acids produced in this process primarily decrease the pH of the intestinal contents [73]. One of the SCFA produced by the microbiota is the butyrate, which stimulates the growth of intestinal epithelial cells, and thus improves nutrient absorption [74]. The ratio of the crypt depths to the villi height is an indicator of the digestive potential of the gut and may indicate the maturity of the intestinal mucosa [1]; the elongation of the villi increases the area of nutrient absorption [26]. Additionally, shallower crypts may indicate a lower loss of enterocytes from the villi surface, which slows down the mitosis of cryptographic cells. In studies conducted on broiler chickens with the use of bioactive substances, Awad et al. [26,27] found that their use in bird nutrition significantly elongated the villi and increased the V/C ratio in the ileum and duodenum compared to the control group. According to the authors, this is of practical and economic importance, because shortening the intestinal villi with the simultaneous deepening of the crypts may lead to a reduction in the absorption of nutrients and in the efficiency of meat chickens. In the experiment there was no beneficial effect of bioactive substances administered in ovo at the V/C ratio in Ross 308 chickens. Therefore, when analyzing the effect of in ovo stimulation with various bioactive substances in Ross 308, it is difficult to explain a significant decrease in the height, width and surface of the villi, as well as the V/C ratio in the ileum in the group stimulated with synbiotics. The most favorable effect of the in ovo application of the bioactive substance on the V/C ratio in the jejunum and ileum of GP chickens was noted in the SYN group; although, in the case of pro- and prebiotics, their positive effect on the intestinal microstructure was also observed. This may prove the beneficial effect of the preparations on the structure of the small intestine of GP chickens. So far, most of the experiments looking at the effect of in ovo stimulation with various bioactive substances have been carried out on broiler chickens [50,53,56,65,75]. However, few studies indicated that probiotics may also positively affect the health of the small intestine mucosa in laying hens [76,77,78]. The studies of Lei et al. [77] and Xiang et al. [78] found, for example, that feeding laying hens a diet supplemented with Clostridium butyricum and a combination of Saccharomyces boulardii, Pediococcus acidilactici and B. licheniformis resulted in a favorable ratio of villi height to the depth of the crypts in the ileum and cecum. 4.3. Effect of Chickens Genotype on Histological Parameters of Small IntestineThe morphometric and morphological parameters of the gastrointestinal tract, in particular the small intestine, may be significantly influenced by the composition of the diet and the supplements used, including pro-, pre- and synbiotics [7,38,79]. Moreover, the genotype of birds is a factor determining the differences in production and physiological parameters in poultry [48]. It was shown that the deposition of pectoral muscles and their chemical composition differed depending on the genotype and sex of the bird [80]. It is well known that laying-type, dual-purpose and meat-type chickens differ significantly in terms of physiological and production parameters. This is due to the intensive selection of birds in terms of the production of eggs or meat. This results in large differences in the growth rates of the various types of poultry. The average body weight of a 6-week-old broiler chicken is 2918 g [81], while the laying-type of chicken weighs, on average, 432 g [82]. In addition, the selection of birds for specific performance traits may affect the structure and functions of the digestive tract and the function of the immune system [57]. This study showed significant differences between two distinct chicken genotypes (Ross 308 vs. GP) stimulated in ovo. The conditions in which the chickens were kept during the experiment were identical for both genotypes. It is known that Ross 308 are characterized by an intensive growth rate and high production parameters. However, the long hatching window and the lack of access to food and water may disrupt the development of the intestinal microflora due to the limited possibility of microbiota inoculation [83]. On the other hand, GP is a general-purpose chicken breed, with low nutritional and environmental requirements, characterized by a much slower growth rate and better disease resistance compared to broiler chickens [61]. These two types of chickens differ significantly in the histological parameters of the small intestine, which is likely due to their genetic background. The longer villi and the deeper crypts in Ross 308 compared to GP chickens correspond with the results obtained by Uni et al. [57]. These authors found more favorable intestinal morphological parameters in meat-type chickens compared to laying-type chickens. The differences in villi length and crypt depth between Ross 308 and GP may be a consequence of the origin of the birds. Broiler chickens and laying hens were created through intensive selection and are adapted to farm rearing, while GP, as an old, native breed, is mainly used for extensive rearing because it forages well and is resistant to disease. This form of maintenance and feeding with fodder of a lower nutritional value higher fiber content than farm fodder may result in the less intensive development of the intestinal microstructure.5. ConclusionsBased on the data obtained, it can be concluded that pre-, pro- and synbiotics administered in ovo have an impact on the production parameters and intestinal morphology in Ross 308 and GP chickens. However, it is difficult to determine which of the compounds used has the best effect on the microstructure of the small intestine. In Ross 308, the effect of pro, pre- and synbiotics is not unequivocal, while in GP chickens the largest effect, taking into account the height of the villi and the V/C ratio, was determined in the groups administered in ovo either with prebiotic or synbiotic. Taking into account the obtained results, it can be concluded that chickens with different genotypes react differently to pro-, pre- and synbiotics; therefore, the substances should be adapted to the genotype/breed.

animals : an open access journal from mdpi

10.3390/ani11051349

PMC8151840

The use of rye in poultry diets is associated with impairments in poultry performance due to the increased content of non-starch polysaccharides (NSPs). The performance of the body also depends on its health status, which can be reflected in structural changes of organs and systems. The gastrointestinal tract is the primary site of possible interaction between various nutrients, so changes in nutrition may be reflected in intestinal morphology. New hybrid rye varieties have been developed with a lower content of NSPs. Xylanase supplementation attenuates the negative effects of NSPs on poultry performance. The current study evaluated the inclusion of modern hybrid rye and xylanase supplementation on the absorptive surface of the small intestine of broilers. The results of the current study showed that the inclusion of modern hybrid rye to a corn–wheat-based diet improved the absorptive surface of the small intestine of broilers, regardless of xylanase supplementation.

The current study investigated the effects of the inclusion of modern hybrid rye (Brasetto variety) to a corn–wheat-based diet, with or without xylanase, on the absorptive surface of the small intestine of broilers. A total of 224 one-day-old male Ross 308 broiler chicks were randomly divided into four experimental groups with seven replicate cages of eight birds/replicate. A 2 × 2 factorial study design was used, with rye inclusion (0% or 20%) and xylanase supplementation (0 or 200 mg/kg of feed) as factors. Inclusion of rye increased duodenal and ileal crypt depth, villi height, the villus-to-crypt ratio and absorption surface area (p < 0.05), and ileal mucosa thickness and crypt width (p < 0.05). Xylanase supplementation attenuated the effects of rye in the duodenum and ileum and decreased the villi height and villus-to-crypt ratio in the jejunum (p < 0.05). Rye and xylanase had no effect on the spatial distribution of claudin 3 and ZO-1 protein, but xylanase supplementation reduced the amount of claudin 3 in the duodenum and jejunum (p < 0.05). The findings of this study indicate that 20% inclusion of modern hybrid rye to the diets of broilers improved the structure of the duodenum and ileum, but these effects were attenuated by xylanase supplementation.

1. IntroductionCorn is the most common energy source used in the diets of intensively reared poultry [1,2]. However, in an attempt to keep up with feed demands, alternative energy sources, including wheat, barley and rye have been investigated. Rye (Secale cereale) is resistant to fungal diseases, has a high tolerance to low temperatures, droughts and irregular soil pH and has a high production yield [3]. Nonetheless, the utilization of rye in poultry diets has been shown to impair poultry performance [4,5,6] due to its high content of non-starch polysaccharides (NSPs), mainly in the form of arabinoxylans [7]. The limited capacity of monogastric animals to digest these NSPs results in increased digesta viscosity, decreased nutrient digestibility, slowed passage of the digesta through the gastrointestinal tract and the increased incidence of wet and sticky droppings [8,9,10].To attenuate the deleterious effects of NSPs, poultry feeds are routinely supplemented with exogenous xylanases, which hydrolyze the complex carbohydrates (NSPs) present in cereal cell walls and consequently decrease digesta viscosity and release nutrients which can then be utilized by the animal [2,11]. The inclusion of xylanase to poultry diets has been shown to improve overall performance indices [2,11,12]. Strategies to improve feed efficiency are particularly important for environmental and economic sustainability, and increasing investments and efforts are made to decrease antinutritional factors in cereal varieties. Recently, new hybrid varieties of rye have been developed, with a lower NSP content, specifically with regards to that of arabinoxylans [13,14,15]. Conflicting results with regards to the effect of xylanase supplementation on the intestinal histomorphometry of broiler chickens, specifically with regards to goblet cell number, villous height and crypt depth, have been observed [16,17]. Thus, it was hypothesized that more complex structural indices combined with epithelial integrity traits would indicate the true effect of modern hybrid rye on broiler small intestine. Therefore, the objective of the current study was to investigate the effects of the inclusion of hybrid rye (of the Brasetto variety) in corn–wheat-based diets, with or without xylanase supplementation, on the intestinal absorptive surface through the assessment of intestinal histomorphometry and the expression and quantification of tight junction proteins (zonula occludens and claudin-3) in broiler chickens.2. Materials and Methods2.1. Birds and Experimental DietsThis study is part of a larger project on modern cereals in poultry and livestock farming. It provides new and additional data and analysis from the perspective of structural changes in the broiler digestive system from previously reported studies [1,11]. A completely randomized study on 224 one-day-old male Ross 308 broiler chicks was performed at the Experimental Station of the National Research Institute of Animal Production in Balice, Poland. Chicks, with an average weight of 41 g, were randomly divided into four experimental treatments, with seven replicate cages of eight birds/replicate. A 2 × 2 factorial study design was used, with rye inclusion (0% or 20%) and xylanase supplementation (0 or 200 mg/kg of feed) as factors. The birds were kept in a wire-floored battery cage with 7800 cm2 total floor space in the cage (length × width × height = 120 × 65 × 50 cm), in an environmentally controlled room in the poultry house. During the study, the temperature in the experimental facility was maintained from 32 °C at 1 d of age to 21 °C at 21 d of age and later, relative humidity cycled from 50 to 60%, air exchange was 1 m3/1 kg of body weight gain (BWG)/1 h and concentrations of CO2 and NH3 were maintained below 2000 and 20 ppm, respectively. Each cage was equipped with four nipple-cup drinkers and a trough feeder along the front of the cage. Water and feed were supplied ad libitum. Feed was supplemented ad libitum as follows: starter (1 to 21 D), and grower-finisher (22 to 42 D) feeding phases. Table 1 presents the composition and calculated nutrient contents of the experimental diets.Nutrient requirements of broilers were met or exceeded, according to poultry feeding standards, by formulated isonitrogenous and isoenergetic experimental diets [19]. The content of non-starch polysaccharides divided into soluble and insoluble fractions along with the content of arabinoxylans in rye grain and experimental diets was analyzed by gas chromatography as previously described [20] (Table 2).The hybrid winter rye cv. Brasetto was used as a partial replacement of corn in the diet. In order to improve the high NSP levels within the rye, xylanase Ronozyme WX (DSM Nutritional Products Sp. z o.o., Mszczonów, Poland) with a minimum xylanase activity of 1000 FXU/g was used as a supplement to the diet. As previously stated, the chemical composition of the rye grain included 9.23% of crude protein, 0.81% of crude fat, 1.46% of crude fiber and 1.51% of crude ash [11].2.2. Tissue Collection and Histomorphometry AnalysisAt the end of the experiment, birds were weighed, and seven representative birds (one per replicate cage with body weight close to the average) from each group were slaughtered by decapitation after electrical stunning, following a 12-h fast. During the necropsy, two 20-mm-long samples of the duodenum (2 cm after stomach), jejunum (from 50% of intestine length) and ileum (2 cm before ceca), were collected and processed for standard histology, as previously described [21,22]. The segments were gently cut open longitudinally along the mesentery line and placed flat (not stretched) into a standard histological cassette. Tissues were fixed in 4% buffered formaldehyde (pH 7.0) for 24 h, washed in tap water and dehydrated in graded ethanol solutions. Samples were further trimmed to achieve four transverse macro sections (4 × 8 mm in dimensions, transverse section on the longer side) from each individual and were processed in Ottix Plus and Ottix Shaper (DiaPath, Martinengo, Italy) with the use of a tissue processor (STP 120, Thermo Scientific, Waltham, MA, USA) to saturate the samples in paraffin. Paraffin blocks were then made using an embedding station (MYR EC-350, Casa Álvarez Material Científico S.A. Madrid, Spain). Two paraffin blocks were made for each chicken for each intestinal segment, containing two 4 × 8 mm tissue samples. The tissue samples were placed to represent sections at successive depths of the intestine 4 mm apart. Twenty cross sections (with 20 µm intervals between each five-slice section), 5 µm thick, were then cut with a microtome (Microm HM 360, Microm, Walldorf, Germany) from each small intestine paraffin block (80 cross sections/bird/intestine segment in total). The samples were stained with Goldner’s trichrome, and bright field microscopic (two-dimensional) images (magnification ×100, ×200) were taken with a confocal microscope (AXIOVERT 200 M, Carl Zeiss, Jena, Germany) and a color digital camera (AxioCam HRc, Carl Zeiss, Jena, Germany) for histology and histomorphometry analysis of intestine structures [21,22,23]. For each bird and intestinal segment, 20 cross-sections were selected that showed the course of the sampled intestinal segment and did not duplicate the assessed structures with respect to villi and crypts. Twelve measurements of each parameter evaluated were taken at each of the selected cross sections. The multiple measurements were averaged. The following parameters were analyzed: the thickness of the serous membrane (magnification ×200), the outer and inner muscle layer (magnification ×100), the submucosa, the lamina muscularis mucosa (magnification ×200) and the mucosa; crypt width (measured in the middle of the crypt depth) and depth (defined as the depth of the invagination between adjacent villi, from the bottom of the crypt to the base of the villus), as well as villi height (from the tip of the villus to the villus–crypt junction) and width (measured in the middle of the villus height) (magnification 100); (measurements were taken on villi and crypt assemblies) absorption surface area and villus length-to-crypt depth ratio, as previously described [22,24]. Histomorphometry measurements were made with the use of graphic analysis software (ImageJ 1.53, National Institutes of Health, Bethesda, MD, USA; available at: http://rsb.info.nih.gov/ij/index.html, accessed on 20 November 2020).2.3. ImmunohistochemistryImmunohistochemical staining was performed on the tissue slices according to a previously described protocol [25]. Heat-induced epitope retrieval was performed using a pressure cooker Rapid Cook (Morphy Richards, Swinton, UK) in sodium citrate buffer (10 mM sodium citrate, 0.05% Tween 20, pH 6.0). Endogenous peroxidase activity was blocked subsequently with a 3% solution of hydrogen peroxide in deionized water for 5 min. After blocking for 30 min in normal serum, sections were incubated with the first antibodies over-night at 4 °C. Two types of antibodies were used to mark tight junctions: rabbit polyclonal anti-ZO-1 (zonula occludens -1) antibody (orb11587, Biorbyt, St. Louis, MO, USA, dilution 1:500) and rabbit polyclonal anti-claudin 3 antibody (AB15102; Abcam, Cambridge, UK, dilution 1:100). The sections were then incubated for 30 min with the second antibody (peroxidase conjugated goat anti-rabbit, #611-1302, Rockland Immunochemicals, Inc. Limerick, PA, USA, dilution 1:500). Negative control sections for each antibody were obtained by identical immunohistochemical staining, excluding the primary antibody. The sections were then developed in 3,3′-diaminobenzidine tetrahydrochloride (DAB D5905; Sigma–Aldrich, St. Louis, MO, USA) used as chromogens, for 15 min at room temperature. Counterstaining was performed with Mayer’s hematoxylin (MHS32-1L; Sigma–Aldrich, St. Louis, MO, USA).2.4. Western-Blot AnalysisTissue samples obtained from relevant segments, adjacent to the above-mentioned histology samples of the small intestine, were collected from every sacrificed chicken and were used for Western-Blot analysis, as previously described previously [26]. Briefly, the samples were homogenized in lysis buffer (125 mM TRIS-HCl pH 6.8; 4% SDS; 10% glycerol; 100 mM DTT), boiled in a water bath for 10 min and centrifuged at 10,000× g for 10 min. The supernatant was then collected. The protein content was determined using the Bradford method [27]. Samples containing 80 µg of protein were separated by 10% SDS-PAGE and then electroblotted onto an Immobilon P membrane (Sigma–Aldrich, St. Louis, MO, USA). The membranes were blocked with 3% low fat milk in PBS for 1 h, after the transfer, and incubated overnight with polyclonal rabbit anti-ZO-1 (orb11587, Biorbyt, St. Louis, MO, USA, dilution 1:1000) or rabbit polyclonal anti-claudin 3 (AB15102; Abcam, Cambridge, UK, dilution 1:1000) antibodies. The membranes were washed three times for 10 min with PBS containing 0.05% TRITON X-100 (Sigma–Aldrich, St. Louis, MO, USA) and incubated for 2 h in the presence of the second antibody (peroxidase conjugated goat anti-rabbit, #611-1302, Rockland Immunochemicals, Inc. Limerick, Pennsylvania, USA, dilution 1:10,000). The membranes were visualized with 3,3′-diaminobenzidine tetrahydrochloride (DAB D5905; Sigma–Aldrich, St. Louis, MO, USA). An anti-β-actin antibody (1:2000, Sigma–Aldrich, St. Louis, MO, USA) was used as the loading control. Densitometry analysis of the obtained blots was performed. A gel analysis function was used to determine the rows of blots and to determine the optimal level of background correction. A background subtraction function was then applied to compensate for background irregularities using the rolling ball algorithm built into ImageJ [28] to determine appropriate thresholds for blot marking. The blots were densitometrically quantified and normalized to their corresponding β-actin bands. Western-Blot analysis was performed in triplicate, and an average for each bird was used for statistical analysis.2.5. Statistical AnalysisData were analyzed by 2-way ANOVA using the GLM procedure of Statistica 13 (TIBCO Software Inc., Palo Alto, CA, USA). The model included diet and xylanase and their interactions as fixed effects. The replicate cage served as the experimental unit. Tukey’s test was used to compare differences between means when the model was declared significant (p < 0.05). The sample size was calculated for a two-sided test with an α of 0.05 and power at 0.8, with an effect size of 1.63 [29].3. Results3.1. Changes in Intestinal HistomorphometryResults from the histomorphometry measurements are presented in Table 3, Table 4 and Table 5. The morphology of the duodenum, jejunum and ileum was affected by both the rye inclusion and xylanase supplementation. Significant main effects of both additives, as well as their interactions, were noted.Duodenal histomorphometry parameters are presented in Table 3. The crypt depth (17, 11 and 9%, all p < 0.001) and villi height (25, 20 and 12%, all p < 0.001) were increased in the duodenum of broilers receiving rye or xylanase, or both rye and xylanase in the feed compared to broilers not receiving rye and xylanase in the feed. In both cases, the effect of rye was attenuated by xylanase (p < 0.001). The absorption surface area was increased in broilers receiving rye in the feed (29%, p < 0.001), and the villus-to-crypt ratio was increased in broilers receiving rye (7%, p = 0.025) or xylanase (8%, p = 0.005) compared to birds without rye and xylanase in the feed. In addition, in both these cases, the effect of rye was attenuated by xylanase, especially with respect to the villi-to-crypt ratio (p < 0.001). There was no response to feed with rye or xylanase or both in other parameters such as serous membrane thickness, outer and inner muscle layer, submucosa and lamina muscularis mucosa (p > 0.05). It was shown that the main effect of the addition of xylanase was a reduction in the thickness of the inner muscle layer by 16%. Although there were no differences between groups for serosa and mucosa thickness, the interaction of rye and xylanase still existed, and xylanase attenuated the response of these individual duodenal elements to rye in the feed (Table 3).Jejunal histomorphometry parameters are presented in Table 4. Feed containing rye and xylanase caused 6% (p < 0.001) increase of jejunal crypts depth compared to groups with xylanase supplementation and without rye or xylanase, whereas 9% (p < 0.001) increase compared to broilers receiving 20% rye in the feed. Unlike in the duodenum, xylanase stimulated deepening of crypts when the feed contained rye. Villi height and villus-to-crypt ratio were reduced by 10% (p = 0.002) and 15% (p < 0.001), respectively, in broilers receiving rye and xylanase in the feed compared to broilers without rye or xylanase in the feed. The observed effect could be strengthened by additive impact of rye, since these two parameters were lowered by 4 and 5% respectively by the main effect of rye content (Table 4). The addition of xylanase caused reduction of villi height by 7% (p = 0.026), villi width by 20% (p = 0.019) and villus-to-crypt ratio by 7% (p = 0.009), compared to broilers without xylanase in the feed. The thickness of the serosa, outer and inner muscle layer, submucosa, lamina muscularis mucosa, mucosa, crypt width and absorption surface area were not affected by the rye inclusion or xylanase supplementation (p > 0.05). Although rye, xylanase, or both had no effect on mucosal thickness compared with broilers not receiving rye or xylanase in the feed, xylanase exacerbated the lowering effect of rye.Ileal histomorphometry parameters are presented in Table 5. The feed with 20% rye inclusion increased the thickness of the mucosa by 10% (p = 0.004), crypt width by 10% (p < 0.001) and depth by 19% (p < 0.001), villi height by 13% (p < 0.001), absorption surface area by 23% (p < 0.001) and villous to crypt ratio by 12% (p = 0.020) compared to feed without this cereal and xylanase. Xylanase addition to the broiler feed increased the crypt depth by 19% (p < 0.001), villi height by 13% (p = 0.006) and villi width by 15% (p = 0.009) compared to the broilers receiving feed without rye and xylanase. The effect of rye inclusion depended on the addition of xylanase in all the parameters mentioned above. Xylanase attenuated the response of the intestinal mucosa, crypts and intestinal villi to rye. The absorption surface area and the ratio of villous to crypt, increased by rye inclusion, were also decreased by xylanase. Only crypt depth was stimulated by both rye inclusion and xylanase addition but without additive effect in broilers receiving rye and xylanase in the feed (Table 5). No changes were noted in the thickness of the serous membrane, outer and inner muscle layers, submucosa and the lamina muscularis mucosa.3.2. Epithelial Barrier Characteristics (Expression and Quantification of Tight Junction Proteins)The immunolocalization of claudin 3 and ZO-1 (zonula occludens tight junction protein-1) proteins was performed through microscopic observation of the small intestine epithelium of the duodenum, jejunum and ileum. The immune reactions with antibodies in the examined parts of the digestive tract were similar in all treatment groups. The reactions were continuous, and claudin 3 and ZO-1 were observed on the pericellular borders of the epithelium cells. The spatial distribution of the proteins assessed was normal, without any signs of epithelial gaps. There were no visible differences in the intensity of protein expression between treatment groups (Figure 1).Quantitative analysis of claudin 3 and ZO-1 proteins was performed using the Western-Blot method. Although there were no visible differences in the immunohistochemistry microscopic assessment, the quantitative analysis of the ZO-1 amount showed a decrease in the jejunum of broilers receiving xylanase (0.70) compared to broilers receiving feed with rye and xylanase (0.76) (p = 0.037), feed with rye (0.79) (p = 0.002) and feed without rye and xylanase (0.80) (p = 0.002) (Figure 2c). Rye normalized ZO-1 protein levels when broilers were fed with xylanase (p = 0.036). Lower amounts of the ZO-1 protein were also detected in the duodenal epithelium of broilers receiving xylanase in the feed (0.78) compared to broilers receiving feed with 20% rye inclusion (0.84) (p = 0.028) (Figure 2b). The amount of claudin 3 was significantly decreased in the duodenum of broilers receiving xylanase (0.82) and rye with xylanase (0.84) compared to the broilers receiving no rye and xylanase in the feed (0.96) and rye inclusion (0.97) (p < 0.001 for both comparisons) (Figure 2b). The amount of claudin 3 was higher in the jejunum of broilers receiving rye (1.19) in the feed compared to those receiving xylanase (1.06) (p = 0.010) and broilers receiving rye and xylanase (1.04) (p = 0.004) (Figure 2c). There were no differences between groups in the amounts of ZO-1 and claudin 3 in the ileum (Figure 2d).4. DiscussionThe current study investigated the effects of the inclusion of modern hybrid rye (Brasetto variety) to a corn–wheat-based diet, with or without xylanase supplementation, on the absorptive surface of the small intestine of broilers. Previous studies have shown that the use of rye, as an alternative energy source in poultry diets, has been associated with increased viscosity of the digesta and thus poor digestibility and absorption of ingested nutrients [8]. Rye inclusion, as well as xylanase supplementation, has yielded conflicting results with regards to their effects on the absorptive surface of the small intestine.The small intestine is the primary site for the digestion and absorption of ingested nutrients [30,31]. Some of the digestive processes and all of the absorptive processes take place in or around the intestinal villi and crypts [32]. In fact, villus height and crypt depth, along with villus height-to-crypt depth ratio and villi absorptive surface area, are key parameters which define the functional capacity of the small intestine [33,34,35]. Various dietary nutrients or additives have been shown to affect the morphology of the intestinal mucosa, and in doing so affect overall nutrient metabolism [33,36,37].In the current study, both the rye inclusion and the xylanase supplementation affected the morphology of the small intestine, with the duodenum and ileum being the most affected, followed by the jejunum. The inclusion of rye in the broiler’s diet had a more pronounced effect on the small intestine histomorphometry compared to the xylanase supplementation. The enzyme-induced changes in villus height, crypt depth and villus height-to-crypt depth ratio in the small intestine of broilers in the current study contrast with previous findings. Wu et al. (2004) observed no significant effects of xylanase supplementation on the villus height, crypt depth and villus height-to-crypt depth ratio in the duodenum, jejunum and ileum of broilers fed a wheat-based diet supplemented with xylanase (1000 XU/kg diet), for a period of 21 days [17]. Similarly, Yan et al. (2017) also observed no significant changes in mid-duodenal and mid-gut villus height, crypt depth and villus height-to-crypt depth ratio following 21 days of carbohydrase supplementation (at 0.05%, providing 1000 units of 1,4-β-xylanase per kg diet) to broilers fed a basal diet containing rye, soft wheat, soybean meal and feather meal [4]. Pekel, Horn and Adeola (2017) also observed no effects of xylanase supplementation (800 xylanase units/kg of diet) on the jejunum villus height or crypt depth of broilers fed corn–soybean meal-based diets for a period of 14 days [38]. Souza et al. (2014) observed a decrease in the crypt depth and an increase in the villus height-to-crypt depth ratio in the ileum of laying hens fed diets based on corn and soybean meal, following xylanase supplementation (100 g/tonne of feed, equivalent to an enzyme activity of 16,000 BXU/kg) for a period of four weeks [39]. Since villus morphology is dependent on the enteral absorption of nutrients, the inconsistencies among previous studies involving exogenous enzyme supplementation may be due to the type of cereal used in the diets or the overall diet composition [38,40].Dietary inclusion of the new hybrid Brasetto rye significantly increased the duodenal crypt depth and villus-to-crypt ratio, as well as the ileal villus height of the broilers in the current study. Since the duodenum is the primary site for nutrient absorption [30], it would be expected that this section of the small intestine of broilers in the current study was the most affected in terms of the diet-induced changes in intestinal morphology. An increase in villus height or in villus height-to-crypt depth ratio has been associated with improved nutrient digestion and absorption [41,42]. The increased villus-to-crypt ratio and increased villus height observed in the duodenum and ileum of broilers in the current study, following dietary rye inclusion, could possibly suggest an improvement in the overall capacity of the small intestine for the digestion and absorption of ingested nutrients, as confirmed by the significant increase in the duodenal and illeal absorption surface area. As the base of the crypts is constantly dividing to maintain the structure of the villi, an increase in crypt depth would produce more developed villi [43]. The increase in the duodenal and illeal crypt depth of broilers in the current study could be indicative of increased proliferative activity within the intestinal mucosa in the region of the villi, allowing for renewal of the villi if necessary and ensuring improved digestion and absorption of the nutrients consumed [44,45]. Previous studies have yielded varying results with regards to the effects of dietary rye inclusion on intestinal morphometry, which were mostly dependent on the section of small intestine examined and the composition of the basal diet to which the rye was added. El-Wahab et al. (2020) observed no significant changes in ileal morphological parameters of broilers following the addition of increasing amounts of rye (5%, 10%, 20%, 30%) to a wheat and soybean meal-based control diet [46]. Van Krimpen, Torki and Schokker, (2017) observed an increase in villus height and crypt depth in the jejunum of broilers following the inclusion of 5% or 10% rye to a maize-based diet from d 14 to 28 [5].In addition to its digestive and absorptive functions, the intestine also serves as an important barrier against the external environment, preventing the entry of toxins, food antigens and harmful microorganisms [47,48,49]. Intestinal barrier dysfunction is associated with altered tight junction protein expression [50]. Tight junctions are the connections between enterocytes which are responsible for regulating the paracellular diffusion of solutes and ions across the intestinal epithelium [51,52]. To assess the integrity of the intestinal barrier of broilers in the current study, the expression of tight junction proteins, claudin-3 and zonula occludens-1, was assessed. The immune reactions with antibodies against the tight junction proteins in the examined parts of the tract were not affected by rye inclusion or xylanase supplementation, with the spatial distribution of the proteins being evenly distributed thorough epithelium and no signs of any gaps within the epithelium barrier. There were also no visible differences in the intensity of protein expression between treatment groups. With regards to the quantitative analysis of claudin-3 and zonula occludens-1, neither the rye inclusion nor the xylanase supplementation had a significant effect on the amount of claudin-3 and zonula occludens-1 in the ileum of broilers in the current study. However, xylanase supplementation decreased the amount of zonula occludens-1 protein in both the duodenum and jejunum and the amount of claudin-3 protein in the duodenum. These findings are not in agreement with previous studies involving the use of xylanase in pigs, which, like birds, are monogastric animals. He et al. (2020) observed increased gene expression of zonula occludens-1 in the intestine of piglets supplemented with 30 or 60 mg/kg (24,000, 48,000 BXU/kg, respectively) of xylanase for 28 days [53]. Tiwari et al. (2018) also observed increased concentrations of claudin, occludin and zonula occludens-1 in the jejunum of piglets fed a corn–soybean meal-based diet supplemented with xylanase (1500 endo-pentosanase unit of xylanase/kg of the diet) [54]. Thus, the effects of xylanase supplementation on tight junction protein expression and in turn the intestinal barrier function need to be further explored.The present study has its limitation since physiological approach and methods would provide greater insight into the effects of rye and xylanase on the nutrient absorption rate and would allow the establishment of intestinal barrier permeability and integrity in the context of rye and xylanase interaction. It would also be an interesting approach to study different doses of xylanase considering the present findings on the effects of the rye and xylanase interaction. The present study, however, indicates that modern hybrid rye varieties can be used as a safe energy source for broiler chickens from the standpoint of structural integrity and small intestinal function.5. ConclusionsIn conclusion, the current study showed that modern hybrid rye of the Brasetto variety can be added to corn–wheat-based diets for broiler chickens, as an alternative energy source, without adversely affecting small intestine morphology. In fact, dietary rye inclusion has the potential to improve the structure of the small intestine and to increase the absorptive surface, but xylanase supplementation attenuates these effects.

animals : an open access journal from mdpi

10.3390/ani13101577

PMC10215249

The science of animal welfare can be approached along a continuum of perspectives. Historically, we considered animal welfare at a distance, through a big-picture examination of population-level parameters (e.g., longevity, reproductive success). In recent decades, scientists and practitioners have advanced the field and optimized animal welfare by incorporating a focused approach examining each individual (e.g., their lived experiences). Population-level welfare evaluations are key to validating parameters used to measure individual animal welfare and have an important role when individual animal welfare cannot be easily measured. However, there are also situations in which individual and population welfare may be in conflict, and managers must consider maximizing population welfare at the expense of individuals. We examine these cases and explore opportunities for the integration of individual and population-level welfare to promote optimal well-being for animals in zoos and aquariums.

Over the last 50 years, animal welfare science has advanced dramatically, especially in zoos and aquariums. A shifting focus from population-level welfare parameters such as reproductive success and longevity (macroscopic, big-picture concepts) to the subjective experience of individual animals (microscopic, focused concepts) has led to more effective animal welfare assessments and improvements in animal welfare. The interplay between individual animal and population welfare for captive animals is critical to the way zoos and aquariums operate to realize their welfare and conservation missions, especially when these missions conflict with one another. In this report, we explore the intersection of individual animal and population welfare in zoos and aquariums and how these two concepts may support one another or be in conflict.

1. IntroductionSocietal interest in animal welfare is not new and has guided the humane care of animals across the spectrum of settings in which humans use animals, from food production to exhibition for educational and recreational purposes. Furthermore, this interest has spurred animal caregivers to scientifically evaluate the welfare implications of those practices. Historically, reproductive success measured at the population level, often in terms of production (e.g., numbers of offspring, gallons of milk, dozens of eggs, pounds of meat, etc.), was considered the key indicator of animal welfare [1]. When populations were producing large numbers of offspring, the welfare of the individuals within that population was assumed to be good. Over time, this emphasis on population-based outputs led to intensive “factory farm” practices in which the welfare of individual animals was not routinely evaluated. Ruth Harrison’s 1964 exposé Animal Machines revealed the reality of these practices and demonstrated that population-based parameters do not adequately protect animal welfare [2]. The subsequent public outcry prompted the formation of what became known as the Brambell Committee and the basis of the modern approach to animal welfare, the Five Freedoms [3]. The Five Freedoms’ assertion that welfare includes the physical and mental states of an individual animal and that animals have a right to specific minimal levels of care was revolutionary at the time [4]. Over the last 50 years since the Brambell Committee, the field of animal welfare science has advanced dramatically from merely measuring population-level production parameters to minimize negative welfare to closely evaluating how an individual animal responds to its environment to promote positive welfare [5]. This shift has been accompanied by accrediting bodies articulating animal welfare principles and guidelines for zoos and aquariums centered on individual animals. The World Association of Zoos and Aquariums (WAZA) defines animal welfare as “a state that is specific for every individual animal; is how the animal experiences its own world and life through its association with pleasant experiences specific for that species such as vitality, affection, safety and excitement, or unpleasant experiences such as pain, hunger, fear, boredom, loneliness, and frustration” [6]. From this definition and building on the Five Freedoms, the Five Domains have emerged as a more recent model to assess animal welfare with particular emphasis on preventing compromises in welfare across a wide range of general needs animals have [7]. This modern concept of animal welfare represents a microscopic approach, focusing on the experiences of individual animals, not just groups or populations of animals residing at a facility, and using animal-based indicators such as behavior to assess welfare. With this definition, zoos and aquariums have made a concerted effort to optimize animal welfare through research, advanced veterinary care, exhibit modifications, behavioral management and enrichment programs, staff education and training, and other approaches [8,9]. Despite this focus on individual animal welfare, zoos and aquariums are responsible for managing populations of animals, both within their own institutions and throughout the larger community of accredited facilities around the world. Accordingly, caregivers must also consider population welfare when making animal management decisions.Although welfare measured at the population level historically focused on production outputs without necessarily understanding the cost to the individuals, a modern concept of population welfare that specifically accounts for the well-being of individuals within that population has recently emerged. In contrast to WAZA’s definition of animal welfare focused on individual animals, population welfare has been defined as “coherence between the adapted needs of a species with critical social and environmental resources” [10]. In other words, population welfare is connected to species conservation and population health and maximized through ensuring a species has an environment that matches its needs. It represents a macroscopic approach to animal welfare centered on the optimal environment for a population to thrive. ‘Group welfare’ has become a widely used term within zoos and aquariums. For the purposes of this paper, we use the term population welfare to refer to the collective physical, behavioral, and psychological well-being of groups of animals housed within zoos and aquariums as well as welfare assessments that utilize indicators measured at the group rather than the individual level. For example, population welfare may refer to the well-being of large groups of fish and invertebrates in aquarium exhibits, antelope housed in extensive enclosures, or other highly social, inter-dependent species such as nonhuman primates, meerkats, and others, and considers the shared needs of the individuals as a species or a group. In this way, population welfare correlates with the conservation mission of accredited zoos and aquariums as they seek to maintain healthy populations of various species not only for continued public display but also as a hedge against extinction in the wild. However, this conservation mission can result in competing interests when individual and population welfare goals are not aligned.The dichotomy of individual (microscopic) and population (macroscopic) approaches to animal welfare has been an important challenge in formulating meaningful welfare assessments. The critical components of an animal’s welfare, their lived experiences (e.g., the Five Domains, the Five Opportunities to Thrive; see [4,11], respectively), are nested within the big-picture concepts typically used in population welfare assessments (Figure 1). As we will discuss, such big-picture concepts can be applied to individuals to gather information about their welfare, but the fine detail of individual animal welfare assessments is difficult to apply to large populations without significant resources. This difficulty has prevented the application of welfare methods developed for individual animals in zoos and aquariums to not only wild animals in their natural habitats (i.e., in situ environments) but also to members of some species within zoos and aquariums. The experimental nature of many approaches to animal welfare assessments, such as intervention effectiveness testing, makes those approaches inherently challenging to apply in environments where animal intervention is avoided and/or confounding variables cannot always be controlled. Subsequently, in situ animal welfare research tends to be theoretical in nature [12]. While the welfare of wild animals is certainly connected to conservation and the mission of the zoos and aquariums, an examination of the population welfare of wildlife is beyond the scope of this paper. The interplay between individual animal and population welfare for captive animals is critical to the way zoos and aquariums operate to realize both their welfare and conservation missions. In this report, we explore the intersection of individual animal and population welfare in zoos and aquariums and how these two concepts may support one another or be in conflict. 2. Synthesizing Individual and Population WelfareZoos and aquariums have always informally assessed the welfare of the animals in their care. Typically, a population-level approach was integrated into the perception of the welfare of individual animals. Early attempts at more formal welfare assessments often focused on “inputs,” or animal care and husbandry parameters provided to the animals. This approach assumed that animals given an optimal environment would experience optimal welfare. Indeed, the development and promulgation of standards at the species (or higher) level has been one important method to systematically improve animal welfare in zoos and aquariums. To this end, the Association of Zoos and Aquariums (AZA)’s Animal Welfare Committee participates in the production of Animal Care Manuals that establish best practices in many areas of animal care and management, including environmental parameters, exhibit design, transport, social environment, nutrition, veterinary care, reproduction, behavior management, and research with the goal of maximizing “welfare potential”, the potential that individual animals will experience good welfare based on the care they receive [9]. Similarly, the European Association of Zoos and Aquariums (EAZA) produces best practice guidelines through various Taxon Advisory Groups with the goal of merging expert husbandry knowledge and making it widely available [13]. While these guidelines correctly recognize that environmental and population-level factors (i.e., inputs) have a large impact on individual animal welfare, such best practices assume all individuals of a species or taxon share a core set of common needs. Yet, because animal welfare is fine-tuned at an individual level, adherence to these guidelines does not guarantee that all individuals within that population will have good or even adequate welfare. The growing study of individual differences highlights the impact of personality and lived experiences on an animal’s ability to cope and thrive within a given environment, from groups of gorillas responding differently to varying visitor volume [14] to the influence rank has on the enriching effect of training sessions [15] and the importance of challenges being appropriate for an individual’s ability and resources [16]. These studies highlight the idea that individuals in the same setting can and do experience various states of welfare, and consideration of individual differences within a group is critical to understanding the welfare of each individual.Beyond the fact that not all individuals of a species have identical needs, the lack of species-specific information regarding the physical and psychological needs of many species is an additional challenge to the use of taxon-level guidelines [17]. Despite the increased interest in animal welfare across all species and the promotion of animal welfare research over the last two decades, there remains a paucity of validated welfare indicators for many species maintained by zoos and aquariums. This is especially true in understudied taxa such as reptiles, amphibians, fish, and invertebrates whose specific natural histories and physical, nutritional, social, and psychological needs may not even be well understood. To address this issue, accredited zoos and aquariums have prioritized research utilizing the individuals in their care to characterize animal needs and key indicators of welfare (e.g., [18]). For these species, welfare assessments may be performed macroscopically simply because we lack the knowledge of individual welfare needs to accurately assess it at the individual level at this time.Although a critical need, identifying and validating physiological, behavioral, and psychological indicators associated with animal welfare requires significant time and expense. As discussed, animal welfare research in zoos and aquariums is particularly challenging because strict experimental conditions cannot be imposed in most real-world situations, and the relatively small numbers of each species housed at a single facility means that generalizing any findings to individuals at other facilities is complicated. For this reason, studies which combine macro- and micro-level approaches to welfare assessments can be critical to identifying and validating welfare indicators, thus improving individual animal welfare. Furthermore, the multidimensional nature of animal welfare requires assessments to be based on scientific knowledge that considers many aspects including provision of resources, caregiver interactions, positive and negative affective states and events, behavior, and others [19]. To this end, the Elephant Welfare Initiative (EWI) was a large-scale, epidemiologic study of elephants in North American zoos that documented the prevalence of positive and negative welfare states in individuals across facilities. The study included nearly 300 elephants in over 70 facilities and used a population-level, epidemiologic approach to determine the environmental, management, and husbandry factors that could impact elephant welfare [20]. The EWI’s population-level findings could then be used to assess and improve the welfare of individual animals. For example, the size of an elephant exhibit was a common resource-based, input-type metric used to assess elephant welfare prior to the EWI. However, the EWI found that exhibit size was not correlated with better foot or musculoskeletal health [21] or reduced stereotypic behaviors [22], which are commonly accepted animal-based indicators of elephant welfare. In other words, results from the EWI suggested that moving an individual elephant displaying stereotypical behavior to a larger exhibit would, as a single intervention, be unlikely to improve that animal’s welfare. In contrast, the EWI found that spending more time in larger social groups was protective against performance of stereotypic behaviors [22], providing caregivers with a science-based intervention to improve the welfare of an elephant displaying stereotypic behavior. As essentially a large-scale epidemiology study, the major challenge of the EWI was that it was limited to elephant care facilities as they existed at the time, thus specific hypotheses for improving elephant welfare could not be tested. Despite this limitation, the EWI generated hypotheses for targeted research to test interventions likely to improve welfare in the future. Given the challenges of studying animal welfare with the small populations and multiple confounding variables found in zoos and aquariums, this type of evidence-based approach is not possible without population-level welfare research.Population-level welfare research of this nature may be even more impactful assisting zoos and aquariums in selecting species most likely to experience good welfare in captivity. The importance of selecting species well adapted for an institution’s physical and climatic environment has been discussed for decades [23]. However, considering specific information regarding the needs of individuals of different species housed in captivity could lead to a paradigm shift in approaches to collection plans, facilities’ master plans, and day-to-day operations of zoos and aquariums. For example, using species-typical behaviors, such as natural hunting behavior, general activity levels, ranging, and territorial patrolling, Clubb and Mason [24] were able to explain the signs of poor welfare (e.g., abnormal behavior and poor reproductive success) seen in some captive carnivore species but not others. As a result, Clubb and Mason make recommendations regarding species likely to experience good welfare in zoos and aquariums and species that should perhaps be avoided [24]. At the very least, population-level research can inform enclosure designs to facilitate improved welfare for the animals who will live there. More importantly, this approach to animal welfare science relies on identifying trends which will likely yield the best results for individuals of a given species or taxon group, but recognizes that individual assessments, when possible, are still critical to forming a complete picture of animal’s welfare in a given setting. While population-level research can guide welfare assessments of individuals, population-level approaches cannot replace animal-based assessments at the micro level, which require direct observation of individual animals [25]. To capture individual differences in the subjective experience of welfare, assessors must have knowledge of and familiarity with an animal as an individual. At the most basic level, this familiarity requires the ability to individually identify each animal in the group. However, in large aquariums in which potentially hundreds of individuals of the same species, with a nearly identical appearance, live in the same enclosure, it may not be possible for caregivers to identify animals as individuals, much less be familiar with an individual’s behavior and temperament, in order to assess the welfare of individuals. A similar challenge might exist in an expansive enclosure in which large herds of hoof stock are managed more extensively than traditional zoos. It is important to note that animals that can be individually identified, even when housed in large enclosures or large groups, should be assessed individually whenever possible. Facilities may choose to house animals in these more naturalistic ways to improve welfare, but these settings complicate individual animal welfare assessments and can make population-level assessments more appropriate. How can facilities meaningfully assess welfare in these situations? Group observations make welfare assessments possible when individual assessments are not feasible, and they are effective when used appropriately [26,27]. Even in population-level analyses, the behavior of the individual is important to note as an individual that looks or acts differently is a potential indicator of a welfare concern. These outliers should be a stimulus for investigation and may warrant subsequent individual assessments to determine the cause of the outlier behavior and the possible welfare implications, for both the individual and the group. For many of the species in which group observations may be utilized, the welfare of each individual is so intricately tied to the other animals in the group that population-level welfare assessments may be more meaningful than individual assessments. For example, many fish spend their whole life as a member of a group, and this schooling behavior has extremely high biological significance affecting a wide variety of adaptive functions [28]. Aquatic invertebrates such as coral and bryozoans that form colonies and are literally connected to other members of the group represent another important example. More complex examples involve animals such as some sea anemones that reproduce by budding and species such as the Mexican topminnow (Poeciliopsis 2 monacha-lucida), an all-female fish species that reproduces by cloning with all individuals sharing the same genotype for thousands of years [29]. Population-level indices such as fecundity, adult survivorship, neonatal mortality, and stereotypic behavior may be useful to indicate general issues with animal well-being within these groups that require further characterization at the individual level. However, it is important to recognize that these parameters are largely affected by negative welfare states. In other words, animals experiencing negative welfare are, in general, likely to have decreased fecundity and adult survivorship and increased neonatal mortality and stereotypic behaviors [30]. Animals experiencing a minimum level of welfare are likely to be free from diseases, injury, and malnutrition, and these factors clearly affect longevity, reproductive success, and other parameters that can be assessed at the population level [17]. A more forward-looking approach to welfare assessments focused on optimization of welfare incorporates indicators of positive welfare such as animal autonomy, play behaviors, positive human–animal relationships, and social interactions [31]. Such positive welfare indicators require validation at the population level to be useful. For population-level welfare assessments to be effective in promoting optimal welfare, the monitoring schedule must be frequent enough with a low enough threshold for intervention to identify specific problems before they cause significant welfare effects [32]. 3. Tension between Individual and Population WelfareAs we have discussed, although the experience of individual animals is critical, population-level considerations can be important tools in the assessment and optimization of animal welfare. Given the historical use of population-level indicators that may compromise individual animal welfare, it is unsurprising that tension between population and individual animal welfare occurs in zoos and aquariums. Importantly, different concepts of animal welfare among caregivers and other stakeholders may be the root of this tension in many cases [33]. For example, Veasey [34] found that veterinary staff prioritized health-centered parameters such as ease of preventive and emergency care and morbidity and mortality rates over a more holistic approach to welfare assessments that included elements relating to biologic functioning, natural living, and affective states of individual animals when assessing animal welfare. Emphasis on certain aspects of welfare over others is expected as a result of the different roles caregivers may have within an institution and the nuanced nature of animal welfare. Achieving consensus through a team approach to evaluations of animal welfare is critical to relieving some tension that occurs as a result of the lack of consensus on how to prioritize different aspects of animal welfare. For this reason, many accredited zoos and aquariums have developed objective scoring systems that require input from a range of stakeholders including veterinarians and animal caregivers to help foster a collaborative approach and to facilitate decision making [34].Conflicts also arise when there are differences in defining and measuring welfare among stakeholders. For example, animal caregivers and even members of the public may form a bond with an individual animal and prioritize the welfare of that individual, whereas veterinarians and administrators charged with managing populations of animals may take a more macroscopic approach to welfare. These conflicts arise because humans have such a profound impact on animals, and humans are increasingly interested in active animal management to mitigate this impact [35]. Obviously, prioritizing the well-being of individual animals best minimizes suffering and maximizes positive welfare states in individuals. However, when caregivers prioritize population welfare, they adopt a broader approach to animal stewardship that emphasizes species preservation as a greater good. Both approaches have merit, and both have their limitations. When possible, the use of scientific knowledge to make animal welfare decisions is critical to at least fostering collaboration among all stakeholders when complete conflict resolution is not possible. However, animal welfare science often relies on tacit ethical judgements and assumptions on what matters in humans’ interactions with animals [36]. In reality, ethical decision-making regarding animal welfare issues is complex, and when science cannot resolve conflicts, animal professionals must use a reasoned approach to animal ethics to communicate their concerns and find a collaborative way to move forward. Although some conflicts may arise from differing concepts of animal welfare, legitimate challenges in which caregivers must weigh the welfare of individuals against the welfare of the population do occur. As we discuss these situations, it is important to note again that animal welfare is individual but decisions regarding individuals have potential consequences for the welfare of other animals in the group and inclusion of those consequences in evaluations of management decisions for an individual is inevitable. Challenges to individual welfare as a result of group dynamics in social species is perhaps the most common situation animal managers need to consider. In the wild, social species develop a hierarchy in which dominant animals require some form of submission from subordinate animals. The negative effects on the welfare of subordinates outweigh the positive effects these animals realize as members of the group (e.g., protection from predators). However, in a zoo and aquarium setting where animals are spatially restricted, subordinates may not have sufficient opportunity to escape from aggression or resources guarded by dominants. Caregivers must recognize the natural history of species, accepting some level of natural aggression towards subordinates, and that intervention to protect these lower ranking individuals may actually cause more deleterious effects on the welfare of the group. In some cases, an individual within a population may have special needs that compromise the welfare of other individuals, especially in social species. For example, in the author’s experience, a baboon with a significant health problem that requires frequent veterinary care may lead to disruptions within the troop, such as displaced aggression on subordinate troop members. Non-target individuals may experience fear, distress, and physical injury when the target individual is captured, removed from, and/or re-introduced to the troop for treatments or other interventions (e.g., [37]). This fear and distress can complicate future efforts to provide treatment to those individuals when they experience a health concern. In other cases, when a social animal requires close observation in the hospital, animal care staff may strategically select a healthy companion as a social partner. The situation inherently decreases the healthy companion’s opportunity for optimal welfare in the short term, but drastically increases the likelihood of a better welfare experience for the animal under veterinary care. In these cases, animal care teams need to explicitly clarify what values and objectives are being prioritized (e.g., individual vs. group welfare) so that all stakeholders understand how and why management decisions were made.Additionally, resources may be limited in zoos and aquariums such that additional expenses for the welfare of an individual animal may preclude other interventions that may provide a benefit to more individuals. Geriatric animals, especially solitary animals that require relatively large exhibits such as tigers, represent another challenge to zoo resources. To meet their conservation goal, zoos need to exhibit reproductively viable animals, but a commitment to lifelong welfare requires facilities to invest in resources to maintain post-reproductive animals. In other cases, an exhibit designed specifically to encourage species-specific natural behaviors could challenge an individual’s welfare as the individual ages. For example, an orangutan exhibit designed to promote species-typical climbing behaviors may need to be altered for a geriatric individual who develops arthritis. While intended to improve the welfare of the geriatric individual, these alterations could lead to the other orangutans in the enclosure spending more time on the ground and an atypical behavior profile for the species. In these situations, caregivers are required to weigh the benefit to one individual against the potential harm to other individuals in the group. Individual animal welfare is contingent on effective population planning, both within and among institutions. Although animal welfare is a core principle of modern zoos and aquariums, their core purpose is conservation [38]. Historically, conservation and animal welfare have occasionally been at odds, as management for conservation often involves activities that may be detrimental to individual animals such as killing of invasive species to promote an indigenous endangered species [39]. In these situations, staff within zoos and aquariums often revert to arguments concerning the benefits to wildlife or the species concerned generally. While important from a utilitarian point of view, these arguments are irrelevant to individual animal welfare. More recently, the term “compassionate conservation” has been coined to encourage those concerned primarily with conservation and those concerned primarily with animal welfare ethics to work together based on a shared commitment to nature (see [40]). There are opportunities for conservationists and animal welfare ethicists to come together. Ultimately, animal welfare ethics differs from the concept of animal rights in that interventions that may be harmful to an individual, such as euthanasia or confinement, are acceptable, provided the individual does not experience “unnecessary pain and suffering” and the resulting benefits outweigh the cost of the suffering. In these cases, conservationists and animal welfare ethicists can agree on a more utilitarian approach to animal welfare to weigh the cost to the individual with the greater good (e.g., long-term viability) the population will realize as a result. Animals in zoos and aquariums are managed intensively with the goal of population conservation. Successful breeding in zoos and aquariums is essential to the long-term conservation of species in captivity, and, more importantly, as a hedge against extinction in the wild. Inevitably, cooperative management programs must balance the goal of conserving a population with the welfare of individual animals when considering transfer, introduction, breeding, and contraception decisions. A transfer between facilities may be the most stressful experience in a captive animal’s life. For example, experiencing an inter-zoo transfer was identified as a risk factor for elephant mortality in zoos in one study [41]. Yet, the transfer of genetic material among institutions is the key component essential for population viability. Alternative approaches to inter-zoo transfers, such as using positive reinforcement to train animals for semen collection and artificial insemination, can improve individual animal welfare by reducing the need for transfers and also achieve the goal of long-term population viability. While perhaps a refinement, even this approach to breeding introduces challenges to individual animal welfare. Perhaps most importantly, denying animals a natural mating opportunity prevents expression of a key species-specific behavior its wild counterparts are highly motivated to perform. Secondly, development of successful artificial insemination procedures requires research, and, in many cases, trial and error. The individuals who participate in these procedures may experience anesthesia, hormone therapy, and other interventions that may have negative effects on their welfare, at least in the short-term. Yet, the greater good of these procedures will ultimately lead to both enhanced individual animal welfare through reduction of the stress of transport and improved population viability through more efficient breeding.With respect to animal acquisition and breeding, aquariums face unique challenges in terms of balancing individual animal and population welfare, as well as their conservation missions. Removing animals from the wild for display in aquariums challenges the welfare of the individuals and, depending on the methods used for capture, threatens the long-term viability of wild populations of many species (see [42] for a more thorough review of these issues). For this reason, the development of culturing techniques for many species has been a priority for accredited aquariums, and recent successes have led to the potential for large-scale acquisition of animals through these captive breeding programs. Eliminating the stress animals experience from capture, transport, and introduction to an aquarium enclosure is a significant improvement for both individual animal welfare and conservation of wild populations. Cultured animals also tend to acclimatize better to human care, and, since these individuals typically present a lesser health risk to established aquarium populations than wild-caught individuals, they may be able to avoid some quarantine procedures and medical treatments that have historically been stressful [43]. Captive fish breeding, though, requires the same considerations for individual and population welfare as other species previously discussed.The ultimate example of compromising individual animal welfare for the greater good of the population is the use of culling “surplus” individuals as a management tool. From a conservation genetics standpoint, it is likely that zoos and aquariums need to produce more offspring than there are existing spaces to ensure long-term population viability [44]. Contraception and other methods used in zoos and aquariums to delay and/or reduce the frequency of breeding have long-term negative effects on fertility in many species and may be associated with deleterious health conditions in treated individuals [45]. Furthermore, physical isolation of animals to prevent breeding may have negative welfare effects on those individuals, especially in social species. In these cases, achievement of optimal welfare for individuals, and populations, may require natural breeding based on a species’ life history, which can result in populations too large for existing facilities. These concerns have led some managers within the zoo and aquarium field to consider euthanasia of healthy individuals as a necessary management tool. While an in-depth discussion of the ethics of management euthanasia, or culling, is beyond the scope of this paper, both AZA and WAZA recognize that humane euthanasia is a tool for managing the demographics, genetics, and diversity of animal populations within zoos and aquariums [46]. For the purposes of this discussion, it assumed that culling methods are consistent with the principles of humane euthanasia, which requires that methods to induce the most rapid, painless, and distress-free death possible are utilized [47]. When an individual is euthanized, or culled, for management reasons, it may be argued that that individual’s welfare is compromised for the good of the population [48]. However, others argue that euthanasia is welfare neutral when performed humanely since it results in the death of the animal, and thus no subjective experience of a positive or negative state [49]. This dichotomy reflects a fundamental difference in stakeholder values. Although an in-depth discussion of the level of self-awareness required for euthanasia to be considered welfare negative is beyond the scope of this paper, the distinction is important when considering the range of species housed in zoos and aquariums that may be affected by management euthanasia. While general aspects of management euthanasia are applicable to both terrestrial and aquatic species, aquarists and invertebrate caregivers are faced with unique considerations when discussing culling as a population management tool. While attempts are made to treat aquatic animals with chronic parasitic or other infectious diseases, culling affected individuals is often a necessary tool if other treatments have failed. Aquarists justify this management approach with a focus on group health and welfare. The cost to individual health and welfare is accepted because infectious diseases are often so virulent within enclosed aquatic habitats that a focus on individual welfare will come at a cost to the group in terms of the potential for other animals to become affected. As discussed above, breeding programs in aquariums are increasing to improve individual animal welfare and minimize stress on wild populations. However, in the authors’ experience, as reproductive rates increase, there is also an increase in juveniles with deformities such as missing fins, misshapen bodies, and underdeveloped swim bladders. These conditions are deleterious to both individual animal and population welfare, and, while controversial, aquarists do utilize euthanasia as a management tool in these cases. Additionally, the management of surplus animals becomes problematic since it is nearly impossible to breed a specific number of animals. Culling these excess individuals, and at what point in the fish development process, to prevent overcrowding and poor welfare is another controversial topic in this growing field. 4. ConclusionsThe field of zoo and aquarium animal welfare science has advanced dramatically over the last 50 years from merely evaluating population-level indicators such as reproductive success to assessing the welfare of individual animals using a variety of parameters indicating negative and positive welfare states. Often, population-level observations and epidemiological research have led to the identification and validation of welfare indicators at the individual level. These population-level assessments have facilitated the development of best practices for the care of individual species and taxa and improvements in individual animal welfare, and, in some cases, make welfare assessments possible for large numbers of animals in zoos and aquariums. Ignoring population-level welfare and assessments limits the advancements of welfare science on an individual level. However, animal welfare is based on an individual animal’s subjective experience. Zoos and aquariums must balance individual animal and population welfare especially regarding breeding programs and management of geriatric and special-needs animals. Despite the advancements in animal welfare science through significant research investigating population and individual welfare, as discussed in this paper, much more work is needed to continue this progress. Population-level studies to validate indicators of welfare for individual animals are essential, especially for under-studied taxa such as invertebrates, fish, amphibians, and reptiles. Approaches to assessing the welfare of large populations, especially when individuals within a group have no identifying characteristics, that do not rely on production-based indicators (e.g., reproductive output) need to be developed and validated. Finally, research to understand individual differences within a species is needed to evaluate and improve best practice guidelines used to care for animals and facilitate animal welfare improvements.

animals : an open access journal from mdpi

10.3390/ani13081390

PMC10135127

MicroRNAs have been actively studied for their diagnostic, prognostic, and therapeutic purposes for various diseases in animals. In this study, we analyzed the changes in miRNA expression under different conditions in the testis and epididymis of male dogs. We identified differentially expressed miRNAs in the testis and epididymis depending on age and the presence of cryptorchidism in male dogs, which suggests that they could help better understand aging and diseases of the reproductive system in male dogs.

In the present study, we aimed to investigate age-, cryptorchidism-, and testicular tumor-related changes in miRNAs in the testis and epididymis of dogs. Twelve healthy male dogs were divided into two groups: young (<1 year, n = 8) and old (>3 years, n = 4). Five dogs with unilateral cryptorchidism, one with a Sertoli cell tumor, and one with seminoma were referred to a veterinary hospital. After surgery, the testes and epididymis tails were collected. A high-throughput miRNA array analysis was performed to identify miRNAs affected by age, cryptorchidism, and testicular tumors. The expression of only cfa-miR-503 was downregulated in the epididymis of younger dogs, whereas the expression of 64 miRNAs was upregulated. Among them, the top five miRNAs were cfa-miR-26a, cfa-miR-200c, cfa-let-7c, cfa-let-7b, and cfa-let-7a. The expression of cfa-miR-148a and cfa-miR-497 was considerably lower in cryptorchid testis than in healthy dog testis. In the epididymis, the cfa-miR-1841 level was significantly decreased. We observed a significant difference in the expression of 26 cfa-miRNAs between testicular tumors and normal tissues. This study demonstrated that aging and cryptorchidism have a causal relationship with miRNA expression. The identified miRNAs may be candidate genes for male reproductive traits and could be applied in molecular breeding programs.

1. IntroductionMale reproductive function declines with age in both humans and dogs. It is well-known that testis volume, germ cell and Sertoli cell number, serum testosterone levels, and sperm viability decrease with age in humans [1]. Reproductive aging is associated with a reduction in the number of Leydig cells and seminiferous tubules, as well as oxidative stress [2,3]. In addition, changes in the expression of genes involved in the regulation of apoptosis and DNA repair have been observed in aging testis [4]. A negative correlation has been reported between age and normal ejaculated sperm characteristics in dogs [5]. A previous study has shown that epididymal sperm quality and fertility decrease with age in male dogs [6]. Furthermore, fresh and thawed semen from old male dogs display decreased sperm motility and mitochondrial function [7]. Therefore, age is one of the most important factors influencing infertility in male animals, including dogs. Cryptorchidism is the most common pathological condition in dogs, in which the testis fails to descend to the base of the scrotum, with an informed morbidity rate of 0.8–10% [8]. Hereditary defects caused by sex-limited autosomal recessive genes are considered an etiology of cryptorchidism in male dogs. Although cryptorchidism is often considered a mild malformation, it can seriously affect the health of dogs, as a very high-risk factor for sterility and testicular tumor. As cryptorchid testes generally have considerably higher temperatures than normally descended testes, unilateral cryptorchids reduce semen quality or cannot produce normal sperm [9]. Cryptorchidism is associated with testicular tumors in dogs [10,11]. The incidence of testicular tumors in undescended testes is approximately 13 times greater than that in normally descended testes [8]. Although testicular tumors are typically malignant, the excess production of endogenous estrogen by the tumor cells can lead to a condition known as feminization syndrome and bone marrow suppression, which can be fatal [12]. MicroRNAs (miRNAs) are a group of short non-coding RNA molecules and are approximately 20–24 nucleotides long; they function by epigenetically downregulating gene expression. miRNAs play a crucial role in controlling both mRNA stability and protein synthesis, thereby influencing the processes of spermatogenesis [13] and sperm maturation during sperm passage through the epididymis [14]. Although the differentially expressed miRNAs in the testicular tissue in cryptorchidism have been investigated in humans and various animal species, including horses, rats, and mice [15], direct correlation studies between miRNAs and cryptorchidism in dogs are limited. miRNAs can be utilized as biomarkers to elucidate changes in tissues affected by several disorders. In our previous study [16], we identified several miRNAs associated with uteropathies and age markers in female dogs. To the best of our knowledge, there are no reports of alterations in miRNA expression within the testes and epididymis of canines of different ages or with cryptorchidism and testicular tumors. The purpose of this study was to (i) assess the relationship between aging and disease status and the expression of miRNAs in male dog testis and epididymis and (ii) determine the potential of miRNAs as biomarkers and therapeutic targets for infertility and various diseases in male dogs.2. Materials and Methods2.1. Collection of Tissue Sample Male reproductive organ tissues were collected from two local animal hospitals and the veterinary teaching hospital of Seoul National University, Korea, at the request of the dog owners when general neutralization surgery or castration was required according to clinical findings, such as cryptorchidism, Sertoli cell tumor, and seminoma, with owners’ informed consent. Testicular tumor diagnosis was based on histological examination (IDEXX, Seoul, Republic of Korea) of the surgically removed tissues. Furthermore, testicular tumor tissue collection was approved only by the Seoul National University Institutional Animal Care and Use Committee (approval number: SNU-200217-3-2). The dogs with testicular tumors were not in the cryptorchidism state. The testes were obtained from 19 male dogs and epididymis cauda tissues (tail part of the epididymis) were collected from 17 male dogs, excluding two dogs with testicular tumors, during castration for the prevention and treatment of disease at the request of the owners, with informed consent obtained from the owners. In dogs, the final diagnosis of cryptorchidism can only be made after 6 months of age [17]. All cryptorchid testes were palpable in the right inguinal area and the left testis was present within the scrotum. Before anesthesia for castration, all dogs underwent appropriate clinical evaluations such as blood chemistry tests for total protein, glucose, blood urea nitrogen (BUN), creatinine, alkaline phosphatase (ALKP), and alanine aminotransferase (ALT) to identify any potential pathological conditions and determine their suitability for systemic anesthesia. The serum chemistry results for the dogs used in this clinical examination are shown in Table 1. This study involved eight healthy male puppies under the age of 1 year, four healthy male dogs over the age of 3 years, five dogs with cryptorchidism under the age of 1 year, one dog with Sertoli cell tumor, and one dog with seminoma over the age of 10 years. The average results and reference ranges of each parameter of the serum chemistry are presented in Table 1. Before the tissues dried or were damaged, each testis and tail of the epididymis were promptly separated after surgery and collected. As the testis and epididymis are surrounded by the tunica vaginalis, removal of the tunica vaginalis was performed after scrotal incision. The immature spermatozoa within the testes gain the ability to move and become capacitated as they pass through the epididymis. Therefore, we collected the testes and tail parts of the epididymis cauda to compare miRNA expression in all tested dogs. To collect the epididymis cauda tissue, the surrounding tissues such as the ligament of the epididymis were excised and separated from the testis. Some of the cryptorchid and descended tissues of the testis and epididymis were stored in neutral buffered formalin for histological examination. In addition, the remaining testis and epididymal cauda tissues from each male dog were collected in RNAlater (Thermo Fisher, Waltham, MA, USA) and stored at −80 °C for miRNA array analysis.2.2. Hematoxylina and Eosin Staining Both cryptorchid and non-cryptorchid sides of the testes and epididymis of dogs with unilateral cryptorchidism were collected for hematoxylin and eosin (H&E) staining and analysis. The testis and epididymis cauda were fixed in neutral buffered formalin, dehydrated with a gradient series of alcohol from 60% to 90%, and embedded in paraffin. According to the standard protocol, H&E-stained 4-µm sections (Leica Microsystems GmbH, Wetzlar, Germany) placed on silane-coated slides were observed under a microscope (BX53; Olympus, Tokyo, Japan).2.3. RNA Isolation and Quality Check, and cDNA Synthesis The total RNA was extracted following the protocol provided by the manufacturer. To extract the total RNA from each tissue sample, weighing a minimum of 50 mg, an easy-spin™ Total RNA Extraction Kit (Intron Biotechnology, Seoul, Republic of Korea) was used to homogenize the samples with 1 mL of RNA lysis solution. To perform gene microarray hybridization, the Agilent RNA 6000 nano kit and 2100 Bioanalyzer (G2939BA; Agilent, Santa Clara, CA, USA) were used to assess both the quantity and quality of the RNA. Only samples that fulfilled the following criteria were selected for microRNA analysis: A260/A280 and A260/A280 > 1.0, concentration > 50 ng/µL, volume > 10 µL, total amount > 0.7 µg, rRNA ratio > 1.0, and RIN > 7.0 with visible small RNA peaks. Before cDNA synthesis, the NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific Inc., Wilmington, MA, USA) was used to assess the quantity and quality of the RNA. The Maxime RT-PCR premix kit (Intron Biotechnology) was used to reverse transcribe the RNA samples (500 ng) to cDNA, with a reaction mixture of a total volume of 20 µL.2.4. MicoRNA Hybridization, Scanning, and Data ProcessingAll procedures were performed in accordance with the guidelines in the Affymetrix Expression Analysis Technical Manual (Affymetrix Inc., Santa Clara, CA, USA). Total RNAs were labeled using the FlashTag Biotin HSR RNA Labeling Kit (Thermo Fisher). The GeneChip® Affymetrix miRNA microarray (Affymetrix Inc.) was used to hybridize the biotin-labeled samples. The Affymetrix GeneChip® scanner was used to examine all arrays, and raw data analysis was carried out using the Affymetrix GeneChip® Command Console® Software (AGCC) (Affymetrix Inc., Santa Clara, California, USA). The measured intensity of each array probe was obtained from CEL files, which contained the raw data images developed by the scanner. A p-value of <0.05 and fold change of >2 served as cut-off criteria to restrict a broad range of differentially expressed microRNAs. 2.5. Statistical AnalysisThe clinical data of the dogs are presented as mean ± standard deviation. An unpaired t-test was used to compare the groups. All statistical analyses were conducted with SPSS software (version 25.0; SPSS Inc., Chicago, IL, USA). The level of statistical significance was defined as p < 0.05.3. Results3.1. H&E Staining of Cryptorchid Testis and EpididymisThe cryptorchid side testis showed severe diffuse tubular atrophy of the seminiferous tubules and only Sertoli cells with germ cell depletion (Figure 1A). However, the other (non-cryptorchid) side testis showed tubular atrophy, which was less severe than that of the cryptorchid-side testis (Figure 1B). Degeneration of germ cells and depletion of elongating spermatids were observed. In the cryptorchid-side epididymis, no spermatozoa showed epithelial tubule degeneration (Figure 1C). Cell debris and germ cell exfoliates were observed; however, no spermatozoa were observed in the lumen of the non-cryptorchid side epididymis (Figure 1D).3.2. MiRNA Expression in the Testis and Epididymis Cauda in the Dogs according to AgeThe testis and epididymis cauda miRNA volume plots for the comparison of immature male dogs younger than 1 year of age versus mature dogs older than 3 years of age are presented in Figure 2. According to the fold change cut-off of >2 (upregulation or downregulation) and p value of <0.05, there were no significantly differentially expressed miRNAs in the testes between immature and mature dogs. In the epididymis, the top five miRNAs with noticeable differential expression were confirmed by considering the fold change, p-value, and volume value. Sixty-five significant miRNAs that were differentially expressed were identified. Among them, the top five meaningful miRNAs, considering fold change and volume values, were cfa-miR-26a, cfa-miR-200c, cfa-let-7c, cfa-let-7b, and cfa-let-7a. The only significant miRNA that was less expressed in mature dogs compared with that in immature dogs was miR-503.3.3. MiRNA Expression of Testis and Epididymis Cauda in Cryptorchid DogsAs shown in Figure 3A, two significantly downregulated miRNA genes, cfa-miR-148a and cfa-miR-497, were found in the testes of cryptorchid dogs compared with age-matched normal dogs. As shown in Figure 3B, cfa-miR-1841 was significantly downregulated in the epididymis of cryptorchid dogs.3.4. MiRNA Expression in the Testis of Dogs with Sertoli Cell Tumor and SeminomaOne hundred and seven miRNAs, with significant differences in expression between Sertoli cell tumors and normal testes, were identified. Sixteen miRNAs were downregulated, and most of the remaining ninety-one miRNAs were upregulated in one case of Sertoli cell tumor compared with those in the normal testis. The top five miRNAs with a fold change cut-off of >2 (upregulation or downregulation) between Sertoli cell tumor and normal testis were cfa-miR-27b, cfa-miR-20a, cfa-miR-93, cfa-miR-502, and cfa-miR-500 (Supplementary Figure S1A). Cfa-miR-27b, cfa-miR-20a, and cfa-miR-93 were highly expressed in Sertoli cell tumors, and cfa-miR-502 and cfa-miR-500 were less expressed. In seminoma, 86 miRNAs, different from those in normal testes, were identified. It was confirmed that 21 miRNAs were downregulated and 65 were highly expressed. The top five miRNAs with a fold change cut-off of >2 (upregulation or downregulation) were cfa-miR-378, cfa-miR-29a, cfa-miR-27b, cfa-miR-16, and cfa-miR-106a (Supplementary Figure S1B). The overlapping and non-overlapping differentially expressed miRNAs between the Sertoli cell tumor and seminoma groups are presented in Supplementary Figure S2 as Venn diagrams. The miRNAs indicated in red were differentially upregulated and those in blue were downregulated. The miRNAs indicated in green were upregulated in one comparison, which showed contradictory results for each comparator group, but downregulated in the other comparison. While there was an overlap between 55 significant miRNAs in “Sertoli cell tumor vs. normal testis” and “Seminoma vs. normal testis,” the expression of 11 miRNAs indicated in green were interestingly discordant in each comparison, indicating that a significant portion of the “tumor type” could affect the miRNA expression pattern. Twenty-nine miRNAs were commonly identified in the comparison between Sertoli cell tumor and normal testis and between seminoma and normal testis. Next, both Sertoli cell tumors and seminomas, regarded as testicular tumors, were grouped and compared to age-matched normal testes. Significant changes in the expression of 28 miRNAs were observed in testicular tumors compared with that in the normal testes. All 28 miRNAs (100%) overlapped, with the expression of the miRNAs frequently changing compared with that in the comparison of Sertoli cell tumors versus normal testes. Two miRNAs, miR-216b and miR-449, were commonly downregulated in the comparisons of testicular tumors versus normal testes and Sertoli cell tumors versus normal testes. The overlapping parts of the three circles represent 26 miRNAs—23 upregulated and 3 downregulated. 4. DiscussionThe results of this study provide conclusive evidence regarding the effect of age and cryptorchidism on miRNA expression in the male reproductive system. The miRNA expression in the epididymis observed in this study was affected by age, with increased expression of cfa-let-7 members, cfa-miR-26a, and cfa-miR-200c in dogs older than 3 years of age. We focused on miRNA expression in dogs with cryptorchidism and testicular tumors as (1) cryptorchidism is the most common hereditary disorder in male dogs and (2) it may be a risk factor for infertility and testicular cancer. Our results showed that in cryptorchid dogs, the expression of cfa-miR-148a and cfa-miR-497 was downregulated in the testes and that of cfa-miR-1841 was downregulated in the epididymis compared with those in age-matched dogs. In addition, testicular tumors showed different miRNA expression levels compared with those in age-matched dogs. In addition to advanced maternal age, paternal age tends to be significantly associated with a decline in undesirable embryonic development and poor pregnancy outcomes because semen volume, sperm motility, and normal morphology can significantly decline with age in humans [18]. Similarly, in various animals, including dogs, ferrets, and cats, age negatively correlates with male reproductive capacity [5,6,19,20]. To the best of our knowledge, there is no report on the miRNA expression patterns in the reproductive organs of dogs across different age groups. In this study, the dogs were categorized into two distinct age groups (<1 year old and >3 years old) for the purpose of evaluating miRNA expression in the testes and cauda epididymis. Interestingly, the miRNA levels were not significant in the testis but were significant in the epididymis, with higher expression of cfa-miR26a, miR-200c, and let 7 family members (let 7a, let7b, and let 7c). These results suggest that the testis and epididymis are different in terms of miRNA expression and processing. The epididymis serves as the location for the post-maturation of testicular sperm, resulting in the acquisition of sperm motility and the capacity to recognize and fertilize oocytes. In this study, epididymal tissue was harvested from the cauda epididymis, where mature sperm are stored until ejaculation. To minimize the influence of prostatic fluid and immature sperm, the cauda epididymis may be a more appropriate tissue to examine the effects of age and cryptorchidism on miRNA expression in dogs. Furthermore, as the incidence of canine prostatic disorders markedly increases with age, the miRNA expression results may have been affected by a mixture of prostatic fluid. miR-26a is a functional miRNA that regulates sperm metabolism and apoptosis. It has been reported that miR-26a can have a major effect on the quality of semen in Holstein bulls as it plays a negative regulatory role in the expression of phosphoenolpyruvate carboxykinase-1 (PCK1) [21]. Additionally, it is suggested that miR-26a is involved in the regulation of bull sperm motility [22]. In boars, miR-26a is associated with decreased sperm motility [23]. A previous study demonstrated that there is a considerable increase in the expression of miR-26a in highly motile frozen-thawed sperm compared with that in low-motile frozen-thawed sperm [24]. Furthermore, the sperm transcript level of miR-26a-5p is lower in men with unexplained infertility than in fertile control, and high phosphatase and tensin homolog (PTEN) expression is associated with ejaculated spermatozoa [25]. PTEN signaling is a major negative regulator of PI3K signaling and is involved in the maintenance of spermatogonial stem cells in mice [26]. Interestingly, our study showed that cfa-miR-26a was highly expressed in the epididymis of mature dogs compared with that in immature dogs. These findings offer compelling evidence supporting the hypothesis that miR-26a may play a role in the regulation of epididymal aging and, consequently, have an effect on sperm metabolism or motility. Let-7 (lethal-7) is among the earliest identified miRNAs. As the expression of let-7 family members gradually increases during development, it is not surprising that high levels were observed in the cauda epididymis of mature dogs compared with those of younger dogs. However, in a previous study, female germ cells did not show changes in let-7 miRNA expression, but male germ cells showed increased expression during development [27]. Boars with low sperm motility and a high percentage of abnormal sperm showed higher levels of let-7a, let-7d, and let-7e miRNAs in their spermatozoa [28], indicating that the let-7 family members may be markers for infertility. Interestingly, although the expression of miR-26a and let-7 family members was increased in the ovaries of dogs with uteropathies in our previous study, significant differences in the expression of these miRNAs were observed in the epididymis of mature dogs in this study. Therefore, miR-26a and the let-7 family members may be related to female reproductive organ disease in dogs as well as the aging of male reproductive organs. In Yorkshire boars, endogenous miR-26a and let-7 have anti-apoptotic and pro-survival functions in sperm cells by targeting PTEN and PMAIP1 [23]. The most abundant miRNAs in the epididymis of bovine species are let-7 family members and miR-200a/b tumor suppressors [29]. miRNA-mediated inactivation of cellular oncogene products could play a role in maintaining the stability of the epididymis, which is an organ with a unique ability to evade tumorigenicity. Consistent with these findings, the let-7 family members and miR-200a/b tumor suppressors, whose expression was confirmed in the epididymis of younger dogs, were not identified in the testicular tumors in the present study. The miR-200 family, which includes miR-141, miR-200a, miR-200b, miR-200c, and miR-429, is the most prevalent family in the miRNA system, and all members are highly conserved. It has been reported that the miR-200 family members regulate epithelial–mesenchymal transition, which is an important step for breast cancer infiltration and metastasis [30]. Furthermore, miR-200 has a functional role in the regulation of cell invasion and migration by targeting PTEN [31]. The expression of miR-200a, miR-200c, and miR-141 in both male and female mouse germ cells is downregulated gradually during development [27]. In a human study, miR-200a-3p and miR-200c-3p were identified as potential biomarkers for male subfertility [32]. As miR-200b and miR-200c accumulate in spermatozoa during passage through the epididymis [33,34], the present study results indicate that sperm maturation in the epididymis cauda may be differentially regulated according to dog age. Cryptorchidism is a congenital defect commonly found in dogs, where one or both testicles fail to descend normally into the scrotum. The retained testicle(s) may remain in the abdomen or become lodged in the inguinal canal, causing potential health issues such as infertility, testicular tumors, and torsion. In recent years, several studies have focused on miRNA expression in the testes of various animals with cryptorchidism, including horses [35], rats [36], and mice [37]. miR-148a was identified as differentially expressed in the seminal plasma extracellular microvesicles of men with oligoasthenozoospermia subfertility compared with that in men with normozoospermic fertility, demonstrating that it may be a marker for male infertility [38]. Similarly, the present study suggests that cryptorchid-side testes and epididymis do not have spermatozoa and cryptorchid-side testes display differential expression of miR-148a. In agreement with the fact that miR-497-5p has been identified only in the testicular tissues of spermatozoa and seminal plasma in humans [39], cfa-miR-497 was found to be expressed at low levels in the testes of cryptorchid dogs. In testicular tumors, miR27b was highly expressed in both Sertoli cell tumors and seminomas; it has been found to be differentially expressed in the mature spermatozoa of infertile men. The high expression of cysteine-rich secretory protein 2 (CRISP2), which is predominantly expressed in the testis, is correlated with the expression of miR-27 in humans [40]. miR-27b expression is downregulated during sheep fetal testis development from D42 to 75 and plays an important role in regulating cellular differentiation [41].A major limitation of this study is that the miRNA expression was analyzed in a limited number of dogs, especially in one case each of Sertoli cell tumor and seminoma. The present analysis of miRNA expression in testicular tumor tissues lays a foundation for more extensive and larger-scale studies. The results of this study should be interpreted with caution. Furthermore, age and breed predisposition may have affected the miRNA expression results of this study. In order to reduce this expected bias, we classified the dogs into two age categories, those under 1 year old and those over 3 years old; evaluated tissue samples obtained only from small dog breeds; and analyzed age-matched dogs as a control group for the comparison of cryptorchidism testes and testicular tumors. 5. ConclusionsIn conclusion, miRNA expression in the male reproductive tissue of dogs with cryptorchidism and testicular tumors was comprehensively analyzed and the effect of age on miRNA expression in male reproductive organs was examined. The present study demonstrated that epididymal cfa-miR-26a, cfa-miR-200c, cfa-let-7c, cfa-let-7b, and cfa-let-7a expression regulated via PTEN may be involved in aging in dogs. Furthermore, cfa-miR-148a and -497 expression were consistently lower in the testes and cfa-miR-1841 expression was lower in the epididymis of dogs with cryptorchidism, suggesting that these miRNAs may be useful biomarkers for cryptorchidism and male infertility. This study provides insights into the development and causes of cryptorchidism and testicular tumors. We hope that our study will help develop new diagnostic methods and preventive medication for spontaneous cryptorchidism and testicular tumors and improve male fertility with aging in dogs.

animals : an open access journal from mdpi

10.3390/ani11113161

PMC8614450

Melatonin is a known antioxidant and anti-inflammatory regime, while in sheep it is broadly used to accelerate the onset of the breeding season. Our recent study showed that melatonin administration during pregnancy in heat-stressed ewes improved fertility rate and number of lambs born per ewe, the redox status of the maternal organism and the produced milk quantity until weaning. In this study, we present the impact of melatonin administration in stressed ewes during pregnancy considering: (a) humoral response of both maternal organism and offspring during the first two days after parturition, (b) chemical composition and antioxidant parameters of colostrum and milk until weaning and (c) redox status of the offspring until weaning. The results indicated that melatonin improved the redox status of the offspring and the quality of colostrum. Moreover, melatonin could be administered as immune-modulatory regime, apart from antioxidant, in prenatally stressed offspring in order to cope with the crucial first days of their life, as the humoral response results suggested.

In this study, the effects of melatonin treatment on growth, redox status and immunity in prenatally stressed newborn lambs were evaluated. Thirty-seven newborn lambs were allocated into two groups (melatonin-MEL and control-CON), based on whether their mothers were treated with melatonin implants or not, respectively. All pregnant ewes were exposed to heat stress. The body weight of lambs was recorded at birth (L0), and then on days 15 (L15) and 40 (L40). Redox biomarkers [total antioxidant capacity (TAC), glutathione (GSH), thiobarbituric acid reactive substances (TBARS)] were assayed in blood samples collected from lambs on days L0, L1, L2, L5, L10 and L40. Chemical analysis and antioxidant capacity were evaluated in colostrum and milk samples collected at the same time points with blood samples. Cytokines (IL-1β, IL-6, IL-10, IFN-γ) and immunoglobulin (IgG) were assayed in blood and colostrum samples collected from ewes on days L0 and L1, and in lambs’ blood on days L0, L1 and L2. The results revealed that body weight gain of newborn lambs did not differ between the two groups (p > 0.05). Better redox status was found in MEL lambs until L2, as well as higher antioxidant capacity in the colostrum of MEL ewes compared to CON ones on day L0 (p < 0.05). In MEL ewes’ colostrum, higher protein content was measured on day L0 and higher fat content on L1 compared to CON group (p < 0.05). The highest level of IL-6 was found in MEL ewes on L1, with a concomitant increase of IL-10 level in MEL lambs in comparison to CON lambs on L2. Moreover, CON colostrum resulted in a higher level of IL-10 within time, coupled with an increased level of IgG found in lambs’ plasma on L2 (p = 0.04). This study indicated that melatonin could be administered as antioxidant and immune-modulatory regime in prenatally stressed offspring in order to cope with the crucial first days of their life. This effect of melatonin was also amplified by crosstalk between IL-6, IL-10 and IgG production, resulting in an improved quality of produced milk.

1. IntroductionSeveral studies have considered the long-term consequences of early life events on physiological processes linked to animal production. It is now well established that the phenotype of an individual can be driven by in utero environmental conditions [1]. Growth evidence supports the concept of “developmental programming” in livestock, which implies that a stimulus or insult acting during critical periods of pre- or post-natal growth and development may result in permanent programmed alterations on health and wellbeing of the offspring. A number of insults, such as maternal nutritional perturbations, heat stress and inflammation have been recognised as prominent causes of developmental programming [2,3], inducing, directly or indirectly, an inhibitory effect on the innate immunity of the offspring [1]. Perturbations to the emerging immune system might have long-term consequences in the physiology and disease risk of the offspring due to programming effects [4,5]. It has been demonstrated that dairy cows being exposed to heat stress during late gestation caused in female offspring a low blood lymphocyte proliferative response [6], thus showing that prenatal treatment influences both passive and acquired immune function in offspring [1]. However, the critical developmental stages of vulnerability of the immune system to environmental programming have not been elucidated so far and probably vary between different species [5].Furthermore, various factors such as infections, undernutrition, stress of transfer and extreme weather conditions can cause an imbalance of reactive oxygen species (ROS) production [7]. One of the predisposing factors that could lead to elevated production of ROS is heat stress (HS) [7]. Thus, during pregnancy, an uncontrolled rise in ROS production in the maternal organism can cause more harm than profit, due to the fact that the defensive antioxidant mechanisms are not capable of reversing the ROS imbalance, which may lead to disruption of pregnancy and abortion or abnormalities to the foetuses [7]. We have recently shown that melatonin can be safely administered in heat-stressed ewes for improving the fertility rate as well as the redox status of ewes during pregnancy [8]. Moreover, melatonin may play a role in prenatally stressed offspring, possibly through the modulation of proinflammatory cytokines via redox-related mechanisms [9].Melatonin (N-acetyl-5-methoxytryptamine) is a small indoleamine produced by the pineal gland predominantly during the dark phase of the circadian cycle [6,10]. Melatonin can be produced by immune cells and in many peripheral tissues, including male and female reproductive organs [11] and exerts pleiotropic bioactivities [12]. Indeed, it is a broad-spectrum antioxidant, free radical scavenger and an anti-inflammatory molecule [13,14,15,16] that regulates a number of physiological processes, including reproduction. As an antioxidant molecule, melatonin acts in two ways. First, it is a scavenger for ROS when it is found in low concentration in blood, and when its concentration is elevated, it induces the expression of antioxidant genes such as glutathione peroxidase (GPx), leading to high levels of GSH. This double role is crucial for embryo development since it reduces the levels of ROS in the placenta and embryo environment, allowing the foetus to develop in an environment where oxidative stress is abundant [11]. Moreover, melatonin has pleiotropic effects on different steps of inflammation, as demonstrated by the proinflammatory role at an early phase of inflammation through activation of cytokines release, such as IL-1 and tumour necrosis factor-alpha (TNF-α) [17].The important role of melatonin in pregnancy and parturition has been well established. Owing to its lipid- and water-soluble character, maternal melatonin crosses the placenta easily and enters the fetal circulation without being modified [18], indicating that it has a direct effect on the embryo by upregulating its antioxidant capacity. This hormone plays a key role in regulation of development of foetal organs, which is critical for prevention of foetal losses and for successful adaptation of the neonate to extrauterine life [9]. Increased maternal melatonin circulating levels have been reported during pregnancy in sheep [19], rats [20] and humans [21,22,23]. The circulating melatonin acts to synchronise physiological functions, including energy metabolism. During pregnancy, the regulation of energy metabolism is crucial for the maintenance of maternal and fetal health [12], including the foetus’ capacity to produce anti-inflammatory molecules. Maternal serum melatonin levels show a diurnal rhythm that is an important signal for the foetus to entrain the light-dark rhythm of the newborns after birth [6]. Moreover, the role of melatonin in foetal programming has been discussed [9,24] and the use of melatonin has been suggested as a reprogramming agent [24].The emerging immune system is vulnerable to insult not only during foetal life, but also through colostrum transfer. The “Lactocrine hypothesis” extends the developmental origins hypothesis beyond the intrauterine environment, through ingestion of colostrum as a conduit for the transmission of signalling molecules from mother to offspring [2]. Furthermore, prenatal stress may affect the acquisition of maternal immunoglobulins (Ig) via colostrum intake, which is prerequisite for the acquisition of passive immunity in sheep and the survival of newborns during their first hours after birth [25]. Apart from Ig, colostrum also provides antioxidants [26], defence cells (including macrophages), lipids and proteins that are sensitive to oxidation and contribute to the function of various antibacterial systems. However, colostrum itself is a source of oxidation-reduction reactions [27] and the Ig contained therein are molecules with high susceptibility to peroxidation [28]. As it was found in calves, the redox balance of the colostrum is closely associated with the IgG absorption [29]. Potential effects of melatonin administration on prenatally stressed offspring have not been studied in sheep. We have recently indicated the positive effects of melatonin on redox status of heat-stressed pregnant ewes. Melatonin treatment not only increased the mean number and body weight of lambs born per ewe but also led to higher milk production during the puerperium [8]. Our hypothesis was that melatonin could play a role in prenatally stressed offspring, possibly through the modulation of proinflammatory cytokines via a redox mechanism [9]. Thus, the aim of this study was to evaluate the role of melatonin administration prenatally as an antioxidant and anti-inflammatory regime in newborn lambs and in the quality of the produced colostrum/milk.2. Materials and Methods2.1. Experimental OverviewA total of 37 Karagkouniko breed lambs were included in the study and allocated into two groups, MEL (n = 18) lambs’ group and CON (n = 19) lambs’ one, based on whether their mothers had been treated with melatonin implants during pregnancy or not, respectively. The MEL group consisted of 5 singletons, 5 twins and 1 triplet, while the CON group consisted of 13 singletons and 3 twins. After the lambs’ birth, no intervention was made.Pregnant ewes (n = 31) were exposed to heat stress for the first 100 days of pregnancy during the summer period in central Greece and were allocated into two groups, the MEL (n = 15) ewes’ group and the CON (n = 16) ewes’ one. In ewes of MEL group, melatonin implants (dose rate: 1 implant per ewe; Regulin, Ceva, Libourne, France) were inserted subcutaneously in the base of the ear 16 days before mating. The same procedure was repeated thrice every 40 days as was previously described by Bouroutzika et al. [8]. In total, each MEL ewe received 4 melatonin to ensure high levels of circulating melatonin throughout pregnancy and basal levels at parturition. After mating, ewes were fed with 300 g of ratio twice a day, 1 kg clover and 2 kg alfalfa hay per ewe, and had access to water ad libitum. From the 100th day of gestation, ewes with singletons were fed with 350 g of ratio twice a day, whereas ewes that bore more embryos were fed with 400 g twice a day. All ewes consumed 1.5 kg of clover and 2 kg of alfalfa hay daily and had access to water ad libitum. After lambing, the consumption of clover was increased to 1.8–2 kg per ewe daily.Lambs’ body weight was recorded at birth, as well as on days 15 (L15) and 40 (L40) after birth. All lambs suckled their mothers until day 20 (L20) of life and then gradually were fed, also, with alfalfa hay and 100 g of ratio. Weaning of lambs took place 40 (L40) days after birth.Conditions prescribed by legislation of the European Union in relation to animal experimentation procedures (Council Directive 86/809/EEC) were met during this work (licence number of experimental works: 41-bio/exp-04, approvals by Veterinary Faculty: 37/2016 and 6/2018).2.2. SamplingBlood samples were collected by jugular venipuncture (EDTA, BD Vacutainer® blood collection tubes, BD, Franklin Lakes, NJ, USA) from ewes at lambing (L0), 24 (L1) and 48 (L2) h later. Moreover, blood samples were collected from lambs at birth (L0), 24 (L1) and 48 (L2) h later and then 5 (L5), 10 (L10) and 40 (L40) days after birth. Blood plasma was separated and stored at −20 °C until assayed.Colostrum and milk samples were collected at the same time points for blood as were for colostrum on L0, L1 and L2 and for milk on L5, L10 and L40 time points. Each colostrum or milk sample was collected in 50 mL conical centrifuge tubes and they were immediately refrigerated at −20 °C.2.3. Blood Antioxidant MethodsThree redox biomarkers, namely, total antioxidant capacity (TAC) as a crude index of the antioxidant potential of the samples, thiobarbituric acid reactive substances (TBARS) as a biomarker of lipid peroxidation and reduced glutathione (GSH) as the most important endogenous antioxidant molecule, were evaluated in all blood samples. All the methods were based on the protocols described by Veskoukis et al. [30,31,32,33].In brief for the TAC assay, 20 μL of each plasma sample was mixed with 10 mM sodium phosphate buffer pH = 7.4 (480 μL) and 0.1 mM 2,2-diphenyl-1-picrylhydrazyl radical (DPPH•) solution (500 μL), and then incubated for 1 h in the dark at room temperature (RT), centrifuged (20,000× g for 3 min at 4 °C) and the absorbance was measured at 517 nm in a spectrophotometer (U-1900; Hitachi, Ltd., Tokyo, Japan). TAC was calculated on the basis of the mmol DPPH• reduced by the antioxidants present in the samples. For the TBARS assay, 100 μL of plasma was mixed with 35% trichloroacetic acid (TCA) (500 μL) and 200 mM Tris-HCl pH = 7.4 (500 μL), then incubated for 10 min at RT and 1 mL of 2 M Na2SO4 and 55 mM of thiobarbituric acid (TBA) were added. Following 45-min incubation at 95 °C, 1 mL of 70% TCA followed. The samples were centrifuged (15,000× g for 3 min at 20 °C) and the absorbance was measured at 520 nm in a spectrophotometer (U-1900; Hitachi, Ltd., Tokyo, Japan). The concentration of TBARS was calculated on the basis of the millimolar extinction coefficient of malonyldialdehyde (156 L/mmol/cm). Finally, for GSH assay 20 μL of erythrocyte lysate treated with TCA was mixed with 67 mM phosphate buffer (pH = 7.95) (660 μL) and 1 mM 5. 5-dithiobis (2 nitrobenzoic acid) (DTNB) (30 μL), then incubated for 45 min in the dark at RT and the absorbance was measured at 412 nm in a spectrophotometer (U-1900; Hitachi, Ltd., Tokyo, Japan). GSH concentration was calculated on the basis of the millimolar extinction coefficient of DTNB (13.6 L/mmol/cm). Haemoglobin concentration of erythrocyte lysate was measured using a commercially available kit.All reagents were purchased from Sigma-Aldrich. Each assay was performed in triplicate and within 3 months of collection.2.4. Colostrum and Milk Antioxidant MethodsABTS•+ Radical Scavenging AssayThe 2,2’-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS•+) radical scavenging capacity (RSC) was determined in all colostrum and milk samples collected, as previously described by Cano et al. [34] with minor modifications. Briefly, 1 mL reactions were prepared in distilled water containing ABTS•+ (1 mM), H2O2 (30 µM) and horseradish peroxidase (6 µM) in 50 mM phosphate-buffered saline (PBS; pH 7.5). The solution was vortexed, followed by incubation for 45 min at RT in the dark. Subsequently, 50 µL of colostrum or milk, at various concentrations, was added and was read at 730 nm on a Hitachi by radio beam spectrophotometer (U-1900; Hitachi, Ltd., Tokyo, Japan). In each experiment, a blank lacking the peroxidase was used, while the ABTS•+ radical solution without colostrum or milk was used as the control. The percentage RSC of colostrum or milk was calculated using the following equation: RSC (%) = [(ODcontrol − ODsample)/ODcontrol] × 100, where ODcontrol and ODsample are the optical density (OD) values of the control and the test sample, respectively. Moreover, the IC50 value indicating the colostrum or milk amount that caused 50% scavenging of the ABTS•+ radical was calculated. All experiments were carried out in triplicate and on at least 3 separate occasions.2.5. Chemical Analysis of Colostrum and MilkColostrum and milk samples were assayed for proteins, fat B, lactose, solids not fat (SNF) and total solids (TS©) by using Milkoscan 133 (A/S N. Foss Electronic, Hillerod, Denmark) at the laboratory of organisation ELGO-Demetra in Larissa, Greece. The colostrum samples were diluted at range 1:10, while the milk samples at range 1:5.2.6. Cytokines Assays in Blood and Colostrum SamplesCytokines’ profile of IFN-γ, IL-10, IL-1β and IL-6 was determined in ewes’ and lambs’ plasma and in colostrum samples after an initial step for defatting by centrifugation at 4 °C for 20 min at 4000× g. For all colostrum samples a 1:2 dilution in PBS was made.The ELISA for IL-10 in plasma and colostrum samples was determined according to Kwong et al. [35], with some modification as previously reported in Ciliberti et al. [36], whereas, IFN-γ evaluation was performed according to Ciliberti et al. [37]. Briefly, 96-well plates (Sterilin, Newport, UK) were coated overnight at 4 °C with 100 μL of anti-bovine IL-10 mAb and with anti-bovine IFN-γ (Serotec Ltd., Oxford, UK; 2 μg/mL) in buffer carbonate (pH 9.6). After blocking non-specific binding with PBS and Tween 20 (PBST, 0.05% Tween 20) with 3% of BSA, IL-10 (Serotec Ltd., Oxford, UK) and IFN-γ (Serotec Ltd., Oxford, UK) standards, serum or colostrum samples were added and incubated for 1 h. Biotinylated secondary anti-bovine IL-10 mAb and anti-bovine IFN-γ antibody (Serotec Ltd., Oxford, UK; 2 μg/mL in PBST/1% BSA) were added for 1 h. Subsequently, streptavidin–horseradish peroxidase (1/500 in PBS, Serotec Ltd., Oxford, UK) was added for 45 min. Finally, 3,3′, 5,5′-tetramethylbenzidine substrate solution was added to each well for 30 min, and the colorimetric reaction was stopped adding H2SO4 (2 M). All incubations were performed at RT, and after each step, the plates were washed 4 times. Plates were read at 450 nm by a spectrophotometer (Power Wave XS, Biotek, Winooski, VT, USA) and data were expressed as nanograms per millilitre for IL-10 and pg/mL for IFN-γ.The levels of IL-6 and IL-1β in plasma samples were determined by sandwich ELISA performed in 96-well microtiter plates, according to Ciliberti et al. [36]. The sandwich was build using specific antibody against sheep IL-6 and IL-1β. All the incubations were conducted at 37 °C for 1 h, and after each step, the plates were washed 4 times. Plasma and colostrum samples were read against a standard curve obtained using scalar dilution of recombinant ovine IL-6 (Cusabio Biotech Co., Wuhan, China) and recombinant bovine IL-1β (Kingfisher Biotech Inc., St. Paul, MN, USA). Data were expressed as nanograms of IL-6 and IL-1β per millilitre. Plates were read at 450 nm by a spectrophotometer (Power Wave XS, Biotek, Winooski, VT, USA). The intra-assay CV was around 10% for IFN-γ, IL-10, IL-1β and IL-6; whereas, the average of the inter-assay calculated for low, medium and high concentration was about 7, 11 and 15% for IFN-γ, 3, 7 and 15% for IL-10, and 4, 7 and 4 for IL-1β and 2, 7, and 4% for IL-6, respectively.2.7. IgG Assay in Blood and Colostrum SamplesIgG level was determined in ewes’ and lambs’ plasma samples and in colostrum samples using Sheep IgG Elisa kit (Wuhan Fine Biotech Co., Wuhan, China) following the manufacturer’s instructions. All samples were diluted in PBS and the plates were read at 450 nm by a spectrophotometer (Power Wave XS, Biotek, Winooski, VT, USA). The intra-assay CV was around 10%.2.8. Statistical AnalysisAll data were checked for normality; when this assumption was not satisfied, a Log transformation was performed. Statistical analysis was performed by SAS statistical program (SAS University Edition).Statistical evaluations of the lambs’ weight, of the plasma (TAC, TBARS, GSH) and colostrum/milk (ABTS•+) redox biomarkers and of chemical components of colostrum/milk (proteins, fat B, lactose, SNF and TS©) samples collected at time-series points were compared within each group (MEL or CON) and between the two groups (MEL and CON) by using two-way ANOVA for Repeated Measures. Multiple comparisons were performed by the Tukey’s test.Cytokines (IFN-γ, IL-10, IL-1β and IL-6) and IgG levels in plasma and colostrum/milk samples collected at time-series points were analysed by ANOVA for mixed models using the MIXED procedure with Tukey’s post-hoc correction. Treatment (melatonin), time of sampling and their interaction were used as fixed effect. Animal was a random factor nested in the treatment.Correlations were performed for each animal in pairs for the blood redox parameters (pairs consisted of TAC-GSH, TAC-TBARS or GSH-TBARS) and within time (L0-L40). In addition, correlations were performed, in each group at the same time point, in IFN-γ or IL-10 or IL-1β or IL-6 or IgG levels between ewes’ plasma and colostrum, as well as between colostrum and lambs’ plasma. Moreover, lambs’ IFN-γ or IL-10 or IL-1β or IL-6 or IgG level from colostrum was evaluated in each group from day L0 to L1 and from L1 to L2.The correlations were considered for every measurement throughout the study. Initially, the Pearson’s correlation coefficients r, were calculated in each group separately. Then, the differences of r coefficients between groups MEL and CON were evaluated separately for each parameter effect by using Fisher r to z transformation.Data are shown as least square means ± standard error of the mean. Significant differences were set at p < 0.05 for all tests, while p < 0.10 was considered a tendency. All graphs were prepared using GraphPad Prism version 7.3. Results3.1. Lambs’ Body Weight GainThe mean body weight of lambs (Figure 1) did not differ between groups within time (4.03 ± 0.12 kg versus 3.96 ± 0.12 kg, p = 0.34; 8.64 ± 0.31 kg versus 8.95 ± 0.39 kg, p = 0.55 and 14.20 ± 1.94 kg versus 15.09 ± 2.10 kg; p = 0.29, on days L0, L15 and L40, respectively).3.2. Antioxidant Biomarkers in BloodTAC values showed significant variation within time and were different between the two groups for the first 24 h of life (L0 and L1, p < 0.001, Figure 2). GSH levels showed variation within time and between the two groups (Figure 3). Specifically, at birth (L0), GSH was higher in MEL lambs compared to CON ones (p = 0.046). In both groups, a decrease was recorded in GSH between L1 and L2 (p = 0.003 for MEL lambs and p = 0.025 for CON lambs), followed by a rise on day L5 (p < 0.0001 and p = 0.0013, respectively). The levels of lipid peroxidation (Figure 4), measured as TBARS, varied within time in both groups (p < 0.0005 and p = 0.008, respectively). TBARS levels were significantly lower at birth (L0) in MEL lambs compared to CON ones (p = 0.049).Clear correlations were found in MEL group in the pair TAC-TBARS on day L0 and L1 (r = −0.50151, p = 0.0143; r = 0.448551, p = 0.0271, respectively). In CON group, correlations were found in the pair TAC-GSH on L2 and L5 (r = 0.215439, p = 0.0067; r = 0.133508, p = 0.0014, respectively), in the pair TAC-TBARS at each time point (p < 0.0385) apart from L2 and L10 (p > 0.05) and in the pair GSH-TBARS on L2 and L5 (r = 0.233137, p = 0.05; r = −0.08572, p = 0.0002, respectively). The only significant difference between the two groups was found in the pair TAC-TBARS at L0 (z = −1.78, p = 0.0375). Moreover, a difference was found in CON group in the pair GSH-TBARS on L1-L2 (z = 1.62, p = 0.05).3.3. Antioxidant Biomarkers in Colostrum/Milk SamplesThe ABTS•+ results (Figure 5) revealed that colostrum of MEL ewes showed greater ability of neutralisation at parturition (L0) compared to colostrum of CON ones (p = 0.047). Moreover, the neutralising ability decreased after day 5 (L5) in both groups (p < 0.0001).3.4. Chemical Analysis of Colostrum and Milk SamplesChemical analysis revealed that MEL-treated ewes had a higher amount of proteins (Figure 6) at lambing and higher fat content 24 h later (p < 0.05) compared to CON ewes. Moreover, both groups showed increased levels of SNF and TS© at lambing and decreased levels of lactose on day L5 (p < 0.05).3.5. Cytokines Secretion in Blood of Ewes and Lambs and in Colostrum SamplesIFN-γ secretion in ewes’ plasma was affected by treatment (p = 0.006) and time of sampling (p < 0.0001), registering on average a lower concentration in MEL than in CON ewes; a decreased level on day L1 as compared to L0 was found in both experimental groups (Figure 7a). No significant differences emerged for both IL-10 (Figure 7b) and IL-1β secretion (Figure 7c). Whereas, the level of IL-6 was significantly affected by treatment (p = 0.006) and time of sampling (p = 0.0008). In particular, the MEL ewes registered the highest IL-6 level on day L1 in comparison with MEL ewes on L0 and CON ewes both on L0 and L1 (Figure 7d).The level of cytokines in colostrum samples is presented in Figure 8. IFN-γ secretion in colostrum was significantly affected by treatment (p = 0.013) and by the interaction between treatment and time of sampling (p = 0.015). On average, colostrum of MEL ewes had lower level of IFN-γ than CON ewes; this reduction was significant on day L1 (Figure 8a). IL-10 showed a tendency for the interaction treatment and time of sampling (p = 0.06) with no significance of treatment (p = 0.85) and of time of sampling (p = 0.13). In particular, colostrum of CON ewes had increased levels of IL-10 on day L1 in comparison with L0 (Figure 8b). Both the IL-1β (Figure 8c) and IL-6 (Figure 8d) did not show any significant effect of treatment, time of sampling and their interaction.The IFN-γ level in lambs’ plasma was significantly affected by time of sampling (p = 0.0431, Figure 9a) as demonstrated by the higher level found on average on L2 than on L0. Furthermore, for IL-10 production a significant effect of treatment (p = 0.01), time of sampling (p = 0.008) and their interaction (p = 0.05) was registered. On average, IL-10 was lower in lambs’ plasma on L0 and increased from L1 to L2. Moreover, the MEL lambs on day L2 showed an increase of IL-10 level in comparison with CON lambs and a higher level than that found at birth in both experimental groups (Figure 9b). On the contrary, IL-1β was affected by time of sampling (p = 0.037), with no significant effect of treatment (p = 0.74) and by the interaction between treatment and time of sampling (p = 0.40, Figure 9c); on average, the level of IL-1β decreased from day L1 to L2. No significant effect for IL-6 secretion was registered (Figure 9d).For IFN-γ, clear correlation was found in MEL group regarding lambs’ IFN-γ intake of colostrum during the transition from L1 to L2 (r = 0.888, p = 0.0016). In the CON group, a clear negative correlation was found between the amount of IFN-γ in the colostrum and the levels of them in lambs’ blood (r = −0.825, p = 0.0059). In the same group, a clear correlation resulted regarding lambs’ IFN-γ intake of colostrum during the transition from L0 to L1 (r = 0.805, p = 0.008). The Fischer r to z transformation revealed significant difference in the compared groups (MEL and CON) for IFN-γ levels between colostrum and lambs’ plasma on L0 (z = 1.64, p = 0.05). Furthermore, the same test showed that on L2, lambs’ ability to absorb the amount of IFN-γ, contained in colostrum of day L1, differed between the two groups (z = 1.86, p = 0.0314). Finally, the same test in CON group showed that the IFN-γ levels between colostrum and lambs’ blood differed within time (L0 and L1) (z = −2.04, p = 0.0207).For IL-1β, none of the correlations performed were statistically significant (p ≥ 0.087). The Fischer r to z transformation revealed significant difference in MEL group within time regarding IL-1β concentration in colostrum on L0 and lambs’ plasma on L1 compared to colostrum on L1 and lambs’ plasma on L2, respectively (z = 2.16, p = 0.0154). Moreover, the same test showed that at L1, lambs’ ability to absorb the amount of Il-1β, contained in colostrum of day L0, differed between the two groups (z = 1.72, p = 0.0427).For IL-10, clear correlations were found in both groups at the same time points (L0 and L1) with regard to IL-10 levels between lambs and colostrum (Mel group: rL0 = 0.664, p = 0.0181; rL1 = −0.607, p = 0.0314; CON group: rL0 = 0.762, p = 0.014; rL1 = 0.703, p = 0.026). Moreover, a clear correlation resulted in lambs’ IL-10 intake on L1 of colostrum produced on L0 in MEL group (r = 0.936, p = 0.0322). The Fisher r to z transformation revealed significant difference in the compared groups (MEL and CON) of IL-10 levels between colostrum and lambs’ plasma on L0 (z = −2.1, p = 0.0179). Furthermore, the same test showed that on L1, lambs’ ability to absorb the amount of IL-10, contained in colostrum of day L0, differed between the two groups (z = 2.82, p = 0.0024).For IL-6, significant correlations were found in CON group at both time points (L0 and L1) with regard to IL-6 levels between ewes’ plasma and colostrum (rL0 = 0.819, p = 0.0065; rL1 = 0.696, p = 0.0413). The Fisher r to z transformation revealed significant difference in the compared groups (MEL and CON) in IL-6 levels between colostrum and lambs’ plasma on L0 (z = −2.52, p = 0.0059) and on L1 (z = −3.9, p = 0.000); and in IL-6 levels between ewes’ plasma and colostrum on L1 (z = −3.85, p = 0.0001). Moreover, the same test in MEL group showed that the IL-6 levels between ewes’ plasma and colostrum differed within time (L0 and L1) (z = 3.64, p = 0.0001).3.6. IgG Levels in Blood and Colostrum SamplesIgG level in ewes’ plasma (Figure 10a) and in colostrum (Figure 10b) was not affected by treatment (p = 0.12 for ewes’ plasma and p = 0.18 for colostrum) and time of sampling (p = 0.18 for ewes’ plasma and p = 0.7347 for colostrum). On the contrary, in lambs’ plasma the level of IgG was significantly affected by treatment (p = 0.001) and the interaction between treatment and time (p = 0.03, Figure 10c). On average, lambs born from CON ewes registered a higher level of IgG than lambs born from MEL-treated ewes; furthermore, in lambs born from CON ewes, the level of IgG increased from day L0 to L2 (p = 0.04).Regarding IgG correlations, none of them were statistically significant (p ≥ 0.110). The Fischer r to z transformation revealed significant differences in MEL group within time; in particular, IgG levels between ewes’ plasma and colostrum differed during the transition from L0 to L1 (z = 2.52, p = 0.0059); IgG concentration between colostrum and lambs’ plasma differed during the transition from L0 to L1 (z = −2.36, p = 0.0091). Moreover, the same test showed that on L1, lambs’ ability to absorb the amount of IgG, contained in the colostrum of day L0, differed between the two groups (z = −1.62, p = 0.0526).4. DiscussionThe present study aimed at the evaluation of the effect of melatonin treatment in prenatally stressed lambs on redox status and immune response, as well as on the quality of produced colostrum/milk. Our data revealed that melatonin exerted an antioxidant effect and an immunomodulatory role during the first crucial hours of newborn lambs’ life. To our knowledge this is the first study that evaluated the positive effect of maternal melatonin treatment throughout embryonic development on redox status in conjunction with immune competence of newborn lambs. Additionally, this study indicated that quality assessment of colostrum should be evaluated along with chemical composition, the IgG levels and redox status.Melatonin can protect animals from oxidative stress, even during heat wave exposition [8,38,39]. The peculiar properties of melatonin are supported in our recent study by the increase of GSH levels in the melatonin-treated pregnant ewes followed with simultaneous decrease of TBARS almost from the beginning of pregnancy until 48 h post-partum [8]. Further evidence on the antioxidant effect of melatonin is provided by its ability to reduce lipid peroxidation, a degradative phenomenon involved in the pathogenesis of many diseases [40]. A result of great significance, in the present study, is that MEL lambs were born with better redox profiles compared to CON lambs, supporting their immune competence. Melatonin is known to promote GSH production, an endogenous antioxidant, by direct action to GPx [41], in order to reverse oxidative stress. This effect was confirmed in MEL lambs at birth, which resulted in a concomitant increase of GSH levels and lower levels of lipid peroxidation. The latter is further supported by the comparison of GSH-TBARS r’s in the two groups within time. Fisher r to z transformation revealed that the available GSH in CON group was as a whole offered to reduce TBARS, whereas in MEL group, modifications within time were not observed.Maternal melatonin crosses the placenta unaltered, being an important factor for entraining circadian rhythms of foetuses. Since melatonin is also detected in milk, it contributes to the newborn’s circadian entrainment as well [42,43,44]. Melatonin is a scavenger of both oxygen and nitrogen-based reactive species, and promotes the production of endogenous antioxidants, which leads to a better adaptation of both maternal organism and embryo to heat stress conditions. The amine is a much more potent antioxidant than many traditionally used antioxidants and has two unique features. First, melatonin does not undergo redox cycling and once oxidised, cannot be regenerated to its reduced form [45]. Secondly, the antioxidant action of melatonin involves the donation of two electrons instead of one; therefore, melatonin does not become a free radical in the antioxidant process [45]. Furthermore, melatonin can be metabolised to both kynurenic acid and to N1-acetyl-N2-formyl-5-methoxykynuramine and N1-acetyl-5-methoxykynuramine [38]. These metabolites are considered very powerful antioxidants, cyclooxygenase-2 inhibitors and potentially selective anti-inflammatory agents [13,14,15]. Melatonin induces the expression of the Nrf2 gene and suppresses that of NF-κB through epigenetic processes [9,46]. In addition, melatonin acts as an anti-inflammatory agent, preventing the translocation of NF-κB to the nucleus, thus reducing the upregulation of proinflammatory cytokines [13].Colostrum is a complex biological fluid composed of water, proteins, carbohydrates, lipids, vitamins and minerals. Colostrum is secreted by the mammary gland immediately after parturition and provides nutrition, immunity and defence, as well as growth factors to the newborn [47,48]. In the present study, the last implant was inserted on day 100 of gestation to ensure high levels of melatonin during lactogenesis-colostrogenesis. Indeed, the antioxidant effect of melatonin was clear in colostrum on L0 in MEL ewes, as well as in the TBARS levels in MEL lambs’ plasma. Given the fact that melatonin enhances the production of antioxidant peptides and their presence in the colostrum [47], it could be assumed that the higher antioxidant capacity of colostrum from MEL ewes was attributed to this effect. Moreover, the colostrum of MEL ewes showed higher fat content 24 h later compared to CON ewes. According to others, higher fat content was found in colostrum of ewes treated with melatonin in their third month of pregnancy but no alteration was found in protein content [49]. Molik et al. [50] have shown that melatonin treatment increases protein and fat content of milk, which is in agreement with our results.It is known that birth weight per se is only a reflection of an insult or multiple insults to the foetus during development, indicating that birth weight is not the clearest reflection of a positive interference’s impact in developmental programming [51,52]. In the current study, no differences were observed in the birth weight between MEL lambs and CON ones, although the majority of MEL lambs were twins or triplets in contrast to CON lambs that were mainly singletons. Moreover, no difference was found in the body weight gain between the two groups until weaning, which is consistent with the results of other studies [53]. This finding may also be due to the higher milk yield measured in MEL-treated ewes than in CON, as we have previously described [8]. Perhaps melatonin treatment throughout embryonic development in MEL lambs improved their birth weight.The immunoglobulins are certainly the most important proteins of colostrum. In ruminants, the placentation types prevent the in utero transfer of maternal immunoglobulins. For this reason, newborn ruminants rely on the ingestion and absorption of maternal immunoglobulins, especially IgG, from colostrum [47,54,55]. They are important for subsequent protection against neonatal infectious diseases and other post-partum environmental challenges [55,56], as the neonate’s immune system is not fully functional. On this basis, colostrum proteins can be divided into two major categories: (i) high abundance proteins, mainly immunoglobulins and caseins, and (ii) a wide range of low abundance proteins (acute-phase proteins, antimicrobial proteins/peptides, cytokines, growth-promoting components) (12–15). Data from the present study demonstrated the passive IgG transfer from colostrum to the lambs in both experimental groups, which is in line with the IgG level found in ewes’ plasma. Although no difference was found in IgG levels in ewes’ plasma and colostrum between the two groups, lambs born from CON ewes exhibited a higher level of IgG than lambs born from MEL ewes; whereas, the MEL lambs absorption capacity was gradually increased from birth to 48 h after birth, probably due to a better response to oxidative stress that occurred at birth. Furthermore, the increased level of IgG in CON lambs could be explained by the concomitant high level of IL-10, as demonstrated by Rousset et al. [57], in which the proliferative effect of IL-10 on B cells activated via stimulation of Ig secretions was ascertained.On the other hand, it could be assumed that melatonin treatment prioritises the presence of antioxidant peptides in the colostrum [47] over other proteins, including IgG, as was confirmed in MEL lambs by the lower levels of IgG and better redox status. According to previous studies, a decrease in IgG was shown three days after melatonin injection in lactating cows, with a concomitant decrease in somatic cell count in milk of cows [58,59] and ewes’ milk [60]. Contrary to the latter study and to the present one, others found a higher amount of IgG but no higher amounts of proteins in colostrum just after parturition in melatonin-treated ewes that lambed males compared to females [49]. Accordingly, although different experimental designs were followed, the lower levels of IgG in MEL lambs versus CON ones of the current study could also be attributed to the higher milk yield of their mothers, as we have recently described [8]. Furthermore, melatonin effects on humoral immune responses could be connected to the time of melatonin administration as reported by Carrillo-Vico et al. [61]. In particular, the immuno-enhancing action of melatonin in vivo was more evident during a condition of immune depression and/or when the melatonin is administered in the late afternoon or evening. This last concept could account for the absence of clear effect of melatonin in modulating the immune system in sheep [62]. In our experimental design, the melatonin release occurred in a slow-releasing manner subcutaneously and probably this type of protocol does not imply a boosted humoral response.Once the lamb is 24 h old, transport of IgG across the intestinal epithelium is virtually complete [63], with no further possibility of significant IgG absorption from colostrum. Fisher r to z transformation revealed that absorption capacity was reduced in MEL lambs 48 h after birth in conjunction with the better redox status found at the first days of life in MEL lambs; whereas, in CON ones, this was absent. Furthermore, the same test revealed that the amount of IgG in colostrum of MEL ewes increased within time, as well as lambs’ capacity to absorb it during the first 24 h of life (p = 0.0059 and p = 0.0091, respectively). These findings are in line with others found in calves in which the redox balance of the colostrum was closely associated with the IgG absorption [29]. Likewise, Kamada et al. [64] indicated that colostrum supplementation with selenium increased IgG absorption because selenium is a confirmed antioxidant. In our study, MEL lambs not only resulted in a better redox profile, but probably the better redox status of the colostrum on L1 contributed to attenuate the absorption capacity of the consumed colostrum.Several studies have demonstrated a decline in immune function during the periparturient period in dairy cows; however, the causes of immunosuppression are not completely understood. It is certain that immunosuppression has been linked to endocrine changes associated with parturition, metabolic stresses associated with lactogenesis and availability of critical nutrients, including vitamin E and calcium [65,66]. Caroprese et al. [67] reported that cell-mediated and humoral immune responses and plasma IL-6 level underwent marked fluctuations in periparturient ewes. In the study of Theodorou et al. [68], two of the total three studied Greek dairy sheep breeds exhibited signs of mild immunosuppression during periparturient period. Notably, immune suppression around calving can be measured in terms of lymphocyte proliferation in vitro, antibody secretion and cytokine release [69,70]. Both cell-mediated and humoral responses are activated and regulated by proinflammatory cytokines, such as IL-6 and IL-1β. Both IL-6 and IL-1β are considered the main mediators of the secretion of other proinflammatory cytokines and of the acute phase protein synthesis by the liver [71,72].IL-6 has a wide range of immunological functions, including stimulation of B cells and cytotoxic T cells [73]. Release and activity of IL-6 are strongly controlled and mainly occur under inflammatory conditions, being partially responsible for the increase of certain acute phase proteins [74]. Winter and Colditz [75] found elevated concentrations of IL-1β and IL-6 in ewes’ milk after experimental mammary infection with Staphylococcus epidermidis. The lack of consistent IL-6 trends in blood and milk could be attributed to the fact that this cytokine is likely to be quickly utilised by lymphocytes after having been secreted or inactivated by soluble receptors as described for human IL-6 [73,76]. Hence, our results highlighted the inopportunity of considering IL-6 secretion in milk as an indicator of the physiological stress connected to parturition, in contrast to IL-6 concentration in plasma, which appeared to be a suitable indicator for this purpose. Moreover, during the transition period, the level of IL-6 peaked at parturition then decreased throughout the post-partum period [67]. However, when flaxseed supplementation was offered to ewes, the levels of IL-6 measured after 14 days post-partum increased compared to CON ewes [77]. Similarly, in the present study, the level of IL-6 of MEL ewes at 24 h post-partum increased in comparison to CON ewes.In a study conducted by Fernandez et al. [78] in healthy lambs, it was reported that the serum concentration of IL-10 increased progressively over time and the concentration peaked at day 28 [78]. Accordingly, our data demonstrated, for MEL lambs, a rise in IL-10 secretion within time. It has been demonstrated that IL-10 exerts a potent anti-inflammatory/immunosuppressive property in vitro [57], with both immunosuppressive and immunostimulatory effects on adaptive immune cell mediators via stimulating B-cell functions [79]. Furthermore, in our previous study, a positive correlation between IgG and IL-10 levels has been observed, further supporting the involvement of IL-10 in IgG synthesis in the mammary gland, as has been previously suggested [80]. This statement could help to explain the increased level of IL-10 in colostrum and the enhancement of IgG level in CON lambs 48 h after birth.The proportion of T lymphocyte subsets changes during parturition into Th2 phenotype expression, favouring to the secretion of Th2 cytokines (IL-10, IL-4, IL-13), and during mid lactation into Th1 phenotype expression favouring the Th1 cytokines (IFN-γ and IL-12) [65,81]. In particular, the Th1 cells can activate cellular immunity and inflammatory responses, whereas the Th2 cells control humoral immunity and promote anti-inflammatory responses. The increased level of IL-10 might have a direct suppressive effect on the Th1 cell response by reducing the secretion of IFN-γ [82]. Accordingly, in the present study, the positive correlation found in IL-10 level between colostrum and lambs’ plasma could explain the low level of IFN-γ in MEL colostrum coupled with the high level of IL-10 found in MEL lambs. Moreover, the IFN-γ intake from colostrum by MEL lambs seems to increase as the time passes according to the correlation mentioned above. The latter pattern is observed for IL-10, too.Interleukin-1 is known as an inflammatory cytokine involved in lymphocyte activation and is implicated in acute-phase response [83], host defence against bacterial and viral infections [75] and stress responses [84]. The production of detectable concentrations of IL-1β arises when there is a strong immune challenge [71,85]. Moreover, other research groups failed to detect IL-1 in milk from cows immunised with Staphylococcus aureus α-toxin [86]. In the present study, the absence of IL-1β concentrations in both ewes’ plasma and colostrum samples may be related to absence of acute infection caused in animals.An interesting result occurred during the tested time period. In the MEL group, there was a tendency for increase in the produced amounts of IgG, IL1-β, IL-10 and INF-γ in colostrum or the absorbed ones by lambs, confirmed either by the correlations or “Fisher r to z transformation”. IL-6 demonstrated the opposite tendency in MEL, but in the CON group no tendency for all cytokines and IgG was found at all. This finding might support the immunomodulatory role of melatonin treatment in the offspring.5. ConclusionsEarly life events, including in utero environmental conditions, affect physiological processes and, as a consequence, animal production. Melatonin treatment prenatally probably assisted the newborn lambs in overcoming the oxidative stress connected to birth by supporting the antioxidant defences, reducing ROS production in lambs’ plasma and improving the free radical scavenger capacity of colostrum. Moreover, melatonin contributed to an improvement of colostrum/milk composition, in terms of protein and fat content. The cytokine profile and IgG secretion and intake further support the immunomodulatory role of melatonin. Based on our recent study [8], it could be assumed that melatonin administration throughout pregnancy in heat-stressed ewes could be a management practice not only for the pregnant females but also for their offspring to overcome the oxidative stress after their birth. These potential benefits for lowering vulnerability to insults of offspring are notable for further research focus.

animals : an open access journal from mdpi

10.3390/ani11113160

PMC8614336

In the European Union, gestating sows are housed in groups in pens that often have slatted floors. Slatted floor can cause injuries to the animals’ legs and hooves, leading to potentially painful lameness. The purpose of our study was to verify if the installation of rubber mats on their floors could limit the occurrence of injuries and lameness in gestating sows. Mats were installed on three commercial farms for use over two consecutive sow pregnancies. The mats limited the occurrence of inflammations around the sows’ leg joints but had no impact on the occurrence of lameness or leg and hoof injuries. The benefit of mats would be greater if they were available throughout the sows’ lives and not just during the gestation period. However, the mat design tested was unsatisfactory because manure did not drain well off the mat and sows were dirtied quickly.

Lameness and foot disorders are major health and welfare issues in intensive swine production systems. They are exacerbated when sows are housed in large groups on slatted concrete floors during gestation. Our study aimed to assess the effect of rubber mats in the lying area of the gestation pen on lameness and leg health in gestating sows housed in large pens in commercial conditions. The study was conducted on three commercial farms over two successive gestations. A total of 582 Large White × Landrace sows, housed in 10 static groups, were enrolled: 5 groups in pens with rubber mats and 5 groups on slatted concrete floors. Lameness, bursitis, leg injuries, claw growth defects and claw lesions were measured at the beginning, middle and end of each gestation period. The rubber mats decreased the risk of suffering from bursitis, but had no effect on the risk of lameness, leg injuries, claw growth defects or claw lesions. Sows housed on rubber mats were heavily soiled compared with those on slatted concrete floors because the mats were not perforated for slurry evacuation. Locomotion disorders and foot lesions remained prevalent despite the rubber mats in the lying area of the gestation pens, but adding rubber mats in service rooms and farrowing crates may produce better results.

1. IntroductionLameness in sows is a major health problem in intensive swine production systems. Studies across Europe have revealed that the prevalence of lameness on farms ranges from 9 to 29% [1,2,3,4,5,6]. This painful disorder can have multiple infectious or non-infectious aetiologies, including foot and leg injuries [7]. Pain due to foot and leg injuries is one of the major welfare risks identified for gestating sows [8]. Lameness is also an economic issue, because lame sows are less productive [9,10] and culled earlier than non-lame sows [3,11,12]. Lameness is also one of the major reasons for on-farm deaths or euthanasia [13,14].Housing on slatted concrete floors, which is the main type of flooring for gestating sows in France [15], has been reported as a main cause of lameness [1,2,4,16,17,18]. Concrete floors can lead to excessive abrasion of claws. Wet concrete floors are also slippery and may cause leg injuries. Claws can get caught between the slats and be pulled off. On the contrary, housing sows on soft floors may reduce leg disorders. As an example, several studies have demonstrated the benefit of straw bedding compared with slatted concrete floors on lameness and foot health of sows and gilts [1,2,4]. However, straw litter, even on just part of the floor, is not compatible with slatted floors and liquid-manure handling systems. Therefore, rubber mats on slatted floors in the lying areas may be a solution to improve floor softness without impairing slurry evacuation by scraping. Mats increase the contact area between the animal’s body and the floor and limit push forces on body parts in contact with the floor [19], thereby improving the comfort of animals lying down on the floor. Using a preference test, Parsons et al. [20] demonstrated that lame sows preferred rubber mats to concrete floors.Positive effects of rubber mats on the reduction of locomotor disorders have already been described for growing pigs [21,22]. In farrowing crates, rubber mats decrease sow shoulder ulcers [23,24,25,26,27,28]. In service crates, they limit claw lesions [29]. In gestation pens, rubber mats improve leg health of sows housed in small groups (four to eight animals), but show inconsistent results on lameness [30,31,32]. In larger groups of gestating sows, rubber matting has showed positive effects on overall leg health [33] and on lying comfort [34]. However, the effect on lameness has not been assessed [34] or has been assessed on small groups (20–25 sows) in experimental conditions only [33]. The assessment of the impact of rubber mats (in the lying area) on lameness in large groups of gestating sows is of importance because group housing—especially large group housing—has been identified as a risk factor for lameness [1]. Given that housing gestating sows in groups is mandatory in the European Union [35], rubber mats may reduce the risk of lameness associated with this housing system.Our study aimed to assess the effect of rubber floor mats installed in the lying area of large gestation pens on lameness and leg health of gestating sows housed in commercial conditions. The sows were followed over two successive gestations. Several parameters on claw lesions, body condition and performance were also monitored.2. Materials and MethodsThe experimental procedure was designed at the officially approved organization for animal experimentation ANSES (official number D22-745-1). The experiment lasted from January 2016 to August 2018 and was then conducted during all seasons and under different thermal conditions, as recommended by EFSA [36].2.1. Study PopulationThe study was conducted on three commercial farrow-to-finish farms located in Brittany, western France. These farms previously encountered lameness issues in sows. The prevalence of lameness in the three farms was 22% (confidence interval of 95% [18,19,20,21,22,23,24,25,26]) at the beginning of the study. Two of them (Farms A and B) had 250 sows and the third (Farm C) had 700 sows. The longitudinal study monitored Large White × Landrace sows housed in 10 static groups, over 2 successive gestations, from 6 weeks before the first service (Week 0) to 1 week before the second farrowing (W33). During the gestation period, the groups were made up of sows at the same stage of gestation, but with different parities: 4 groups of 50 sows on Farm A, 4 groups of 45 sows on Farm B and 2 groups of 95 sows on Farm C. During service, from 1 week after weaning to 4 weeks after insemination, sows were housed in individual stalls floored with concrete slats. During gestation, sows were housed in group pens as described below in the experimental design. During the farrowing and suckling periods, sows were housed in individual farrowing crates with standard slatted metal flooring. Piglets were weaned at 28 days old.All sows present within the groups were observed. Some sows were removed from the groups and from the experiment due to reproductive failure or culling. Conversely, nulliparous sows were added to the groups at the second gestation cycle. Consequently, group composition changed over cycles. Over the 58 visits carried out on the farms, 582 sows were observed at least once: 220 sows on Farm A, 186 on Farm B and 176 on Farm C. Due to feasibility constraints, not all sows could be observed during each visit and the number of observations varied among sows. When the study started, 17% of the sows were primiparous and 11% had a parity of greater than five.2.2. Experimental DesignRubber mats were tested during the two gestation periods, from W0 to W12 and from W21 to W33 (Figure 1). Monitored gestating sows were housed in 10 group pens on the 3 farms: 4 on Farm A (4 m × 65 m), 4 on Farm B (4 m × 65 m) and 2 on Farm C (2 m × 140 m). In half of the group pens, evenly distributed across farms, the lying area was covered with rubber mats. This area represented 40% of the floor in the group pens. The rest of the area remained a fully slatted concrete floor with a permeability less than 15% in accord with the European regulation [35]. The floor of the other pens, considered control pens, was fully slatted concrete. The interval between two batches of sows was three weeks. During the first monitored gestation period, groups of sows were allocated to rubber mat or control treatments alternately every 3 weeks. Thus, five groups of sows were housed in pens partially lined with mats (“rubber mat”–RM treatment) and five groups were housed in pens with fully slatted concrete floors (“control”–C treatment). Each group of sows was allocated to a pen and stayed on the same type of floor during the two gestation periods. The animals were housed at a density defined by Directive 2008/120 [35] and fed using an electronic feeder (liquid feed for farms B and C and solid feed for farm A). Water was freely available from nipple drinkers.The rubber mats (BioretAgri, Nort-sur-Erdre, France) were made of a foam underlayer for suppleness, and a rubber upper layer (thickness: 10 mm) (Supplementary file 1). The rubber layer was grooved to provide a non-slip surface and contained woven thread to improve durability. Mats were attached in pens’ laying boxes using baseboards nailed to the partitions and threshold bars nailed to the slatted floor in the open part of the box. The mat was not perforated and it had a slight slope (3%) to favour slurry runoff.2.3. Measures and Data CollectionThe sows were observed in gestation and farrowing pens and during transfers between rooms, the most convenient places and times for observations. Sow lameness was scored during the transfer between the service room and the gestation pen at W0 and W21 (Tb, beginning of gestation time), inside the gestation pen at W6 and W27 (Tm, mid-gestation) and during transfer between the gestation pen and the farrowing pen at W12 and W33 (Te, end of gestation) (Figure 1). Transfer corridors had a concrete floor. The sows were observed while they walked a distance of at least 15 m, except very lame sows, which were not forced to walk. Lameness was scored according to a four-category table based on the Welfare Quality protocol [37]. Non-lame sows (0) were distinguished from slightly lame sows (1) who had difficulties walking but were still using all theirs legs. A score of two corresponded to animals with a severe lameness; they put a minimum weight on the affected limb; a score of three was given to animals unable to walk. For statistical analysis, scores of two and three were aggregated in the “severe” class. On the same occasions than lameness observations, wounds on the legs and manure on the body were measured according to the Welfare Quality three-category scales, scored on both sides of the sows. For the wound score, the scale varied from zero (no visible injury or up to four lesions) to two (more than 15 visible lesions). Scores for manure on the body ranged from zero (up to 10% of the body surface soiled) to two (more than 30% of the body surface soiled). Scores of two and three were aggregated in the “very soiled” class for statistical modelling. Bursitis was counted on all four legs. Body condition was assessed using a three-category score derived from the Welfare Quality (zero—thin, one—normal condition, two—fat).Claw lesions were visually scored on the hind legs when the sows were lying in farrowing pens on four occasions (W-6, W12, W15 and W33) for the convenience of the observations of the four legs. The three-category scoring system for claw lesions was based on the lesion scoring table proposed by Enokida et al. [38]: zero (no lesions), one–two (superficial cracks and/or overgrowth), and three (deep cracks or wrenching). The heel, the junction between the heel and the sole, the white line (junction between the sole and the wall), the wall, the growth of major claws and the growth and integrity of dewclaws were scored. Scores for the heel, junction, white line and wall of the two hind claws were summed to obtain an erosion score (from 0 to 12). An erosion score of zero or one was considered “no erosion”, a score between three and six “mild erosion” and a score over six “severe erosion”. Scores for the growth of major claws and dewclaws were summed to obtain a growth score for each sow (over 24).Measurements were taken by six trained assessors. Training sessions of observations were carried out for ensuring inter-observer agreement at the beginning of the study. To control observation bias, each assessor monitored the same sows, equally distributed in RM and C groups within each farm, from the beginning to the end of the experiment. Reproductive performance, in terms of number of piglets born, number of piglets born alive and number of weaned piglets, were recorded for all sows over the two gestation periods.2.4. StatisticsScores obtained at the beginning of the Cycle 1, before affecting sows to treatments, were considered as baseline for ulterior comparisons. Lameness, bursitis, claw defects and body condition are shown in terms of frequencies of observation and of numbers of sows affected at least once over the study period. Distributions of erosion score and claw growth score are given. All statistical analysis were performed in R version 3.6.2 [39]. Factors considered for statistical analysis were Treatment (C and RM), period of gestation (Tb, Tm for lameness and bursitis and Te), cycle of gestation (Cycle 1, Cycle 2) and parity of the sow when enrolled (first gestation, parity 2 to 5 and parity >5). These four factors were introduced as fixed effects in generalized linear mixed models (GLMM). A random component was introduced in the models accounting for the effects associated with a sow, within a pen, within a farm (nested effects). The Treatment × Cycle interaction was tested for all models and removed if non-significant (p > 0.05). Risks of suffering from bursitis, leg injuries, scratches, and abnormal body condition (thin, fat) were modelled using a mixed-effect logistic regression model (binary response as affected vs. non-affected); the function glmer from the lme4 package [40] was used. Other measures related to lameness, claw defects and body cleanliness were categorized on a three-score scale (absence–mild–severe) according to score frequencies. The risks of suffering from mild and severe conditions were modelled using an ordinal regression based on a cumulative link mixed model (function clmm from the ordinal package [41]). Results from the GLMM models are reported as odds ratios adjusted on the fixed effects with their confidence intervals at 95%. A factor with an odds ratio higher than 1 (i.e., the lower limit of the OR’s confident interval at 95% is higher than 1) increases the risk of suffering from the studied condition. In contrast, a factor with an odds ratio lower than one (upper limit of the OR’s Confident Interval at 95% is lower than 1), can be considered as a protective factor. Odds ratios and their confidence intervals are shown in figures and the complete results of the models are provided (Supplementary file 2). Reproductive performance traits were analysed using ANOVA mixed models with Treatment, Cycle and Parity as fixed effects and Pen, nested in Farm, as a random effect (lmer function from lme4 package). Results are shown as least square means with standard error. Post-hoc comparisons of means were performed using the Tukey correction (package emmeans [42]) for multiple comparison adjustments.3. ResultsAt the beginning of the study, the prevalence of lameness was similar within treatments: 23% ([17,18,19,20,21,22,23,24,25,26,27,28]) in treatment RM vs. 21% ([16,17,18,19,20,21,22,23,24,25,26]) in treatment C (p = 0.33). No Treatment*Cycle interaction (p < 0.05) was observed for any of the parameters under study, suggesting that the effect of rubber mat was constant over the two observed gestation periods.3.1. LamenessLameness was reported for 28% of the observations (663/2361), from a moderate state (17% of the observations) to a severe state on 39 occasions (2% of the observations, Figure 2). From the 495 sows observed for lameness scoring (no data available for 87 sows due to feasibility constraints), 256 were lame at least once (52%) and 7 sows exhibited severe lameness. The risk of suffering from mild or severe lameness increased during the gestation period: the odds ratio (OR) was 2.1 (CI 95% [1.6–2.8]) when comparing Tm with Tb (p < 0.001) and was 2.5 ([1.9–3.3], p < 0.001) when comparing Te with Tb (Figure 2). No effect of RM treatment was observed on the risk of suffering from lameness (RM vs. C, OR = 0.50 [0.13–2.0], p = 0.33).3.2. Leg ConditionBursitis was noted on 21% of the observations, affecting 226 sows at least once (39% of the sows, Figure 3). Up to five bursitis were observed on a sow on three occasions, but 55% of the observations corresponded to one bursitis affection only. The risk of being affected by bursitis (at least one bursitis) was significantly lower at the end of the gestation period for the sows on rubber mats only: in the RM treatment, the OR associated with Te vs. Tb was 0.42 [0.20–0.60] (p = 0.03). On the contrary, the frequency of bursitis was unchanged over the gestation period for sows in the C treatment (OR associated with Te vs. Tb = 0.8 [0.55–1.21], p = 0.35). Primiparous sows were less frequently affected with bursitis (OR = 0.39 [0.22–0.68], p = 0.001) than older sows. The risk of suffering from bursitis turned out to be lower during the second gestation cycle than the first one (OR = 0.71 [0.55–0.93], p = 0.01)). Presence of leg injuries was reported for 142 observations out of 1650 (9%); 115 sows (20%) exhibited wounds on least at one visit. The probability that a sow suffers from leg injury was lower at the end of the gestation period compared with the beginning (OR = 0.55 [0.38–0.80], p = 0.002), but was not influenced by the rubber matting (OR = 1.4 [0.16–11.7], p = 0.77) in comparison with concrete floor.3.3. Claw ConditionDewclaws exhibited lesions more frequently (54% of the observations) than major claws (12%). Out of 576 sows, 174 (30%) showed a claw growth defect score (sum of scores on dewclaws and major claws) higher than 3 out of 6 (severe) at least upon one observation; only 159 sows (28%) had no claw defects (score 0) over the whole study period. Heels were more heavily worn than the other parts of the hind claws (Figure 4). No erosion (score 0 or 1) was observed for 11% of the observations, mild erosion (score [2,3,4,5,6]) for 66% of the observations and severe erosion (score over 6) for 23% of the observations. Overall, 224 sows (out of 576, 39%) had a severe erosion score on at least one occasion, whereas 27 sows (5%) had a “no erosion” score over the whole study period. The presence of a rubber mat had no effect on the claw growth-defect score (OR = 0.92 [0.67–1.25], p = 0.59) or on erosion score (OR = 0.65 [0.24–1.81], p = 0.41) (Figure 5). The probability of observing claw growth defects was greater (OR = 1.6 [1.3–2.0], p < 0.001) during the second gestation cycle than the first one. The risk of “mild” or “severe” erosion of claws was higher at the end of the gestation period than at the beginning (OR = 1.7 ([1.4–2.1], p < 0.001). Claws of sows with parity >5 at the beginning of the study were more worn (OR =2.4 [1.7–3.5], p < 0.001) than those of younger sows.3.4. Body ConditionAbout two-thirds of the sows were scratched (756/2361 observations, 68%), including 4% heavily scratched sows. Two-thirds of the sows were scratched on at least one occasion (68%, 336/495). The risk of being scratched increased during the gestation period (OR associated with Te vs. Tb = 7.7 [5.8–10.5], p < 0.001) (Figure 6). No effect of the RM treatment was observed (OR associated with RM vs. C = 0.73 [0.19–2.9], p = 0.66). Scratches were less frequent during the second cycle than the first one (OR = 0.52 [0.42–0.66], p < 0.001). Sows were judged as clean in 58% of the observations (1377/2360 observations) whereas scores for lightly soiled (1), soiled (2) and heavily soiled (3) sows accounted for 29%, 12% and 0.4% of the observations, respectively. State-of-cleanliness strongly varied over the gestation period and among treatments, as shown by the significant Time*Treatment interaction. Sows in the RM treatment became soiled more quickly and more heavily than those in the C treatment: at Tm, the OR associated with RM vs. C was 4.3 [2.5–7.6] (p < 0.001) and at Te, the OR associated with RM vs. C was 3.5 [2.0–6.3] (p < 0.001). The older sows (parity >5 at the beginning of the study) were less at risk of being soiled than the other sows (OR = 0.70 [0.49–0.99] in comparison with sows with parity 2–5, p = 0.05). Conversely, primiparous sows at the end of the gestation period were dirtier than the other sows (OR = 1.6 [1.2–2.2], p < 0.001). Thin sows were reported for 108 observations over 2353 (5%) and 15% of the sows (73/495) were judged as thin at least once. Forty-three percent of the sows (213/495) were judged as fat at least at one visit, accounting for 24% of the observations (561/2353). Frequencies of abnormal body condition (thin or fat) were not affected by the type of floor. The risk of thinness (in comparison with normal condition) was lower during the second cycle of gestation (OR = 0.43 [0.26–0.72], p < 0.001).3.5. Reproductive PerformanceThe numbers of born piglets, viable piglets at birth and weaned piglets per sow were not affected by the floor lining in the gestation pen (Supplementary file 2). The number of weaned piglets per sow was higher for Cycle 1 (12.6 [11.5–13.7], p < 0.001) than Cycle 2 (12.0 [10.4–13.7]) and for primiparous sows (12.8 [12.4–13.2]) or parity 2 to 5 (12.4 [12.1–12.7]) in comparison with older sows (11.7 [11.3–12.1], p < 0.001).4. DiscussionThe aim of this study was to assess the impact of a rubber mat floor lining on lameness and foot health of gestating sows housed in large groups in commercial conditions. Adding a rubber mat floor lining in the gestation pens protected sows from bursitis development during the gestation period. However, it did not improve lameness, claw state or body integrity. Additionally, the mats, which were not perforated, impaired slurry evacuation. Sows on rubber mats became more quickly and more heavily soiled than sows kept on fully slatted floor. Previous studies reported results on the impact of rubber mats on foot health and on the risk of lameness in sows [21,23,27,29,30,32,33]. However, our study provides new results obtained on pregnant sows housed in large groups and under real production conditions, which have never been studied before.No protective effect of the rubber mats was observed for lameness, claw erosion, claw defects, leg injuries, body condition or reproductive performance. In previous studies, rubber matting has shown a beneficial effect on locomotor disorders in experimental conditions, particularly during periods when sows were housed in crates with rubber mats [30,31,33]. In commercial conditions, the beneficial effects of rubber mats would possibly have been observed if the mats had been installed over the entire area of the pen. Prior to this study, we tested groups pens fully lined with perforated rubber floor mats during a pilot study. However, the perforations in the mats did not line up with those of the slatted floor and problems of slurry evacuation occurred. It was impossible to produce perforated mats that perfectly matched with the slats of the floor because the slatted floors were different in the three studied farms. Slatted floors directly covered with a layer of soft material can be considered to cover the whole surface of the gestation pen but this solution implies changeing the slatted floor. Better results may be possible if the sows were to spend more time on a more comfortable floor, i.e., in their service rooms and farrowing pens. Sows were kept in gestation pens for only half of the production cycle (12 out of 21 weeks). Nevertheless, poor locomotion scores remained high on both floor types, as already described in previous studies [31,43]. The prevalence of mild and severe lameness was the highest at the end of the gestation period, as in previous studies [31,43]. Conversely, lameness decreased between the two gestation periods, probably because the sows were housed in individual stalls during farrowing and service and could no longer walk [43].Rubber mats in the resting areas significantly decreased the risk of suffering from bursitis. Bursitis is an inflammation of the bursa surrounding a joint. Inflammation is caused by repeated pressure or friction at the joint and can be a source of pain [44]. Bursitis is also considered a risk factor for lameness [45]. Bursitis count is one of the control points in the Welfare Quality protocol for assessing floor comfort. In our study, lying on a softer floor reduced the number of potentially painful bursitis-affected joints and was more favorable to the welfare of the animals, as demonstrated previously [30].Occurrence of claw growth defects and state of claw erosion were not influenced by the presence of rubber mats. The frequency of leg injury was also unaffected by the type of floor. Previous experimental studies reported mixed effects of rubber mats on the foot health of gestating sows [32,46]. In a study focused on the beginning of gestation, when sows are mixed in a pen, Elmore et al. [32] observed a reduction of lesions on the bodies of sows housed on rubber mats in comparison with sows housed on concrete floor. However, no difference was observed for leg injuries. Similarly, Jais et al. [46] reported no effect of rubber mats on leg injury of sows followed during two years (up to five gestations), but claw-horn erosion was less severe for sows on rubber mats. In this study, rubber mats did not improve claw state in terms of horn fracture and growth defects. We observed no significant effect of rubber mats on the severity of claw erosion and on the occurrence of claw defects in our study, but the study period (two gestation cycles) may not be long enough to observe the long-term effects of rubber matting on claw health. Erosion and claw defects increase when sows get older, as shown in our study for sows with a parity greater than five.Regarding body condition, the risk of being scratched was as high in sows on rubber mats as in sows on concrete slatted floor. Most of the scratches observed were due to fights when sows were grouped in gestation pens. No influence of the type of floor was observed on sow body condition, in line with results obtained on lactating sows provided with rubber mats [27]. In contrast, body cleanliness was poor for sows on rubber mats at the end of the gestation period. The gentle slope on the rubber mat surfaces was not sufficient to ensure optimal slurry evacuation. In fact, the mats were so dirty that we could not assess their state of wear during the gestation periods. However, body cleanliness is an important factor for welfare, in particular for the thermal comfort of the sows (Welfare Quality, 2009). Perforated rubber mats are highly recommended to maintain the body cleanliness of sows [30,31]. In addition, several mats were replaced between the two gestation cycles because the sows had chewed them—in particular, on one farm where the sows were not provided with enrichment material during the first gestation, despite the experimental design and the mandatory practices stipulated in Directive 2008/120. Foraging and chewing are important activities in normal pig behavior. In absence of chewable material, this behavior may be redirected to material present in the pen, such as the rubber mats. Therefore, providing enrichment material, even sub-optimal material such as metal chains, is needed to improve sow welfare and prevent the excessive wear of the mats.Reproductive performance traits were similar between sows on rubber mats and sows on slatted concrete floors, in terms of litter size, number of piglets born alive and number of weaned piglets per sow. In the Lagoda et al. study [47], gilts housed on rubber mats experienced less intense aggression when grouped in the gestation pens than the gilts housed on slatted concrete floors. As a consequence, the number of dead-born piglets was lower for the sows on the rubber mats at the second farrowing. This may be due to the less stressful environment for those sows. However, the overall productivity of the sows was not affected by the type of floor lining, as in the present study. In fact, sow lameness seldom affects reproductive performance and piglet health [2]. As rubber mats in the laying area did not affect the occurrence of lameness in sows in the present study, it was unlikely to observe an increase in reproductive performance and in piglet performance. Given that improvement in reproductive performance traits is expected to be low in sows on rubber mats, longitudinal cohort studies on a small number of farms may not be the most appropriate study design from which to draw conclusions on these parameters. Large surveys based on performance data are likely to be more statistically powerful for that purpose. The number of weaned piglets was higher for the second cycle of gestation in comparison with the first gestation cycle. The first gestation cycle was from June to October, whereas the second gestation period was from December to March in the three studied farms. Sow fertility and prolificacy are known to decrease during summer [47]. Hot temperatures may have negatively affected reproductive performance during the first gestation cycle. Other factors might decrease reproductive performance, like the parity rank. Primiparous and aged sows usually exhibit lower reproductive performance than females with parity ranks between two and five [47], as observed in the present study.5. ConclusionsHousing pregnant sows on floors partially lined with rubber mats instead of a fully slatted floor had some positive effects, such as reducing the occurrence of bursitis. However, the overall effect of the rubber mat lining was limited on lameness, foot and claw health and therefore on sow welfare. Locomotion disorders and foot lesions remained prevalent despite the installation of rubber mats. Better results may be expected with the use of rubber mats in service rooms and farrowing crates also, i.e., during the whole servicing period of the sows. The interest of rubber mats should therefore be tested in a more global approach, covering the entire servicing period and integrating other parameters such as the provision of adequate chewing material. The design of rubber mats also needs to be improved (more efficient perforations for slurry evacuation, improved resistance to chewing) to develop their use in commercial pig farms.

animals : an open access journal from mdpi

10.3390/ani11113082

PMC8614437

Waste management strategies which favour the value of the organic waste instead of its disposal should be developed. One of the insects able to convert biowaste into high valuable protein and fat is the black soldier fly (Hermetia illucens). This article describes a preliminary assessment of the larval growth and biowaste conversion of Hermetia illucens larvae reared on digestate (biogas slurry), pig manure and vegetal residue-based waste in order to select most promising organic waste for further research. Larval growth was highest when reared on “Swill” (catering waste) and was almost twice as high as on the reference substrate “Chicken feed”. Other biowaste sources tested in this experiment with potential for further research were “Pig manure liquid mixed with chicken feed” and “Pig manure solid”. Most promising organic waste sources from this experiment were selected for a follow-up experiment to study the effect of different combinations of organic waste on biowaste conversion by Hermetia illucens larvae.

Insects can play an important role to upgrade waste streams into high-grade proteins and fats as food and feed ingredients or non-food products. The aim of this research was to assess the feasibility to use waste streams with a low value for direct application as animal feed as substrates to grow BSF larvae in terms of larval growth rate, waste reduction index, and efficiency of conversion of ingested feed. The growth of black soldier fly (BSF), Hermetia illucens larvae and conversion of biowaste was assessed in triplicate in biowaste substrates: chicken feed (CF; reference diet), pig manure solid (PMS), Betafert® solid (BTFS), swill (SW), olive pulp (OP), pig manure liquid mixed with chicken feed (PMLCF), and silage grass (SG). Per kilogram fresh substrate 2500 starter (8-days-old, second instar) larvae were incubated in 21 plastic containers (75 × 47 × 15 cm). The BSF larvae were fed according to a batch feeding system. Highest growth rate was found in larvae reared on SW (13.4 mg/d). Larval growth rate was even higher than in larvae reared on the reference substrate CF (7.2 mg/d). Growth rate in larvae reared on PMLCF (7.3 mg/d) did not differ from CF, whereas growth rate of larvae reared on PMS (3.2 mg/d) was lower than on CF. Growth rate of larvae reared on BTFS, OP and SG was very low (0.6, 0.2 and 0.7 mg/d, respectively). Waste Reduction Index (WRI) was highest on SW (11.3), followed by PMLCF (9.3), and both were higher than WRI on CF (8.5). Waste Reduction Index further decreased in descending order from PMS, SG, BTFS to OP (7.6, 4.0, 2.9 and 1.7, respectively). The Efficiency of Conversion of Ingested substrate (ECI) was highest on SW (0.31), followed in descending order by PMLCF, CF and PMS (0.25, 0.21 and 0.18, respectively). The substrates OP, BTFS and SG (0.16, 0.15 and 0.14, respectively) resulted in a lower ECI than other substrates. Highest CO2 and lowest NH3 concentrations were found above substrates with the highest larval growth performances. This study showed that BSF larvae can be reared on different biowaste substrates; the growth rate of the larvae was extremely high on SW. The effects of chemical composition and physical properties of the substrates on larval growth and gas emissions should be further considered.

1. IntroductionRoughly one-third of food produced for human consumption is lost or wasted globally, which amounts to about 1.3 billion tons per year [1]. In the European Union, despite the production of 89–100 million tons of biowaste every year, only around 3 million tons are recycled as livestock feed due to consumer and farmer concerns related to food safety and disease control [2]. Waste management strategies, which favour the value of the organic waste instead of its disposal, should be developed. Animals may be used to provide a significant contribution to waste treatment, in addition to the well-known aerobic bacterial and anaerobic degradation of biowaste during composting and biogas production. Biowaste that cannot be used directly as feed ingredient may be fed as a first step to insects to upgrade this waste into high-grade proteins and fats as food and feed ingredients or non-food products. Biowaste that is suitable and allowed to feed directly to livestock animals should be fed directly, otherwise sustainability of the production chain is not improved. Insects associated with manure and biowaste can play a key role for the sustainable valorisation of biowaste streams [3]. One of the insects able to convert biowaste into high valuable protein and fat is the black soldier fly (BSF; Hermetia illucens L.) [4,5].The conversion potential of biowaste by BSF larvae is heavily dependent on the nature of substrates provided to these larvae [6,7,8,9]. These authors implicate that the concentration and ratio of protein and carbohydrates affect development and nutrient composition of the larvae. Moisture content in the substrates also affects development and harvesting of the larvae. Residue from food waste containing 70% or 75% moisture could be effectively separated from insect biomass by using a 2.36 mm sieve but this was not feasible for residue from food waste with 80% moisture [10]. The presence of excessive moisture content in the waste can hinder its decomposition rate and the resulting residue with a sticky consistency is known to result in a clumpy material difficult for further processing [11]. Texture of the substrate is also important for mobility of the larvae and accessibility to nutrients, but so far literature is scarce on this issue. This study offers a preliminary assessment of the development and growth of Hermetia illucens larvae reared on digestate (biogas slurry), pig manure and vegetal residue-based waste varying in dry matter content and texture. The waste streams were selected, based on the current economic value, as those are currently used as a fermentation source or offered for incineration, with the aim to assess the feasibility to use these waste streams as substrates to grow BSF larvae in terms of larval growth rate, waste reduction index, and efficiency of conversion of ingested feed. Converting these substrates into high-grade insect products will highly contribute to a circular and climate-neutral society, and after this study on development and growth of Hermetia illucens larvae, the most promising waste streams will be selected for further optimizing by mixing in follow-up experiments.2. Materials and MethodsBlack soldier fly (BSF; Hermetia illucens) larvae were obtained from the commercial insect rearing company Bestico B.V. (Berkel en Rodenrijs, The Netherlands). The larvae were 8 days of age at arrival. In total, seven rearing substrates were tested: Chicken feed (CF; reference diet, broiler grower diet, Agri Retail B.V.), pig manure solid (PMS), Betafert® solid (BTFS), swill (SW), olive pulp (OP), pig manure liquid mixed with CF (PMLCF), and silage grass (SG). Chicken feed (Agruniek Rijnvallei Voer BV, Wageningen, The Netherlands) was used as a reference diet (19.8% crude protein, 5.3% fat, 6.2% crude fiber, and 5.2% crude ash). Chicken feed was also used as a reference diet in other experiments [12,13]. Pig manure solid were sow faeces provided by Van Beek SPF Varkens B.V. (Lelystad, The Netherlands). Betafert® is digestate (biogas slurry) from the SuikerUnie biomass digester in Dinteloord (The Netherlands) and was delivered by AgriBioSource Europe B.V. (Nijmegen, The Netherlands). The digestate is produced from sugar beet residues (beet pulp, leaves and root tips) after solid-liquid separation. Catering swill was provided by Tekagroup BV (Barneveld, The Netherlands) and mainly consisted of pasta, potatoes and vegetables (no animal products). Olive pulp (also called olive pomace, olive mill waste or olive cake) is what remains after olive oil extraction (pulp, skins, kernels and water) and was obtained from ACRRES (Lelystad, The Netherlands). Pig manure liquid was pig slurry (mixed faeces and urine) (same source as the solid pig manure) and was mixed with CF. Grass silage was nature grass obtained from Staatsbosbeheer (The Netherlands) fermented by ensiling at ACRRES (Lelystad, The Netherlands).Substrates were obtained one week prior to the start of the rearing cycle and kept at 4 °C until further use. Different quantities of substrates were provided in the containers to realise a substrate layer of approximately 5 cm (Table 1). A batch feeding strategy was applied, which means that the substrates were placed in the plastic containers one day prior to placing the larvae at the start of the experiment. This allows the substrates to heat up until the start of the experiment. The substrates were not pre-treated other than chicken feed (CF) and olive pulp (OP) substrates were mixed with water (1:1 w/w). Pig manure liquid was mixed with CF (1:1 w/w). Particle size of the different substrates was not recorded. On top of each substrate, 2500 starter (8 days-old) larvae per kilogram (wet) substrate were incubated in 21 plastic containers (75 × 47 × 15 cm) (Table 1). Each substrate was tested in triplicate in a climate chamber. The temperature was 30.1 ± 0.5 °C and relative humidity (RH) was 46.4 ± 3.7% at day 1 and 60.6 ± 1.6% from day 2 until the end of the experiment. The photoperiod was 0:24 h light:dark. The plastic containers were randomly arranged in three piles with vents between the containers. On day 3, water (450 mL/container) was added to the containers with PMS because from visual observation it was concluded that this substrate was too dry for consumption by larvae. The duration of the experimental period for CF, PMS, SW and PMLCF was 8 days and the duration for BTFS, OP and SG was 11 days. Experimental durations varied according to the substrate consumption estimated from the 21 containers and/or with the appearance of 10% prepupae. Dry matter content of the substrates at the start of the experimental period and of the substrate/frass and BSF larvae at the end of the experiment were determined by oven-drying for 48 h at 105 °C. Mean individual start wet weight of the larvae (11.43 mg) was determined from three samples of ca. 400 larvae. The containers were placed in three boxes (120 × 100 × 60 cm) mostly at random but not completely as the containers with highest moisture content were placed at the bottom to avoid escaping larvae falling down in containers below them. Duplicate samples of substrate, frass and larvae were collected from the remaining substrate in each container and larvae were separated from substrate and frass, counted and weighed. The number of larvae collected in these samples varied between 45 and 756 and mean larval wet weight was calculated from these samples. Survival of the larvae could not be determined because removing the top-layer of the substrate/frass included an unknown number of larvae. The method used to calculate the larval growth rate (g/d) was according to [14]:Larval growth rate = (final larval average weight − initial larval average weight)/number of days of the trial.(1)To evaluate larval efficiency in consuming and metabolizing the growing substrates, the total final biomass (larvae + prepupae) and the residual substrates were weighted. Waste reduction index describes the larval ability to reduce feeding substrates, taking into account the number of days the larvae were fed on the substrates; therefore, higher values show a greater ability to reduce the organic matter. The conversion efficiencies are based on dry matter because efficiencies on a fresh matter basis can be obscure, as considerable variation is present in the dry matter contents of the substrates. Waste reduction index (WRI) and the efficiency of conversion of the ingested feed (ECI) were calculated for the determination of the waste consumed by the larvae and the conversion efficiency of the substrates into insect biomass. The following indexes were calculated:Waste reduction index (WRI) = ((W − R)/W)/days of trial (d) × 100(2) where W= total amount of substrate provided; R = residual of the substrate, and Efficiency of conversion of the ingested food (ECI) = B/(W − R)(3) where B = total larval + pupal biomass at the end (g); W = total amount of substrate provided; R = residual of the substrate.CO2 and NH3 gas concentrations in the climate chamber were measured manually at different times per day. CO2 and NH3 concentrations were also measured at substrate level by use of plastic buckets and a calibrated Innova 1412A-5, a photo-acoustic infrared detection method from LumaSense Technologies. The air quality sensor detects gases through chemical reactions, producing a current that is directly proportional to the concentration of target gas present and shows good responses to various gas concentrations over a wide range of ambient conditions. The buckets were placed on the substrates in the containers and gases were measured three times during a period of 30 min at day 8. Dry matter content, larval growth and bioconversion data were analysed by analyses of variance using the statistical program Genstat, 19th Edition (https://www.vsni.co.uk/software/genstat/, accessed on 17 November 2020) [15] in a randomized block design. Each rearing box served as a statistical unit and a pile of seven plastic containers was considered as block. Data are presented as the mean and considered significant if p ≤ 0.05 and a tendency if 0.05 < p ≤ 0.10.3. ResultsAt the end of the experimental period containers with CF still contained residual substrate in addition to the frass. Rearing on PMS resulted in a lot of dry granular frass with dry non-converted crusty substrate material on top. Rearing on BTFS resulted in a dry top-layer, while frass was found at the bottom of the containers with BTFS. SW was completely converted into larval biomass and frass and no substrate was left. The larvae were sticky, i.e., the larvae were covered with an oily substance. Rearing on OP resulted in a solid granular top-layer while some frass was found at the bottom of the containers with OP. Rearing on PMLCF resulted in an almost similar remaining substrate as CF, but with a solid top-layer. Rearing on SG resulted in a dry top-layer with a moist bottom-layer. Dry matter concentrations of the substrates at the start and end of the experiment are presented in Table 2.Initial dry matter content of OP was highest, subsequently followed by CF, PMLCF, PMS, SG, BTFS and SW. During the experiment, dry matter content of the substrates increased. Highest final dry matter content was determined in CF and PMLCF, subsequently followed by OP and PMS, SW, SG and BTFS. Dry matter content of larvae increased along with higher larval growth rate during the trial. The highest dry matter content was found for larvae on SW, subsequently followed by CF and PMLCF, OP, PMS, BTFS and SG. Dry matter content of larvae on BTFS, PMS and SG was very low.Highest growth rate was found in larvae reared on SW (Table 3). Larval growth rate was significantly higher than in larvae reared on the reference substrate CF and PMLCF. Growth rate of larvae reared on PMLCF did not significantly differ from CF, whereas growth rate of larvae reared on PMS was significantly lower than on CF. Growth rates of larvae reared on BTFS, OP and SG were very low and significantly lower than other substrates. Waste Reduction Index (WRI) was highest on SW followed by PMLCF, and both were higher than WRI on CF. Waste Reduction Index decreased further in descending order from PMS, SG, BTFS to OP. The Efficiency of Conversion of Ingested substrate (ECI) was highest on SW followed by PMLCF, CF and PMS. The substrates OP, BTFS and SG resulted in a lower ECI than other substrates. Gas concentrations were measured in the entire climate chamber, which means that gas concentrations cannot be related to one of the substrates separately. CO2 concentrations increased during the first five days with a peak at five days of age, followed by a decrease during the last three days (Figure 1). The maximum measured CO2 concentration was 3140 ppm at day 5 of the experiment.NH3 concentrations were at a low level until day 4 (Figure 2). NH3 concentrations increased very fast to 63 ppm at day 5. NH3 concentration remained at a level between 40 and 60 ppm after day 5.CO2 and NH3 concentrations measured above the different substrates at day 8 are presented in Figure 3 and Figure 4, respectively, and are presented relative to the concentrations measured at CF.The highest CO2 concentrations were measured at day 8 above the substrates SW, CF, PMLCF and PMS in descending order. Lowest CO2 concentrations were measured above the substrates OP, SG and BTFS.The highest NH3 concentrations were measured at day 8 above the substrates SG, OP, PMLCF and BTFS in descending order. Lowest NH3 concentrations were measure above the substrates PMS, CF and SW.4. DiscussionThis assay investigated how seven substrates affect BSF larvae bioconversion in terms of larval growth, waste reduction and conversion efficiency of ingested substrates. The larval density was 2500 starter larvae per kilogram wet substrate and was similar in all containers because from literature it is clear that larval density affects larval growth and development [16,17]. Water was added to the substrates CF and OP to make the substrates more optimal for growth and development of BSF larvae. There is a lack of literature data on the most suitable moisture content of substrates in BSF bioconversion for effective residue separation [10] but these authors showed that the residue can be effectively separated from the insect biomass by sieving at 70% and 75% moisture content. For the current experiment, it was aimed to create a substrate containing about 65% moisture. Therefore, CF and OP substrates were mixed with water (1:1 w/w) and liquid pig manure was mixed with CF (1:1 w/w) to create PMLCF. Also, 450 mL water was added in each container with PMS because the substrate was assumed to be too dry for ingestion. The dry matter content at the start of the experiment was in all treatments around 30–35% except for OP (50.0%). The experiment ended at 8 days for the fast-growing larvae in CF, PMS, SW and PMLCF substrates because at 8 days no substrate was left at SW. First larvae on SW reached the prepupae stage at the end of the experiment. Containers with CF still contained residual substrate. Containers with PMS, BTFS, OP, PMLCF and SG had a dry top-layer and before determining the dry matter content of the remaining substrate the dry top-layer at PMS, BTFS, OP and SG was removed. The substrate below this top layer was stirred prior to sampling. Dry matter content in CF and PMLCF were above 80% at the end of the experiment and dry matter content in other substrates varied from 62% in PMS to 38% in BTFS. Dry matter content of larvae was positively correlated with growth performance. The highest dry matter content was found for larvae on SW (38%), subsequently followed by CF, PMLCF, OP, BTFS, PMS and SG (14%). In other papers, dry matter content of fresh larvae has been reported between 20% and 44% [12,18], and depends on both substrate and larval stage [19], because dry matter is higher in the later instars. Highest larval growth rate (13.4 mg/d) was found in larvae reared on SW and was higher than in larvae reared on the reference substrate CF (7.2 mg/d) and PMLCF (7.3 mg/d). The final larval weight (69 mg) on the reference substrate was in the range of larval weights obtained on a chicken feed in another experiment [13]. Larval growth rate (13.4 mg/d) of larvae fed SW containing pasta, potatoes and vegetables in the experiment was higher than the VEGFRU or FRU substrates [20]. The authors [20] studied the effect of different rearing substrates (vegetable–fruit waste–VEGFRU-celery 43.4%, oranges 28.9% and peppers 27.7%; fruit waste–FRU-apples 47.8%, oranges 15.5%, apple leftovers 13.8%, strawberries 7.1%, mandarins 4.8%, pears 4.1%, kiwis 3.4%, bananas 1.9% and lemons 1.6%; winery by-product–WIN-containing grape seeds, pulp, skins, stems and leaves; brewery by-product–BRE-Barley brewers’ grains wet) on the development of BSF larvae. Each substrate was ground with a 3 mm die meat mincer and mixed prior to placing the substrates in the containers. Larval growth rate was highest in BRE followed by FRU, VEGFRU and WIN (14 ± 0.9, 7 ± 0.7, 6 ± 1.8 and 6 ± 0.9 g/d, respectively). A large variation in larval growth and development on different substrates such as poultry feed, restraint waste, faeces, vegetables and flesh were also reported [18]. Differences in growth rate may be explained by the chemical composition of the substrates, but these were not determined in this experiment because the aim was to select the most promising substrates for growth performance. The most optimal substrates from this experiment were selected and will be tested again or mixed as test substrate to grow BSF larvae in a follow-up experiment. Waste Reduction Index (WRI) was highest on SW followed by PMLCF and both were higher than WRI on CF. Waste Reduction Index decreased subsequently further from PMS, SG, BTFS to OP. Food wastes, such as swill in the current experiment, provide good conditions for larval growth and therefore, BSF composting could be a good option [21]. Waste reduction was evaluated by growing BSF larvae on okara (soy pulp or fiber obtained from tofu production), wet brewers’ spent grains (from the beer industry), and maize distiller’s grains (from spirit or ethanol production) and a laying hen diet as a reference [22]. Waste reduction index in this experiment increased from 3.0, 3.2, 4.5 to 4.9 in the brewer’s grain, maize distillers, hen diet and okara, respectively. The WRI on CF in the current experiment was also 4.5 and the WRI on the vegetal substrates in the current experiment was 0.5, 1.2 and 1.9 for OP, BTFS and SG which is lower than in the vegetal substrates tested in the experiment of Bava et al. [22]. Waste reduction of PMS was slightly higher than the vegetal substrates in the current experiment, but pig manure still contains undigested dietary components, such as cellulose and lignin and BSF larvae cannot ingest these components [23]. The Efficiency of Conversion of Ingested substrate (ECI) was highest on SW followed by PMLCF, CF and PMS. The substrates with OP, BTFS and SG resulted in a lower ECI than other substrates. Substrates that contain a high proportion of easily available carbon, but a low content of nitrogen, such as OP or SG in this experiment, do not support larval development and thus the efficiency of the process is reduced [21]. The CO2 concentration in the climate chamber peaked at day 5 of the experiment. During the first five days CO2 concentrations increased followed by decrease after day 5 of the experiments whereas NH3 concentrations were low during the first four days of the experiment and increased very fast at five days. This course of the CO2 and NH3 concentrations during the experiment was also found in an experiment by Parodi, et al. [24]. These authors observed a clear peak in CO2 production between day 5 and day 6. They indicated that the drop in CO2 concentration observed on day 5 was caused by the physiological response of BSF larvae to either limited availability or accessibility of fresh feed. The authors concluded also that microbial metabolism in the substrate contributed to 34% of the overall CO2 emissions. The results demonstrate that the contribution of microbial respiration to the overall CO2 production during the rearing period is substantial. CO2 is the major gas product resulting from BSFL treatment of organic waste and is related to the respiration rate of associated microorganisms and BSF larvae, which can indirectly indicate the biodegradation rate of the substrate [25,26,27]. In the experiment of Parodi et al. [24], the NH3 concentration also had a defined temporal pattern. NH3 was produced from day 5 onwards, right after the peak of CO2. The timing of NH3 emissions might be explained by the high excretion rates of uric acid during the larval metabolic peak, followed by the microbial breakdown of uric acid into NH4+, and the subsequent volatilization of NH3. Considering that the occurrence and intensity of NH3 emissions are associated with the timing and total production of CO2, it is likely that NH3 emissions are the result of changes taking place in the substrate when larval metabolism is high (e.g., changes in temperature, moisture, pH and microbial activity) [24]. At the end of the experiment the highest CO2 concentrations were determined above the substrates SW, CF, PMLCF and PMS and the lowest concentrations above the substrates OP, SG and BTFS. Highest NH3 concentrations, however, were measured above the substrates SG, OP, PMLCF and BTFS and lowest concentrations above the substrates PMS, CF and SW. A direct link has been observed between gas concentrations and growth performances, WRI and ECI of BSF larvae. Highest CO2 concentrations and lowest NH3 concentrations have been determined at substrates resulting in the highest growth performance, WRI and ECI. The BSF larvae in the suboptimal substrates have a less efficient nitrogen metabolism, where the nitrogen is not built into insect protein and subsequently is broken down into NH3. These findings were confirmed by Parodi et al. [24]. Further research for a deeper understanding of this process of microbial activity in the substrates, larval metabolism and performance in different substrates varying in dry matter, chemical composition and physical properties is required.5. ConclusionsBiowaste conversion of seven substrates by black soldier fly (BSF) larvae was assessed to screen the potential to upgrade these substrates into valuable resources. BSF larvae showed the highest growth rate when reared on SW. Growth rate of larvae on SW was almost twice as high as on the reference substrate CF. Other substrates with potential were PMLCF and PMS. Dry matter content of BSF larvae was highest of larvae grown on the substrates with the highest potential illustrating a direct relationship between larval development stage and dry matter content. The substrates with the highest potential resulted also in the highest WRI and ECI. Indicative measurements of CO2 and NH3 in the climate chamber demonstrated a peak in CO2 concentration at day 5 and after the peak in CO2 concentration the NH3 concentration increased. Highest CO2 concentrations and lowest NH3 concentrations were measured above the substrates SW, CF, PMLCF and PMS which are the substrates that resulted in the highest growth performance, WRI and ECI. The relation between dry matter and chemical composition of the substrates and larval development and growth performance will be further studied in follow-up experiments.

animals : an open access journal from mdpi

10.3390/ani11072033

PMC8300117

Apart from feeding with forages, dietary supplementation with concentrate and rumen bypass fat is one of the feeding strategies to enhance nutrient availability and improve buffalo performance and productivity. This review paper thoroughly discussed the utilization of concentrate and bypass fat as dietary supplementation in buffalo feeding, and discussed the effects on performance, fermentation characteristics and general health of buffaloes to give better insight about the potential and challenges of dietary supplementation in buffalo diet. Based on the literature studies, it can be summarized that supplementation of concentrate and bypass fat in buffaloes may overcome the nutritional problems and improve the growth performance, health status, rumen environment and carcass traits.

With the increase in the global buffalo herd, the use of supplementation in the ruminant feeding has become an important area for many researchers who are looking for an isocaloric and isonitrogenous diet to improve production parameters. In order to improve the performance of the Asian water buffalo, the optimal balance of all nutrients, including energy and protein, are important as macronutrients. Dietary supplementation is one of the alternatives to enhance the essential nutrient content in the buffalo diet and to improve the rumen metabolism of the animal. Researchers have found that supplementation of concentrate and rumen bypass fat could change growth performance and carcass traits without causing any adverse effects on the buffalo growth. Some studies showed that dry matter intake, body condition score and some blood parameters and hormones related to growth responded positively to concentrate and rumen bypass fat supplementation. In addition, changes of feeding management by adding the supplement to the ruminant basal diet helped to increase the profit of the local farmers due to the increased performance and productivity of the animals. Nevertheless, the effects of dietary supplementation on the performance of ruminants are inconsistent. Thus, its long-term effects on the health and productivity of buffaloes still need to be further investigated.

1. IntroductionThe Asian water buffalo (Bubalus bubalis) is an important animal resource in a minimum of 67 developing countries. Many people rely on this species for their livelihoods [1]. It provides economic value from its meat, milk and leather, and especially draft power [2]. An estimation by Scherf [3] revealed that more than 2 billion people depend on approximately 200 million heads of buffaloes, which is more than any other domesticated animal. The majority of buffaloes operate in close association with humans on small farms, for whom these animals are also their largest capital asset. Based on morphological and behavioral criteria, there are two subspecies of domesticated water buffaloes, the swamp (Bubalus bubalis carabanesis) and the river or Murrah (Bubalus bubalis bubalis) buffaloes [4,5]. Crossbreds between Murrah and Swamp (Bubalus sp.) buffaloes are found in some parts of Asia, especially in China, Indonesia, the Philippines, Thailand, Vietnam and Malaysia [6].Currently, buffalo management practices are intrinsically correlated to the status of buffalo health, production and welfare. In order to ensure the optimum health of the animals and the organized production of high-quality and safe animal products (meat, milk and dairy products), a proper diet ration arguably represents the easiest strategy that can be implemented by farmers at the farm level. Both the meat and the dairy buffalos’ industries have made significant advances in animal management, husbandry, genetics and nutrition. However, the current climate change phenomenon has caused a reduction of the availability of rangeland pastures and forages, especially for animals reared under the free-grazing system [7]. According to Henry et al. [8], extreme and fluctuating temperature may impair the optimum health and quality of life of animals. Furthermore, changes of the economic patterns around the world due to COVID-19 issues and the increasing demand for livestock as well as animal products from the developing countries trigger all the stakeholders involved in ruminant production to reconsider the strategic use of nutrition for enhancing animal health and production. Indeed, the livestock industry must find alternative nutritional strategies which meet the demand of consumers for economical animal products that are produced in clean, halal, green and ethical manners [9]. From our literature review, we found an abundance of scientific trial studies on animals (in vitro and in vivo) that indicated reliable and cost-effective approaches for increasing ruminant profitability through optimizing the composition of the feed nutrients and with the addition of supplementation [7].Nowadays, the buffalo industry in Malaysia is starting to gain some attention as there are currently more studies on buffalo production, especially on how to improve it. This is in line with what has been carried out elsewhere, especially in other Southeast Asian countries. We therefore propose an approach using a suitable diet of a combination of basal diet with supplementation aimed for growing buffaloes. Hence, in this review article, we discussed the challenges of the buffalo industry, the requirements for energy and protein for buffalo growth, the potential of supplementation in the buffalo diet and the effects of the supplemented diet on rumen fermentation characteristics, growth performance, quality of the buffalo meat and the economics of the feed ration in ruminants. Our findings would allow specific dietary supplementation-based strategies to be established, which could efficiently enhance the health, welfare and longevity of the buffalo.2. Characteristics of Asian Water Buffalo2.1. Morphology and GeneticsIn the field, river and swamp buffaloes can be differentiated based on their morphology and behavior. Swamp buffaloes are ash or dark grey with a white chevron line on the neck, either one or two stripes, and have socks, while the tip of the tails and the horns are swept backwards [10,11]. They prefer to wallow in the marshland and mud, and have large feet with slow steady movement that make them well-suited for paddy land preparation in swampy waterlogged rice fields [12]. On the other hand, river (Murrah) buffaloes have black bodies with tightly and forwardly curled horns. They prefer to wallow in clean water [10]. The crossbred buffaloes have the same morphology as the Murrah but are smaller than the Murrah and bigger than the Swamp buffaloes.The chromosome number also differs between Swamp and Murrah, with river 2n = 50 and swamp 2n = 48 [11], owing to the fusion of chromosomes 4p and 9 [13]. The river and swamp buffaloes are also genetically distinct, as confirmed by the variations in allozymes, sex-linked, autosomal DNA markers, mitochondrial DNA sequences, microsatellite variation and a comparison with protein-coding loci [11,14,15]. Malaysian swamp buffaloes from the peninsula and Borneo region such as Sabah and Sarawak states were studied in this work. The peninsular and the Borneo swamp buffaloes were found to be genetically distinct, hence paving the way for the possibility of crosses between them to improve the Malaysian swamp buffaloes [6]. In addition, a prior study in Malaysia had reported that phylogenetic tree and mtDNA analysis on Swamp buffaloes were genetically conserved and the crossbreds were dominantly Swamp according to the maternal lineage using d-loop mtDNA [6].On the other hand, crossbred buffaloes are the result of a combination of the two genetic types, but showing 80% dominant characteristics of the Murrah breed [6]. Crosses between river and swamp buffalo are fertile, despite having 2n = 49 chromosomes in F1 and F2 offspring. The crossbred buffaloes show better growth, larger body size and much improved draft power compared to the local swamp buffaloes [16]. Furthermore, the crossbred buffaloes are capable of yielding extra milk production, with an average of between 4.0 and 1.94 kg/day [17]. In fact, crossbred buffaloes are much more improved in terms of birthweight, age at maturity and first calving, duration of heat and period of inter-calving [18]. Indeed, with improved feeding, the crosses were recorded to grow 40% faster, with significantly improved meat quality than the Swamp buffaloes [19].2.2. Distribution and UseThe indigenous breed of buffalo in East and Southeast Asia is the Swamp buffalo (Bubalus bubalis carabanesis). It is largely concentrated in Southeast Asia and Southern China (Figure 1). In China, the Swamp buffaloes have adapted to a range of climates, altitudes and temperatures [20]. Therefore, the Swamp buffaloes could be found in both low- and high-lands. Traditionally, the Swamp buffalo is used mainly for draught power, particularly for ploughing paddy fields and transportation, but it is also used to supply meat for human consumption. Until now, some oil palm estates are still using Swamp buffaloes as draught animal to pull carts carrying oil palm bunches. Occasionally, Swamp buffaloes’ milk is used to make dairy products such as yoghurt and mozzarella cheese.River or Murrah buffaloes (Bubalus bubalis bubalis) are mainly found in South Asia, with the highest distributions in Pakistan, India, the Middle East and Italy (Figure 2). They are primarily reared for their milk typified by the high contents of fat and dry matter. Furthermore, buffalo milk has lower cholesterol content, but compared to cow’s milk, it has more calories and fat. Thus, it is used to produce high-value thick and creamy cheese, yoghurt and ghee [20]. Murrah buffaloes are also used to improve buffalo milk production in many other countries such as Egypt and Bulgaria [22]. According to Hamid et al. [23], the Murrah buffaloes have been described as the “Asian tractors” and serve the purpose of meat, milk and work. In contrast to the Swamp buffaloes, river buffaloes are more aggressive, with temperamental instability [24].The multipurpose crossbred buffaloes are mainly found in several parts of Asia, especially in China, Indonesia, the Philippines, Thailand and Vietnam. They are used to provide draught power and meat in rice-growing areas and milk in other regions [25]. Now, crossbreeding of buffaloes is practiced in almost all countries where Swamp buffaloes are found, such as China, Burma, Thailand, the Philippines, Malaysia and Sri Lanka, with the aim of improving the milk yield and the animal size for work in the field [26].3. Challenges in Buffalo Production SystemsAn alarming decline in the buffalo population of Southeast Asia has happened over the past two decades at an average rate of 1.2 percent per year. This is due to several factors: (i) poor market demand for buffalo products [27], (ii) high rate of slaughter coupled with insufficient input for research and development [28], (iii) increased agricultural mechanization that made the Swamp buffalo redundant, (iv) a myth against the quality of buffalo meat [29], (v) poor reproductive performance coupled with poor responses to the biotechnology currently available, such as embryo transfer technology and artificial insemination, which prevented sufficient proliferation of the buffalo [17], and above all, (vi) a lack of knowledge on farm and feed management, resulting in a rapid decline in the number of buffaloes.Furthermore, a few other factors have been cited as possible constrains that had contributed to the low output from beef and mutton producers. These factors include the inadequacy of land suitable for grazing to sustain significant livestock breeding populations, low supply of quality breeding stocks, erratic supply of high nutritional value feed and lack of an effective system of marketing. Taking cues from the poultry industry, the beef industry must promote ready supplies for the breeding and fattening of the production stock, ensuring the continuous supply of reasonably priced buffalo feed and creating an effective marketing network [30].In Asia, in accordance with the environment, soil and socioeconomic opportunities, buffalo production systems vary widely [31]. The semi-intensive production system practiced by smallholder farmers is currently focused upon by many developing countries for the ruminant industry, mainly for cattle and buffalo. According to Saadullah [31], buffaloes in Asia that are mostly under the semi-intensive system are kept mainly for specific purposes, such as for meat and milk production. Approximately, 11%, 5% and 84% of the smallholder farmers reared buffalo for milk, meat and for both milk and meat, respectively [32]. Recently, the extensive production system is used through integrating ruminants, including buffaloes with oil palm or rubber plantations [30], where large-scale pasture lands and green forages or grasses are available [33] that allow the animals to graze for an average of 6 to 8 h daily [34]. However, most buffalo farms practice the semi-extensive grazing system in oil palm or paddy field areas without supplementation [35].South and Southeast Asia have many marshlands and rivers which are suitable for raising buffaloes. Moreover, improvements in feeding management have influenced the growth performances of buffaloes in countries such as India, Brazil, the Philippines and Malaysia [35,36]. In general, indigenous Swamp and crossbred buffaloes in Southeast Asia are low in numbers, and this has affected the production of dairy and meat. Furthermore, longer puberty age, seasonality of breeding, longer calving interval, high calf mortality and poor genetic pool, nutrition and management practices [37,38,39] have all influenced the productivity.4. Feeding and Nutritional Management for Buffalo Production4.1. Nutrient Requirements and Utilization of BuffaloLike other ruminants, buffaloes obtain their energy and protein in the form of volatile fatty acids and microbial proteins from fermentation end products. Based on research studies on the nutritional requirements and digestive physiology of buffaloes, it was concluded that buffaloes underwent relatively higher ruminal degradation of both protein and fiber [40] as compared to cattle [41]. This unique ability, particularly the better fermentation of fibers in buffaloes in temperate countries, is the consequence of adaptation following years of being fed with low-quality and highly fibrous diets [42]. According to Manish [43], the protein and energy demands of buffaloes in Asia were being met by feeding roughages containing high levels of lignocellulosic materials, namely cellulose, hemicellulose, lignin and low levels of fermentable carbohydrates and proteins. Indeed, the irregular and inadequate availability of quality feedstuffs and their utilization had been reported as the main causes of the poor performance of buffaloes in Asia [44]. In contrast, developed countries that produce a large number of meat and dairy animals place much emphasis on improving energy and protein levels of animal feed as well as on developing a specific model of nutrient requirements in buffaloes. These were reported by various studies, where the levels of energy and protein requirements varied in buffalo diets during lactation and growth [41,42,45]. Nevertheless, limited strategic studies were performed to establish the protein and energy needs of buffaloes in Asia, particularly on assessing their effects on different physiological stages and on the performances of buffaloes of various breeds.In general, buffaloes need optimal nutrient requirements such as protein, fat, vitamins, minerals and water in order to maintain life, to reproduce and to enhance productivity. These requirements are influenced by many factors [46,47]. They include:Animal-based factors, such as the physiological status (growth), age and body weight of animal, production line, traits of digestive system and health status [48,49].Ration-associated factors, including the feeds that are used in the ration and the nutritional, physical and chemical composition status of the feed [50].Environmental-related factors, particularly the climate, air temperature and feeding system [51].Currently, there is paucity of information regarding the energy requirements of growing, breeding, lactating or working buffaloes [52]. Therefore, the nutrient requirements for cattle have always been adopted for buffalo feeding following the recommendations by NRC [53] and AFRC [54].Energy is typically derived from carbohydrates, namely starch, cellulose and fat [47]. The nature of the buffalo digestive system physiology makes the cellulose in roughages, a rather cheap energy source, important [55]. The recommended values of energy requirements for buffaloes differ depending on their stages and physiological conditions. Estimations of the energy requirements of buffalo gain in the literature varied from 0.78 to 2.23 TDN/g gain [52], and for the lactating stage it was 1.97 TDN/g gain [48]. In addition, the amounts of feedstuffs offered to buffaloes to meet their requirements were also linked to various characteristics, such as the type, quantity, quality and presentation of the feed [52]. Therefore, besides feeding with low-quality roughage, the high demand for energy by growing buffaloes should be fulfilled by supplementing with a mixture of quality roughage and grains that contained an abundance of energy [56]. Furthermore, adequate supplementation of fat was capable of increasing the concentration of energy in the animals, which could also enhance the percentage of fat in the milk as well as the quality of the meat [57].Protein is an important substance for growth and development of muscles, nerves and other tissues. It is also important for the repair of aged tissues, fetus development and the production of meat and milk [58]. Ammonia is required for the growth of rumen microorganisms before optimal microbial protein synthesis is achieved by supplementation with adequate levels of protein and non-protein compounds [59]. Other studies reported earlier that there was a large variation in the values for protein requirements in buffaloes [48,52]. The estimated range of digestible crude proteins needed by buffalo for maintenance and growth was 1.11 to 5.05 g/kg metabolic body size 0.75 and 0.30 to 0.45 g/g gain, respectively [52]. Following a low level of protein or energy supply to buffaloes, the demand for proteins was met by a low supply of medium-quality pastures and fodder. Therefore, growing, pregnant and lactating buffaloes should be fed with meadow grass and leguminous forage supplemented with concentrate, grain or oil seed cakes [46]. This could prevent protein insufficiency that might lead to declines in appetite and feed consumption of the animals, a negative utilization of feed and a reduction in cellulose digestion [55].4.2. Roughage FeedingIt is clear that buffaloes’ main diet consists of roughages such as grass, legumes and straw. Most of the buffaloes are fed on tropical grasses as the primary source of nutrients. The roughages are fed either fresh as the grazing pasture or dried such as in a cut and carry system or conserved as hay or silage. Thus, a relatively low cost of digestible energy from grasses is provided to the buffaloes, mainly in the form of fibrous compounds [60]. Nevertheless, the roughage that forms the basis of a feed ration should be of good quality, have both nutritional and hygienic qualities and be capable of meeting at least the total maintenance requirements. Unfortunately, tropical grasses for grazing buffaloes infrequently represent a balanced diet, since they have constraints on one or more nutrients that may limit the intake of forage, digestibility or the metabolism of the absorbed substrates [61]. Therefore, an addition of concentrate in the diet should be practiced in buffaloes’ feeding to ensure a balanced ration, and nutrients are provided to meet the buffaloes’ requirements [62].To improve buffalo performance, the utilization of pastures in any season as the main nutrients is not considered to be optimal [61]. Thus, selecting mature, dried foliage and stems of grasses ensures supply of a low protein level of less than three percent of crude protein. Furthermore, the grasses have been variably leached of soluble components, including minerals, proteins, sugar and starchy carbohydrates that are needed for efficient fermentative digestion in ruminants. On the other hand, too much intake of non-fibrous feed can change the environment of the rumen, and in the long term, leads to serious feed digestion problems, including reducing feed intake, which leads to ketosis, weight loss and a reduction in milk yield. According to Figueiras [61], unbalanced energy to protein ratios were recorded in tropical grasses, with relative energy surplus. Thus, the use of this poor-quality forage without a supplementary diet contributed to the low production of the ruminant meat industry in developing countries. In fact, a study in Brazil and Australia revealed that using a proper feed supplementation program improved the body weights of ruminants [60]. For this reason, there is a need to recognize tropical pastures as having limitations of nutrients and to solve this or to change to feeding regimes that may lead to improved performances of the animals and the overall production efficiency [63].4.3. Supplementation StrategyLow growth performance, poor reproductive performance and milk yield have been reported in buffaloes in other studies [64,65]. Indeed, the poor performance reported by those studies correlated with poor dry matter intake and weight gain and longer calving intervals [64,65]. In many parts of Asia, inadequate and irregular availability of quality feedstuffs and their utilization are the main causes of the poor performance of buffaloes [44]. A report stated that buffaloes in Southeast Asia were mainly fed with hay or forages high in lignin and low in fermentable protein and carbohydrate contents [44]. Many strategies have been trialed in order to improve the nutrient supply and utilization in buffaloes, with varying degrees of success. These are: (1) utilization of available feed resources such as local plants that have high crude protein or energy content, (2) use of industrial and agricultural by products, (3) dietary addition of concentrate, fermentation modifiers and vitamins and (4) usage of ruminally protected dietary fat and protein sources, which have shown significant potentials to improve growth, reproduction and milk yield of buffaloes [44]. However, in order to choose the best strategy to improve buffalo performance, the farmer should identify the main problem causing the low growth performance of the animal.In the buffaloes’ diet, the roughage should often be complemented with concentrate, grains or agro-industrial products as supplements. The supplements should be fed only to fulfil the additional requirements for improving growth, pregnancy and milk production. Therefore, feed supplementation programs should concentrate mainly on the establishment of a diet that contains balanced nutrients by increasing the energy content in the diet as well as by increasing the dry matter intake through the addition of supplements. Indeed, a supplemented diet in the tropics should focus primarily on protein and fat supplementations in order to provide optimum energy for better growth performance of the animals [63,66]. This would allow for utilization of the relative excess of energy from the supplemented diet to be converted into body weight gain [67]. Balancing the fat and protein nutrition through supplementation is one of the strategies for increasing production in ruminants with high-energy diet requirements, such as young post-weaning animals, animals in the last trimester of pregnancy and lactating animals.In most regions of developing countries, it is not practical to identify the deficient micronutrients and macronutrients in pastures or other forages, as these vary from site to site and year to year. Furthermore, they are also influenced by the pattern of fertilizer application and the weather conditions. The alternative is through providing buffaloes with a supplemented diet, and the supplementation involves providing energy and protein to satisfy the requirements for efficient digestion in the rumen. Diet with a proper supplementation is able to provide optimal ammonia nitrogen and good fatty acid in the rumen. Palatable and tasty feed with a well-balanced ratio of protein and fat as additional energy sources is the best way to increase milk production and live weight, maintaining health and enhancing fertility. This has influenced the changes in supplementation utilization, since a lack of knowledge on feeding management would affect animal production and nutritional performance [63,66]. However, comparative analyses regarding the combination of energy and protein supplementation on buffalo performance in the tropics remain scarce. Therefore, extensive studies are needed to assess the impacts of supplements on grazing buffaloes in the tropics, particularly on the intake, ruminal fermentation pattern and the quality of the meat.4.4. Types of Supplementation in Buffalo DietsSupplemented feed offers a promising way to enhance the overall health and performance of buffaloes. In contrast to a nutritionally complete ration, supplemental diets are intended to provide an additional source of energy and protein when forage quantity and quality are inadequate to meet the desired performance [68]. Nutritional husbandry of domestic buffalo often contains high energy and protein supplements in combination with roughage to increase the growth rate of sub-adult animals [69] and to enhance the digestibility of forage diets [70]. Supplementation strategies are essential in designing the feeding programs for this species. In fact, supplementation with larger amounts of energy-rich feeds with a source of protein and fat could reduce the time taken to prepare buffaloes for the market, thus increasing profitability, such as those reported for cattle that consumed low-digestibility forages with energy and protein supplements [71].Concentrate is one of the dietary supplements that supply protein to animals. Many experiments have demonstrated the benefits of supplementing dietary protein meals or concentrate to ruminants that are fed poor-quality forage [72,73,74]. In fact, in developing countries, low cattle and buffalo productions are primarily due to limited supplies of nutrients in high forage-based rations [75]. Therefore, it is important to identify new feed sources and technologies for cattle and buffalo production systems. Recently, there are several varieties of concentrate ingredients that have been used by smallholder farmers, namely maize meal, cassava powder, rice bran and mixtures of these feedstuffs in ruminant production [75], such as cattle and buffalo. However, due to inadequate information on the nutritional value, digestibility and characteristics of the rumen fermentation, the benefits of using these feeds for buffalo are not well-understood. This is especially true since previous studies had reported that different proportions of concentrate feeds differed substantially in their rumen fermentation characteristics [76]. Nevertheless, a high starch content in the concentrate is important in ruminant nutrition because it is cost-effective, contains protein sources and has been proven able to influence rumen function and digestion of nutrients [77,78].The technology of bypass nutrients such as rumen bypass fat has been implemented in feed management through passive rumen manipulation [79]. It is also known as rumen-protected fat, inert fat or rumen bypass fat [80]. Bypass fat is the supplement that escapes rumen degradation as it is being protected from microbial fermentation, biohydrogenation and remains insoluble at normal rumen pH. However, it is easily digested and absorbed in the lower gastrointestinal tract at the abomasum and small intestine, respectively [81]. Therefore, it might be beneficial for ruminants to be fed with rumen bypass fat as a supplementary diet that is absorbed readily from the lower digestive tract. There are two types of rumen bypass fat supplements commercially used by farmers, namely natural bypass fat (e.g., cotton, roasted soybeans, sunflower and canola whole oil seeds) and chemically prepared bypass fat (e.g., crystalline or prilled fatty acids, formaldehyde-treated protein-encapsulated fatty acid, fatty acyl amide and calcium salts of long-chain fatty acids) [82]. The calcium salt-coated method on fatty acid of vegetables has been reported to be a more accessible bypass fat to all types of farmers. It has also been proven to be a cost-effective technology compared to other rumen bypass fats [83]. A few studies reported improvements on the growth and nutrient utilization of buffalo calves after being fed with bypass nutrients [84,85]. Integration of fat supplement into the diet enhances the growth potential of buffalo calves [79]. Studies have reported that high fat supplementation, such as RPF, could also enhance fiber digestibility of various fibrous feedstuff due to the high hydrolysis rate in rumen (85% to 95%) [81,82]. This supplement can also improve energy efficiency, as a result of reduced production of methane from the rumen and direct use of long-chain fatty acids [86]. Many studies recommended that the ration of high producing and growing animals should contain between 3% to 6% fat in the total ration DM, in order to obtain the beneficial effects [82]. Meanwhile, feeding more than 9% rumen bypass fat would not be beneficial for the growth (feed intake) and lactation (milk yield) of the animals [82].5. Effect of Dietary Supplementation on Buffalo ProductionThe advantages of high energy and protein in dietary supplements can be the explanation for the improvements in the growth and fattening of ruminants. Indeed, it has been shown that apart from improvement of the growth performance upon supplementation of concentrate and bypass fat, the improvement in dry matter intake may further enhance the average daily gain, growth hormones (e.g., GH and IGF-I), body condition score, carcass traits, meat quality and breeding performance of buffaloes [21,79,87]. In addition, the supplementation of both concentrates and bypass fat did not cause any adverse effects on serum biochemical profiles, rumen fermentation and microbial population [21,28,33,34,35]. An overview of the role of these concentrate and bypass fat supplementations in buffalo nutrition is presented in Figure 3.5.1. Growth PerformanceThe body weight gain and the body condition score (BCS) of buffalo calves after weaning represent the growth vigor of the animal. This is a substantial feature in the selection of animals [88]. The body condition score of a buffalo is classified as a subjective scoring method to assess the outer appearance of the animal, which interacts with its body fat to provide a better understanding of the biological relationships between body fat, reproductive performance and production of milk. These could assist in the implementation of optimal management practices to achieve maximum production and to preserve better health status [87]. Indeed, the BCS may also provide an immediate evaluation of the body condition of the animal and can be readily integrated into operational decision-making [89]. The use of BCS started in studies of ewes, beef cattle and Holstein dairy cows in 1961, 1976 and 1989, respectively. It used a 0 to 5 scale with a chart developed for references of body condition scoring [87]. In Pakistan, there was a study on Nilli Ravi buffalo that assessed the BCS by using a linear scale of 1 to 9 (1–3 for thin, 4–6 for average and 7–9 for fat), where the scoring was performed visually by assessing the covering of fat over the tail head, rump, sacral bone and loin and withers area [90]. However, the BCS of buffaloes was improved and established in India [87] using a 1 to 5 scale for assessing the animals (Figure 4). India has the highest buffalo population in the world and shows dramatic increases of population numbers yearly [91]. In addition, it is the native tract for the best buffalo breeds of the world [92]. The improved chart for BCS of buffalo with a 1 to 5 scale using 0.25 increments has been widely used in Asian countries [92]. The BCS score assessment was carried out by taking into consideration the anatomical features and amounts of fat reserves at various skeletal checkpoints. Validation of the precision of the BCS score had been carried out via ultrasonic measurements of subcutaneous fat [92]. Thus, the use of the BCS 1 to 5 score is suitable for assessing the reproduction and production status of buffaloes. Furthermore, the most widely used criterion is also to determine the growth of animals by assessing their body weights. Body weight is significantly associated with the types of feed and ration offered.According to Vahora et al. [79], improvements in the average daily weight gain, body length, height and heart girt in buffaloes fed with a basal diet were significantly associated with the incorporation of protein and fat supplements. Furthermore, offering supplementation at 0.6 kg/animal/day to grazing buffaloes after weaning for a period of two years enabled the calves to reach an average of 578.38 kg body weight, with improved body condition scores [36]. Similarly, supplementing beef heifers with dietary energy supplements increased the average daily gain and utilization of energy from native forage which contained low-quality nutrients [93]. Sawyer et al. [94] also showed that supplementing with a low ratio of energy at 40 g/day of crude protein might potentially replace greater quantities (160 g/day of crude protein) while still maintaining rumen function. However, animals grazing on low-quality dormant range and fed with a supplementation had no change in body weight during pregnancy, breeding and longevity compared to those feds grazing without supplement or a lower rumen undegradable plant-based protein supplement [95]. However, adding supplemental fat in the ration at a rate of 5–7% dry matter (DM) resulted in an improved lamb weight at 15% to 20% [96]. Ngidi et al. [97] reported that the apparent digestibility of fat increased, whereas true digestibility lessened when fat was added at up to 8% of diet DM. Meanwhile, Kumar and Thakur [98] concluded that supplementation of bypass fat at 2.5% to 4% of dry matter intake increased average daily gain and feed conversion ratio in buffalo calves and improved the growth performance without an adverse effect on nutrient utilization. It was concluded that addition of 2% to 4% of fat potentially stimulated feed intake and increased ruminant’s digestibility energy intake [99].5.2. Serum Biochemistry and Hormone ProfilesThe main function of blood in the body is to maintain the physiological equilibrium, but many physiological conditions may alter this equilibrium [100]. Blood contains a myriad of constituents that provide a valuable medium for clinical investigations and nutritional evaluations of an organism [101]. Serum biochemicals can be affected by age, nutrition, physiological status, sex, genetics, environmental factors and stresses, such as diseases and transportation [102]. The consequences of these variables can be measured by evaluating the physiological responses, since it is known that environmental and nutritional factors predispose animals to the occurrence of disease and decrease animal productivity. Such deviations from normal alter animal body constituents, especially the body fluids, thus health risk conditions can be well-understood by evaluating blood components. Disease incidence and malnutrition could be diagnosed by analyzing the deviations of the various hematological and serum biochemical parameters from the normal reference values [103]. In fact, these values are used to compare and interpret the metabolic state or condition of animals as a reference point [104].In the ruminant industry, analysis of blood metabolic profiles for assessing the nutritional and health status of goats, cows and buffaloes are not widely used [105], although the health and nutritional status of animals are important. However, the conventional and common practices used to evaluate the nutritional status of animals include the body condition scoring and the gain of body weights. Therefore, the use of blood metabolites is less common in assessing nutritional status of ruminants, especially among smallholder farmers in developing countries [106,107]. Needless to say, there are some blood metabolites that are related to the nutritional status of ruminants, and they reflect the animal’s response to nutrition. They include blood total protein, cholesterol, triglyceride, glucose and urea.Blood metabolites could be used regularly, objectively and reliably to assess the nutritional status of animals. However, the use of blood metabolite analysis in field buffalo and cattle is rare, particularly in developing countries such as Southern Africa and India due to the high cost of analyzing the samples, lack of equipment and expertise [106,107]. Similarly, the use of blood metabolites is quite uncommon in the field of buffalo management. This is unfortunate since several factors, namely the animal physiological status, nutrition, breed, age and season, are found to affect the blood metabolites, and in combination with data from body condition scores and body weights, blood metabolite analysis increases the accuracy of assessing the nutritional and welfare states of the ruminant population [107]. The success of the blood metabolite profile test alone is limited because several non-dietary factors also influence the concentrations of blood metabolites, such as herd origin, lactation stage, milk yield and season of the year [108].According to Campanile et al. [109], the buffalo heifers fed Brachiaria hay with concentrate (high-energy diet) showed similarities in non-esterified fatty acid and triglyceride levels when compared to the group fed a low-energy diet (without concentrate) (0.53 vs. 0.47 mmol/L, 17.1 vs. 18.7 mg/dL, respectively), but there were significant increments in glucose, total cholesterol and high-density lipoprotein (90.5 vs. 73.6 mg/mL, 80.4 vs. 58.7 mg/dL, 64.0 vs. 45.4 mg/dL, respectively). Bertoni et al. [110] have revealed that buffaloes fed isonitrogenous diets with different energy contents showed constant blood urea content regardless of the different diets, while cattle with similar treatment showed a significant decrease in blood urea with increasing dietary energy. This indicated the decline in ammonia content in the rumen of cattle as a result of the limited ability to recycle blood urea into the rumen. On the other hand, blood urea nitrogen of buffaloes would be at optimum range following feeding with different energy content diets that ranged between 7.00 and 8.50 g/dL [111]. Tiwari et al. [112] reported that the normal glucose levels in growing buffaloes and Holstein cattle were 51 to 64 and 74 to 76 mg/dL respectively, when provided concentrate and roughages at an equal ratio. Other studies reported that Murrah buffalo fed a basal diet with concentrate supplementation and a mixture of concentrate with bypass fat supplementation had no effect on the blood urea nitrogen (BUN) (49.30 vs. 50.41, mg/L), total protein (7.57 vs. 7.75, g/dL), albumin (2.71 vs. 2.84, g/dL), globulin (4.86 vs. 4.91, g/dL) and cholesterol (106.27 vs. 106.36, mg/dL) levels [113]. However, the buffaloes that were fed with supplement bypass fat showed slightly increased high-density lipoprotein and calcium levels as compared to animals fed without the bypass fat (65.55 vs. 57.22 mg/dL and 7.03 vs. 5.70 mg/dL, respectively) [113,114]. Nevertheless, the buffaloes that were supplemented with bypass fat showed a slight decrement of blood glucose, non-esterified fatty acid and low-density lipoprotein levels as compared to animals fed without the bypass fat (63.50 vs. 65.98 mg/dL, 0.66 vs. 0.93 mmol/L, 33.66 vs. 37.49 mg/dL, respectively) [113].Similarly, hormonal profiles can also be used to determine the health and nutritional status of ruminants. Animal growth is influenced by many hormones, blood metabolites and growth factors acting both in an endocrine and an autocrine manner and requires the coordinated actions of several hormones, such as growth hormone (GH) and insulin-like growth factor-I (IGF-I) [115]. The somatropic axis is the important hormonal system for growth development of animals. It consists of GH, IGF-I, carrier proteins and receptors [116]. The hormones of GH and IGF-I have both independent characteristic and combined impacts on ruminant metabolism and production. The growth hormone is synthesized in the pituitary gland and acts directly on the liver and adipose tissue to regulate gluconeogenesis, protein synthesis, lipogenesis, lipolysis and insulin secretion by binding to the growth hormone receptor (GHR) [117,118,119]. On the other hand, IGF-I is a critical somatomedin that is synthesized in the liver. It plays an important role in some physiological processes, contributes to improved feed conversion rate and increases protein synthesis [120]. The IGF-I binds to insulin-like growth factor binding protein-3 (IGFBP-3) to influence the growth, development and reproduction in animals [121]. Meanwhile, the existence of the axis between GH and IGF-I has played a vital role in the regulation of metabolism. GHR combines with GH to stimulate a series of metabolic activities by producing IGF-I in the target tissues, especially in the liver [122,123].In some cases, nutritional factors are critical regulators of IGFs [124]. Deficiency in either energy or protein intake significantly decreases the IGF-I levels [124]. Similarly, Clemmons et al. [125] reported that low energy in the diet caused the IGF-I level to decrease 4-fold. In fact, limiting the energy in the diet increases the GH levels and reduces the IGF-I secretion [126,127]. Meanwhile, over-consumption increases the IGF-I, although excess calories are not nearly as strong a stimulus as nutritional restriction [128]. A short-term feeding study on protein deprivation revealed a potent and independent role of protein on the IGF-I levels. Deficiency of essential amino acids has a severe depressing effect on the IGF-I levels [124]. However, a high-carbohydrate diet increases the IGF-I levels relative to a high-fat diet, possibly by maintaining hepatic sensitivity to GH [129]. Several researchers have recorded that yaks and buffaloes have evolved and developed complex strategies to respond to the deficiencies of nutrition and hypoxia stress [130,131,132].According to Campanile et al. [109], buffalo heifers fed with Brachiaria hay and supplemented with concentrate (high-energy diet) showed increases in GH and IGF-1 levels compared to buffalo fed with a basal diet without concentrate (low-energy diet) (6.3 vs. 5.6 pg/mL, 95.5 vs. 79.1 ng/mL, respectively). Other studies also revealed that Murrah buffalo increased IGF-1 and remain constant in GH levels when fed a diet supplemented with a mixture of concentrate with bypass fat when compared to being fed with a diet supplemented solely by concentrate (119.10 vs. 116.24 ng/mL, 4.91 vs. 4.93 ng/mL, respectively) [113,133]. Even though a few studies have shown the effects of dietary fat and protein supplements on the serum biochemistry profiles as well as the hormonal profiles in the blood, unfortunately, limited work has been performed on the comparison of blood and hormonal profiles between buffalo breeds.5.3. Rumen Fermentation PatternRumen digestion is fundamentally a fermentation process within the rumen by various types of bacteria, protozoa and fungi [134]. These microbes are responsible for the breakdown of feed and water intake into volatile fatty acids (VFA), gases (i.e., methane, carbon dioxide) and microbial proteins that are useful for the animal. For the rumen microbes to function properly, the rumen environment parameters including pH, temperature and moisture must be maintained [134]. Therefore, the outcome of rumen fermentation depends on adequate nutrition with respect to composition and quality of feedstuffs, which is reflected in the voluntary intake and digestibility of ruminants [135].Buffaloes are like cattle which utilize micro-organisms in the rumen to digest the feed. It has been reported that many farms until today fed their buffaloes using cow requirements as a reference point [136]. This might be due to the similarities in rumen fermentation characteristics between buffalo and cow. According to Smith et al. [137], both animals have similarities in heat tolerance, milk composition and ability to utilize highly fibrous feed [138,139]. Nevertheless, studies also revealed that buffaloes indeed have higher forage digestibility due to higher populations of cellulolytic bacteria, total fungi and lower protozoa numbers in their rumen, as compared to cattle rumen [140,141]. Therefore, it is important to understand the rumen fermentation and the ruminal microbial differences between buffaloes and cows when formulating a feeding regime for them [138].Changes in diet, from forage to high-protein diet, can affect the fermentation process of ruminants such as cattle and buffalo. The increase in dietary crude protein (CP) leads to increased concentration of total rumen volatile fatty acid (VFA), which is consistent with the improved degradability following increased bacterial populations and microbial enzyme activities [142,143]. A study by Kang et al. [144] reported that Swamp buffalo fed with a high concentrate (160 g/kg DM) supplement ratio had constant ruminal pH (average 6.59 to 6.61) and temperature (average 39.1 to 39.3 °C), but had increased values of ammonia, total VFA, propionic acid, butyric acid of 9.8 vs. 13.8 mg/dL, 20.1 vs. 26.2 mol/100 mol and 10.7 vs. 12.3 mol/100 respectively, when compared to animals fed with low concentrate (120 g/kg DM).Carbohydrate in the diet is mainly characterized by the proportion of non-structural (NSC) and structural carbohydrate [145,146]. In the rumen, the NSC are initially utilized by the rumen microbes and are degraded quickly to produce volatile fatty acids [147]. Sutton et al. [148] confirmed that high NSC in the concentrate diet resulted in higher propionate concentration, while McCarthy et al. [149] also concluded that increasing the content of ruminal fermentable starch enhanced the total volatile fatty acid (TVFA) concentration. In fact, decreased ruminal pH was also reported following high intake levels of dietary crude protein, and this was attributed to the increased ruminal total VFA output [150,151]. Ruminal ammonia-nitrogen (NH3-N) is primarily derived from ruminal degradable proteins and is used for the synthesis of microbial protein [152,153]. An increase in ruminal NH3-N content occurs following an increase in dietary CP levels [152] and is largely attributed to the increased ruminal CP degradability [154]. However, an increase in dietary concentrate or carbohydrate is not a successful strategy to mitigate either the enteric methane production or ammonia emission from the manure. Therefore, incorporating supplemented concentrate with bypass fat has the potential of reducing the methane and ammonia productions.Unfortunately, a study by Budi et al. [155] found that the increased level of bypass fat significantly reduced the in vitro dry matter degradability, but the levels of TVFA and ammonia nitrogen remained constant, while Naik et al. [156] revealed that following in vitro fermentation, the levels of TVFA and ammonia nitrogen were much higher in animals fed with a diet with concentrate and bypass fat compared to those fed without the supplement. Saijpaul et al. [157] recommended that the high level of bypass fat supplementation could substitute up to 40% of natural fat in a concentrate mixture (6% natural fat) contained in total mixed rations (50:50 roughage to concentrate) with no changes in in vitro fermentation parameters of TVFA, total nitrogen and ammonia. Other studies also reported that supplementation of bypass fat between 5% and 15% in buffalo diet had no adverse effect on the in vivo rumen fermentation characteristics such as pH, NH3-N and VFA levels [158,159,160]. The specific ingredient of dietary buffer in calcium salt bypass fat functioned to maintain ruminal pH and to minimize the rate of dissociation of calcium salts in the rumen [82], thus the rate of biohydrogenation was potentially reduced. In summary, supplementation of solely concentrate in a basal diet could affect the buffalo rumen fermentation characteristics but no adverse effects were found on the reported parameters when buffaloes fed given the mixture of concentrate and bypass fat in the diet.5.4. Rumen Microbial PopulationsIt is also known that the feed and feeding regime in ruminants may also significantly affect the ruminal microbial community. Indeed, it has been demonstrated that the composition of the rumen bacterial population significantly correlated with feed efficiency [161,162]. A study showed that changes in the structure of the ruminal microbial population potentially promoted feed efficiency, intake and the average daily gain of ruminants [163]. The microorganisms also often provided the host ruminant with nutrients to produce energy [164]. Thus, increased ruminant production could be achieved by using proper feeding formulation and management that were able to manipulate the ruminal microbes and ecosystem. For example, a proper ratio of supplemented feed in the diet that provided adequate energy and protein, allowed for an optimal ruminal fermentation process that maximized production efficiency while decreasing energy loss such as methane that polluted the environment [165]. Indeed, there were also reports on the effects of dietary changes involving protein and energy on rumen microbial population and the rumen environment in ruminants [166].The effects of dietary energy and protein supplementation on the rumen microbial population have been studied extensively in sheep, goat and cow, but less so in buffaloes. Indeed, a study by Dahllöf et al. [167] has reported that dietary modifications in cattle feeding have major effects on the communities of rumen bacteria. Faniyi et al. [168] also stated that a shift in the ruminant diet from forage-based to a high-concentrate diet resulted in significant alterations of the ruminal environment and rumen bacterial population structure. Indeed, various studies also showed that there were clear changes in the structure of the rumen bacterial population as the dietary forage to concentrate ratio gradually increased from 80:20 to 60:40 or even to 20:80, with increases in Proteobacteria and decreases in Firmicutes [168,169,170,171,172]. The increased abundance of Proteobacteria during high-concentrate diets was suggestive of an increased need for bacterial species that could metabolize the newly available fermentable carbohydrates [169], thus favoring the growth of amylolytic and other starch-digesting bacterial species and reducing the number of cellulolytic bacteria. This suggested that when animals were shifted from a forage diet to a high-concentrate diet, the microbial diversity in terms of the different species numbers remained but the composition or the species makeup changed drastically in order to adapt to the new rumen environment.Fibrobacter succinogenes, a fibrolytic bacterium that digests fiber, was reported to be gradually decreased as animals were adapted to a high-concentrate diet, and their numbers were 40-fold lower than in those animals fed on hay [169]. A study by Tajima et al. [173] reported a 20-fold decrease in the Fibrobacter population size by day 3 and a 57-fold decrease by day 28 in animals on high-concentrate diets. The population of Butyrivibrio fibrisolvens, another fibrolytic bacterium with high affinity toward maltose and sucrose, also declined 20-fold during adaptation to a high-concentrate diet [172]. Due to the ability of this species to use both cellulose and starch, the Butyrivibrio fibrisolvens population showed a slight decrease in the rumen environment [169]. However, the drop in the population of Butyrivibrio fibrisolvens during a diet with a high proportion of concentrate (30:80) might be because of pH changes due to the increased number of fermentable substrates present within the rumen. This was consistent with the findings of a recent study which showed that the population of Butyrivibrio fibrisolvens increased in high-fiber diets and decreased in high-energy diets [174,175].Changes in bacterial populations with increasing dietary crude protein were attributed to the increased ruminal amino acids, peptides, branched chain VFA and NH3-N [176,177]. Moreover, it was reported by Wang et al. [178] that increased dietary crude protein would enhance ruminal microbial growth, particularly of the bacterial populations in the rumen fluid that consisted of Ruminococcus albus, Ruminococcus flavefaciens, Butyrivibrio fibrisolvens, Prevotella ruminicola, Fibrobacter succinogenes and Ruminobacter amylophilus. In general, the improved microbial enzyme activity has a significant relationship with the increase of the bacterial populations in the rumen [143]. In particular the activities of cellobiase, xylanase, pectinase, carboxymethyl-cellulase, pectinase, protease and α-amylase increased following an increase in dietary protein. However, another researcher showed that lambs fed with a diet containing crude protein levels from 111.7 to 143.6 g/kg DM did not show any effect on the rumen microbial population of R. albus, R. flavefaciens and F. succinogenes [179]. These contradictory findings might be due to the differences in the animals studied and the dietary composition [151,179].5.5. Qualities of Carcass and Buffalo Meat and Their Implications on Human HealthNutrition is a dominant component of livestock production systems that influences several aspects of meat quality. Consumer demands on meat quality have motivated the meat producers to focus on the nutritional aspects of livestock rearing since carcass and meat qualities are affected by the amount and type of nutrient intake. These include dressing yield, meat to bone ratio, protein to fat ratio, fatty acid composition, caloric value, color, physicochemical and processing properties, shelf life and sensory attributes [180].Dressing percentage is one of the important parameters that reflects the potential meat yield from an animal. Usually, the weight of hot carcass is used to compute the dressing percentage. When the weight of cold carcass is used, the dressing percentage is less due to the chilling shrinkage that ranges between 3% and 4.5% of the initial weight of the hot carcass for buffaloes at the age of 6 months up to 4 years [181]. Other than chilling, a feeding diet with supplementation might inconsistently influence the carcass quality. Anjaneyulu et al. [182] reported that supplemented dietary protein did not affect the carcass composition of male buffalo. However, the dressing percentages and yields of the lean meat were higher when buffaloes were fed a high-energy diet compared to those fed a low-concentrate diet [112]. Nevertheless, based on the current information, it was concluded that feed supplementation had little effect on the carcass quality of buffaloes.Meat quality is a major factor that is used for marketing the product [183]. The meat quality is evaluated through physical, biochemical, histological and sensory analyses. The nutritional composition and pH of meat are parts of the biochemical analyses used in assessing the meat quality, which contribute to the edibility or the desirability of the product [184]. Three crucial factors can affect the quality and composition of the meat produced [185]. They are: (1) the feedstuffs and proportion of the diet used for feeding the animal, (2) types of diet, supplement, breed or genetic cross of the animal and (3) the age at which the animal is slaughtered. Meanwhile, diet has been shown to be one of the most important environmental factors that influences the carcass yield, meat cutability and quality [186,187]. The effects of feeding on meat quality are generally studied in terms of the content and composition of the lean and fat tissues, and the subsequent effects on the nutrient content of protein, fat, energy and moisture content.In particular, the total fatty acid content and the types of fatty acid found in meat have major influences on the meat quality and acceptability of the meat by consumers [187]. Feeding a high-energy diet potentially affects the rate of conditioning and consequently, the carcass composition, conformation, meat yield and meat and fat quality [188].Carabeef, also known as buffalo meat, is considered to be a highly nutritious and valuable food. It is a source of high biological value of protein, omega 3 and omega 6 fatty acids and low in fat and cholesterol levels [189]. However, different nutrient contents of buffalo meat have been reported due to different feeding regimes [190]. Lambertz et al. [191] proposed a concentrate supplementation at the rate of approximately 1.5% of body weight to enhance the carcass characteristics of Swamp buffaloes via expressing superior dressing percentage, better muscling and redder meat, with higher contents of protein and fat. Furthermore, addition of a fat supplement enables facilitated absorption of liposoluble nutrients, making it possible to modify the meat fat composition according to consumers’ demand [192]. Moreover, the high energy levels allow the increase of pulp proportion in the diet of fattening animals, which act as precursors of the fatty acid responsible for the lack of consistency of fat from carcasses [193].6. ConclusionsFrom the available literature, it can be summarized that supplementation of concentrate and bypass fat in a potential buffalo diet is very important to overcome the problem of negative energy balance without adversely affecting the dry matter intake, rumen fermentation, blood metabolites and rumen microbial populations. Supplementation of concentrate and bypass fat provides additional benefits due to improved ruminant body weight, body condition score and the economics of the ruminant industry. Further research is necessary to find out the effects of supplementation with concentrate and bypass fat on growing buffaloes fed with various types of basal rations at different productive levels.

animals : an open access journal from mdpi

10.3390/ani13101642

PMC10215283

Host genetics plays a significant role in the effectiveness of immune responses against pathogens. Disease severity for an individual or population is associated with dissimilarity in the levels of host gene expressions. In this study, the variations in the effects of host immune responsiveness between Taiwan Country and White Leghorn chicken breeds were investigated by next-generation sequencing. Overall, immune response-related genes between Taiwan Country chicken and White Leghorn chicken were expressed differently against live attenuated infectious bronchitis virus vaccination. The major histocompatibility complexes and cytokines were determined as significant players in determining the pattern and magnitude of immune responses following an infection. This study demonstrated the host genetic influences on the development of adaptive immune response between the Taiwan Country and the White Leghorn chickens after infectious bronchitis virus vaccination.

This study aims to identify the immune-related genes and the corresponding biological pathways following infectious bronchitis virus vaccination in Taiwan Country and White Leghorn chicken breeds. Transcriptomic analyses of the spleen of these two breeds were conducted by next-generation sequencing. Compared to White Leghorn chicken, Taiwan Country chicken showed a significantly higher level of anti-infectious bronchitis virus (IBV) antibodies at 14 and 21 days pos vaccination. At 7 days post vaccination, in the Taiwan Country chicken, higher expression of mitogen-activated protein kinase 10, Major histocompatibility complex class 1, and V-set pre-B cell surrogate light chain 3 were found. In contrast, the White Leghorn chicken had a high expression of interleukin 4 induced 1, interleukin 6, and interleukin 22 receptor subunit alpha 2. These findings have highlighted the variations in immune induction between chickens with distinct genetic background and provided biological pathways and specific genes involved in immune responses against live attenuated IBV vaccine.

1. IntroductionInfectious bronchitis (IB) is a highly contagious disease in chickens, substantially affecting the poultry industry worldwide. This disease is caused by avian corona infectious bronchitis virus, a member of Gammacoronavirus [1]. Most clinical signs are related to the upper respiratory, urinary, and reproductive systems [2]. The mortality rate of IB infection ranges from 0% to 82%, depending on the viral strain, health status, and age of birds [3]. The virus primarily replicates in the trachea and causes ciliary stasis, leading to secondary bacterial infection, such as Escherichia coli and Mycoplasma gallisepticum infection [4]. This results in respiratory distress and poor growth performance [5]. In addition, the disease can cause significant loss due to a drop in egg production and poor egg quality [6,7].Several studies have noted the influence of host genetic background on the immune response following inoculation of virulent or avirulent (vaccine) poultry viruses. Different breeds and sublines showed the variation of genes expression following infection, affecting the level of susceptibility to diseases [8,9,10,11]. The polymorphism of MHC genes was found to be particularly associated with resistant traits against pathogenic bacteria and viruses [12,13]. In addition, several genes were reported as disease-resistant genes in chicken, including Cyclophilin B (PPIB), MX Dynamin Like GTPase 1 (MX1), and 2’-5’-Oligoadenylate Synthetase Like (OASL) [14,15]. Vaccination studies also revealed the direct effect of host genetic background on variation of innate, mucosal, cellular, and humoral immune responses [16,17,18,19].The spleen is an important organ in chickens, especially for development of both cellular and humoral immune responses [20]. The white pulp of the spleen contains ellipsoids, peri-ellipsoid sheaths (PESS), and lymph nodes, which are the main sites for initiating the humoral immune response by activating and differentiating B cells against blood-borne antigens [21]. This process leads to the secretion of large amounts of antibodies into the bloodstream and the development of memory B cells [22]. T lymphocytes are responsible for cell-mediated immune responses, and their activation occurs in the periarteriolar lymphoid sheaths (PELS), which allow for the monoclonal expansion and differentiation of naive T cells into effector T cells [23]. Gaining an understanding of the biological mechanisms underlying gene expression in the spleen could provide valuable insights into the effects of genetics on the development of immune responses in chickens.Taiwan Country chicken (TCC) is a native breed in Taiwan, which was selected over 30 generations at National Chung Hsing university for prodigious characteristics including high growth rate and feasible adaptation. On the other hand, White Leghorn chicken (WLC) is a Mediterranean breed with high rate of egg production and outstanding feed efficiency. Previous studies have reported a variation in susceptibility between TCC and WLC responding to infectious diseases such as Newcastle disease, Marek’s disease, and Leucocytozoonosis [24,25,26]. The use and comparative analysis of these two genetically distinct breeds of chickens have the potential to elucidate the host genetic components that are associated with differential susceptibility to infections.The aim of this study is to enhance our knowledge on the influence of host genetics on immune responses after IBV vaccination. RNA sequencing was used to cross-compare the immune-related genes of IBV-vaccinated Taiwan Country chickens (TCCs) and White Leghorn chickens (WLCs). The findings of this study would increase the availability of effective disease control strategies.2. Materials and Methods2.1. Animals and Tissue SamplingIn this experiment (Figure 1), B strain TCCs and WLCs were obtained from the National Chung Hsing University and Taiwan Hubbard GP Farm, respectively. Thirty-six day-old chicks of each type were obtained and raised in a disease-free facility with ad libitum feed and water. At 3 weeks old, the chickens of each breed were separated into two groups (18 each in the control and IBV groups). The control group received an IBV-free diluent intranasally, whereas the IBV group received live attenuated IBV, variant strain 4-91 (Nobilis IB 4-91, 7.2 log10 EID50, MSD Animal Health, Boxmeer, The Netherlands) intranasally.Nine birds from each group of TCC and WLC were bled through the wing vein at 0 (before inoculation), 7, 14, and 21 days after the sham or IBV inoculations. After 2 h, the blood was centrifuged at 10,000 rpm for 10 min, and separated sera were stored in a freezer at −20 °C. At 7 days post vaccination, nine chickens of each group were humanely killed through cervical dislocation and spleens were collected. Each sample was transferred into a microcentrifuge tube filled with tissue storage reagent (RNAlater, Sigma-Aldrich, Singapore), and then directly frozen in a freezer at −80 °C.2.2. Enzyme-Linked Immunosorbent AssayAll serum samples were tested for the antibody titer response to IBV by using the IB enzyme-linked immunosorbent assay kit (IDEXX IBV, Invitrogen, Taipei, Taiwan) following the manufacturer’s protocol. The optical density (OD) values were measured using a microplate spectrophotometer (Epoch, BioTek, Taipei, Taiwan). Antibody titers were determined following the manufacturer’s standard protocol using the sample-to-positive (S/P) ratio method and were reported with a positive cut-off value of 0.2.2.3. RNA Extraction and cDNA SynthesisFifty milligrams of spleen from each sample was used for RNA extraction using total ribonucleic acid (RNA) isolation reagent (TRIzol Reagent, Invitrogen, Taipei, Taiwan) following the manufacturer’s protocol. The quality and concentration were determined using the microplate spectrophotometer (Epoch, BioTek, Taipei, Taiwan). Nine total RNA samples from each group were randomly pooled into three samples of equal amounts (three RNA pools in each of the three samples). In addition to using the total RNA for RNA sequencing, some RNAs were converted into complementary deoxyribonucleic acid (cDNA) by using the cDNA synthesis kit (RevertAid First Strand cDNA Synthesis kit, Thermo Scientific, Taipei, Taiwan) following the recommended protocol for quantitative real-time polymerase chain reaction (qPCR).2.4. Library Construction and RNA SequencingRNAs extracted from spleen samples were analyzed through RNA Sequencing (RNA-Seq) with NGS, which was conducted by Biotools Co., Ltd. (New Taipei, Taiwan). RNA purity and quantification were checked using SimpliNano-Biochrom Spectrophotometers (Biochrom, Holliston, MA, USA). RNA degradation and integrity were monitored using Qsep 100 DNA/RNA Analyzer (BiOptic Inc., New Taipei, Taiwan). In total, 1 μL of total RNA per sample was used as input materials for RNA sample preparation. Sequencing libraries were generated using the KAPA mRNA HyperPrep Kit (KAPA Biosystems, Roche, Basel, Switzerland) following the manufacturer’s recommendations, and index codes were added to the attribute sequences of each sample. Briefly, messenger RNA (mRNA) was purified from the total RNA by using magnetic oligo-dT beads. Captured mRNA was fragmented through incubation at a high temperature in the presence of magnesium in the KAPA Fragment, Prime, and Elute Buffer. The first strand of cDNA was synthesized using random hexamer priming. A combination of second strand synthesis and A-tailing converted the cDNA–RNA hybrid into double-stranded cDNA (dscDNA), incorporated deoxyuridine triphosphate (dUTP) into the second cDNA strand, and added dAMP to the 3′ ends of the resulting dscDNA. The dsDNA adapter with 3′-dTMP overhangs were ligated to library insert fragments to generate library fragments carrying the adapters. To select cDNA fragments of 300–400 base pairs (bp), the library fragments were purified using the KAPA Pure Beads system (KAPA Biosystems, Roche, Basel, Switzerland). The library carrying the appropriate adapter sequences at both ends was amplified using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, Roche, Basel, Switzerland) and library amplification primers. The strand marked with dUTP was not amplified, allowing strand-specific sequencing. Finally, PCR products were purified using the KAPA Pure Beads system, and library quality was assessed using the Qsep 100 DNA/RNA Analyzer (BiOptic Inc., New Taipei, Taiwan). All libraries were sequenced using the NovaSeq 6000 platform for paired-end 150 bp sequencing.2.5. RNA-Seq Data Analysis and Go and KEGG Enrichment AnalysisIn this study, the low-quality regions in the reads were removed using Trimmomatic based on the following criteria: THREADS:4; PHRED:33; ILLUMINACLIP: TruSeq3-PE. fa: 2:30:10; LEADING:3; TRAILING:3; SLIDINGWINDOW: 4:20 and MINLEN:36 [27]. The output files were further analyzed using FastQC [28] and MultiQC [29] to ensure the quality of the data. Clean reads were mapped to the Gallus gallus reference genome (GRCg7b) obtained from the National Center for Biotechnology Information by using HISAT2 software. Prior to the differential expression (DE) analysis, low-expression genes were excluded. Then, DE analysis between groups was performed using DESeq2 [30]. The p-values were corrected using the Benjamini–Hochberg procedure to control the false discovery rate (FDR). Transcripts that passed the cut-off criteria of |log2 (FoldChange)| > 1 and with a p-value < 0.05 were considered as differentially expressed genes (DEGs). Candidate genes were used to assess the biological pathway by using the Gene Ontology (GO) database and Kyoto Encyclopedia of Genes and Genomes (KEGG) database.2.6. Quantitative Real-Time PCR VerificationThe RNA-Seq results were validated through qPCR analysis with six selected genes, namely, interleukin 6 (IL-6), interleukin 22 (IL-22), C-C motif chemokine ligand 19 (CCL-19), C-X-C motif chemokine receptor 4 (CXCR4), C-X-C chemokine ligand 13-like 2 (CXCL13L2), and CD 34 molecule (CD34). These immune-related genes were selected based on their involvement in the GO and KEGG pathways and the availability of the sequences in the National Center for Biotechnology Information (NCBI) database. qPCR was performed using SYBR Green qPCR assays. The total volume of the reaction mixture was 10 µL, which comprised 5 µL of qPCR Master mix (PowerUP SYBR Green Master Mix, Appliedbiosystems, Taipei, Taiwan), 1 µL of cDNA from spleen extract, 3.8 µL of nuclease-free water, and 0.2 µL of forward and reverse primers (Supplementary Table S1). Then, each sample was placed into a qPCR analyzer (Step One Plus, Appliedbio systems, Taipei, Taiwan). In the analysis step, Step one software version 2.3 was used, followed by a qPCR cycle consisting of a Fast cycling mode (Primer Tm 60.0 °C) involving 40 cycles of denaturation (95.0 °C for 15 s), annealing (60.0 °C for 30 s), and elongation (60.0 °C for 30 s). In this study, we selected glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as the endogenous gene due to its stable expression in our samples, and all qPCR reactions were performed in triplicate. Relative mRNA expression was calculated using the 2−ΔΔCt method [31].2.7. Statistical AnalysisThe differences in antibody titers against IBV between treatment and control groups in each breed were analyzed by t-test using the TTEST procedure. All statistical analyses were conducted using SAS software (SAS, 2012).3. Results3.1. Anti-IBV ELISA TitersIn this study, following the vaccination of the 3-week-old birds, there were no clinical signs. The mean anti-IBV ELISA titers in each breed of birds is presented in Table 1. Compared to WLCs, TCCs showed earlier and higher antibody production. Compared with the control group, the antibody titers of the vaccinated TCC and WLC were significantly higher at 7, 14, and 21 or 14 and 21 days post vaccination, respectively. When comparing the IBV-inoculated TCC and WLC birds, significantly higher antibody titers were observed in TCCs at 14 and 21 days post vaccination.3.2. Splenic Transcriptome SequencingThe results of all 12 splenic transcription data (68.12 Gb), with the GC content at approximately 44–49% and the percentage of Q30 at >93% (Supplementary Table S2), indicated that the data could be used for further analysis. In addition, the comparison efficiency of the total reads compared with the GRCg7b reference genome of the 12 samples was 93–95% (Supplementary Table S3), and the comparison efficiency of the percentage of read and the reference genome was approximately 87–93%.3.3. Differentially Expressed Gene ProfilingTo identify the effect of genetics in IBV vaccination, vaccinated and unvaccinated chickens of the same breed were compared (Table 2). In TCCs, 257 upregulated and 186 downregulated genes were found. Upregulated significant immune-related genes involved in the early immune response and B-cell activation were macrophage mannose receptor 1-like 2, mitogen-activated protein kinase 10 (MAPK10), transforming growth factor beta receptor 3, immunoglobulin superfamily DCC subclass member 4, MHC, and class I and V-set pre-B cell surrogate light chain 3. In contrast, WLCs showed 49 and 72 upregulated and downregulated genes, respectively. The upregulated genes in response to vaccination were the genes for interleukin 4 induced 1 (IL-4I1), IL-6, and interleukin 22 receptor subunit alpha 2 (IL22RA2).In the unvaccinated groups between breeds (Table 3), 577 and 1118 genes were up-regulated and downregulated, respectively. TCCs highly expressed IL-4I1, IL-6, IL-22, C-X-C, motif chemokine ligand 14, and leukocyte immunoglobulin-like receptor subfamily B member 5. Upregulated genes in WLCs were the genes for interleukin 1 receptor-like 2, MAPK10, T cell-interacting, activating receptor on myeloid cells protein 1-like, and tumor necrosis factor (TNF) receptor superfamily member 19.Regarding the vaccinated groups between breeds (Table 3), 470 and 416 genes were upregulated and downregulated, respectively. Immune-related genes highly expressed in TCCs were IL-8L1, IgG Fc-binding protein-like, c-type lectin domain family 2 member L-like, class I histocompatibility antigen F10 alpha chain-like, and MHC class II beta chain BLB1. In WLCs, highly upregulated genes were IL-6, IL-22 receptor subunit alpha 2, T cell surface glycoprotein CD8 alpha chain-like, granzyme K, MHC class I antigen YF5, and MHC class II beta chain BLB2.3.4. GO and KEGG Databases of DEGsTo assess crucial biological processes, the pathways were analyzed according to GO and KEGG database. Significant pathways in each comparison from TCCs and WLCs are shown in Supplementary Tables S4–S7.Regarding the comparison between treatment and control groups, the GO database of TCCs (Supplementary Table S4) revealed significant differences in terms of response to chemicals, response to hormones, and taxis. In addition, differences were found in terms of cytokine–cytokine receptor interaction, cytosolic DNA-sensing pathway, and influenza A in the KEGG pathway analysis (Supplementary Table S5).On the other hand, the GO pathway analysis of WLCs (Supplementary Table S6) indicated the enriched pathway in term of IL-6 production, positive regulation of T cell proliferation, and positive regulation of B cell activation. Likewise, the KEGG pathway analysis (Supplementary Table S7) showed that the significant enriched pathways responding to vaccination in WLCs were those related to neuroactive ligand–receptor interaction, taurine and hypotaurine metabolism, phenylalanine metabolism, and cytokine–cytokine receptor interaction.Comparing the vaccinated groups of the two breeds (Table 4), the significant GO enriched pathways in TCCs were those related to cell–cell adhesion through plasma-membrane adhesion molecules, cell differentiation, chemotaxis, and regulation of leukocyte migration. Meanwhile, significant GO pathways of WLCs were those involving cell proliferation, acute inflammatory response, cell differentiation, regulation of cell proliferation, and positive regulation of cell adhesion. From the KEGG pathway analysis (Table 5), B strain TCCs and WLCs were those related to cell adhesion molecules (CAMs), cytokine–cytokine receptor interaction, toll-like receptor (TLR) signaling pathway, salmonella infection, and influenza A.3.5. Validation Based on qPCRIn this study, significantly, DEGs related to innate and adaptive immune responses, including IL-6, IL-22, CCL-19, CXCR4, CXCL13L2, and CD34, were selected by using qPCR assay to confirm the creditability of the RNA-sequencing technique. The expression patterns of all DEGs from qPCR analysis were concomitant with those from RNA-Seq data analysis (Figure 2).4. DiscussionGenetics play a crucial role in immune responses to infectious pathogens and vaccines [32]. Vigorous innate immune responses, rates of macrophage differentiation and activation, and MHC haplotype were well recognized as essential factors associated with disease resistance and susceptibility [33]. Between the breeds, variations in the MHC haplotype and immune-related genes define the outcomes of immune responses and survival ability. In this experiment, the comparison of transcriptomics between distinct genetic line chickens could provide better understanding on transcriptional responses in particular breeds and it revealed significant immune-related genes and molecular mechanisms against IBV vaccination between vaccinated and control groups, as well as between TCC and WLC.4.1. Immune Responsiveness in Taiwan Country ChickensFrom week 2 post vaccination, vaccinated TCCs showed higher antibody levels compared with the controls within 1 week after vaccination. At the same time, with the rise of systemic anti-IBV immunoglobulin, numerous genes involved in innate immune responses, B-cell accumulation and activation were highly expressed. The significant upregulated genes in TCCs responding to immunization were MAPK10, macrophage mannose receptor 1-like 2, Gallus gallus immunoglobulin-like receptor CHIR-B2-like, and V-set pre-B cell surrogate light chain 3. The Gallus gallus immunoglobulin-like receptor CHIR-B2-like is mainly expressed on B cells and its main function is inhibitory receptor-related with B cell proliferation process [34]. In addition, V-set pre-B cell surrogate light chain 3 has a higher expression response to IBV vaccination, which is significant in the B cell maturation and plays a crucial role in Pre-B cell receptor formation [35,36]. The high expression of these genes represented the accumulation of B cells within the spleen in response to vaccination compared with the control group.GO and KEGG database analyses between vaccinated and control groups of TCCs (Supplementary Tables S4 and S5) showed significant pathways related to inflammation and pathogen recognition in response to vaccination. The remarkable enriched pathways from the KEGG database were those involving cytokine–cytokine receptor interaction, nucleotide oligomerization domain (NOD)-like receptor signaling pathway, and TLR signaling pathway. By identifying pathogen-associated molecular pattern (PAMP), NOD-like and Toll-like receptor signaling pathways are the important connector between innate and adaptive immune responses. Additionally, from the analysis from GO database, vaccinated TCCs were highly enriched in response to chemicals, response to hormones, taxis, and cellular response to peptide. These biological processes are essential mechanisms for the host to respond to external stimuli and initiate the adaptive immune response. In addition, hormone and taxis pathways are related to the attraction and accumulation of immune cells to the spleen, allowing T and B cell response to antigens in the circulatory system [37].4.2. Immune Responsiveness in White Leghorn ChickensWLCs showed only 49 upregulated genes and 72 downregulated genes in response to vaccination (Table 2). The significantly upregulated immune-related genes were involved in the innate immune response and activation of adaptive immune responses, including IL-6 and IL-4I1. IL-6 is a crucial multifunctional cytokine related to the proinflammatory process and the activation and differentiation of T and B lymphocytes. A study found that IL-6 is crucial for mice survival after infection with influenza through the optimization of T cell regulation and the migration and phagocytic activities of macrophages [38]. However, the presence of IL-6 and TGF-β are also related to immune tolerance by promoting the expression of regulatory T cells [39]. In human studies, IL-4I1 plays an important role in the limitation of side effects from adaptive immune response by inhibiting IFN -γ production and differentiation of effector T cells and turning naive T cells into regulatory T cells [40].According to the GO and KEGG databases, the important GO pathways in WLCs (Supplementary Table S6) were those involving the regulation of IL-6 production, IL-6 production, positive regulation of T cell proliferation, and B cell activation. The main gene involved in these enriched pathways was IL-6. This evidence stated the important role of IL-6 in immune response modulation against IBV vaccination in WLCs. For the KEGG pathway analysis (Supplementary Table S7), the significant enriched pathways were involved in the inflammatory process, lymphocyte proliferation and differentiation, including cytokine–cytokine receptor interaction, transforming growth factor-beta signaling pathway, and phagosome.From human study, the transforming growth factor-beta (TGF- β) signaling pathway plays a critical role on the divergent function of T cell. On the one hand, TGF-β plays a role in supporting the activation of functional T cells by promoting chemotaxis of immune cells. TGF-β can induce chemotaxis of CD4+ T cells towards the CXCL12 and promote the migration of antigen-presenting cells towards lymphatic vessels and lymph nodes [41,42]. On the other hand, TGF-β also controls the immune tolerance by inhibiting T-cell receptor signaling and promoting the differentiation and function of the regulatory T cells (Tregs) [39]. The highly expressed IL-4I1 and enriched transforming growth factor-beta signaling pathway in WLCs could limit the side effects from overstimulation of the adaptive immune response. However, lower levels of effective T cells could also reduce the host’s capability to eradicate the invasive pathogens. A previous study indicated that high expression of regulatory T cells is related to susceptible traits in chicken against Marek’s infection [43].4.3. Differences of Immune Responsiveness between BreedsTo investigate the effect of genetics between breeds, the control groups between TCCs and WLCs were compared. TCCs had highly expressed cytokine-related genes involved in immunological tolerance, including IL-4I1, IL-6, and IL-22. As previously described, IL-4I1 plays a significant role in the limitation of immunopathology mediators by controlling the effector Th1 and Th17 cells [44]. Additionally, IL-22 is secreted by activated dendritic cell and Th 17 cells. It functions as a proinflammatory and regenerative factor and is crucial for the protection and regeneration of barrier organs such as the lungs and gastrointestinal system [45]. In addition, immunoglobulin superfamily member 1-like 6, MHC class I polypeptide-related sequence A, and class I histocompatibility antigen, F10 alpha chain-like were enriched. An immunoglobulin superfamily member, recognized as an adhesion molecule, plays a crucial role in the mediation of cell surface interaction and pathogen perception [46]. MHC class I polypeptide-related sequence A, class I histocompatibility antigen, and F10 alpha chain-like are related to the antigen presentation process through MHC class I.The upregulated genes in unvaccinated WLCs were those related to T cell-interacting, activating receptor on myeloid cell protein 1-like, and nuclear factor of activated T cells (Table 3). T cell-interacting, activating receptor on myeloid cells protein 1 is a triggering receptor on macrophages and neutrophils related to TNF-α and IL-8 production against pathogen infection [47]. Likewise, the nuclear factor of activated T cells is a transcriptional factor that involves many normal body processes and is well known as a crucial player in T cell activation and the determination of the fate and function of the T cell population [48]. Moreover, WLCs also highly expressed interleukin 1 receptor-like 2 and MAPK10. The main function of interleukin 1 receptor-like 2 is inhibiting IL-1 activity, which can inhibit the excess production of proinflammatory cytokines.Comparison between vaccinated groups of the two breeds (Table 3) revealed that the genes highly expressed in TCCs were those encoding IL-8L1, C-type lectin domain family 2 member L-like, IgG Fc-binding protein-like, C-type lectin domain family 2 member L-like, class I histocompatibility antigen, F10 alpha chain-like 3, class I histocompatibility antigen, F10 alpha chain-like, MHC class I polypeptide-related sequence A, and MHC class II beta chain BLB1 response to vaccination. The IL-8L1 plays a crucial role in neutrophil activation through the promotion of cell adhesion, transendothelial migration, and killing process. Furthermore, the function of the IgG Fc-binding protein is the modulation of the adaptive immune response through fusion with specific antigenic proteins [49]. Higher expression of the above genes in the TCCs in comparison to WLCs shows the role of these genes in activation of B and T cells in subsequent IBV vaccination.Among the vaccinated groups, WLCs showed high expression of IL-6, IL-22 receptor subunit alpha 2, T cell surface glycoprotein CD8 alpha chain-like, granzyme K, MHC class I antigen YF5, and MHC class II beta chain BLB2. The high expression of these genes in WLCs relates to T cell development and activation process responding to IBV vaccination. The T cell surface glycoprotein CD8 alpha chain-like was related to CD8 T cell accumulation and T cell activation within the spleen. In addition, granzyme K was secreted by activated macrophage, natural killer cell, and cytotoxic T lymphocyte, which functionally promotes the cytotoxicity of invader and infected cells [50]. The higher expression of these genes compared with TCCs suggests that T cell development and activation were among the major immune processes in the spleen responding to vaccination in WLCs.From the KEGG pathway analysis (Table 5), TCCs and WLCs shared several immune-related pathways responding to vaccination, including cell adhesion molecules (CAMs), cytokine–cytokine receptor interaction, toll-like receptor (TLR) signaling pathway, salmonella infection, and influenza A. Interestingly, the gene members in each pathway were expressed differently between the two breeds of chicken. The major gene members in TCCs were IL-8L1 and BLB1, whereas the main members in WLCs were IL-6 and BLB2. BLB1 and BLB2 are class II MHC genes that are highly polymorphic and responsible for transducing signals during the B cell activation process. The results in Table 1 show that TCCs had a higher capability to produce more immunoglobulin in response to IBV vaccination compared with WLCs. Based on this experiment, we could assume that the different expression of MHC class II between TCCs and WLCs might play a significant role on varying antibody production levels against IBV vaccination. However, further investigation is needed to confirm these findings.5. ConclusionsThis study provides a comprehensive understanding of immune related genes and biological pathways against IBV vaccination of TCCs and commercial WLCs. On day 7 post inoculation, the vaccinated TCCs group exhibited higher levels of antibody production compared to the control group. Additionally, a set of differentially expressed genes related to innate immune responses, as well as B cell proliferation and development, were highly expressed. These genes included MAPK10, macrophage mannose receptor 1-like 2, Gallus gallus immunoglobulin-like receptor CHIR-B2-like, and V-set pre-B cell surrogate light chain 3. On the contrary, the significant enriched immune-related genes in vaccinated WLCs were IL-6, IL-4I1, and IL22RA2, which related to proinflammatory process and the activation and differentiation of T and B lymphocytes. In addition, the data analysis revealed that significant biological pathways of both two breeds were related through cell adhesion molecules (CAMs), cytokine–cytokine receptor interaction, toll-like receptor (TLR) signaling pathway and influenza A against live attenuated IBV vaccination.

animals : an open access journal from mdpi

10.3390/ani11051324

PMC8148191

The pygmy hog is one of the world’s rarest suids and classified as an endangered species. Efforts are being made to breed them in captivity and reintroduce them into the wild. In this study, we examined reproductive hormones in captive pygmy hogs using a non-invasive method by collecting 785 fecal samples from five females and two males for 12 months. High-pressure liquid chromatography was performed to examine the presence of immunoreactive progesterone and testosterone metabolites in the fecal samples. We standardized and validated enzyme immunoassays (EIA) for fecal progesterone and testosterone metabolites. Using progesterone EIA, we were able to detect pregnancies in four females and estimate the relevant gestation period. We also recorded 172 births from the captive breeding center and found strong seasonality patterns in births. In males, fecal testosterone metabolite concentrations were higher in the breeding season than in the non-breeding season as evidenced by elevated testosterone concentrations during breeding season. A significant difference in fecal progesterone metabolites concentration was observed between non-pregnant and pregnant females. This study can directly help in monitoring the reproductive status of reintroduced hogs both in the wild and in conservation breeding programs in India and elsewhere.

The pygmy hog (Porcula salvania), until recently was classified as a critically endangered suid facing the threat of extinction due to habitat degradation. Efforts are being made to protect the pygmy hog from extinction and breed them in captivity under the Pygmy Hog Conservation Programme (PHCP). However, very little information is available on the reproductive physiology of pygmy hogs. Therefore, the present study aims to standardize enzyme immunoassays (EIAs) for monitoring pregnancy and reproductive status using progesterone and testosterone metabolites. A total of 785 fecal samples were collected from five females and two males over a period of one year from the PHCP Research and Breeding Centre, Guwahati, Assam. High-pressure liquid chromatography (HPLC) analysis revealed the presence of immunoreactive progesterone and testosterone metabolites in feces. Mating was observed in all five females, and four of them gave birth successfully. We were able to detect pregnancy using fecal progesterone metabolites. The mean gestation period, based on mating and parturition, was estimated to be 153.25 days from the four females studied. The breeding center recorded 172 births between 1996 and 2000 and found strong seasonal patterns in the birth rate, with most of the births occurring between May and June. In the males, fecal testosterone metabolites were significantly higher in the breeding season than in the non-breeding season. This is the first study on the subject and will help with future breeding programs in other captive breeding centers and with reproductive monitoring of reintroduced populations.

1. IntroductionThe pygmy hog (Porcula salvania) is the world’s rarest and smallest wild suid belonging to the family Suidae [1]. It was listed as critically endangered by the IUCN Red List until 2019, but it has recently been downgraded to endangered [2] due to the conservation breeding and reintroduction efforts of the Pygmy Hog Conservation Programme (PHCP). It continues to be listed under Schedule I of the Indian Wildlife (Protection) Act, 1972. The pygmy hog is considered an indicator species of the healthy grassland ecosystem andalso suffers from poor wildlife management practices, persistent burning and other anthropogenic disturbances [3,4]. Once widespread across tall wet grassland in a narrow strip south of the Himalayan foothills from Uttar Pradesh to Assam (India) across Nepal and Bhutan, the pygmy hog population declined in the last century. By the early 1990s, it was reduced to a single global population of 400–500 individuals in the Manas National Park, India. The pygmy hog population has declined due to degradation and loss of grassland, the rapid expansion of human settlements and agricultural encroachments, flood control schemes, and improper management of grassland ecosystems [5,6,7]. Furthermore, planting trees in grasslands andthe indiscriminate use of fire to create an opening and to promote fresh grass are other major threats to the pygmy hog’s habitat [8]. Interestingly, pygmy hog habitats are shared by other endangered animals that include the one-horned Indian rhinoceros (Rhinoceros unicornis), tiger (Panthera tigris), hispid hare (Caprolagus hispid), water buffalo (Bubalus arnee), Bengal florican (Houbaropsisbengalensis), and Assam roofed turtle (Kachugasylhetensis).Efforts are being made to save the species from extinction, which include conservation breeding and reintroduction. Initial efforts in 1971 and 1976 failed to yield any success due to the nonscientific method of breeding [5]. In 1996, the Assam Forest Department, Durrell Wildlife Conservation Trust, and IUCN/SSC Wild Pig Specialist Group, along with Eco-Systems-India, set up a research and breeding center to breed pygmy hogs in captivity and release them into the wild to replenish natural populations. Until 2020, the program had successfully produced 683 individuals from tenwild-caught originalpygmy hogs. Between 2008 and 2018, a total of 116 captive-bred individuals were released periodically into three reintroduction sites at SonaiRupai Wildlife Sanctuary, Rajiv Gandhi Orang National Park, and Barnadi Wildlife Sanctuary in Assam [8]. In 2020, 14 hogs were released in the eastern ranges of Manas National Park, where less than 100 hogs may now survive in the central range. Thus, 130 captive-born individuals have been released into the wild as part of the continuing recovery program, which has put increased stress on the efforts to restore and manage suitable grasslands in their former range.Pygmy hogs eat a wide range of food, including roots, tubers, shoots, insects, earthworms, eggs, and carrion. They are foragers and spend six to eight hours searching for food by digging and turning up litter and topsoil using their snout [4]. They live in groups of 4–6 individuals, primarily adults with their young. Adult males weigh about 8–10 kg with a head-body length of 61–71 cm, while females weigh 6–8 kg with a head-body length of 55–62 cm [6]. Most of the mating in captivity occurred between December and February, and births were recorded before the monsoon (May to September). The litter size ranged between 2–7 but was mostly in the range of 4–6 in captivity [6].Reproductive seasonality is characteristic of many mammalian species. However, seasonality is a result of various intricate factors formed by physiological mechanisms. The physiology of species is significantly influenced by environmental factors such as climate, temperature, humidity, photoperiod, nutrition, foraging conditions, and social interactions between the conspecifics [9,10,11]. The physiological control of seasonal breeding is driven by the central circadian regulatory system situated in the suprachiasmatic nucleus (SCN), which involves modulation of the neuroendocrine mechanism using the hypothalamus and the pituitary and pineal glands to regulate the breeding season. Most species show strong seasonal reproductive variation evidenced by increasing levels of sex steroids, including long-tailed macaques (Macacafascicularis) [12], plains zebras (Equus quagga),springboks (Antidorcas marsupialis) [13], Iberian red deer (Cervus elaphushispanicus) [14], Père David’s deer (Elaphurusdavidianus) [15], coyotes (Canis latrans) [16], and camels (Camelus dromedarius) [17]. The wild boar (Sus scrofa), a close relative of the pygmy hog, exhibits seasonal polyestrous, while the domestic pig is known to breed throughout the year [18].Understanding basic reproductive function is crucial for successful conservation breeding programs of endangered species, and it can be studied by monitoring circulating hormones [19,20,21]. Hormones can be measured in a variety of biological samples such as feces [22,23,24], urine [25], blood [26], saliva [27], milk [28], and hair [29,30,31]. Although circulating hormones in the blood give an accurate measurement, blood sampling for long-term monitoring of wild animals is challenging and stressful. As an alternative method, estimating the hormone metabolites in feces as a non-invasive method is feasible since circulating hormones metabolize in the liver and are excreted through the feces. In many species, no, or very little, native hormone is present in the feces, and most of the hormones are metabolized. The excreting metabolites vary considerably between even closely related species and in some species even between the sexes. Therefore, each assay needs to be validated with biologically relevant concentrations of the hormone and its metabolites in the feces [20]. Fecal steroid analysis has been used to assess the reproductive status and endocrine function in various captive and free-ranging wild species, including Asian elephants [32,33], musk deer [34], red pandas [35], primates [36,37], big cats [22,38], birds [39], and chelonians [40].Despite the successful breeding program, the reproductive physiology of this species is poorly understood; the ongoing conservation breeding program provides an exceptional opportunity to understand the reproductive biology of the species, particularly reproductive physiology of endangered suids. The present study aimed (1) to characterize fecal hormone metabolites using high-pressure liquid chromatography (HPLC), (2) to biologically validate enzyme immunoassays for progesterone and testosterone metabolites, (3) to monitor the pregnancy and reproductive status in captive pygmy hogs using fecal steroid hormone analysis, and (4) to examine the seasonality of reproduction. This is the first report on monitoring the reproductive status of pygmy hogs in India using a non-invasive method.2. Materials and Methods2.1. Sample CollectionA total of 785 fecal samples were collected from seven captive pygmy hogs (two males and five females) from the Pygmy Hog Research and Breeding Center, Basistha, Guwahati, Assam. Males and females were caged separately and adjacent to each other. The males were allowed into the female enclosures during the breeding season (based on 10 y of breeding and mating records in the center) for mating. The captive pygmy hogs were fed daily with a balanced diet with a wide range of variety of tubers, cereals, pulses, fruits, vegetables, and eggs. Furthermore, they were allowed to forage for natural vegetation and soil invertebrates such as earthworms, termites, ants, and beetles. The enclosures were planted with Saccharumnarenga and Phragmitieskarka grasses, which are known to occur in pygmy hog habitats. The temperatures in this region range from 11° C (January) to 33 °C (July), and June to September is the rainy season with peak rainfall during July.Samples were collected three to four days in a week during one year (July 2015 to July 2016). Due to space restrictions, samples collected for some individuals were discontinued over some periods. Freshly collected fecal samples were dried in a hot air oven at 70 °C; pulverized; and stored in zip lock bags with date, individual IDs, etc. at 4 °C until further extraction. Observations, if any, on mating and other reproductive behaviors (nudging, mounting, squeaking, soft grunting) were recorded on a daily basis during the sample collection period. Details of the age, sex, individual IDs and the number of samples collected are given in Table 1.2.2. Birth and Gestation DataTo examine the seasonality of births in pygmy hogs, the data on births from April 1996 to July 2020 at the Pygmy Hog Research and Breeding Centre, Basistha, Guwahati, Assam were collected and analyzed. Data on mating observations and parturition were also collected from the center’s records for estimating the length of gestation.2.3. Extraction of Fecal Steroid MetabolitesFecal samples were extracted using the previously described procedure with minor modifications [41,42]. The dried fecal powder was sieved and weighed to 0.2 g in a 15 mL falcon tube, and 2 mL of 80% methanol was added and vortexed for 20 min. Furthermore, samples were then kept at 4 °C overnight and centrifuged at 2000× g for 10 min, and supernatants were stored in −20 °C for further analysis.2.4. Hormone AssaysFecal progesterone was measured using the monoclonal anti-progesterone antibody (CL425; provided by Dr. Coralie Munro, University of California, Davis, CA, USA). The progesterone antibody had 100% cross-reactivity with progesterone and a variety of 5α- and β-reduced pregnane [43]. Fecal testosterone was measured using the polyclonal anti-testosterone antibody (R156/7; provided by Dr. Coralie Munro, University of California, Davis, CA, USA). The testosterone antibody had 100% cross-reactivity with testosterone; 57.4% with dihydrotestosterone; <0.3% with androstenedione; and <0.1% with androsterone, dihydroepiandrosterone, β-estradiol, and progesterone [44].2.5. Enzyme Immunoassay ProcedureEnzyme immunoassays (EIAs) for fecal progesterone and testosterone were performed as described previously [32,34]. The 96-well Nunc–Maxisorp microtiter plate was coated with 50 µL of antibody per well, diluted in coating buffer (0.05 M sodium bicarbonate buffer, pH 9.6) and kept at 4 °C for overnight incubation. The plate was washed four times with washing buffer (0.15 M NaCl, 0.05% Tween 20). Added to each well was 50 µL of fecal extract diluted in EIA buffer (0.1 M PBS, pH 7, and 1% BSA) or standard followed by 50 µL of conjugated HRP (horseradish peroxidase), incubated at room temperature for 2 h. The plate was then washed 4 times with washing buffer; then, 50 µL of TMB (Tetramethyl benzidine/H2O2, Genei, Bangalore) was added to each well and kept in the dark for 5–10 min for color development. The reaction was stopped using 50 µL of stop solution (1M Hydrochloric acid (HCL), and optical density (absorbance) was then measured at 450 nm using an ELISA reader (Thermo Multiskan Spectrum Plate Reader, version 2.4.2; Thermo Scientific, Helsinki, Finland).2.6. High-Performance Liquid ChromatographyTo evaluate the immunoreactivity of fecal progesterone and testosterone with corresponding antibody and separation of fecal steroid metabolites, high-performance liquid chromatography was performed using the Shimadzu CTO-10AS system (Shimadzu Corporation, Tokyo, Japan). Steroid specific reverse-phase C-18 column was used (waters column, symmetry C-18, 4.6 3 20 mm, 3.5 mm, intelligent speed (IS)column to identify the steroid metabolites from fecal samples. Before HPLC analysis, pooled fecal extracts were passed through Sep-Pak C18 cartridges (Waters, Milford, MA, USA) for purification and eluted with 3 mL of absolute methanol. The purified fecal extracts were dried using nitrogen gas and resuspended in 100 µL of absolute methanol as described previously [34,45]. The protocol running time was 8 min using a gradient flow of 20–64% acetonitrile (ACN):water (H2O) at a flow rate of 1 mL/min, and steroid hormones were detected at the 190 to 400 nm wavelength. Fractions were collected from progesterone and testosterone standards and pooled fecal extracts manually, about 250 µL every 15 s (4 fractions/minute), and vacuum dried. The dried fractions were resuspended in 100 µL of EIA buffer and used in the assay.2.7. Data AnalysisData are represented as mean ± standard error of the mean. Correlation analysis for parallelism was carried out using Pearson’s correlation analysis. The fecal testosterone metabolite data are presented using descriptive statistics because of the small sample size. The differences between mean progesterone metabolite concentrations of pregnant and non-pregnant samples were analyzed using a Wilcoxon signed-rank test, as data were not normally distributed (using Shapiro–Wilk test). All statistical analyses were carried out using SPSS 17.0.3. Results3.1. Enzyme Immunoassay ValidationProgesterone and testosterone enzyme immunoassays were validated by demonstrating the parallel displacement curves between the pooled serial dilution ofpygmy hog fecal extracts and their respective standards to determine the immunological activity of fecal hormone and standard with the corresponding antibodies used in the assays (Figure 1). Assay sensitivity was calculated with 90% binding and found to be 0.39 and 1.17 pg./well for progesterone and testosterone, respectively. The intra- and inter-assay coefficient of variations (CV) were 6.6 and 11.6% for progesterone and 6.8 and 11.1% for testosterone. Recovery and accuracy of a known amount of unlabeled steroid hormones in fecal extracts were 81.4 ± 4.4% for progesterone and 83.1 ± 11.42% for testosterone. The correlation (r2) and slope (m) values for the recovered exogenous steroids were r2 = 0.99, m = 0.87 and r2 = 0.98, m = 0.80 for progesterone and testosterone, respectively. The presence of fecal progesterone and testosterone confirmed by HPLC profiles and eluted fractions showed the immunoreactivity with corresponding EIAs. However, fecal extracts showed a single large peak due to close immunoreactivities. Moreover, standard concentrations of progesterone and testosterone were higher as compared to pooled fecal steroid metabolites (Figure 2).3.2. Reproductive MonitoringA total of 785 samples were collected from five adult females and two adult males for a one-year period. All five females were found mating with males between January and April, and four of them delivered young, although one died due to unknown reasons (PH368) (Table 1).Overall, individual fecal progesterone metabolite concentrations ranged from 172 to 2590 ng/g (Figure 3). The pregnant females had significantly higher fecal progesterone metabolite concentrations compared to their non-pregnant values (Wilcoxon signed rank test, p < 0.001 for all five animals; Figure 4). All the pregnant females showed similar progesterone profiles throughout their pregnancies.Based on observation of mating and parturitions of four females, the gestation period ranged from 148 to 157 days with an average of 153.25 days, which correlates to previous data recorded by the breeding center that ranged from 148 to 161 days with a mean of 154.40 days (n = 30).Two adult males, those involved in successful mating with the females, were also monitored for fecal testosterone metabolites, and they showed elevated fecal testosterone concentrations between September and December (Figure 5), which is about two to three months before the mating observations. Overall, the fecal testosterone metabolite concentrations ranged from 36 to 888 ng/g, and the elevated values were recorded during the pre-mating period (September–December).A total of 172 births were recorded between 1996 and 2020 in the PHCP, Guwahati. All of the births were recorded between March and October, and about 74.71% of births were observed in May and June, showing a strong seasonality in the births (Figure 6).4. DiscussionThe present study reports on the standardization of enzyme immunoassays (EIAs) for fecal progesterone and testosterone metabolites and the endocrine patterns of reproductive hormones in endangered captive pygmy hogs using a non-invasive method. For the firsttime, long-term monitoring of the reproductive hormones in pygmy hogs was undertaken in a captive population. As expected, we found immunoreactive progesterone and testosterone metabolites in fecal samples of pygmy hogs using HPLC analysis. The progesterone metabolites in the fecal extracts could be monitored using a monoclonal antibody EIA (CL425) developed against progesterone (UC Davis, USA). This antibody reported high cross-reactivity with five alpha and beta pregnane metabolites excreted in the feces of a variety of species [43]. The progesterone EIA (CL425) has been previously standardized to detect pregnancy in a wide range of animals, such as the Himalayan musk deer (Moschus chrysogaster) [34], dugong (Dugong dugon) [46], maned wolf (Chrysocyonbrachyurus) [47], black rhinoceros (Dicerosbicornis) and white rhinoceros (Ceratotheriumsimum) [48], giant anteater (Myrmecophagatridactyla) [49], giraffe (Giraffa camelopardalisrothschildi) [50], Nile hippopotamus (Hippopotamus amphibius) [51] and red brocket deer (Mazama americana) [52]. In this study, we were able to successfully monitor pregnancies in pygmy hogs and were also able to distinguish between pregnant (>3000 ng/g) and non-pregnant values using fecal progesterone. This finding has a direct implication on the successful breeding and monitoring of reproduction in one of the most endangered mammals in the world.Of the five females, four were observed to successfully mate and conceive, as evidenced by the delivery of their litters (size = 3–4). One of the pygmy hogs (PH 368) died during the study period and was found to be pregnant, as four fetuses were discoveredduring the post-mortem. The mean gestation period was estimated to be 153.25 days based on mating and delivery observations. During pregnancy, the fecal progesterone metabolite concentrations were elevated in all females until parturition. The fecal progesterone concentrations dropped to baseline values within a few days of parturition. The observed gestation period was also within the range of data from the breeding center, which was between 148 to 161 days from 30 females studied. However, previous reports suggested that the mean gestation period was 120 ± 5 days, and it ranged between 110 and 130 days based on the behavioral observations [53,54,55]. Interestingly, the gestation periods in the Suidae family ranges widely from 115 days in wild boar (Sus scrofa) to 170 days in the common warthog (Phacochoerus africanus) [56]. The present observation is within the range of the Suidae family’s gestation period. Furthermore, the present study shows that pygmy hogs are seasonal breeders as evidenced by most of the births being recorded within a few months before the monsoon, while their related species, the domestic pig, in this region breeds throughout the year [18].Previously, testosterone EIA (polyclonal antibody, R156/7) has been reported for monitoring fecal testosterone metabolites in a wide range of animals, including the pronghorn (Antilocapra americana peninsularis) [57], red river hog (Potamochoerusporcus) [58], and polar bear (Ursus maritimus) [59]. Fecal testosterone metabolite levels of two monitored pygmy hogs did not show aclear cycle; however, there were elevated concentrations during the September–December period for both males. Most of the mating was observed between December and February, which is about one to two months after the elevated fecal testosterone metabolites in males. Fecal testosterone metabolites elevation in mammals is directly related to reproductive preparedness and sperm production. Overall, the elevated testosterone metabolite concentrations were related to male fitness in breeding, as evidenced by mating with the females during December and January.Previous studies have shown that the analysis of fecal steroid metabolites could be conductedin other members of the Suidae family, including the red river hog (Potamocherusporcus), common warthog (Phacochoerus africanus), babirusa (Babyrousababyrussa) [60] wild boar (Sus scrofa) [18], and collared peccary (Pecaritajacu) [61]. However, this is the first report of the validation and standardization of enzyme immunoassays (EIAs) for reproductive monitoring in pygmy hogs using non-invasive methods. Since the pygmy hog is considered one of the most endangered mammals globally, this study maydirectly help breeding management in captivity. Furthermore, this method could be used forfertility monitoring and pregnancy detection in pygmy hogs in captivity and in the wild as well as inreintroduced populations.5. ConclusionsThis is the first study on reproductive hormone (progesterone and testosterone) monitoring in endangered pygmy hogs using a non-invasive method. Fecal progesterone and testosterone EIAs can be used to detect the pregnancy and fertility status in pygmy hogs. This study mayfurther facilitate reproductive monitoring of breeding programs in captivity and also assist in the management of wild and reintroduced populations.

animals : an open access journal from mdpi

10.3390/ani11092678

PMC8466337

China is the world’s largest producer of food fish, and Chinese consumers have a preference to buy live fish. Live transport of fish is, therefore, a common procedure in aquaculture and is a potential animal welfare hazard. Little has been published on current fish transportation practices in China or the knowledge and attitudes of stakeholders in this industry. Our qualitative study aimed to obtain original information about live transport processes from a cross-section of aquaculture stakeholders in China by conducting individual interviews. Stakeholders were interviewed about their knowledge of live transport and their attitudes towards the welfare of fish. Self-described knowledge of live transport varied between participants with different job types. Most participants had heard of and understood the concept of “animal welfare”, but many understood it to only refer to terrestrial livestock, not fish. This suggests that knowledge of fish welfare in the industry may be less than for other farm animals. The findings of this pilot study contribute to a better understanding of live fish transport from a stakeholder point of view. The findings will also assist in informing, educating, and sensitizing stakeholders to the importance of fish welfare during live transport.

China is the largest food fish producer in the world. Chinese consumers normally purchase fish that are still alive to ensure freshness. Therefore, the live transport of fish is important in China’s aquaculture, although it carries potential risks for animal welfare. This study investigated the attitudes and knowledge of stakeholders within Chinese aquaculture towards the live transport and welfare of fish. Semi-structured interviews were conducted with 12 participants who were involved with the aquaculture industry in China. Most participants self-rated their transport-related knowledge as moderate and had some understanding of animal welfare, although this term was generally considered only relevant to terrestrial animals. Participants’ responses indicated that the live transport of fish occurs frequently in China, generally using sealed tanks, plastic bags, and foam boxes, in purpose-built vehicles. Seasonal changes, such as changes in ambient and water temperature, are considered to be important contributors to successful live transport, as well as sufficient oxygen supplies and stocking density. The use of anesthetics was not commonly reported, particularly in food fish, and fish capture is predominantly by conventional dipnets. The health status of transported fish is determined mostly by morphology (body injury, body or eye color, and fin condition), as well as vigor and swimming ability. Our results indicate that live transport poses a number of welfare risks to fish but that participants in the process associated welfare concerns more with terrestrial animals, not fish.

1. IntroductionFish is an important dietary protein for many people in the world, and global fish consumption has continuously increased over recent years [1]. With the rapid global growth of aquaculture production, like other farmed terrestrial animals, concern for the well-being of farmed fish has also gained attention among consumers, animal protection activists, researchers, and producers [2].Road transport of live fish by vehicle (henceforth, live transport) is a common practice in aquaculture, but it can lead to detrimental effects on fish well-being. Fish are often transported between farms or to markets for on-growing or sale [3]. There are two main methods for transporting live fish in water. The first is by using water-filled containers equipped with an outside oxygen source (e.g., oxygen tanks), and the second is by using sealed plastic bags filled with oxygen prior to transport, described as the open system and the closed system, respectively [4,5]. Live transport includes pre-transport procedures (grading, crowding, netting, fasting, handling, and loading/packing), during transport procedures, and post-transport processes (unloading and handling), which are potentially stressful to fish [6,7]. Common stressors associated with live transport are inappropriate handling, air exposure, food deprivation, poor water quality, inappropriate transport densities, sudden changes in water temperature, and rapid water movement [3,5,8,9,10]. It is challenging to maintain the health and well-being of live fish during transport, particularly because of the large numbers of animals in many varied transport situations. Transported fish show physiological responses indicative of stress, such as elevated glucocorticoid (e.g., cortisol) levels and blood glucose content, and excessive physiological stress is known to reduce fish vitality and increase mortality [7,8,11].China has led global freshwater aquaculture production since the 1990s [1,12,13]. Inland aquaculture, particularly freshwater farming of finfish, is the main component of the Chinese aquaculture industry, comprising more than 50% of the country’s total aquaculture production in 2019 [11,12]. The majority of this production is in the provinces of Hubei, Guangdong, Jiangsu, Hunan, Jiangxi, Anhui, and Zhejiang [11], which largely fall in South and East China where water is generally abundant. Cyprinid species (e.g., grass carp Ctenopharyngodon Idella and silver carp Hypophthalmichthys molitrix) are predominant in most of the production in China [2,11,12,13]. Although production methods used in Chinese freshwater aquaculture are highly diverse, pond culture is the most common rearing method [11,12].In China, consumers prefer to purchase fish while still alive for later cooking, which is believed to be healthier for the consumer and better tasting than fish that are killed earlier or preserved [14]. Live fish from domestic markets are preferred to frozen and processed products [14,15]. A substantial proportion of aquatic products are transported between provinces in China, largely by road [12]. However, transportation and sale of live fish can have challenges for meeting health regulations, quality standards, and animal welfare requirements. The mortality of transported live fish is one of the biggest concerns for Chinese aquaculture, particularly transport companies [16]. It is estimated that around 7% of farmed fish die annually due to live transport [17], and this is attributed to the transport time, inappropriate transport procedures, and inadequate monitoring technology [14,18]. Meanwhile, freshwater fish farming is also geographically imbalanced in China [13], which increases the duration of transport time to some regions.Animal welfare is defined as “the physical and mental state of an animal in relation to the conditions in which it lives and dies” [19], which is an important consideration for farm animal production. Animal welfare can be assessed against a variety of standards, such as the “Five Freedoms” [20], as well as the Five Domains Model, which was developed and updated to incorporate advances in animal welfare science [21]. The Five Domains Model assesses animal welfare from the perspectives of (1) nutrition, (2) physical environment, (3) health, (4) behavioral interactions, and (5) mental state [21]. In Europe, many organizations have issued standards or guidelines to improve and ensure farmed fish welfare. For example, the World Organization for Animal Health (OIE) publishes the Aquatic Animal Health Code that provides guidelines for the health, welfare, and international trade of farmed fish [22]. Similarly, the Royal Society for the Prevention of Cruelty to Animals (RSPCA) in the United Kingdom has standards for two farmed fish species—Atlantic salmon (Salmo salar) and rainbow trout (Oncorhynchus mykiss)—which includes guidelines for maintaining fish welfare during transport. China only has one national standard (GB/T 27638-2011) that contains some recommendations relevant to live fish transport [23]. This code classifies live transport into road transport with water, road transport with little or no water (waterless transport), and vessel transport [23]. For live transport with water, the code outlines procedures for selecting healthy fish, pre-transport food withdrawal (1–2 d), pre-transport stocking density (suggested to be 20–45 kg/m3), and acclimation, control of water quality during both acclimation and transport, transport tools and methods, oxygen supply (>8 mg/L), and maximum transport time (<40 h). Unlike in other welfare standards mentioned previously, this code does not include welfare requirements for any specific fish species, transport density, or training for transportation staff, although these are known to affect fish welfare during live transport [8,24]. Moreover, the code only generally describes live transport procedures and lacks specific descriptions, such as loading and unloading processes, responsibilities of transport staff, and other transport-related welfare issues [23]. Lack of species-specific standards or regulations of live transport may cause transport-related welfare issues for fish.Live transport procedures for fish have been described in the English scientific literature, but most are not connected to Chinese contexts [4,5,25]. For example, the RSPCA UK issued welfare standards for Atlantic salmon [26] and rainbow trout [27], which are commonly farmed fish in Europe, but Asian carps are the most popular farmed species in China [12]. Fish welfare is starting to become a concern in China, as evidenced by the appearance of reviews on that topic since 2009 [25,28,29]. In China, current research on live fish transport published in English focuses on waterless transport techniques [11,16,30], the efficacy of fish anesthetics [31,32], transport density [30,33], and physiological stress responses of transported fish [7,34]. Although several reviews on fish transport [19,35,36] have also been written in Chinese, their contents are very similar to each other and do not connect to welfare issues.In livestock production, industry stakeholders, including farmers and service providers (e.g., retailers and transporters), are usually responsible for the welfare of their animals, as well as responsible to consumers for food quality and safety [37]. There is a need to communicate with stakeholders to understand existing and emerging issues relating to Chinese aquaculture, particularly around live transport of fish, as there is currently limited information available. The qualitative research method is useful when limited data have been published on a topic [38], and it contributes to a rich understanding of the human condition through their various experiences and observed circumstances [39]. Grounded theory commonly uses face-to-face interviews to investigate a particular phenomenon or a less-known issue or situation [40]. Moreover, an interview allows researchers to have a deeper discussion with participants [39]. This study aimed to obtain initial information on the knowledge of Chinese stakeholders from within the aquaculture industry or working in fishery-related positions relevant to live transport procedures. We also aimed to garner an in-depth understanding of the attitude of stakeholders towards fish welfare and key factors that may affect fish welfare during live transport, using semi-structured interviews. This information will identify perceived and potential fish welfare issues for live transport in China and inform future research on this important issue that affects many animals each year. This preliminary study is part of a larger project that investigates the knowledge and attitudes of stakeholders and current practices for live transport of fish in the Chinese aquaculture industry. This report conforms to the Standard for Reporting Qualitative Research (SRQR) guidelines [35], with slight modifications.2. Materials and MethodsA series of one-to-one interviews of Chinese stakeholders in the aquaculture industry (n = 12) was conducted between June and September 2020.2.1. ParticipantsA purposive sampling strategy [36,41] was used to identify potential participants if they (i) were adults (at least 18 years of age) who spoke fluent Mandarin and (ii) had worked in the Chinese aquaculture industry or were involved with fishery-related jobs for at least one year (in total, even if they had changed their job or retired). We aimed to identify 1–2 participants from several job categories we identified. The initial recruitment had 13 people from within our existing network; however, one participant subsequently declined to be interviewed due to time constraints. Previous literature suggests an adequate sample size for qualitative research methods varies from 5 to 50 participants [42]. Text analysis was first carried out among the 12 participants to check saturation, which has been a “gold standard” for determining an adequate sample size in qualitative research [43,44,45]. Subsequently, no more recruitment was undertaken, and 12 participants in total were interviewed in this study.Participants had different degrees of exposure to, or experience with, the aquaculture industry and held different fishery-related positions in China (Table 1). The twelve participants included nine males and three females. Nine participants lived in urban environments and three in suburban or rural regions. Various job categories across the aquaculture industry were represented, and some participants occupied more than one role. The three participants with the longest involvement in the industry (more than 15 years) all worked in fish sales. Three of the twelve worked at fish farms, two as employees (management positions) and one being self-employed (a farmer). Two participants worked in an animal protection/welfare organization as volunteers, one was a student who majored in ornamental fish rearing, and the other had experience with terrestrial animals and aquatic mammal protection. We also included two participants from fish research/academia. One of the remaining participants worked for a delivery and logistics company that delivered live and chilled aquatic products either by road or air. The last participant was a restaurant owner who sold live fish and seafood.2.2. InterviewsSemi-structured interviews, lasting approximately 30 min each, were conducted individually either face-to-face (4) or by telephone (8), depending on the geographic distance and travel restrictions due to COVID-19. The lead researcher (Y.Y.) conducted the majority of interviews (11), and another volunteer conducted the remaining interview. Respondents participated in interviews based on an open-ended preliminary interview guideline (see Appendix A). Thirty-nine questions were included in that guideline, but participants were offered the opportunity to skip any questions or to indicate that they did not know or did not understand a particular question. All interviews followed a similar structure between individuals, with slight variations depending on their involvement in the industry. All interviews were conducted in Mandarin Chinese and audio recorded (iPhone7, Apple Inc., Cupertino, CA, USA). The recordings were subsequently transcribed into written Chinese by a second researcher (T.W.) and checked by the lead researcher, both of whom are fluent in Chinese. Notes were also taken during the interviews for evidence of emergent, preliminary concepts. Participants were asked to describe:i.Their demographic information and employment statusii.Their experience and knowledge on live transport of fishiii.Variables that are monitored during live transportiv.Factors that affect the success of live transport, as evidenced by fish mortality levels and fish well-beingv.Methods or criteria that are used to assess fish health after live transportvi.Their understanding and opinion of “animal welfare” and whether this was a familiar term2.3. EthicsEthical approval was obtained from the University of Queensland Human Research Ethics Committee before commencing the interviews (human ethics approval number: #2020/HE001290). Before each interview, participants reviewed and signed a participant consent form, indicating that the anonymity of the participant’s information would be retained and that participants had the right to withdraw from the study at their discretion.2.4. Qualitative AnalysisThematic analysis was aided by the use of NVivo 12 Plus software (QSR International Pty Ltd., Melbourne, VIC, Australia). The interview texts were coded automatically as nodes (questions) and cases (participants). The number of questions answered by each participant was counted, and questions that were answered by almost all of the participants (n > 10) were selected for analysis. Long answers with important emerging information or welfare- and transport-related terminology was extracted to identify themes and direct quotations (indicated by double quotation marks “…”) from participants’ conversations were used to interpret identified themes. Short answers such as “yes” or “no” were excluded from direct quotations as supporting evidence but were included in the analysis. Lastly, we grouped the 14 questions answered by all participants into three main themes: live transport processes, fish health and care, and animal/fish welfare. Capital letters (A–L) were randomly assigned to each participant as a unique anonymous identifier, and these appear with each quotation in the results section of the manuscript. Verbatim responses are displayed in quotation marks. In order to preserve the original meaning of the interviews, the transcript from each participant was first analyzed in Chinese and was subsequently translated into English by a bilingual volunteer. Subjects in Chinese language were often derived from the context of the direct quotes; they are added in parentheses for clarity. Chinese characters are also displayed with specific words mentioned by participants for a better understanding of both Chinese and Chinese-speaking researchers. Two researchers (Y.Y. and T.W.) reviewed the translations thoroughly for quality assurance and to ensure that the reports accurately conveyed the intention of the participants.3. Results3.1. Live Transport Processes3.1.1. Experience with Different Fish Species (Question 10)The fish species that participants had experience with was dependent on their jobs. All participants indicated that they had worked with either freshwater fish, marine fish, or other aquatic animals. Freshwater fish species were the most commonly mentioned (n = 12). Among the freshwater fish, five participants reported that they had experience with the “four major Chinese carps” (四大家鱼): grass carp, silver carp, bighead carp (Hypophthalmichthys nobilis), and black carp (Mylopharyngodon piceus). Other freshwater fish species, such as crucian carp (Carassius auratus), mandarin fish (Siniperca chuatsi), and bass (Lateolabrax japonicus), were also mentioned, because many participants (n = 8) worked across several fish species groups. In addition to freshwater fish, four participants also worked with other live seafood, including marine fish and shellfish. Groupers were reported as one of the most popular seafood products in our study—for example, tiger grouper (Epinephelus fuscoguttatus). The two participants from aquaculture research/academia not only worked with commonly farmed fish species but also had experience with local fish species such as Amur minnow (Phoxinus lagowskii) or nationally protected wild fish, such as Tsinling lenok trout (Brachymystax lenok tsinlingensis) and Sichuan taimen (Hucho bleekeri).3.1.2. Transport Destinations and Their Areas (Question 11 and Question 12)Participants were asked what sort of destinations they usually transported live fish to and where these places were located. They reported wholesale markets, supermarkets, wet markets, laboratories, restaurants, residential communities, and private residences as popular destinations. Wholesale markets were the most common destination mentioned (n = 6) for transporting food fish: (Participant A) “One of the most popular destinations is a large aquatic wholesale market” and (Participant E) “Fish are first transported to a wholesale market and they can subsequently be distributed to supermarkets or other shops.” In addition to markets, fish were also directly transported between fish producers and private buyers. According to participant A “[…] another [sales target] is the private buyer” and H “[…] there are some casual buyers, such as households from a residential community, we also deliver live fish to them.” Except for direct human consumption, fish are also transported alive for research purposes to laboratories. Participants D and G respectively noted, “We transport [wild-caught] fish from mountain areas to a laboratory” and “Fish are transported to our [university] laboratory for experiments.”According to participants’ answers, the connecting and final destinations were mainly in urban areas (n = 8), with some in suburban areas (n = 2). Most destinations were reported to be provincial cities (e.g., Hangzhou, Changsha, and Changchun) or larger cities (e.g., Shanghai). Participant E said that large wholesale markets are gradually moving into suburban areas: “Nowadays, this sort of fresh market is to the extent possible near to the edge of the [Changsha] city, clustered between the city and suburb, where those transport trucks can get in and out.” The two animal advocates were not directly involved with live transport, but they stated that they purchase live fish in supermarkets and fresh markets, which are near to their (urban) residence (in Shenyang and Dalian). Live transport also occurs frequently within a province or between adjacent provinces, for instance, “[from a farm in Hangzhou to the transport destinations], within this area of Jiangsu, Zhejiang and Shanghai [up to 500 km approximately]” (Participant A) and “[live transport] usually occurs within the province, for example, from Jilin to Changchun [approximately 120 km], similar to that, maybe 2–3 h of trip” (Participant G).3.1.3. Proportion of Live Transport (Question 14)Participants were asked how many fish are generally transported alive as a percentage of the fish transported in total. Their responses indicated that the live transport of fish in China is close to 100%, particularly for freshwater fish species. Most participants (n = 9) said that freshwater fish are mostly transported alive, for example, (Participant A) “From our experience, the fish transported are all alive, throughout the process of catching to putting into the transport tank” and (Participant G) “Fish all need to be alive for the transport.” While many marine fish species or other aquatic animals, such as crustaceans, can also be cold preserved (still alive) or directly frozen (dead) before transport, Participant J reported that “Our seafood here, it is live transported. There is also some seafood that is dead [due to capture], how is that transported? Under normal circumstances after being caught from the sea, they are directly frozen and iced [for transport].”3.1.4. Seasonal Effects on Live Transport (Question 15)Interviewees were asked whether seasonal changes have an impact on the success of live transport. According to their responses, live transport occurs more in cool-weather conditions and less in summer, and the majority (n = 10) believed that seasonal changes have a great impact on the success of live transport. Water and ambient temperature were considered particularly important factors, for example, Participant A reported that “[…] when the ambient temperature is high, the water temperature will also be high, and the fish may experience hypoxia. So basically, the proportion of live fish transported in summer is very small.” Participants also suggested that fish mortality is higher in summer compared with other seasons. According to Participant G, “[…] under hot weather conditions, the [fish] survival rates are little lower. Then after they arrive at the destination, the fish condition apparently does not look good, and they need more time to recover, [during the recovery], even have a few deaths.” Similarly, Participant F stated, “[…] fish require constant water temperature during the whole transport [to survive]. In summer, you need to add ice [into the water]. In the winter you probably need to increase water temperature.”In addition to the difference in ambient/water temperature, demand for fish species also varies by time of year and important events such as holidays or festivals: (Participant H) “This [live transport] will not change [due to seasons], it is mainly to do with workdays, or related to holidays, but for seasons, there is not that much relationship. […] for long-distance transport during holidays, the highway will be packed with traffic, and this is not good for live transport.” Live transport is also associated with fish production cycles throughout the year. Fish of different sizes and life stages are transported in different ways, as suggested by Participant C: “[…] in springtime, actually most transported fish are fry, then the rest in other seasons. Live transport of market size fish occurs more frequently.”3.1.5. Types of Transport Vehicle (Question 18)During the interviews, we asked about what sort of vehicles are commonly used for live transport. Interviewees reported that the size and volume of the vehicle used in fish transport vary, dependent on transport purpose and distance. Commonly, purpose-built vehicles were used, as well as vehicles modified for live transport, such as trucks/lorries, vans, and private cars. According to Participant A, “Transport vehicles (活水车) must be professionally modified and equipped with oxygen supply, as well as water storage systems.” Modified trucks were typically used for transferring larger volumes of fish, as indicated by Participant F:” They are modified vehicles that have a tank with a circulation system, refrigeration and heating equipment.” In contrast, research fish can be transported using private cars because of their small volumes and smaller fish size. Participant G mentioned that “For fish transport vehicles, basically it is just us transporting them in the private cars that we drive, […] If we do not transport lots of fish, we just transport them back in our own private car[s]. Sometimes we find a van [for live transport] if there are more fish.”3.1.6. Transport Methods/Containers (Question 22)Participants were asked about the most commonly used methods of transporting live fish. Based on their responses, transport tanks, plastic bags, and foam boxes were commonly used. Transport tanks made with polyvinyl chloride (PVC) plastic were commonly reported to be used for large volumes of fish or over long distances, with modified vehicles. For example, Participant A stated that “For large-scale transport, it has to be transport trucks with [PVC] tanks. For small-scale transports like we do, well, within a certain limit, we use plastic bags or foam boxes.” Participant E also mentioned: “Transport vehicles all use compartmented tanks made from that sort of PVC material, with oxygen added.” Small volumes of fish or short-distance deliveries were often carried out using aerated bags: (Participant G) “For transport within the province, about 2–3 h, fish are all in bags, fish transport bags.” Participant E also described that “If we are talking about after arriving at wholesale markets and transporting separately to grocery stores, or restaurants, then after arriving at wholesale markets they use foam boxes and aerated bags. At wholesale markets, they use that sort of foam box with aerated bags inside. This can also be used to transport live fish.” One participant (L) transported live fish regularly using a carrying pole (扁担). Fish were placed in homemade baskets with sealed plastic bags for transporting from his house to the local market on foot.3.1.7. Use of Fish Anesthetics (Question 24)The use of chemical anesthetics in the live transport of food fish was not reported by participants in this study to be common. Most (n = 7) said that no fish anesthetics are used during live transport of food fish based on their experience, but other methods such as the addition of ice, a high concentration of sea salts, or vitamin C were applied to control water quality and reduce transport stress in the fish. Participant H: “We never use chemical anesthetics [for food fish], but sometimes we add a little vitamin C or sea salt.” The use of fish anesthetics seems to be species-specific in our study, evidenced by “There are some fish for which we will use anesthetics, such as yellow catfish because their body has a lot of spines and sharp bones” and “I know anesthetics have been used for marine fish. In terms of farmed fish, either freshwater or seawater, as long as they are relatively valuable fish types, they will usually use anesthetics”, from participants A and C, respectively. Two participants (D & G) answered that fish for research purposes were occasionally transported with anesthetics, as those are nationally protected species that are easily stressed by handling and transport processes.3.1.8. Loading and Unloading Processes (Question 25)Question 25 obtained information on loading and unloading processes and their time points. According to most participants (n = 9), fish were caught by manual methods using dipnets or trawls. It is suggested by Participant A that “Fish are loaded and unloaded traditionally […] using a so-called ‘fishing net’ (拖网), or a fully sealed bucket with water, and [fishermen] will use that for loading and unloading.” Two participants mentioned that fish can be transferred by pumping and cage trapping. For example, participants C and J, respectively, stated that “One way is just directly to use a net to scoop up some fish […]. Another way, for relatively smaller fish, will be a sort of water pump that directly sucks the fish out” and “[…] in summer, you cannot pull fish out with a net. Generally, they use cages to trap fish, then to pull them up.”Most stakeholders (n = 8) indicated that loading may occur several hours before transport starts, and fish were unloaded shortly after arriving at their destination. However, Participant F suggested that unloading may also occur at different times of the day, depending on traffic conditions and the opening hours of a market: “Fish do not have to be immediately unloaded [after arrival]. Basically, fish are unloaded in the afternoon or evening. Sometimes when the market has not yet opened, if a transport vehicle arrives [at the market] a little early, they will wait a bit. It is normal.”3.1.9. Knowledge around Live Transport (Question 34)We asked all participants to self-rate their knowledge level and understanding in terms of live transport of fish on a five-point scale:(i.)Very low level of knowledge/very poor understanding(ii.)Low level of knowledge/not much understanding(iii.)Moderate level of knowledge/general understanding(iv.)High level of knowledge/rich understanding(v.)Very high level of knowledge/very rich understandingFive participants indicated they had a moderate level of knowledge/general understanding. Four participants said their knowledge level was low. Two reported a high level of knowledge/rich understanding, and only one participant self-rated a very high level of knowledge/very rich understanding.3.2. Fish Health and Care3.2.1. Oxygen Supply during Transport (Question 26)Participants were asked how oxygen was typically provided during live transport. Oxygen was considered necessary in both open and closed transport systems, according to participants’ answers. For an open transport system, liquid oxygen cylinders and air generators were used to provide dissolved oxygen (n = 11). Sufficient oxygen was aerated into plastic bags, e.g. by aerating from a liquid oxygen tank, when fish were transported in a closed transport system; therefore, no oxygen was supplied during live transport (n = 2).3.2.2. Challenges during Live Transport (Question 28)Participants were asked to identify potential challenges that arise when transporting live fish. The control of water temperature, oxygen levels, transport density, water quality, vehicle vibration, the selection of transport distance, transport time, fish species, capture method, and driver experience were all mentioned by different participants as potential challenges during live transport. All participants identified more than one challenge during live transport, and water temperature was mentioned by 8 of the 12 respondents. The second most reported challenge (n = 7) was the maintenance of dissolved oxygen levels in transport containers. Transport density was also identified by three stakeholders, while transport time, fish species, travel time, and vehicle vibration were each mentioned twice or less in the study.3.2.3. Methods to Assess the Health Status of Fish (Question 31)Fish outward appearance was a commonly reported indicator for assessing the health and marketability of transported fish. Key measures included the presence of bodily damage (injury or bleeding), a change of body color (red/white spots), eye color, and the secretion of skin mucus. For example, Participant A stated that “You must rely on experience to assess whether or not fish are healthy. You can observe whether or not fish bodies are descaling, whether they are bleeding, whether there are any red spots.” Similarly, Participant E reported: “Firstly, when fish arrive at their destination, one point is observing whether their eyes are clear (眼睛是否清澈), then the fish body, even checking whether their color is normal.” In addition to appearance, fish equilibrium, vigor, and swimming ability were also used to assess fish health after live transport. Participant D, for example, stated that they looked for whether fish showed a “floating head” (the fish ventral region was upward).Assessment measures were reported to be more complicated for research fish, as evidenced by Participant G, “[…] we first observe the body color of the fish. Any fish that show abnormal body color will be excluded from our formal experiments. Furthermore, uneven fish sizes—too large or too small ones—we will not use them as well. For some high-value and expensive fish, we may do a microscopic examination to see whether they have any parasites, viruses or bacteria. […]. For the appearance check of fish, we only do simple checks, for instance, to see whether there is any body damage or mechanical injury to the fish.”3.3. Animal/Fish WelfareUnderstanding of Animal Welfare (Question 33)Nine participants had previously heard of the phrase “animal welfare” and also were able to explain their understanding of the term. The remaining three did not know this term or were not sure of its meaning.Some participants mentioned that pre-slaughter and slaughter conditions were influential for animal welfare, and animals should be treated well before or during slaughter. For example, “[…] animals should be treated as calmly as possible during slaughter” was suggested by Participant B. Some participants also believed that animal welfare affects product quality, in particular meat quality, as evidenced by Participant E: “[…] during the slaughter process, if animals experience that sort of excessive fear or shock, their bodies will produce acidic substances. It probably will result in worse meat quality, or fish quality. [animal] Welfare is probably just like this.” Some participants suggested that the husbandry and slaughter of farmed or aquatic animals should be standardized within China to improve animal welfare. They also indicated that animals should be kept in a good environment and that their mental state should be considered. Participant D suggested that “no matter the farming or processing practices, we should follow a principle that reduces pain and fear to animals.” Similarly, Participant G stated: “We should treat farmed animals as, how to say it, as humans. We should pay attention to their mental states and relevant issues. If we do not treat animals well during husbandry, they do not keep in a good mood or a good state. Their own condition can also affect their product quality.”Three participants said that they were familiar with this term being applied to terrestrial livestock, but it was novel to describe fish or other aquatic animals in this way. For example, Participant A stated: “[…] it is quite fanciful for me that the term of animal welfare can be used in aquaculture.” Another participant (K) thought that aquatic animals did not have welfare, as she suggested that aquatic animals were not sentient like livestock and humans. This perspective differed between interviewees, as another participant (G) stated that fish should be treated the same as other farmed animals and that all food animals should have good welfare, regardless of the species.The attitudes of stakeholders towards different animals could affect their welfare. Participant C gave an example that “[…] animal welfare is relative to our kinship distance (亲缘关系) and determined by the degree to which our lives are similar. As for non-food animals, for example, those ornamental fish, their welfare is probably a little better compared to food animals who are usually seen as food to prepare, so they probably do not have good welfare.” Animal welfare specifically in relation to transport was also mentioned by two participants. For example, Participant H mentioned that a low fish mortality rate meant better fish welfare during transport from a producer’s perspective. Participant E suggested that a good environment should be provided during transport.4. DiscussionThe results of this study are not fully representative of the entire Chinese aquaculture industry, but the findings provide insight into the industry in China and reveal fundamental information on fish welfare during live transport. We discuss live transport processes, fish health and care, and the animal/fish welfare implications of our findings.4.1. Live Transport Processes in ChinaOur results confirm that live transport remains a key component of the Chinese aquaculture industry, in line with previous literature [14,46], particularly for freshwater fish. Various fish species were mentioned, including Chinese carps, which are dominant within Chinese freshwater aquaculture [13,15,47], and non-native fish species, such as tilapia, bass, and catfish [48,49]. Based on participants’ answers, both fish for food and research are transported alive in sealed containers or plastic bags using purpose-built trucks or private cars. Methods used for live transport are highly customized depending on transport volume, purpose, and fish species. Live transport occurs most frequently over short distances within a province (destined for a provincial city, up to 500 km approximately) or between adjacent provinces (up to 700 km approximately), based on the results presented in our study. Geographically, the Yangtze River region contains one of the highest concentrations of aquaculture producers in the central-eastern part of the country [11]. Participants from Zhejiang province also mentioned that live transport often occurs in this region between adjacent provinces: Jiangsu, Shanghai, and Anhui, all of which belong to the Yangtze River Delta, with an approximate distance from 170 to 460 km.Participants indicated that fish are transported to various locations, but wholesale markets, often located in suburban areas, appear to be the most common commercial destination for food fish. Sales channels for aquaculture in China generally include producers, processors, traders (wholesalers), retailers, consumers, and restaurants [50]. Fish at wholesale markets can be directly sourced from individual farmers or from distributors who buy products from different farms [43]. Primary and secondary wholesalers are the main market operators who handle aquatic products before they reach smaller retailers, restaurants, and consumers [50]. Therefore, it seems that wholesale markets are an interim stop for live fish, and welfare concerns may still occur from a wholesale market to a final destination. In addition to large-volume transport, small volumes of fish are also transferred to private buyers for their home consumption [50].Seasonal effects on the occurrence and success of live fish transport are substantial. Heat stress may be an influential factor on fish welfare during live transport, as most participants indicated that live transport occurs less frequently on hot weather days. Acute increases in water temperature are known to adversely affect fish physiology, with negative implications for fish welfare [51]. If fish must be transported in hot weather, then appropriate temperature reduction is necessary to maintain the welfare and survival of fish. For example, cooling of the transport water by 5–7 ℃, compared with the water from rearing systems, is a common protocol for live transport of salmon [44]. Methods such as ice cooling are often used to reduce water temperature during live transport [45], and these are similarly noted in the current study - the reported addition of ice into water. Lower water temperatures can also help to maintain water quality during transport [9]. For example, in a previous study, ammonia nitrogen concentrations were lower when largemouth bronze gudgeons (Coreius guichenoti) were transported in cool water [32].Apart from water temperature, the maintenance of appropriate oxygen levels during live transport was also reported as one of the key controlling factors in our study. Sufficient oxygen supply is an essential component of the transport process [4,47]. Low dissolved oxygen levels not only alter fish behavior and physiology but also reduce their growth, and, more seriously, result in increased mortality [52]. In addition to these issues, participants also reported a variety of other potential challenges, many of which align with the previous literature, such as inappropriate transport density, transport time, poor handling and acclimation, and impact from mechanical vibrations [8,53,54].From participant responses, it is clear that a variety of transport methods/containers are used to transport live fish, including transport tanks, plastic bags, and foam boxes. Plastic bags were noted to be more commonly used for the short-distance transport of a small volume of fish. This aligns with the literature from outside of China, which notes that market-sized fish are often transported in truck-mounted, high volume tanks over long distances [4,55], while smaller fish, such as juveniles and fingerlings, or some ornamental fish of high value, are transported in plastic bags [6,7,38,56].From our interviews, chemical anesthetics were rarely used in live transport of most food fish but might occasionally be used for valuable fish (e.g., ornamental fish), research fish, and some food fish that are challenging to handle, although specific drugs were not mentioned in this study. Tricaine methane sulphonate (MS-222) is a common sedative that is approved as a food fish anesthetic by the US Food and Drug Administration (FDA), but it requires a 21-day withdrawal period before sale [57]. No anesthetics have been approved in China for fish handling and live transport of food fish [58]. The safety of aquatic products evokes concern from the public [58], and nowadays, there are more than 40 standards or regulations that control drug residual levels in aquatic products in China [59]. One previous survey reported that common fish anesthetics, including eugenol and isoeugenol, have been used for sedation of transported fish before being sold in Chinese markets, but the residual levels from investigated flesh samples were not high enough to be of risk to human health [58].Capture, loading, and unloading methods noted in these interviews were still traditionally manual methods of moving fish, such as using dipnets. Chinese freshwater farming systems are typically carried out as a polyculture in pond systems, which enhances the utilization of all the available food resources in the pond ecosystem [12]. In this system, it is challenging to identify and only capture a single fish species without handling and manual sorting. Thus, the use of dipnets allows farmers to select specific fish species for their purposes. Another reason that traditional capture methods are still popular on Chinese fish farms could be the smaller scale of aquaculture companies. The proportion of small- and medium-sized aquaculture enterprises remains high in China, compared with European countries [59]. The costs of using advanced techniques on pre-transport procedures could be prohibitively expensive.4.2. Fish Health and CareParticipants mentioned that the appearance, swimming ability, vitality, and mortality of fish were often checked after transport to estimate their health status and sale value. Physical damage to a fish looks unsightly and may lower the value of the fish [60]. Consumers often avoid unhealthy or injured fish because this indicates a lack of freshness [61]. In terms of the application to animal welfare science, these parameters are important outcome-based indicators that can be used to assess the welfare of individual fish, although the indicators are mostly species-specific [47]. Changes to body color are observable indices of welfare; for instance, they are observable stress indicators for salmon [24]. Changes in eye color and eye damage are also reliable and relatively easy welfare indicators from direct observation via the glass aquarium or checking individual fish on-site; however, they tend to be species-specific. For example, stressed Nile tilapia show eye darkening [62], while largemouth bass show corneal clouding after live transport [53]. Body injuries, including the presence of blood and loss of scales, are commonly used to check the health, welfare, and value of an individual fish [24,54]. Behavior can also be an observable indicator of fish health or welfare, although this may not affect fish marketability. Changes in fish behavior are considered welfare indicators. For instance, rainbow trout spend a longer time at the bottom of the tank, occupy a smaller number of tank sections, reduce swimming activity, and increase the number of abnormal movements after experiencing transport stress [63]. Variatus platy (Xiphophorus variatus), an ornamental fish, show increased occurrence of biting and freezing behaviors post-transport, which may increase the risk of injury [56].4.3. Awareness and Understanding of Animal WelfareThis research provides insight into how stakeholders in Chinese aquaculture view animal welfare. Previous studies indicated that animal welfare is still in an early stage of development in China, reporting that about half of respondents had never heard of this term [64,65]. These studies mainly targeted the general public and showed that they are concerned for animal welfare because of its importance to food quality and safety [65,66,67]. Although industry professionals in our study also mentioned that animal welfare is related to product quality, they emphasized more heavily the connection between specific practices and animal welfare, for example, pre-treatment during slaughter and slaughter/handling standards. Therefore, the results from previous research with consumers may not be fully generalizable to industry stakeholders in the current study.The concept of “animal welfare” has attracted growing attention within Chinese social media and researchers in recent years, although it was only introduced to mainland China in the 1990s [68]. There has been some positive transition in attitudes towards animals in China, which is due to economic development, increased concerns about food safety, and changes to human relationships with animals [43,69]. Food producers may therefore have more opportunities to be exposed to the concept of animal welfare, and their understanding could be enhanced by consumer feedback. This may give us some insight into why fish wholesalers and producers (farm managers) in our study were more familiar with the concept of animal welfare, as we expected. Consumers’ preferences for food safety is highly determined by socio-demographic variables in urban China [66]. A recent study suggests that Chinese urban consumers in large cities (e.g., Beijing or Shanghai) show a strong preference for choosing products that have good animal welfare and environmental stewardship, which is believed to be associated with better taste and safety of pork [70]. Therefore, education levels, concerns around food safety, and feedback between producers/retailers and consumers are potential ways to enhance positive attitudes towards the welfare of livestock and farmed fish.Some participants felt that the concept of animal welfare refers more to the welfare of terrestrial animals, rather than fish. Currently, the main welfare focus for Chinese researchers, producers, and government authorities is to improve conditions for livestock, specifically for pigs [71,72] and chickens [73]. The publicity around the concept of animal welfare has also promoted the welfare of livestock in China, for example, the establishment of the International Collaborative Committee for Animal Welfare (ICCAW) in 2013, which is now the leading farm animal welfare organization in China [74]. This contributes to an increasing number of Chinese people, particularly younger generations, who show positive attitudes and behavior towards the welfare of livestock, as well as zoo animals [70,75,76,77]. These changes are also evident in the development of several relevant livestock standards. For example, the first enterprise-level welfare standard for dairy cattle was published by China Mengniu Dairy Corporation (Inner Mongolia Mengniu Dairy Group Co., Ltd., Hohhot, China) in 2020, which filled the gap in dairy cattle welfare management standards in China [78]. At legislative levels, national standards for the slaughter of livestock are now available and have been updated in China (GB/T 19479-2019).However, farmed fish welfare has not received the same attention as terrestrial livestock from the general public and industry stakeholders in China. Legislation and welfare standards for farmed fish are already available in some European countries, while China as a major fish producer has paid little attention to fish welfare [28]. The recent developments for livestock welfare may be why some participants in our study felt that animal welfare referred to livestock species and not fish. The attitudes displayed by some participants towards fish welfare may in part be due to a lack of understanding of the needs of fish compared with other species. For example, one participant questioned whether fish are animals (like humans and livestock). Both in and outside of China, fish are often considered to be less important or less evolved than mammals [79,80]. For example, Callahan et al. [81] found that the American public ranked mammals as having the highest capacities of cognitive and emotional traits, while fish were ranked the lowest. Historically, it has been debated whether fish can suffer, although abundant evidence shows that fish are sentient and can consciously experience pain [79,82,83]. Fish are ectotherm (cool-blooded) animals that live in aquatic environments completely different to those of humans, and this difference may create barriers to understanding their feelings and welfare needs [47]. One participant mentioned that fish are less of a concern to them because of a lack of human–animal bond compared with land animals and the low genetic proximity between fish and humans. One study provided evidence to support this perception that mammals are the most emotive and cognitive species due to the “closeness” of their relationship with humans [81]. For some mammalian species, such as dogs and cats, people can observe and even interact with them. There may be a positive effect on these animals when interacting with humans because the social bond with the owner may be rewarding or the joint activities may be enjoyable in line with domain 4 in the Five Domains Model (behavior interactions) [21]. However, this bond is very challenging to achieve for most aquatic species, such as farmed fish who are part of a large production system [28]. It is also challenging to prepare welfare standards for each farmed fish species because of the diversity of species [28] and the polyculture systems on Chinese farms [12]. Currently, specialized welfare information is only available for around 20 species; therefore, the scientific literature on aquaculture welfare is still emerging and developing, and the issue needs on-going exploration [84]. Each fish species has different ecological and behavioral demands and varying physiological capacities, so information about one species cannot necessarily be translated to others. More effort is needed to develop appropriate standards for farmed fish in China, especially for polyculture systems.4.4. LimitationsParticipants in this study were a small cross-section of people specifically approached because they represented different stakeholders within the Chinese aquaculture industry. While we collected detailed answers from them in response to a range of free-text questions, the participants cannot be considered representative of the industry. The findings from this research will contribute to the development of a larger questionnaire that aims to be more representative in investigating fish welfare during live transport in China. Additionally, in this study, we not only aimed to investigate stakeholders’ attitudes towards animal welfare but also their knowledge on specific transport processes, to gain insight on this topic in China, as little is currently published. The two participants who were interviewed because of their role in animal welfare advocacy were not directly involved in fish transport and so were less likely to provide detailed contextual information compared with others who were directly involved with the process (e.g., retailers and farmers). However, they provided valuable insight into attitudes and perceptions around animal welfare. Therefore, a future study could be potentially developed to further examine similar insights from a range of non-industry stakeholders, such as the public and consumers, animal rights advocates, and aquaculture students. Although most participants indicated their willingness to participate in all the questions, one interview occurred in a market, and the participant was not able to answer all questions due to time limitations. The interview was interrupted by the participant’s business; therefore, the length of this interview was shorter than the rest.5. ConclusionsParticipants in this study confirmed that the live transport of fish (mainly farmed freshwater species) is common in the current Chinese aquaculture industry. Fish are transported using various methods and different vehicle types. The effect of seasonal changes on live transport is considered important, and transport is less commonly carried out in hot weather. Fish anesthetics are used to sedate some fish species but seldom used for food fish. Trawling and netting are still commonly used to catch fish from water due to the dominance of polyculture systems and many small-scale farms in Chinese aquaculture. Oxygen is always provided during live transport, and other challenges of keeping fish healthy are similar to those reported in the scientific literature. Physical appearance and fish vigor are used to measure fish well-being and marketability, according to participants’ answers. The self-estimated knowledge level of live transport was at a moderate level because most participants were not directly involved with live transport as a fish transporter. This study also provides information on the understanding of animal welfare and attitudes towards fish during live transport in China, as identified by a small sample of stakeholders in the industry. Although most stakeholders had heard of animal welfare and could provide their understanding, this term is considered more relevant to livestock or poultry, rather than fish. Future research should identify key welfare issues during live transport with a broader range of stakeholders and should investigate their attitudes towards fish welfare.

animals : an open access journal from mdpi

10.3390/ani13091517

PMC10177492

This study aimed to investigate the occurrence of paratuberculosis (Johne’s disease), caused by Mycobacterium avium subsp. paratuberculosis (MAP), in Austrian sheep and goats by testing 22,019 blood samples. Furthermore, detailed investigations in five MAP-infected goat herds were carried out to detect the disease in blood and faecal samples. The detected animal MAP seroprevalence was 2.0% for goats and 0.7% for sheep (calculated true prevalence 3.5% and 1.2%, respectively). Herd-level apparent MAP seroprevalence was 11.1% for goat herds and 8.9% for sheep flocks. Herds with a more intensive production system had a significantly higher risk of being infected, as well as farms with frequent trading of animals or where other ruminant species were kept on the same premise. In the five goat farms investigated, 21.8% (11.7%–28.0%, calculated true seroprevalence 38.6%) of the animals were found to be infected and 12.3% (5.0%–24.7%) of the animals were shedding the bacterium with their faeces. It was further possible to identify the bacterium using boot swab samples from the stable environment in each of the five herds. The results indicated a moderate paratuberculosis infection rate in small ruminants in Austria.

This study aimed to investigate the prevalence of Mycobacterium avium subsp. paratuberculosis (MAP) in small ruminants in Austria by testing 22,019 serum samples with ELISA for the presence of specific antibodies. Furthermore, detailed investigations in five MAP-infected goat herds were carried out by ELISA, qPCR and bacterial culture. The found animal-level apparent MAP seroprevalence was 2.0% for goats and 0.7% for sheep (calculated true prevalence 3.5% and 1.2%, respectively). Herd-level apparent MAP seroprevalence was 11.1% for goat herds and 8.9% for sheep flocks. Significant risk factors for seropositivity in goat herds were: herd size, animal trading, farmed as a dairy herd, Animal Health Service membership and cohabitation with farmed game. For sheep flocks, seroprevalence was significantly higher in flocks with animal trading and where cattle or goats were kept in the flock, respectively. The overall apparent within-herd MAP seroprevalence in the five goat farms investigated was 21.8% (11.7%–28.0%, calculated true seroprevalence 38.6%) and an overall rate of MAP shedding of 12.3% was detected (5.0%–24.7%). It was possible to identify MAP by culture using boot swab samples in each herd. The results indicated a moderate MAP infection rate in small ruminants in Austria.

1. IntroductionParatuberculosis (Johne’s disease, JD) is a chronic intestinal disease caused by Mycobacterium avium subsp. paratuberculosis (MAP), affecting ruminants with a global distribution [1]. Infections in sheep and goats are mainly acquired at an early age by faecal–oral transmission of MAP, followed by a long subclinical period. The main clinical sign of JD in small ruminants is progressive weight loss [2]. Caprine and ovine paratuberculosis is known to cause considerable economic losses in affected herds [3].In contrast to cattle, the MAP prevalence in sheep and goats is unknown in many countries and the prevalence appears to be underestimated [1]. Additionally, prevalence studies carried out among sheep and goats in Europe are scarce. Due to different study design, sampling frames and the uncertainty of the diagnostic tests, MAP prevalence among small ruminants is difficult to estimate [4]. A few studies describe several risk factors associated with MAP prevalence in small ruminants [5,6,7,8,9,10,11], but further research is needed to confirm their validity for the local small ruminant population. The enzyme-linked immunosorbent assay (ELISA) is a suitable cost-effective and commonly used diagnostic test for MAP prevalence studies [12]. For herd-level diagnostics and control programs in dairy herds, culture and/or polymerase chain reaction (PCR) of boot swab (BS) samples and pooled faecal samples have been applied successfully [13,14], but, to the authors’ knowledge, the application of BS sampling has not been verified for sheep and goats so far. In the past, cattle in Austria were predominantly affected by JD. However, MAP was also isolated from goat, sheep and wild ruminants (red deer, roe deer and mouflon) [15]. The herd-level MAP seroprevalence of Austrian cattle was observed to have increased over the years to 19.1% [16,17], but more recent studies utilising BS sampling in a region in western Austria showed a much lower MAP prevalence in dairy cattle of 0.97% [18]. In 2006, clinical JD became a notifiable disease in cattle, sheep, goat and farmed deer in Austria. Based on the compulsory national paratuberculosis control program, suspicious animals that test MAP positive have to be culled. Moreover, preventive hygienic measures have to be implemented in affected farms [19].Sheep and goat are minor species in Austria and the production is characterised by smallholders. The Austrian national goat herd comprises 92,800 goats distributed in approximately 10,000 herds, with an average herd size of 9 animals (only 3% of the herds have more than 50 animals). About 16,000 sheep flocks hold 394,000 sheep, corresponding to an average sheep flock size of 25 animals; 13% of the flocks hold more than 50 sheep. Since the year 2011, the number of goats kept in Austria increased by about 28% and the number of sheep by about 9%, respectively [20]. According to the national animal health information system, goats are mainly kept for milk production (33%), whereas sheep are predominantly kept for meat production. The high number of animals grazing on common mountain pastures during summer months (112,000 sheep (28%) and 13,000 goats (14%) in 2020) underlines the importance of small ruminants for alpine landscape preservation in Austria [21].The aim of the present study was to investigate the current herd-level MAP seroprevalence in Austrian sheep and goat holdings and to identify risk factors associated with a positive MAP status. Furthermore, subsequent investigations in five infected dairy goat herds were carried out to gain information about the within-herd MAP seroprevalence and bacterial shedding and whether boot swab or pooled faecal sampling may be suitable for MAP herd-level detection in goats.2. Materials and Methods2.1. Study Design, Animal Sampling and Data Collection 2.1.1. Serological ScreeningIn total, serum samples from 6434 goats in 638 herds and from 15,585 sheep in 1032 flocks were analysed in this study (Table 1). Serum sample numbers were consistent with the totality of the 2020 national annual risk-based sampling plan for the maintenance of the Brucella melitensis-free status in Austria [22]. In this sampling plan, herds were selected using stratified random sampling, with sample sizes proportional to the number of herds in the respective provinces (see Figure 1 and Figure 2). A sampling probability proportional to the natural logarithm of the herd size was used to select farms. In addition, dairy herds, farms with increased domestic or international livestock trade, farms using common pastures and farms with previous brucellosis history were assigned a higher selection probability. The number of samples tested for each selected herd depended on the herd size to ensure a minimum level of herd sensitivity: In herds with fewer than 12 animals, all animals were sampled. In larger herds, the sample size increased slightly. A maximum sample size of 22 animals was achieved in herds with more than 164 animals. Only sheep and goats older than 6 months were included in the sample scheme. The median herd size of the study population was 42 animals per sheep flock and 11 animals per goat herd, respectively, while the median sample size per herd was 16 samples for sheep and 8 samples for goats. Further production parameters, such as information about cohabitation with other ruminant species, common alpine pastures (yes/no), organic farming (yes/no) and Animal Health Service membership (yes/no) were obtained from the national animal health information system for the herds participating in the study (Table 2). The Animal Health Service supports its members (livestock owners) regarding animal health and welfare, providing consulting, diagnostic services, training courses and control programs. 2.1.2. Herd-Level Examination in Selected Dairy Goat HerdsIn five dairy goat herds, either detected as seropositive during serological screening or during previous investigations by the National Reference Laboratory (NRL) for Paratuberculosis, detailed herd-level examinations were carried out. Herds were selected based on herd size and the willingness of the owner and the responsible veterinarian to participate in the study. Dairy goats were housed in stalls and at pasture (n = 4) or housed indoors only (n = 1). In two holdings, the herd was divided into two physically separated groups. The predominant breed was the Saanen Goat, other breeds were the German Improved Fawn, the German Improved White and the Chamois Coloured Goat. Animals did not have access to common pastures, but billy goats originating from external herds were introduced periodically to the herds. A separated kidding area was provided in three herds; moreover, kids were separated from their does after ingestion of colostrum during the first days postpartum (n = 4) or kids were separated immediately after birth from their does and fed with artificial colostrum (n = 1). In addition, two herds had dairy cattle kept on the farm.Individual blood and faecal samples were taken from all animals older than 12 months in three herds and from a representative random sample in two herds. Blood samples were taken from the external jugular vein, according to Baumgartner and Wittek [23], using a vacuum system with serum tubes containing a clot activator (Greiner bio-one, Kremsmünster, Austria). Faecal samples were taken from the rectum; in 8 animals, no faecal sample could be collected, because faecal material was absent in the rectum at the time of sampling. Moreover, environmental BS samples and pooled faecal samples (5 animals each) were collected. BS samples were collected mainly in stable locations with high animal traffic and manure concentration in a meandering pattern, as described for cattle elsewhere [24]. Individual faecal samples were tested by qPCR, whereas BS samples and pooled faecal samples were tested by culture and qPCR, as described below. Herd size and numbers of samples per herd are shown in Table 3. For each animal, the age was documented in years, according to owners’ records, and the body condition score (BCS) was determined by a trained veterinarian, as described elsewhere [25]. Samples were transported to the laboratory for further processing immediately after collection. At the Austrian NRL for Paratuberculosis, samples were stored and refrigerated and processing started on the same working day.2.2. Serological AnalysesBlood samples were first centrifuged for 5 min at 2325 g. Serum samples were tested for the presence of MAP specific antibodies, using a commercial ELISA test kit (ID Screen® Paratuberculosis Indirect Screening test, ID.Vet, Grabels, France) following the manufacturer’s instructions, using the short incubation protocol. According to the data provided by the technical datasheet, the kit detects antibodies in cattle sera with a sensitivity (Se) of 56.86% and a specificity (Sp) of 99.63%; these values were applied for true prevalence (TP) calculation. Samples with positive and questionable test results were confirmed by ID Screen® Paratuberculosis Indirect Confirmation test (ID.Vet, Grabels, France).2.3. qPCR of Individual Faecal SamplesAccording to the prescribed method in the Austrian NRL for Paratuberculosis, faecal samples underwent a pre-treatment: 3 g of faeces each was put into an EZ-Drop bottle (ID Gene® Easy preparation for faeces samples, ID.vet, Grabels, France), pressed carefully with a spatula and mixed for 30 s in a vortex mixer. A total of 1.5 mL of the homogenate was transferred to a 2 mL tube and centrifuged for 10 min at 13,000× g. Then, the supernatant was discarded and 1 mL of PowerBead Solution (Qiagen GmbH, Hilden, Germany) and 1 µL IC-DNA (bactotype® MAP PCR Kit, Indical Bioscience GmbH, Leipzig, Germany) were added to the sediment. The entire suspension was transferred to a 2 mL tube with a screw cap, containing 300 mg glass beads, size 0.1 mm (Retsch GmbH, Haan, Germany), and homogenised twice for 30 s at 6800 rpm on a Precellys 24 (Bertin Technologies, Montigny Le Bretonneux, France). Then, the tube was centrifuged for 1 min at 20,000× g and 200 µL of the supernatant was used for the extraction. DNA was extracted using the IndiMag® Pathogen Kit (Indical Bioscience GmbH, Leipzig, Germany) and the KingFisher™ Flex Purification System (Thermo Fisher Scientific, Waltham, MA, USA) or IDEAL™ 32 Extraction Robot (ID.vet, Grabels, France), depending on the number of samples, following the manufacturer’s instructions. Extracts were subjected to a commercial qPCR protocol targeting the IS900 DNA fragment of MAP (bactotype® MAP PCR Kit, Indical Bioscience GmbH, Leipzig, Germany) using the following detection systems: CFX96 Touch Real-Time PCR Detection System and CFX Maestro V1-1 Software (Bio-Rad Laboratories, California, USA), Applied Biosystems 7500 Fast Real-Time PCR System and 7500 v2.3 Software (Thermo Fisher Scientific, Waltham, MA, USA), Quantstudio 6 Pro Real-Time Systems and Software 1.3.1 (Thermo Fisher Scientific, Waltham, MA, USA).2.4. Culture and qPCR of Boot Swab and Pooled Faecal SamplesFor analysis of the BS, a pre-treatment was applied: BS samples were homogenised for 1 min using a Laboratory Blender Stomacher 400 (Seward LTD, Worthing, UK) together with 50 mL bi-distilled water. The dissolved faeces were transferred to a 50 mL tube, centrifuged for 15 min at 2938× g and the supernatant was discarded. Thereafter, 3 g of pooled faecal sample or the BS manure sediment was mixed together with 30 mL 0.75% HCP solution for 30 min at 150 rpm on a laboratory shaker. Subsequently, gross particles were sedimented for 5 min. A total of 15 mL of the supernatant was transferred to another tube and incubated in the dark for 48 h at room temperature. Then, after centrifugation for 15 min at 2938× g, the supernatant was discarded and the sediment was re-suspended with 1 mL HPC solution. Finally, four HEYM tubes (Becton-Dickinson, Heidelberg, Germany), one M7H10 agar slope (Sigma-Aldrich, Vienna, Austria) and one liquid culture medium M7H9C, manufactured in-house as described elsewhere (Australian and New Zealand Standard Diagnostic Procedure, July 2015), respectively, were inoculated with 200 μL of each sample and incubated at 37 °C. Tubes were incubated for 12 weeks and monitored periodically for bacterial growth and fungal contamination. After four weeks of incubation, one HEYM tube of each sample was rinsed with 400 μL bi-distilled water and the liquid obtained was examined by MAP qPCR. DNA was extracted using the IndiMag® Pathogen Kit and the KingFisher™ Flex Purification System and qPCR was carried out using the bactotype® MAP PCR Kit, as described above. After 12 weeks, the remaining HEYM and M7H10 tubes underwent the visual evaluation. Colonies suspected to be MAP were confirmed by PCR, as described elsewhere [26]. The liquid culture samples were evaluated after 12 weeks by MAP qPCR. 2.5. Statistical AnalysesStatistical data analysis was performed using the IBM SPSS Statistics v27 (IBM, New York, NY, USA) software. Potential risk factors associated with herd-level MAP seropositivity were estimated using chi-square test and logistic regression analyses and are listed in Table 2. The level of significance was set at p < 0.05. Operating chi-square test, the age and BCS parameters were dichotomized: BCS regular to poor were grades 1–2; BCS good to optimal were grades 3–5. Ages were also merged into two groups (age 1–4 years and 5 years or older). For the analysis of serological screening data, 10 sheep flocks with doubtful ELISA results were excluded from the dataset.3. Results3.1. Serological ScreeningThe investigation revealed an animal-level apparent MAP seroprevalence of 2.0% for goats and 0.7% for sheep (TP 3.5% and 1.2%), respectively. In 71 goat herds and 92 sheep flocks, at least one seropositive animal was detected, leading to a herd-level apparent MAP seroprevalence of 11.1% for goat herds and 8.9% for sheep flocks. In 24 goat herds, two or more samples tested positive (34%), whereas in sheep flocks 13 (14%) flocks had more than one positive sample (Table 1).From the serological screening (ID Screen® Paratuberculosis Indirect Screening test), 33 sheep and 5 goat samples showed a doubtful result. Out of these samples, 7 samples originating from sheep were confirmed positive, 11 samples remained doubtful (10 sheep and 1 goat sample) and 20 tested negative (16 sheep and 4 goat samples) by the second confirmatory testing (by ID Screen® Paratuberculosis Indirect-Confirmation test). All samples testing positive at the serological screening were confirmed by the second test method.animals-13-01517-t001_Table 1Table 1Results of the serological screening for Mycobacterium avium subsp. paratuberculosis using ID Screen® Paratuberculosis Indirect Screening and Confirmation test (ID.Vet, Grabels, France). Animals (n)Seropositive Animals (n)Animal-Level Apparent Seroprevalence (%)Animal-Level Calculated True Seroprevalence (%)Herds (n)Seropositive Herds (n)Herd-Level Apparent Seroprevalence (%) Goat 64341262.0 (95% CI = 1.6–2.3)3.5 (95% CI = 3.1–4.0)6387111.1 (95% CI = 8.9–13.8) Sheep 15,5851100.7 (95% CI = 0.6–0.9)1.2 (95% CI = 1.0–1.4)1032928.9 (95% CI = 7.3–10.8) Total 22,0192361.1 (95% CI = 0.9–1.2)1.9 (95% CI = 1.7–2.1)16701639.8 (95% CI = 8.4–11.3)Logistic regression showed significant association between herd size and MAP seropositivity for goat herds (p < 0.001), but not for sheep flocks (p = 0.895). For goat herds, seropositivity rate was significantly higher in herds with animal trading; in dairy herds, if herd owners were Animal Health Service members and if farmed game was kept alongside goats in the herd (Table 2). For sheep flocks, seroprevalence was significantly higher in flocks with animal trading and if cattle or goat were kept together in the flock, respectively (Table 2). Depending on the region, seroprevalence ranged from 2.9 to 20.9% for goat herds and from 3.1 to 14.6% for sheep flocks, respectively (two regions with an unrepresentative sample size, each <22 herds, were not included). The prevalence in the region with the highest prevalence in both goat and sheep herds was significantly higher when compared to the prevalence in the other regions together (p < 0.001 and p < 0.004). animals-13-01517-t002_Table 2Table 2Description of data and chi-square test p-values of potential risk factors for Mycobacterium avium subsp. paratuberculosis seropositivity in goat and sheep holdings in Austria. Goat Herds Sheep Flocks Variable a Apparent seroprevalence in %, absolute numbers in brackets bp-valueApparent seroprevalence in %, absolute numbers in bracketsp-value Animal trading c Yes No16.8 (25/149) 9.4 (46/489)0.012 *11.8 (48/406) 7.1 (44/616)0.011 * Dairy herd Yes No18.6 (32/172) 8.4 (39/466)<0.001 *6.1 (4/66) 9.2 (88/956)0.388 Grazing on common pastures Yes No10.5 (9/86) 11.2 (62/552)0.83310.6 (32/302) 8.3 (60/720)0.249 Grazing on alpine pastures Yes No9.9 (11/111) 11.4 (60/527)0.65310.7 (34/319) 8.3 (58/703)0.213 Animal Health Service membership Yes No18.9 (25/132) 9.1 (46/506)0.001 *8.9 (24/270) 9.0 (68/752)0.940 Organic farming Yes No13.6 (23/169) 10.2 (48/469)0.2328.0 (22/274) 9.4 (70/748)0.511 Cohabitation with other animal species Cattle Yes No8.7 (23/265) 12.9 (48/373)0.09711.8 (34/287) 7.9 (58/735)0.047 * Goat Yes No--13.0 (40/308) 7.3 (52/714)0.003 * Sheep Yes No10.1 (28/278) 11.9 (43/360)0.456-- Farmed game Yes No33.3 (7/21) 10.4 (64/617)0.001 *0.0 (0/10) 9.1 (92/1012)0.318 South American camelids Yes No20.8 (5/24) 10.7 (66/614)0.12310.0 (2/20) 9.0 (90/1002)0.875a variables were chosen based on their availability in the national animal health information system. b (number of seropositive herds/total number of herds). c herd entry of one or more animals in the year 2020 for replacement or breeding purposes. * p < 0.05 = significant.3.2. Herd-Level ExaminationDetailed investigations in five infected dairy goat herds (432 animals) revealed an overall apparent within-herd MAP seroprevalence of 21.8% (ranging from 11.7 to 28.0%); the calculated overall TP was 38.6%, with an overall rate of MAP shedding of 12.3% (ranging from 5.0 to 24.7%). The results are shown for each herd in Table 3 in detail. In 6 out of the 52 faecal PCR positive animals (11.5%), no seroconversion could be determined by ELISA. The potential risk factors of body condition score and age were analysed in terms of seropositivity and MAP shedding (data shown in Table 4). Both factors, BCS and age, were not identified as significant risk factors in the logistic regression model nor in the chi-square test (dichotomized variables as described above). However, for animals of three years old, a high frequency of MAP seropositivity (35.5%) and shedding (31.1%) was observed.It was possible to identify MAP by culture from BS in each of the five investigated MAP positive herds. In total, 15 out of 16 BS samples tested positive (94%) on HEYM agar. Using pooled faecal samples (five animals each), 7 out of 15 samples tested positive (47%) on HEYM agar (Table 3). A minimum of one pooled faecal sample tested positive in every herd. Because of an insufficient sample amount, M7H10 was carried out for 13 BS samples (9 tested positive, 2 were contaminated) and 12 pooled faecal samples (5 resulted positive) only; M7H9C was conducted for five boot swab samples (all five positive) and nine pooled faecal samples (four with positive results; of those, two tested negative on HEYM and M7H10). animals-13-01517-t003_Table 3Table 3Herd-level testing for Mycobacterium avium subsp. paratuberculosis in five dairy goat herds by ELISA on serum samples, individual faecal sample qPCR and culture and qPCR of boot swab and pooled faecal samples.Herd NumberHerd Size (n)Serum Sample Size (n)Faecal Sample Size (n)ELISA Positive aPCR Positive aBoth ELISA and PCR Positive aBoot Swab Samples (HEYM) dPooled Faecal Samples (HEYM) d 1 114114112 c20.2 (23)10.7 (12) 8.9 (10)3/31/3 2 126126 b122 c24.0 (30)10.7 (13)10.7 (13)4/41/3 3 57575722.8 (13)10.5 (6)8.8 (5)3/32/3 4 6007573 c28.0 (21)24.7 (18)23.3 (17)2/32/3 5 550606011.7 (7)5.0 (3)1.7 (1)3/31/3 Total 43242421.8 (94)12.3 (52)10.9 (46)15/167/15Abbreviation: HEYM—Herrold’s Egg Yolk agar slants with Mycobactin J. a results in %, absolute numbers in brackets. b 1 serum sample resulted doubtful. c in 8 animals, no faecal sample could be collected. d positive samples/number of samples.animals-13-01517-t004_Table 4Table 4Mycobacterium avium subsp. paratuberculosis ELISA and PCR results of dairy goats in different BCS classes and age groups in %, absolute numbers in brackets. Number of AnimalsELISA PositivePCR Positive Age, years 120.5 (88)18.2 (16)5.7 (5)219.6 (84)13.1 (11)8.3 (7)314.5 (62)35.5 (22)30.6 (19)410.0 (43)16.3 (7)18.6 (8)5 or older35.4 (152)25.0 (38)8.6 (13)Total100 (429)21.9 (94)12.1 (52) BCS 10.9 (4)75.0 (3)75.0 (3)217.4 (75)25.3 (19)12.0 (9)357.4 (248)20.6 (51)11.7 (29)423.8 (103)20.4 (21)10.7 (11)50.5 (2)0.0 (0)0.0 (0)Total100 (432)21.8 (94)12.3 (52)4. DiscussionThis study gives an overview about the current epidemiological situation of MAP in the Austrian small ruminant population. The investigation revealed a herd-level apparent MAP seroprevalence of 11% for goat herds and 9% for sheep flocks, respectively. The animal-level apparent MAP seroprevalence was 2.0% for goats and 0.7% for sheep. In contrast, a study conducted in Austria in 2003 reported an animal-level apparent prevalence of 0.6% in sheep and 0.0% in goats, concluding that MAP was not widespread in the small ruminant population [27]. In that study, serum and tissue samples from 169 sheep and 80 goats were investigated for MAP by ELISA and culture. One sheep was positive by ELISA and a second sheep showed positive results for MAP in PCR and culture; all the goats tested negative. The considerable difference in MAP prevalence may be partially explicable by differences in study design (the study population in the present study is more representative) and variation in the diagnostic tests used. Nevertheless, the results of our study show, that the MAP prevalence has increased in small ruminants over the last years. In Germany, the herd-level seroprevalence was estimated to be 65% in sheep flocks and 71% in goat herds [8]. According to a global review, MAP herd-level prevalence estimation determined by laboratory testing was higher than 10% in 5 out of 11 countries for sheep and in 7 out of 12 countries for goats, respectively. More than 40% of herds being affected by MAP were reported in two countries for sheep and four countries for goats [1]. However, as the number of prevalence studies carried out among sheep and goats is low and as they differ in study design and diagnostic tests used, MAP prevalence in small ruminants is still unknown in many countries and the prevalence appears to be underestimated [4]. The results of our study indicate a moderate MAP seroprevalence and compared to prevalence reported in other countries, the Austrian MAP prevalence seems to be relatively low. The large serological screening sample size of the present study allows flocks to be selected randomly, considering a proportional provincial distribution. As the samples originated from the national Brucella melitensis sampling scheme, it was not required to take blood samples for this prevalence study, which implied a benefit for animal welfare. However, large flocks, dairy herds and herds with increased animal movement were selected more frequently. As the statistical analysis showed significant association between MAP seropositivity and the risk factors’ herd size, dairy herd and animal trading in goat herds, prevalence of caprine paratuberculosis might have been overestimated in this study. The serological tests used in our study are validated for small ruminant sera. However, since no particular test accuracy data for sheep and goats are available for these tests, Se/Sp indicated for cattle sera had to be applied to calculate TP. The calculated TP therefore should be interpreted cautiously. MAP diagnosis using ELISA is challenging, especially in young stock, since the immune response varies between the different stages of infection [28] and between individuals [29]. Nielsen and Toft [30] reviewed test accuracies of several ELISAs for serological MAP diagnostic and concluded that test Se/Sp values differ between species. It appears that ELISA is more accurate in the detection of infection in goats than in cattle (Se ranging from 0.63 to 0.84). In contrast, Se in sheep appears to be poor (0.16–0.44). Consequently, MAP prevalence in sheep is likely to be underestimated in this study. Furthermore, test accuracy may only be valid for the target population in which a test has been validated [31]. Therefore, more research on validation of serological tests for small ruminant species is needed. The fact that a representative number of samples was investigated per herd (the median sample size per herd was 16 samples for sheep and 8 samples for goats) enabled the identification of infected flocks despite the low test sensitivity, assuming a common within-herd MAP seroprevalence of >10% in infected flocks [1]. However, infected flocks with a very low seroprevalence might have been classified as false negative.The present study revealed a significant association between herd size and MAP seropositivity for goat herds; the same effect was not seen in sheep flocks. This finding is in contrast to what was described previously, where flock size was found to be associated with increased MAP prevalence in sheep only [8,9]. It might be assumed that association between herd size and seropositivity in goats is related also to the fact that larger goat herds in Austria are also applying an intensive production system. For sheep, the overall moderate MAP seroprevalence in Austrian flocks and the general poor test sensitivity in sheep may circumvent the detection of a potential association. We could demonstrate significant differences in flock-level MAP prevalence between regions for both species too, as described elsewhere [5,7]. Prevalence differences between regions in Austrian goat herds may be associated with an increased percentage of herds applying a more intensive production system in some geographic areas, whereas prevalence differences between regions in sheep could probably be related to a variable regional incidence of cohabitation with cattle and/or goats. Therefore, region might be considered a confounding variable. To answer this question, further studies, with a sampling scheme specifically designed for the detection of MAP, should be performed. Goat herds were significantly more often affected by MAP if they were categorised as flocks with animal trading, as a dairy herd and if herd owners were Animal Health Service members, all factors synonymous with elements of an intensive production system. These results are consistent with current knowledge: contact with other flocks [10] and intensive production systems [5,11] were reported as risk factors associated with an increased MAP seroprevalence. Furthermore, seropositivity rate was significantly higher in the present study in goat herds with cohabitation with farmed game. To the authors’ knowledge, a similar correlation has not been described before, though Verdugo et al. [32] found high flock prevalence of 46% in deer herds. For sheep flocks, the seropositivity rate was significantly higher in flocks with animal trading and if cattle or goats were kept together on the farm, respectively. These findings enable the suggestion of a possible cross-species transmission of MAP between cattle, sheep, goat and farmed game in Austria. An important tool to reconstruct infection chains and confirm MAP cross-species transmission is the bacterial strain genotyping of MAP isolates, which should be a consideration for future studies [33]. Cross species infection is suspected to occur between ruminant species [34,35,36,37,38]. Consequently, the infection of small ruminants should also be considered as a possible risk factor in the epidemiology of MAP in cattle herds. Furthermore, there is evidence for interspecies transmission between wild and domestic ruminants [15,39,40]. In an Austrian study, four out of seven MAP genotypes detected in wild ruminants were also present in local cattle and sheep herds [15]. In another study, 15 MAP genotypes across multiple wild and domestic ruminant host species were identified, leading to the conclusion that wild ruminants can act as reservoirs in the transmission of MAP to ruminant livestock [40]. Several risk factors, such as breed, kidding area, management by batches, ventilation and lactation, found to bias MAP prevalence in other studies were not considered in this study because of absent data due to the retrospective study design [5,6,10,11]. Furthermore, widespread MAP infection has been reported in goat flocks seropositive to CAEv (caprine arthritis encephalitis virus) [5]. As control programs for CAE have been implemented in Austria for many years and most herds are free of the infection, no attempts were made to survey the CAE status in participating herds in the course of our study. Both factors of age and BCS were not significant in terms of seropositivity and MAP shedding in goats in the present study, although weight loss was the main clinical sign of paratuberculosis. However, the number of investigated herds and individual animals was not sufficient to gain consolidated findings. In the present study, it was possible to isolate MAP by culture using BS samples in all five investigated herds (15/16 samples tested positive). Using pooled faecal samples, the detection rate was comparatively low (9/15), but all herds were detected as MAP positive by a minimum of one pooled faecal sample. According to Eamens et al. [41], pooled faecal culture is suitable for herd diagnosis if the animals are moderate or high shedders of MAP but not in herds with a few low shedders only. As two samples were positive on M7H9C, but not on HEYM in this study, it should also be considered for future investigations to use both media types for caprine samples, as goats can be infected by both MAP-S strains (sheep-type) and MAP-C strains (cattle-type) [42]. Environmental sampling is mainly used in cattle holdings as an effective and cost-efficient surveillance method [43]. Combined culture and PCR of BS samples has been applied successfully for large-scale herd-level testing in a few control programs for dairy cattle herds [13,44]. The results of the present study indicate that BS sampling may be suitable to detect MAP infection in goat herds. However, further studies are needed to validate the sensitivity of this diagnostic tool in goat holdings.To prevent a future spread of MAP among herds and between species, a nationwide control programme should be established for sheep and goat holdings, including herd-level monitoring (e.g., by repeated environmental sample testing) and on-farm control and biosecurity measures. Some regions in Germany and Austria are currently applying such an approach in cattle [18]. Moreover, the purchase or movement of livestock should rely on herd status certification combined with an individual animal test result in order to reduce the risk of between-herd transmission of MAP by infected livestock. Goat production in Austria is a growing sector (since the year 2011 the number of goats kept has increased by about 28%), with the trend for larger flocks and more intensive production systems. Furthermore, according to experimental infection, goats have been found to be more susceptible to paratuberculosis than cattle and sheep [45]. As the prevalence was quite high in Austrian dairy goat herds, it could be judicious to focus on caprine paratuberculosis control in this production group. Windsor [2] mentioned three main approaches to control ovine and caprine paratuberculosis: management changes to decrease transmission, test and cull strategies and vaccination. Few countries are also using vaccination for disease control [1] and a meta-analysis showed vaccination reduces MAP shedding, production losses and pathology [46]. On the other hand, MAP vaccination can interfere with testing for bovine tuberculosis (TB). Austria is officially free of bovine TB, but a wildlife reservoir for Mycobacterium caprae in red deer exists in some geographic areas in western Austria and serves as source of infections for domestic cattle grazing on Alpine pastures during the summer [47]. In addition, vaccination may interfere with MAP control based on test-and-cull programmes using immunological tests, as ELISA is not able to differentiate between antibodies produced by infection and by vaccination [12]. Therefore, vaccination against paratuberculosis is prohibited for all ruminant species in Austria. 5. ConclusionsMycobacterium avium subsp. paratuberculosis infection is present in Austrian sheep and goat holdings, with a herd-level apparent seroprevalence of 11.1% for goat herds and 8.9% for sheep flocks. Identified risk factors for MAP seropositivity in goat herds were herd size, animal trading, dairy farms, Animal Health Service membership and cohabitation of farmed game; for sheep flocks, identified risk factors were animal trading and cohabitation with cattle or goat, respectively. Furthermore, prevalence differences were observed between different regions for both species. At the herd level, average apparent seroprevalence was 21.8% (calculated true prevalence 38.6%) and MAP shedding was 12.3% in dairy goats. It was possible to identify MAP by culture from boot swab samples in each of five MAP infected herds. It can be concluded, that the seroprevalence of MAP in the Austrian small ruminant population is moderate and below reported prevalence in other countries. Goat herds with an intensive production system were identified to have an increased risk to be affected by MAP; moreover, interspecies transmission seemed to occur. The use of BS sampling to identify MAP positive goat flocks may be promising but needs further investigation. Based on these results, a close monitoring of the MAP prevalence in Austrian small ruminants as well as a national control program, especially for dairy goats, seems to be advisable.

animals : an open access journal from mdpi

10.3390/ani13081303

PMC10135088

The aim of this study is to investigate the effects of mono-lactate glyceride on growth performance and the morphology and function of the intestine in weaned piglets, which provided a theoretical basis for its practical application as a new feed additive. Dietary supplementation with 0.6% mono-lactate glyceride (LG) essentially decreased diarrhea rate and the contents of malondialdehyde (MDA) and hydrogen peroxide (H2O2) in the ileum and jejunum, increased the expression of intestinal tight junction protein (Occludin) and the activities of superoxide dismutase (SOD) and catalase (CAT) in the ileum and colon, and improved the intestinal morphologic structure. In addition, mono-lactate glyceride supplementation could enhance intestinal mucosal growth, promote intestinal mucosal water and nutrient transport and lipid metabolism, and enhance antiviral and immune function and antioxidant capacity. Overall, these results suggested that dietary supplementation with mono-lactate glyceride could decrease the diarrhea rate.

Mono-lactate glyceride (LG) is a short-chain fatty acid ester. It has been shown that short-chain fatty acid esters play an important role in maintaining intestinal structure and function. The aim of this study is to investigate the effects of mono-lactate glyceride on growth performance and intestinal morphology and function in weaned piglets. Sixteen 21-day-old weaned piglets of similar weight were distributed arbitrarily to two treatments: The control group (basal diet) and the LG group (basal diet + 0.6% mono-lactate glyceride). The experiment lasted for 21 days. On day 21 of the trial, piglets were weighed, and blood and intestinal samples were collected for further analysis. Results showed that dietary supplementation with 0.6% mono-lactate glyceride decreased (p < 0.05) the diarrhea rate and the contents of malondialdehyde and hydrogen peroxide in the ileum and jejunum and increased (p < 0.05) the expression of intestinal tight junction protein (Occludin) and the activities of superoxide dismutase and catalase in the ileum and colon. In addition, mono-lactate glyceride supplementation could enhance intestinal mucosal growth by increasing (p < 0.05) the mRNA levels of extracellular regulated protein kinases, promote intestinal mucosal water and nutrient transport and lipid metabolism by increasing (p < 0.05) the mRNA levels of b0,+ amino acid transporter, aquaporin 3, aquaporin 10, gap junction protein alpha 1, intestinal fatty acid-binding protein, and lipoprotein lipase, enhance antiviral and immune function by increasing (p < 0.05) the mRNA levels of nuclear factor kappa-B, interferon-β, mucovirus resistance protein II, 2’-5’-oligoadenylate synthetase-like, interferon-γ, C-C motif chemokine ligand 2, and toll-like receptor 4, and enhance antioxidant capacity by increasing (p < 0.05) the mRNA levels of NF-E2-related factor 2 and glutathione S-transferase omega 2 and decreasing (p < 0.05) the mRNA level of NADPH oxidase 2. These results suggested that dietary supplementation with mono-lactate glyceride could decrease the diarrhea rate by improving intestinal antioxidant capacity, intestinal mucosal barrier, intestinal immune defense function, and intestinal mucosal water and nutrient transport. Collectively, dietary supplementation with 0.6% mono-lactate glyceride improved the intestinal function of weaned piglets.

1. IntroductionSocial, environmental, and nutritional changes will reduce piglet feed intake during the critical period of piglet adaptation to the initial diet. [1]. After weaning, the diet is changed from highly digestible liquid milk to more indigestible and complex solid feeds, which can impair the structure and function of the intestine [2]. Thus, the weaning period is one of the most critical developmental stages of the digestive tract of piglets. Symptoms caused by weaning profoundly affect the health of piglets, resulting in reduced growth performance and sometimes death [3]. These effects have resulted in economic losses to pig production and increased public health risks due to the production of pathogenic bacteria-infected pork [4]. Therefore, it is urgent to develop high-quality and safe antibiotic replacement products to improve adverse impacts caused by weaned piglet syndrome and promote piglet intestinal health.Short-chain fatty acid esters are low molecular weight chemicals formed by the esterification of short-chain fatty acids (SCFAs) and alcohols under acid catalysis, i.e., the substrates are common with chain lengths of less than ten carbon atoms. It is an important substance to provide energy and fat metabolism to the animal body [5]. When the body is in a state of energy demand, short-chain fatty acid esters are gradually hydrolyzed by lipases to free fatty acids and glycerol, released into the blood, and oxidized and utilized by other tissues [6]. In recent years, it has been shown that dietary supplementation with tributyrin can improve the growth performance of weaned piglets and promote the development of immune organs and small intestine of weaned piglets. [7]. Previous research reported that glyceryl butyrate attenuated the inflammatory responses in ETEC-challenged piglets by inhibiting NF-κB/MAPK pathways and modulating gut microbiota, thereby improving the intestinal health of piglets [8].Studies have shown that SCFA inhibits inflammatory responses by inhibiting immune cell chemotaxis and reducing the release of pro-inflammatory cytokines and reactive oxygen species [9]. As fatty acids, acetic acid, propionic acid, and butyric acid play an important role. Butyric acid can play an anti-inflammatory role by inhibiting the release of IL-12, IL-1β, TNF-α, and NO in monocytes, promoting the expression of IL-10 and reducing the activity of NF-κB [9]. Acetic acid and the propionic acid act as antimicrobial agents by promoting the secretion of defense peptides in the host [10]. Butyrate can increase the secretion of antimicrobial peptides that are intrinsically immune in the host, clearing Salmonella enterica before an inflammatory response is triggered [11]. SCFAs are primarily produced not only from food sources but also from microbial fermentation of non-digestible sugar in the colon and cecum [12].As a short-chain fatty acid ester, mono-lactate glyceride (LG) is water-soluble. When administered parenterally, mono-lactate glyceride is rapidly hydrolyzed to glycerol and free fatty acids in the small intestine [6]. It has been shown that SCFAs can change chemotaxis and phagocytosis of immune cells, induce reactive oxygen species (ROS), and alter cell proliferation and function, which has anti-inflammatory, antitumorigenic, and antimicrobial effects [13]. Therefore, dietary supplementation with SCFAs can effectively replace antibiotics and improve the growth performance of weaned piglets. We hypothesize that LG can attenuate weaning-induced intestinal oxidative stress and inflammatory responses, thereby improving intestinal function in weaned piglets. However, the effects of mono-lactate glyceride on growth performance and intestinal function in weaned piglets remain unclear. In this experiment, the effects of LG supplementation on growth performance and intestinal function in weaned piglets were investigated by detecting the growth indexes, plasma biochemical parameters, intestinal histomorphology, antioxidant parameters, gene expression levels, and protein expression levels in piglets.2. Materials and Methods2.1. Experimental Animals and DesignThe animal experiment for this research was approved by the Animal Care and Use Committee of the Hubei Province (WPU201508001). The sixteen healthy crossbred female piglets (Duroc × Landrace × Yorkshire) with similar body weights were weaned at 21 days of age. Each piglet was individually housed in a 1.20 × 1.10 m2 steel metabolic cage with eight replicate cages per treatment. After a period of 3 days of adaptation, piglets (average body weight of 6.59 ± 1.19 kg) were assigned randomly into the two treatments: Control group (piglets were fed with the basal diet) and LG group (piglets were fed with the basal diet supplemented with 0.6% mono-lactate glyceride). The basal diet was prepared according to the nutritional needs of NRC (2012) pigs (8~20 kg), and the basal diet nutritional levels were consistent in each treatment group, and its composition and nutritional content are shown in Table 1. On days 7 and 14 of the trial, blood samples were collected from the anterior vena cava of piglets. On day 21 of the trial, each pig was anesthetized with 10% pentobarbital sodium by intramuscular injection at a dose of 80 mg/kg BW and slaughtered 15 min later. Then, the pig‘s abdomen was incised from the sternum to the pubic bone to expose the entire gastrointestinal tract. Blood, intestine, and intestinal contents were collected and stored at −80 °C until assay [14].2.2. Plasma Biochemical IndicesOn the 21st day of the experiment, piglets in each group were aseptically bled from the anterior vena cava 1 h after feeding D-xylose, and the blood samples were allowed to stand for 15 min and centrifuged (3000 rpm, 10 min), and the supernatant was taken after the end of centrifugation, that is, plasma was separated, and the blood samples were aliquoted and placed in a −80 °C freezer for testing [15]. Plasma biochemical Indices (ALT, AST, TBIL, TP, ALB, CHOL, BUN, ALP, CK, and GGT) were measured by the Testing and Analysis Center of the Hubei Institute of Pharmaceutical Industry.2.3. Intestinal Morphology and Intestinal Redox StatusTo investigate intestinal morphology, paraformaldehyde-fixed jejunum, ileum, and duodenum were dehydrated and embedded in paraffin. Next, 4-µm sections were cut and then stained with hematoxylin and eosin stain. Intestinal morphology was carried out with a light microscope (Leica, Solms, Germany) with the Leica Application Suite image analysis software (Leica, Solms, Germany) [14]. There are 6 villus and crypts that were counted per histological cutting. Intestinal villus height, crypt depth, and villus surface area were measured to calculate the ratio of villus height to crypt depth. The activities of GSH-Px, SOD, and CAT and the contents of MDA and H2O2 were determined by using commercially available kits (Jiancheng Bioengineering Institute, Nanjing, China).2.4. Expression Levels of ProteinThe expression of proteins was analyzed by using Western blotting as described by Hou et al. [16]. The primary antibodies used in this study included Villin, Occludin (rabbit, 1:1000; Cell Signaling Technology, Inc., Danvers, MA, USA), Caspase-3, Bax, MAX1, and β-actin (mouse 1:2000; Sigma-Aldrich Inc., St. Louis, MI, USA). The secondary antibodies used in this study included Anti-mouse (mouse, 1:5000; Zhongshan Golden Bridge Biological Technology Co., Ltd., Beijing, China) and Anti-rabbit (rabbit, 1:3000; Zhongshan Golden Bridge Biological Technology Co., Ltd., Beijing, China). Blots were developed using an enhanced chemiluminescence kit (Amersham Biosciences, Uppsala, Sweden) and then analyzed by an imaging system (Alpha Innotech, New York, NY, USA).2.5. Expression Levels of GenesThe quantification of gene mRNA levels was performed by the real-time PCR method, as described by Yi et al. [17]. The primers used in this study were shown in Table 2. The reference gene was ribosomal protein L 4 (RPL4). SYBR® Premix Ex TaqTM (Takara, Dalian, China) was used for real-time PCR 7500 System Fast Real-Time RT-PCR (Applied Biosystems, Foster City, CA, USA). Results were analyzed by the 2−ΔΔCt method as described [1].2.6. Statistical AnalysisAll data were analyzed using Student’s t-test, and data were expressed as mean ± standard deviation. All results were analyzed using SPSS (Version 17.0, SPSS Inc., Chicago, IL, USA). A p-value ≤ 0.05 was considered statistically significant.3. Results3.1. Growth PerformanceDuring the experimental period, the average daily gain (ADG), average daily feed intake (ADFI), feed to gain (F/G), and diarrhea rate (DR) were shown in (Table 3). Although there was no significant difference (p > 0.05) in ADG and ADFI between control and LG groups, dietary LG supplementation had a tendency to increase ADG. Moreover, compared with the control group, dietary supplementation with 0.6% LG reduced (p < 0.05) DR between days 0 to 10, days 10 to 21, and days 0 to 21.3.2. Plasma Biochemical IndicesPlasma biochemical indicators were shown in Table S1 to reflect the metabolic function of piglets. Compared with the control group, alkaline phosphatase (ALP) and blood urea nitrogen (BUN) on day 7 and alanine aminotransferase (ALT) on day 21 in plasma were increased (p < 0.05) in the LG group, total protein (TP), total cholesterol (CHOL), and glutamyl transpeptidase (GGT) were decreased (p < 0.05) in the LG group on day 7, total bilirubin (TBIL), albumin (ALB), creatine kinase (CK), and glutamyl transaminase (GGT) were decreased (p < 0.05) in the LG group on day 14, and creatine kinase (CK) were decreased (p < 0.05) in plasma on day 21.3.3. The Effects of Dietary Supplementation with LG on Piglet Intestinal MorphologyResults showed that the villus surface area and the ratio of villus height to crypt depth in the jejunum and the ratio of villus height to crypt depth in the ileum were increased (p < 0.05) in the LG group, whereas the crypt depth in the jejunum and ileum and villus surface area in the ileum were decreased (p < 0.05), compared with the control group (Table 4).3.4. Intestinal Redox StatusCompared with the control group, LG supplementation increased (p < 0.05) the activities of SOD in the ileum and CAT in the colon and decreased (p < 0.05) the activity of GSH-Px in the duodenum and jejunum and the contents of MDA in the jejunum and ileum, and H2O2 in the jejunum (Table 5).3.5. Protein AbundancesCompared with the control group, piglets in the LG group exhibited an increase (p < 0.05) in the protein abundances of Occludin and MX1. There were no significant differences in other protein abundances, including Villin, Caspase-3, and Bax (Figure 1).3.6. Gene ExpressionCompared with the control group, LG supplementation increased (p < 0.05) the mRNA levels of ERK in the jejunum while decreasing (p < 0.05) the mRNA levels of Bax in the jejunum in the LG group (Table 6).Compared with the control group, piglets in the LG group exhibited obvious increases (p < 0.05) in the mRNA levels of b0,+AT in the colon, and AQP3 in the ileum and colon, GJA1 in the jejunum, and AQP10 in the ileum (Table 7).Compared with the control group, LG supplementation increased (p < 0.05) the mRNA levels of NF-κB, IFN-β, MX1, MX2, TLR4, and OASL in the jejunum and TFF3, NF-κB, IFN-α, IFN-β, MX2, and OASL in the ileum (Table 8).Compared with the control group, piglets in the LG group exhibited significant increases (p < 0.05) in the mRNA levels of IL-1β and IFN-γ in the jejunum and IL-1β, IL-4, CCL-2, and IFN-γ in the ileum, while also exhibiting a decrease in (p < 0.05) the mRNA levels of CXCL-9 in the ileum, and of IFN-γ in the colon (Table S2).Compared with the control group, LG supplementation increased (p < 0.05) the mRNA levels of LPL, Nrf-2, and GSTO2 in the jejunum and LPL and Nrf-2 in the ileum and INSR in the colon, while also decreasing (p < 0.05) the mRNA levels of I-FABP in the jejunum and I-FABP and PCK1 in the ileum and I-FABP and NOX2 in the colon (Table 9).4. DiscussionEarly weaning causes stress and diarrhea in piglets, which is one of the most challenging problems in the pig industry [1]. Previous research suggested that dietary supplementation with SCFAs could promote intestinal health by improving intestinal absorption and immunity in piglets [18]. Previous studies suggested that dietary supplementation of calcium butyrate significantly reduced diarrhea rates in piglets [19,20]. In good agreement with these studies, our results demonstrated that dietary supplementation with 0.6% LG effectively reduced the diarrhea rate.Plasma protein synthesized by the liver can be used as an indicator of protein metabolism function, and an increase in total protein in plasma can indicate enhanced immune function [14,21]. In the present study, although dietary LG supplementation decreased the total protein content in plasma on day 7, there was no significant difference at a later stage, which may be because LG helps to improve piglet immunity and promote body protein synthesis. The level of enzyme activity in plasma is an indicator of tissue damage. Alkaline phosphatase (ALP) is a ubiquitous membrane-bound glycoprotein involved in protein phosphorylation and plays a role in the transport of intestinal epithelial cells [22]. The results showed that LG intervention increased plasma ALP activity and may promote protein synthesis in piglets. In addition, ALT, AST, AST/ALT, and ALP in plasma are indicators of liver function, and liver dysfunction causes an increase in AST/ALT activities, which are gradually decreased when liver function is repaired [23,24,25]. In this study, LG intervention decreased AST/ALT activities at day 21, indicating that LG may help repair liver function. Total bilirubin is an indicator of liver function. It has been shown that TBIL content in the plasma is decreased when liver function is enhanced [25]. In good agreement with these studies, our results showed that there was a significant decrease in TBIL levels in the plasma of piglets in the LG group. GGT is an essential enzyme for protein and amino acid metabolism, and it could reflect the injury of various cells by oxygen free radicals [14]. The elevated plasma GGT levels might be an indicator of oxidative stress and liver damage [26]. We found that LG intervention reduced GGT activity in plasma on days 7 and 14, thereby alleviating the stress experienced by piglets at the early weaning stage. Creatine kinase plays an important role in the process of energy metabolism, and its level is often used as an indicator of cardiac and skeletal muscle disease [27]. A previous study suggested that the CK level was increased in blood when the skeletal muscle was compromised. [28]. In this study, we found that LG intervention decreased CK level in plasma on days 14 and 21, indicating that LG may help repair bone damage caused by stress in piglets. These results showed that supplementation with LG could regulate the metabolism of proteins, improve the immune level of piglets, and relieve stress in piglets, which plays a role in protecting functions of the liver and skeletal muscle in piglets.Insulin receptor (INSR) is a single-pass transmembrane receptor with tyrosine kinase activity, which is primarily involved in cell growth and metabolic homeostasis. Its main function is to mediate IGF-2 and insulin signaling pathways and then regulate the metabolic activity of the body [29]. Lipoprotein lipase (LPL) is one of the key enzymes in the systemic partitioning and metabolism of lipids. It plays an important role in lipid metabolism, transport, and energy metabolism and affects the growth and development of animals [30]. Intestinal fatty acid binding protein (I-FABP) is a key protein in lipid transport and can transport lipids from the intestinal lumen to enterocytes, bind excess fatty acids, and maintain a stable fatty acid pool in epithelial cells [31]. In this study, we found that LG intervention could regulate lipid metabolism, transport, and fat deposition and activate the insulin signaling pathway. Aquaporins (AQPs) are a family of membrane channel proteins, of which AQP3 and AQP10 are important aquaporins, which can rapidly absorb water in the intestinal cavity into the blood and alter the endocrine environment of the intestinal cavity [14,32]. It has been shown that intestinal absorption of basic amino acids mainly transports and absorbs basic amino acids and cystine into epithelial cells through the b0,+ system at the brush border, and b0,+AT plays an important role in the b0,+ system [33]. GJA1 is a gap junction protein that plays an important role in the exchange of nutrients, ions, and cellular regulators between cells [34]. In this study, dietary supplementation with LG significantly increased the mRNA levels of AQP3, AQP10, GJA1, and b0,+AT, suggesting that dietary LG supplementation may improve the transport of water and nutrients in intestinal mucosa, promote the intestinal water metabolism, and effectively relieve diarrhea in piglets.Indicators of intestinal morphology, such as villus height, surface area, crypt depth, and the ratio of villus height to crypt depth, are commonly used to reflect intestinal morphological development and intestinal morphological integrity. Generally, the decrease in crypt depth and the increases in villus height, and the ratio of villus height to crypt depth reflect improved healthy intestinal development and nutrient absorption [35]. We found that the crypt depth in the jejunum of piglets in the LG group was decreased to a greater extent than the villus height, possibly because LG intervention increased the number of mature cells in the intestinal mucosa and promoted the complete development of the intestinal mucosa, while the growth and proliferation of cells or the cell maturation rate in the intestine had a great relationship with the crypt depth and the increase of the cell maturation rate could reduce the crypt depth. Bax is a representative pro-apoptotic protein in the Bcl-2 family, and Bcl-2 protein can block the apoptosis signal transmission system, thereby inhibiting apoptosis [36]. ERK1/2 is involved in processes such as cell proliferation, growth, and apoptosis [37]. These results showed that LG inhibited intestinal mucosal cell apoptosis by decreasing the relative expression of the Bax gene in the jejunal mucosa. Moreover, the ERK1/2 signaling pathway was activated, and intestinal mucosal growth was promoted by regulating the relative expression of the ERK1/2 gene.NOX2 is widespread in phagocytes and tissues where it can be activated to induce increased ROS and is one of the major sources of ROS [38]. A previous study found that Nrf2 improves abnormal oxidative stress by increasing the expression of antioxidant-related genes [39]. GSTO2 can affect the expression of corresponding active proteins by changing the transcriptional activity of related genes, thereby regulating the activity of related antioxidant enzymes and reducing the negative effects of oxidative stress [40]. In this study, LG intervention significantly increased the relative expression of Nrf-2, GSTO2 gene in the jejunum, and Nrf-2 gene in the ileum and significantly decreased the relative expression of the NOX2 gene in the colon, suggesting that LG can improve the antioxidant function of the body by regulating the expression of antioxidant related genes. Superoxide dismutase and catalase are antioxidant enzymes, both of which are involved in neutralizing ROS reactions, thereby protecting tissue cells from oxidative damage [41]. MDA is a major product of polyunsaturated fatty acid peroxidation, which can induce toxic stress in cells and is a marker for the assessment of oxidative stress levels in biosomes [42]. H2O2 is the main product of oxidative stress in the body [43]. We found that dietary supplementation with LG increased the activities of SOD in the ileum and CAT in the colon and decreased the contents of MDA in the jejunum and ileum and H2O2 in the jejunum. These results are consistent with Yu et al.‘s study that supplementation of 500 mg/kg B. licheniformis in the diet of weaned piglets enhanced antioxidant capacity [44]. In summary, dietary supplementation of 0.6% LG enhanced the antioxidant capacity of the intestine by regulating the expression of antioxidant-related genes and increasing the activity of intestinal antioxidant enzymes. Of note, dietary LG supplementation improved the activity of SOD in the ileum but decreased the activity of GSH-Px in the duodenum and jejunum. This may be due to the existence of a dynamic balance mechanism in the body’s antioxidant system. When one mechanism is activated, the other may be inhibited [45].Intestinal trefoil peptide (ITF, i.e., TFF3) plays an important role in maintaining and repairing mucosa, inhibiting tumors, and regulating cell growth and apoptosis in animals [46]. It has been shown that secretion of TFF3 contributes to improving intestinal mucosal morphology, reduces the generation of inflammatory cells, and repairs and maintains intestinal mucosa [47]. Our results are consistent with that study. IFN-α and IFN-β belong to type I interferons, which can induce cells to produce antiviral enzymes to interfere with viral transcription and translation, thereby achieving the effect of inhibiting viral proliferation [48]. MX protein has a wide range of antiviral effects and GTPase activity, MX1 protein inhibits myxovirus replication, and MX2 protein has a strong inhibitory effect on vesicular stomatitis virus [49,50,51]. Zhou et al. found that porcine Mx1 has activity against classical swine fever virus (CSFV) [52]. TLR4 is an important member of TLR, which plays an important role in innate immunity and inflammation by sensing pathogen-associated molecular patterns [53]. It has been shown that TLR4 signaling in macrophages can activate hundreds of genes that contribute to the protection against bacterial infection [54]. Furthermore, 2‘-5’ oligoadenylates synthesis (OAS) is an antiviral protein induced by interferon, of which OASL belongs to this class of proteins [55]. NF-κB can induce the expression of inhibitors of apoptosis (IAP) and certain members of anti-apoptotic Bcl2 by activating the transcription of genes involved in the inhibition of cell death through intrinsic and extrinsic pathways [56]. Occludin protein is an extremely important protein in the TJ, which promotes tight junctions in the intestinal epithelial cell space [57]. In this study, dietary supplementation with LG increased the mRNA levels of TFF3, NF-κB, IFN-α, IFN-β, MX1, MX2, TLR4, and OASL, as well as the protein expression of Occludin and Mx1. These results supported the notion that mono-lactate glyceride could regulate mucosal protection and repair, regulate apoptosis and anti-virus, and then improve intestinal immunity and intestinal barrier function.IL-1β is a pro-inflammatory cytokine produced by cells of the innate immune system and is essential in host defense responses [58]. It has been shown that short-chain fatty acids are beneficial in increasing the abundance of IL-1beta and IL-6 in the small intestine without producing intestinal inflammation [59]. IL-4, a Th2 cytokine, is an important regulator of the humoral immune response, which can regulate B cells and other non-immune cells and reflect the cellular and humoral immunity of animals [60,61]. These results showed that LG significantly up-regulated the relative expression of IL-1β in jejunal mucosa and IL-4 and IL-1β genes in ileal mucosa and then regulated Thl/Th2 immune balance, thus, LG played an inflammatory regulatory role. IFN-γ is the only member of the type II interferon family and is mainly produced by activated T cells, and has immunomodulatory functions [62]. Chemokine 9 (C-X-C motif 9, CXCL9), a member of the CXC family of chemokines, has the induction and chemotaxis of T cells and monocytes [63]. The C-C motif chemokine ligand 2 (CCL2) is a crucial mediator of immune cell recruitment during microbial infections and tissue damage [64]. Ferrari et al. found that IFN-γ had a synergistic effect on the secretion of CCL2 [65]. In this study, LG intervention significantly up-regulated the relative expression of CCL2 in ileal mucosa and IFN-γ gene in jejunoileal mucosa, indicating that glyceryl mono-lactate can regulate the immune function of the body.5. ConclusionsDietary supplementation with 0.6% LG significantly reduced diarrhea by improving intestinal histomorphology, maintaining intestinal integrity, and promoting the intestinal antioxidant capacity and mucosal barrier function, thereby improving intestinal function. Taken together, our results demonstrate the importance of LG in improving gut health in weaned piglets.

animals : an open access journal from mdpi

10.3390/ani11051380

PMC8152034

Obesity and related diseases are common problems for dogs and inappropriate feeding during development is a contributor to life-long weight issues. Judging the right amount of food to give a growing puppy is challenging and providing a simple recommendation to owners is essential. However, differences in dog size, activity, and many other factors such as neutering can all have a role in impacting the actual energy requirements for growth. Yet, the current feeding guideline for growth (NRC 2006) does not accommodate these factors. Therefore, this study investigated how much a small breed (Norfolk Terrier puppies) requires to maintain growth and a healthy body condition score (BCS) through their first year of life. We found that they required significantly less than suggested by the NRC. Changes in the assessment of appropriate feeding during development are required and this study supports the need to revise the NRC (2006) equation for small breed dogs.

An appropriate energy intake for healthy growth can reduce the risk of obesity and co-morbidities, such as orthopaedic diseases. The 2006 National Research Council (NRC) universal equation calculates the energy requirement of growing dogs based on predicted adult body weight, but evidence suggests a revision may be required. This study investigates the energy requirements of seventeen Norfolk terrier puppies over their first year (10 to 52 weeks). Puppies were individually fed complete and balanced diets in amounts to maintain an optimal body condition score (BCS), recording intake daily and body weight and BCS weekly. To monitor health a veterinary examination, haematology and plasma biochemistry and serum measures of bone turnover were undertaken every 12 weeks. Skeletal development was assessed using dual-energy X-ray absorptiometry (26 and 52 weeks). Puppies were clinically healthy with normal skeletal development and healthy growth throughout. The energy intake to achieve this was significantly lower than that predicted by the NRC (2006) equation at all time points, with largest mean difference of 285 kJ/kg0.75 per day at 10 weeks. If fed according to the NRC 2006 equation, dogs would have been in positive energy balance, possibly leading to obesity. These data support a revision to the NRC (2006) equation.

1. IntroductionObesity has been declared one of the greatest health challenges of the 21st century in our global pet population. A number of studies have revealed the scale of the problem in different regions of the world with the prevalence of overweight or obese dogs ranging from 20% to 50% [1,2,3,4,5,6]. This excessive bodyweight is linked to a myriad of associated conditions such as orthopaedic diseases [7,8,9,10], cardiorespiratory diseases [11,12], neoplasia [13,14] and diabetes mellitus [15,16,17]. As a result, the health-related quality of life and the expected lifespan are detrimentally affected [18,19].Obesity is, however, preventable and can be controlled through managing energy intake, especially during early growth. Studies in humans have shown that atypical growth patterns during this critical window of life can predispose to obesity [20,21,22]. This predisposition to obesity has also been documented in cats where there are recommendations for energy intake to be tightly regulated after neutering to prevent unnecessary weight gain during early life. The authors demonstrate that if a healthy body weight and condition can be achieved during early life, then the risk of obesity in adult life is dramatically reduced [23]. Similarly, a dog study has shown that 83% of dogs that were overweight by early adulthood had crossed at least two centile lines when tracking their bodyweight through puppyhood using the WALTHAM Puppy Growth Charts [24]. Furthermore, studies have demonstrated that rapid weight gain during the first year of life is detrimental to skeletal development [25,26,27,28]. In a similar vein to cats, neutering also increases the risk of obesity in dogs in addition to sex, breed and owner characteristics/behaviours [2,3,4,29,30,31,32,33,34,35]. As such, healthy growth is essential during this critical timeframe and needs to be closely regulated.Guidelines for the energy requirements during growth in puppies are provided by the National Research Council (NRC) in 2006 [36]. This was first suggested by Blanchard, Grandjean [37] and is in agreement with the equation suggested by Meyer and Zentek [38]. However, this is a single universal equation that does not take breed, sex, temperament, coat quality or neuter status into account. Previous studies demonstrated that the NRC [36] equation can result in an overestimation of their energy requirements, especially in younger puppies across a range of breeds [27,39,40]. These studies highlighted clear breed specific differences in energy requirements and a need for breed-specific feeding guides.The objective of this study is to investigate the energy requirements for healthy growth in a small dog breed, the Norfolk terrier, from ten weeks to one year of age. This was achieved by feeding puppies to an optimum body condition score (BCS) and monitoring their growth trajectory, using WALTHAM™ Puppy Growth Charts. This was then compared to the NRC (2006) equation to determine whether this calculation is suitable to determine the energy requirements for healthy growth in this breed of dog.2. Materials and Methods2.1. Animals and HusbandryThis work was approved by the WALTHAM Animal Welfare and Ethical Review Body and conducted under the authority of the Animals (Scientific Procedures) Act 1986. A total of seventeen Norfolk Terriers from eight litters took part in the study. All puppies underwent a physical examination by a veterinary surgeon at the start and end of the study. Puppies were housed with their mother until weaning at 8 weeks of age, in litter groups until 10 weeks of age, and in pairs thereafter. In all cases, housing consisted of environmentally enriched kennels with constant access to an outdoor area. All puppies received socialisation and training sessions daily and access to large outdoor play areas. Free access to drinking water was provided at all times. Male puppies were neutered around 26 weeks and females remained entire throughout.2.2. Diet ManagementPuppies were offered a commercially available dry format diet (Royal Canin Yorkshire Terrier Junior; Mars Petcare) from multiple batches. Between the ages of 10 and 26 weeks, puppies were offered their daily ration in 3 × 30-minute meals equally spaced out between 8 am and 4 pm. From 27 to 52 weeks of age, the puppies received their daily intake across two meals across the same time frame. Diets underwent nutritional analysis (Eurofins, UK) to ensure compliance with the NRC and FEDIAF guidelines. In summary the average nutrient composition of the diets fed was as follows: Moisture = 20.6 g/4184 kJ; Protein = 72.4 g/4184 kJ; Fat = 48.8 g/4184 kJ; Ash = 17.3 g/4184 kJ; Crude fibre = 4.9 g/4184 kJ. Nutritional analysis results were used to calculate the average predicted metabolisable energy content of the diets (1701 kJ/100 g), which in turn was used to calculate the actual energy intake of the dogs.2.3. Bodyweight ManagementFood intake was recorded immediately following each meal as the mass of food offered minus the mass of food refused. Initial feeding allowances were determined by a veterinary surgeon through calculating each puppy’s resting energy requirements (RER) multiplied by three. After this point the amounts consumed were adjusted weekly, as needed (see below), with the aim of maintaining puppies at an optimum body condition score (BCS) throughout the study. Bodyweight was measured once per week, on the same weekday, using calibrated scales (Mettler-Toledo Ltd; Leicester; UK) throughout the trial. Although not fully validated in puppies, BCS was evaluated every week using a 9-point scale [41] by the same two assessors to maintain consistency. If the BCS of any puppy increased or decreased from an ideal BCS (score 4 or 5 out of 9), then the dietary amount was recalculated in order to achieve an ideal BCS. Dietary changes consisted of either a 5% increase if the BCS decreased from ideal (3 or less on 9-point BCS) or a 5% decrease if the BCS increased from ideal (6 or above on 9-point BCS). In addition, WALTHAM™ Puppy Growth Charts, a validated tool for healthy growth [42], were used to ensure a healthy growth trajectory of all of the puppies. Weekly bodyweights were plotted, and the growth trajectory monitored to stay within two centile lines. If the growth trajectory crossed two centile lines in either direction, then a 5% dietary increase or decrease was implemented.2.4. Sample Collection and AnalysisAt 3, 6, 9 and 12 months of age, fasted (>12 h) jugular blood samples were collected (2.2 mL total volume). Lithium–heparin-treated blood was centrifuged and the resulting plasma used for the determination of standard biochemistry parameters; total protein, albumin, inorganic phosphorus, alkaline phosphatase (ALP), alanine transaminase (ALT), aspartate aminotransferase (AST), calcium, cholesterol, urea, creatinine, triglycerides, sodium, potassium, chloride and glucose using an AU480 (Beckman Coulter (UK) Ltd; High Wycombe; UK) analyser. EDTA treated blood was collected for the measurement of standard haematology parameters using a 3-part differential automated haematology analyser (Mythic 18 Vet, Orphée, Geneva, Switzerland). Parameters measured were total leukocyte count, leucocyte counts as a number and percentage (lymphocytes, monocytes and granulocytes), total erythrocyte count, haemoglobin concentration, haematocrit, mean corpuscular volume, mean corpuscular haemoglobin, mean corpuscular haemoglobin concentration, erythrocyte distribution width, platelet count, and mean platelet volume. Markers of bone turnover were analysed in the serum: bone-specific alkaline phosphatase (BAP) and carboxy-terminal telopeptide cross-links (CTx) using MicroVue™ Quidel ELISA (TECO medical Group; Sissach; Switzerland) and CartiLaps® ELISA (Immunodiagnostic Systems Ltd, Boldon; Tyne and Wear; UK.). Skeletal development was assessed at 26 and 52 weeks of age by means of Dual-energy X-ray absorptiometry (DXA; total body software package; Lunar Hologic QDR-1000 W; GE Healthcare; Chicago; USA). Prior to the scan, dogs were fasted at least 16 h and sedated with Torbugesic (0∙1 mg/kg; Pfizer Animal Health; Walton Oaks; Surrey; UK) and Dexmedetomidine (300μg/m2; Pfizer Animal Health; Walton Oaks; Surrey; UK) and reversed with Atipamezole (0∙1 mg/kg; Pfizer Animal Health, Walton Oaks; Surrey; UK).2.5. Statistical AnalysisThe predicted maintenance energy requirements were calculated, using the following NRC (2006) puppy energy requirement predictive equation (below), assuming that the adult bodyweight (kg) was that measured at 52 weeks of age. MER(kcal / kg0.75) = 130 × 3.2(e−0.87×(BW observed/BW52weeks) − 0.1)(1)MER: maintenance energy requirements as recommended by the NRC 2006 guidelines, BW: Body weight.Thereafter, kJ intake, NRC estimated requirements and the difference between them were modelled using linear mixed effects models. These models had fixed effects of age, sex and neuter status, and two-way interactions between neuter status and sex, and age. A random effect of dog nested in litter was also included to account for repeated measurements. Using these models mean values were estimated for each age, sex and neuter status combination. Likelihood ratio tests were used to test for the inclusion of the two-way interactions. Whilst their inclusion in the model was detected as statistically significant, visual inspection of the differences showed no biologically relevant difference. Means were, therefore, also estimated for each age averaged across levels of neuter status and sex to give an overall estimate. For the difference model (actual vs. NRC), all individual time point estimates were tested for a statistically significant difference from zero. All analyses were performed using R v3.5.1 [43] and the lme4 [44], multcomp [45] and lmerTest [46] libraries. Single step multiple comparisons correction was performed to maintain a familywise error rate of 5%.3. ResultsAll dogs remained healthy during the study, with no skeletal abnormalities observed, as judged by clinical examination and DXA. Haematological and biochemical parameters remained within normal ranges for all dogs (data not shown) and body weights increased with time for all dogs (Figure 1). Although some dogs deviated from an optimal BCS (4 or 5 out of 9) to a score of 3 or 6, this was corrected through dietary increases or decreases (Figure 2). At the end of the study, 1 male (K) and 1 female (A) were scored with a BCS of 6, while 15 dogs tracked at 4 or 5.3.1. WALTHAM™ Puppy Growth ChartsGrowth was also tracked and recorded using the WALTHAM™ Puppy Growth Charts to ensure all dogs were growing in a healthy trajectory throughout the study (Figure 3). Only one dog, between 40 and 50 weeks (Figure 3E), crossed two centiles in an upward direction but this was managed through dietary intake reduction to bring the trajectory back to between two centiles. Although this is normal for some dogs, it can be indicative of rapid or compensatory growth, which could be relevant here as the BCS of this dog was the only BCS to drop to a 3 for a period of two weeks at week 26 and 27.3.2. Energy IntakeThe results indicate that the NRC (2006) equation overestimates the energy requirements throughout the first year (Figure 4). No impact of neuter status was observed on actual or predicted energy intake. When the data were split for sex difference (data not shown), the over estimation of energy intake was greater in males due to the NRC (2006) using the 52-week weight and adult males being heavier in comparison to females. Statistically significant differences (p < 0.05) were detected at all ages between the actual intakes and NRC estimates, when averaging across sex and neuter status. The maximum difference between the actual and predicted energy intake was a mean of 286 kJ/kg0.75 per day at 10 weeks of age. The difference reduced with age but the energy to maintain optimal body condition score always remained significantly less than the NRC predicted equation with the lowest difference being 71 kJ/kg0.75 per day at 50 weeks of age. (Table 1).4. DiscussionThe data presented here demonstrate that the NRC (2006) equation predicting energy requirements for growth consistently overestimates the energy requirements for Norfolk terrier puppies and is therefore not consistent with the energy required to maintain healthy development. Specifically, the data indicate that the NRC overestimates the daily energy needed for both male and female growing Norfolk terriers from 10 to 52 weeks of age. The energy intake needed to maintain a healthy growth trajectory and optimal body condition score was similar in both males and females, across the 52-week period. Neutering at 26 weeks did not significantly alter the energy intake needed for growth in male dogs. However, as all female dogs remained entire during this study, it cannot be determined whether neutering would significantly affect the energy requirements of female Norfolk terrier puppies. Sex differences and the interaction between neutering and risk of weight gain and obesity is currently unclear. Neutering can alter the level of circulating sex hormones, which have been shown to affect appetite regulation and metabolic rate, as well as altering levels of appetite-related hormones such as leptin and ghrelin [47,48,49,50,51,52,53]. However, most of the studies were conducted in cats. Canine specific studies are less numerous and somewhat contradictory with studies showing increased trends of food intake and body weight following neutering [54,55,56,57] and others showing no effect on food intake or bodyweight [58,59]. This could be explained through the difference in male and female sex hormones [60], which could result in differing food intake effects between sex post-neutering. Notwithstanding this, the incidence of obesity in adult dogs that are neutered is much higher than that of entire dogs [3,29,30,31,32,33,61]. However, many other factors are also related to canine obesity, such as age [2,31,33,61], breed [3,32], activity level [31,33,62] and owner characteristics and behaviour [62,63,64]. It is difficult to isolate or quantify the significance of each individual risk to obesity.The energy intake data for the Norfolk terriers in this study is consistent with that previously reported for toy breed dogs living in a colony environment [23]. The estimated median MER was reported as 473 kJ/kg0.75 per day and the 52-week mean MER in the current study is 461 kJ/kg0.75 per day. Similarly, the average MER of pet dogs was demonstrated to be 519 ± 159 kJ/kg0.75 per day [65]. Importantly, the data presented here are consistent with studies that demonstrate collectively that the NRC recommendation overestimates the amount of energy required for healthy growth across multiple breeds [27,39,40,66,67]. Indeed, breed specific differences in energy requirements, especially between different sized breeds, have been documented, with Alexander et al. (2017) showing differences between the energy requirements of Yorkshire terriers, miniature schnauzers and Labrador retrievers. The energy intake per kg0.75 in Yorkshire terriers was significantly lower than both Labrador retrievers (until 29 weeks) and miniature schnauzers between (16 and 25 weeks). Furthermore, Dobenecker et al. (2013) showed a difference in energy requirements between foxhound-boxer-Ingelheim Labrador mixed breed puppies and beagle puppies up to 28 weeks of age. A large-scale study of client owned puppies reported that weaned puppies younger than 6 months had energy intakes that were approximately 80% of the NRC (2006) recommendation and in older puppies approximately 88% of this recommendation [39]. Collectively these studies suggest that breed differences in energy requirements should be considered when recommending feeding amounts during growth and the NRC (2006) universal equation is not suitable for this purpose.Obesity is the fastest growing disease in both people and pets with an estimated 1.9 billion adult humans being overweight and a third of those obese [68]. Studies have drawn correlations between obese pet owners and obese pets [4,69,70] and with canine obesity being the number one health concern in dogs worldwide [33] interventions and management are now paramount. One critical intervention is to provide pet owners with accurate feeding guidelines to ensure adequate nutrition and energy intake. This is especially important to ensure healthy growth rates. Managing the energy requirements of dogs, especially during early growth, will also provide a healthy growth trajectory and body weight which will reduce the risk of obesity in adult life.Throughout this study body condition scoring was used weekly to ensure the Norfolk terriers were being fed the correct energy requirements to maintain an optimal body condition. Although this method is widely used by veterinarians, researchers and some pet owners, due to the non-invasive nature and low cost, it is important to note that these scales are not validated for use in puppies and are a subjective tool. To reduce the subjectivity in this study, the assessment was conducted by the same two experienced assessors each week and a validated clinical tool for monitoring healthy growth was used in conjunction with BCS: the WALTHAM™ Puppy Growth Charts. These were developed using statistical modelling of the bodyweight and age data from 50,000 healthy dogs attending Banfield Pet Hospitals [42]. A recent study has shown that dogs who have crossed two centile lines on the growth charts, in either direction, were either over- or underweight by early adulthood, demonstrating the potential of this as a clinical tool for monitoring healthy growth in dogs [24]. Using these tools, this study provides additional evidence to demonstrate that feeding dogs to an optimal BCS results in healthy growth for the first year of life and also that the growth trajectory of the colony-held Norfolk terriers in this study is akin to client-owned dogs that the charts were modelled on.A limitation of this current study is that we did not have a control group of puppies that were fed to the NRC (2006) equation for growth. The choice to not include a control group of this type was because previous evidence suggested that following the NRC recommendations would provide energy in excess of requirements to growing dogs possibly leading to overweight or obesity. Furthermore, this study shows that it is likely that if the dogs on this study had been fed to the NRC (2006) equation for growth, they would have been offered food in amounts providing excessive energy intake. This could have led to faster growth, likely at least partly due to increased fat deposition and resulted in possible harm to skeletal development and obesity [26,71,72,73]. The combined techniques of feeding to an optimal body condition score and using a WALTHAM™ Puppy Growth Chart to monitor weight development appeared to prevent or address any periods of rapid/slow growth before they become detrimental to the animal and thereby ensured healthy growth.5. ConclusionsThe data further support previous studies on energy requirements during growth in dogs and indicates the unsuitability of the NRC (2006) equation for the calculation of puppy energy requirements for Norfolk terriers. A re-evaluation of the NRC (2006) equation is required and this study supports the need for breed specific feeding guides for growth. Pet owners should be provided with the correct feeding guidelines for their pet to ensure adequate nutrition for a healthy life. The WALTHAM Puppy Growth Charts are an additional tool that could support pet owners and enable them to see their puppies’ growth trajectory in real time, again guiding pet owners towards providing healthy growth for their pet.

animals : an open access journal from mdpi

10.3390/ani11123477

PMC8698158

Migratory bats species are among the most heavily impacted by the erection of wind energy facilities, with many individuals killed at wind turbines each year. Bat carcasses may be collected and used for a variety of biological studies. In this paper, we review the use of intrinsic markers—chemical signatures in bat tissues that can provide information about that animal’s life history—to study bat movements across the landscape. In doing so, we aim to provide our audience with a better understanding of the currently available literature and, more importantly, the areas of this field that need expansion. We emphasize the applications of intrinsic markers that have not been used extensively to study migratory bat species (i.e., trace elements, contaminants, strontium isotopes), and provide a workflow for researchers interested in conducting studies of this type.

Mortality of migratory bat species at wind energy facilities is a well-documented phenomenon, and mitigation and management are partially constrained by the current limited knowledge of bat migratory movements. Analyses of biochemical signatures in bat tissues (“intrinsic markers”) can provide information about the migratory origins of individual bats. Many tissue samples for intrinsic marker analysis may be collected from living and dead bats, including carcasses collected at wind energy facilities. In this paper, we review the full suite of available intrinsic marker analysis techniques that may be used to study bat migration, with the goal of summarizing the current literature and highlighting knowledge gaps and opportunities. We discuss applications of the stable isotopes of hydrogen, oxygen, nitrogen, carbon, sulfur; radiogenic strontium isotopes; trace elements and contaminants; and the combination of these markers with each other and with other extrinsic markers. We further discuss the tissue types that may be analyzed for each and provide a synthesis of the generalized workflow required to link bats to origins using intrinsic markers. While stable hydrogen isotope techniques have clearly been the leading approach to infer migratory bat movement patterns across the landscape, here we emphasize a variety of lesser used intrinsic markers (i.e., strontium, trace elements, contaminants) that may address new study areas or answer novel research questions.

1. IntroductionGlobally, many bat species move substantially across the landscape [1], although the dominant patterns in these movements are understudied. Many species engage in swarming and hibernation behaviors at sites distinct from their summer roosts [1,2,3,4,5,6,7], and the associated movements among these sites are frequently at a regional scale, with some long-distance movements consistently reported (if apparently rare) [3,8,9,10,11]. Other species have more frequently been reported migrating long distances (>1000 km); these movements may be to and from hibernation sites [12,13,14,15] or following ephemeral food resources [16,17]. At least some members of several North American species migrate substantially across latitudes, e.g., [18,19,20], likely to the more temperate overwintering locations and perhaps to forgo hibernation, but see [21]. There is substantial evidence for partial or differential migratory systems within bat species [22], with variation among populations, sexes, and individuals of various species. Protection of significant habitat is a key component in wildlife conservation. The paucity of knowledge about bat movements makes identification of significant habitat for these increasingly imperiled species challenging. The difficulties in tracking migratory movements of individual bats are well-documented. The small size and nocturnal nature of insectivorous bat species present challenges associated with capturing and re-capturing individuals, making the effective use of most extrinsic marking techniques problematic e.g., [23,24]. More recent technological innovations such as small-size satellite tags [21] and radio telemetry arrays [8,25,26,27] have increased the potential for tracking bat migration but have so far been used rarely. Further, such techniques still require a capture and release event, which is resource intensive.A suite of techniques for tracking animal movements based on intrinsic markers (“biomarkers”) also exist and have been applied to investigations of bat movement systems. The guiding principle for most of these techniques is that bats incorporate various naturally occurring chemical signatures into their tissues, and these signatures are representative of the landscape where the tissue was formed. Examples of intrinsic markers include the stable isotope composition of “light” isotopes of elements such as hydrogen, oxygen, carbon, nitrogen, and sulfur; “heavy” isotopes of elements such as strontium and lead; and the relative compositions of trace elements and contaminants. Given a comprehensive understanding of chemical variation across the landscape, such signatures can be used to make origin assignment estimates of migratory individuals. One benefit of intrinsic marking techniques is that they can be used on tissues from pre-deceased animals, such as those in museum collections [20,28,29,30] or those killed at wind energy facilities [19,31,32]. The bats most frequently killed by wind turbines are those species typically considered long distance or latitudinal migrants [32,33,34]. Mortality rates of Lasiurus cinereus over the past two decades are likely leading to unsustainable population declines in this species [35,36]. The substantial number of bats collected under wind turbines each year provide a valuable source of tissue samples that may contribute to, among other uses, intrinsic marker analyses. These can aid in further elucidation of the origins and associated migratory patterns of highly mobile bat species.Intrinsic markers provide an indirect source of data about animal origin, and thus require researchers to make a series of assumptions and interpolations e.g., [37]. For example, researchers must consider the natural variation of chemical markers in the environment, the mechanisms through which the markers are incorporated into animal tissues, the resulting concentration of the marker of interest, and the timing of tissue formation and turnover. Further, there is often an offset between the value (“signature”) of the intrinsic marker in the tissue and that in the local environment (requiring a transfer/rescaling function). Limitations in these steps and analytical processing have restricted the use of the full suite of intrinsic markers for investigations of bat biology. There is a substantial body of literature detailing the use of stable hydrogen isotope analyses of fur to investigate bat migration, although there is still much to be achieved in this area. Studies using other types of intrinsic markers are less frequent in the field of bat migration science, but see [38,39,40,41], as are studies that use samples from non-fur tissues, but see [41,42,43]. Further development in this field will improve our ability to learn about the biology of individual migratory bats. Combinations of different intrinsic marker analyses on tissues samples from the same bat can narrow origin estimates [40] and analyses of various tissues can provide information about different times in an individual bat’s life, e.g., [41,42,43]. The over-arching goal of this paper is to review the full suite of available intrinsic marker analyses, and the tissue types that may be analyzed for each, within the framework of investigating bat movement systems. Our objectives are threefold. Firstly, (i) we will describe the types of intrinsic markers that may be used to learn about migrant origin and summarize the body of literature that has used these markers to study bat migration, highlighting areas for future research; (ii) we will further list the tissues that may be used for various intrinsic marker analyses and describe bat-specific considerations for each; and finally, (iii) we will provide a synthesis of the generalized workflow required to use intrinsic markers for linking bats to origins with an emphasis on identifying research (and knowledge gaps) that explicitly address that workflow. Because there is already a significant body of literature dedicated to reviewing the use of stable carbon, nitrogen, and hydrogen isotopes for studying bat migration [44], we provide a more cursory treatment of these and a more detailed focus on the lesser used markers.2. Intrinsic Markers in Studies of Bat Migration2.1. Using the Stable Isotopes of Hydrogen and Oxygen to Study Bat MigrationPredictable, continental-scale variation in the stable hydrogen and oxygen isotope compositions of precipitation make these markers particularly well suited for investigations of long-distance migration systems. Stable hydrogen isotope techniques are widely used to study the migratory systems of extant animals, usually through analyses of keratinous (feathers and fur) or chitinous (insect) tissues, and several reviews deal with this topic, e.g., [37,45,46]. Stable oxygen isotope techniques can also be informative in movement studies but are most frequently used for this purpose in modern or paleoecological studies using analyses of calciferous tissues, e.g., [47,48]. The heavy isotopes of both hydrogen and oxygen preferentially condense through Rayleigh distillation [49] when precipitation forms from meteoric water vapor. Therefore, the stable hydrogen and oxygen isotope compositions of precipitation vary accordingly with continental climatic patterns, including with latitude in many parts of the world, with season and temperature, across elevation, with distance from the coast, and with relative humidity [50]. Sources of hydrogen in animal tissues include diet and environmental water. The stable hydrogen isotope composition of tissues (δ2Htissue) is governed by complex physiological processes including both catabolic processes and evaporative water loss [51,52]. There is some evidence for a trophic effect on the δ2H values of animal proteins, e.g., [53], but the role of δ2H values as a trophic marker is still under investigation [51]. There is substantial evidence for variation in the δ2H values of organisms with aquatic- and terrestrial-based diets, e.g., [54]. Different bat species may route water from different sources (i.e., insectivorous bats may source more hydrogen from environmental water compared to frugivorous bats which may source water from their diet) [55]. In addition to food and water sources, δ18Otissue has the third influence of inhaled O2. The proportion of δ18Otissue derived from inhaled O2 depends on the volume of drinking water consumed [56], with implications for the relationship between δ18Oprecip and δ18Otissue. Additionally, fractionation of the stable isotopes of oxygen among trophic levels is complicated by the many sources (i.e., drinking water, diet, oxygen in breath), and terminuses of oxygen (i.e., exhaled breath, urine, feces) in a single individual [57]. Stable hydrogen isotope composition is the most frequently used intrinsic marker to study bat migration and most studies have focused on North American and European migratory systems, but see [30]. The continental scale variation of δ2Hprecip values is most suited for research questions investigating largescale movements across latitudes. Some research on bird migratory systems have successfully used stable hydrogen isotope techniques to detect smaller scale movements of organisms across elevations, e.g., [58,59], but attempts to do this with bats have so far had limited success [40,60,61]. Researchers have used stable hydrogen isotope techniques to estimate the origins of individual bats captured or collected at important features such as wind energy facilities, e.g., [31,34], or hibernacula, e.g., [62,63]. Others seek to identify continental scale patterns in migratory movements by sampling bats across locations, often using museum specimens, e.g., [28,29,30]. Research in both the U.S. and Germany has shown that bat fatalities at wind energy facilities include both local and migratory bats in varying proportions [19,31,32]. Research at hibernacula and swarming sites has identified sites with greater and lesser catchment areas [62,64] and has revealed changes over time in bat migratory habits [63]. There is clear evidence that many bat species have partial and differential migratory patterns, with variation among sexes [19,20,29,63], age groups, e.g., [65,66], and among bats with varying anatomy [63]. The transfer function linking the stable hydrogen isotope composition of bat fur to that of local precipitation has also been developed for many individual species (Appendix A) [67], as well as generically using data from sedentary species [30,40]. The use of species-specific transfer functions is ideal, as interspecific variation in these functions may impact origin assignment [67]. While there are strong transfer functions for many species, there is still substantial variation in the δ2Hfur values of bats within and among species at common locations [68,69]. Mean δ2Hfur values may vary significantly among proximate roosts [55,69] and reproductive females may have fur that is depleted of 2H compared to juveniles [66,69]. Variation among species can be even more significant. Voigt et al. [55] reported 65‰ variation among neotropical bats of 36 species and much of this variation may be caused by a trophic effect of discrimination [55,60,70], as well as substantial differences in the δ2Hfur values of bats consuming aquatic and terrestrial prey [54]. Understanding the sources of variation in the δ2Hfur values of resident bats can improve both the accuracy and precision of origin estimates, e.g., [66,67]. Stable hydrogen isotope analyses of organic tissues are limited by the presence of a proportion of hydrogen that is exchangeable with atmospheric water vapor [71,72]. All samples must be analyzed alongside matrix-matched standards with known non-exchangeable δ2H values. Samples and standards must be treated identically throughout preparation and analysis, including an equilibration process. Currently, internationally recognized keratin samples exist [73], and standards for other tissue types must be developed in house. As a result, most studies use keratinous tissues; usually fur, but occasionally claws [70]. Recent work on monarch butterflies has used the stable hydrogen isotope composition of lipids (which have no exchangeable hydrogen) to investigate animal origin [74], and there is further much potential for the compound-specific analyses of the stable hydrogen isotope composition of fatty acids [75]. This is surely an area for future development in bat research. Stable oxygen isotope techniques have not been widely used to investigate bat migration. In temperate climates, the δ18O signature varies with precipitation type (i.e., snow vs. rain) and thus forms predicable seasonal variation in tissues such as teeth and bones [56]. Stable oxygen isotope techniques are used most extensively in multi-isotope studies investigating movements and the life histories of extant and extinct ungulates, e.g., [47,48,76,77], as well as to explore land use strategies in other modern mammals, e.g., [78]. Although some researchers have sought to use δ18O to study migratory bird movements [79,80,81,82], there is a relatively weak correlation between δ18Oprecip and δ18Ofeathers compared to δ2H [82]. Additionally, there is no international keratin standard for stable oxygen isotopic analysis [82]. For more information on δ18O analytical methods, see Appendix B, Table A1.2.2. Using the Stable Isotopes of Carbon and Nitrogen to Study Bat MigrationStable carbon and nitrogen isotope techniques are used widely in studies of bat biology, most often inferring diet and habitat use, e.g., [83,84,85,86,87]. While many of these studies have examined stable isotope signatures in bat tissues, there is also a significant body of literature that describes stable isotope analyses of contemporary and subfossil bat guano collected beneath roosting colonies, e.g., [88], usually to investigate paleoenvironment [89]. There is further a growing amount of literature investigating the stable carbon isotope signature of bat breath, e.g., [90]. Applications using stable carbon and nitrogen isotope compositions for studying migration systems are limited by the lack of predictable largescale variation in the markers of interest across the landscape. Ecosystem variation in δ13C is largely driven by variation in trophic level [91], as well as in the δ13Ctissue values of plants with different photosynthetic pathways [92]. δ15Ntissue values vary predictably with trophic level [91] and other physiological characteristics of individual organisms [93]. As a result, these two markers are typically most effectively used to infer movement among locations where there is known variation in the prevalence of C3, C4 and Crassulacean Acid Metabolism (CAM) photosynthesizing plants (δ13C), e.g., [94,95] or variation in other environmental factors (e.g., drought) [96]. Both markers are frequently used in combination with other markers in studies of migration [95,97,98]. Fleming et al. [17] conducted the first study investigating bat migratory movements using stable isotope techniques and successfully identified migratory movements of nectarivorous Leptonycteris curasoae based on dietary shifts between C3 and CAM plants as the bats moved across landscape. Segers and Broders [64] used stable carbon and nitrogen isotopes to identify highly variable summer origins of bats captured at swarming sites in Nova Scotia, Canada. Other applications of stable carbon and nitrogen isotope techniques to study bat movement have used them in combination with other stable light isotopes e.g., [40,41,65]. In a multi-isotope study, Voigt et al. [61] found that the stable isotopes of nitrogen and carbon were particularly useful in tracking seasonal elevational movements of Miniopterus natalensis at Mount Kilimanjaro [61]. 2.3. Using the Stable Isotopes of Sulfur to Study Bat MigrationStable sulfur isotopes are among the lesser used stable light isotopes for studying animal ecology and have been used infrequently to study bat movements. Four stable isotopes of sulfur exist, but the most common ratio studied is 34S/32S (or δ34S), reported relative to the international standard, Vienna Canyon Diablo Troilite (VCDT). Sources of sulfur in the environment include (1) the oceans, i.e., oceanic sediments and seawater, (2) soils and lithology (depending on rock type and age), (3) the atmosphere, in the form of dust, pollution, and sea spray, (4) freshwater aquatic environments, and (5) biological materials, i.e., decaying organic matter and fossil fuels [99]. Additionally, agricultural landscapes may influence the local δ34S signature, as sulfur is a common soil amendment in both inorganic and organic fertilizers [78]. δ34S is incorporated into organic tissues via amino acids, most commonly in cysteine and methionine, which both have sulfur in their side chains [99]. The δ34S composition of many tissues has been studied, e.g., [100], and the method of δ34S incorporation is often related to the tissue’s amino acid content. Sulfur incorporation into fur and feathers, specifically, is well understood because keratin is a structural protein and therefore contains relatively large amounts of sulfur (up to 5%) [99]. Additionally, there is minimal fractionation of δ34S between trophic levels [99]. Due to the abundance of sulfur in fur keratin and the resulting small sample needed for analysis (Appendix B, Table A2), δ34S can be easily incorporated into intrinsic marking studies of bat migration. Stable sulfur isotopes are less common than other intrinsic markers in studies of migration because the variation of δ34S across the terrestrial landscape is still largely undescribed, but see [101], and the analytical methodology is not standardized (Appendix B, Table A2), see [57]. Stable sulfur isotopes are most commonly used in combination with 87Sr/86Sr in archaeological studies, e.g., [102,103,104] and δ2H, δ15N, and δ13C in studies of modern migratory vertebrates, e.g., [78,105,106,107]. We know of only two studies that have incorporated δ34S into studies of bat movement ecology and neither specifically used δ34S to investigate migration. Cryan et al. [68] used δ2H, δ15N, δ13C, and δ34S to investigate habitat use and prey selection of two roosting colonies of Eptesicus fuscus. Later, Dechmann et al. [108] used radio telemetry in combination with δ34S, δ13C, and δ15N analysis of fur and feces to investigate differences in diet, foraging behavior, and body condition between sexes of Nyctalus noctula. Studies which have solely used δ34S to map domestic livestock movements (i.e., sheep, cattle) across the landscape have been successful e.g., [109,110], and there is great potential to expand these techniques to investigate bat migration. Due to the distinct and uniform marine δ34S signature (+20.3‰) [99], many studies have used stable sulfur isotopes to differentiate between marine and terrestrial origin, e.g., [105,109,111], and this has clear application to bat migration systems. For example, Cryan et al. [19] provided evidence that some L. cinereus migrate longitudinally between inland and coastal areas. Stable sulfur isotope analyses of fur samples from this species could further investigate this assertion. Similarly, tracking migrations along coastlines via proximity to sea is important for some European bat species that may be impacted by offshore wind energy facility development e.g., [39]. Studies of other taxa also utilize other sources of variation in δ34S across the terrestrial landscape, including lithology, e.g., [106,110], and agricultural fertilizers, e.g., [78]. A significant knowledge gap is the need to describe δ34S variation across landscapes in tissues of sedentary bat species or using known-origin individuals. 2.4. Using Strontium Isotopes to Study Bat MigrationStrontium isotopes (87Sr/86Sr) are radiogenic, meaning they are formed by the decay of a secondary element. The relatively heavy isotope 87Sr is formed when an isotope of rubidium (87Rb) radioactively decays see [112]. Both strontium and rubidium can substitute for calcium and potassium, respectively, in minerals [113]. Therefore, 87Sr/86Sr signatures in the landscape are largely related to (1) the 87Sr/86Sr concentration in the underlying geology, considering the age of rock and 87Rb, 86Sr, and 87Sr concentrations at the time of formation; (2) the 87Sr/86Sr concentration in the soil; (3) 87Sr/86Sr in the atmosphere in the form of dust, pollution, or sea spray; and (4) the 87Sr/86Sr concentration of surface waters [112,113,114]. Biological incorporation of these strontium isotopes occurs through the diet and water consumption of the organism of interest [115,116].Researchers in the fields of palaeoecology and archaeology have extensively used strontium isotopic patterns in the landscape to delineate prehistoric movements of various species, e.g., [48,117] including humans (although that is beyond the scope of this review, see [112,115,118]). These studies commonly analyze calciferous tissues (e.g., bones and teeth), which have relatively high concentrations of Sr, with Sr2+ substituting for Ca2+ in those tissues. Strontium isotope techniques have rarely been used to study migration of modern aerial vertebrates but see [39,119,120], likely because of multiple challenges associated with the technique. The method for strontium incorporation into keratin is not well understood, but see [121], and the relative concentration of strontium in keratin is low, so analysis requires large samples of fur and feathers (Appendix B, Table A3) [122,123]. Also, sample preparation and analyses are time and technique intensive [121,123,124]. Finally, migratory origin analysis using strontium may require the development of a unique bioavailable 87Sr/86Sr isoscape, but see [114]. Therefore, only a handful of studies have used strontium isotopes to track avian migration, with fewer studies in recent years [119,120,125,126]. We know of one study that used strontium isotope techniques to investigate the movements of migratory bats. Kruszynski et al. [39] coupled δ2H and 87Sr/86Sr to infer migratory pathways of Pipistrellus nathusii in Europe. Using δ2H, this study successfully identified movement pathways across Europe, but the combined use of δ2H and 87Sr/86Sr warrants further investigation in the context of bat migration, as there was not agreement between the probable origin maps for these two isotopic systems [39].There is little to no strontium discrimination across trophic levels [127], so strontium isoscapes are not always constructed using the study species, e.g., [128,129,130,131]. However, Kruszynski et al. [39] reported a discrimination factor of 0.0028 ± 0.0002 between bioavailable 87Sr/86Sr and 87Sr/86Sr in the fur of P. nathusii and suggested further analysis of keratin structures in modern mammals to investigate a possible trophic discrimination factor between biologically available 87Sr/86Sr, and the 87Sr/86Sr signature in mammal fur. Therefore, a first step in further applying strontium isotope techniques to bat migratory systems is the generation of strontium isoscapes for the species and area of interest using samples taken from known-origin individuals during their summer residency, e.g., [28], or sedentary bat species occupying a similar niche to the migratory species of interest, e.g., [40]. Future research by bat biologists may focus on regions of the world with extensively developed strontium isoscapes, e.g., Europe and North America, or regions with large variation in bedrock type and age, e.g., Alaska and Spain. Additionally, strontium isoscapes may be particularly useful for recreating migratory pathways or demonstrating natal philopatry using tissues with differing turnover rates (studies involving teeth, bones, and fur are most promising). Migratory studies of modern taxa using strontium isotope techniques frequently do so in combination with one or more stable light isotopes, but see [126,132]. The most common second isotope is δ18O or δ2H, e.g., [39,47,76,120], but δ13C [119], δ15N [133], and/or δ34S may be included. Using multiple isotopes, a number of studies have successfully differentiated between local and non-local mammals within a predetermined area of interest, e.g., [77,134,135]. Fewer studies have sought to identify origin or piece together pathways of migratory taxa, e.g., [39,47,117,136]. Others have used patterns in 87Sr/86Sr to pose questions about behaviors, including natal dispersal and philopatry [137], niche occupancy [138], dietary calcium sources [125], and changes in migratory behavior over time [133]. Many of these applications are highly relevant to bat migratory systems. Specifically, the distinction between local and non-local is a useful one in studies of bats at congregation sites such as hibernacula, swarming sites, or large roosts.The use of strontium isotope techniques includes several important analytical considerations. Due to the relatively low concentration of 87Sr/86Sr in keratin and the lesser understood method for incorporation, the initial method development may be required for analysis of keratinous tissues. There is a good foundation of literature to build on for this work, i.e., [123,124,139]. When live individuals are sampled, thermal ionization mass spectrometry (TIMS) may be the preferred analysis mechanism, as it relies on a smaller sample size than the more traditional multi collector inductively coupled plasma mass spectrometry method (MC-ICP-MS; Appendix B, Table A3). Proper preparation of biological samples is necessary to ensure the 87Sr/86Sr signature recorded after analysis is reflective of the 87Sr/86Sr signature of the tissue of interest during the time of formation. Feather and fur samples may contain exogenous (“superficial”) strontium, which is not incorporated into the internal keratin structure, and should be removed before analysis. Exogenous strontium likely originates from atmospheric or lithospheric strontium (i.e., soil and dust particles) as opposed to dietary strontium (i.e., food and water) [123,124]. In studies of bat migration, the removal of exogenous strontium is particularly important when analyzing unknown-origin fur collected outside of the summer residency period. In these cases, the location where the bat was captured may be distinct from the location where the fur was formed, and there is potential for exogenous strontium to contaminate the endogenous signal, contributing to additional noise in the 87Sr/86Sr signature, as seen in [39]. This extrinsic signature can be problematic but may also provide an opportunity, e.g., [124]. Future studies of bat migration may investigate whether the extrinsic signature could provide valuable land-use information about time periods when fur is not growing (e.g., hibernation) and may help identify the general location of important roost structures or hibernacula.2.5. Using Trace Elements and Contaminants to Study Bat MigrationTrace elements and contaminants are used extensively to study migration in birds but have received little use by bat biologists for the same purpose, but see [38]. Trace elements may be referred to as trace metals, but the terms are not interchangeable; trace elements can refer to both metals and metalloids found at low concentrations (0.1%) in the earth’s crust while trace metals should only refer to rare cations [140]. Both are believed to be naturally incorporated into the biosphere via soil and water, and artificially via pollution [140]. Typically, trace elements present in the landscape via pollution or other anthropogenic activities are referred to as contaminants. However, the term contaminants can also be used to refer to organic pollutants or pesticides (e.g., organochlorides, dichlorodiphenyltrichloroethane, dichlorodiphenyldichloroethylene, polybrominated diphenyl ethers). Some studies were successful in mapping the distribution of contaminants across the landscape, e.g., [141]. Other studies have paired contaminant analyses with stable isotopes to address questions of contaminant exposure in avian systems (e.g., δ34S, δ13C, δ15N, δ2H, and Hg in Phalacrocorax auratus [142]). Contaminants have most commonly been used to track animal dispersal or migration by studying movements to and from highly contaminated areas, such as environmental contamination sites (e.g., heavy metal contaminant exposure at the Savannah River Site [143]), the Arctic (i.e., persistent organic pollutant bioaccumulation via atmospheric transport and deposition), e.g., [144,145,146], and some parts of Asia (e.g., polychlorinated biphenyl (PCB) exposure in southern Asia [147]). This is achieved by linking contaminant bioaccumulation to specific areas, e.g., [144,145,147], and pairing contaminant analyses with stable isotope techniques, e.g., [142]. While contaminants have been more thoroughly explored by bat biologists than trace elements [148], they have only been investigated under the lens of toxicity and contaminant exposure (likely via diet and drinking water), e.g., [149,150,151,152,153,154,155]. To our knowledge, contaminants have not yet been used to study migratory bat behavior or assign probable origin. Existing studies that demonstrate bioaccumulation of contaminants in bat tissues at sites near point sources of contaminants (e.g., chemical plants [151], urban centers [152], mines [155]) provide a framework that could be used in the future to track bat movements to and from these sites (e.g., questions of fidelity to maternity colonies or hibernacula). Of the potential contaminants, atmospheric mercury (Hg) is a promising place to start for studies of bat migration; it is correlated with mercury in the fur of some bat species (i.e., Myotis lucifugus, M. septentrionalis, E. fuscus), and its distribution can be mapped across the landscape [141], but see [38]. Trace element concentrations do not reliably vary at the landscape scale (i.e., with latitude, longitude, elevation), but see [38], making large-scale origin assignments impractical. Nonetheless, there can be substantial variation in trace element concentrations at relatively small scales. The development of more efficient extraction and analytical techniques in recent decades has allowed researchers to quantify the concentrations of many trace elements in small samples (Appendix B, Table A4; e.g., Donovan et al. [156] measured 62 trace elements in each 2 mg feather sample using ICP-MS techniques). The results of this approach can subsequently be narrowed down into “predictor elements”, or the elements that show enough variation to discriminate among the different groups of samples (usually achieved using a principal component or discriminant analysis). The predictor elements often depend on the study area, but magnesium (Mg) is often a common predictor [38,156,157,158,159,160,161,162,163]. Previous research using this technique to track bird migrations has shown that trace element profiles can differentiate among sites that are less than 4 km apart [157,159]. While these studies are unable to identify migratory origin across a large geographic landscape, they can pinpoint previously identified habitat, or assign origin across small landscapes, e.g., [157,159,162,163]. Bat-specific applications of trace element analyses may include making assignments on a regional or local scale, for example, when differentiating among breeding colonies [158]. In a recent and innovative development, Wieringa et al. [38] created a distribution map using 14 trace elements in soils across eastern North America, a much larger area than has previously been used to study migratory movements. They sampled fur from museum specimens of Lasiurus borealis to create a base map of the distribution of trace elements across the landscape and to assign known-origin bats to locations of origin based on the trace element profiles in their fur [38]. The study showed ~80% accuracy in the training dataset with 50% precision [38]. Wieringa et al. [38] emphasized accuracy over precision, and the results were less precise and accurate when compared to studies using stable light isotopes (especially when compared with δ2H [164]). Future research by bat biologists could expand on the methods established by Wieringa et al. [38] to map trace element distribution across the landscape, as well as improve the accuracy and precision of origin assignments using this method. Additionally, researchers could expand the use of these methods to migratory systems in regions outside of North America, bearing in mind that it is best practice to characterize the abiotic (e.g., soil) trace element distribution across the landscape before expanding to biotic systems (e.g., bats). Researchers should also consider pairing this technique with more broadly understood intrinsic marking techniques (e.g., δ2H; see Section 2.6).2.6. Using Paired Techniques to Study Bat MigrationAlthough intrinsic marking techniques have many benefits, their biggest drawback is the low resolution at which origin assignments can be made. For species that commonly migrate large distances, and for questions addressing minimum distance traveled, assignment resolution may not hinder the research objectives, e.g., [20,28]. However, for species moving regionally, e.g., [64,68], across longitudes, e.g., [120], or in habitats with high homogeneity of the marker of interest, e.g., [30], the degree of specificity in origin assignments may contribute to the success of the study. Additionally, as studies of bat migration become more commonplace, complex questions (e.g., those addressing both migratory movements and dietary needs) may also become more common, e.g., [90,108]. In these instances, using multiple intrinsic marking techniques or paired intrinsic and extrinsic marking techniques may be the most appropriate approach.The use of multiple isotopes to identify migratory origin can improve both accuracy and precision of assignment. Popa-Lisseanu et al. [40] used three stable light isotopes (δ2H, δ13C, and δ15N) to identify probable origin locations of migratory bats in Europe and found the accuracy of assignments increased from 47.4% (using only δ2H), to 86–89.5% (using δ2H/δ15N and δ2H/δ13C, respectively), to 93% (using all three) [40]. Bataille et al. [165] used 87Sr/86Sr, δ34S, and δ18O to assign probable origin of canine teeth from archaeological remains in Brittany, France. The researchers showed increasingly precise assignments with two and three isotopes when compared with 87Sr/86Sr, δ34S, or δ18O alone [165]. While these studies incorporated paired stable and radiogenic isotope techniques, there are many more intrinsic marking techniques to consider. Migratory bird studies have incorporated trace elements, e.g., [166,167], contaminants, e.g., [142,168], genetics, e.g., [31,95], song dialects, e.g., [169], phenotypic characteristics (“biometrics”), e.g., [158], flight direction, e.g., [170], and a priori knowledge about the study species, e.g., [171] to investigate migratory pathways or seasonal and/or natal origin, in addition to population structure and habitat use. While some of these (i.e., genetics) are beyond the scope of this review, they are helpful tools to consider for certain research objectives. Although most multi-isotope studies of bat migration use some combination of δ2H, δ13C, and/or δ15N e.g., [30,40,61,65], recently, researchers have begun to incorporate δ34S, 87Sr/86Sr, and trace elements [38,39,68] into studies of bat migration. Additional information incorporated by bat biologists via a priori knowledge has included species range [63]; niche occupancy [41]; density based on museum records [19]; preferred elevation [63]; previous records of dispersal distance [172]; and previous migratory flight bearings [32]. Dietary preference is likely also important; for example, the stable hydrogen isotope composition of aquatic insects are distinct from sympatric terrestrial insects [41,172]. Ultimately, the most appropriate pairings of intrinsic markers will depend on the biology and behavior of the study species, the heterogeneity of the landscape, and the research question. It is clear that a deep knowledge of study species biology can improve the accuracy and precision of origin assignments based on intrinsic markers.Recent advances in Passive Integrated Transponder (PIT) tag and radio/satellite transmitter technology have reinvigorated the use of extrinsic markers to study bat migration, e.g., [173,174]. These advances have expanded the available techniques that may be paired with intrinsic markers to study migratory behavior. Many studies have paired extrinsic and intrinsic marking techniques to study migratory birds, and this is an approach that holds much promise in bat biology. The most common combination of intrinsic and extrinsic markers is stable light isotopes and band recovery data, e.g., [175,176,177], but see [63]. By combining these techniques, researchers can infer both migratory pathway and origin, while also decreasing the bias associated with using band recovery data alone [178]. Despite the promise of this combination, the previously documented low recapture rates for marked bats and decreased survivability associated with banding of certain bat species may hinder the widespread feasibility of these coupled techniques for bat biologists [9,24,179]. Recently, however, researchers have developed passive detection mechanisms for PIT tags that result in increased recapture rates [180] without implicating flight behavior of otherwise affected species [181]. Another important and newly developed mechanism for inferring migratory direction is the circular release box for bats (CRBox), which allows inferences to be made about orientation behavior and flight direction [182,183]. Additionally, the Motus Wildlife Tracking System (“MOTUS”) was established on a continent-wide scale in 2014 to passively track radio-tagged aerial organisms (i.e., birds, bats, insects) via remote receiver stations [25]. Since its introduction, several species of bats have been tracked using concepts employed by the Motus network e.g., [26,27,184]. Future directions for biologists interested in using multiple techniques to study bat migration are vast. In studies of swarming and hibernating species, researchers could use a combination of intrinsic markers and PIT tag readers to address both long term (since summer fur replacement) and short-term (among swarming site) movements of bat species. The combination of intrinsic marker techniques and the MOTUS network or CRBox during fall movements of long-distance migratory bat species may allow researchers to both identify migratory origin and track future migratory distance and direction. Finally, the combination of flight direction with isotopic analyses could improve origin assignment precision, especially if combined with extrinsic techniques (i.e., radio/satellite tracking, PIT tags) and/or MOTUS. 3. Tissue Selection for Intrinsic Marker Analysis in BatsIntrinsic marker analyses may be conducted on various tissue types, and the selection of the most suitable tissue(s) is a critical step in any study [37]. Important factors include whether the tissue type is metabolically active or inert; the period in the animal’s life that is reflected by the chosen tissue (related to tissue turnover rate and the timing of growth); the quantity of the marker of interest within the tissue (depending on the sample mass required for analysis); and the invasiveness of sampling different tissue types [41]. In bats, some common tissues can be sampled non-lethally for intrinsic marker analysis (e.g., wing membrane [185], blood [186], fur [43] and claw [70]) while the processes for sampling others are highly invasive or lethal (e.g., liver, muscle, and bone collagen) [17,84,187].Most tissues are either metabolically inert or active. Metabolically active tissues continually regenerate and thus have a chemical composition that is continuously changing. Examples include the blood, muscles, liver, and wing membrane [187,188,189]. Comparatively, metabolically inert tissues are fixed after formation and are reflective of the conditions during that development period (e.g., fur) [187,188,189]. Breath, while not a tissue, is frequently sampled for intrinsic marker analysis and shares salient characteristics with tissues, so will also be discussed here [42,190,191]. Figure 1 presents a graphical summary of the tissue types that may be used for intrinsic marker investigations of bat ecology.3.1. Metabolically Inert Tissues in BatsIn studies using intrinsic markers to investigate bat migration, fur is the dominant tissue type used. Bat fur is usually assumed to be replaced annually through molting. The typical molting pattern in temperate bats is an annual molt during the summer–fall before migration, but factors such as sex, age or migratory behavior may contribute to some bats molting outside of the usual timeframe [28,192] For example, molting may be postponed until after lactation or disrupted during pregnancy, reproduction, or other energy demanding processes and will vary with age and sex [28,192]. Typically, fur samples are taken from the upper dorsal region between the shoulder blades [43,188]; growth may be asynchronous between ventral and dorsal surfaces [28]. Understanding the molting cycles helps to further the accuracy and predictability of fur isotopic composition and to gain a deeper understanding of bat behavior.Other metabolically inert tissues are frequently used in studies of other taxa, but have received little attention in modern bats, include teeth and bone collagen [43,188]. Bone collagen is developed early in life [189] and is metabolically active initially but with age the turnover rate slows to a negligible rate. The teeth consist of: (1) enamel, a hard outer layer, (2) primary dentine, an inner layer beneath enamel, both formed during infancy above the gum line; (3) secondary dentine, which continually forms new layers, and (4) cementum, an outer layer, both continuously formed at the root of the tooth below the gum line [193]. Many bats have deciduous teeth which they shed at variable frequencies during their infancy and are replaced with their adult teeth [194]. Both tooth and bone collagen could provide information about individual bats when they were juveniles or subadults because these tissues are active during their growth and inert when formed.Inert tissues that grow continuously over longer time periods present an opportunity for time series analyses. For example, in some mammals, individual hair strands can be sampled at varying locations along their length to gain information about the animal at various points in its lifetime [195,196,197,198]. We do not know of any bats that have continuously growing fur, but the hind claws of bats may potentially be sampled at varying lengths to achieve the same goal. There is little information about the growth patterns and timing of bat claws, but Ethier et al. [199] provide a useful summary of patterns in mammalian claw growth. To date, claw tissue has been infrequently used to study bats, likely because of their size and the invasiveness of claw removal, but see [70,200].3.2. Turnover of Metabolically Active Tissues in BatsThe rate at which a chemical marker in a metabolically active tissue is replaced by the same marker from another source is known as a tissue turnover rate. The timing of tissue growth and turnover is critical information, as these factors inform the time period about which markers are providing information. Turnover rates may range from minutes to years [187]. Tissue turnover is often quantified as the half-life (t50) of the marker of interest, i.e., when turnover occurs in half of the markers in the tissue [189]. The turnover rate of an intrinsic chemical marker varies among tissues, and among different markers within the same tissue. This latter variation occurs because of metabolic decoupling, change in diet/nutrients, or variation in nutrient routing (e.g., carbon sourced from protein or carbohydrate) [201]. In most cases, research on the turnover rates of metabolically active tissues has focused on the turnover rates of carbon and nitrogen, because of predictable discrimination factors of ~0.2‰ and 2.2–3.4‰ between trophic levels, respectively [43,87,188], and the turnover rates of other intrinsic markers are a substantial knowledge gap. Diet-switching studies on captive animals provide most of the information on turnover rates [190,201]. Less commonly, in wild populations, variation over time in the intrinsic marker composition of metabolically active tissues can be used to infer turnover rate [42,90]. There have been several studies that have explicitly investigated tissue turnover rates in bats [43,188,191,201]. In our summary below, we will report bat-specific findings where possible, and findings from other taxa when not. A more complete summary is included in Table 1. The turnover rate of carbon in CO2 in breath is widely studied because breath samples can provide information on very recent (minutes to hours) dietary patterns, with t50 turnover rates of 27.3 ± 6.4 min in Noctilio albiventris [191], 18.6 min in Desmodus rotundus [210], and 10.9 ± 7.5 min in Carollia perspicillata [190]. Diet switching experiments show that the variance occurring in breath turnover rates is likely due to the different ratios of proteins and sugars consumed [190]. We are unaware of reported liver turnover rates in bats, but Tieszen et al. [187] recorded the t50 of carbon in Meriones unguiculatus (referred to as M. unguienlatus) liver to be 6.4 days. Depending on which components are used, the turnover rate of carbon and nitrogen in blood varies. Reported values include 24 to 39 days for Glossophaga soricina (whole blood cells) [201], 120 to 126 days for G. soricina and L. curasoae (whole blood cells) [43], 2.9 days (plasma), and 29.8 days (cellular) for Corvus brachyrhynchos [211]. The t50 of carbon in M. unguiculatus muscle tissue is 27.6 days [187].Wing membrane is a tissue that is unique to bats, and one that is commonly sampled in a relatively minimally invasive way using a biopsy punch. Following sampling, reports of wing membrane regeneration include 3 to 4 weeks in L. curasoae and G. soricina [43] and 27.3 ± 12.2 days (wing membrane) or 18.3 ± 4.3 days (tail membrane) in E. fuscus [185]. Voigt et al. [43] suggest that biochemical processes help the wing tissue regenerate following injury, resulting in regrowth faster than the actual turnover rate of the tissue. They report a t50 of carbon in wing membrane to be between 102–134 days and suggest that the low turnover rate of wing membrane could be due to high concentrations of bone collagen found within the wing membrane. Roswag et al. [188] observed the wing membrane turnover rate of N. noctula to be 7 weeks. Although most data on wing membrane turnover rates come from laboratory studies, Frick et al. [42] documented seasonal (winter to spring) turnover rates in the wing membrane of Antrozous pallidus.Turnover rates of specific intrinsic markers in the same tissue type may vary substantially, likely related to variation in metabolic rate associated with diet change or changes in energetic requirements (e.g., during migration) [211,212]. Bats eating a diet with a lower C:N ratio had a slower carbon turnover rate in blood than those consuming a diet with high C:N, while the nitrogen turnover rate remained similar [201]. During periods with high energy requirements, bats may increase both food consumption and metabolic rate with corresponding shifts in nutrient routing [42,90,203,209]. The seasonal availability of certain foods may cause changes in nutrient routing, with some intrinsic markers being immediately metabolized, while others are incorporated into new tissues [42,43,190].The effects of torpor on the incorporation of intrinsic markers into tissues, and the tissues’ turnover rates, are unknown. However, frequent use of torpor by bats undoubtedly plays an important role in tissue turnover rates. Torpor alters the metabolic rate of bats allowing for the conservation of energy, especially during cold periods or periods when endogenous energy stores are low [18]. Because metabolic activity has a direct relationship with tissue turnover rate [211], the tissues of torpid bats would be expected to turnover more slowly than non-torpid bats. Males and non-reproductive females enter torpor more frequently than reproductive females [18] potentially leading to intraspecific variation in tissue turnover rates.3.3. Discrimination FactorsVariation in diet may also result in variation in diet-tissue discrimination, which has been best illustrated using stable isotopes of carbon and nitrogen but is likely relevant for other markers. The carbohydrates within plant-based foods are typically metabolized quickly and the resulting CO2 is exhaled, while the small amount of protein within the plant is used in tissue catabolism [190]. In omnivorous bats, carbon in the wing membrane mainly originates from protein in the insect portion of the diet while the carbon in breath originates from carbohydrates in fruit [42,190]. As a result of their high protein diet, insectivorous bats often have higher δ13Ctissue than nectarivorous or frugivorous bats, but similar δ13Cbreath. Therefore, there is a direct relationship between the trophic level and the difference between breath δ13C and tissue δ13C [190]. Turnover rates of δ15N can vary with dietary source, as protein can be sourced both externally, via diet, and internally through the nitrogen cycle [203,213]. Internal nitrogen is enriched because it has been previously metabolized [203]. The nitrogen cycle has several reservoirs of nitrogen, and other biological processes, such as pancreas secretion, that can contribute to endogenous nitrogen sources [213]. 3.4. Approaches to Tissue SamplingThe mass of each sample needed for isotopic analysis is a critical consideration because the small size of most bats limits the quantity of tissues that can be sampled non-lethally. The key considerations are the amount of the marker of interest in the tissue; the sensitivity of the laboratory equipment to detect the marker of interest; and the quantity of tissue that may be taken from an individual bat. Table 1 and Appendix B summarize sample masses that have been used for various intrinsic marker analyses of a range of tissues. A small amount of literature exists discussing specific practices for sampling blood, wing membrane, and fur from bats. The sampling of blood has been particularly evaluated; Baer and McLean [214] originally suggested the removal of 0.1–0.2 mL of blood from the jugular vein of small bats (in this case Tadarida brasiliensis), although more recent studies have suggested an order of magnitude smaller. Wimsatt et al. [215] sampled 58 ± 12 µL from the interfemoral vein in E. fuscus under anesthesia without impacting survivability. Smith et al. [216] sampled blood from the brachial and propatagial veins in eight species of microbats and suggested 6 µL/g of body mass. This study was quickly refuted by Racey et al. [217] who suggested sampling from the interfemoral vein to avoid impacting flight. Eshar and Weinberg [186] suggested the removal of blood ≤ 1% of total body weight from either the interfemoral or cephalic vein (providing detailed instructions for sampling blood in bats, using Rousettus aegyptiacus as an example). The sampling of wing membrane has been evaluated to a lesser extent; both Faure et al. [185] and Pollock et al. [218] studied propatagium sampling techniques in E. fuscus and suggest sampling tail membrane tissue over wing membrane tissue; the increased vasculature in tail membrane causes wounds in the tail to heal significantly faster compared to the wing. Finally, Fraser et al. [192] details considerations when sampling fur from various bat species, accounting for differences in molting patterns and timelines.4. Overview of WorkflowWhile some intrinsic markers (e.g., stable hydrogen isotopes) have been used extensively to study bat migration, others are in their infancy for this purpose. Because intrinsic marker analyses of tissues provide indirect evidence of bat movement, the use of any markers for migration research requires significant background knowledge, modelling, and assumptions. Vander Zanden et al. [37] presented a generalized workflow for designing a study to track animal movement using stable isotope analyses of tissue samples. In Figure 2, we present a modified version of this workflow that can be applied to any of the intrinsic markers discussed in the present paper. We provide important questions for consideration at each stage and a summary of existing bat-specific literature (where appropriate) that has explicitly addressed the methodological considerations associated with each step. This summary highlights the volume of work that has been conducted in this area, as well as the knowledge gaps. Aligned with the greater volume of work that has used stable hydrogen isotope techniques to study bat migration, there has been significant attention to the methods associated with this technique. Bat-specific rescaling functions and associated isoscapes for markers that have been used less frequently (e.g., strontium, sulfur) are less prevalent or entirely absent, but see [38,39], and intrinsic and extrinsic marker techniques have not been combined as frequently as in avian research. The majority of work has focused on analyses of fur, but there is great potential to analyze multiple tissues to learn about different time periods in an individual bat’s life. e.g., [42,43]. Conducting this work well requires further investigations of tissue growth and turnover rates, as well as laboratory work to modify and develop analytical techniques (e.g., stable hydrogen isotope analyses in non-keratinous tissues). 5. ConclusionsThere is close to a thirty-year history of using intrinsic markers to study bat migration [17] and in the past fifteen years, applications have particularly proliferated. The ability to make origin estimates of individual migratory bats has furthered our understanding of migratory patterns, as well as the migratory ecology of these elusive animals. Stable hydrogen isotope techniques have been the leading approach, but marker choice is dependent on both the research question and the study area. Recent innovations in analytical techniques have made lesser used intrinsic markers (e.g., trace elements/contaminants, strontium) and the combined analysis of intrinsic markers increasingly accessible and informative, although logistical challenges still exist. There is clearly much important methodological innovation to be achieved in the applications of these lesser used intrinsic markers in making inferences about bat movements, especially if the goal is to estimate probabilities of origin. Combinations of intrinsic marker analyses can be particularly powerful in estimating migratory origin and, even in the absence of clearly defined isoscapes, can allow researchers to address simple but important questions about whether congregating groups of bats consist of individuals from few or many locations, e.g., [64]. As extrinsic marking technologies advance and become more accessible to bat research, there is further potential to combine these with intrinsic marking techniques.

animals : an open access journal from mdpi

10.3390/ani13071212

PMC10093662

Endometrosis is a major cause of infertility in mares and involves the excessive deposition of extracellular matrix in the mare’s endometrium, such as collagen and α smooth muscle actin (α-SMA). Collagen is formed by activated fibroblasts, which are mainly stimulated by transforming growth factor β1 (TGF-β1). Alterations in fibroblast phenotype are linked with epigenetic alterations. Unlike genetic alterations, epigenetic alterations are changes in gene function without DNA nucleotide sequence modification. Epigenetic changes can be reversed and are therefore extremely promising for therapeutic use. DNA methylation analysis is one of the most used methods to detect epigenetic changes. It can be assessed by measuring DNA methylating enzymes (DNMT1, DNMT3A, and DNMT3B). Thus, the aims of this study were to investigate the in vitro epigenetic regulation of mare endometrial fibrogenesis through DNMTs transcription and the effect of the epigenetic inhibitor 5-aza-2′-deoxycytidine (5-aza-dC or decitabine) on collagen expression in mare endometrial fibroblasts challenged with TGF-β1. It was observed that TGF-β1 upregulated DNMT3A, COLs, and α-SMA transcripts and COLs secretion. The increase in DNMT3A and COLs (transcripts and protein) after TGF-β1 stimulation of equine endometrial fibroblasts was reduced after treatment with the demethylating agent 5-aza-Dc, suggesting an epigenetic regulation of mare endometrial fibrosis.

Endometrosis negatively affects endometrial function and fertility in mares, due to excessive deposition of type I (COL1) and type III (COL3) collagens. The pro-fibrotic transforming growth factor (TGF-β1) induces myofibroblast differentiation, characterized by α-smooth muscle actin (α-SMA) expression, and collagen synthesis. In humans, fibrosis has been linked to epigenetic mechanisms. To the best of our knowledge, this has not been described in mare endometrium. Therefore, this study aimed to investigate the in vitro epigenetic regulation in TGF-β1-treated mare endometrial fibroblasts and the use of 5-aza-2′-deoxycytidine (5-aza-dC), an epigenetic modifier, as a putative treatment option for endometrial fibrosis. Methods and Results: The in vitro effects of TGF-β1 and of 5-aza-dC on DNA methyltransferases (DNMT1, DNMT3A, and DNMT3B), COL1A1, COL3A1, and α-SMA transcripts were analyzed in endometrial fibroblasts, and COL1 and COL3 secretion in a co-culture medium. TGF-β1 upregulated DNMT3A transcripts and collagen secretion. In TGF-β1-treated endometrial fibroblasts, DNA methylation inhibitor 5-aza-dC decreased collagen transcripts and secretion, but not α-SMA transcripts. Conclusion: These findings suggest a possible role of epigenetic mechanisms during equine endometrial fibrogenesis. The in vitro effect of 5-aza-dC on collagen reduction in TGF-β1-treated fibroblasts highlights this epigenetic involvement. This may pave the way to different therapeutic approaches for endometrosis.

1. IntroductionEndometrosis is responsible for infertility in mares and is characterized by excessive deposition of collagen (COL) in the endometrium, with collagen type I (COL1) and type III (COL3) being the most abundant. The periglandular deposition of collagen in the endometrium contributes to the formation of fibrotic nests, which may impair glandular flow secretion [1,2]. Indeed, it was recently demonstrated by proteomic analysis of uterine lavage fluid that the secretion of essential proteins is affected in mares with endometrosis, due to endometrial glandular function impairment [3]. Moreover, the decreased number and area of healthy glands results in a deficient nutrient exchange between the placenta and the conceptus, and therefore may hinder its viability [4,5,6]. Therefore, all these endometrial alterations may result in pregnancy failure, delayed placental development, retarded foetal growth, or abortion [2,4,7]. Transforming growth factor β1 (TGF-β1) is over expressed in several fibrotic tissues [8,9,10,11] and induces COL production in cultured fibroblasts, regardless of their origin [10]. It not only regulates cell growth, development, and tissue remodelling, but it also participates in the pathogenesis of tissue fibrosis. In the equine endometrium, the activity of TGF-β1 is correlated with endometrosis [12,13], as it is in human endometriosis [14]. Endometriosis in women is a disease characterized by the presence of endometrial tissue within the serosa of the abdominal or pelvic cavities, which is not the same condition as equine endometrosis [15]. Nevertheless, fibrosis is consistently present in all disease forms of human endometriosis and contributes to the classic endometriosis-related symptoms of pain and infertility [16]. In tissues other than the uterus, TGF-β1 induces differentiation of many cell types into myofibroblasts. These cells are characterized by α-smooth muscle actin (α-SMA) expression and the ability to deposit excessive amounts of extracellular matrix (ECM) components. Increased expression of α-SMA in fibroblasts is therefore widely interpreted as a marker of fibroblast activation [17]. Moreover, aberrant expression of TGF-β1after injury stimulates the expression of α-SMA [18] and ECM [19] in fibroblast-like cells. Fibroblasts are key effector cells in tissue remodelling. They remain persistently activated in fibrotic diseases, resulting in the progressive deposition of ECM. Although fibroblast activation may be initiated by external factors, prolonged activation can induce an “autonomous”, self-maintaining profibrotic phenotype in myofibroblasts [20]. Accumulating evidence suggests that epigenetic alterations play a central role in establishing this persistently activated pathologic phenotype of fibroblasts [20]. Epigenetic changes, unlike genetic alterations, can be reversed and are therefore extremely promising for therapeutic use [21]. DNA methylation, considered a stable epigenetic marker, can be assessed through the action of DNA methyltransferases (DNMTs: DNMT1, DNMT3A, and DNMT3B), and commonly mediates gene repression [22,23]. The most used epigenetic treatments aim to alter either DNA methylation or histone acetylation [24,25]. To date, seven agents have been approved by the USA Food and Drug Administration for the treatment of different diseases, but many more are undergoing clinical trials [26,27,28,29,30]. DNMT inhibition is considered as an efficient approach for the prevention of DNA hypermethylation alterations [31]. The ability of DNMT inhibitors to reverse epimutations is the basis of their use as novel strategies for cancer therapy [32]. These medications act like nucleotide cytosine and incorporate themselves into DNA while it is replicating [33,34,35,36,37,38]. Demethylating agents are currently used to treat myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML). They are also being experimentally used in solid tumours in low dose administration, with very promising results [39,40]. Recent studies have shown an epigenetic involvement in several human fibrotic disorders [41,42,43,44,45]. Moreover, epigenetic involvement in equine endometrial fibrosis has been demonstrated in our previous studies [46,47]. As such, we have hypothesized that endometrial fibroblasts might be under epigenetic regulation.Therefore, our objective was to analyze the effect of TGF-β1 on methylating enzymes and the effect of the licensed demethylating agent 5-aza-2′deoxycytidine (5-aza-dC, 5-aza or decitabine) on TGF-β1-stimulated equine endometrial fibroblasts. For that purpose, we first evaluated DNA methylation through the expression pattern of DNMT1, DNMT3A, and DNMT3B on TGF-β1-stimulated mare fibroblasts to determine if equine endometrial fibroblasts were under epigenetic regulation. Further, we aimed to confirm whether the TGF-β1-induced alterations on COL1 and COL3 expression by equine endometrial fibroblasts could be reversed by the action of 5-aza-dC. Thus, we determined the transcription levels of DNMT enzymes, ECM components, and α-SMA, as well as COL1 and COL3 protein concentration, before and after fibroblasts were treated with TGF-β1, 5-aza-dC, or both.2. Materials and MethodsUteri (n = 5) were obtained post mortem from cyclic mares at a local abattoir (Rawicz, Poland) from April to June, according to the protocols approved by the local institutional committee for animal care and use. The mares were declared clinically healthy by independent official government veterinary inspectors. Immediately before death, peripheral blood samples were collected into heparinized tubes for subsequent progesterone (P4) analysis. The animals were slaughtered for meat, as part of the routine breeding and slaughter of animals, and in agreement with the European mandates (EFSA, AHAW/04-027). The internal genitalia (uteri and ovaries) were retrieved within 5 min of animal death. In this study, uteri from mares in the follicular phase of the estrous cycle were used. The follicular phase was identified based on the macroscopic observation of ovaries and P4 analysis of blood plasma. This phase was characterized by the absence of an active corpus luteum (CL) and the presence of follicles larger than 35 mm in diameter, with a concentration of P4 < 1 ng/mL [48]. Samples of endometria were placed in 4% buffered formaldehyde for histological examination and for endometrial categorization, according to Kenney and Doig [49].2.1. Isolation and Culture of FibroblastsThe fibroblasts isolated from healthy endometria (Kenney and Doig’s category IIA endometria) were isolated according to Szóstek-Mioduchowska et al. [50]. In the laboratory, the uterine lumen was washed three times with 10 mL of sterile Hanks’ balanced salts (HBSS; H1387; Sigma-Aldrich, Saint Louis, MO, USA) containing 0.01% of antibiotic/antimycotic (AA) solution (AA5595; Sigma Aldrich, Saint Louis, MO, USA). The uterine horns were split open with scissors to expose the endometrial surface. Endometrial strips were excised from the myometrium layer with a scalpel, washed once with sterile HBSS containing 0.01% of AA solution, and sliced with a scalpel into small fragments (1–3 mm). A single digestion of the minced tissues was performed by agitation for 45 min, in 100 mL of sterile HBSS with 0.05% (w/v) collagenase I (C2674, Sigma-Aldrich, Saint Louis, MO, USA), 0.005% (w/v) DNase I (11284932001; Roche-Sigma Aldrich, Saint Louis, MO, USA), 0.01% AA, and 0.1% (w/v) bovine serum albumin (BSA; A9418, Sigma Aldrich, Saint Louis, MO, USA). Afterwards, to extract undigested tissue fragments, filtration of the cell suspension was accomplished with 70 μm and 40 µm filters. The filtrate was mixed gently with 1 mL of Red Blood Cell Lysing Buffer Hybri-Max™ (R7757; Sigma-Aldrich, Saint Louis, MO, USA) to lyse red blood cells. Afterwards, the filtrate was washed three times by centrifugation (4 °C, 100× g, 10 min) in HBSS supplemented with antibiotics and 0.1% (w/v) BSA. The final pellet of endometrial cells was resuspended in FBMTM Basal Medium (CC-3131, LONZA, Basel, Switzerland) supplemented with FGMTM-2 SingleQuotsTM, ascorbic acid (100 ng/mL; A4544; Sigma-Aldrich, Saint Louis, MO, USA) and 0.01% of AA solution. A hemocytometer was used to perform cell count. The trypan blue exclusion test was employed to assess the viability of endometrial cells, which was higher than 95%. The immunofluorescent staining for vimentin was utilized to evaluate the homogeneity of fibroblasts, based on the protocol described [51] (Figure 1). The cells were then independently seeded, at a density of 1 × 105 viable cells/mL and incubated at 38.0 °C in a 5% CO2 atmosphere. The medium was replaced 18 h after plating, to purify fibroblast population. At that time, the selective attachment of fibroblasts had taken place and elimination of other types of endometrial cells was possible. The medium was replaced until the cells achieved confluence, every 48 h. Fibroblast purity after isolation was approximately 96%. After reaching 90% of confluency, the cells were cryopreserved, as described previously [52].2.2. Preliminary StudiesTo determine the most adequate protocol to be used in our study, preliminary studies were performed. The dose of 10 ng/mL of TGF-β1 was chosen based on other studies, as the treatment has previously shown to maximally activate myofibroblasts [13,53,54]. Transforming growth factor β1 treatment of 48 h was initially chosen, based on other studies in which a maximum increase in collagen expression was achieved at that specific incubation time [13,20,54]. However, based on the analysis of preliminary studies’ results, treatment with TGF-β1 for 96 h was preferred.To establish the appropriate dose of 5-aza-dC, a preliminary study was performed, in quadruplicate, with 0 µM, 1 µM, or 5 µM, n = 4. The 1 µM dose achieved the same results in collagen reduction as 5 µM (Supplementary Figure S1). Therefore, the rationale was to choose the lowest concentrations due to the toxic side effects of 5-aza-dC (in clinical use) and based on other studies that achieved the same results [20,32,55]. A preliminary study was also performed to determine the protocol and the duration of 5-aza-dC treatment. The experiments were done separately, but with the same endometrial fibroblasts, in quadruplicate and with n = 5 for both protocols. As such, some endometrial fibroblasts were incubated with vehicle (control), 5-aza-dC (1 µM), TGF-β1 (10 ng/mL), or 1 µM 5-aza-dC + 10 ng/mL TGF-β1 at the same time for 48 h (protocol #1), and others with vehicle (control), 5-aza-dC (1 µM), TGF-β1 (10 ng/mL) for 96 h, or TGF-β1 (10 ng/mL) for 96 h + 5-aza-dC (1 µM) added after 48 h from the beginning of the experiment (protocol #2), with a total cell incubation time of 96 h. With protocol #1, no reduction was observed in COL1 and COL3 mRNA levels and protein expression or α-SMA mRNA levels in fibroblasts incubated with 1 µM 5-aza-dC and 10 ng/mL TGF-β1 simultaneously, for 48 h (Supplementary Figure S2). In contrast, in protocol #2, both mRNA and protein concentration of COLs were reduced. Hence, protocol #2 was chosen and is presented here, since it also mimics the clinical conditions in which it may be used (treatment after fibrosis development and not its prevention). The duration of 5-aza-dC treatment was chosen as the minimum time (48 h) at which positive results were achieved in other studies [54,55].The effect of TGF-β1 and of 5-aza-dC on cell viability was also analysed. After reaching confluence in a 96-well plate, fibroblasts were exposed to TGF-β1 (10 ng/mL) or 5-aza-dC (1 µM or 5 µM) for 48 h. Fibroblast viability was measured using In Vitro Toxicology Assay Kit, MTT based (TOX-1KT, Sigma Aldrich, Madison, WI, USA). None of the doses used showed toxic effects (Supplementary Figure S3).2.3. Treatment of Cultured FibroblastsThawed fibroblasts were seeded at a density of 1 × 105 viable cells/mL on T75 cm2 cell culture flasks. After reaching 90% confluence, fibroblasts were seeded on 24-well plates. When fibroblasts from passage 1 reached the desired 80% confluence for 48 h treatment, the culture medium was replaced with fresh Dulbecco’s Modified Eagle’s Medium/Nutrient Mixture F-12 Ham (DMEM/Ham’s F-12; D2906; Sigma-Aldrich, Saint Louis, MO, USA) supplemented with 0.01% of AA solution, 0.1% (w/v) BSA, and ascorbic acid (100 ng/mL). The cells were incubated at 38.0 °C in a 5% CO2 atmosphere. Then, fibroblasts were treated as follows: (i) vehicle (control); (ii) with 10 ng/mL of TGF-β1; or (iii) with 1 µM of 5-aza-dC, at 37 °C, 5% CO2 for 96 h. Forty eight hours after the beginning of the experiment, 1 µM 5-aza-dC was added to TGF-β1 group for another 48 h, under the same conditions. Since the half-life of 5-aza-dC is very short, the medium was changed every 24 h and fresh 5-aza-dC was added daily for 48 h. Fibroblasts were incubated alone (control), with 1 µM 5-aza-dC or 10 ng/mL TGF-β1, for 48 and 96 h, as controls. The cells and the conditioned media were collected at 48 h and 96 h and stored at −80 °C. The collection of conditioned media, for ECM determination, was performed with 1.5 mL tubes. The disruption of cells, after incubation, was performed with 1 mL of lysis buffer RTL (1015750; Qiagen GmbH, Hilden, Germany) and stored at −80 °C for RNA extraction and PCR.2.4. Total RNA Isolation, cDNA Synthesis and qPCRTotal RNA was extracted using Qiagen RNeasy® mini kit (74104; QIAGEN, GmbH, Hilden, Germany) according to the manufacturer’s information, including a DNase digestion step, and the samples were stored at −80 °C. The concentration of RNA was assessed spectrophotometrically, and its quality by agarose gel electrophoresis. The absorbance ratio at 260 and 280 nm (A260/280) was approximately 2. The QuantiTect Rev. Transcription Kit (no. 205313; QIAGEN, GmbH, Hilden, Germany) was used to perform the reverse transcription of RNA (1 mg) into cDNA, following the manufacturer’s instructions, and stored at −80 °C. The ABI Prism 7900 sequence detection system with 384-well plates with SYBR Green PCR master mix (Applied Bio-systems, Foster City, CA, USA) was used to perform real-time PCR. The amplified genes were α-SMA, COL1A1, COL3A1, DNMT1, DNMT3A, and DNMT3B. Specific primers and the reference gene were designed (Table 1) using the Internet-based program Primer-3 [56] and Primer Premier software (Premier Biosoft Interpairs). SDHA was chosen as the most stable internal control gene, among four validated reference genes, as described [57]. All primers were manufactured by Sigma-Aldrich (Saint Louis, MI, USA). The total reaction volume (10 mL) was composed of 3 mL cDNA (1 ng), 1 mL of forward and 1 mL of reverse primers (500 nM), and 5 mL of SYBR Green PCR master mix. Real-time PCR was performed by initial denaturation (2 min at 50 °C; 10 min at 95 °C), followed by 42 cycles of denaturation (15 s at 95 °C) and annealing (1 min at 60 °C). Then, after each PCR reaction, the melting curves were achieved by gradual increases in temperature from 60 °C to 95 °C to guarantee single-product amplification. Agarose gel (2%) electrophoresis was performed to confirm product specificity. To quantify relative mRNA expression levels, data were analyzed using the equation [1/(1þE)Ct] described by Zhao and Fernald [58], where the average cycle threshold (Ct) of each sample was related to the primer efficiency (E). Transcription of the target gene was normalized to that of the reference gene and relative expression values were calculated. Relative mRNA levels of control samples were compared with treated fibroblasts data.2.5. Collagen Protein QuantificationELISA techniques were performed to quantify COL1 and COL3 concentrations, in conditioned media from cultured cells. The determination of COL1 concentration in conditioned medium was accomplished by Enzyme-Immunosorbent Assay Kit for Collagen Type I (COL1) (SEA571Eq; Cloud-Clone Corp., Katy, TX, USA). The standard curve for COL1 ranged from 3.12 to 200 ng/mL. The average of intra- and inter-assay coefficients of variation (CVs) were 11.5% and 9%, respectively. The determination of COL3 in conditioned media was accomplished by Enzyme-Immunosorbent Assay Kit for Collagen Type III (COL3) (SEA176Eq; Cloud-Clone Corp., Katy, TX, USA). The standard curve for COL3 ranged from 1.56 to 100 ng/mL. The average of intra- and inter-assay CVs were 10% and 9%, respectively. The concentrations of COL1 and COL3 in control samples were compared with treated fibroblasts data, in conditioned medium.2.6. Statistical AnalysisData are shown as the mean ± SEM. For each analysis, the Gaussian distribution of results was tested using the Shapiro and Wilk normality test (GraphPad Software version 9; GraphPad, San Diego, CA, USA). Significance was considered when p < 0.05. A two-way ANOVA was performed to analyse the effect of time (48 and 96 h), treatment (TGFB or 5-aza-dC), and their interaction (time × treatments) on COL1A1, COL3A1, α-SMA, DNMTs genes, and COL1 and COL3 protein expression. One-way analysis of variance (ANOVA) followed by Tukey’s multiple comparison test was used to analyse the effect of different treatments on the expression of the same genes (COL1A1, COL3A1, α-SMA, DNMTs) and COL1 and COL3 proteins at 96 h. The treatment groups were analyzed with respective control. As such, the treated groups 5-aza-dC, TGF-β1, and TGF-β1 + 5-aza-dC were compared to untreated fibroblasts (control C). In addition, TGF-β1 + 5-aza-dC treatment was compared to TGF-β1, as previously used in other studies [59,60].3. Results3.1. TGF-β1 Upregulated Collagen Type I, III and α-SMA Expression in Endometrial FibroblastsThe effects of treatment time, TGF-β1 treatment, and their interaction (time × treatments) were studied. Significant interactions between time and TGF-β1 treatment were observed for COL3A1 (p = 0.0008) and α-SMA (p = 0.0213) mRNA levels and for COL3 protein abundance (p = 0.0035). No interactions were found either for COL1A1 transcripts (p= 0.1078) or COL1 protein concentrations (p = 0.2443). Treatment of endometrial fibroblasts with TGF-β1 increased mRNA levels of COL1A1, COL3A1, and α-SMA at 96 h (p < 0.001, p < 0.0001, and p < 0.01, respectively) (Figure 2A–C). It also increased protein concentration of COL1 at 48 h and 96 h (p < 0.01 and p < 0.0001, respectively) (Figure 2D) and of COL3 only at 96 h (p < 0.0001) (Figure 2E). There was a rise in COL3A1, α-SMA mRNA levels, and COL3 protein concentration between 48 h and 96 h (p < 0.001, p < 0.01, and p < 0.01, respectively) (Figure 2B–E). The same was not observed in COL1A1 mRNA levels or COL1 protein concentrations (Figure 2A,D).3.2. TGF-β1 Upregulated DNMT3A Expression in Endometrial FibroblastsWe examined DNMT1, DNMT3A and DNMT3B gene mRNA levels in TGF-β1-treated endometrial fibroblasts to determine whether DNMTs regulate the collagen expression through DNA methylation, at 48 h and 96 h. TGF-β1 upregulated DNMT3A at 48 h and 96 h (p < 0.05 and p < 0.01, respectively) (Figure 3B), while no differences were observed for DNMT1 or DNMT3B mRNA levels (Figure 3A,C). There were also no differences in DNMTs mRNA levels between 48 h and 96 h (p > 0.05). No interactions were found between time and TGF-β1 treatment for any of the DNMTs transcripts (DNMT1, p = 0.37; DNMT3A, p = 0.19; DNMT3B, p = 0.36).3.3. 5-aza-dC Downregulated Collagen Type I and III Expression Induced by TGF-β1 in Endometrial FibroblastsDemethylating DNMT inhibitor 5-aza-dC was used to test whether epigenetic regulation is involved in collagen expression. Endometrial fibroblasts were treated with 5-aza-dC, TGF-β1, or TGF-β1 + 5-aza-dC. TGF-β1 upregulated COL1A1 and COL3A1 mRNA levels (p < 0.01 and p < 0.001, respectively). The administration of 5-aza-dC to the TGF-β1-treated fibroblasts (TGF-β1 + 5-aza-dC) downregulated their expression (p < 0.001 and p < 0.01, respectively) (Figure 4A,B). The same pattern was observed for COL1 (p < 0.05 and p < 0.01, respectively) and COL3 protein concentration (p < 0.05 an p < 0.01) (Figure 4D,E). However, the TGF-β1-induced increase of α-SMA mRNA levels in endometrial fibroblasts was not reduced with the 5-aza-dC treatment (Figure 4C) (p > 0.05). There was also a reduction in COL1 protein after fibroblast treatment with TGF-β1 + 5-aza-dC, with respect to non-treated fibroblasts (control—C) (p < 0.05; Figure 4D).3.4. 5-aza-dC Downregulated DNMT3A Expression in TGF-β1-Treated Endometrial FibroblastsAfter administration of 5-aza-dC to the TGF-β1-treated fibroblasts, a decrease was observed in DNMT3A mRNA levels (p < 0.001) (Figure 5B). No alterations were found either on DNMT1 or DNMT3B mRNA levels after administration of 5-aza-dC to the TGF-β1-treated fibroblasts (Figure 5A,C).3.5. 5-aza-dC Down-Regulated COL1A1 and Upregulated α-SMA Expression at 96 hThe effects of treatment time, 5-aza-dC treatment, and their interaction (time × treatments) were also studied. Significant interactions between time and 5-aza-dC treatment were observed for COL1A1 (p = 0.0388) and COL3A1 (p = 0.0078) mRNA levels. No interactions were found for α-SMA transcripts (p = 0.0746), COL1, or COL3 protein concentrations (p = 0.0623; p = 0.5382, respectively).We also aimed to study the effect of demethylating 5-aza-dC in endometrial fibroblasts. When administered alone, 5-aza-dC downregulated COL1A1 at 96 h (p < 0.05) and upregulated α-SMA mRNA levels at 96 h (p < 0.01) (Figure 6A,C), while no differences were observed for COL3A1 mRNA (Figure 6B). Regarding COL1 and COL3 protein concentrations, only COL1 increased at 48 h (p < 0.05), while no other differences were found at 96 h or in COL3 (Figure 6D,E). There was a decrease in COL1A1 and a rise in COL3A1 transcripts between 48 h and 96 h (p < 0.05; p < 0.01 and p < 0.01, respectively) (Figure 6A,B).3.6. 5-aza-dC Downregulated DNMT1, DNMT3A and DNMT3B ExpressionThe effects of treatment time, 5-aza-dC treatment, and their interaction (time × treatments) in the mRNA levels of methylating enzymes DNMT1, DNMT3B, and DNMT3A were also studied. Significant interactions between time and 5-aza-dC treatment were observed for DNMT1 (p = 0.0006), DNMT3A (p = 0.0342), and DNMT3B (p = 0.0161) mRNA levels and for COL3 protein abundance (p = 0.0035).There was a decrease in mRNA levels of DNMT1 and DNMT3B at 96 h (p < 0.01; p < 0.001, respectively) and of DNMT3A at 48 h (p < 0.05) (Figure 7A–C). An increase of DNMT3A mRNA levels (p < 0.01) and a decrease of DNMT1 and DNMT3B mRNA levels between 48 h and 96 h treatment (p < 0.001) were also observed (Figure 7A–C).3.7. TGF-β1 Induced Collagen Type I and III, and DNMT3A Expression in Equine Endometrial Fibroblasts and These Effects Were Reversed by 5-aza-dCThere was an increase in mRNA levels of DNMT3A in TGF-β1-treated endometrial fibroblasts (p < 0.05; Figure 8C), as well as a rise in collagen type I and III mRNA levels (p < 0.01; p < 0.001, respectively; Figure 8A,B)), and COL1 and COL3 protein concentration (p < 0.001 and p < 0.01, respectively; Figure 8D,E). After 5-aza-dC treatment of TGF-β1-induced fibroblasts, a reduction in DNMT3A mRNA (p < 0.001) was observed (Figure 8C), simultaneously with a decrease in collagen type I and III transcripts (p < 0.01; Figure 8A,B) and COL1 and COL3 protein concentrations (p < 0.0001 and p < 0.001, respectively; Figure 8D,E).A study between all control groups at different time points was performed for all the mentioned genes, to determine if the observed changes could be happening without any treatment. However, no differences were observed for any of the genes under study during the different incubation time periods (hours).4. DiscussionWe have previously reported increased DNMT3B mRNA levels in equine endometrial fibrosis [46], and increased concentrations of COL1 and COL3 proteins with the degree of endometrosis [61]. We have also demonstrated epigenetic modulation of equine endometrial fibrosis by hypermethylation of the promoter region of the anti-fibrotic MMP2 and MMP9 genes [47]. Thus, to evaluate what was taking place at the cellular level, we aimed to study the epigenetic mechanisms associated with TGF-β1 action in endometrial fibroblasts. Regulation of collagen expression has been extensively studied and a plethora of evidence has indicated that TGF-β1 is an important regulator of ECM metabolism in different organs [62].Our findings indicate that in equine endometrial fibroblasts, TGF-β1 upregulated the expression of collagen type I, collagen type III, and α-SMA mRNA levels and COL1 and COL3 secretion at 96 h, and only COL1 secretion at 48 h. The same increase in COL1A1 mRNA has been observed in TGF-β-treated human fibroblasts from a dermal cell line [63]. Moreover, TGF-β1 can also facilitate epithelial-mesenchymal transition (EMT) by downregulating cell junction expressions and facilitating cell motility and proliferation of human endometrial cells [14,64].Furthermore, TGF-β1 induced an increase in DNMT3A expression at 48 h and 96 h. This agrees with other studies on humans, which have shown increased DNMTs expression in lung fibrosis [65], cardiac fibroblasts [66], and skin fibroblasts from systemic sclerosis patients [20]. In addition, in human nasal epithelial cells, DNMT3A might be the most affected by TGF-β1 [67], as happened in our study. It was suggested in the same study that DNMT inhibitors suppress the progression of chronic rhinosinusitis pathology by regulating DNA methylation. In primary mouse renal fibroblasts, DMNT1 expression was induced by TGF-β1 [68]. Nevertheless, in contrast, TGF-β1 downregulated DNMT1 and DNMT3A and upregulated COL1A1 mRNA expression and COL1 secretion in cardiac fibroblasts [54]. Interestingly, treatment with 5-aza-dC abrogated the effects of TGF-β1-induced myofibroblasts in human cardiac cells [69].These different results may be explained by the passage number of the cells used in each study. Moreover, it may also be ascribed to the type of cells used (cardiac cells vs. renal cells), since it has been established that methylation patterns vary in different tissues and individuals [54,70]. Thus, TGF-β1 can induce both hypermethylation or hypomethylation in genes, illustrating the complexities of the pathways that control and alter methylation patterns.To confirm if the increase of methylation, collagen, and α-SMA mRNA expression in endometrial fibroblasts after TGF-β1 treatment could be reverted by an epigenetic drug, a demethylating epigenetic modifier, 5-aza-dC, was used. The administration of 1 µg of 5-aza-dC for 48 h to TGF-β1-stimulated equine endometrial fibroblasts (for 48 h) was effective in reducing the increased COL1 and COL3 expression (mRNA levels and protein concentration) to normal levels (control), but not for a-SMA transcripts. The same results were observed in human cardiac fibroblasts [59]. Our data also agree with other studies on humans, where it was observed that 5-aza-dC mitigates renal [68,71], cardiac [59,72], and pulmonary [60,73,74] fibrosis by reducing hypermethylation of genes associated with fibroblast activation. In another study, it was found that hypermethylation contributes to renal fibrosis and inhibition of DNMTs suppresses chronic unilateral ureteral obstruction-induced renal fibrosis [17]. A similar reduction in the upregulation of COL1 transcripts and protein expression was reported after administration of 5-aza in TGF-β1-induced human dermal fibroblasts [20]. Furthermore, in a study on human hepatic stellate cells, it was observed that 5-aza-dC inhibited their differentiation into myofibroblasts [75]. In addition, 5-aza suppressed fibrogenic changes in human conjunctival fibroblasts [55]. On the contrary, treating TGF-β1-treated rat lung fibroblasts with 5-aza-dC stimulated a-SMA gene expression by inhibiting DNMTs [32].In our study, the administration of 5-aza-dC alone to equine endometrial fibroblasts for 48 h decreased DNMT3A mRNA levels, but not DNMT1 or DNMT3B transcripts. When fibroblasts were exposed to 5-aza-dC for 96 h, it reduced DNMT1 and DNMT3B mRNA levels, but not DNMT3A. Neveu et al. [60] also reported a decrease in DNMT1 expression after treatment with 5-aza-dC alone in lung fibroblasts. In our study, there was a reduction of COL1A1 mRNA levels and an increase of a-SMA mRNA levels at 96 h after the treatment of endometrial fibroblasts with 5-aza-dC alone. However, COL1 protein concentration increased at 48 h, but no difference was found at 96 h, despite its decreased mRNA levels at 96 h. As a demethylating agent, 5-aza-dC may have led to hypomethylation of the a-SMA gene, thereby increasing its expression. The reason for COL1A1 mRNA levels decreasing after exposure to 5-aza-dC (96 h) is not clear. There are many contradictory results regarding the effect of 5-aza-dC on ECM components. Some studies have shown an upregulation of COL1A1 mRNA levels after treatment with 5-aza-dC alone [54], while others found no differences [59,60]. This might be explained by different experimental conditions and/or types of cells. However, further investigation is needed.In the present study, the endometrial fibroblasts were challenged with TGF-β1 for 48 h and then treated with 5-aza-dC for a further 48 h, so the treated group was not exposed to 5-aza-dC for the total duration of the experiment (96 h). It appears that a longer exposure to 5-aza-dC might provoke bigger changes in both DNMTs, COLs, and a-SMA expression. However, the 48-h incubation period was enough to reduce the increased collagen expression induced by TGF-β1 in endometrial fibroblasts, although the same did not happen for a-SMA. Due to the toxic effects of demethylating agents, the minimal dose that produced the desired effect was used as a rationale for future clinical trials. Some limitations of this study include the lack of information of DNMTs and α-SMA protein expression and DNMTs activity. Likewise, soluble COL1 and COL3 were only assessed in the culture medium by ELISA, and no immunoblots were performed for their detection in the fibroblasts. Although the rationale for using COL1 and COL3 Elisa kits was to use equine-specific antibodies, the western blotting could help to assess changes within the ECM. The stimulation of endometrial fibroblasts with TGF-β1 increased COLs (mRNA levels and protein concentration), a-SMA, and DNMT3A mRNA levels, and the treatment with 5-aza-dC decreased their expression, except for a-SMA gene expression, suggesting an epigenetic regulation through the alteration of DNA methylation.In summary, although epigenetic alterations have been implicated in the development of many types of cancer [76,77], the role of epigenetic changes in fibrosis, particularly in equine endometrial fibrosis, needs much further investigation, since this research is relatively preliminary. Based on our findings, along with the evidence of other studies linking DNA methylation and fibrosis, one may suggest that DNA methylation plays a role in the pathogenesis of endometrial fibrosis. Therefore, pharmacological modulation of this process may result in an effective treatment for endometriosis.5. ConclusionsThe increase in DNMT3A and COLs (mRNA and protein) after TGF-β1 stimulation of equine endometrial fibroblasts was reduced after treatment with a demethylating agent (5-aza-dC), suggesting an epigenetic regulation of mare endometrial fibrosis.

animals : an open access journal from mdpi

10.3390/ani11082210

PMC8388428

Composition of the bacterial community in a newborn’s gut plays a role in their early development and immune system function. Understanding relationships between the bacterial communities of cows and their offspring can help identify which communities have a greater influence on bacterial community development. We examined bacteria at various sites of the cow at birth and bacteria in their calf’s gut throughout early life to understand their relationship. We found that bacteria in the cow’s reproductive tract, gut, and even milk all served as predictors for calf gut bacteria from birth up to 60 d old. Further exploration of these relationships as well as examining relationships of these bacterial communities with illness could help to prevent disease in calves.

Body systems once thought sterile at birth instead have complex and sometimes abundant microbial ecosystems. However, relationships between dam and calf microbial ecosystems are still unclear. The objectives of this study were to (1) characterize the various maternal and calf microbiomes during peri-partum and post-partum periods and (2) examine the influence of the maternal microbiome on calf fecal microbiome composition during the pre-weaning phase. Multiparous Holstein cows were placed in individual, freshly bedded box stalls 14 d before expected calving. Caudal vaginal fluid samples were collected approximately 24 h before calving and dam fecal, oral, colostrum, and placenta samples were collected immediately after calving. Calf fecal samples were collected at birth (meconium) and 24 h, 7 d, 42 d, and 60 d of age. Amplicons covering V4 16S rDNA regions were generated using DNA extracted from all samples and were sequenced using 300 bp paired end Illumina MiSeq sequencing. Spearman rank correlations were performed between genera in maternal and calf fecal microbiomes. Negative binomial regression models were created for genera in calf fecal samples at each time point using genera in maternal microbiomes. We determined that Bacteroidetes dominated the calf fecal microbiome at all time points (relative abundance ≥42.55%) except for 24 h post-calving, whereas Proteobacteria were the dominant phylum (relative abundance = 85.10%). Maternal fecal, oral, placental, vaginal, and colostrum microbiomes were significant predictors of calf fecal microbiome throughout pre-weaning. Results indicate that calf fecal microbiome inoculation and development may be derived from various maternal sources. Maternal microbiomes could be used to predict calf microbiome development, but further research on the environmental and genetic influences is needed.

1. IntroductionBacterial colonization of the newborn gut during and after parturition influences intestinal development and immune system function [1,2]. Previous studies have demonstrated microbiota in meconium, or calf feces present at birth, are similar to those in feces in calves up to 24 h of age [3]. There is a dramatic shift in the fecal microbiota at 24 h, demonstrating the calf fecal microbial community is influenced very early in life [3]. These early influences can include bacteria from the dam as well as the environment, but the extent of their influence is not yet fully understood. Understanding the sources of colonization and their influence on gut development is key in determining what the “expected” microbiome is, as deviations in the gut microbiome can reflect an animal’s response to environmental or physiological stressors [4,5]. However, the “expected” or “normal” calf gut microbiome is not yet fully understood and may not be the same for every animal.The dam’s microbiomes from the uterine environment, vaginal canal, feces, saliva, and colostrum are a major influence on calf digestive system microbial colonization. While bacteria in the feces are not fully representative of those in other portions of the gastrointestinal tract, like the rumen, changes in the fecal microbiome have reflected calves’ response to their environment and can predict risk of dysbiosis [6,7,8]. The early calf fecal microbiome is dominated by bacteria present in the vaginal microbiome of the dam, as the vaginal microbiome shared the most bacteria with calf feces from 30 min to 48 h after birth when compared to dam feces or colostrum [9]. Rumen microbiota differed based on mode of birth (vaginal vs. cesarean section), which demonstrates the vaginal canal as a major influence on the entire gastrointestinal tract [10]. Cow feces and colostrum do influence calf digestive development, as both shared abundant bacteria with calf feces during the first 24 h post-partum [10]. There is also evidence that bacteria from these sources influence the microbiome up to 21 d of age [11]. However, many bacteria in calf feces have not been found in dam vaginal, fecal, or colostrum microbiomes. Consumption of bacteria is the most common method of gut inoculation, but bacteria also have the ability to cross into the blood and lymphatic system and could potentially inoculate other systems. The entero-mammary pathway is one proposed mechanism in which bacteria travel from the gut to inoculate the mammary gland [12]. It is unclear if a similar pathway exists for other body systems and what their relationship is with the gut. Characterizing other maternal sources and routes of inoculation might identify the origin of these bacteria and further explain colonization of the calf gut and other systems.Another potential source of inoculation is the upper reproductive tract of the dam. While previously considered sterile, recent evidence in multiple species has identified microbiomes distinct to locations within the upper reproductive tract and in the fetus itself [13,14,15]. These bacteria could have migrated from the lower reproductive tract or the dam’s gut, as bacteria may cross the intestinal epithelium and travel to the uterus during periods of intestinal hyperpermeability or “leaky gut” [12,16]. The uterus contains a lumenal environment and epithelium distinct from the intestine, which would support its own unique bacterial community and is available to inoculate the calf gut [17,18]. Bacteria have been identified in multiple locations of the pregnant tract of dairy cattle and these additional microbiomes could serve as a source of calf gut colonization [14]. It would be difficult and potentially dangerous to collect samples from the post-partum uterus, but the placenta could be representative of the upper reproductive microbiome. However, potential relationships between the placental and calf microbiomes have yet to be described. Further to this, due to the known influence of other maternal microbiomes, the placenta’s potential influence cannot be studied independently.Various biological system microbiomes have been linked to health, fertility, and efficiency and have been shown to influence the microbial ecology of other systems. Therefore, environmental or genetic changes in one biological system, such as the digestive system, may unintentionally affect other microbiomes; thus, it is critical to elucidate the interrelationships of these systems within individuals and between mothers and their progeny. The objectives of this study were (1) to characterize the maternal and calf fecal microbiomes during peri-partum and post-partum periods and (2) examine the influence of the maternal microbiome on calf fecal microbiome development during the pre-weaning phase. We hypothesized that dam reproductive, fecal, and colostrum microbiomes would all play significant roles in calf gut colonization.2. Materials and Methods2.1. Animal Observation and Sample CollectionAnimal procedures were approved by the Virginia Polytechnic Institute and State University Institutional Animal Care and Use Committee (protocol #17-187-DASC). Multiparous, pregnant Holstein cows (n = 13) were enrolled in the study 12 to 14 d prior to expected calving date and housed in individual box stalls. Box stalls were bedded with sawdust and re-bedded after each calving to avoid contamination across dams. Close-up dry cows were fed a total mixed ration twice daily at 0900 h and 1900 h and were provided ad libitum access to water. The Moocall calving alert system sensor (Moocall Ltd., Dublin, Ireland) was placed on the dam’s tail 7 d prior to expected calving to alert when calving began. Sterile, flocked swabs (Puritan, Guilford, ME, USA) were used to sample vaginal fluid from the dam’s caudal vagina within 24 h prior to parturition and snap frozen in cryotubes using liquid nitrogen.At parturition, calves (n = 13; bulls = 9, heifers = 4) were immediately separated from dams and transferred to a clean 111.28 × 55.40 × 46.13 cm3 plastic container containing fresh wood shavings to prevent environmental contact. The container was rebedded between each calving. Calves were weighed immediately after birth. Sterile, flocked swabs (Puritan, Guilford, ME, USA) were used to collect meconium from newborn calves before passage out of the body and oral samples from the left and right buccal wall of the dam immediately after parturition. These samples were snap frozen in cryotubes using liquid nitrogen. Dam fecal samples were removed from the rectum using a clean palpation sleeve and sterile flocked swabs were used to collect samples before being snap frozen in cryotubes using liquid nitrogen.Representative colostrum samples were aseptically collected before milking and frozen at −20 °C. Remaining colostrum was collected using a stainless-steel portable bucket milking machine. Colostrum was required to have a Brix score ≥22% using a Brix refractometer (VEE GEE Scientific, Vernon Hills, IL, USA), which correlates to ≥50 g/L of immunoglobulin G (IgG) in the colostrum. If colostrum did not achieve a Brix score ≥22%, the dam–calf pair were removed from the study. One dam and one bull calf were removed due to failure to meet colostrum requirements. Calves were assigned individual bottles and nipples at colostrum feeding to be used for the remainder of the study. Calves were bottle fed 4 L of their dam’s colostrum within 1 h post-birth. Antibiotics used to treat common bacteria-associated calf morbidities might influence gut microbiome composition. In order to mitigate the use of antibiotics in the study, calves were bottle fed an additional 2 L of colostrum at 12 h post-calving.Sections of placenta were collected within 6 h post-birth after passage through the vagina but before coming in contact with the ground using a sterile scalpel. Cotyledon tissue was snap frozen in cryotubes using liquid nitrogen.After their initial colostrum feeding, calves were moved to individual, sawdust-bedded hutches and remained there through the end of the study. Sterile, flocked swabs (Puritan, Guilford, ME, USA) were used to collect calf fecal samples at 24 h post birth. Blood was collected from each calf 24 h post birth via jugular venipuncture using Monoject blood tubes with no additive (Covidien, Mansfield, MA, USA). Blood was stored at 4 °C for 12 h and then centrifuged at 2000× g for 20 min at 4 °C to isolate serum.Calves were fed 4 L of 27.0% CP, 20.0% fat milk replacer (Cow’s Match® ColdFront® Medicated (67 mg/kg lasalocid sodium), Land O’Lakes® Animal Milk Products Co., Shoreview, MN, USA) twice daily at 600 h and 1800 h beginning approximately 24 h post-birth. Calves were fed using individually-assigned bottles and nipples to avoid cross-contamination. Calves were allowed ad libitum access to water at 1 d of age. All calves were vaccinated with INFORCE™ 3 (Bovine Rhinotracheitis, Parainfluenza 3, Bovine Respiratory Syncytial Virus Vaccine; 2 mL intranasally; Zoetis Inc., Kalamazoo, MI, USA) at 4 d of age. At 28 d of age, calves were given ad libitum access to a 22% CP starter grain (Intensity 22% Textured Calf Starter Medicated, Cargill Animal Nutrition, MN, USA). Step down weaning began at 42 d of age, with calves fed 3 L of milk replacer twice daily from 42 to 49 d and 2 L of milk replacer twice daily from 50 to 56 d. Calves were completely weaned at 57 d and removed from the study at 60 d. Water and starter refusals were measured at each feeding. Calves were observed at each feeding for symptoms of scours. Calves were weighed weekly approximately 1 h prior to evening feeding. Sterile, flocked swabs (Puritan, Guilford, ME, USA) were used to collect calf fecal samples at 7 d, 42 d, and 60 d.2.2. Serum and Colostrum IgGColostrum and calf serum IgG concentrations were measured using a commercial Bovine IgG ELISA (Bethyl Laboratories, Inc., Montgomery, TX, USA) according to the manufacturer’s protocol in order to confirm successful passive transfer (serum IgG ≥ 1000 mg/dL). Plates were read at 450 nm (BioTek Instruments, Winooski, VT, USA) and data were analyzed using a four-parameter logistic curve software (MyAssays Ltd., Brighton, UK). Samples with an intra assay CV of <10% and inter assay CV of <15% were used to determine IgG concentration.2.3. DNA Extraction and SequencingThe mis-estimation of calving events led to incomplete sample sets from eight of the cow–calf pairs. Of these eight, six cow–calf pairs were removed from the study because they were missing two or more key pre-calving microbial samples, while two cow–calf pairs yielded nearly complete sample sets, only missing pre-birth vaginal samples. One dam and one bull calf were removed due to failure to meet colostrum requirements. This left the study with six cow–calf pairs for microbiome analyses, with three heifers and three bull calves.Bacterial DNA was extracted from all oral, fecal, and vaginal swab samples using the QIAamp BiOstic Bacteremia DNA kit (Qiagen, Germantown, MD, USA). Bacterial DNA was extracted from placenta and colostrum samples using the Qiagen Mini Stool Kit (Qiagen, Germantown, MD, USA). Colostrum was initially centrifuged at 12,000× g for 30 min at 4 °C in order to pellet bacteria before DNA extraction. Before DNA precipitation, each sample was treated with 20 µg RNAse A at room temperature for 3 min to remove any potential RNA contamination. Qubit 2.0 Fluorometer and Qubit dsDNA HS Assay kit (Invitrogen, Carlsbad, CA, USA) were used to measure DNA quality and quantity before sequencing.Samples were submitted to the Virginia Bioinformatics Institute Genomics Research Laboratory (Blacksburg, VA, USA) for library preparation and sequencing. 16S rDNA amplicons covering variable region V4 were generated using primers 515F–806R (reverse barcoded: FWD: GTGCCAGCMGCCGCGGTAA; REV: GGACTACHVGGGTWTCTAAT) [19]. Amplicons were pooled and purified using a Pippin Prep 1.5% gel cassette (Sage Science, Inc., Beverly, MA, USA). Amplicon libraries were sequenced using 300 bp paired end sequencing via Illumina MiSeq (Illumina, San Diego, CA, USA).2.4. Bioinformatics Analysis2.4.1. Taxonomic ProfilingTaxonomic profiling was performed using CLC Genomics Workbench Microbial Genomics Module version 12.0 (Qiagen, Germantown, MD, USA). Amplicon sequences had adapters removed and were filtered to remove reads with a Phred score <30. Filtered reads were aligned to the 97% Greengenes database version 13.8 to be separated into operational taxonomic units (OTU). These OTU were aligned using Multiple Sequence Comparison by Log-Expectation (MUSCLE, version 3.8.31) [20] with a maximum of 16 iterations and a minimum combined abundance of 10 across samples. A phylogenetic tree was constructed using aligned OTU with a Neighbor Joining method, General Time Reversible nucleotide substitution model, and Whelan and Goldman (WAG) protein substitution model [21,22,23].2.4.2. Alpha and Beta DiversityAlpha diversity, the microbial diversity within a sample, was measured using Shannon entropy, Simpson’s index, and phylogenetic diversity (PD) based on the constructed phylogenetic tree. PD=∑i=1nbi I(pi>0) where n was the number of branches within the phylogenetic tree, bi was the length of branch I, pi was proportion of taxa descending from branch i, and the I (pi > 0) assumed the value of 1 if any taxa descending from branch i were present in the sample or 0 otherwise. A Kruskal–Wallis H test was used to measure differences in alpha diversity measures based on sample type. A p-value ≤ 0.05 was considered significant.Beta diversity, diversity in microbial community structure between samples, was measured using weighted Unifrac distances (d(W)) based on the constructed phylogenetic tree. d(W)=∑i=1nbi|piA−piB|∑i=1nbi(piA+piB) where n was the number of branches in the phylogenetic tree, bi was the branch length i, and piA and piB were the proportion of taxa descending from branch i in samples A and B. A permutational multivariate analysis of variance (PERMANOVA) was used to measure difference in Beta diversity based on the main effects of sample type and calf sex [24]. A p-value ≤ 0.05 was considered significant. A Bonferroni p-value ≤ 0.05 was considered significant when multiple pair-wise comparisons were made between various sample types.2.4.3. Microbiome AssociationsSpearman ranked correlations were performed among maternal microbiomes, among each calf fecal microbiomes, and between maternal microbiomes and calf fecal microbiomes based on genera relative abundance using cor.test function in the package stats in R version 3.6.1 [25]. A p-value ≤ 0.05 was considered significant.A negative binomial regression model was created using genera count data to evaluate the ability of dam’s placental, colostrum, vaginal, fecal, and oral microbiomes to predict calf fecal microbiomes at each timepoint. The following model was created in R version 3.6.1 [25] and the glm.nb function within the MASS package version 7.3-51.5 [26]:lnμ=β0+β1x1+β2x2+β3x3+β4x4+β5x5 where μ is calf fecal bacteria count at a given time point, β0 is the intercept, x1–x5 are the dam placental, colostrum, vaginal, oral, and fecal bacteria count, respectively, and β1–β5 are the expected change in ln μ if xi changes by 1. Maternal microbiome predictors were considered significant if p ≤ 0.05.3. Results3.1. Descriptive StatisticsTwelve dams gave birth to calves that met our criteria (bulls = 8, heifers = 4; Table 1). There were no signs of dystocia and calvings did not require assistance. Serum IgG concentrations indicated successful passive transfer of immunity in all calves (calf serum IgG = 2997 ± 251 mg/dL, Table 1). Calves had no signs of scouring or illness during the experiment.3.2. Bioinformatics AnalysesAcross all samples, a total of 18,852 OTU were identified using 11,777,504 reads (Table 2). Shannon entropy and Simpson’s index indicated the 24 h calf fecal microbiome had reduced diversity compared to other calf fecal microbiomes [(p ≤0.010) (Figure 1A,B, Table S1)]. Placenta and colostrum had low phylogenetic diversity compared to other dam and calf microbiomes (Figure 1C). Beta diversity indicated placenta and colostrum samples clustered independently from other samples (Figure 2). There was a difference in beta diversity based on sample type (p < 0.001), but further pairwise comparisons did not indicate a difference between specific sample types (p ≥ 0.097; Table S2). No difference in beta diversity was observed based on calf sex (p = 0.842).The predominant phylum in colostrum, placenta, vagina, dam oral, and calf 24 h fecal samples was Proteobacteria (96.15%, 47.70%, 57.84%, 69.33%, and 85.10%, respectively; Figure 3, Table S3). The predominant phylum in dam fecal, meconium, calf 7 d, 42 d, and 60d fecal samples was Bacteroidetes (48.81%, 42.55%, 43.36%, 49.35%, and 45.58%, respectively; Figure 3). At the genera level, no one genus was dominant across all maternal or calf sample types (Figure 4; Table S4). An unidentified genus within the family Pasteurellaceae dominated the vaginal microbiome (55.31%) and Stenotrophomonas dominated the colostrum microbiome (42.72%; Figure 4A). Other maternal microbiomes did not contain one genus with a relative abundance >27.98%. Meconium, 42 d calf fecal, and 60 d calf fecal all had Prevotella as the most abundant genus (11.56%, 30.23%, and 27.83%, respectively; Figure 4B). The 24 h calf fecal sample was dominated by an unknown genus in the family Enterobacteriaceae (83.94%; Figure 4C).3.3. Microbiome AssociationsSpearman ranked correlations were performed among maternal microbiomes, among calf fecal microbiomes, and between maternal microbiomes and calf fecal microbiomes using genera relative abundance. All correlations were significant (p ≤ 0.001). The dam fecal microbiome had a moderate correlation with the vaginal microbiome (Table 3). From 24 h to 60 d, there was moderate to strong correlation between calf fecal microbiomes of subsequent timepoints (Table 3). Correlations between calf fecal microbiomes and dam and vaginal microbiomes increased with age (Table 3).Negative binomial regression models were created to estimate predictive ability of maternal microbiomes on calf fecal microbiomes (Table 4). Each maternal microbiome was a significant predictor for at least two time points. None of the maternal microbiomes were significant predictors for all calf fecal microbiomes.4. DiscussionThe objectives of this study were to characterize the various maternal and calf fecal microbiomes during the peri-partum and post-partum periods and examine the relationship of the maternal microbiome with calf fecal microbiome development during the pre-weaning phase. Using 16S amplicon sequencing, we identified unique microbiomes within the dam’s placenta, vagina, colostrum, feces, and oral cavity and the calves’ feces. Genera in the dam oral microbiome had a moderate positive correlation with genera in the early calf fecal microbiomes. All maternal microbiomes were a significant predictor for the calf microbiome during at least 2 time points during pre-weaning. No maternal microbiome was a significant predictor at every time point.Inoculation of the calf microbiome can stem from many sources; (1) the dam before and during birth, (2) the diet, and (3) the environment. Previous research has investigated the influence of the dam on early rumen or intestinal inoculation (birth to 7 d) or exclusively diet on rumen microbiome development in dairy calves [11,27,28]. Our study was the first aimed at associating how the maternal microbiomes, including placental, vaginal, colostrum, oral, and fecal, are related to the calf gut microbial development throughout the pre-weaning phase (birth to 60 d).4.1. Early Changes in the Calf Fecal MicrobiomeDramatic changes occur in the neonatal calf fecal microbiome between birth and 24 h of age. In the current study, the fecal microbiome at 24 h of age was almost entirely made up of Proteobacteria, compared to meconium collected at birth, which mainly comprised Bacteroidetes, Proteobacteria, and Firmicutes. The 24 h fecal microbiome also had a reduction in alpha diversity measures compared to meconium, indicating reduced diversity in the microbial community structure. This has been seen previously in a recent study investigating the composition of the perinatal intestinal microbiome in Holstein and Ayrshire calves [27]. Others have observed the neonatal gut microbiota as an unstable community due to its rapid variation and colonization by facultative anaerobes, specifically Proteobacteria [29]. Proteobacteria play an important role in preparing the neonatal gut microbiota for successive colonization by strict anaerobes by consuming oxygen, altering pH, lowering redox potential, and producing carbon dioxide and nutrients [29,30,31]. Proteobacteria have been observed as a dominant phylum in many environmental niches, including soil [32], plants [33], freshwater [34], seawater [35], and the atmosphere [29,36], suggesting that the high prevalence of Proteobacteria in the fecal microbiome of calves at 24 h of age could be a result of the calf’s first environmental exposure. This suggests significant environmental effects on calf gut microbiome within a short period of time.The most abundant genera in the young calf fecal microbiome may play a role in microbiome composition and calf response to disease. Prevotella and an unclassified Enterobacteriaceae genus were the most abundant genera in calf feces from during the first 24 h after birth. In mice, increased abundance of some species of Prevotella led to decreased acetate and increased butyrate in the large intestine and increased production of inflammatory cytokines [37]. In calves, gut inflammation followed by prolonged dysbiosis caused by Enterobacteriaceae has resulted in calf diarrhea [38]. It is possible that increased abundance of these genera could alter the newborn gut microenvironment and exacerbate calf illness. Further research examining specific inclusion and exclusion of these microbes alongside immune response measurements could elucidate host–microbe interactions in the calf gut.4.2. Variation between Maternal SourcesThe current study examined how bacteria from various maternal sites inoculated the calf gut, but one important aspect not within the scope of this study was how those maternal microbiomes were initially inoculated. Consumption of bacteria and passage from the oral cavity through the gastrointestinal tract could explain inoculation of the gut microbiome and its development, but it does not account for the colostrum or reproductive tract microbiomes. One potential method of bacterial colonization of these sites is through an entero-mammary pathway. In this proposed pathway, bacteria in the maternal intestine permeate the intestinal epithelium and enter the lymphatic or circulatory system, allowing them to travel to the mammary gland or reproductive tract [12]. There is little evidence in the dairy cow demonstrating the existence of this pathway, but common gut bacteria like Ruminococcus and Bifidobacterium have been identified in mammary secretions, blood, and feces within the same dam [16]. In mice, Enterococcus, Streptococcus, Staphylococcus, and Propionibacterium were cultured from umbilical cord blood [39]. Additionally, pregnant mice were orally inoculated with genetically labeled Enterobacterium faecium that was then identified in amniotic fluid [39]. However, the murine placenta is very different in both structure and transport function from the ruminant placenta [40,41,42]. Further research in dairy cattle using similar labeled bacteria methods would be needed to support the existence of this pathway and explain how these maternal sites are inoculated.The microbial composition and community structure between maternal sites provides some insight on how these various sites are related and could potentially influence each other. In the current study, dam vaginal, oral, and fecal microbiomes were moderately correlated with one another. However, the principal coordinate scatter plot demonstrated dam fecal samples tightly clustered while vaginal and oral samples were not. Previous literature shows similar results and provides some insight on how various maternal microbiomes are inoculated, but these results also point towards the difficulty of determining bacterial contamination versus inoculant [27,43]. The broader clustering of the vaginal and oral microbiomes makes sense, as these sites not only contain bacteria typically commensal to that location, but they are also consistently exposed to sources of new bacteria, like feed for the oral cavity or feces and bedding for the vagina, which would increase beta diversity of the microbiome [27,44]. Compared to the oral cavity or vagina, the cow’s colon is exposed to fewer external sources of bacteria; therefore, microbial diversity between fecal samples is expected to be reduced.Similarly, sections of the reproductive tract, like the vagina, cervix, uterus, and oviduct, support the growth of specific subsets of bacteria, but the microbial composition and diversity of each section could be influenced by those adjacent to it [45,46]. We observed a moderate correlation between the placental and vaginal microbiomes, but we also observed lower phylogenetic diversity in placental samples compared to vaginal samples and separate clustering of placental and vaginal samples in the principal coordinate scatter plot. This is expected, as bacteria in the vagina may enter the uterus throughout pregnancy, but the difference between vaginal and uterine/placental environments might support the abundance of certain bacteria over others [17,18,47]. There is still the possibility that bacteria utilize a pathway similar to the entero-mammary axis to reach the uterus or that some bacteria found in the placenta are contaminants from the vagina. Research utilizing both fluorescent in situ hybridization (FISH) and 16S amplicon sequencing on multiple samples throughout the pregnant reproductive tract could provide insight into the route bacteria use to colonize the uterus, if certain bacteria have a location preference within the reproductive tract, and help differentiate between commensal and contaminant bacteria.4.3. Microbiome HeritabilityOne potential component that shapes microbiomes which we were unable to account for is heritability. A core rumen microbiome has been identified in beef and dairy cattle with an estimated narrow sense heritability of ≥0.15 [48,49]. This heritable subset of rumen bacteria has also been associated with feed efficiency and methane emissions [50]. This could mean the fecal microbiome is also heritable and could influence cow performance. However, a much larger study examining various maternal sources of calf fecal bacteria are needed to estimate their heritability.Instead of direct passage from parent to progeny, microbiome heritability may be due to genetic influence on tissue morphology. Which taxa dominate a particular location is influenced by the available proteins, metabolites, and molecular substrates, as certain bacteria are more efficient at surviving in a particular environment than others [17,18,47]. In an animal’s body, organ luminal environment influences and is influenced by tissue morphology, including type of cells, abundance of each type, and level of activity within these cells [51]. In humans, genomic markers have been associated with tissue morphology, including skeletal muscle, pancreas, and reproductive tissues [52]. This genetic influence on morphology would then influence the tissue environment and subsequently the microbiome. We observed in the principal coordinate scatter plot that samples were clustered based on location within the dam’s body, with placenta, vagina, oral cavity, udder, and large intestine all having distinct morphology. It is possible there is a genetic influence on this morphology and the microbiomes of each sample type, but further research is needed to examine the genetic influence on morphology in cattle as well as its association with the various microbiomes.4.4. Study Limitations and Future DirectionsEach sample type from the dam seems to predict the calf fecal microbiome during at least two time points and no one maternal microbiome seems to be the sole influencer of calf fecal development. This supports our hypothesis that each maternal microbiome plays some role in calf gut inoculation and development. However, our sample size was limited to only six calf–dam pairs. Additionally, it is likely that the neonatal calf microbiome is influenced by the birth environment. The neonatal calf is born with a diverse microbiome that is immediately subject to rapid changes due to exposure to the environment [27]. One limitation of our study is the unknown microbial community within the birth environment (calving pen) and housing environment (calf hutches). All cows were calved in stalls bedded with fresh shavings three to four days prior to birth, but the degree of fecal, aerial, or other bacterial contamination in that short pre-calving window certainly varied. Additionally, bacteria in the water made available to the calves and within each of the calves’ bottles would contribute to development of the calf gut microbiome. Research in pigs has demonstrated bacteria acquired from the environment can influence microbial composition at the gut surface [53]. We attempted to limit potential environmental contamination during and following parturition, but analyzing the microbiome from environmental sources like water or bedding would allow separation of source contributions to the calf microbiome. Another limitation to our study was that all animals were from the same location. This removes variation due to location, but identifying a core maternal microbiome that is the main influence for calf microbial development may require calf–dam pairs from various locations to account for this. Future studies with increased sample size and accounting for further sources of environmental variation would support mathematical modelling to predict calf microbiome development.5. ConclusionsThe current study supports our hypothesis that maternal microbiomes, including fecal, oral, colostrum, and reproductive microbiomes, play a role in the calf gut microbiome inoculation and development. All dam microbiomes measured were predictive of the calf fecal microbiome through the preweaning phase, with dam fecal and oral microbiomes having the largest correlation. Studies further validating relationships between these microbiomes as well as other maternal or calf microbiomes are necessary in order to use these microbiomes as a tool for monitoring calf response to environmental stressors.

animals : an open access journal from mdpi

10.3390/ani11041107

PMC8070139

This study was conducted to elucidate the effects of dietary soluble extract hydrolysates obtained from fishery by-products, such as shrimp soluble extract (SSE), tilapia soluble extract (TSE) and squid soluble extract (SQSE). Furthermore, we used a nucleotide, inosine monophosphate (IMP), as an additive in different concentrations along with shrimp soluble extract to understand their effects on growth, immunity and disease resistance in juvenile Nile tilapia. Our results demonstrated that dietary SSE could improve growth performance, non-specific immune responses and disease resistance against pathogenic bacteria Aeromonas hydrophila in juvenile Nile tilapia. Moreover, IMP did not add further benefits to the SSE diet. Further research is needed to better understand the effects of fishery by-products and IMP on fish diets.

We performed an 8-week feeding trial to evaluate dietary soluble extract hydrolysates from fishery by-products, such as shrimp soluble extract (SSE) with or without inosine monophosphate (IMP), tilapia soluble extract (TSE) and squid soluble extract (SQSE), in juvenile Nile tilapia. A diet without feed additives was used as the control diet (CON); and five other experimental diets were formulated with 2% soluble extracts consisting of 100% SSE, 98% SSE + 2% IMP (SSEP2), 96% SSE + 4% IMP (SSEP4), 100% SQSE and 100% TSE. The diets were fed to 4.9 ± 0.07 g (mean ± SD) juvenile Nile tilapia in triplicate groups. The weight gain and specific growth rates of fish fed the SSE, SSEP2 and SSEP4 diets were significantly higher than those of fish fed the CON and SQSE diets. The superoxide dismutase activity levels of fish fed the SSE and SSEP4 diets were significantly higher than those of fish fed the CON, SSEP2, SQSE and TSE diets. Myeloperoxidase activity levels of fish fed the SSE and SSEP4 diets were significantly higher than those of fish fed the CON, SSEP2 and SQSE diets. Lysozyme activity levels of fish fed the SSEP4 and SQSE diets were significantly higher than those of fish fed the SSE and SSEP2 diets. Feed efficiency, protein efficiency ratio, survival rate, whole body proximate composition and hematological parameters were not significantly different among the groups. After ten days of challenge = against Aeromonas hydrophila, the cumulative survival rate of fish fed the SSE diet was significantly higher than those of fish fed the CON, SQSE and TSE diets. In conclusion, dietary shrimp soluble extract could improve the growth performance, non-specific immune responses and disease resistance in juvenile Nile tilapia, and inosine monophosphate did not add further benefits to this ingredient.

1. IntroductionAquaculture is considered as one of the fastest growing food producing industries, which supplies over 50% of global fish production [1]. However, the future development of aquaculture is limited by the excessive use of unsustainable marine ingredients in aquafeed. A significant number of research studies have been conducted on alternative protein sources, mostly with plant origins [2,3,4,5]. However, plant ingredients often lack the essential amino acids for fish and/or possess low digestibility and palatability [6]. Fishery by-products which contain essential nutrients can be utilized as useful ingredients in aquafeed production [7]. According to previous studies, protein hydrolysates derived from fishery by-products have been considered as beneficial ingredients in feed for fish because of their nutritional, functional and cost-effective properties [8].Hydrolysis of feed ingredients through the enzyme processes has been used as one of the important methods for the processing of fishery by-products [8,9]. This processing method results in small molecular weight compounds and hydrolysates with considerably diverse amino acid profiles—potential feed additives and fishmeal replacers for aquaculture [10]. Crustacean hydrolysates, such as shrimp soluble extract (SSE), have been used in aquafeed as protein sources [11,12] and as dietary supplements for improvements to palatability and acceptability for fish [13]. Likewise, hydrolysates of fishery by-products such as tilapia soluble extract (TSE) and squid soluble extract (SQSE) have essential amino acids that are required for the fish growth and survival, and have positive effects, such as enhancing immune response and palatability [14,15,16,17]. SSE, TSE and SQSE contain high levels of amino acids, especially in free form, and active peptides that can be digested and assimilated by aquatic organisms [18,19]. Furthermore, the use of SSE, TSE and SQSE could reduce environmental problems by processing and using the inedible parts of fishery products in the diets of cultured fish.Nile tilapia, Oreochromis niloticus, is one of the extensively cultured fish species with high economic importance for the aquaculture industry because of its faster growth, higher survivability in high stocking densities and higher disease resistance compared to the other freshwater fish species [20]. Nile tilapia has become an iconic freshwater-cultured fish species which contributed about 4.5 million tons to the market in 2018 based on the global aquaculture production report by FAO [1]. However, with the expansion of intensive aquaculture, tilapia farms have been more susceptible to disease outbreaks. For example, Aeromonas hydrophila is a bacterium that has caused massive rates of mortality in tilapia farms around the world [21]. Additionally, due to the use of low marine ingredients (e.g., fishmeal) in the diet of this species, the growth, feed conversion ratio (FCR) and protein efficiency ratio in fish are often affected negatively [21,22,23]. These negative effects of feed ingredients in fish may occur due to lower palatability and/or absence of key compounds such as nucleotides [24]. Inosine monophosphate (IMP), a key compound in purine nucleotide metabolism, has been shown to benefit physiological and nutritional functions in different animals as a dietary supplement [25,26]. According to previous research results, the growth performance, feed intake, immune responses and disease resistance were significantly improved in fish when IMP was used alone or combined with some free amino acids in the diet [27,28,29,30]. The non-specific immune response in terms of lysozyme activity, myloperoxidase and nitro-blue-tetrazolium activities was improved with dietary supplementation of IMP in olive flounder (Paralichthys olivaceus) [28]. In Nile tilapia, final body weight, growth-related gene expression, feed utilization performance and immune responses have been improved by the addition of IMP in the diet [29,30].In our previous studies, Moniruzzaman et al. [2] and Jo et al. [12] reported that dietary supplementation of SSE has positive effects on growth and immune responses in freshwater fish such as rainbow trout. Based on the previous findings, the aim of the present study was to determine the effects of dietary soluble extract hydrolysates from fishery by-product, such as shrimp soluble extract (SSE), with or without the addition of inosine monophosphate (IMP), in comparison to the diets supplemented with tilapia soluble extract (TSE) and squid soluble extract (SQSE) in terms of growth performance, hematology, innate immune responses and disease resistance in juvenile Nile tilapia Oreochromis niloticus.2. Materials and Methods2.1. Experimental DietsFishery by-products, such as shrimp soluble extract, squid soluble extract and tilapia soluble extract, were provided by VNF Company (Vietnam Food Joint Stock Company, Ho Chi Minh City, Vietnam). The fishery by-product production process goes through several steps to eliminate extraneous matter, and then they are shredded and pressed to obtain a liquid extract. Later, this liquid extract went through a centrifugal process to acquire its purest form. The extract is then sent to chemical processing area where the protein is broken into peptides and digestible amino acids. Chemically processed extract is refined to create products that will meet various levels of quality standards according to customer demands. Refined soluble is mixed and added with flavor-preservation additives, to maintain the product’s unique flavor.Six experimental diets were formulated to have the same crude protein (33%) levels. The ingredients and proximate compositions of the six experimental diets are shown in Table 1. A basal diet without feed additives was used as the control (CON); the other five diets were formulated to include 2% of soluble extracts: 100% shrimp soluble extract (SSE), 98% shrimp soluble extract +2% inosine monophosphate (SSEP2), 96% shrimp soluble extract +4% inosine monophosphate (SSEP4), 100% squid soluble extract (SQSE) and 100% tilapia soluble extract (TSE), replacing the total of 2% consisting of soybean meal, wheat flour and soybean oil from the CON diet to balance the nutritional compositions of the diets. Fish meal, soybean meal, rapeseed meal, meat and bone meal, poultry by-product meal and squid liver powder were used as the protein sources; soybean oil and fish oil were used as the lipid sources; and wheat flour was used as the carbohydrate source in the experimental diets. The feed preparation procedure was followed described elsewhere by Hamidoghli et al. [31]. Pellets were air-dried for 48–96 h, broken and sieved to get the desired size and stored at −20 °C until use.2.2. Experimental FishThe juvenile Nile tilapia were obtained from a private hatchery (Docheon Aquafarm, Changnyeong, Korea), and the experiment was conducted at the laboratory facilities of the Feeds and Foods Nutrition Center (FFNRC), Pukyong National University, Busan, Korea. Prior to the execution of experiment, fish were fed a commercial diet for two weeks to be acclimated to the experimental environment. Three hundred sixty fish averaging 4.9 ± 0.07 g (mean ± SD) were randomly distributed into 18 tanks (20 fish/tank) of 30 L volume of filtered freshwater. Each experimental feed was fed to triplicate groups of fish up to apparent satiation twice daily (09:00 and 18:00 h) for 8 weeks. Water temperature and pH were maintained at 27 ± 0.5 °C and 7.5 ± 0.3 and aeration was supplied for sufficient dissolved oxygen to each tank.2.3. Sample Collection and AnalysesAt the end of the 8-week feeding trial, fish were starved for 24 h, and the total number and weight of fish in each tank was determined for calculations of weight gain (WG), specific growth rate (SGR), feed efficiency (FE), protein efficiency ratio (PER) and survival rate. Three fish per tank (nine fish per treatment) were randomly selected and stored at −20 °C for whole-body proximate composition. Three additional fish per tank were randomly sampled, individually weighed and then dissected to obtain liver and viscera samples for the determination of hepatosomatic index (HSI), viscerosomatic index (VSI) and condition factor (CF). Three fish per tank were randomly captured and anesthetized with ethylene glycol phenyl ether (200 ppm) and blood samples were obtained via caudal vein puncture using a non-heparinized 1 mL syringe. Blood samples were allowed to clot at room temperature for 30 min. Then, the serum was separated by centrifugation at 5000 g for 10 min and stored at −80 °C for the analysis of non-specific immune responses, including superoxide dismutase (SOD), myeloperoxidase (MPO) and lysozyme activity.2.4. Proximate Composition Proximate composition, including moisture, crude protein, crude lipid and ash of the diets and whole-body samples were measured based on AOAC [32]. Briefly, parts of the diets and fish samples were dried at 135 °C for 2 h to obtain the moisture contents. Ash contents were determined by incineration at 550 °C for 3 h in muffle furnace. Crude lipid contents were achieved by Soxhlet extraction process (Soxtec system 1046, Tecator AB, Hoganas, Sweden) using ethyl alcohol as organic solvent, and crude protein contents were analyzed by the Kjeldahl method based on nitrogen concentrations in the samples (Nx6.25) after the digestion, distillation and titration.2.5. Hematological ParametersBlood plasma glucose (GLU), total cholesterol (T-CHO), aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were measured by a chemical analyzer (Fuji DRI-CHEM3500i, Tokyo, Japan).2.6. Non-Specific Immune ParametersSerum SOD was measured using an assay kit (Sigma-Aldrich, St. Louis, MI, USA, 19160), following the manufacturer’s guidelines. This method is based on inhibition against WST (Water soluble tetrazolium dye) and determination of SOD enzyme activity. The absorbance was read at 450 nm after incubating samples for 20 min at 37 °C. Lysozyme activity was used for determination of serum lysozyme level by the method described by Hultmark et al. [33] with slight modifications. Lysozyme activity was determined by reaction against Micrococcus lysodeikticus and microplate reader (Sunrise TECAN, Männedorf, Switzerland) analysis with 450 nm. Myeloperoxidase (MPO) was measured according to Quade and Roth [34]. Briefly, 20 µL of serum was diluted with Hanks balanced salt solution (HBSS) without Ca2+ or Mg2+ in 96-well plates. Then, 35 µL o f 3.3′5.5′- tetramethylbenzidine hydrochloride (TMB, 20 mM) (Sigma-Aldrich) and H2O2 (5 mM) were added. The color change reaction was stopped after 2 min by adding 35 µL of 4 M sulfuric acid. Absorbance was read at 450 nm in the micro-plate reader.2.7. Challenge TestIn the challenge test with Aeromonas hydrophila, bacteria were incubated at 27 °C for 24 to 48 h in BHI broth medium and then suspended in sterile distilled water at 1 × 107 CFU/mL from Department of Biotechnology, Pukyong National University (Busan, Korea). The methods for bacteria culture, CFU estimation and concentration adjustment were followed as previously described by Hasan et al. [35]. Fifteen fish (triplicate groups of five fish per tank) were distributed according to their dietary treatment groups into a 50 L tank for the challenge test without water exchange. After injecting 0.1 mL of the suspension into the peritoneal cavity (1 × 106 CFU/ per fish), the survival rate of Nile tilapia was investigated according to elapsed time without feeding, and the experimental group and the control group were compared and analyzed.2.8. Statistical AnalysisAfter confirming normality and homogeneity of variance using Leven’s Test for equality of variances, data were analyzed by one-way ANOVA using the IBM SPSS 26 statistics program. The least significant difference (LSD) multiple range test was used as a post-hoc method, and the significance level was set at p < 0.05. Cumulative survival rate was presented with a Kaplan–Meier plot using the GraphPad Prism 8.4.0 (GraphPad Software Inc., San Diego, CA, USA) application. Survival curves were compared by log-rank (Mantel-Cox) for trend analysis, and Gehan–Breslow–Wilcoxon tests.3. Results3.1. Growth Performance and Survival RateAfter eight weeks of feeding trial, growth performance and survival rates of fish fed the different experimental diets were evaluated (see Table 2). The weight gain (WG) and specific growth rates (SGR) of fish fed SSE, SSEP2 and SSEP4 diets were significantly higher than those of fish fed CON and SQSE diets (p < 0.05). However, there were no significant differences in the WG and SGR of fish fed the SSE, SSEP2, SSEP4 and TSE diets (p > 0.05). Total feed intake (FI) for fish fed the SSE and SSEP4 diets was significantly higher than for fish fed the CON diet. However, there were no significant differences in FI for fish fed the SSE, SSEP2, SSEP4, SQSE and TSE diets (p > 0.05). Feed efficiency (FE), protein efficiency ratio (PER) and survival percentage were not significantly different among fish fed any of the experimental diets (p > 0.05). Hepatosomatic index (HSI), viscerosomatic index (VSI) and condition factor (CF) showed no significant differences in all the experimental groups (p > 0.05).3.2. Whole-Body Proximate CompositionWhole-body proximate compositions of Nile tilapia fed the six experimental diets are shown in Table 3. There were no significant differences in moisture, crude protein, crude lipid or crude ash for fish fed the experimental diets (p > 0.05).3.3. Hematological ParametersThe results of hematological parameters are presented in Table 4. There were no significant differences in aspartate aminotransferase (AST), alanine aminotransferase (ALT), glucose (GLU) or total cholesterol (TCHO) contents of fish fed the experimental diets (p > 0.05).3.4. Non-Specific Immune ResponsesNon-specific immune responses, including the myeloperoxidase (MPO), superoxide dismutase (SOD) and lysozyme activity of fish fed the six experimental diets, are presented in Figure 1. The SOD activity levels of fish fed SSE and SSEP4 diets were significantly higher than those of fish fed CON, SSEP2, SQSE and TSE diets (p < 0.05). MPO activity levels of fish fed SSE and SSEP4 diets were significantly higher than those of fish fed CON, SSEP2 and SQSE diets (p < 0.05); however, there was no significant differences among the former two and the TSE group (p > 0.05). The lysozyme activity levels of fish fed SSEP4 and SQSE diets were significantly higher than those of fish fed SSE and SSEP2 diets (p < 0.05). However, there were no significant differences in the lysozyme activity levels of fish fed SSEP4, SQSE, CON and TSE diets (p > 0.05).3.5. Bacterial Challenge TestCumulative survival rates of Nile tilapia fed six experimental diets and challenged with Aeromonas hydrophila (1 × 106 CFU/fish) are presented in Figure 2. At 10 days after pathogenic bacteria injection, the cumulative survival rate of fish fed the SSE diet was significantly higher than for fish fed CON, SQSE and TSE diets (p < 0.05). However, fish fed the SSE diet showed no significant differences in cumulative survival rate as compared to fish fed SSEP2 and SSEP4 diets (p > 0.05).4. DiscussionExperimental diets prepared for this research were well-accepted by tilapia, and almost no remaining feed was observed in aquaria one hour after feeding. In the present study, the results showed that the SSE, SSEP2 and SSEP4 diets could improve the growth performance compared with the control group. Interestingly, total feed intake in fish fed the SSE and SSEP4 diets was significantly higher than for the control diet, which could be attributed to the high palatability of the feeds ingested by the fish. This was reflected in terms the higher growth rate of those fish compared to those on other diets. Growth performance is an important factor with which to evaluate the palatability of feed ingredients. In agreement with the present study, Leal et al. [36] reported that dietary shrimp protein hydrolysate could improve the growth performance of Nile tilapia. Furthermore, Plascencia-Jatomea et al. [11] reported that 10% dietary shrimp head silage protein hydrolysate could improve the growth performance and feed utilization of Nile tilapia. From our previous studies, we found that supplementation of 2% SSE in animal or plant protein sourced diets had positive impacts on the growth and palatability of feeds in rainbow trout and pacific white shrimp [2,3,12]. Robert et al. [37,38] reported that high quality protein hydrolysates can be obtained from tilapia and shrimp wastes. Moreover, Hung [39] found that SSE may contain a large amount of free amino acids that act as a feed attractant which is collected from the shrimp industry. Research on some other species showed similar results using shrimp protein hydrolysates. For example, Khosravi et al. [40] reported that dietary shrimp hydrolysate could improve the growth performance in low fishmeal diets for red sea bream, Pagrus major. Similarly, Leduc et al. [10] reported that 5% dietary shrimp hydrolysate could improve growth performance, villi length and goblet cell number, while using a low-fishmeal diet in European sea bass, Dicentrarchus labrax. In sea bass larvae, dietary shrimp hydrolysate stimulated larval growth compared to the control group [41]. The functionality of nucleotides as low molecular weight compounds is well-known in terms of increasing diet palatability, immunity and disease resistance in fish; they ultimately enhance aquaculture [23]. Dietary supplementation of nucleotides including IMP showed an improvement in the growth performance of different fish species, such as grouper (Epinephelus malabaricus), rainbow trout (Oncorhynchus mykiss), Atlantic salmon (Salmo salar) and red sea bream (Pagrus major) [26,27,42,43]. In the present study, dietary IMP did not seem to affect the growth performance of tilapia. In contrast to the aforementioned studies that observed the positive effects of IMP on growth performance, Zhang et al. [44] reported no significant differences in the growth performance of gibel carp (Carassius auratus) fed IMP. Inconsistent results for the effects of IMP on fish growth could be related to different feed formulations, culture conditions and physiological characteristics of fish species. Furthermore, in the present study, dietary supplementations of high IMP (4%) together with SSE significantly increased the feed intake in fish compared to the control diet, which endorsed the high palatability of the feeds for juvenile Nile tilapia. In agreement with our study, Hossain et al. [27] found high feed intake in supplementing IMP in the diets for red sea bream. However, contrary to the present study, Kader et al. [30] could not find any positive effect of IMP on feed intake in juvenile Nile tilapia.In this study, the whole body proximate composition of Nile tilapia was not significantly affected by SSE. Khosravi et al. [45] reported that dietary shrimp hydrolysates did not affect whole body proximate composition in olive flounder, Paralichtys olivaceus. Gisbert et al. [46] also reported that dietary shrimp protein hydrolysates did not affect whole body proximate composition in European sea bass, Dicentrarchus labrax. According to these findings and the results of the present study, dietary shrimp protein hydrolysates might not have influenced the whole-body composition of Nile tilapia because the administered level was not very high (2% of the diet), and the proximate compositions of diets were almost the same.Hematological parameters are useful indicators for evaluating the physiological parameters and health status of fish [47]. Based on the results of this study, SSE did not affect the alanine aminotransferase (ALT) or aspartate aminotransferase (AST) of Nile tilapia. Similarly, Khosravi et al. [40] reported that dietary shrimp hydrolysates could not affect AST and ALT in red sea bream. The present study also demonstrated no significant differences among dietary treatments regarding serum glucose (GLU) and total cholesterol (TCHO), which is in agreement with the study conducted by Khosravi et al. [48].Fishery by-product protein hydrolysates have been reported to improve antimicrobial [38,49], antioxidant [50] or antihypertensive activities [51]. Non-specific immune responses such as superoxide dismutase, myeloperoxidase and lysozyme activity are useful parameters for evaluating health status in fish [52]. In the present research, fish fed the SSE and SSEP4 diets improved serum MPO and SOD activities compared to fish fed the CON, SSEP2 and SQSE diets. Additionally, it is worth mentioning that SQSE and TSE diets resulted in higher SOD and MPO activity as compared to the CON diet. Khosravi et al. [40] reported that dietary shrimp hydrolysates could improve SOD activity in low-fishmeal diets for red sea bream, Pargus major. However, in the present study, SSEP2 resulted in significantly lower SOD, MPO and lysozyme activity compared to SSE and SSEP4. This could have been caused by an error during the sampling or analysis, and therefore, further considerations are required in this regard. However, the lysozyme activity of fish fed the SSE and SSEP4 diets showed no significant differences with fish fed the CON diet. Gisbert et al. [46] reported that having 5% of a diet be dietary shrimp protein hydrolysate could improve serum lysozyme activity in European sea bass, Dicentrarchus labrax. The inconstancy in results of the present study and previous findings could be explained by different protein hydrolysates used, various administration dosages and target fish species.This experiment indicated that disease resistance against A. hydrophila was improved by supplementation of SSE in tilapia diet. Likewise, Bui et al. [53] reported that dietary krill protein hydrolysate could improve disease resistance against Edwardsiella tarda in red sea bream, Pagrus major. In another study, Khosravi et al. [40] reported that dietary shrimp hydrolysate could improve disease resistance against Edwardsiella tarda in red sea bream. Additionally, Khosravi et al. [48] postulated that dietary tilapia, krill and shrimp hydrolysates can significantly enhance disease resistance against Edwardsiella tarda in olive flounder, paralichthys olivaceus. In the present study, it could be corroborated that dietary crustacean hydrolysates such as shrimp soluble extract with low molecular weight hydrolysates, could improve disease resistance in juvenile Nile tilapia.5. ConclusionsTaken together, the results of the present study demonstrated that dietary shrimp soluble extract could improve growth performance, non-specific immune responses and disease resistance in juvenile Nile tilapia. Moreover, inosine monophosphate did not add further benefits to the SSE diet. Further research is warranted to better understand the effects of the additives based on nutrigenomic approaches.

animals : an open access journal from mdpi

10.3390/ani11123475

PMC8697953

The black soldier fly (BSF), Hermetia illucens (Diptera: Stratiomyidae), is renowned for its bioconversion of organic waste into a sustainable source of animal feed. Gut microbes play an essential role in aiding their host during the digestion of complex substrates by possessing metabolic properties that the insect lacks. Microbes that survive the gut passage are candidates for microbes that contribute more to larval development, besides just being a nutrient source. Insect larvae cohabit in some form of symbiosis with microbes. Here, a preliminary experiment was performed to explore the dynamics of the H. illucens gut microbiota and the changes in the composition of the bacterial community in organic waste with six different functional strains of the larval feed during rearing. The results showed that the increase in the abundance of Lysinibacillus in the experimental group that was exposed to Lysinibacillus sphaericus was significantly different to the other groups (p < 0.05). The results indicate that H. illucens larvae have a stable gut microbiome that does not change significantly during larval development, whereas bacterial communities in the feed residue with the addition of certain bacteria can be slightly affected by rearing.

Black soldier fly (BSF) larvae, Hermetia illucens (Diptera: Stratiomyidae) have emerged as an efficient system for the bioconversion of organic waste. Intestinal microorganisms are involved in several insect functions, including the development, nutrition, and physiology of the host. In order to transform the intestinal bacterial community of BSF directionally, six different potential functional strains (Lysinibacillus sphaericus, Proteus mirabilis, Citrobacter freundii, Pseudocitrobacter faecalis, Pseudocitrobacter anthropi, and Enterococcus faecalis) were added to aseptic food waste, and aseptic food waste was used without inoculants as a blank control to evaluate the changes in the intestinal microbiota of BSF under artificial intervention conditions. These six strains (which were isolated from the larval intestinal tract in selective media and then identified and screened) may be considered responsible for the functional characteristics of larvae. The results imply that the increase in the abundance of Lysinibacillus in the experimental group that was exposed to Lysinibacillus sphaericus was significantly different to the other groups (p < 0.05). The results revealed that it is feasible to transform the intestinal microbiota of BSF directionally; there are differences in the proliferation of different strains in the intestine of BSF.

1. IntroductionThe study of insect gut microbes is not only conducive to the development and utilization of insect resources, but it is also beneficial to obtaining bacterial resources with specific functions from the environment of the insect gut. Previous studies have confirmed that the insect gut (of various species) can be used as an effective source for separating important enzymes in industry, such as proteases and other productive enzyme strains [1,2,3,4]. There has been much research on the intestinal microbes of insects, including that of silkworms [5,6], termites [7], and long-horned beetles [8].In the study and utilization of insects, there are many reports of insects that combine microorganisms to degrade various organic wastes. Relatively few reports focus on changes in the structure of microbial intestinal microorganisms to promote waste degradation. Qi et al. [9] used Trichoderma viride, Saccharomyces cerevisiae, and Musca domestica to transform crop straws, evaluated their impact on housefly rearing performance, and optimized their utilization. Their results showed that the use of T. viride and S. cerevisiae to ferment crop straw can enhance the biotransformation of crop straw and improve the rearing capacity of housefly larvae.The microbes in the fly larvae gut have multiple functions that are important to larval development [10]. The functions of gut microbiota impact the development, pathogen resistance, nutrition, and physiology of the host. So far, there is little in-depth understanding of the unique intestinal biotransformation system of insects, particularly regarding the functions of the various symbiotic microorganisms in the intestine. The relationships between insects and symbiotic microorganisms, and the potential science and application values have been rationalized; however, regarding the research into mammalian gut bacteria [11,12,13], there remains much room for development concerning the compounds from insect gut microbes. Enzymes are the most important driver of diet decomposition. There are few reports on the screening of high-efficiency enzyme-producing strains from insect intestinal microbes and the degradation of various organic wastes in combination with insects [10,14,15]. Our focus, with respect to substrate composition, was on the organic compound, whereas, because bacterial communities in the feed residue are affected by the addition of certain bacteria, cellulase, protease, and lipase-secreting bacteria were selected and isolated from the BSF larvae using selection media.Black soldier fly (BSF) larvae, an important environmental insect, are saprophytic and have a wide range of food sources. BSF larvae can quickly convert organic waste (such as food waste [16,17], poultry manure [18,19,20], straw [21,22], sewage sludge [23], and organic leachates [24,25]) into stable biological fertilizers and their biomass. Additionally, BSF larvae were found to produce broad-spectrum antimicrobial peptides with different antibacterial activities according to the diet [26], which can reduce a variety of pathogens [27]. Changes in diet can also cause BSF larvae to form different bacterial communities in their intestinal tracts [28,29]. BSF larvae can improve the efficiency of conversion when the feed substrate is inoculated with single strains or mixtures of bacteria [30]. For example, inoculating Lysinibacillus boronitolerans, Kocuria marina, or Proteus mirabilis into chicken manure produced larger larvae and reduced manure residue [31]. Fly larvae are symbiotic with microorganisms in a certain form [10]. In addition to being a source of nutrition, microorganisms that survive in the intestinal tract may also greatly contribute to the development of larvae. However, few studies have evaluated their ability to colonize, or have explored the effects of isolated functional bacterial strains on intestinal microbiota.The intestinal symbiotic bacteria of BSF larvae contribute significantly to the degradation of organic waste [32]. However, our understanding of the intestinal bacteria basic symbiotic capability of BSFL remains fundamental. In the present study, we hypothesized that the H. illucens larvae have a relatively stable gut microbiome, whereas bacterial communities can be affected by rearing the feeding substrates with the isolated functional strains from BSFL gut microbiota. Therefore, the objective of this study is to ascertain the early colonization and microbial communities of insect gut bacteria, as affected by some isolated strains, based on 16S rRNA gene surveys.2. Materials and Methods2.1. Source of BSF and Food WasteThe BSF eggs, 10-day-old BSF larvae, and the food waste were provided by Younong Environmental Protection Industry Technology Co., Ltd. (Taizhou, Jiangsu Province, China). The food waste was sterilized at 121 °C for 15 min and then cooled to room temperature for subsequent testing.2.2. Isolation and Screening of Enzyme-Producing StrainsFrom each batch (of three), guts from 10 randomly selected (of approximately 1000) 10-day-old BSF larvae, were used for bacterial isolation. All isolation procedures were implemented in aerobic conditions. The surface of the 10-day-old BSF larvae was disinfected with 75% alcohol, washed with sterile water several times, and then surgically dissected. At least 10 larvae guts were sampled and homogenized. We took 0.1 g of the intestinal contents and performed a gradient dilution treatment from 10−3 to 10−8. Then, the dilution plate method and the streak plate method were used to isolate the strains. Carboxymethyl cellulose (CMC)-agar medium (CMC 0.2%, peptone 0.5%, beef extract 0.5% NaCl 0.5%, agar 2%, pH 7.0) was used for screening the cellulolytic bacteria. Milk protein medium (skimmed milk powder 1.5%, peptone 1%, beef extract 3%, NaCl 0.5%, agar 2%, pH 7.0) was used for screening the proteolytic bacteria. Neutral red oil medium (olive oil polyvinyl alcohol emulsion 12%, peptone 1%, beef extract 0.5%, NaCl 0.5%, MgSO4 0.05%, 1.6% neutral red 0.1%, agar 2%, pH 7.0) was used for screening the lipolytic bacteria. These strains were cultured separately, inoculated into 300 mL LB medium in a 500 mL Erlenmeyer flask, and cultured for 48 h at 37 °C. The cultured LB media (with bacteria) were centrifuged at 10,000× g to isolate the bacterial cells. The cells collected after centrifugation were suspended in PBS and diluted to the concentration of 108 CFU/mL in distilled water (as described previously by Yu et al. [32]) for further use in the experiments with BSF larvae. Finally, the isolation and identification mainly focused on aerobic bacteria, because anaerobic bacteria are not easy to culture in future industrial applications.2.3. Characterization and Identification of the Enzyme-Producing StrainsAfter isolation and screening, the DNA was extracted with a DNA kit (Omega Bio-tek, Inc., Ltd., Norcross, GA, USA) and then the 16S rRNA genes were amplified with polymerase chain reaction (PCR) using the pair of universal primers: 27F (5′-AGAGTTTGATCCTGGCTCAG-3′) and 1541R (5′-AAGGAGGTGATCCAGCCG CA-3′), according to previous research [33,34]. The PCR mixture (25 μL) consisted of 12.5 μL 2× Phanta Max Master Mix (Vazyme Biotech Co., Ltd., Nanjing, China), 9.5 μL ddH2O, 0.2 μm primers and 10 ng of template DNA. Thermocycling parameters included: initial denaturation at 98 °C for 1 min; 30 cycles (98 °C for 10 s, 50 °C for 30 s, 72 °C for 60 s); a final elongation at 72 °C for 5 min. The amplified products were detected using 1% agarose gel electrophoresis. The sequence determination was conducted by Sangon Biotechnology Company (Shanghai, China), and the strains were compared and detected using the tool’s (BLAST) website at the National Center for Biotechnology (NCBI) at https://novopro.cn/blast/blastn.html, accessed on July 1 2021. The nucleotide sequence data have been submitted to GenBank and the accession numbers are OK053813 to OK053818.2.4. Insect RearingBSF eggs were maintained in a constant temperature (28 °C) incubator with a relative humidity of 60–70% for hatching [35]. Approximately 2 g of BSF eggs were laid on a sterile gauze grid on sterile boxes (120 × 90 × 80 mm) containing the relevant diet. After hatching, the BSF larvae fell into the sterile food waste and were dosed with six different bacterial culture solutions (A to G, wherein A is a control; Table 1). They were kept at 28 °C with a relative humidity of 60–70% and were fed with sterile food waste until 6 days old [35]. Seven diets were used in the current study: sterile food waste was used as a control; the other six groups were fed with sterile food waste dosed with Lysinibacillus sphaericus, Proteus mirabilis, Citrobacter freundii, Pseudocitrobacter faecalis, Pseudocitrobacter anthropic, and Enterococcus faecalis (labeled A to G, respectively (Table 1)). These strains were isolated from the previous steps. Each experimental group with the bacterial strains was inoculated with 20 mL of distilled water containing 108 CFU/mL of bacterial cells, while 20 mL of distilled water without bacterial cells was added into the control group. The diets and bacteria were homogenized and the BSF larvae fell freely into them through the gaps in the gauze on each box for the 12 days spent in each container.2.5. Sample Collection and Experimental WorkAt least 500 samples were used per treatment group. Samples “A” could be collected for 7 days as a control (A1, A2, A3, A4, A5, A6, and A7), three larvae at each time (a total of 21 larvae were obtained), while the others were also taken over 7 consecutive days. A1 represents the gut microbiome of 6-day-old larvae fed with sterile FW. Meanwhile, B1, C1, D1, E1, and F1 samples were collected from 6-day-old larvae intestinal bacteria fed with sterile FW that was inoculated with Lysinibacillus sphaericus, Proteus mirabilis, Citrobacter freundii, Pseudocitrobacter faecalis, and Pseudocitrobacter anthropi, respectively. Samples “G” were fed with sterile food waste dosed with Enterococcus faecalis. After 6 days, the BSF larvae were too small to operate. Therefore, samples were taken from G3 (8-day-old larvae) and a total of 15 larvae were obtained from three replicates. Guts from three larvae per sample were pooled for DNA extraction, resulting in a total of three biological replicates per time point for each treatment. A total of 47 samples were subsequently submitted for sequencing.2.6. DNA Extraction and 16S rRNA SequencingThe DNA of all samples was extracted using E.Z.N.A.® Bacterial DNA Kit (Omega Bio-tek, Inc., Ltd., Norcross, GA, USA), following the manufacturer’s protocol and normalized to equal concentrations before downstream processing. Before DNA extraction, the collected larvae were starved for 24 h to empty their gut to reduce the feed and excrement with non-symbiotic bacteria. For each batch, a total of 141 larvae from 47 sampling points were rinsed with sterile water after alcohol cleaning, and anatomized to extract the total DNA from the gut. The DNA products from three independent batches were mixed in equal concentrations and aliquoted for analysis.The DNA samples were studied by sequencing the V3–V4 regions of bacterial 16S rRNA (Sangon Biotech, Shanghai, China) for the community profiling of the intestinal microbiotas using the 341 F (5′-CCTACGGGNGGCWGCAG-3′) and 805 R (5′-GACTACHVGGGTATCTAATCC-3′) primer set [36,37]. The PCRs were conducted using the following program: 3 min of denaturation at 94 °C, 25 cycles of 20 s at 94 °C, 20 s for annealing at 55 °C, followed by 30 s for elongation at 72 °C, and a final extension at 72 °C for 5 min. The PCR mixture (30 μL) consisted of 15 μL 2× Hieff® Robust PCR Master Mix, sterilized ddH2O, 0.2 μm primers, and 10 ng of template DNA. The resulting PCR products were extracted from a 2% agarose gel and further purified using the Hieff NGS™ DNA Selection Beads (Yeasen Biotechnology Co., Ltd., Shanghai, China) and quantified using a Qubit3.0 Fluorometer (Invitrogen, Carlsbad, CA, USA), according to the manufacturer’s protocol. According to the standard protocols provided by Sangon Biotech (Shanghai, China) Co., Ltd., purified amplicons were pooled in equimolar concentrations and sequenced on an Illumina MiSeq platform (Illumina, San Diego, CA, USA) in PE300 mode.2.7. Bioinformatics and Statistical AnalysesThe bacterial 16S rRNA gene amplicon libraries were prepared using Illumina Miseq™ and were converted into sequenced reads using base-calling analysis. PRINSEQ (Argonne National Laboratory, Argonne, IL, USA) was employed to filter, reformat, and trim the genomic sequence data [38]. Low-quality sequences (Q < 20) were discarded. Quality trimming, chimera checking, singleton removal, and assignment of the obtained sequences to operational taxonomic units (OTUs) at 97% similarity level were conducted using Usearch v.11.0.667 [39]. The taxonomic affiliation of the resulting OTUs was identified by using RDP classifier [40]. A representative sequence for each OTU was filtered for deeper annotation. The microbial diversity, the standard diversity, and the richness indices (Ace, Chao1, and Shannon indices, respectively) were investigated using the Mothur software [41]. Intergroup comparisons were performed using a one-way analysis of variance (ANOVA), followed by Scheffe’s post hoc test (p < 0.05). STAMP software 6.0 [42] and R software 5.5 were adopted to analyze the bioinformatic sequence data sets. Downstream analysis was performed with Mothur [41] and the R package Agricolae (v.3.6.0). OTUs that were differentially abundant among the seven groups were assessed using the LEfSe approach [43].3. Results3.1. Isolation of Organic Compounds Degrading BacteriaCellulase-, protease-, and lipase-secreting bacteria were selected and isolated from the BSF larvae using selection media containing each organic compound. Six strains were identified by using 16S rRNA sequence alignment as follows: the strain for lipase and cellulase activities was identified as Lysinibacillus sphaericus with 100% identity; the three strains for protease activities were identified as Enterococcus faecalis with 99.79% identity, Proteus mirabilis with 99.79% identity, and Citrobacter freundii with 99.72% identity; two strains for cellulase activities were identified as Pseudocitrobacter faecalis with 99.58% identity and Pseudocitrobacter anthropi with 99.93% identity (Table 1).To obtain the optimal pH for bacteria to grow, the strains were grown in LB medium with a pH between 2.0 and 8.0. There were three replicates for each strain and each pH value. Alternatively, the pH for Lysinibacillus sphaericus growth was 2.0 to 8.0. As shown in Figure 1, Lysinibacillus sphaericus grew better under alkaline conditions. The strong acid environment was more suitable for Pseudocitrobacter faecalis growth, while the other strains were more adaptable to a neutral environment (Figure 1).3.2. Taxonomic Composition of Bacterial CommunitiesThe resulting libraries contained an average of 86,139 16S rDNA sequences per sample, with an average length of 428 bases. Out of the bacterial phyla that were identified (Figure 2A), Proteobacteria, Firmicute, and Bacteroidetes were the most dominant phyla that were associated with BSF of all samples. The phyla of the bacterial communities in Group A consisted of Proteobacteria (83.80%), Firmicutes (14.00%), and Bacteroidetes (0.66%). The phyla that were found in Group B were Proteobacteria (82.66%), Firmicutes (16.75%), and Bacteroidetes (0.18%). The microorganism compositions of the other groups with other strains are listed in Table S1.Out of the bacterial genera that were identified (Figure 2B), 19 bacterial genera that shared the seven larval groups were identified. Ignatzschineria, Providencia, Proteus, Klebsiella, and Vagococcus were the most dominant genera that were associated with the BSF larvae of all samples. In Group A, the proportions of these five bacteria represented 49.69, 14.93, 9.20, 5.02, and 3.08% of the total, respectively (Table S2). The contents of Proteus, Morganella, Bacillus, Paenalcaligenes, and unclassified Enterococcaceae in Group B were 12.3, 5.01, 1.09, 1.63, and 1.61%, respectively, which were all higher than the blank control (Group A) and other groups. In Group C, the top five strains were Ignatzschineria, Providencia, Proteus, Vagococcus, and Klebsiella. Ignatzschineria and Providencia were the two most dominant strains in all of the experimental groups. In Group D, Enterococcus was the third most abundant bacteria, rather than Proteus (as was the case in the other groups). In addition, the number of Ignatzschineria and Enterococcus in Group D was the largest among all groups. Furthermore, the abundance of Proteus in Group F was the highest.3.3. Differences in Intestinal Bacterial Communities over TimeAt the genus level, Ignatzschineria was the least abundant among the seven groups on the first day of sampling. In the first two days of sampling, Proteus and Klebsiella were the main components of the intestinal bacteria (Figure 3). As feeding days changed, the number of Ignatzschineria in Group B and D showed a rising trend from day 1 to day 4, and then decreased to day 7. Alternatively, it demonstrated the same trend in the first five days in other groups as it did in the previous two groups, but there was a rapid increase on the sixth and seventh days, until it reached a maximum on the seventh day.Using Welch’s t-test and ANOVA significant difference analysis, the six groups of colonizing strains were compared with the blank experimental Group A. The comparison found that the amount of the other five strains did not change significantly, with the exception of Lysinibacillus sphaericus in Group B—Lysisnbacillus in Group B was 0.66% and, in the other groups, it was below 0.07% (Figure S1). At the same time, the characteristics of the bacteria were found to be significantly different between the seven groups (Figure 4) in the linear discriminant analysis effect size (LEfSe) analysis. As there was no significant difference between Group D and G and the control group, it is not shown in Figure 4. Consistent with the results of Welch’s t-test significant difference analysis, Lysinibacillus sphaericus was found to be a particular component of Group B. Based on 16S rRNA sequencing and OTU contributions from the abundant phyla and others for facultative, anaerobic, and aerobic bacteria according to BugBase analysis, the microorganisms in the larvae gut were revealed (Figure 5). Most of the genes were obtained on aerobic and facultatively anaerobic bacteria. Generally, facultative anaerobes can grow under conditions with or without oxygen, but prefer aerobic conditions.4. DiscussionIn this study, six functional strains were isolated in total: Lysinibacillus sphaericus, Proteus mirabilis, Citrobacter freundii, Pseudocitrobacter faecalis, Pseudocitrobacter anthropi, and Enterococcus faecalis. The colonization experiments showed that Lysinibacillus sphaericus may be grown and accumulated effectively in the gut of BSF larvae (Figure 3). In other words, the colonization method we adopted was proven to be a potential method for certain microbes.Lysinibacillus sphaericus (formerly known as Bacillus sphaericus) is a Gram-positive, spore-forming bacterium that has been used in the biological control of mosquitoes and bioremediation [44,45], and was initially isolated from an adult black fly in Nigeria [46]. Lysinibacillus fusiformis was previously isolated from the eggs of the BSF colony, and could dominate the larval microbiota and increase larval weight and survival [30]. Lysinibacillus sphaericus can be found in a variety of environments, such as soil, water, and animal intestines. Chantarasiri et al. [47] reported that the Ligninolytic bacterium JD1103 was isolated from soil samples that were collected from the wetland ecosystems in Rayong Province, Thailand and was identified as Lysinibacillus sphaericus JD1103 based on 16S rRNA sequence analysis. Similarly, it was reported that lignin-degrading bacteria are not well understood. In their research, an effective lignin-decomposing bacterial strain, BR2308, was isolated from the coastal wetland ecosystem and named Lysinibacillus sphaericus BR2308 [48]. Most reports showed that Lysinibacillus sphaericus can produce insecticidal proteins that have high activity against mosquito larvae [49,50]. However, it is one of the functional cellulase-producing strains that we screened in the BSF larvae gut using CMC-Na solid medium. Others have isolated cellulose-producing Lysinibacillus sphaericus MTCC No. 9468 from the gut of earthworms (Eisenia foetida) [51].Several researchers have found that environmental and feeding sources have a significant influence on the overall composition of the insect microbial community [14], such as Hermetia illucens intestinal microbiota [28,52,53] and Drosophila melanogaster gut microbiota [54]. Thus, before sequencing data analysis, it was supposed that all of the group samples would have more target bacteria in them because the larvae were fed sterile food waste with different target bacteria. The inoculation of the substrate with substrate-associated microorganisms affected larval performance and caused major changes in larval and substrate microbiota, whereas egg-associated microorganisms did not influence performance [30]. De Smet et al. [55] found that BSF larvae can exhibit delayed performance on sterile substrates and some bacteria participate in intestinal digestion and nutrient utilization. However, the substrate has a significant effect on larval gut bacterial community composition.To reduce the external influence, larvae that were fed with autoclaved food waste were used as the control in this study. Yang et al. [56] found that the BSF larval gut microbial community structure was significantly influenced by starving, even over a short time (e.g., 24 h). To reduce the influence of food residues in the intestine on intestinal microorganisms, according to a recent study [57], the collected larvae were starved for 24 h to allow for egestion of their ingested contents before DNA extraction.Interestingly, after conducting colonization experiments of six functional strains, only Lysinibacillus in Group B showed a significant increase in bacterial communities. The others were similar to the blank control group (Group A), and the gut microbiome remained more stable. In particular, the content of Lysinibacillus sphaericus in Group B was found to account for 0.66% of the total in all sampling sites, which was significantly higher than the other groups (p < 0.05). Bacteria serve directly as food for fly larvae and help decompose macronutrients [10]. Such decomposition of microbes via gut-based mechanisms (including pH, enzymes, and antimicrobial proteins) can explain the selective inactivation of microbes, as reported for fly larvae [10]. Escherichia coli and Bacillus subtilis through the gut passage were identified as having been completely inactivated by using the fluorescent bacteria, and Enterococcus faecalis for BSF larvae caused low reductions in both the larvae and residue [10]. Microbe inactivation by fly larvae depends on the specific microbe and strain. Microbes that survive the gut passage are candidates for application. Others reported that microbial colonization depends on different physico-chemical conditions in the lumen of different gut compartments, which shows extreme changes in pH and oxygen availability [14]. The optimum pH for the growth of Lysinibacillus sphaericus, Proteus mirabilis, Citrobacter freundii, Pseudocitrobacter faecalis, Pseudocitrobacter anthriopi, and Enterococus faecalis was approximately 8.0, 6.0, 7.0, 4.0, 7.0, and 6.0, respectively. It can be seen from the above data that Lysinibacillus sphaericus is alkali tolerant. In addition, after the experiment, it was found that Lysinibacillus sphaericus is aerobic and the others are facultative anaerobes. The results of 16S rRNA sequencing predicted that the aerobic bacteria in the gut of BSF larvae account for more than 50% (Figure 5). According to the previous research, the anterior region of the mid-gut of BSF larvae has an acid luminal content, the mid-region presents a strongly acidic pH, and the posterior region has an alkaline luminal content [28]. The environment of the posterior region allows Lysinibacillus sphaericus to grow well. In addition, although many insects will shed the exoskeletal lining of the foregut and hindgut during their molts, most of the changes do not cross into the space that is adjacent to midgut epithelial cells [14]. Moreover, many insect guts display specialized crypts or paunches that promote microbial persistence [14]. Taking the evidence into account, it was inferred that that this may be the reason that Lysinibacillus sphaericus could colonize in the gut of the larvae, while the other strains cannot.Proteobacteria, Firmicutes, and Bacteroidetes were the dominant phyla (Figure 2) in the larval gut, which is consistent with the main taxa of the intestinal microbes of BSF that have been found elsewhere. Others have assessed the effects of different diets and their microbial community on the mid-gut microbiota of BSF larvae and found that, at the phylum level, Proteobacteria, Firmicutes, Bacteroidetes, and Actinobacteria were the most important microbes in the gut [28]. In previous studies, Actinobacteria, Firmicutes, Bacteroidetes, and Proteobacteria were also found to represent the most abundant phylum-level compositions in the gut of larvae [10,53]. The dominance of bacterial communities among the groups of BSF larvae that were fed different diets showed that the type of food and the feeding time could slightly influence bacterial diversity (Figure 2). Previous studies show that the dominating genera (mainly Bacterioides, Dysgonomonas, Morganella, Enterococcus, Providencia, Klebsiella, and Bacillus) have a large variability between BSF larvae and studies, which is likely due to the variability in diet [14,53,58,59]. In the present work, similar results were found in that Ignatzschineria, Providencia, Proteus, Klebsiella, and Vagococcus were in the top five at the genus level. Compared to the intestinal microorganisms of other insects, these bacterial communities are unique [60]. Chaobing Luo et al. [61] discovered that Lactococcus, Enterococcus, Bacillus, Citrobacter, Vagococcus, and Serratia are the main components of the bamboo snout beetle Cyrtotrachelus buqueti intestinal bacteria. One et al. [62] found that, in mealworms that were fed with wheat bran, the majority population in the mealworm gut included Enterococus, Clostridium, Erwinia, and Lactococcus. In addition, in the mid-gut of Aedes aegypti, Pseudomonas, Pseudoalteromonas, Luteibacter, and Rhodanobacter were the most abundant and dominant populations [63]. The composition and abundance of bacterial communities in the gut of insects are not only affected by diet, but also by the species of insect. Therefore, it is important for us to study the composition and function of the intestinal microorganisms of BSF larvae. In this study, the structure of the intestinal microorganisms of BSF larvae was scarcely affected by the colonization of functional strains.Others have reported that exogenous bacterial inoculation oxidized fiber and assisted the joint action of BSF larvae and the gut microbiome, thereby increasing the rate of bioconversion [64]. Others have conducted similar experiments with endogenous bacteria to assess the co-conversion efficacy in BSF larvae after the development of soybean curd residues with Lactobacillus buchneri [65]. They inoculated Lactobacillus buchneri into the feed for fermentation and then used it to feed the BSF larvae to study the changing parameters, such as the reduction rate of dry matter and the biotransformation rate after feeding. On the contrary, in order to better digest, decompose, and utilize various organic wastes by BSF larvae, a co-conversion technique was established. The result provides a practical and promising method for converting organic wastes into BSF larval biomass.5. ConclusionsThe functional bacterial strains that were derived from the intestinal tract of the insects were isolated and screened. We then enriched and cultivated the specimens in vitro, and then colonized the intestines of BSF larvae. The colonization effect was evaluated based on changes in the abundance of intestinal microorganisms after sequencing. Endogenous bacteria were used to construct a synergistic transformation system of ‘BSF larvae-functional microorganisms’ through the ‘directed transformation’ of the intestinal microorganisms of BSF larvae. We inoculated bacteria and fed the BSF larvae at the same time to determine the effects of endogenous bacterial agents on the intestinal microorganisms of BSF larvae. Finally, Lysinibacillus sphaericus may be allowed to colonize the gut of BSF larvae. The results indicate that the inoculation method may be a potential pathway to transform the intestinal microorganisms of the BSF directionally. Further research (e.g., of the gastrointestinal tract) is needed to investigate the cause of the partial variability of intestinal bacteria and the colonization of some bacteria.

animals : an open access journal from mdpi

10.3390/ani13061049

PMC10044252

Human guardians and companion animals develop special and unique bonds. Therefore, witnessing the terminal illness and subsequent death of a companion animal can be a stressful experience for human guardians. Professionals in the veterinary and psychological sciences can support human guardians through the caring and grieving processes. The aim of this research was to validate in the Italian context the HHHHMM Quality of Life Scale, which is specifically used to help human guardians assess companion animals’ quality of life. To this end, other scales and open-ended questions were adopted to test hypotheses and deepen understanding of the grieving experience. The results confirmed the usefulness of the scale and highlighted important correlations between age, bereavement, and attachment. Further, a thematic qualitative analysis revealed the importance of the relationship between the human guardian and the veterinarian as well as the need for social support after the loss. The findings clearly showed that the bond between a human guardian and a companion animal does not cease after the loss of the animal; rather, it continues in new forms. Overall, the present research confirmed the importance of the veterinary and psychological sciences working together to provide complete support for human guardians.

Witnessing a companion animal’s death can be a stressful psychological experience for human guardians, affecting their ability to grieve. The veterinary and psychological sciences offer useful tools for supporting human guardians during their companion animal’s terminal illness. Accordingly, the present study aimed to validate the HHHHMM Quality of Life Scale in the Italian context. The study followed a mixed-methods design and involved 314 participants. The Mourning Dog Questionnaire (MDQ), Lexington Attachment to Pets Scale (LAPS), Pet Bereavement Questionnaire (PBQ), and open-ended questions were adopted to test the research hypotheses and qualitatively explore the grieving experience. The results showed that the model’s fit was partially adequate, with all parameters being significant and over 0.40. Moreover, human guardians’ anger levels were high when their companion animal’s quality of life was poor, and greater levels of grief were associated with higher levels of attachment. Gender differences were observed only with the LAPS, and a negative correlation with age was found with the LAPS and PBQ. A thematic qualitative analysis revealed four themes: continuing bonds, coping strategies, shared moral values, and perceived support. Thus, the research reaffirmed the importance of adequate veterinary and psychological support for human guardians experiencing the loss of companion animals.

1. IntroductionBereavement is a normal psychological experience that is prompted by the death of a significant one [1] and is characterized by complex, multifaceted feelings of grief as well as different mourning practices [2]. In modern times, the ecological cohabitation of humans and other animals has increased to the degree that companion animals represent fundamental members of the domestic space. Indeed, people often develop deep emotional connections with companion animals, and almost all human guardians in Western societies consider them family members [3]. Therefore, recent studies have recognized that human guardians may experience considerable grief over the loss of companion animals [4,5].Gerwolls and Labott [6] found the grief after the loss of a companion animal and the grief after the loss of a human being to have comparable intensities, whereas Hunt and Padilla [7] stated that the bereavement associated with the loss of a companion animal may even be even greater than that experienced following the death of a person [8]. Moreover, the quality of the relationship and, more specifically, the degree of attachment between a human guardian and a companion animal [9] is a key factor determining the psychological burden the human guardian experiences due to companion animal loss [10]. However, despite the frequency and intensity of the grief that people experience due to companion animal loss, this phenomenon lacks proper social recognition, and there is little opportunity for adequate support [11]. As a matter of consequence, the bereavement process usually assumes the form of disenfranchised grief—that is, grief which is not socially acknowledged and becomes a source of additional suffering for the bereaved [5]. Cordaro [12] argued that this disenfranchised grief has three major causes: considering the bereavement over a companion animal unacceptable, believing that the individual can quickly cope with grief and easily replace the lost companion animal, and not considering the mourning experience as authentic. An eloquent example is the dog–guardian relationship. The literature shows that human guardian–dog relationships provide the same, or even greater, sense of comfort, security, and affection that close human relationships do [3]. Several variables influence people’s experiences upon the loss of their dog, including the nature and quality of their attachment bond, the quality of social support received for the bereavement, and the circumstances under which the death occurred [13]. It should be noted that dogs live much shorter lives than humans. Consequently, human guardians may experience multiple such losses and repeated grief, which may overwhelm their normal ability to adapt.Since grief due to the loss of a companion animal can be a painful experience, it is of the utmost importance that experts in the psychological and veterinary fields develop targeted psychological instruments to support the bereaved [4,5]. In recent years, there have been increasing calls for veterinarians to provide tools to assess the quality of life (QoL) of elderly or terminally ill companion animals and thereby prevent or ease their suffering. Veterinary medicine now comprises a wide range of technologies and treatment options that make it possible to prolong a companion animal’s life. However, due to the diversity of beliefs that influence veterinarians and human guardians, they are increasingly confronted with ethical dilemmas regarding the appropriateness of available procedures [14]. Continuous progress in veterinary medicine has led to the optimization of decision-making processes affecting the last stages of companion animals’ lives. The veterinarian no longer replaces the client in decision-making but takes on the role of educator and consultant to maintain a balance between animal welfare and client perspective. This mirrors the American Veterinary Medical Association guidelines [15].Managing a companion animal’s prolonged illness can be complex and time consuming. Recent research shows that the caregiver burden—the distress that emerges when providing care for an individual with an illness [16]—is a frequent occurrence among human guardians and those who care for animals with a chronic or terminal illness [17,18]. This burden is linked to multiple negative psychosocial consequences, including high levels of stress, symptoms of depression and anxiety, and a low quality of life. The importance of understanding clients’ experiences with caring for elderly or sick animals is evident. Caregiver burden appears not only to be a cause of stress for clients but also to play an active role in influencing euthanasia decisions [19]. When clients feel overwhelmed, it is necessary to corroborate their experiences, both in the long-term duration of the companion animal’s illness and in considering euthanasia.There is currently a lack of studies on QoL assessments for pets at the end of their lives [20]. One such assessment is the HHHHHMM (hurt, hunger, hydration, hygiene, happiness, mobility, more good days than bad) Quality of Life Scale developed by Villalobos [21]. The tool guides people who are attached to their companion animals in considering the state of their companion animal’s health and in assessing whether they are able to provide sufficient care for their sick companion animal. It provides an overall assessment of the daily life of a sick animal suffering from a chronic and progressive disease, evaluating aspects such as pain, hunger, hydration, hygiene, and movement. This is an accessible and easy-to-understand tool for assessing the QoL of terminal animals.The present research had three specific objectives. The first was to validate the HHHHMM scale in the Italian context. The second was to identify the predictors of QoL based on the characteristics of the companion animal and the impact of the relationship between QoL, attachment, and human guardian/companion animal characteristics and human guardians’ bereavement-related distress. Finally, a qualitative perspective was adopted with the aim of exploring participants’ experiences with companion animal loss.2. Materials and Methods2.1. Aims and HypothesesThe purpose of the present study was to validate the HHHHMM Quality of Life scale [21] in the Italian context. In addition, the research aimed to identify the predictors of QoL based on the characteristics of the companion animal and its living environment, investigated using questionnaires. Moreover, the research aimed to assess the impact of QoL and attachment together with human guardian and animal characteristics on human guardian distress due to the death of a companion animal. Finally, open-ended questions were qualitatively analyzed to explore participants’ experiences with companion animal loss.Upon considering other measurement tools (see below), it was hypothesized that human guardians’ levels of emotional attachment to their companion animals constitute the main predictor of bereavement experiences and that the level of emotional attachment mediates the relationship between the human guardian’s gender and bereavement.2.2. Study DesignA mixed-methods design was adopted for this study [22], bringing together the potential of quantitative and qualitative inquiry. This design integrates quantitative and qualitative methods to gain a deeper, more sensitive understanding of the phenomenon under investigation. This paper first presents the quantitative results and then illustrates the qualitative results obtained from the thematic analysis.The questionnaires for the study were formed from the HHHHMM Quality of Life Scale and three other scales.The first questionnaire used was the Mourning Dog Questionnaire (MDQ) [23], which consists of two sections. The first section is to gather the demographic data of the human guardian, such as gender, age, education level, marital status, occupation, and presence of children. The second section focuses on information related to the deceased dog, specifically age, gender, cause of and time since death, length of cohabitation with the human guardian, and whether the human guardian was living alone at the time of the dog’s death.Item example: Which of the following best describes the role played by this animal in your relationship? Partner/friend/source of protection/animal from work/family (child)/family (brother/sister)/simply an animal.2.The second questionnaire was from the Lexington Attachment to Pets Scale (LAPS) [24]. This is a 23-item scale developed by Johnson et al. in 1992 with the aim of creating a reliable instrument to assess human guardians’ level of emotional attachment to their companion animals. Participants answered the questions by expressing their degree of agreement on a three-point Likert scale (from “strongly agree” to “strongly disagree”) for each of the following factors: general attachment, animals substituting people, and animal rights/welfare. The latter factor assesses the perception of a companion animal’s moral status within the household. In the present study, the focus was only on this scale’s total score (LAPS total alpha = 0.916).Item example: I love my dog because he/she never judges me.3.The Pet Bereavement Questionnaire (PBQ) is a questionnaire designed by Hunt and Padilla [7] that consists of 16 items with a four-point Likert response scale (0 = strongly disagree, 3 = strongly agree). The PBQ assesses grief related to the death of a companion animal by considering three different factors: grief (items 2, 3, 5, 7, 10, 12, 15), anger (items 1, 4, 11, 13, 14), and guilt (items 6, 8, 9, 16). Reliability indices were good for this instrument, with Cronbach’s alpha coefficients ranging between 0.67 and 0.86 (PBQ grief alpha = 0.84; PBQ anger alpha = 0.67; PBQ guilt alpha = 0.77; PBQ total alpha = 0.86).Item example: I am angry with the veterinarian because he/she failed to save my dog.4.After completing the MDQ, LAPS, and PBQ questionnaires, the study participants filled out the HHHHHMM Quality of Life Scale [21]. This scale is divided into seven criteria which form the acronym of its name: hurt, hunger, hydration, hygiene, happiness, mobility, and more good days than bad. It helps human guardians assess their companion animal’s QoL using a scale from 1 to 10 (1 = unacceptable, 10 = excellent) and make informed decisions about the therapeutic treatments to follow.Item example: HYGIENE: Did the dog have bedsores?Finally, the participants were asked to answer four open-ended questions, which were analyzed using a bottom-up thematic analysis [25]. The bottom-up approach involves taking the empirical data as the starting point of the analysis, meaning that no preexisting categories or theoretical concepts are used to analyze the data. Then, the analysts gradually generate categories and themes based on the participants’ words, constantly and recursively checking their adherence to the participants’ perspectives [22,25]. For the present study, two members of the research team independently performed the analysis, which comprised three fundamental phases. During the first phase (creation of codes or coding), analysts created codes using the participants’ words, paying particular attention to ensuring that the codes were strongly linked to the data. During the second phase (creation of categories from codes), the analysts worked directly with the codes and matched them with self-organized categories based on pertinence and similarity criteria [22,25]. During the last phase (thematic generation), the two analysts worked together, and a third member of the research team acted as a judge when any disagreements occurred. The open-ended questions were as follows: Could you describe the changes that occurred in your daily routine, in the short and long term, after the loss of your companion animal?Do you plan to welcome another companion animal into your life, or have you already done so? If yes, can you indicate the reasons for your choice?How would you describe your relationship with the veterinarian who cared for your companion animal, and his/her approach?How did the people closest to you receive the news of your dog’s death? From whom did you receive support? From whom would you have expected more/greater closeness?2.3. ParticipantsThe present study involved individuals residing in Italy who were owners of companion animals that had been affected by chronic, progressive diseases in their later lives. The following inclusion criteria were adopted: having experienced the death of a companion animal and being over 18 years of age. Each eligible participant received a clear explanation of the research objectives and procedure. Participation was voluntary, and participants were given the option of withdrawing at any time. Research participants were recruited through the database of the San Marco Private Veterinary Clinic (Padova District, Veneto Region). A total of 314 adult participants responded positively; among them, 257 were females, 52 were males, and five chose the option “Other” for their gender. Thus, the sample consisted mainly of women (81.8%). The average age of the participants was 44.42 years (SD = 12.15), ranging from 19 to 77 years, and 83.4% were from Northern Italy (262 subjects), while the remaining 16.6% (52 subjects) were from Central and Southern Italy. Out of the total participants, 295 completed the HHHHHMM scale, and 311 completed the PBQ.The participants were sent a link through which they could access an online survey lasting about 20 min. A total of 242 participants completed the survey. It could be filled out comfortably using either a PC or a smartphone. The research involved a combination of four standardized questionnaires for the factors stated in the objectives.The study followed the American Psychological Association Ethical Principles as well as the Declaration of Helsinki. Moreover, it was approved by the University of Padova Ethics Committee (Ethical Code AAE32A070588F8595C8F06988799321C).3. Results3.1. Quantitative Analysis3.1.1. Confirmatory Factor AnalysisThe HHHHHMM Quality of Life Scale comprises seven items to measure animals’ wellbeing. A confirmatory factor analysis (CFA) was performed with these seven items to test a one-factor model. The model showed a partially adequate fit, and all parameters were significant (p < 0.001) and over 0.40. The standardized parameter estimates for the seven items were 0.48, 0.79, 0.75, 0.43, 0.72, 0.73, and 0.69, respectively. An examination of modification indices indicated two error correlations: one between items 2 and 3 (r = 0.48) and the other between items 3 and 4 (r = 0.49). The two error correlations were included in the modified model because the correlation between these items was supported by the common theme of nutrition for item 2 and item 3 and by the common reference to sores for item 3 and item 4. The new model including these two error correlations showed a good fit. Cronbach’s alpha for the scale was 0.78, indicating good internal consistency. The mean total score was 41.44, and the standard deviation was 15.10. The scale distribution was close to a normal distribution; in fact, it was symmetric but with a slight deviation of the tails (skewness = 0.09 and kurtosis = −0.60). For most human guardians, the dogs’ QoL in the period immediately preceding their death was adequate; for 20% of the participants, it was unacceptable (Table 1).3.1.2. Correlations between the Study VariablesThe scale QoL scale showed (Table 2) only a low negative correlation with anger in the PBQ (r = −0.11, p = 0.057), indicating that the human guardians felt more anger when their dogs’ quality of life was poor (Table 2).The LAPS and PBQ results showed small to moderate positive correlations. In particular, the human guardians experienced greater levels of grief upon their dog’s death when their attachment levels were high (r = 0.47, p < 0.001). With regard to human guardians’ characteristics, gender differences were observed only in the LAPS results, and a negative correlation with age was seen in the LAPS and PBQ results. In particular, females showed greater attachment levels than males (t = 3.76, df = 312, p < 0.001), and older owners had low attachment levels (r = −0.13, p = 0.019) and experienced less distress over their dog’s death (r = −0.16, p = 0.005 for grief; r = −0.18, p = 0.002 for anger; r = −0.31, p < 0.001 for guilt; and r = −0.26, p < 0.001 for PBQ total score). Although the results indicated that the human guardian’s gender had no direct effect on the PBQ, the indirect effect of gender on the PBQ, through the LAPS, was always significant (beta = 0.26, p = 0.001 for grief; beta = 0.13, p = 0.007 for anger; beta = 0.09, p = 0.024 for guilt; and beta = 0.21, p = 0.002 for PBQ total score). Moreover, the possibility of living in a house with a garden was found to increase dogs’ quality of life (t = 2.33, df = 293, p = 0.021) and reduce human guardians’ guilt during bereavement (t = −2.06, df = 309, p = 0.040), with respect to those who did not have this opportunity.Regarding dogs’ characteristics, only one correlation involving sexual status was observed in the LAPS results; human guardians of desexed dogs were found to have greater attachment levels than human guardians of intact dogs (t = 2.11, df = 312, p = 0.036). Furthermore, a correlation between dogs’ QoL and the practice of euthanasia was noted; specifically, euthanasia practice was associated with low QoL (t = −2.17, df = 293, p = 0.031). Several correlations were observed in the PBQ results: Anger was negatively correlated with dog’s age at death (r = −0.16, p = 0.005) and with time spent together by the human guardian and the dog (r = −0.11 p = 0.046), indicating that the human guardians experienced more anger when the dog died at a young age and when they had spent less time together. Further, grief was associated with the dog’s age at acquisition, with human guardians experiencing high levels of grief when dogs were young at the time of acquisition (less than three months; t = 2.30, df = 309, p = 0.022). Differences in anger and in the PBQ total score were observed based on whether the dog’s death was expected; the human guardians experienced greater distress when their dog’s death was unexpected than when it was expected (t = 3.14, df = 309, p = 0.002 for anger; t = 2.00, df = 309, p = 0.046 for the PBQ total score). Finally, differences were noted in the guilt factor and PBS total score based on the practice of euthanasia; lower scores were obtained when euthanasia was practiced than when it was not (t = −3.68, df = 309, p < 0.001 for guilt; t = −2.25, df = 309, p = 0.025 for PBQ total score).3.1.3. Regression Model to Explain the PBQThe results of the first regression analysis model confirmed that attachment to one’s dog is always a significant predictor of the PBQ total score and individual factors, with high attachment levels associated with high distress (Table 3).The human guardian’s age was found to be a significant predictor of the PBQ total score and of guilt; higher ages were associated with lower distress levels. The practice of euthanasia was a significant predictor only of guilt, with low levels of distress experienced when euthanasia was practiced. Dog’s age at acquisition was a significant predictor only of grief, with higher distress levels associated with younger ages. The results of the second regression analysis model showed that three factors had significant interactions with human guardians’ attachment levels: the human guardian’s age, whether the dog’s death was expected, and the dog’s age at death. In particular, the slope analysis of the LAPS for human guardian’s age showed a high impact of LAPS on all PBQ measures for younger human guardians (beta = 0.66, p < 0.001 for grief; beta = 0.50, p < 0.001 for anger; beta = 0.42, p = 0.003 for guilt; beta = 0.66, p < 0.001 for total score) than for older guardians (beta = 0.41, p = 0.007 for grief; beta = −0.08, p = 0.638 for anger; beta = −0.23, p = 0.140 for guilt; beta = 0.17, p = 0.292 for total score). The LAPS slope analysis also showed a higher impact on grief and PBQ total score when the dog’s death was unexpected (beta = 0.55, p < 0.001 for grief; beta = 0.47, p < 0.001 for total score) than when it was expected (beta = 0.37, p < 0.001 for grief; beta = 0.26, p = 0.002 for total score). Finally, the LAPS slope analysis for the dog’s age at death showed a higher impact on anger for younger ages (beta = 0.47, p < 0.001) than for older (beta = 0.01, p = 0.937).3.2. Qualitative AnalysisThe qualitative thematic analysis revealed four major themes: continuing bonds, coping strategies, shared moral values, and perceived support.3.2.1. Continuing BondsThis theme indicates that the death of a companion animal affects multiple dimensions of the human guardian. Indeed, the companion animal continues to be seen and heard, perceived through an eternal bond of love, and its death changes the human’s management of time, space, and behavior. This theme is composed of three categories: spirituality, emotions, and behaviors. The first refers to how death shapes the form and intensity of feelings over time. The following is an extract expounding on this category:“It is difficult to describe what I feel. I lost my little girl, my soulmate. I have two other dogs that I love madly, but none will ever be like my Naira; she was, and she is, full stop. We all suffered at home. Luckily, I had the other dogs and my son near me; otherwise, I would have had a really bad time. For weeks, I kept calling her even after she was gone, looking for her, seeing her shadow in front of the door, preparing her bowls. I have her urn next to me in bed, and she still sleeps there with me and like that forever. My routine has not changed much because, anyway, as I said, I have two other dogs and, to a greater or lesser extent, the things I do with them, I also did with her. The routine I changed most was at the beginning when Raoul, one of the two, went into a crisis. I could no longer keep him alone at home when he went out because he barked with pain. Gradually, our daily life started again, but a huge piece of me left with her. I am sure that my love for her will not end in nothingness; she is definitely with me. Indeed, the night she left, the tap in the bathtub turned on by itself. I will miss her for all my life.”The “emotions” category refers to how participants have spoken about their intimate feelings after the death of their companion animal. A giant void characterizes all of their words.“After Mila’s death, I tried to go on taking care of the other dog, Samvise. It was very hard. Sam lost the will to eat. But I accepted her [Mila’s] departure, despite feeling an emptiness inside. Sometimes, I feel like she has been dead for a minute—so much it hurts—sometimes in another life. I just prepare a bowl; I feel like I have nothing to do. I no longer worry about the thunderstorms that used to scare her.”Finally, the “behaviors” category highlights the short- and long-term behaviors of the human guardians after the death of their companion animal.“The first few days after her loss, I went into a state of apathy. After dropping the children off at school and kindergarten, I would come home, make myself a cup of tea, and sit on the sofa doing nothing. Slowly, the pain became less intense, and I was able to resume doing small chores. After seven months, it is better, and life is back to its usual pace and rhythm. But I feel her absence, and my children mention her often.”3.2.2. Coping StrategiesThis theme emerged from the participants’ views on the possibility of adopting another companion animal after the loss. Two prevailing strategies were highlighted: (i) the orientation towards a new adoption (this has already happened for some participants) as a way of supporting the management of mourning, or (ii) an open rejection, perceived as permanent or temporary, carried out of respect for the animal that has just died and is considered irreplaceable or out of the need for solitude in the experience of grief. The theme is composed of two categories: desire and rejection. The desire for another companion animal demonstrates a positive orientation characterized by the anticipation of new and positive emotions (happiness and joy) as well as a need to dampen negative emotions (to fill a void and manage psychological distress). The refusal to adopt another companion animal is a negative orientation for various reasons, such as the presence of other animals, the need for family reorganization, a sense of guilt, or the irreplaceability of the dead animal; thus, adopting a new companion animal is not viewed as a way of forming new bonds but as a loss of continuity of the relationship with the dead companion animal. The following extracts from the participants’ answers illustrate desire:“After a few months, I adopted a new adult dog. My life has always been with animals, and because I could not get over the pain of losing Baloo … a new presence helped me to react … to commit my time to a new creature.”“Yes, after about a month, another little dog arrived. I was sick; I missed my life partner too much. It did not replace him, but it helped me partly to feel better.”Rejection is well exemplified by the following extract:“Not for now. I do not think I would be able to take care of another dog. Plus, I would feel guilty trying to replace him with another animal.”3.2.3. Shared Moral ValuesThis theme emerged from the responses describing the emotional consonance that can be generated between a human guardian and a veterinary surgeon when the same values are shared, particularly with respect to the meaning of the bond between an animal and a person and the quality of life and death. This theme is composed of two categories: technical competencies and relational competencies. The category “technical competencies” is exemplified by the following extracts:“I waited so much during the first visit. After a few minutes, the dog was literally ripped out of my hands. I did not have time to say goodbye to him. Very confusing diagnosis. Initially, it seemed solvable, and then I was told that I would have to put him down. He died on his own the morning of the day I decided to put him down. I hope he was accompanied.”On the other hand, the category “relational competencies” highlights the importance of creating a professional and personal bond with the human guardian:“The vet who had been looking after my dog for one year had a lot of patience, especially with me. He was always willing to listen [and was] understanding and attentive to my dog’s needs.”3.2.4. Perceived SupportThis theme refers to a human guardian’s perceived support from others when processing the loss of a companion animal. It emerged from descriptions of the reactions of others to the loss, both positive and negative, as perceived by the respondents, and the respondents’ expectations of support from their close circles (family members, real friends, social friends, and veterinarians). It is composed of two categories: others’ emotional reactions and personal grief. The first category highlights the importance of receiving significant emotional support from one’s social circle. In contrast, the category “personal grief” highlights the fact that the significance of an experience of loss is fundamentally personal. The following two statements exemplify the category “others’ emotional reactions”.“The people next to me suffered a lot after the loss of the dog, especially my husband. I received support from my sister and niece. I expected more support from friends.”“The dog died in my arms with all the family people around. It couldn’t have been better than that. All friends who own animals have all been very supportive.”The following is an example from the category “personal grief”:“All the people I love have been deeply saddened, but each of us has experienced it alone.”4. DiscussionThe present study aimed to validate the HHHHHMM scale, a tool that human guardians can use to assess their companion animals’ QoL, in the Italian context. This instrument can be used to improve bereavement counselling, research, and strategies to help human guardians cope with grief and adjust to the loss of a companion animal. The HHHHHMM (hurt, hunger, hydration, hygiene, happiness, mobility, more good days than bad) Quality of Life Scale is a reliable tool that can be used by guardians of companion animals with a terminal disease. With regard to the research hypotheses, the following arguments can be made:In relation to the CFA, it has been suggested that RMSEA values less than or equal to 0.05 are good, values between 0.05 and 0.08 are adequate, values between 0.08 and 0.10 are mediocre, and values greater than 0.10 are unacceptable. The comparative fit index (CFI) results in values ranging from zero to one, with higher values indicating a better fit. In addition, SRMR fit values between zero and 0.05 and TLI index values greater than 0.97 indicate a good fit [26]. When using a single-factor model with the total score and the seven items of the HHHHHMM scale, the standardized parameters were all significant (p < 0.001), so the model was partially adequate overall.With regard to the correlations between the instruments and human guardian characteristics, a higher attachment to companion animals was found among women than men in all factors of the LAPS scale. Previous studies have found similar results for displays of affection and caregiving, which have been reported to be more prevalent among women than men [27]. In the present study, it was hypothesized that the LAPS scale measurement items could influence the relationship between human guardians’ gender and the PBQ. Accordingly, independent of the direct effect of gender on the PBQ, an indirect effect—through LAPS—of gender on the PBQ was expected. Thus, although the results indicated that no direct effect between gender and the PBQ exists, the impact that occurs through the LAPS scale is always significant. Women were found to have high levels of attachment to their companion animals; the greater the attachment, the greater the symptoms of grief and suffering experienced following the death of a companion animal. Therefore, women are at greater risk of complicated grief than men after the death of their companion animals. However, due to the insufficient number of male participants, these results cannot be further generalized and need to be studied in more detail.Negative correlations were found between participant age, attachment, and grief symptoms following the loss of a companion animal. Younger people were found to have higher scores for both attachment and grief symptoms following the death of their dogs, whereas adults and older people had lower scores in both constructs. This finding is consistent with the literature. Indeed, Jarolmen [28] found significant differences in the averages of bereavement symptomatology, with higher values seen among young people than among adults. An individual’s role in the family reaffirms these differences; in fact, children report more feelings of guilt, anger, and distress than parents.Based on the results, it was hypothesized that the LAPS scale items moderate the effects of human guardians’ age on the PBQ. Furthermore, examining the effect of age at the various levels of the LAPS scale revealed a negative correlation between age and the PBQ at medium and high levels of attachment. This means that younger people exhibit more grief symptoms, such as distress and anger, than older people do. Moreover, at low attachment levels the same negative correlation between age and the PBQ was seen for the factor measuring participants’ feelings of guilt. Young individuals would have few experiences of companion animal loss and may form deep attachment bonds with different companion animals. The lack of experience may make them feel guiltier because of potential idealistic tendencies and the feeling that they should have been able to do more to help their companion animal. In contrast, adult individuals have a lower tendency to feel guilt, perhaps because they spend more time caring for their companion animals than other family members or because their experiences are reflected in more realistic expectations [23].Family composition was also found to be positively correlated with the LAPS scale’s measurement of attachment to dogs, specifically with the animal rights/welfare factor; it showed that people living alone have a greater tendency to humanize companion animals, specifically dogs, with traits related to social connectedness [29,30]. This may mean that people who do not have a spouse or are not engaged in a romantic relationship feel closer to their companion animals because animals are sources of emotional fulfilment [31]. Another potential reason for this is that keeping a companion animal has a positive effect on happiness and self-esteem and reduces stress, loneliness, and depression [32]. However, it could also impede one’s search for social support from other people, thus having negative effects.The participants’ anger levels were higher when the companion animal’s death was unforeseen than when they were already aware and thus prepared for the death. These results confirm previous reports in the literature that the unexpected death of a companion animal is associated with high scores for the anger factor of the PBQ [5]. This may be related to the experience of anticipatory grief, which can act as a protective force that allows a person to be better prepared for the death of a loved one, thus reducing the duration and intensity of the postmortem grieving process and preventing complicated grief. Although death is sometimes thought to be more easily accepted when expected because it can facilitate anticipatory grief, when people experience the brevity of the process of death without having the time to prepare in advance, the positive effects of anticipatory grief do not occur. Therefore, low levels of anger are more likely to be related to an individual’s relationship with a veterinarian and the information and preparation obtained. Indeed, when death is anticipated, as in the case of a dog’s terminal illness, the intervention of veterinarians can be very helpful in diminishing human guardians’ feelings of responsibility, validating their decisions, and allowing them to know that they did their best to help their precious companion [33]. The more information a person is given, the better he or she can prepare for what is to come; knowledge of what to expect at the end of a companion animal’s life can decrease fear by containing uncertainty. Therefore, all aspects of impending death should be discussed, including what to expect as the disease progresses and what options are available to manage the disease and provide an adequate QoL for the dog [34].Participants’ feelings of guilt were lower when death was expected; in these cases, euthanasia was considered a viable option to alleviate the dog’s suffering. In contrast, a good QoL during the dog’s final stages of life made the option of euthanasia more difficult to consider. The dog’s age at death and time spent with the owner were associated with less anger and distress after death, which is consistent with the literature on the MDQ. This could be explained by the fact that a long relationship is able to reduce feelings of anger after death, as has been found in cases of marital bereavement [35]. In light of these results, it was hypothesized that QoL mediates the effects of the dog’s age at death, the presence of a garden at home, and the decision to euthanize based on PBQ scores. The direct effect of the dog’s age at death on the “suffering” factor of the PBQ was found to be significant and negative.Furthermore, owning a house with a garden was found to have a significant positive impact on the dog’s quality of life, while the euthanasia was significantly negatively correlated with QoL. The direct effect of QoL on the PBQ factor “anger” was significant and negative. This means that a dog’s quality of life was considered good if the human guardian had a house with a garden; in turn, providing their dogs with a satisfactory QoL led to the human guardians reporting fewer feelings of anger at the time of death. Human guardians who assessed their dog’s quality of life as unacceptable and chose to alleviate its suffering by administering euthanasia reported fewer feelings of anger after the animal’s death. This may be explained by the fact that many owners may not have the time to prepare for a companion animal’s death or may not have received adequate support [23]. Notably, although it does not prevent the effects of depression, receiving detailed information about the companion animal’s health condition from a veterinarian can reduce both the anger and guilt felt by human guardians [5].5. ConclusionsThe aim of the present study was to validate, in the Italian context, an easy-to-use scale for assessing the quality of life of companion animals with terminal illnesses. Companion animal loss can elicit significant grief responses comparable to those caused by the death of a loved one. The main symptoms related to grief for humans, namely guilt, grief, anger, and intrusive thoughts, often occur after the loss of a companion animal. Therefore, people may be at risk of complicated grief responses to the death of their companion animals. In addition, grief resulting from the death of a companion animal is among the forms of grief that are delegitimized, causing a lack of social support for the bereaved. Veterinarians need to be aware of the peculiarities of this type of grief to readily accommodate, support, and acknowledge the legitimacy of human guardians’ suffering. Finally, although this was limited to the Italian context, similar results would likely emerge from the use of the scale in social and cultural contexts wherein the domestic relationship between companion animals and human guardians is diffused.The HHHHHMM Quality of Life scale is a reliable tool that can be used by individuals to make end-of-life decisions for their companion animal, preventing regret and guilt after the companion animal’s death. This tool can be used to implement coping strategies based on rationalization to improve the well-being and resilience of individuals and families. It is of paramount importance to provide psychological counselling during decision-making processes related to chronic illnesses and end-of-life care for companion animals. There is growing awareness in Italy of the need to pay more attention to companion animal bereavement and end-of-life issues in veterinary medicine, given the important role that companion animals play in people’s lives.However, this research has some limitations. First, the sample of respondents was decidedly biased in favor of the female gender. The generalizability of the results is limited because the sample in this study consisted mainly of middle-aged, married, and well-educated women. In the future, it will be necessary to recruit a balanced number of male and female participants. As with all surveys that only include people who use the internet, this study may have had a sampling bias compared to the general population, as internet use is not uniform across different demographic, cultural, and geographical groups. Finally, the instruments were used retrospectively to measure participants’ attachment and grief symptoms in relation to past events.

animals : an open access journal from mdpi

10.3390/ani12030369

PMC8833703

Disease factors and mortality etiologies of free ranging wild cetaceans such as the harbour porpoise (Phoceona phocoena) are difficult to study. However, stranded animals and carcasses can provide invaluable information on the health and biology of this species. Post-mortem examinations performed on 128 stranded harbour porpoises collected over 15 years from Swedish waters examined general health, disease findings and cause of death. The main cause of death was bycatch in fishing gear (31%, confirmed or suspected). Disease, most often pneumonia, was also a frequent cause of death (21%). Porpoise population health may mirror the overall health and stability of marine ecosystems and the effects of human activities on coastal environments. Monitoring health, diseases and causes of death of porpoises allows for identification of threats to these animals, to other animals, to humans and to the environment.

Harbour porpoises (Phocoena phocoena) are useful indicators of the health of their wild populations and marine ecosystems, yet their elusive nature makes studying them in their natural environment challenging. Stranded porpoises provide an excellent source of data to study the health and biology of these animals and identify causes of death, diseases and other threats. The aim of this study was to document pathology, and where possible, cause of death in porpoises from Swedish waters. Post-mortem examinations were performed on 128 stranded porpoises collected from 2006 to 2020. Overall, bycatch including definitive and probable cases was the most common cause of death (31.4%), followed by disease (21.3%), predominantly pneumonia. In adults, infectious disease was the most common cause of death. Bacteria with zoonotic potential such as Erysipelothrix rhusiopathiae and Brucella sp. were documented for the first time in porpoises from Swedish waters, as was the porpoise-adapted group B Salmonella enterica ST416/ST417. Three of four deaths from non-infectious diseases involved parturition complications. Four cases of suspected predation were documented, but further analyses are required to confirm these findings. Our results are consistent with those from other regions in Europe and serve as a reference for future monitoring for changing patterns of health and disease of porpoises and their environments.

1. IntroductionHarbour porpoises (Phocoena phocoena) are small, coastal cetaceans that inhabit subarctic and temperate waters of the Northern hemisphere. They are the only cetacean species that resides in Swedish waters year-round. Globally, the harbour porpoise is classified as a species of least concern [1]. In Sweden, porpoises are common off the west and southwest coasts where the North Sea and Belt Sea populations reside. However, the Baltic Sea population is classified as critically endangered with an estimated 497 animals left (95% CI 80–1091) [2].Porpoises are top predators in marine ecosystems. They have a relatively short life span compared to other top predators such as seals, dolphins and whales [3] and a comparatively more intensive reproductive cycle including earlier maturation and more frequent reproduction when compared with other odontocetes [4]. Additionally, their near-shore habitat makes them vulnerable to anthropogenic activities, which include incidental bycatch in fisheries, pollution, noise and marine traffic [5,6]. Porpoises therefore also can serve as excellent indicators of the health of our marine environments. Their aquatic habitat and shy nature make it challenging to obtain live animal data on porpoise health and biology so stranded animals provide an important source of information for this elusive species. Data from stranded porpoises in other parts of northern Europe and North America have been used to investigate cause of death, disease and other threats in these populations [6,7,8,9,10], but to our knowledge, similar data have not been published for stranded porpoises in Sweden. The purpose of this study was to collate data collected from post-mortem examinations of stranded harbour porpoises (n = 128) from 2006 to 2020 to provide information on cause of death, pathology and general health status for these animals. Comparison of these findings with other populations provides a baseline for future comparison to evaluate health trends of the species and their environment. We also identify knowledge gaps, potential health indicators and highlight areas for future investigation.2. Materials and Methods2.1. AnimalsIn 2008, the National Veterinary Institute, Sweden (SVA) and the Swedish Museum of Natural History (NRM) began a collaboration to perform post-mortem examinations on harbour porpoises to improve knowledge on the health, biology and threats to this species. Animals found in the field and deemed suitable for necropsy and sampling (i.e., were judged to be at most moderately decomposed, or could provide valuable samples for life history, environmental contaminant and genetics studies) were collected and stored frozen at −20 °C until necropsy sessions, which were typically held 1–2 times a year. Necropsy sessions facilitated examination of a larger number of animals even though freezing resulted in artifacts (e.g., colour change, histological artifacts) that needed to be considered during pathologic evaluation. A total of 128 stranded porpoises were examined by necropsy from 2008–2020, including three porpoises that were collected in 2006 and 2007 and stored frozen until this study began.2.2. Sample and Data CollectionStandardized protocols were followed to facilitate systematic data and sample collection. Evidence of human interaction was documented using the protocol in Read and Murray [11] and necropsy examination followed Kuiken and Garcia Hartman [12]. Not all data could be collected from all animals and protocols were modified depending on the state of decomposition and/or scavenging of the carcass. Morphometrics were recorded and animals were weighed, photographed and examined externally for pathology, human interaction, scavenging and other abnormalities. State of decomposition was scored as mild (1), moderate (2), severe (3) or disintegrating/mummified (4), corresponding to decomposition condition codes 2–5 in Kuiken and García Hartman [12], respectively. Body condition was scored subjectively as emaciated (1; moderate to severe epaxial muscle atrophy, prominent depression dorsally between head and body, thin blubber, no internal fat), poor (2; mild epaxial muscle atrophy, i.e., mild concavity to dorsolateral silhouette when viewed from behind and no fat internally along lateral margins of the lungs), normal (3; convex dorsolateral silhouette when viewed from behind, some internal fat along lateral lung margins), and robust (4; convex dorsolateral silhouette, abundant nuchal fat and internal fat along lateral lung margins, thick blubber). Sex was recorded (male, female, undetermined) and animals were assigned to an age class (neonate, calf, juvenile or adult) based on Lockyer [13] and van Elk et al. [6]. Neonates were up to 91 cm long. Calves (young of the year assuming a July 1 birthday) included all animals with a total length >91 cm but ≤118 cm. Juvenile males were ≥118–129 cm long and juvenile females ranged from ≥118–139 cm in length. For animals that were 118 cm long, they were assigned as calves if found from April–June or juveniles if found from July–October. All males ≥130 cm long and females ≥140 cm were classified as adults.Blubber was measured at standardized sites (dorsally, laterally and ventrally at the level of the axilla, cranial and caudal insertions of the dorsal fin and at the anus as depicted in Koopman et al. [14]) before blubber was removed to facilitate internal examination. Gross lesions were documented and when suitable, tissues were collected in 10% neutrally buffered formalin for microscopic examination. Depending on gross lesions or when needed to help determine cause of death, tissues were collected and submitted for ancillary diagnostic testing that included bacterial culture and molecular screening for viruses. Reproductive organs were measured, ovaries were assessed grossly for evidence of follicles, corpus luteum or corpora albicans and the uterus was opened and examined for the presence of a foetus. Since 2017, direct smears of testes, epididymis and the cervix were collected from animals and examined microscopically for the presence of sperm. Mammary glands were assessed for evidence of lactation. A standard set of tissues were collected for SVA’s biobank (muscle, lung, kidney, liver, spleen, brain, colon, blubber) and NRM’s environmental specimen bank for other studies including environmental contaminant analyses, and teeth, stomach contents and reproductive organs were collected for life history studies. Parasites were described, but not routinely collected throughout the study and documentation of presence or absence of a parasite in a given tissue was not consistently recorded for all animals over the entire study. For nematodes in the lungs and heart and trematodes in the liver, parasite burden was semi-quantitatively scored (none, mild, moderate, severe) for 116 and 101 porpoises, respectively.2.3. Microscopic ExaminationFormalin-fixed tissues were processed routinely and embedded in paraffin. Sections (3–4 µm) were stained using Mayer’s haematoxylin and eosin [15].2.4. BacteriologyAll bacteriological analyses were carried out by the Department of Microbiology at SVA. Throughout the study, tissues with lesions suggestive of bacterial infection (e.g., evidence of inflammation including change in tissue colour or texture, or presence of purulent, fibrinous, necrotic or caseous material) were submitted for routine aerobic culture (n = 24 porpoises) and starting in October 2019, grossly normal lungs from an additional 14 porpoises with a decomposition score of moderate or less were submitted for routine aerobic culture for screening. In total, tissues from 38 porpoises were cultured. Samples were inoculated onto blood agar plates with 5% horse blood and bromocresol purple lactose agar plates and held at 37 °C under aerobic conditions. In addition, a second blood agar plate was held at 5% ± 1% CO2. Plates were inspected after 24 h and 48 h for bacterial growth. Any Salmonella sp. isolates were further characterized as described in Sandholt et al. [16]. Additionally, one porpoise had lesions suggestive of Brucella infection in the testis and tissue was submitted for selective Brucella culture. The testis sample and a positive control were inoculated in parallel onto non-selective (5% horse blood agar, bromocresol purple agar and Trypticase soy agar plates) and selective (Farell agar plates) media. Plates were held at 37 °C under aerobic conditions as well as in an 5–10% CO2 incubator and inspected for growth after 3 and 7 days. To further investigate this case, formalin-fixed, paraffin-embedded testis tissue was also submitted for Brucella species PCR. Four 6-µm-thick sections of paraffin blocks containing testis were submerged in 250 µL 0.5% Tween-20 solution. The paraffin was melted by heating the samples at 90 °C for 10 min followed by 55 °C for 5 min. The samples were centrifuged at 10,000× g for 15 min and then placed on ice. The hardened paraffin was removed and 195 µL of the DNA-containing Tween-solution was extracted together with 5 µL seal herpes virions using EZ1 DNA Tissue kit (Qiagen, Hilden, Germany). Real-time PCR was carried out using the PCR-assay for Brucella genus (IS711) and seal herpes (internal control) as described by Boskani et al. [17] with the following modifications; each 15 µL PCR reaction contained PerfeCTa qPCR Toughmix (Quantabio, Beverly, MA, USA), 500 nM och each primer, 100 nM of each probe and 2 µL DNA template. Real-time PCR was performed using Applied Biosystems 7500 Fast thermal cycler (Thermo Fisher Scientific Inc., Waltham, MA, USA). The PCR program comprised of an initial denaturation step of 3 min at 95 °C, followed by 45 cycles of 3 s at 95 °C and 30 s at 60 °C.2.5. VirologyAnimals with unknown cause of death or that had pneumonia or other bacterial infection, and had tissues in the biobank, were analysed for the presence of morbillivirus. Pooled spleen, lung and brain were submitted to the Moredun Research Institute Surveillance Unit, Scotland for morbillivirus screening by real time RT-PCR (n = 55) following methods described in Dagleish et al. [18]. Lung swabs from porpoises collected in 2020 with a decomposition score of <4 (n = 28) were analysed at the Department of Microbiology, SVA for SARS-CoV-2 using real-time RT-PCR targeting nucleoprotein, envelope and RdRp protein genes as described by Corman et al. [19].2.6. ChemistryVaginal calculi found in the cervix of one porpoise were submitted to the Department of Chemistry, Environment and Feed Hygiene, SVA for chemical composition analysis. Analyses for environmental contaminants and biotoxins were not performed within the scope of this study.2.7. DiagnosesAnimals often had more than one significant pathological finding, which resulted in multiple diagnoses for many animals. Using a system similar to Fenton et al. [10], diagnoses were categorized as primary or secondary. A primary diagnosis was defined as the diagnosis that most likely resulted in the series of events leading to stranding or death. Secondary diagnoses were defined as significant findings that may have contributed to the demise of the animal but were not deemed to be the immediate cause of death on their own. Although some degree of parasitism was a common finding, parasitic infection was only considered to be a secondary diagnosis if infections were scored as more than just mild and/or inflammatory lesions were associated with the infection. Primary diagnoses were further classified into the following categories: Bycatch, Probable bycatch, Infectious disease, Non-infectious disease (excluding emaciation), Emaciation, Trauma, Abandoned, Undetermined or Unsuitable material. ‘Bycatch’ was restricted to those animals with characteristic net marks on the head, body or extremities and froth in airways. ‘Probable bycatch’ was assigned if no net marks were evident or presence of net marks could not be assessed due to sloughed skin or scavenging, but the animal was in otherwise normal to good nutritional condition and had froth in the airways, variable levels of subcutaneous haemorrhage and had no other signs of disease or cause of death. ‘Abandoned’ was only assigned to neonates with empty gastrointestinal tracts and no other sign of disease. ‘Unsuitable material’ was assigned to carcasses where extreme decomposition and/or heavy scavenging with extensive loss of tissues precluded comprehensive examination of the carcass. These animals were often brought in for other reasons than establishing cause of death. In cases where no cause of death could be determined, the primary diagnosis was set as ‘Undetermined’. In cases where the primary diagnosis was ‘unsuitable material’ or ‘undetermined’, the animal may have had secondary diagnoses.3. Results3.1. AnimalsStranded animals were collected and examined from the west and south-west coasts of Sweden (Figure 1). No animals were collected from the core area of the critically endangered Baltic Sea population and only six animals were collected from the overlapping area of suggested population management borders for the Belt Sea and Baltic Sea porpoise populations.Of the 128 porpoises examined, 64 were females, 63 were males and sex could not be determined in one animal with missing tissues. There were 21 neonates (16.4%), 40 calves (31.3%), 26 juveniles (20.3%) and 41 adults (32%). Porpoises were found during all months of the year, but there appeared to be a smaller peak of found dead animals in April and a larger peak from July to September (Figure 2). Almost two thirds (63%) of adults were found from July to September. While almost all neonates were found from June to August, two neonates were found May 24 and September 4, respectively. Detailed data for each animal is presented in Table S1.3.2. Ancillary Diagnostic Analyses3.2.1. BacteriologyOf the 38 animals from which tissues were submitted for bacterial analyses, bacteria associated with significant pathological lesions could be documented in 12 cases (Table 1). For the majority of other cases, mixed bacterial flora typical of post-mortem bacterial overgrowth were cultured, or other underlying causes for the inflammation were identified (fungal or parasitic infections).No Brucella bacteria were cultured from the porpoise with granulomatous orchitis. However, Brucella spp. could be demonstrated by PCR on the fixed testicular tissue from this animal.3.2.2. VirologyOf the 55 porpoises tested retrospectively for morbillivirus, the sample quality of 17 was too poor for reliable analysis and results were inconclusive. No morbillivirus could be detected in the remaining animals (n = 38). Genetic material from SARS-CoV-2 was not detected in any of the 28 porpoises analysed.3.3. Diagnoses and Causes of Death3.3.1. Primary DiagnosesA cause of death could be determined for 88 animals (68.8%) (Table 2). In this case, 20 (15.6%) were unsuitable for post-mortem examination and no cause of death could be assigned to the remaining 20 (15.6%). The proportion of animals assigned to diagnoses ‘undetermined’ and ‘unsuitable material’ increased with increasing decomposition code (Table S1). Animals that were classified as unsuitable were excluded from further evaluation of primary diagnoses.Excluding cases that were unsuitable for primary diagnosis, bycatch (12.0%) and probable bycatch (19.4%) when considered together were the most common primary diagnosis in our sample (31.4%), followed by undetermined cause of death (18.5%), infectious disease (17.6%), emaciation (10.2%), trauma (10.2%), abandoned (8.3%) and non-infectious disease (3.7%). Primary diagnoses according to age class are presented in Table 2. The most commonly known cause of death per age class was abandonment for neonates, bycatch or probable bycatch for calves and juveniles, and disease (infectious and non-infectious) for adults. Bycatch or probable bycatch was the next most common cause of death for adult porpoises.Of the 13 porpoises that were clearly bycaught, 12 (92.3%) were classified as in normal to robust body condition. Animals assigned to the probable bycatch category were, by definition, in normal to robust body condition. Here, 12 of the 34 porpoises (35.3%) with a primary diagnosis of bycatch or probable bycatch had at least one secondary diagnosis, the vast majority of which was nematode parasitism in the lungs, often associated with granulomatous pneumonia.Disease was the second-most common cause of stranding or death in animals suitable for examination and of these 23 cases, infectious diseases predominated (n = 19). In this case, 13 (68.4%) of the 19 porpoises that succumbed to infectious disease were in poor to emaciated nutritional condition. Infectious diseases were caused by bacterial infections (n = 10), parasitic infections (n = 6), fungal infection (n = 2) and brain inflammation (encephalitis) of undetermined cause (n = 1). Of the bacterial infections, seven manifested as pneumonia. All seven of these animals also had a moderate to severe lungworm burden and severe thrombosis was evident in one of these cases. One calf suffered from sepsis as a sequela to chronic, infected bite wounds and another had a fibronosuppurative pericaditis, myocarditist and lymphadenitis (Figure 3). In the six animals diagnosed with primary parasitic infections, severe parasitic pneumonia was seen in five animals, including one animal with a large clot that obstructed airways. The sixth parasitic infection was a locally extensive, severe trematode infection morphologically consistent with Campula oblonga in the liver, which caused biliary obstruction, leading to liver failure and icterus (Figure 4). The fungal infections were pneumonia caused by Aspergillus fumigatus. Tissues from the porpoise with encephalitis were analysed for morbillivirus, but tissues were too autolysed and results were inconclusive. In total, pneumonia of various causes made up 14 of the 19 cases of infectious diseases. With respect to non-infectious disease, three of the four cases were related to parturition (one case of dystocia and two cases of stillbirth). In the fourth case, the animal suffered from severe ulcerative esophagitis and gastritis, but the underlying cause was not determined.In cases of trauma, two had wounds consistent with ante-mortem predation (i.e., large blubber defects over the head and thorax with sharp edges accompanied by haemorrhage and regular punctures or scratches consistent with teeth or claw marks) and two other animals had wounds suspicious of predation. However, confirmatory analyses have not been performed. One post-parturient female had a ruptured uterus and another animal died from acute peritonitis following a penetrating wound into the abdomen.3.3.2. Secondary DiagnosesSecondary diagnoses (n = 140) were assigned in 67 animals, meaning some porpoises had more than one secondary diagnosis (Table S1). The vast majority (61%) of all secondary diagnoses were parasitic infections and associated inflammatory tissue changes. These included lungworms and associated granulomatous pneumonia (n = 36, including the six animals that were diagnosed with primary bacterial pneumonia), cholangitis caused by biliary trematodes morphologically consistent with Campula oblonga (n = 27), similar trematode infection of pancreatic ducts (n = 3), gastric ulceration caused by Anisakis sp. nematodes (n = 10) and infection of the tympanic cavity by nematodes morphologically consistent with Stenurus minor (n = 9). Other secondary diagnoses included mild to moderate inflammation of the lymphatic tissues, liver, urinary tract, central nervous system, blubber or peritoneum of unknown etiology (n = 15), ulceration of the gastrointestinal tract not associated with parasitism (n = 11), poor to emaciated nutritional condition (n = 9), adrenal gland cortical hyperplasia, cyst or adenoma (n = 4), hepatic lipidosis (n = 3), renal lipidosis (n = 2), struvite calculi in the cervix (n = 2), pox-like skin lesions (n = 2), granulomatous orchitis caused by Brucella sp. (n = 1) (Figure 5), congenital heart defect (n = 1), blunt trauma (n = 1), healed rib fractures (n = 1), pulmonary edema (n = 1), liver fibrosis (n = 1) and isolation of group B Salmonella enterica ST416/ST417 from the lung (n = 1).3.3.3. ParasitismIn this case, 70 of 116 porpoises (60%) had at least one nematode in the lungs and 36 of 101 porpoises (36%) had at least a mild infestation of trematodes in the liver morphologically consistent with Campula oblonga. Parasitic infection generally increased in frequency and severity in older age classes. Although 16 porpoises were recorded as having nematodes in the stomachs and 17 had nematodes in the tympanic bullae morphologically consistent with Stenurus minor, data was not consistently or systematically collected and recorded, precluding further interpretation of the occurrence and significance of these parasites.4. Discussion4.1. AnimalsWhile stranded animals should not be considered an accurate sampling of disease incidence in the population, they provide an excellent source of data for the identification of diseases and other threats to porpoises and the potential for determining shared “One Health” factors for humans and the environment. Given the elusive nature of porpoises, stranded animals still provide access to samples for studies on the biology and population demographics of this species.In this study, stranding locations generally reflected porpoise occurrence and densities in Swedish waters, with almost all animals originating from areas inhabited by the more abundant North Sea and Belt Sea populations (Figure 1). Given the small estimated population size of the critically endangered Baltic Sea population, it is not surprising that no dead porpoises were reported from the eastern areas of the Baltic Proper where only this population resides (Figure 1). Six porpoises in this study were collected from the overlapping area of suggested population management borders for the Belt Sea and Baltic Sea porpoise populations (Figure 1) and genetic analyses are ongoing to determine the origin of these animals. While results from this study originate primarily from animals of the North Sea and Belt Sea populations, findings are also similar to previous findings in porpoises from other parts of the Baltic Sea [25]. Our findings therefore likely give indications of the causes of morbidity and death for the Baltic Sea population, but comparisons should be carried out cautiously as habitats and conditions differ between regions. Information on the health, biology and threats to the critically endangered population in the Baltic Proper is still incomplete and targeted efforts to examine all dead animals found within their range in parallel with population genetic analyses are highly warranted. Additionally, as sample size increases, the shared morbidity and mortality factors of the North Sea and Belt Sea populations can serve as guides and where appropriate, proxies for the Baltic Sea population health concerns.The apparent seasonal variation in stranding with more animals found in April and the summer months requires further investigation. These peaks may reflect seasonal activities (e.g., specific coastal fisheries) and vulnerable periods (e.g., peri-parturition and the first few months after birth), increased effort to detect carcasses during summer months when more people are outdoors, or likely a combination of both. Stranding data can give an estimate of frequency of species within a given region and changes in frequency of reports of dead animals can provide insights into changing population demographics or geographical shifts [26]. In future, systematic monitoring of all reported dead porpoises in Sweden is recommended because these data can complement other data sources used to monitor porpoise populations.4.2. Diagnoses, Disease, Other Threats4.2.1. Bycatch and Probable BycatchIn this study, confirmed bycatches made up 12% of the stranded animals suitable for examination. Bycatch is extremely difficult to diagnose with certainty in stranded animals and is almost surely underestimated [10]. Tell-tale net marks disappear or become obscured as skin sloughs or is removed through processes of decomposition, beaching, handling and scavenging, and not all fishing gear leave net marks. There is a delay between when animals are first reported dead and when they are collected and examined, and scavenging and autolysis proceed rapidly. To maximize the chance of detecting net marks, close coordination with field volunteers is necessary. The majority of porpoises brought in for necropsy in this study were first photographed in the field to evaluate level of decomposition. Standardized protocols for additional photographs from the field would increase the likelihood of detecting net marks.Probable bycatch was a frequent primary diagnosis in this study. While a small proportion of animals may have been mistakenly assigned to this diagnosis class if cause of death was missed during post-mortem examination, our conservative definition of ‘probable bycatch’ likely led to the incorrect exclusion of a number of animals from this category. Recent studies of bycaught animals show that these porpoises often have concurrent morbidities and/or are in compromised nutritional condition [25,27]. Thus, even cases of probable bycatch reported here are likely an underestimate of the true number in our sample.If bycatch and probable bycatch from human interaction are considered together, they represent the most frequent cause of death in this study. Almost one third (31.4%) of all stranded animals suitable for examination were assigned to this category. This is lower than the 38% described for stranded porpoises on English and Welsh coasts [9], but higher than the 15% described for Belgian and French coasts from 1990–2000 [8]. However, in a more comprehensive summary of causes of death of stranded porpoise deaths from 1990–2017 in Belgium, definitive bycatch made up 28% of the cases and this increased to 35% if probable bycatches were also included [28]. In other studies from German waters, bycatch was the most common cause of death of porpoises from the Baltic Sea whereas it was infrequently documented (7% of cases) in animals from the North Sea [7,29]. Although proportion of bycaught porpoises in the stranded animal sample varied between studies, bycatch accounts for a significant number of stranded animals and confirms that stranding data can be a useful indication of fishery mortality in nearshore waters. Further estimation of the magnitude of bycatch mortality requires well-designed at-sea fishery observer programs [30]. While comparison of fisheries activity (e.g., season, effort, gear used and species targeted) with porpoise strandings was beyond the scope of this study, harbour porpoises previously have been reported as bycatch in nets and trawls for a number of species such as cod (Gadus morhua), lumpfish (Cyclopterus lumpus), pollock (Pollachius pollachius), Atlantic mackerel (Scomber scombrus), spiny dogfish (Squalus acanthias) and langoustine (Nephrops norvegicus) in Swedish waters (Swedish Museum of Natural History records, unpublished data). Comparison of stranding records of cetaceans with fisheries activity will help provide insight into impacts of fisheries on porpoises in Sweden.4.2.2. Infectious DiseaseInfectious disease was the next most frequent known cause of death in porpoises suitable for examination. As in other studies, pneumonia predominated infectious diseases [6,8,9]. In this study, bacterial pneumonia was always seen in association with lungworm infection. Bacteria responsible for pneumonia are consistent with reports from other European countries [6,8,31,32] and streptococcal bacteria were most common as described by Swenshon et al. [33]. Notably, a porpoise-adapted strain of Salmonella enterica (group B Salmonella enterica ST416/ST417) previously documented and definitively confirmed only in porpoises from British waters [34] was isolated for the first time in Sweden in two separate cases [16]. Host-adaptation resulted in the loss of numerous genes typically associated with increase pathogenicity, but serious, opportunistic infection was documented in one of the porpoises in this study [16]. The zoonotic potential of this Salmonella species is not known. We also document the first case of Staphylococcus aureus myocarditis and pericarditis in porpoises from Sweden, adding to the two cases previously described by Siebert et al. [35] in German waters. According to these authors, this bacterium is not commonly isolated from porpoises. In all three documented cases, the heart was targeted by infection, suggesting that that it may be a predilection site for Staphylococcus aureus infections in porpoises. The zoonotic bacteria Erysipelothrix rhusiopathiae has been documented in a number of marine mammal species [36] and has now also been documented on two separate occasions in porpoises from Sweden. In both cases, infection was associated with severe pneumonia. The other notable bacterial infection detected was Brucella sp. in the testis of a mature male harbour porpoise. Although the Brucella species was not identified, it is most probably Brucella ceti, a marine Brucella that infects cetaceans. This is the second documented case of orchitis in harbour porpoises caused by Brucella. Upon detection of the first case in a mature male porpoise from British waters, Dagleish et al. [37] raised the possibility of sexual transmission and concern for effects on reproductive success. Terrestrial Brucella species often cause reproductive disease and B. ceti has been associated with abortion in two bottlenosed dolphins (Tursiops truncatus) [38]. Closer monitoring of Brucella in reproductive tracts of porpoises is needed to investigate any potential impact on reproduction in porpoises in Swedish waters.Here we report on bacterial infections deemed significant for the host because of associated inflammatory lesions. However, evaluating the significance of bacteria cultured from porpoises can be challenging. Some pathogenic bacteria have been isolated from marine mammals without causing apparent disease and a myriad of other bacteria have been cultured from porpoise tissues, but their significance is poorly understood because data on associated pathology are unavailable. Histopathological examination coupled with routine bacterial culture is recommended for all porpoises with a decomposition code of 1 or 2 to better understand the significance of bacteria isolated from porpoises.Fungal pneumonia caused by fungi morphologically identified as Aspergillus fumigatus was also determined to be the cause of death in two animals. The frequency of Aspergillus sp. infection may be increasing in porpoises in the Netherlands [6]. If this represents a true increase, impaired immunity is hypothesized to be the reason [6] and monitoring of fungal infections in porpoises may be a useful indicator of changes in general health status. Increased surveillance of Aspergillus sp. infection in Swedish porpoises along with identification of environmental factors that may facilitate exposure to Aspergillus sp. or impact host immune response is also warranted.As in other studies [6,7], nematodes in the airways, pulmonary vessels and the heart were a common finding in this study and heavy infections can lead to severe pneumonia which is often complicated by bacterial infection. Lungworms have also been implicated in the transmission of certain bacteria in porpoises [39]. Certain nematodes such as Pseudalis inflexus [6,39] are known to be particularly pathogenic for porpoises. Lungworms were not routinely identified in this study but simply assessing lungworm burdens and associated inflammation may serve as a useful indicator of porpoise health status. For example, porpoises in waters from Norway, Iceland and Greenland had milder lungworm parasitism associated with less pathology than animals from German waters, reflecting differences in host populations and/or environmental circumstances [29]. In this case, 60% of porpoises examined for presence of lungworms in this study had at least a mild burden. This is higher than the 46% reported in porpoises from German waters by Reckendorf et al. [40], but similar to prevalences of 63.8% and 69.4% of Pseudalius inflexus and Torynurus convolutes, respectively, in porpoises from the Baltic Sea population [41]. Identification and systematic recording and scoring of lungworm infection are needed to follow trends in parasitism in Swedish waters and compare findings with other porpoise populations.The only other case where cause of death was attributed to parasitism was a locally extensive, severe biliary infection with Campula oblonga trematodes that blocked outflow of bile. Although 36% of porpoises examined for presence of biliary trematodes had at least a mild burden, infections were generally considered incidental findings. These findings are similar to the 42.2% prevalence reported from porpoises from British waters [42] but differ from bycaught porpoises examined in northern Norway where 90% of animals examined were parasitized with Campula oblonga and two thirds exhibited severe associated cholangitis and hepatitis [25].Different viruses including morbillivirus and herpesviruses have been detected in porpoises [43,44] but, with the exception of pox-like dermatitis in two animals, no evidence of viral infection was found in this study. However, because morbilliviruses are known to impair immunity and predispose to other infections, porpoises with no known cause of death or with pneumonia or other bacterial infection were therefore screened for morbillivirus infection. No morbillivirus was detected. Following the emergence of SARS-CoV-2, cetaceans were predicted to be susceptible to infection through wastewater [45]. To rule out infection with SARS-CoV-2, porpoises that were found dead during the pandemic were screened. No evidence of infection was found.Increased environmental contaminant burdens have been associated with immune suppression and increased risk for infectious disease in cetaceans [6,46]. Analyses of environmental contaminants in porpoises from Swedish waters, including a subset of animals from this study, are on-going to be able to assess the significance of these compounds on porpoise health.4.2.3. Non-Infectious DiseaseThree of the four cases (one mature female and two neonates) with a primary diagnosis of non-infectious disease died because of birthing complications. Neonates made up a relatively large proportion (16.4%) of the animals examined in this study and reproductive complications may have contributed to the deaths of neonates assigned to other primary diagnosis categories. Similar reproductive complications are not uncommon in porpoises from British waters and environmental contaminants may play a role [47]. Further investigation and monitoring of reproductive status and failure in porpoises in Sweden would serve as a useful health indicator for these populations.Non-infectious diseases also comprised a number of the secondary diagnoses. Gastrointestinal ulceration was the most common and some cases were likely manifestations of stress.No pathological evidence of intoxication was seen but analyses for biotoxins were not carried out in this study. It is possible that cases of acute toxicosis from biotoxin exposure were missed. However, all porpoises examined in this study were from single stranding events and there was no history of concurrent mortality events in other species, for example fish or seabirds. Samples for biotoxin analyses are now collected and archived routinely, and future evaluation of biotoxin exposure in porpoises from Swedish waters is highly warranted.4.2.4. TraumaPredator-related trauma was suspected to be the cause of death in four porpoises in this study. Grey seals (Halichoerus grypus) have emerged as significant predators of harbour porpoises in the North Sea [48,49] and grey seals and porpoise range overlap in Swedish waters, particularly in the Baltic Sea. Lesions observed in these four cases are consistent with those described for grey seal attacks, but other predators to consider include the group of killer whales (Orcinus orca) now regularly sighted off the Swedish west coast and sharks. Predation from other marine species has not been documented in Sweden and, if confirmed through genetic analyses (environmental DNA analyses of wounds), may represent a new threat to porpoises.4.2.5. Undetermined or Unsuitable MaterialThe proportion of animals with a primary diagnosis of ‘undetermined’ or ‘unsuitable material’ increased as degree of decomposition increased, reflecting that fact that comprehensive necropsy examination is precluded by advanced autolysis. Despite this, a primary diagnosis could still be assigned in just over half of the animals that were severely autolyzed (decomposition code of 3). Severely autolyzed animals (especially adults and those potentially originating from endangered populations like the Baltic Sea) provide important samples and data for other studies (e.g., life history and population genetics). Based on our findings, there is some value in also performing necropsy examinations on these animals despite the limitations associated with advanced autolysis.5. ConclusionsThis study provides an important first description of causes of death, diseases, pathology and potential population threats in porpoises inhabiting Swedish waters and serves as a reference for future monitoring of trends. Findings presented here are generally consistent with findings from other regions in the North Atlantic and a number of diseases were documented for the first time in porpoises from Sweden.Bycatch and probable bycatch when considered together was the most common cause of death, confirming that fishery interactions are still a common threat to porpoises in Swedish waters. Infectious disease was also common, particularly in adult animals. Animals with impaired immunity are more susceptible to infections. Impaired immunity in porpoises may be associated with viral infections, malnutrition or environmental contaminants [6]. We found no evidence of underlying viral infections in our study. Although two thirds of animals with a primary diagnoses of infectious disease were in poor or emaciated nutritional condition, it is difficult to assess whether compromised nutritional status led to impaired immunity and disease, or whether it was the result of disease. Environmental contaminant burdens were not investigated in this study. Given that reproductive failure may also be related to environmental contaminants, comparison of health and reproductive data with environmental contaminant burdens in Swedish porpoises will help fill the knowledge gaps on the effects of contaminants on harbour porpoises in Swedish waters.Stranded animals in this study represent a combination of ill or debilitated animals (e.g., those with disease or that are emaciated) and animals more representative of the general population (e.g., those that died from acute trauma or in fishery interactions but are otherwise without significant disease). In order to better understand the health and biology of harbour porpoises, both types of animals are needed. Sick and debilitated animals provide information on pathogens and other disease conditions of porpoises and the threats that they are exposed to whereas animals more representative of the general population are needed to follow trends in body condition, growth, reproduction, dietary habits and environmental contaminants. Since porpoise health mirrors health of the ecosystem in which they live, changing patterns of health and disease often signal ecosystem change. Long-term monitoring is needed to detect these changes.

animals : an open access journal from mdpi

10.3390/ani12050590

PMC8909886

The Ridden Horse Pain Ethogram (RHpE) comprises 24 behaviours; a RHpE score ≥8 reflects the presence of musculoskeletal pain. An association between the RHpE score and performance has been shown for 5* three-day event horses. The aim of the study was to apply the RHpE to horses performing a dressage test at British Eventing (BE) 90, 100 and Novice one-day events and to compare the scores with competition results. Data were collected for 1010 competition starts. Overall, the most frequent (median) RHpE score was 4/24. The median RHpE score was higher (5/24) for BE 90 competitors, compared with 100 (4/24) and Novice (3.5/24). Horses placed first, second or third had a lower median RHpE score (2/24) compared with other horses which completed. The proportion of horses with a RHpE score ≥8/24 was lowest (2%) in those placed first to third, followed by horses with lower finish placings (9.9%), and highest in those that were eliminated, retired or withdrawn (11.3%). The overall low median RHpE score supports the social licence to compete, but 9% of starters had a RHpE score ≥8/24, which merits concern. Veterinary investigation of these horses and appropriate treatment and management may improve both welfare and performance.

The Ridden Horse Pain Ethogram (RHpE) was applied to 1010 competition starts at British Eventing (BE) 90, 100 and Novice one-day events and compared with performance. The overall median RHpE score was 4/24 (IQR 2,6; range 0,12). There were moderate positive correlations between RHpE scores and dressage penalties (Spearman’s rho = 0.508, 0.468, 0.491, all p < 0.001 for BE 90, 100 and Novice, respectively). There were weak positive correlations between RHpE scores and final placings (Spearman’s rho = 0.157, p = 0.033, BE90; rho = 0.263, p < 0.001, BE 100; rho = 0.123, p = 0.035, Novice). In showjumping, 1.7% of starters were eliminated or retired, compared with 9.8% of cross-country starters. Horse or rider falls occurred in 2.6% of cross-country starts. Horses placed first, second or third had lower median RHpE scores (2/24, IQR 1,4; range 0,8) than other horses that finished (p < 0.001), those that were eliminated or retired (p < 0.001) or were withdrawn (p < 0.001). The RHpE score was ≥8/24 for 9.3% of starters; horses with a RHpE score ≥8/24 had higher total penalty scores (p < 0.001) than horses with a RHpE score <8/24. The overall low median RHpE score supports the social licence to compete, but 9% of starters had a RHpE score ≥8/24. Investigation and treatment of these horses may improve both welfare and performance.

1. IntroductionA Ridden Horse Pain Ethogram (RHpE) comprising 24 behaviours (Table S1) was developed [1], and it was shown that a RHpE score of ≥8/24 is likely to reflect the presence of musculoskeletal pain [1,2,3,4,5,6]. In a previous pilot study assessing horses (n = 35) warming up for dressage at a 4* (now 5*) three-day event in 2018, horses with a RHpE score of ≥7 were more likely to be eliminated or retire in the cross-country phase than horses with a RHpE score <7 [7]. The pilot study highlighted that in a cohort of highly trained event horses competing at 5* level, it may be useful to use a slightly lower total RHpE score as an indicator of possible influence on performance, rather than the score of ≥8/24, previously identified as a reliable score for differentiating sports horses with and without musculoskeletal pain.The RHpE was subsequently applied to all horses (n = 137) competing at two 5* three-day events in 2019 [8]. There was a significant association between the RHpE score during warm-up for dressage and both dressage penalties and final placing. Horses with a RHpE score of ≥7 were more likely to be eliminated or retire during cross-country than horses scoring <7. There was an association between lameness or gait abnormalities in canter and a RHpE score ≥7. There was good consistency of results for horses which competed at both events. It was concluded that the use of the RHpE may help to identify horses which might benefit from investigation and treatment to both improve performance and enhance equine welfare.The RHpE has also been applied to video recordings of horses competing in Grand Prix dressage competitions at elite World Cup level [9] and sub-elite level [10]. There was a negative correlation between the RHpE score and the judges’ total percentage scores.The median RHpE score for non-lame three-day event horses was 3/24 (range 0, 9), whereas horses which showed transient lameness or gait abnormalities in canter had a significantly higher median RHpE score of 5/24 (range 1, 9) [8]. Elite World Cup Grand Prix dressage horses had a median RHpE score of 3/24 (range 0, 7) [9], whereas sub-elite Grand Prix horses had significantly higher scores (Hickstead Rotterdam Challenge median 4/24 (range 0, 8); British Dressage National Championships median 6/24 (range 1, 9), in association with a higher frequency of occurrence of lameness or abnormalities of canter [10].British Eventing (BE) one-day events comprise dressage, showjumping and cross-country phases, with the maximum height of cross-country fences being 0.90 m, 1.0 m and 1.10 m for BE 90, 100 and Novice classes, respectively [11]. For BE 90 competitions, horses must be at least 132 cm in height and 5 years of age (4 years of age after 1st July); for BE 100 and Novice competitions horses must be at least 142 cm in height and 5 years of age. A horse may be withdrawn from the competition before the start of any phase. A horse fall or unseated rider in any phase results in elimination. In the dressage phase, a rider may be eliminated because of three errors of course, the appearance of blood on the horse or severe lameness. In the showjumping phase, three cumulative refusals or resistance for >20 s result in elimination. In the cross-country phase, three refusals at a single fence or three (four at BE 90 or 100) cumulative refusals result in elimination. In the dressage phase, the judge awards a score of 0 (not executed) to 10 (excellent) for each movement, and four sets of ‘collective marks’ for overall quality of paces, impulsion, submission and rider position and effectiveness, for a maximum total of 200. The summed total expressed as a percentage is subtracted from 100 to give the penalty score. Penalties are awarded for knocking a fence down (4), run outs or refusals (4 and 8 respectively, for first and second refusals in showjumping; 20 and 40 respectively, for first and second refusals in cross-country), or for exceeding (showjumping and cross-country) or going under (cross-country) the optimum time. The optimum cross-country speeds are 450 m/min, 475 m/min and 520 m/min for BE 90, 100 and Novice, respectively. A rider may elect to retire a horse during any phase, usually because it is not performing well.The objectives of the study were: 1. to apply the RHpE to horses competing in the dressage phase at BE 90, 100 and Novice one day events; 2. to document the frequency of occurrence of each behaviour of the RHpE and 3. to compare the RHpE scores with performance. It was hypothesised that there would be an association between higher RHpE scores and poorer performance results.2. Materials and Methods2.1. Data AcquisitionThe study was approved by the Royal College of Veterinary Surgeons Ethics Review Panel (2020–26); data were collected at public events, therefore informed rider consent was not required. Data were collected from a convenience sample of BE one-day events at BE 90, 100 and Novice levels. At each level the horses performed a set BE dressage test in a 40 m × 20 m arena. This included halt, medium walk, free walk on a long rein, working trot and working canter at all levels, and medium trot and canter, counter canter ± rein back and leg yield at Novice level. The tests were selected by the event organiser from those available at each BE level; the duration of each test was approximately five minutes. Each section was judged by a single trained judge, listed by British Dressage as qualified to officiate at that level. All horses in each randomly selected section were assessed. Horses were identified by the rider’s number. At preselected events, with the cooperation of the organisers, competitors were offered the opportunity to opt out of the study prior to the day of the competition, but none elected to do so. Ridden Horse Pain Ethogram data were collected by a single trained assessor (SD, an equine veterinarian with 42 years of experience of lameness investigation) who stood approximately 1–3 m from the dressage arena at the MC corner (i.e., 10 m to the left of the official judge who was positioned on the centre line). The RHpE data were recorded manually (binary yes/no scoring) on individual purpose-designed score sheets.Additional subjective data were recorded (for example, forelimb lameness, short stepping forelimb gait, hindlimb lameness, lack of hindlimb impulsion and engagement in trot and/or canter, canter lacks a suspension phase). Behaviour 20 of the RHpE includes both repeated bilateral hindlimb toe drag or repeated stumbling. The presence of bilateral hindlimb toe drag or single or repeated stumbling were also recorded independently. For those Novice tests that included rein back, behaviours such as head behind vertical, mouth opening with separation of the teeth, head tossing, ears back and refusal to step backwards were also recorded independently. These behaviours were not included in the RHpE scores if not fulfilling the RHpE definitions (for example, head above vertical ≥30°, but <10 s).Snaffle bridles were required for dressage; the use of spurs was optional. The type of noseband used (cavesson, crank cavesson, flash, crank flash, Micklem, grackle, drop), the use of a nose net and whether the rider used spurs were documented. Weather conditions, the footing and the levelness of the arena were also recorded.Dressage, showjumping, cross-country and total penalties, and final place were collected from each event’s website. Reasons for elimination were recorded. Each horse’s age, breed and sex information were collected from the BE website.2.2. Data AnalysisData were recorded in an electronic spreadsheet (Microsoft Excel, version 2010; Microsoft Corporation, Redmond, WA, USA) and statistical analysis was carried out using commercial statistical software (STATA: IC version 13; StataCorp. LLC. 2017. Stata Statistical Software: Release 15. College Station, TX, USA).The distribution of continuous variables (horse age, dressage, showjumping, cross-country and overall penalties) was formally evaluated using the Shapiro-Wilk test, in combination with visual assessment of histograms, with overlaid kernel density plots. All continuous variables were determined not to have a normal distribution (Shapiro-Wilk p-value < 0.05) and alongside ordinal variables (RHpE score and final placing) were described as medians with interquartile range (IQR) and range (minimum to maximum). Categorical variables (horse sex and breed, noseband type, use of spurs, presence of gait abnormalities and RHpE behaviours (yes/no), completion status (placed in top three, unplaced, eliminated/retired or withdrawn), elimination/retirement during a specific phase of the competition and competition level (BE 90, 100 or Novice) were summarised as proportions and expressed as percentages. Associations between horse signalment and competition level were described but not statistically assessed. This is because some horses had repeated observations within and across levels, and the focus was on assessing behaviour at competition starts rather than at horse level.Relationships between categorical variables were assessed using the Chi-square (χ2) or Fisher’s exact test when observed counts in any comparison group were <5. These included the presence of gait abnormalities and RHpE behaviours, completion status, elimination/retirement during a specific phase of the competition and competition level. Where significant relationships were identified between categorical variables in 2 × 3 contingency tables, an additional Cramer’s V test was calculated to assess the strength of the association/estimate effect size [12]. Qualitative interpretation of Cramer’s V was performed according to Rea and Parker [13], with <0.20 signifying a weak association, ≥0.20 to <0.40 signifying a moderate association and ≥0.40 signifying a strong association. The Bonferroni correction was used to adjust for multiple comparisons where the significance level of the α test (p = 0.05) was divided by the number of tests/comparisons.The Mann-Whitney U test was used to assess the relationship between continuous/ordinal variables and categorical variables (e.g., overall penalties and RHpE category [<8 vs. ≥8]). Overall median differences in RHpE scores and competition level and completion status were assessed using the Kruskal-Wallis test, with a further post-hoc Dunn’s test with Sidák adjustment to assess pairwise comparisons between groups.Correlations between RHpE scores and dressage, showjumping and cross-country penalties and overall placing at each competition level were assessed using the Spearman rank correlation coefficient.3. Results3.1. Overall ResultsData were collected at 20 competition days between 17 April 2021 and 29 October 2021 at venues in Bedfordshire, Cambridgeshire, Leicestershire, Lincolnshire, Norfolk, Northamptonshire, Suffolk, Surrey and Sussex. This included 34 sections: Novice n = 12 (356 competition starts), BE 100 n = 15 (450 competition starts) and BE 90 n = 7 (204 competition starts), comprising a total of 1010 competition starts by 841 horses and 708 riders. The dressage tests took place on grass arenas for 20 sections, including 588 (58.2%) tests, or on all-weather arenas at two venues for 14 sections, including 442 (41.8%) tests. The weather was variable among venues and times of day, and included wind, rain, sun, cool and occasionally warm/hot.3.1.1. Horse DataThe median age for all horses (n = 841) was nine years (IQR 7, 11; range 5, 22), with 563 (66.9%) geldings, 5 (0.6%) stallions and 273 (32.5%) mares. Breeds included Irish Sports Horse n = 335 (39.8%), Warmblood n = 188 (22.4%), Warmblood cross n = 153 (18.2%), Thoroughbred or Thoroughbred cross n = 9 (1.1%), Other crossbred/unknown n = 103 (12.3%), and Pony n = 53 (6.3%).3.1.2. Nosebands and SpursData for noseband type and the use of spurs were not available for the first section evaluated, n = 28. Noseband types (n = 982 of competition starts) included: grackle n = 248 (25.3%), crank flash n = 231 (23.5%), flash n = 164 (16.7%), Micklem n = 137 (14.0%), crank cavesson n = 92 (9.4%), cavesson n = 91 (9.3%) and drop n = 19 (1.9%). A nose net was used in 7 of 1010 (0.7%) competition starts. Spurs were used in 719/982 (73.2%) of competition starts, but not in 263 (26.8%). Tail swishing in synchrony with the application of spur cues was not observed.3.1.3. Gait AbnormalitiesThe frequencies of occurrence of gait abnormalities in trot and canter are summarised in Table 1. Overall, there was a low frequency of occurrence of overt lameness (8.6%), but poor hindlimb impulsion and engagement were observed in 38.1% of competition starts, and canter was abnormal in the majority (61.0%).Teeth grinding or chomping repeatedly were observed in 62 (6.1%) of competition starts. Rein back was performed at seven competitions (Novice 111 (2010) test) and was performed poorly (relative to the guidelines for dressage judges [11]) in 45/197 (23%) competition starts (not including horses that stepped back crookedly or took an incorrect number of steps). Errors included refusing to step backwards, the front of the head being considerably in front of or behind a vertical position, opening the mouth widely, ears back, tail swishing, head tilt and head tossing. However, the duration of these abnormal behaviours was generally less than the RHpE definition.3.1.4. Ridden Horse Pain Ethogram Score and PerformanceOverall, the median RHpE score was 4 (IQR 2, 6; range 0, 12). The median dressage penalty score (n = 1009, because of 1 elimination) was 33 (IQR 30.5, 35.8; range 18.5, 56.8). The median showjumping penalty score (n = 980 because of withdrawals, eliminations and retirements) was 4 (IQR 0, 4; range 0, 52). The median cross-country penalty score (n = 851 because of withdrawals, eliminations and retirements) was 4 (IQR 0, 14; range 0, 139.2). The median total penalty score was 41.7 (IQR 35.3, 54.8; range 20.5, 170.5).The median RHpE score for horses placed first, second and third in a section was 2 (IQR 1, 4; range 0, 8), compared with a median RHpE score of 4 (IQR 2, 6; range 0, 12) for all other horses that completed. Horses placed in the top three had significantly lower (p < 0.001) median RHpE scores compared with horses which completed but were not placed in the top three.The median RHpE score for horses eliminated or retired in the showjumping or cross-country phases (n = 112) was 4.5 (IQR 2, 6; range 0, 12). One horse was eliminated in the dressage phase. The median RHpE score for horses withdrawn (n = 47) was 6 (IQR 3, 7; range 0, 8). Twelve horses were withdrawn before show jumping and 35 were withdrawn before cross-country. Seventeen of 997 (1.7%) showjumping starters were eliminated or retired. Ninety-four of 945 (9.8%) cross-country starters were eliminated or retired. There was a total of 25 unseated riders (2.5%), including two in the showjumping phase. There were 25 horse or rider falls in the cross-country phase, representing 2.6% of cross-country starters. This included two horse falls (0.2% of cross-country starters).There were significant differences in the median RHpE scores between horses placed in the top three and unplaced horses (p < 0.001), horses that were eliminated/retired (p < 0.001) and horses that were withdrawn (p < 0.001). There was also a significant difference in the median RHpE scores between unplaced horses and withdrawn horses (p = 0.04). However, no differences in median RHpE scores were identified between unplaced horses and eliminated/retired horses (p = 0.687) nor between eliminated/retired and withdrawn horses (p = 0.297).The RHpE score was <8 for 916 (90.7%) competition starts but was ≥8 for 94 (9.3%) competition starts. The proportion of horses with a RHpE score ≥8 was lowest in those placed first to third (2.0%), followed by horses with lower finish placings (9.9%), and highest in those that were eliminated, retired or withdrawn (11.3%) (p = 0.01). Considering horses that completed, horses with a RHpE score ≥8 had significantly higher total penalty scores (median 47.8, IQR 40.2, 62.0; range 31.8, 116.8) than horses with a RHpE score <8 (median 41.1, IQR 34.8, 53.5; range 20.5, 170.5) (p < 0.001).3.2. Results Presented for Each Level Independently3.2.1. Horse DataThere was a high proportion of Warmblood horses in BE 100 and Novice sections relative with BE 90, while the BE 90 sections had a relatively high proportion of ponies (Table 2).Age and sex data are summarised in Table 3. The median age was highest for horses competing at BE 90 level. Geldings predominated at all levels.3.2.2. Gait AbnormalitiesThe distribution of gait abnormalities at each level is summarised in Table 1. There was a significant but weak relationship (χ2 p < 0.001; Cramer’s V < 0.20) between competition level and forelimb lameness, hindlimb lameness and lack of hindlimb impulsion and engagement, and a moderate association between competition level and abnormal canter (Cramer’s V 0.23). The frequency of occurrence of these gait abnormalities was highest at BE 90 and lowest at BE Novice competition level.3.2.3. Ridden Horse Pain EthogramWhen considering each level of competition separately, at BE 90 the median RHpE score was 5 (IQR 3, 7; range 0, 12), compared with a median RHpE score of 4 (IQR 2, 5; range 0, 12) at BE 100 and a median RHpE score of 3.5 (IQR 2, 5; range 0, 11) at Novice. There were significant differences in the median RHpE scores between BE 90 and both BE100 (p < 0.001) and Novice (p < 0.001), but not between BE 100 and Novice (p = 0.859) (Figure 1).The frequency of occurrence of the 24 behaviours of the RHpE overall and at each competition level are documented in Table 4. There was a significant and weak to moderate relationship (χ2 p ≤ 0.001; Cramer’s V < 0.30) between 10 of the RHpE behaviours and competition level, with repeated movement of the head up and down, head in front of the vertical, repeated side to side movement of head, ears behind vertical, an intense stare, mouth opening with separation of the teeth, bit pulled through to one side, repeatedly crooked and repeated bilateral hindlimb toe drag and/or stumbling being most frequent in BE 90 competitions. Spontaneous change of gait was most frequently observed at Novice competitions.There was a significant and moderate relationship (χ2 p < 0.001; Cramer’s V 0.27) between stumbling or bilateral hindlimb toe drag and competition level, with frequency highest at BE 90 competitions (50.5%) followed by BE 100 competitions (45.1%), and frequency lowest at Novice competitions (20.2%). Bilateral hindlimb toe drag or repeated stumbling was observed more frequently on all weather surfaces (43.6%) compared with grass (37.4%) (χ2 p = 0.001).3.2.4. Competition PerformanceThe proportions of completions, eliminations in any phase, retirements (in showjumping or cross-country) and withdrawals (before show jumping or cross-country) are summarised in Table 5. A relationship between completion status and competition level was not identified (χ2 p = 0.06).The proportions of eliminations or retirements in the showjumping and cross-country phases at each competition level are summarised in Table 6. A relationship between discipline and eliminations or retirements for each competition level was not identified (showjumping χ2 p = 0.386; cross country χ2 p = 0.502).3.2.5. British Eventing 90 Level, n = 204When considering the relationship between RHpE scores and performance, there was a moderate positive correlation (Spearman’s rho = 0.5083, p < 0.001) between the RHpE scores and the dressage penalty scores (Figure 2).There was no correlation between RHpE scores and showjumping (Spearman’s rho = 0.069, p = 0.327) or cross-country (Spearman’s rho = −0.043, p = 0.561) penalties for horses that completed each phase. However, there was a weak positive correlation (Spearman’s rho= 0.157, p = 0.034) between the RHpE scores and final placing for 182 completions (Figure 3).3.2.6. British Eventing 100 Level, n = 450When considering the relationship between the RHpE scores and performance, there was a moderate positive correlation (Spearman’s rho = 0.468, p < 0.001) between the RHpE scores and the dressage penalty scores (Figure 4).There was no association between the RHpE scores and showjumping (Spearman’s rho = 0.088, p = 0.065) or cross-country (Spearman’s rho = 0.098, p = 0.057) penalties for horses that completed each phase. However, there was a weak positive correlation (Spearman’s rho = 0.263, p < 0.001) between the RHpE scores and final placings for 375 completions (Figure 5).3.2.7. British Eventing Novice Level, n = 356When considering the relationship between the RHpE scores and performance at Novice level, there was a moderate positive correlation (Spearman’s rho = 0.491, p < 0.001) between the RHpE scores and dressage penalties for 355 competition starts (one horse was eliminated) (Figure 6).There was no association between the RHpE scores and the showjumping (Spearman’s rho = 0.053, p = 0.331) or cross-country (Spearman’s rho = 0.014, p = 0.809) penalties for those horses that completed each phase. However, there was a weak positive correlation (Spearman’s rho = 0.123, p = 0.035) between the RHpE scores and final placings for 294 completions (Figure 7).4. DiscussionIn accordance with our hypothesis, there was a relationship between RHpE scores and performance, with significant correlations between RHpE scores and both dressage penalties and final placings of the horses that completed. Horses that were placed first to third had lower median RHpE scores than other finishers, and horses with RHpE scores ≥8 were over-represented in non-completing and lower-placed horses. However, there was no correlation between the dressage phase RHpE scores and either showjumping or cross-country penalties for those horses which completed. At BE 90 and 100 levels the height of the fences is small, and the course designs are straightforward, with riders often riding more positively when showjumping and riding cross-country than in the dressage phase [14]. Moreover, the release of endorphins and adrenaline when jumping [15,16] may enhance horses’ performances by masking musculoskeletal discomfort. The completion proportion was similar at all competition levels, despite amateur riders predominating at BE 90 level, whereas at BE 100 and Novice levels there was a combination of both amateur and professional riders, including Olympic, European and World Championship level riders. Performance may be influenced by numerous factors including the course, the talent and physical aptitude of the horse and rider [17], as well as musculoskeletal pain.4.1. Frequency of Gait Abnormalities and LevelThe frequency of occurrence of lameness (overall 17%, BE 90 32%, 5* three-day events 13%), lack of hindlimb impulsion and engagement (overall 38%, BE 90 48%, 5* three-day events 7%) and abnormalities of canter (overall 61%, BE 90 75%, 5* three-day events 28%) was highest for horses competing at BE 90 level in the current study and higher than previously recorded for horses competing at 5* three-day events [8]. The horses competing at BE 90 also had higher median RHpE scores, probably reflecting discomfort.The overall high frequency of occurrence of head behind the vertical ≥10° for ≥10 s (59%), poor hindlimb impulsion and engagement (38%) and abnormal canter (61%), often characterised by lack of a suspension phase, also introduces the question of the relative roles of training and riding ability versus discomfort, and of the potential adverse consequences of inappropriate training on musculoskeletal health [18]. Improved gait quality was often seen in medium trot and canter compared with working gaits in Novice tests [14], suggesting that with more positive or less defensive or restrictive riding there was the potential for improvement in gait quality. There was a much larger spectrum of riding ability seen at BE 90 compared with Novice levels, reflecting the higher proportion of professional riders at Novice level [14].4.2. Manifestations of the RHpE and Competition Level4.2.1. Comparison within LevelsThere was a higher frequency of occurrence of some behaviours of the RHpE seen in horses competing at BE 90 level compared with BE 100 and Novice. These included repeated movement of the head up and down or from side to side, the head in front of vertical ≥30° for ≥10 s, ears back ≥5 s, an intense stare ≥5 s, repeated bilateral hindlimb toe drag or repeated stumbling and the bit pulled through to one side repeatedly. This may reflect the higher frequency of gait abnormalities at BE 90 level, which was associated with a higher median RHpE score. It may also, in part, reflect rider skill, as observed in a previous study which compared RHpE behaviours when horses were ridden by two riders of varying skill [19]. There was no difference in the total RHpE scores when ridden by the two riders, but the behaviours exhibited varied according to rider skill. A more skilled rider has a stable phase synchrony with the horse [20,21,22], a more consistent trunk and limb position [23,24], superior ability to control the position of the horse’s head [25] and the ability to create more propulsion [26] compared with less-skilled riders. Less-skilled riders may have less independent control of the arms and hands compared with more skilled riders [21,27], and a lack of ability to steer or straighten the horses with other aids. These factors may contribute to an unstable head position, the bit being pulled through to one side and hindlimb toe drag.Spontaneous changes of gait were observed more frequently at Novice level than at lower levels. Some of the movements were biomechanically more challenging at Novice level compared with BE 90 and 100, for example counter canter, which may have predisposed to more errors. Spontaneous changes of gait were observed in a similar proportion (17.5%) of sub-elite Grand Prix dressage horses [10] compared with only 8.8% of elite Grand Prix dressage horses [9]. The sub-elite group had higher RHpE scores and a higher proportion of gait abnormalities than the elite Grand Prix horses.4.2.2. Comparison with 5* Three-Day Events and Grand Prix DressageWhen the overall frequency of occurrence of specific behaviours of the RHpE observed in the current study was compared with horses competing at 5* three-day events [8], clear differences were observed. Head in front of a vertical position ≥30° for ≥10 s, head up and down repeatedly, ears back ≥5 s, bit pulled through repeatedly, moving on three tracks and repeated spontaneous changes of gait occurred more frequently in the lower-level horses. The explanation may be multifactorial, reflecting the higher frequency of pain-related gait abnormalities at the lower levels, an overall lower skill level of riding and inferior training. However, repeated tail swishing was seen more often in the 5* level event horses [8]) compared with horses in the current study, and was also a frequent observation in Grand Prix dressage horses [9,10]. This may be a reaction to stronger application of leg and spur cues by the riders, or the horses experiencing more difficulty with movements requiring a greater level of collection.Persistent positioning of the head >10° for ≥10 s behind a vertical position was seen with similar frequency in this study (59%) and in 5* three-day event horses (64%) [9], and was also observed in elite (67%) [9] and sub-elite (77%) [10] Grand Prix dressage horses. This is contrary to judging guidelines [28,29] but appears to be inadequately penalised. There are limited scientific data concerning head and neck position and the kinetic and kinematic effects on the limbs and thoracolumbosacral region. The immediate effects of short-term alterations in head and neck position in a small number of non-ridden [30,31] and ridden [32,33,34] riding horses [30] or well-trained dressage horses [31,32,33,34] on a treadmill at walk and trot have been investigated. The data generated cannot be used to determine the long-term effects of regular overground ridden exercise with the head behind vertical at all paces from a young age. However, clinical observations indicate adverse consequences on the optimal development of the pelvic and hindlimb muscles, the abdominal ‘core’ muscles, the muscles of the thoracic sling, the cervical muscle and the epaxial and hypaxial muscles of the thoracolumbosacral regions, and for the establishment of correct movement patterns of the forelimbs, the hindlimbs and the thoracolumbosacral region [18,35,36,37], factors which may have the potential to predispose to injury.Mouth opening with separation of the teeth for ≥10 s was observed in only 28% of competition starters in the current study, compared with 44% of non-lame sports horses [6], 45% of 5* three-day event horses warming up for dressage [8] and 81% [10] and 68% [9], respectively, of sub-elite and elite dressage horses during Grand Prix tests. Mouth opening may be a non-specific response to musculoskeletal discomfort [1,2] or reflect oral discomfort secondary to the buccal mucosa being pressed against the sharp edges of the teeth [38] or other oral lesions [39], excessive rein tension [40,41], movements of the rider’s hands [42] or the type and size of the bit relative to the size and shape of the horse’s oral cavity and tongue [43,44]. The lower frequency of occurrence of mouth opening in the current study compared with previous studies [6,8,9,10] may reflect the use of only snaffle bits rather than double bridles.To what extent mouth opening is promoted by, or restricted by, potentially restrictive nosebands or nosebands that are tightened excessively is subject to debate [45,46,47,48,49,50,51], with limited fact-based information concerning pressure effects of nosebands [52,53,54]. In the current study, despite the relatively low frequency of occurrence of mouth opening, the majority of horses (92%) had potentially restrictive nosebands. However, the tightness of the nosebands was not evaluated objectively, nor was there any legislative assessment or control of noseband tightness. In an observational study of 750 competition horses in Ireland, Belgium and the United Kingdom, objectively evaluated noseband tightness was highest in event horses compared with dressage horses and show hunters [47]. It nonetheless seems unlikely that the use of a potentially restrictive noseband was a major causal factor of mouth opening in the current study.4.3. Use of SpursSpurs were used in a high proportion (73%) of competition starts in the current study. There is limited documented information about spur use in event horses. In an observational study of 3143 dressage, showjumping, event and endurance horses in competition in Denmark spurs were used in 77%, however event horses and ponies comprised only 3.3% of the study population and it was not possible to determine spur use specifically related to event horses [55]. In an online questionnaire-based study in the United Kingdom with 628 responses, 12 of 33 (36%) event riders used spurs [56]. In a similar Australian-based questionnaire study in 2012 with 1101 respondents, including 50 event riders, overall, 41% of riders reported the regular use of spurs [57].Contrary to observations in elite Grand Prix dressage horses [9], no tail swishing in synchrony with spur use was observed in the current study. This may reflect either the absence of the application of spur cues or less forceful use of spur cues among the lower-level event riders compared with elite dressage riders.4.4. Failure to CompleteIn the current study, the non-completion proportion rose from 11% at BE 90 to 17% at Novice level. This compares with a non-completion proportion of 19% from 42,810 entries across all levels in 2007 [58]. In the current study, horse falls comprised 0.2% of all cross-country starts compared with 0.04% of 576 cross-country starts in a convenience sample of events ranging from Novice one day events to 4* (now 5*) three-day events in 2001 and 2002 [59]. In the latter study, the risk of a horse fall was significantly higher at three-day events compared with one-day events, particularly at Advanced level. In the current study, horse falls or unseated riders comprised 2.6% of all cross-country starters compared with only 0.83% of all starters in BE competitions for the years 1996–1999 [60] and 0.76% in 2000 [61]. In the latter study it was noted that amateur event riders were approximately 20 times more likely to fall than professional riders. The current results appear to reflect a disturbing trend of an increased proportion of unseated riders at the lower levels. These results are consistent with the most recent BE Safety report (2019) [62], which recorded horse falls or unseated riders in 2.4% of approximately 66,000 cross-country starts across all levels (BE 80 to Advanced, including 4* [now 5*] three-day events), with the largest proportion of starts being at BE 90, 100 and Novice levels. In a study of Fédération Equestre Internationale international competitions, including European and World Championships and Olympic Games, from 2008 to 2018, of 187,602 cross-country starts there were 1.5% horses falls and 3.5% unseated riders [63]. There were mildly increased odds of a horse fall (1.1) or unseated rider (1.1) if the dressage penalty score was >50 compared with ≤50.4.5. Social Licence to CompeteThe social licence to use horses in competition is increasingly being questioned [64,65,66,67,68]. The overall low median RHpE score observed in the current study supports the continuing use of horses in affiliated eventing. However, a RHpE score ≥8/24 was documented in 9% of competition starts, and this merits attention. Several dressage judges commented in conversation after the event that they considered that some horses looked clearly uncomfortable, but they felt powerless to intervene. Even when overt lameness was observed, judges commented that they felt reluctant to advise competitors to withdraw, although it was within their remit to do so [11], because of previous adverse experiences. On some occasions competitors had sought the advice of the event veterinarian, who only evaluated their horse moving in hand, and no lameness had been observed. This had resulted in complaints to the event organisers about the dressage judges. It must be borne in mind that there is a considerably higher frequency of occurrence of lameness in ridden horses compared with horses assessed in hand [6,69].Education of riders and coaches/trainers is required to recognise both gait abnormalities that reflect discomfort and ridden horse behaviours that are a manifestation of pain, and to understand the potential consequences of incorrect training on long-term musculoskeletal health. The relationship between RHpE scores and performance highlights the importance of recognition and management of pain for optimising performance. Riders and their coaches/trainers also need to learn to consider all reasons why a horse performed poorly, rather than attribute blame to rider errors, ground conditions, the uncooperative nature of the horse or the difficulty of the course.4.6. Dressage Penalties and RHpE ScoresThe dressage tests at BE 90, 100 and Novice level are straightforward, are not biomechanically demanding and should be relatively easy for a pain-free equine athlete that has been trained and ridden correctly. Nonetheless, there was a large range of dressage penalties and RHpE scores, although considerable clustering of dressage penalties, with a large proportion being between 30 and 40 (Figure 2, Figure 4 and Figure 6). This is likely to reflect the limited range of marks used for each movement; a penalty score of 30 equates to a mean mark of 7 (fairly good) (on a scale of 0 [not executed] to 10 [excellent]) per movement, whereas a penalty score of 40 equates to a mean mark of 6 (satisfactory) per movement [28]. Although there was a moderate correlation between the RHpE score and dressage penalties at all competition levels, there were some notable outliers. For example, at BE 100 there was a horse with a RHpE score of 6 (Figure 2) and a dressage penalty score of 27.8, despite lack of hindlimb impulsion and engagement; marked repeated bilateral hindlimb toe drag; mouth opening with separation of teeth ≥10 s; repeated tail swishing and a stiff stilted canter, lacking suspension. This draws into question the accuracy of some judging, as has been previously observed [70,71,72]. According to the guidance to judges, the marks are assessed based on ‘the gaits (‘The trot is free, supple, regular and active. The canter is united, light and balanced’), impulsion (‘…the engagement of the hindquarters, originating from a lively impulsion. The hindquarters are never inactive or sluggish’), and submission (‘…Harmony with rider, lightness of movements and acceptance of the bit with submissiveness/thoroughness without any tension or resistance.’)’ [28,29]. Riders have commented that ‘harsh, forceful training practices were sometimes rewarded by judges’ [16]. It has previously been observed that ‘…despite the rigorous training that judges receive, they do not protect horses from poor riding or poor welfare. This could be addressed by providing better training to allow judges to recognise and mark down behavioural signs that are indicative of conflict or underlying pain’ [67].4.7. Rein BackRein back was only included in the dressage test at Novice level, but was executed poorly by 23% of competition starters relative to judging guidelines [28]. Major errors in rein back were also observed in Grand Prix dressage competitions [9,10]. This presumably reflects either inadequate training and practice or conflict behaviour [73]. In a small study (n = 32) of dressage horses warming-up before a test, rein back was rarely performed [74]; whether rein back was included in the subsequent test was not documented. However, it is acknowledged that rein back is ‘the severest test of the coordination between driving and restraining influences’ [75] and ‘proof of the degree of suppleness, the action of the rein through the body and obedience’ [76]. The rules indicate clearly that the front of the head should remain vertical and resistance to or evasion of the contact are serious faults [26]. While the movement has clear practical utility, for example being required to open a gate while out hacking, training of this movement in a dressage arena needs to be improved and may be facilitated by early ground work [77,78] and when ridden, accepting one or two steps initially, before progressively asking for more steps [76,79,80,81]. With improved performance of rein back, competitors could gain valuable additional marks.4.8. Limitations of the StudyThe study had some limitations. Not all features of the RHpE could be assessed for some test designs and test locations. For example, for some tests it was not possible to assess straightness in canter on either one rein or both reins, because the assessor was positioned in one standardised location. Strong wind influenced tail carriage, so under some weather conditions the straightness of the tail could not be assessed. Long grass on occasions prohibited accurate determination of the presence or absence of a toe drag. A behaviour was only determined to be present if this was an unequivocal observation. The observer could not be blinded to horse or rider identity, with the potential for bias, however the horse’s subsequent performance could not be predicted, and all statistical analyses were performed completely independently. The duration of the tests was approximately 5 min, the lower end of the spectrum for accurate application of the RHpE [82]. The BE 90 and 100 tests did not incorporate 10 m diameter circles in trot, which are more biomechanically demanding than 20 m diameter circles, and effective in highlighting gait abnormalities and influencing behaviour [82]. Several judges commented that ‘they found it difficult to mark down professional riders’. There are a variety of factors which may adversely influence dressage scores in addition to lameness, including rider skill [17,19], tack fit for horse and rider [6] and how the horse has been trained [83]. Jumping performance may also be influenced by rider skill, confidence and fitness, the athletic capability of the horse, the difficulty of the course, the weather and the terrain and ground conditions [18]. Despite these limitations, consistent results were acquired, with a large data set, across a wide range of venues and competitors.5. ConclusionsThere were significant associations between RHpE scores and performance for horses competing at BE 90, 100 and Novice one-day events. Horses placed in the top three had significantly lower median RHpE scores compared with horses which completed but were not placed in the top three. This indicates that although the quality of performance in one-day events is affected by many factors, musculoskeletal pain is likely to be influential in some horses. Although the median RHpE score was low, supporting the social licence to compete, 9% of competition starters had a RHpE score of ≥8/24, indicating the presence of musculoskeletal pain. Horses with a RHpE score of ≥8/24 performed less well than those with a RHpE score <8. Clinical investigation of horses with pain-related gait abnormalities and instigation of appropriate treatment and management may enhance both welfare and performance. Further education for riders, coaches/trainers and dressage judges is required to facilitate the recognition of signs reflecting pain-related gait abnormalities.

animals : an open access journal from mdpi

10.3390/ani13091477

PMC10177294

More than 10 million tons of coffee are consumed annually in the world, generating two kg of wet spent coffee grounds per kg of coffee consumed, which are considered food waste. Despite the interesting nutritional value of spent coffee grounds for ruminant feeds, their fibre fraction is very high, which presents a limitation for including this alternative ingredient in animals’ diets due to its low digestibility. This study considered thermal and mechanical treatments combined with enzymatic hydrolysis to improve the spent coffee grounds’ nutritive value and digestibility. The main conclusions are that the effect of enzymatic treatments is overwhelmed by the action of ruminal bacteria and that diminution of the particle size is the best strategy to improve the spent coffee grounds’ digestibility.

Lignin in animal diets is a limiting factor due to its low digestibility. This study assessed the effects of thermal or mechanical pre-treatments and enzymatic hydrolysis on spent coffee grounds’ (SCG) nutritional value and digestibility. A first trial studied the effect of thermal pre-treatment and hydrolysis with removal of the liquid part and a second trial studied mechanical pre-treatment and hydrolysis with and without removal of the liquid part. Autoclaving did not improve the enzymatic performance nor the nutritional value. Hydrolysis reduced the digestibility of the solid phase and impaired its ruminal fermentation efficiency. Hydrolysates without removing the liquid part improved its nutritional value, but not compared with unprocessed SCG. Grinding increased crude protein and reduced crude fibre and protein, which led to greater fermentation and in vitro digestibility. Thus, grinding emerges as the most promising valorisation strategy to improve SCG nutritional characteristics and their use for animal feed, contributing to the circular economy.

1. IntroductionCoffee is one of the world’s most important commodities and its consumption is widespread across the planet. According to the International Coffee Organization [1], about 10.2 million tons of coffee were consumed worldwide during the 2020/2021 period (coffee year: October–September). This amount represents an increase of 3.27% compared with 2019/2020, despite the influence of the COVID crisis on food consumption. Europe is the region with the highest consumption worldwide, at 3.3 million tons of coffee.The hotels, restaurants, and catering industry (HORECA) is one of the most important sectors responsible for this consumption since its activity consists basically of preparing and serving food and beverages. However, coffee consumption involves the production of different organic wastes. The most important of these are spent coffee grounds (SCG), which are the insoluble parts that remain after making coffee. Each kg of coffee consumed produces two kg of wet SCG that are considered food waste (about 6.6 million tons of SCG) and should be managed in the best possible way [2].The most extensively used method of managing SCG in Europe is landfilling [3], which is limited by a current European directive (Directive EU 2018/850). In any case, the management of SCG in landfill involves a carbon footprint of about 1716 million kg CO2eq/year, associated with an environmental cost of 0.26 kg CO2eq/kg of food waste [4]. Therefore, it is necessary to find a global solution for the reintroduction of this by-product into the value chain, avoiding the environmental impact of its management as waste.Several potential alternatives have been considered for the recycling of SCG, such as the production of pellets as an energy source or as a substrate for biodiesel production [5,6]. However, these alternatives could be considered of low value according to the prioritisation hierarchy for the best use of surplus food, as established by the EU Waste Framework Directive 2008/98/EC: first reduce food surplus, followed by its use for human consumption or livestock feed, and finally for bioenergy such as biogas. Furthermore, if the nutritional properties of this raw material are considered, they are suitable for higher value applications such as animal feed. The high content of cellulose, hemicelluloses, proteins, fats, polyphenols, and minerals makes SCG interesting for the livestock sector [7,8].A previous study proposed the use of the existing logistic routes for HORECA waste, such as those for used oil, to collect the SCG. This framework established a collection period of no more than 4 days depending on microbiological stability. In addition, it stipulates periodic cleaning of the containers and their placement in areas not intended for garbage [2].According to the European Feed Manufacturers’ Federation (FEFAC), farm animals in Europe consumed an estimated 701 million tons of feed in 2021 [9], 22% produced by compound feed manufacturers. In 2020, compound feed production in the European Union reached 150.2 million tons of feed, excluding petfood [10].The main cost factor in livestock activity is animal feeding, with up to 55% for poultry, 32% for pigs, and 14% for cattle [10]. In addition, feed costs have increased more than production prices in recent years. Therefore, farmers need to improve their productivity to maintain the sustainability and profitability of livestock activity in the future.Within this framework, despite the interesting nutritional value of SCG, its fibre fraction is very high and its acid detergent lignin (ADL) content is around 27.83% [2]. This ADL content presents a limitation for including this alternative ingredient in animals’ diets due to its low digestibility, as reported in previous studies [2,11,12]. Therefore, it is necessary to degrade lignocellulosic bounds to allow a higher level of inclusion of SCG ingredients in animal feed.In this context, thermal and mechanical pre-treatments are presented as effective strategies to break the lignocellulosic bounds in the fibre fraction, while increasing the surface area of the material and facilitating contact with enzymes [13]. This would lead to an increase in the digestibility of the ingredients [14]. However, the intensity and duration of thermal treatments have an important effect on the final digestibility [15]. Mechanical pre-treatment aims to reduce the particle size of substrates, which normally leads to more digestible ingredients [16,17,18]. However, defining the optimal particle size of SCG is of utmost importance to achieve its optimal nutrient and energy digestibility as an ingredient.Meanwhile, enzymatic hydrolysis has the potential to increase digestibility by degrading fibre fractions [19]. Hydrolysis involves the breaking of bonds to obtain fibres of different sizes. This process must always be adapted to the characteristics of both the initial product to be hydrolysed and the final product to be obtained. Thus, the optimum conditions of the hydrolysis treatment are critical and must be defined.The objective of the present study was to determine the best process to improve the digestibility of SCG, with the aim of increasing their inclusion in ruminants’ diets. Thus, this study focused on evaluating the effects of two different pre-treatments (autoclaving and grinding) and enzymatic hydrolysis using different enzymes on the nutritive value, in vitro organic matter digestibility (IVOMD), and short chain fatty acids (SCFA) production of SCG from the HORECA sector.2. Materials and Methods2.1. Experimental DesignsThe SCG samples used in this study were obtained from HORECA industry in northern Spain (Basque Country). The initial samples were divided into 500 g bags and were kept frozen (−20 °C) until processing. Two different experiments were designed and performed: the first was to evaluate the effects of thermal pre-treatment of the SCG and hydrolysis with four different enzymes removing the liquid phase, whereas the second studied the effects of mechanical pre-treatment and hydrolysis with a mix of two enzymes selected accordingly to the results of the first experiment with and without removing the liquid phase.2.1.1. Thermal Pre-Treatment and Enzymatic HydrolysisIn the first experiment, different cellulolytic enzymes were used to degrade the SCG fibre fractions. Thermal pre-treatment was also evaluated as a method to facilitate the availability of cellulose and hemicellulose fractions to the enzymes.The trial was designed as a factorial design (2 × 5) including two factors: thermal pre-treatment (with and without) and enzymatic hydrolysis (EH) (CTR: unprocessed SCG; 1: Celuclast®; 2: Ultimase®; 3: Viscozyme®; 4: Ultraflo®).The initial SCG sample was divided into two. Half of the sample was preserved for further analysis. The other half was subjected to thermal pre-treatment (autoclaving) at 121 °C for 15 min. Both subsamples were again divided into two halves. One half of each subsample was preserved unmodified for further analysis. The other half was divided into four subsamples which were hydrolysed by four different enzymes.Enzymes were provided by Novozymes (NovozymesA/S, Bagsvaerd, Denmark). Viscozyme® is an endo-beta-glucanase that hydrolyses (1,3)- or (1,4)-linkages in beta-D-glucans with high mannase activity. Celluclast® and Ultimase® are cellulases that hydrolyse (1,4)-beta-D-glucosidic linkages in cellulose and other beta-D-glucans. Ultraflo® is an endo-beta-glucanase that hydrolyses (1,3)- or (1,4)-linkages in beta-D-glucans and a xylanase that hydrolyses (1,4)-beta-D-xylosidic linkages in xylans.Hydrolysis conditions were stablished based on the technical data sheets for the enzymes provided by Novozymes: pH 5, 55 °C, 20 h, 250 rpm, ratio 1:1 SCG:water, and 1% (v:w) of enzyme with respect to fibre. Hydrolysis was performed using Sell Symphony 7100 Bathless Dissolution Distek equipment (Distek Inc., North Brunswick, NJ, USA), controlling and monitoring temperature, time, and stir speed. The pH of each run of the experiments was controlled manually and adjusted with NaOH 1 M in a final volume of 500 mL. The hydrolysis processes were ended by enzyme inactivation at a temperature of 90 °C for 15 min. Then, the samples were centrifuged (2650× g; 15 min; room temperature), and two fractions were recovered: the solid sample (the one intended for animal feed) and the liquid fraction (not considered for animal feed in this study; this fraction was obtained for all treatments except for the CTR which was unprocessed). This procedure was performed three separate times.After all the treatments, samples were freeze-dried and kept in closed plastic bags until physicochemical analyses and IVOMD determination.2.1.2. Mechanical Pre-Treatment and Enzymatic Hydrolysis with and without Removing the Liquid PhaseIn the second experiment, considering the results obtained in the first experiment, two of the enzymes used in the first trial were selected to perform the hydrolysis of the SCG (Viscozyme® and Ultimase®). Additional evaluation assessed particle size reduction by means of grinding as pre-treatment to facilitate the availability of cellulose and hemicellulose fractions to the enzymes.In addition, with the aim of testing the effect on IVOMD of the potential release of soluble components of SCG to the liquid fraction in the hydrolysis process, a hydrolysate sample without separation of the solid and liquid fraction was also evaluated in this experiment. Thus, the hydrolysis treatments evaluated in this experiment were: unprocessed SCG, SCG hydrolysed with the mixture of the two described enzymes and with the liquid fraction removed, and SCG hydrolysed with the mixture of the two described enzymes without removing the liquid fraction.The trial was designed as a factorial design (2 × 3), with two factors: grinding (unground sample; coarse grinding and fine grinding) and hydrolysis (without hydrolysis, hydrolysis without removing the liquid fraction, and hydrolysis with removal of the liquid fraction).The initial SCG sample was divided into three. One of the samples was preserved for further analysis. The other two were subjected to grinding in a Comitrol® Processor Model 1700 (Urschel, Chesterton, IN, USA). One subsample was ground using a knife head with 160 blades to achieve a final estimated particle size of 250 µm, and the other subsample was ground using a knife head with 260 blades to achieve an estimated final particle size of 100 µm.Each subsample was again divided into three. One of the samples was preserved unmodified for further analysis. The other two were hydrolysed by the mixture of two different enzymes (Viscozyme® and Ultimase®, Novozymes A/S, Bagsvaerd, Denmark). Hydrolysis conditions were established based on the technical data sheets for the enzymes provided by Novozymes: pH 5, 55 °C, 20 h, 250 rpm, ratio 1:1 SCG:water, and 1% (v:w) of each enzyme with respect to fibre. The hydrolysis process, inactivation and centrifugation were performed as explained in Section 2.1.1. Then, all the treated samples were freeze-dried and kept in closed plastic bags until physicochemical analyses and IVOMD determination.2.2. Rumen In Vitro Digestibility DeterminationEach of the samples obtained in the first and second experiments were used as a substrate in a short-term in vitro batch fermentation trial as described by Pell and Schofield [20], to test the effects of the different treatments on the rumen IVOMD of the SCG and the fermentation characteristics.Each of the samples were incubated in triplicate, in four different incubation runs performed in different weeks.In each of the runs, rumen fluid was collected from one multiparous Latxa ewe slaughtered for production purposes. Before slaughtering, ewes were fed fescue hay ad libitum for 3 weeks and had free access to fresh water. Ruminal fluid was collected before the morning feeding and strained through four layers of cheesecloth into a pre-warmed thermos flask.Approximately 500 mg of solid samples from the three independent processing runs were weighed into 125 mL serum bottles, 50 mL of culture fluid was added (1:4 ruminal fluid and phosphate–bicarbonate buffer, respectively) [21], and bottles were crimp sealed. Bottles were incubated at a constant temperature (39 °C) in an incubator for 24 h. Gas production was released at 2, 4, 6, 8, 10, 12, and 15 h post-inoculation to avoid pressure exceeding 48 kPa in the bottle headspace, as suggested by Theodorou et al. [22]. After 24 h of incubation, bottles were put into the fridge for 15 min to stop fermentation before subsequent sampling for SCFA determination.IVOMD was calculated as described by Pell and Schofield [20]. In this process, 45 mL of a neutral detergent solution was added to each bottle and warmed at 105 °C for 1 h; then, the bottles were cooled, filtered through glass filter crucibles (Porosity 2) and washed with distilled water, ethanol, and acetone. The remaining sample was dried at 100 °C overnight and then burned in a muffle furnace at 525 °C and weighed to obtain true IVOMD values according to the difference from the weight of the incubated organic matter (OM) [20].2.3. Chemical AnalysesThe physicochemical composition was analysed only in the solid fractions of both experiments. SCG was analysed for dry matter (DM, method 934.01), ash (method 942.05), and nitrogen (method 984.13) content following the Association of Official Analytical Chemists [23]. Neutral detergent fibre (NDF) was determined by the UNE EN ISO 16472 method with use of an alpha amylase but without sodium sulphite, and was expressed free of ash. Acid detergent fibre (ADF) and ADL, expressed exclusive of residual ash, were determined by AOAC method 973.18. Neutral detergent insoluble protein (NDICP) and acid detergent insoluble protein (ADICP) were determined by analysing the NDF and ADF residues, respectively, for Kjeldahl nitrogen. Total reducing sugars (TRS) were determined by the dinitrosalicylic acid reagent method [24] adjusted for the microplate (Thermo Fisher Scientific, Roskilde, Denmark) assay procedure [25].The antioxidant activity of samples was measured using the DPPH (2,2-Diphenyl-1-picrylhydrazyl, D9132 Sigma Aldrich, Steinheim, Germany) radical scavenging activity method [26]. DPPH in methanol (40 ppm) was prepared and 280 μL of this solution was added to 20 μL of sample solution. The mixture was incubated at room temperature in the dark for 30 min. Absorbance was measured at 515 nm. The standard comprised of water–methanol (50% v/v) and different concentrations of trolox (218940050, Acros Organics, NJ, USA). The antioxidant capacity was expressed as mg trolox equivalent antioxidant capacity (TEAC) per g of DM, using the calibration curve. TPC was measured using the Folin–Ciocalteu method [27]. Initially, 30 μL of Folin–Ciocalteu (J/4100/08, Fischer Scientific, Loughborough, UK) solution was added to 140 μL of sample, blank, or standard and 140 μL of Na2CO3 7% (w/v) (Sigma Aldrich, Steinheim, Germany). The mixture was incubated at room temperature in the dark for 1 h and the absorbance was measured at 750 nm. Gallic acid (G7384, Sigma Aldrich, Steinheim, Germany) was used as standard at a concentration range of 1.4–20 ppm, and results were expressed as mg gallic acid equivalent (GAE) per g of DM sample.For caffeine analysis, 1 g of previously homogenized sample was weighed into an Erlenmeyer flask and 2 g of magnesium oxide was added. The samples were diluted with milli Q water without exceeding a volume of 100 mL and boiled for 15 min. The samples were then made up to the volume of 100 mL, filtered through 0.45 m PTFE filters and placed in vials for subsequent caffeine analysis. Caffeine was measured using chromatographic assays on an Agilent Technologies 1200 series HPLC system (Santa Clara, CA, USA) equipped with a UV/Vis photodiode array detector, a quaternary pump, and a degasser system. The column used was a Nova Pak C—18 3.9 × 150 mm (Waters, Etten-Leur, The Netherlands) at 40 °C. The mobile phases used were acetonitrile 12% and water 88% in isocratic elution, and were pumped at 1.0 mL/min. In this process, 20 µL of each sample and standard were injected and absorbance was measured at 274 nm of wavelength. The standard used was caffeine and the constructed calibration curve ranged from 10 to 70 ppm. In the first experiment, TRS, caffeine, TEAC, and TPC were analysed only in the liquid fraction in order to quantify the release of compounds to the liquid fraction. In the second experiment, they were analysed only in the solid fraction in order to quantify their concentrations in the final ingredient intended for animal feed.The analysis of the SCFA (acetic, propionic, butyric, isobutyric, valeric, and isovaleric) was performed by gas chromatography using a flame ionization detector as described by Goiri et al. [28]. Briefly, a volume of 4 mL of ruminal incubation medium mixed with 1 mL of a solution of 20 g/L of metyl-valeric acid as an internal standard in 0.5 N HCl was centrifuged (462× g for 20 min at 4 °C) and microfiltered (premium syringe filter regenerated cellulose, 0.45 µm 4 mm, Agilent Technologies, Madrid, Spain), and 0.5 µL of liquid phase was directly injected into the apparatus (Agilent 6890 N, Agilent, Spain). Data are expressed in mmol/100 mmol.2.4. Calculations & Statistical AnalysisData distribution was tested for normality using the Shapiro–Wilk test, and the Levene test was used to assess the equality of variances.The data of the physicochemical characterization of the SCG solid fractions and the TRS, TPC, caffeine, and TEAC of the liquid fraction were processed by analysis of variance using the GLM procedure of SAS [29], including the fixed effects of the pre-treatment, hydrolysis, and the interaction between them.In the first experiment, the total number of observations for the IVOMD trial comprised 3 runs of processing (hydrolysis) × 2 pre-treatment conditions × 5 enzymatic procedures × 4 in vitro incubation runs × 3 laboratory replicates = 360; however, after averaging the incubation runs and laboratory replicates, the remaining 30 observations were subjected to analysis of variance using the GLM procedure [29]. The statistical model included the fixed effects of the thermal pre-treatment, the enzymatic hydrolysis, and their interaction.In the second experiment, the total number of observations for the IVOMD trial comprised 3 runs of processing (hydrolysis) × 3 grinding treatments × 3 hydrolysis procedures × 4 in vitro incubation runs × 3 laboratory replicates = 324; however, after averaging the incubation runs and laboratory replicates, the remaining 27 observations were subjected to analysis of variance using the GLM procedure [29]. The statistical model included the fixed effects of grinding, hydrolysis, and their interaction.Least squares mean values for treatments are reported. Treatment means were separated using Tukey adjustment, and significant effects were declared at p < 0.05 with a tendency at p < 0.10.3. Results3.1. Thermal Pre-Treatment and Enzymatic HydrolysisThere was no interaction between thermal pre-treatment and enzymatic hydrolysis for TRS, TPC, caffeine, and TEAC, as shown in Table 1.No differences were observed for TPC, TEAC, and caffeine concentration among enzymes (Table 1). The mean losses of polyphenols, caffeine, and antioxidant compounds to the media were 5.6, 0.79, and 8.6 mg per gram of initial dry SCG, respectively.Regarding TRS, a significant effect of the enzymatic hydrolysis was found, where Viscozyme® released more TRS to the liquid fraction during hydrolysis than Celluclast® (p = 0.013) or Ultraflo® (p = 0.003), and Ultimase® tended to release more TRS than Ultraflo® (p = 0.076) (Table 1).There was no significant interaction between thermal pre-treatment and enzymatic hydrolysis, nor a thermal pre-treatment effect on the chemical composition variables of the solid fractions of SCG (Table 2). Regarding the enzymatic hydrolysis treatments, no effect was observed for DM, CP, NDF, ADL, or NDICP in the SCG solid fraction (Table 2). However, enzymatic hydrolysis with Celluclast®, Ultimase®, and Viscozyme® reduced the content of ash by 29% (p = 0.003), 29% (p = 0.003), and 33% (p < 0.001), respectively, compared with CTR, but no differences were observed for Ultraflo® compared with CTR. All enzymes increased the content of ADICP (from 27 to 34%; p ˂0.001) compared with CTR without any significant differences among them. Viscozyme® and Ultraflo® also increased the content of ADF by 11% and 13%, respectively, compared with CTR (p < 0.05), whereas Celluclast® and Ultimase® showed only a tendency to increase it compared with CTR (p < 0.1).Regarding SCG solid fraction in vitro digestibility values (Table 2), neither significant interaction between thermal pre-treatment and hydrolysis nor a thermal pre-treatment effect were observed. However, an effect of enzymatic hydrolysis on the IVOMD of SCG was observed (p = 0.001). In Table 2, it can be observed that all the enzymes reduced the IVOMD between 21% and 32% compared with the CTR, with no significant differences among enzymes.Although enzymatic hydrolysis led to a decrease in IVOMD, SCFA concentration in the in vitro medium remained unaffected compared with CTR (p = 0.927; Table 3). There was no interaction between thermal pre-treatment and enzymatic hydrolysis for any of the measured fermentation products (Table 3). Enzymatic hydrolysis showed a tendency to increase acetic acid proportions; only Viscozyme® significantly increased this parameter compared with CTR (62.5 vs. 60.8 mmol/100 mmol, p = 0.025), and all the tested enzymes decreased the propionic acid proportions (p < 0.001) by around 7 to 10% compared with CTR. All enzymes increased the acetic to propionic acid ratio (p ˂ 0.001) compared with CTR. Similarly, the thermal pre-treatment increased the acetic to propionic acid ratio (p ˂ 0.001) by 12% compared with CTR.3.2. Mechanical Pre-Treatment and Enzymatic Hydrolysis with and without Removing the Liquid PhaseAn interaction between the grinding pre-treatment and the hydrolysis process was observed to affect the concentrations of bioactive compounds in the SCG (Table 4).Regarding TPC of the SCG samples, it was observed that the grinding process did not have a significant effect on TPC when the SCG samples were hydrolysed removing the liquid phase, whereas both fine grinding and coarse grinding increased TPC when SCG were not subjected to any hydrolysis, and fine grinding increased TPC when SCG were hydrolysed without removing the liquid phase (Table 4).Regarding TEAC, similar results were observed in the SCG without hydrolysis where both fine and coarse grinding presented higher TEAC. When SCG were hydrolysed removing the liquid fraction, only fine grinding increased TEAC. When SCG were hydrolysed without removing the liquid fraction, no effect on TEAC was observed for fine grinding compared to unground, and coarse grinding even decreased it.Caffeine content in SCG was significantly (p < 0.001) affected by the grinding pre-treatment and hydrolysis processes, although there was no significant interaction between them. The unground sample had a higher caffeine content than the fine (0.56 vs. 0.41%) and coarsely ground samples (0.56 vs. 0.42%). No differences were found between the fine and coarse grinding processes. Hydrolysed SCG samples with the liquid phase removed showed a lower caffeine content compared with non-hydrolysed ones (3.5 vs. 5.2 mg/g DM) and compared with SCG samples hydrolysed without removing the liquid phase (3.5 vs. 5.1 mg/g DM).Regarding the TRS concentration of samples, an interaction between the grinding pre-treatment and the hydrolysis process was observed. When SCG samples were not hydrolysed, no differences between grinding processes were found. When samples were hydrolysed with and without removing the liquid phase, unground samples had lower TRS concentration than coarse or fine ground samples and coarse ground samples had lower TRS concentration than fine ground ones. The highest TRS was observed in the samples in which the liquid phase had not been removed (p < 0.001). A significant interaction between grinding and hydrolysis was observed for the ash content of the samples (Table 4). When the SCG were hydrolysed removing the liquid fraction, both grinding processes reduced the ash content compared with the unground samples, and fine grinding reduced it compared with coarse ground samples. However, when the samples were not hydrolysed both grinding types reduced ash to the same extent compared with unground samples, and when samples were hydrolysed without removing the liquid fraction only fine grinding reduced the ash content.No interaction between grinding and hydrolysis processes was found for CP, NDICP, or ADICP content, but both the grinding and the hydrolysis process showed a significant effect. The unground SCG sample showed lower (150 g/kg DM) CP content compared with both fine (153 g/kg DM, p < 0.001) and coarse (152 g/kg DM, p < 0.001) grinding. However, the unground SCG sample (61 g/kg DM) showed greater CP content in the NDF fraction compared with fine (56 g/kg DM, p = 0.031) and coarse (54 g/kg DM, p = 0.003) grinding. A similar trend was observed for the CP content in the ADF fraction, where unground samples showed greater content compared with either fine (30 vs. 26 g/kg DM, p = 0.002) or coarse (30 vs. 27 g/kg DM, p = 0.046) grinding. The hydrolysis process with removing the liquid phase resulted in a reduction of the CP content compared with non-hydrolysed samples (150 vs. 153 g/kg DM, p < 0.001) and hydrolysed samples without removing the liquid phase (150 vs. 153 g/kg DM, p < 0.001), and no differences were observed between the two latter samples (p = 0.987). In addition, the hydrolysis process removing the liquid phase resulted in a greater NDICP fraction compared with hydrolysis without removing the liquid phase (33 vs. 27 g/kg DM, p = 0.004) and samples without hydrolysis (33 vs. 24 g/kg DM, p < 0.001), the difference being significant between the latter two (27 vs. 24 g/kg DM, p = 0.004). In addition, the hydrolysis process removing the liquid phase resulted in a greater NDICP fraction compared with hydrolysis without removing the liquid phase (66 vs. 55 g/kg DM, p < 0.001) and the samples without hydrolysis (66 vs. 55 g/kg DM, p < 0.001), the difference being non-significant between the latter two (p = 0.980).Regarding the fibre fractions, only hydrolysis processes affected NDF content. Non-hydrolysed SCG samples showed greater NDF content compared with hydrolysis without removing the liquid phase (624 vs. 566 g/kg DM, p < 0.001), but lower content compared with hydrolysis removing the liquid phase (624 vs. 646 g/kg DM, p = 0.028). There were significant differences between hydrolysis removing the liquid phase and without removing the liquid phase (p < 0.001). Hydrolysis and grinding processes affected the ADF content in the SCG. Non-hydrolysed SCG samples showed lower ADF content compared with hydrolysis removing the liquid phase (375 vs. 454 g/kg DM, p < 0.001) and hydrolysis without removing the liquid phase (375 vs. 423 g/kg DM, p < 0.001). There were significant differences between hydrolysis removing the liquid phase and hydrolysis without removing the liquid phase (p < 0.001). The grinding process reduced ADF content after both fine (409 vs. 440 g/kg DM, p < 0.001) and coarse (402 vs. 440 g/kg DM, p < 0.001) grinding compared with unground samples, and the differences between fine and coarse grinding were not significant (p = 0.623).A significant interaction was observed for ADL content only when the samples were hydrolysed removing the liquid phase; lower ADL contents were found when the sample was coarse ground compared with fine (p < 0.001) or unground samples (p = 0.018). Regarding IVOMD, Table 5 reveals that no significant interaction between grinding pre-treatment and hydrolysis was observed. However, both grinding pre-treatment and hydrolysis affected the IVOMD values of the SCG.Table 5 shows that hydrolysis of SCG removing the liquid fraction reduced IVOMD compared either with unprocessed SCG (272 vs. 350 g/kg OM, p < 0.001) or hydrolysed SCG without removing the liquid fraction (272 vs. 364 g/kg OM, p < 0.001). However, no differences were found between non-hydrolysed samples and hydrolysed samples without removal of the liquid phase (p = 0.403). Fine grinding (429 g/kg OM, p < 0.001) and coarse grinding (297 g/kg OM, p = 0.003) processes both resulted in increased IVOMD compared with the unground sample (261 g/kg OM). In addition, fine grinding improved IVOMD compared with coarse grinding (p < 0.001).Similar results were observed for fermentation parameters, where no significant interaction between grinding and hydrolysis was observed for SCFA production. Fine grinding increased SCFA production compared with either coarse grinding (4.52 vs. 3.85 mmol/100 mL, p < 0.001) or unground samples (4.52 vs. 3.73 mmol/100 mL, p < 0.001).A significant interaction between grinding and hydrolysis was found for the main SCFA produced during fermentation of the samples.Regarding acetic acid, no significant differences between grinding conditions were found when the samples were not subjected to hydrolysis. However, when hydrolysis removing the liquid phase process was applied, both fine and coarse grindings reduced the acetic proportions compared with the unground sample. When the hydrolysis without removing the liquid phase process was applied, only fine grinding reduced the acetic proportions compared with the unground sample. Propionic molar proportions increased in the fine ground samples regardless of the hydrolysis process. Butyric molar proportions were not affected by the grinding process when hydrolysis processes were applied either with or without removing the liquid phase. However, when not subjected to hydrolysis, butyrate molar proportions were reduced after samples were subjected to fine grinding, compared with either coarse or unground samples. Fine grinding resulted in a lower acetic:propionic acid ratio compared with either coarse grinding (2.12 vs. 2.48, p < 0.001) or unground samples (2.12 vs. 2.55, p < 0.001), with differences between the latter not being significant (p = 0.106). Subjecting SCG samples to hydrolysis without removing the liquid phase resulted in a reduced acetic:propionic acid ratio compared with hydrolysis removing the liquid phase (2.32 vs. 2.42, p = 0.002) or samples without hydrolysis (2.32 vs. 2.42, p = 0.007), and the differences between the latter were not significant (p = 0.929).4. DiscussionSpent coffee grounds have been included in ruminant rations at doses up to 10% of the concentrate (3% of total diet) for dairy sheep [11] and up to 5% of the concentrate (2% of total diet) for dairy cows [2] without impairing productive performance. In addition, De Otalora et al. [11] observed some improvement on productive performance with the SCG diet, which could be related to the effect of the secondary compounds present in the SCG on the rumen microbial populations [12].Although these results are promising, the aim of a “circular economy” approach should be to maximize the inclusion of this by-product in ruminants’ rations. However, other studies have reported that the low fibre digestibility of SCG impaired animal performance when this by-product was included in the ration in a much higher dose [30,31,32].To overcome these constraints, different valorisation strategies were tested in the present study with the aim of attacking the lignocellulosic bonds and fibre fraction of the SCG and thereby improving their digestibility.In the first experiment, contrary to other studies in the literature [33,34], thermal pre-treatment did not improve SCG nutritive value. Autoclaving of the SCG in the conditions described in this experiment (121 °C, 15 min) appeared to be ineffective for breaking the lignocellulosic bounds in the SCG. These results could be due to a low rate of cellulose hydrolysis and, therefore, low TRS release at the autoclaving temperature used. Other authors working with lignocellulosic biomass have reported that reaction temperature when applying thermal pre-treatments significantly affects the characteristics of the solid product obtained [35]. Therefore, it is possible either that the temperature applied in the present study was not high enough or the time was not long enough to obtain an effect from the thermal pre-treatment.In contrast, enzymatic hydrolysis affected the nutritive value and digestibility of SCG. The hydrolysis processes carried out involved addition of water that was subsequently removed when the hydrolysis was finished. It was noteworthy that according to the data measured in the liquid fraction, the enzymatic hydrolysis process did not seem to compromise the concentration of bioactive compounds in solid SCG intended for animal nutrition. This issue is of interest, as mentioned previously, due to the potential beneficial effects of bioactive compounds present in SCG on animals’ productive performance and health [11].Release of TRS to the liquid medium was observed to differ between the various enzymes used for the hydrolysis processes. These differences between enzymes in the release of TRS are probably related to the higher enzymatic activity of Viscozyme ® and Ultraflo ®.However, as the liquid fraction is removed, it is expected that this release of TRS to the liquid fraction during the hydrolysis processes would lead to a loss of TRS in the solid fraction intended for animal nutrition compared with the unprocessed SCG (CTR). In the first experiment, TRS in the solid fraction were not measured, but this hypothesis is corroborated by the changes observed in the proportions of other components of the solid fraction. Indeed, a concentration of insoluble and more recalcitrant compounds (ADF and ADICP) in the solid part was observed, which could be partially explained by the release of soluble compounds in the liquid phase followed by its subsequent elimination. Such an effect is not surprising, since studies dealing with the improvement in TRS release to the liquid phase in biomass-to-bioethanol processes using lignocellulosic materials have shown the efficiency of enzymatic hydrolysis in releasing TRS to the liquid media [14,36,37,38,39]. Moreover, a lower concentration of ash was observed after the hydrolysis, which could be due to the draining of minerals to the liquid phase during the hydrolysis process and their loss when this fraction was removed.Digestibility of feedstuffs is an important issue to consider when formulating a diet, and is known to be closely related to their physicochemical composition. In this sense, the results obtained for the physicochemical composition of the solid fraction of the SCG and the TRS concentration of the liquid phase after the hydrolysis processes are in line with those observed for the digestibility of the solid fraction.Fibre concentration is one of the main important factors affecting dry matter digestibility, especially in relation to its level of lignification [40,41,42]. Reducing concentration of sugars in a feedstuff is also of interest, since these are rapidly fermented in the rumen, yielding microbial cells, organic acids, gas, and microbial glycogen. For example, glucose and fructose were completely fermented within 4–6 h in the rumen [43] and TRS concentration in a feedstuff is therefore related to its digestibility.Although reduced digestibility was observed with the enzymatic hydrolysis, production of total SCFA during ruminal fermentation was not affected. This is surprising, since SCFA production during fermentation is positively associated with the amount of organic matter fermented by ruminal microorganisms [44]. However, Hvelplund [45] observed that in situations where microbial synthesis efficiency was low, the fermentation products increased in relation to the amount of substrate digested. Therefore, it could be said that when the SCG in the present study were hydrolysed, the microbial growth in the in vitro systems may have been limited, and this issue also affected the digestibility values.The physicochemical characteristics of a feedstuff also influence the rumen fermentative process and, therefore, the SCFA produced as a consequence. In general, raw material with an elevated ADF value leads to a fermentation process in the rumen with acetic acid as a main final product in detriment of propionic production, which corroborates the results observed in this experiment.The effects observed for the enzymatic hydrolysis are relevant in the context of ruminants’ nutrition, since a decrease in digestibility and a shift towards less efficient fermentation routes with proportionally more acetic and less propionic acid is not desirable.Therefore, taking into account the results of this first experiment, a different pre-treatment method (grinding) was tested in a second experiment to try to break the lignocellulosic bounds present in the SCG fibre fraction. Furthermore, considering the influence of the release of soluble compounds to the liquid fraction on the physicochemical characteristics and digestibility of the solid fraction, an enzymatic hydrolysis process that did not include removal of the liquid fraction was also tested.In order to study the effect of the enzymes, eliminating the dilution effect caused by solid–liquid separation, Ultimase® and Viscozyme® were selected for the hydrolysis because they showed higher release of TRS to the liquid fraction associated with higher enzymatic activity.In this second experiment, grinding pre-treatment and EH showed an effect on SCG physicochemical characteristics and digestibility. Moreover, an interaction between them was observed for some of the measured variables.Regarding the proportion of secondary compounds in the SCG, contrary to the results observed in the first experiment, effects of the grinding pre-treatment and hydrolysis process on the concentrations of bioactive compounds in the SGC were observed in the second experiment.The effect of grinding on the caffeine content in the brew and, therefore, in the SCG has previously been analysed in the literature [46], with conclusions in line with our results that the finer the grinding the more caffeine appears in the brew and the less in the remaining SCG. Concerning EH, the results suggested that some of the caffeine may have been released to the liquid fraction during the hydrolysis process and was removed in the hydrolysed samples when the liquid phase was removed, and that this effect disappeared when the liquid phase was not removed after the hydrolysis.The concentrations of TPC and TEAC indicated an interaction between the grinding pre-treatment and EH. The mechanical pre-treatment increased their concentrations in the SCG, but this effect disappeared when the liquid fraction was removed after the hydrolysis process. This confirmed the hypothesis of the first experiment concerning loss of compounds by solubilisation in the liquid during EH.Coffee polyphenols have been variously studied for their antioxidant properties [47]. Polyphenols in plant materials are closely connected to the plant cell wall structure [48] and any attempt to break the lignocellulosic bounds of the cell wall may cause a release of these bioactive compounds. In this sense, grinding pre-treatment seemed to achieve this objective thereby improving the antioxidant capacity. However, TEAC results observed for hydrolysis with and without removing the liquid phase were not so clearly related to the results obtained for TPC.There is no clear explanation for these data observed in the hydrolysis processes. Antioxidant compounds have different mechanisms of action correlated with structural specificity [49]. Therefore, depending on the type of compounds present in the sample, the results may differ due to the method of analysis [50]. In this sense, several studies have shown that there is not always a linear relationship between antioxidant capacity and TPC determined by the Folin–Ciocalteu method [51]. Another possible explanation is the presence in the SCG of non-phenolic compounds with TEAC that were not analysed in the current study, such as melanoidins. Melanoidins are compounds with recognized antioxidant capacity that are formed due to Maillard reactions during the processing of coffee [52]. These melanoidins are not fully extractable nor digestible, which may interact with their release to the liquid fraction during hydrolysis processes [53].Regarding the nutritive value of SCG, the interaction observed between mechanical pre-treatment and EH for TRS concentration indicates that grinding pre-treatment before the hydrolysis process was effective in increasing the surface area of the SCG and facilitating contact with enzymes [13], therefore increasing the TRS content in the SCG samples. Some of these TRS were released to the liquid fraction, and thus the SCG sample hydrolysed without removing the liquid fraction showed the highest content of TRS per g of DM. Moreover, this also indicates that EH has the potential to increase digestibility by degrading fibre fractions, as reported in the literature [19].The results concerning SCG ash content agreed with those obtained in the first study and with the hypothesis that some minerals were solubilized in the liquid fraction during the hydrolysis processes and removed from the solid fraction. These results also indicate that as the grinding became more intense the release of minerals increased, especially in the hydrolysed samples.Mechanical pre-treatment showed other results of interest for animal nutrition. The grinding pre-treatment could increase the CP of SCG intended for animal feeding and reduce the amount of CP that it is attached to the fibrous fraction and is more difficult for the animal to digest.However, EH exerted the opposite results. The slight decrease in CP and increase in protein linked to fibre observed with the hydrolysis process is not positive from an animal nutrition point of view, as previously mentioned, but these negative effects disappeared when the liquid fraction was retained after the hydrolysis.Regarding the fibre fractions, EH affected NDF content. No such effect was observed in the first experiment, when NDF or CP were not affected by the hydrolysis. It may be that the two selected enzymes used together could digest the SCG samples to a greater extent, leading to less fibre-associated protein and releasing more simple carbohydrates to the media and concentrating fibre fractions in the solid phase. It is possible that this effect was counterbalanced when the samples were reconstituted with the liquid fraction.Results obtained for the physicochemical composition of the solid fraction of the SCG after the grinding pre-treatments and the hydrolysis processes are in line with the IVOMD results observed. Moreover, as stated previously, the physicochemical characteristic of a feedstuff also influences the rumen fermentative process and, therefore, the SCFA produced as a consequence. In this context, the effects on the composition of the fibre fraction observed in this experiment could have led to the results obtained for individual SCFA proportions during in vitro fermentation.Results obtained in the second experiment showed that the grinding pre-treatment contributed to the breakdown of the lignocellulosic bounds present in the SCG fibre fraction, and that this effect depended on the particle size. Previous studies have proven the efficacy of grinding as a pre-treatment for lignocellulosic materials [54]. From the animal nutrition point of view, the decrease in more recalcitrant fibre components and its influence on improving the digestibility of SCG and the fermentation products obtained suggest that such pre-treatment may be a very interesting option for valorisation of this by-product. Furthermore, the results observed for the hydrolysis processes indicate that the release of TRS and other substances of interest to the liquid phase during the hydrolysis led to a SCG solid fraction intended for animal nutrition that had poorer physicochemical characteristics and lower digestibility, as was observed in the first experiment. This is not desirable, and it opposes the main objective of this work. Conversely, when the liquid fraction of the hydrolysis process was not removed, these negative effects were avoided. In this case, the physicochemical composition of the obtained SCG was more interesting in terms of animal nutrition than the SCG without processing. However, these changes in the physicochemical composition did not lead to greater IVOMD as had been expected, with IVOMD values similar to those of the unprocessed SCG.These results could be explained by the inherent enzymatic activity of the rumen microorganisms. Ruminant animals harbour a diverse and complex microbial ecosystem capable of digesting and fermenting feedstuffs rich in fibre. These rumen microbes have developed the ability to efficiently use complex plant polymers such as, for example, cellulose and hemicellulose. It is known that degradation and fermentation of structural carbohydrates is accomplished by a cascade of activities carried out by the diverse microbial enzymes that exist in the rumen (cellulases, xylanases, β-glucanases, pectinases, amylases, proteases, phytases, tannases, etc.) [55]. Therefore, in light of these results, the hydrolysis of fibre components under these experimental conditions prior to ruminal fermentation did not improve ruminal digestibility, because rumen microorganisms could counterbalance the benefits of this hydrolysis with their enzymatic activity to attain similar digestibility values. However, this enzymatic process could be of interest for monogastric animals, which lack this type of effective microbial digestion in their guts. Thus, this could be a topic of interest for future works.Although an interaction between grinding pre-treatment and the hydrolysis process was found for many of the measured variables, the limited results observed with the hydrolysis processes minimize the practical opportunities for combined application of these treatments. However, grinding alone could be an interesting strategy for SCG valorisation. Maximizing the efficiency of plant cell wall material degradation in the rumen has become an important goal in modern livestock production [55]. It is known that the insolubility, structural complexity, and initial inaccessibility of cell wall components often limit the extent to which they are fermented in the rumen [56], and the grinding pre-treatment seemed to succeed in improving this accessibility.5. ConclusionsThis study determined the effect of thermal and mechanical pre-treatments combined with enzymatic hydrolysis to improve spent coffee grounds’ nutritional value as an ingredient for ruminants’ diets.The hydrolysis process with different cellulolytic enzymes involved a release of valuable compounds, such as sugars, polyphenols, and other elements with antioxidant activity into the liquid fraction, resulting in a less valuable raw material. In addition, the action of the enzymes on the solid fraction was counteracted by the action of the ruminal bacteria. Thermal pre-treatment of the SCG appeared to be ineffective in improving the breakdown of lignocellulosic bonds and thus improving digestibility. In contrast, grinding pre-treatment improved the coffee grounds’ digestibility and the fermentative process in the rumen.In conclusion, extra grinding is presented as the most powerful technological choice to improve the digestibility of spent coffee grounds for their reintroduction into the value chain.

animals : an open access journal from mdpi

10.3390/ani12020163

PMC8772576

Sheep sperm is extremely sensitive to reactive oxygen species (ROS) and can produce a large amount of ROS during chilling storage, leading to a decline in semen quality. Adding antioxidants is an important method to improve semen quality. Chlorogenic acid (CGA) is a kind of plant extract with an antioxidant capacity, which can effectively eliminate free radicals and improve the antioxidant capacity of semen. However, its role in the chilling storage of Hu ram semen is not clear. Therefore, CGA with different concentrations was added to chilling storage extender to investigate its effect on chilled ram sperm. The results of this study revealed that CGA with proper concentration had a positive effect on chilled Hu ram sperm and 0.8 mg/mL CGA had the best effect.

The purpose of this study was to investigate whether the addition of chlorogenic acid (CGA) to a sheep semen extender could improve the quality of chilled sheep sperm. Ejaculates (n = 80) were collected from five Hu rams with an artificial vagina. The ejaculates were mixed and divided into five equal parts, diluted with a CGA-free Tris–egg yolk extender (control), or supplemented with 0.2, 0.4, 0.8, and 1.2 mg/mL. The sperm kinematic parameters (viability, progressive motility), functional integrity of plasma membrane and acrosome, adenosine triphosphate (ATP) concentration and antioxidant parameters (Catalase (CAT), Superoxide dismutase (SOD) activity, total antioxidant capacity (T-AOC), ROS level and Malondialdehyde (MDA) content) were evaluated during storage of the semen. The results indicated that: PM, plasmatic membrane integrity and acrosomal integrity in 0.8 mg/mL CGA were higher (p < 0.05) from day 1 to 5. The ROS level in CGA groups was lower than the control (p < 0.05). CAT, SOD, ATP, and T-AOC were highest at 0.8 mg/mL concentration within 1 to 5 days. The above results indicated that the right concentration of CGA improved the quality of Hu ram sperm during chilling storage.

1. IntroductionHu sheep is a valuable sheep breed in China, with high-quality meat and skin. It has the advantages of early sexual maturity, fast growth, four season estrus, and two offspring a year [1]. It can effectively make up for the defects of sheep fattening in autumn and winter, and fully meet people’s demand for mutton if breeding on a large scale. Natural mating is influenced by factors such as geography and the spread of reproductive diseases, which inhibits the reproductive potential of dominant male varieties. Therefore, artificial insemination (AI) technology is very important in large-scale breeding farms. The quality of semen storage is key to the effect of artificial insemination. When stored at room temperature, sperm motility decreases sharply in a short time and cannot meet the needs of long-distance transportation [2]. Otherwise, cryopreservation of semen causes serious damage to sperm, which decreases the ability of sperm fertilization and affects the potential fertility of artificial insemination [3]. The preservation of semen at low temperature can effectively slow down the metabolism of sperm and prolong its survival time [4], which is of great significance for overcoming geographical limitations and the cultivation of excellent animals’ genetic characteristics. Liu et al. found that the blastocyst rate of cryopreserved sheep sperm after fertilization was low (29.12 ± 3.01) [5]. Studies revealed that the rate of ewes fertilized with frozen-thawed sheep sperm was 4% [6]. It was reported that the pregnancy rate of ewes fertilized with frozen semen (preserved at 15 C for 6 h) was 52% [7]. Therefore, chilling storage technology for semen has been widely used by sheep farmers and breeding enterprises.Sperm are susceptible to external factors such as temperature, pH, osmotic pressure, and internal factors such as their own metabolic products [8,9] during storage. Excessive ROS can combine with PUFAs in sperm plasma membrane, causing lipid peroxidation and finally destroying the membrane structure and function decline of sperm plasma [10]. In addition, lipid peroxidation can interact with related proteins in mitochondrial electron transport chain, which leads to the production of lipid aldehydes, and then produces more ROS, eventually damaging DNA in the sperm nucleus and leading to sperm death [10,11]. The lipid composition of the sheep sperm membrane is different from that of somatic cells, and it is rich in PUFAs, which makes sperm susceptible to physical, chemical, and oxidative damage caused by the accumulation of ROS during storage [4,12]. Sperm cells are deficient in endogenous antioxidants, which leads to the decline in integrity of the sperm plasma membrane [5]. Therefore, adding exogenous antioxidant substances has become one of the effective methods to improve the quality of semen preservation and pregnancy rate. Studies have shown that bioactive peptides isolated from natural herbs or mammalian organs effectively eliminated the production and accumulation of ROS [5]. At present, it has been reported that vitamin E [13], CoQ10 [14], reduced glutathione (GSH) [15,16] astaxanthin [17], and melatonin [18,19] have positive effects on sperm of sheep, humans, dogs, boars, and rats. Most antioxidants, however, tend to have both positive and negative effects on sperm: the appropriate concentrations of antioxidants protect sperm, whereas a high concentration has a toxic effect [20].Chlorogenic acid (CGA), a polyphenol compound with antibacterial, antioxidant, and anti-inflammatory properties, has been applied to the food, cosmetics, and pharmaceutical industries. In addition, it also has the ability to eliminate free radicals [21]. Recent research findings revealed that CGA significantly inhibits the expression and secretion of IL-8 mRNA in mouse intestinal epithelial Caco-2 cells caused by oxidative stress [22,23]. It has been reported that the sperm count of epididymis increased by 20% after 5 weeks of CGA administration in rats [24]. CGA improved the quality of cooled and frozen-thawed boar sperm [25,26,27]. Furthermore, CGA improved the antioxidant capacity of human sperm in vitro and during the frozen-thaw stage [28]. A variety of antioxidants have positive effects on chilled ram semen, such as argan oil [29], royal jelly [30], and Mito-TEMPO [31]; however, there are few reports on the study of CGA on chilled sheep semen. This study was designed to investigate the effects of CGA with the proper concentration on sperm quality and ability to attenuate oxidative stress of Hu sheep during chilling storage, which can provide basic references for semen reservation in Hu sheep.2. Materials and Methods2.1. Animals, Semen Collection and Processing ProceduresThe five rams were kept in a facility at the Agriculture of Yangzhou University Agriculture. They are given straw, hay, and mixtures. All the procedures for animal treatment and sample collection were approved by the Ethical Committee of Experimental Animal of Yangzhou University, Jiangsu, China (license number: SYXK[Su]2017-0044). Ejaculates (n = 80) were collected from five rams by an artificial vagina. The semen samples were taken to the lab, and examined for volume, concentration, and viability. Only ejaculates with a volume ≥ 0.5 mL, concentration ≥ 2.0 × 109/mL, and viability ≥ 0.8 were included in this study. After passing evaluation, fresh semen from five rams was mixed to reduce errors due to individual differences. The mixed semen was divided into 5 equal fractions and diluted with CGA without antioxidant and different concentrations; finally, the samples were stored at low temperature for subsequent tests.2.2. ChemicalsChlorogenic acid was purchased from Beijing Solarbio Science & Technology Co., Ltd. (Beijing, China). CGA (120 mg) was dissolved in DMSO (1 mL) to obtain CGA basic mother liquor. Unless otherwise specified, the rest of the chemicals were purchased from Sangon Biotech (Shanghai) Co., Ltd. (Shanghai China).2.3. Semen Processing and EvaluationTris (3.07 g), fructose (2.00 g) and citric acid (1.64 g) were dissolved in 100 mL distilled water. After the basic extender (90 mL) was added with 10 mL of egg yolk, the mixture was fully stirred until completely dissolved, stored at 4 °C overnight, and centrifuged at 13,000× g for 15 min to collect the supernatant for later use. Each mixed semen sample was divided into five equal aliquots to diluted with 0.2, 0.4, 0.8 and 1.2 mg/mL CGA. Semen was diluted ten times with an extender containing Tris, and the final sperm concentration was adjusted at 100 × 106/mL. The semen sample was stored at 4 °C in a refrigerator. Sperm kinematic parameters, plasmatic membrane integrity and acrosome integrity were assessed every 48 h for 7 days. Total antioxidant capacity(T-AOC), Superoxide dismutase (SOD), Catalase (CAT) activity, adenosine triphosphate (ATP) and Malondialdehyde (MDA) content were determined at the 1st, 3rd, and 5th days. ROS level was evaluated at 5th day. 2.4. Sperm Kinematic ParametersSperm kinematic parameters were evaluated using Mailang sperm automatic analysis system ver5.0 (Instrument number: ML-608JZ II, Mailang, Nanning, China), including viability, PM, Curvilinear velocity (VCL, µm/s), Straight line velocity (VSL, µm/s), Average path velocity (VAP, µm/s). Briefly, 20 µL of chilled semen sample was diluted five times with extender, and incubated at 37 °C for 2 min. A total of 10 µL of incubated semen sample was added to the sperm count plate for evaluation of sperm kinematic parameters.2.5. Sperm Plasma Membrane Integrity AssessmentSperm plasma membrane integrity was evaluated using a hypotonic swelling test (HOST). A 10 µL semen sample was mixed with 100 µL hypo-osmotic solution (consisting of 0.90 g fructose and 0.49 g sodium citrate dissolved in 100 mL ultrapure water), the mixture was incubated at 37 °C for 30 min. The percentage of tails coiled in 200 sperm was counted using a 400× phase contrast microscope.2.6. Sperm Acrosomal Integrity AssessmentThe integrity of sperm acrosomal was evaluated using coomassie brilliant blue G-250 staining. Briefly, a 50 µL semen sample was mixed with 1 mL 4% paraformaldehyde to fixed for 10 min, the mixture was centrifuged at 1500× g for 5 min to obtain precipitate sperm cells, and made a smear. The smear was stained using Coomassie brilliant blue G-250 dye solution (Coomassie brilliant blue G-250 (0.10 g) was dissolved in 95% ethanol (50 mL), then 85% phosphoric acid (100 mL) was added, and the volume of the final staining solution was adjusted to 1 L). The percentage of the acrosome stained blue in a total of 200 sperm was counted using a 1000× oil immersion.2.7. Determination of ROS in SpermROS level was evaluated using a ROS assay kit (Solarbio, Beijing). Briefly, 50 µL chilled semen sample was washed with 500 µL PBS, the semen sample was treated with 300 µL 10 µmol/L DCFH-DA working solution, and incubated at 37 °C for 30 min. The mixture was centrifuged at 1000× g for 10 min to obtain precipitated sperm cells, the precipitate was washed with PBS three times to remove DCFH-DA from the non-entering sperm. The level of ROS was expressed as absorbance at an excitation wavelength of 488 nm and an emission wavelength of 525 nm for a microporous multimodal detection system.2.8. Evaluation of Sperm Protein ConcentrationSperm protein concentration was evaluated using Bradford protein concentration determination kit (Beyotime institute of Biotechnology, Shanghai). A 50 µL sample was centrifuged at 550× g for 10 min to remove the supernatant, the semen sample were resuspended in lysis buffer. The lysate was centrifuged at 12,000× g for 5 min at 4 °C to obtain the supernatant. G250 staining solution (detergent compatibility, 300 µL) was added to the sample (10 µL), the absorbance was measured at 595 nm, and the protein concentration was obtained according to the standard curve [32,33].2.9. Evaluation of T-AOC of Seminal PlasmaT-AOC of seminal plasma was measured using Total Antioxidant Capacity Determination Kit (Nanjing Jian Cheng Institute of Biological Engineering, Nanjing). Simply put, the standard Trolox solution (10 mM) was diluted using distilled water to 0.1, 0.2, 0.4, 0.8, 1.0 mM to obtain a standard curve. A total of 0.1 mL semen sample was centrifuged at 1500× g for 10 min to obtain supernatant. A total of 10 µL of supernatant was added into each well of the 96-well plate, and the corresponding reagents were added according to kit instructions. The mixture reacted at room temperature for 6 min, and the absorbance at 405 nm was obtained by a microporous multimode detection system. Since the extender contained egg yolk, a control only with extender was set to eliminate the effect of egg yolk. The results were calculated according to the standard curve.2.10. Evaluation of CAT Activity in Seminal PlasmaCAT activity of seminal plasma was evaluated using Catalase Assay Kit (Nanjing Jian Cheng Institute of Biological Engineering, Nanjing). The basis for evaluating CAT activity is that ammonium molybdate could quickly terminate the reaction of catalase to decompose hydrogen peroxide, and H2O2 could react with ammonium molybdate to produce a pale yellow complex. A total of 0.2 mL diluted semen sample was centrifuged at 1500× g for 10 min to take the supernatant; the corresponding reagents were added in turn according to the kit instructions. Since the extender contained egg yolk, a control only with the extender was set to eliminate the effect of egg yolk. The absorbance of mixture was measured at 405 nm using a microporous multimode detection system.2.11. Assessment of MDA Content in SpermMade a standard curve is based on the lipid oxidation (MDA) detection kit (Beyotime institute of Biotechnology, Shanghai). After resuspension, a 50 µL semen sample was lysed to fully release the MDA in the sperm. The lysate was centrifuged at 12,000× g at 4 °C for 5 min. After centrifugation, 0.1 mL sample was mixed with 0.2 mL MDA detection working solution, the mixture was treated with a boiling water bath for 15 min, cooled to room temperature, and centrifuged at 1000× g for 10 min. The absorbance of mixture was obtained at 532 nm using a microporous multimode detection system. The MDA content was obtained according to the standard curve, and the protein concentration was measured according to the procedure in Section 2.8. MDA content in sperm was presented in nm/mg protein.2.12. Determination of SOD Activity in SpermA 50 µL semen sample was washed with PBS; 300 µL SOD liquid sample preparation was added to fully crack the cells, and the lysate was centrifuged at 12,000× g at 4 °C for 5 min to take the supernatant. In strict accordance with a SOD activity detection kit (Beyotime institute of Biotechnology, Shanghai) manual operation, the semen sample was incubated at 37 °C for 30 min, the absorbance of mixture at 450 nm was measured, and the protein concentration was measured as in Section 2.8. The results were expressed in unit/mg protein.2.13. Determination of ATP ContentThe semen sample was washed with PBS and centrifuged at 1500× g for 5 min to obtain precipitation cells, 500 µL double steaming hot water was added to the sample, then placed in a hot bath homogenate and broken. The mixture was treated in a boiling water bath for 10 min, and centrifuged at 4000× g for 5 min to take supernatant based on the instructions of ATP assay kit (Nanjing Jian Cheng Institute of Biological Engineering, Nanjing). The absorbance at 636 nm was obtained using a microporous multimode detection system, and the determination of protein concentration was shown in Section 2.8. The results were presented in µmol/mg protein.2.14. Statistical AnalysisThis study was replicated six times. The experimental data were analyzed by Statistical Package for the Social Sciences (SPSS, IBM, version 24.0). The Shapiro–Wilk tested normal distribution of data. All experimental results were presented as Mean ± SEM and compared using Duncan’s multiple range tests by one-way analysis of variance procedures. p < 0.05 indicated significant difference.3. Results3.1. Effects of CGA on Sperm Viability and PMViability and PM rate of 0.8 mg/mL and 1.2 mg/mL groups was higher than other groups (p < 0.05) on the 1st day. On the 3rd day, PM of 0.4 mg/mL and 0.8 mg/mL groups was higher than control group (p < 0.05). The viability and PM of all CGA groups were higher than control group (p < 0.05), the 0.8 mg/mL CGA group was highest from day 5 and 7. On the 3rd day, the VSL, VCL and VAP of all the CGA groups had no significant differences compared with the control group. Compared with the control group, the VAP of sperm in all CGA groups was higher (p < 0.05) on the 5th day. From day 5 to 7, the value of VCL and VAP in 0.8 mg/mL group was the highest (Table 1).3.2. Effects of CGA on Sperm Plasma and Acrosomal Membrane IntegrityCompared with the control group, the sperm plasma membrane of all CGA supplementation groups was higher (p < 0.05) within 3 to 5 days. On the 7th day, the sperm plasma membrane integrity of 0.8 mg/mL group was the highest. On the 3rd day, the sperm acrosome integrity of 0.4 mg/mL and 0.8 mg/mL groups was higher than other groups (p < 0.05). On the 5th day, the sperm acrosome integrity in all CGA groups was higher than control group (p < 0.05), 0.8 mg/mL group was the highest (Table 2).3.3. Effects of CGA Supplementation on Sperm ROS ContentThe ROS levels of CGA-treated groups decreased than the control group (p < 0.05), the value of 0.8 mg/mL group was lowest, and no significant difference was observed between 0.4 mg/mL and 1.2 mg/mL groups (Figure 1).3.4. Effects of CGA on CAT Activity in Seminal PlasmaThe CAT activity of 0.8 mg/mL group was highest from day 1 to 5 (p < 0.05). On the 5th day, the CAT activity of CGA supplementation groups was higher than the control group (p < 0.05, Figure 2).3.5. Effects of CGA on T-AOC in Seminal PlasmaCompared with the control group, T-AOC of CGA added groups was higher (p < 0.05), 0.8 mg/mL group was the highest from day 1 to 5. No significant difference was observed between o.4 mg/mL and 1.2 mg/mL group on the fifth day (Figure 3).3.6. Effects of CGA on MDA in SpermOn the 1st day of semen preservation, the MDA content of all CGA added groups was lower than control group (p < 0.05), and that of 0.4 mg/mL and 0.8 mg/mL group was lower than 0.2 mg/mL group (p < 0.05). On the 3rd day, no significant difference was observed among 0.4~1.2 mg/mL groups. On the 5th day, MDA content in CGA added groups was lower than control group (p < 0.05, Figure 4).3.7. Effects of CGA on SOD Activity in SpermThe SOD activity of 0.4 mg/mL and 0.8 mg/mL groups was higher than control group from day 3 to 5 (p < 0.05), and the value of 0.8 mg/mL group was higher than other groups at different time points (p < 0.05, Figure 5).3.8. Effects of CGA on ATP ContentOn the 1st day of semen preservation, ATP concentration in all CGA supplementation groups was higher than the control group (p < 0.05). The ATP concentration of 0.8 mg/mL group was the highest from day 1 to 5 (Figure 6).4. DiscussionWhen the balance of ROS production and elimination was disrupted, excessive ROS caused damage to sperm [22,34]. ROS can not only lead to DNA chain break or sister chromatid exchange, but also oxidize key enzymes in the methylation process, leading to DNA methylation [35,36]. In addition, lipid peroxidation can also damage sperm. Experiments have proved that adding antioxidants to the semen extender of goats [37], cattle [38], pigs [39] and horses [40] can effectively improve sperm motility and eventually improve the effect of artificial insemination [41], which is of great significance for the development of animal husbandry.In sperm kinematic parameters, VCL, VSL and VAP are positively correlated with sperm motility, whereas VCL is highly correlated with sperm fertilization ability [42]. A factor that may contribute to the decline of sperm kinetics (VCL, VSL and VAP) is its density. In addition, higher values for VCL and VAP were measured in extenders containing low-density lipoprotein, compared with extender egg yolk [43]. These results may be helpful to explain the lower sperm velocity in current experiment.The current experiment indicated that CGA could significantly reduce the content of ROS and MDA during low temperature storage of semen, which was consistent with Pereira’s research results on the effect of CGA on pig semen [25]. The content of MDA in sperm increased with the prolongation of semen chilled storage in vitro. This is consistent with the discovery of the harmful effects of liquid storage on the level of MDA [44]. In vivo and in vitro studies have verified that CGA can chelate with aluminum ions and reduce lipid peroxidation and oxidative stress damage caused by aluminum ions [45].In this experiment, with the prolongation of sperm preservation time, the integrity of sperm plasma membrane decreased significantly, which may be concerned in the destruction of sperm plasma membrane and protein distribution by ROS [46]. The sensitivity of sperm to cryoprotectants and the difference of compound concentration may affect the functional integrity of sperm. In the present study, 0.8 mg/mL CGA significantly maintained high plasma membrane integrity and acrosome integrity of chilled ram sperm. It has also been reported that the difference in the number and type of phospholipids between sperm types and regions could intervene in the stability of sperm membranes during low temperature storage [47]. In present study, the plasma membrane integrity of CGA supplementation groups was higher than control group, which is consistent with the result of Namula’s research on the effect of chlorogenic acid on semen quality of freeze–thaw pigs [27].CAT and SOD are antioxidant enzymes widely existing in sperm cells. SOD can transform O2− into H2O2 through disproportionation reaction, and H2O2 is converted into H2O through CAT to eliminate the influence of ROS. In the current study, CAT activity of 0.2–0.8 mg/mL CGA groups was higher on the 1st day, and that of 0.8 mg/mL group was higher on the 5th day, which indicated that 0.8 mg/mL CGA mitigated oxidative stress. It was found that the activity of SOD in chilled semen was positively correlated with sperm motility [48]. In this study, the value of SOD activity in 0.8 mg/mL CGA group was higher than control group at different time points, which indicates that 0.8mg/mL CGA has a positive effect on SOD activity. Treatment of ram sperm with BSA conjugated to OA has been reported to result in increased SOD activity after 48 h of storage at low temperature [12]. The results of this study are consistent with it.Sperm requires ATP produced from the middle of the mitochondria to main motility [49]. However, due to species, its production and metabolism process is not fully understood. Some researchers have found correlation between the fertility in different bull breeds [50] and the motility in starlet [51], whereas other reports on mammals have found that there is no correlation between fertility and ATP content of sperm [52,53], but it is related to the motility parameters [52]. In the present study, the ATP concentration in 0.8 mg/mL group was higher than that of other groups on 5th day of semen preservation, which was corresponding to viability and PM during semen preservation and consistent with stallion [51]. With the prolongation of semen preservation time, sperm ATP concentration decreased gradually, which was corresponding to viability and PM.High concentrations of antioxidants may damage the functional integrity of acrosome and membrane of sperm [54,55]. In the present study, the chilled ram sperm plasma membrane and acrosomal integrity in 1.2 mg/mL group was lower than 0.8 mg/mL group on the 7th day of semen preservation, possibly due to the increase in cell membrane permeability, which resulted in decreased sperm quality.5. ConclusionsIn conclusion, the ram semen extender containing CGA improved chilled ram sperm kinematic parameters, plasma membrane integrity, acrosome integrity, total antioxidant capacity, CAT, SOD activity, decreased the accumulation of ROS and MDA, and contained the ATP content. The optimum CGA concentration in the semen extender was determined to be 0.8 mg/mL. The reproductive potential of chilled ram sperm needs further study.

animals : an open access journal from mdpi

10.3390/ani11051346

PMC8151611

The welfare of animals in human care should be taken into consideration by both the scientific community and members of the public who are able to view animals in a zoo setting. One component of individual welfare is how groups of animals and the individuals within groups may be affected by the presence of zoo guests. At the Buffalo Zoo in New York, we studied the behavioral differences of six gorillas in two conditions: heavy guest presence and the complete absence of guests. We found few significant behavioral differences between the conditions, with the greatest behavioral change observed in the adult silverback male. We stress the importance of not over-generalizing our findings, and suggest an inter-institutional study of the nuanced ways that zoo guests interact with gorillas in managed care.

Research conducted on the effects that zoo visitors have on primate behavior has yielded inconsistent patterns. This study aims to contribute to the growing body of literature regarding visitor effects on zoo-housed primate’s activity budgets, with the purpose of quantifying the behavioral variability under two conditions: guest presence and guest absence. Due to the COVID-19 pandemic, many zoos were closed to the public for varying lengths of time. The Buffalo Zoo was closed to guests for an 18-week period including the summer of 2020, which allowed us to effectively control for zoo guest presence. This case report compares data on the zoo’s gorilla troop from the same time period in 2019. We found inconsistent results, similar to prior studies conducted with zoo-housed gorilla troops. Most gorillas were observed foraging less and exhibiting more inactivity in 2020, whereas the adult male silverback showed the opposite pattern. Abnormal or undesirable behaviors were performed less frequently when guests were absent however, these differences were not significant. We encourage others to compare behavior patterns during the pandemic shutdown to add to our knowledge base of visitor effects. We suggest that researchers do not try and generalize their individual and troop results to the entire population of gorillas in managed care, as both intrinsic and extrinsic factors contribute to individual differences in behavioral response.

1. IntroductionThe welfare of animals in managed care has received considerable attention in recent years [1,2,3]. One condition that may influence welfare is the presence of zoo guests and the unpredictable variability that zoo guests bring to exhibit spaces [4]. Investigating how visitors may or may not influence animal behavior in a zoo setting can help institutions better understand how the environment affects the animals’ welfare. Zoos can use this information to adapt husbandry practices such as the amount of time animals spend on exhibit or how exhibits are designed. It can also help provide a deeper comprehension of general human–animal relationships.Each individual animal is likely to express its own idiosyncratic and variable responses to zoo guests, which is why the study of the zoo visitor effect [4,5] is important in quantifying visitor impacts to animal behaviors. The zoo visitor effect shows that species—and individuals within each species—may express varying welfare impacts from long-term exposure to unfamiliar and unpredictable zoo visitors. For some species, visitors can be viewed as positive and novel enrichment [4]. However, for other species across a plethora of taxa, including harbor seals (Phoca vitulina), koalas (Phascolarctos cinereus), and hornbills (Bucorvus abyssinicus), the presence of visitors can lead to negative welfare impacts that elicit stereotypic behaviors and even prolonged stress in individuals [6,7,8]. These studies often provide the baseline knowledge for the inception of new and improved welfare standards in zoos.Primates have been particularly well-studied with respect to possible impacts of zoo visitors on individuals and intra-troop interactions. Black-capped capuchins (Sapajus apella) have shown a decrease in aggression and abnormal behaviors when viewing windows of their exhibit were obstructed [9]. Although not in a zoo setting, chimpanzees in a sanctuary open to visitors were found to locomote more when visitors were given access to viewing areas, though they showed inter-individual differences in reactions to sanctuary visitors [10,11]. Visitors were more likely to influence orangutan (Pongo spp) behavior when crowds were large, visitors were close to the individual orangutans, and when visitors possessed food [12]. White-handed gibbons (Hylobates lar) were more likely to engage in territorial displays (via song or increased brachiation), self-scratching behaviors, and social bonding behaviors such as allogrooming in periods of high noise levels and large crowds [13].The impacts of zoo visitors are well-documented in Western lowland gorillas (Gorilla gorilla gorilla) as well, and these zoo-housed troops have been shown to exhibit variable responses to zoo guests. Their responses tend to show considerable individual variation independent of factors such as age, sex, or rearing history. In a study of four gorilla troops comprising both family and bachelor groups, no troop consistently performed undesirable or abnormal behaviors when crowd levels were high [14]. They also found that sex, age, and rearing history were not determinants of behavioral responses; however, personality factors may have had an influence on individuals’ crowd responses.In two studies where visual barriers were installed to block direct view with zoo visitors, individuals were less likely to engage in intra-troop aggression, visitor directed aggression, and stereotypy; however, no significant, patterned changes in the gorillas’ activity budgets attributed to guest presence were found in either study [15,16].High crowd levels have been associated with a decrease in foraging behavior [17,18] and an increase in inactivity and resting behaviors [19,20,21]. Undesirable behaviors, such as overgrooming, plucking, regurgitation and reingestion (R/R), and pacing can increase with denser crowds [19,22,23,24]. When installing a privacy screen on an exhibit window, one study found that a single gorilla ceased to engage in R/R behaviors altogether [17]. Although inconsistent, aggression towards visitors and intra-troop contact and non-contact aggression have been shown to increase with high crowd levels [17,19].The goal of this study is to corroborate the findings of similar studies that attempt to quantify the effect of the presence of zoo visitors on zoo-housed Western lowland gorillas (Gorilla, gorilla, gorilla). We collected behavioral data on one gorilla troop at the Buffalo Zoo consisting of six individuals. Beginning March 14th of 2020, the novel coronavirus pandemic presented us with a unique experimental condition which removed guests from the gorilla’s environment for a three month period. With no additional external changes to the gorilla’s environment, zoo staffing, daily routines, or any changes within the troop, we opportunistically compared these two conditions, with the only substantive change being the presence or absence of visitors. Thus, this case study allows us to compare the data collected during the pandemic shut-down to our archived data from the same period in 2019 when visitors were present.2. Materials and Methods2.1. Study SiteThe study was conducted at the gorilla exhibit at the Buffalo Zoo in Buffalo, NY, USA. All the subjects were housed in an indoor exhibit measuring approximately 185 m2 containing a climbing structure and alcoves that allowed the gorillas to be out of view from visitors and observers if they chose. Visitors to the zoo were able to view the gorillas’ exhibit on days that the zoo was open between 10:00 a.m.–4:00 p.m. Four glass viewing windows permitted visual access. The exhibit included two alcoves that led to the off-exhibit holding area. The gorillas had access to the alcoves at all times, and could not be seen by visitors when in this location. During the period of closure in 2020, the gorillas often had access to their off-exhibit holding areas, though access varied periodically. Normal routines continued for feeding and provision of enrichment.2.2. SubjectsWe have been collecting behavioral data since 2009 on the group of western lowland gorillas (Gorilla gorilla gorilla) housed at the Buffalo Zoo. The family group includes a mature adult male named Koga (hereafter designed as adult male, “AM”), two adult females named Sidney (adult female 1, “AF1”) and Lily (“AF2”), and three offspring: a sub-adult female named Amari (subadult female, “SAF”), a juvenile female named Nyah (juvenile female, “JF”) and a juvenile male named Kayin (juvenile male, “JM”) (Table 1).2.3. ProceduresBehavioral data were collected using a standardized ethogram by members of the Margulis Lab research team at Canisius College in Buffalo, NY. In 2019, we collected data in one or two sessions each day in the morning and/or afternoon during the zoo’s open hours. We collected data from three to seven days each week throughout the year based on availability of student researchers. We conducted twenty-minute instantaneous focal observations [25] on each of the six gorillas in the exhibit, randomizing the order in which we observed each individual. Every minute, the focal subject’s behavior, location, and neighbors were entered into a spreadsheet using the ExcelTM app. For the purposes of this investigation, we focus only on selected state behaviors (Table 2). We omitted behaviors that occurred too rarely for analysis unless they were particularly relevant to the study. The behaviors we included in analyses were locomotion, inactivity, forage, self-care, regurgitation/reingestion, pluck, and social play. We also evaluated time spent out of view.For the 2020 data, the zoo was closed to the public from 14 March through 1 July. One of the authors (SWM) was given access to the zoo’s remote cameras to permit observations during a portion of the closed period. All methods of data collection were kept consistent with in-person observations. These data were collected between 26 May 2020 and 1 July 2020 and comprised 36 h of data.We used data collected during the summer of 2019, from 27 May 2019 through 2 Sept 2019 (the “busy” summer season) as our comparison. These data provided approximately the same amount of data on each subject (Table 1).2.4. Data AnalysisBefore conducting behavioral analyses, we first removed “out of view” observations from the raw data. This included fully removing all observations where the individual was out of view for more than half (11 or more scans out of a possible 20) of the total scans recorded during that session, and including only scans where the animal was visible. All remaining occurrences of an individual being out of view were removed from analyses. There were no significant differences amongst individuals or between the two years in percent time spent out of view.Data were analyzed in R Studio [26], version 4.0.3. To analyze behavioral change between the two conditions, we conducted Mann–Whitney U Tests using the Bonferroni correction to account for multiple comparisons. p-values were adjusted accordingly.3. ResultsWhen data on all six gorillas were combined to give us a whole-troop comparison, we found no significant differences in behavior between the two study periods (Figure 1). We then broke analyses down to study potential individual differences in behavior, and found few significant differences individually. Some non-significant but notable differences were observed in particular behaviors, including foraging, inactivity, locomotion, and autogrooming behaviors. While visitors were absent, five of the six gorillas showed a decrease in foraging with concurrent increases in inactivity. The AM showed opposing patterns, nearly doubling his foraging time and showing notable decreases in inactivity when zoo guests were not in attendance.Mann–Whitney U tests showed very little statistical difference in behaviors for all six study subjects (Table 3; Figure 2). The AM demonstrated the most notable changes in behavior, however only autogroom behavior decreased significantly between the two conditions (U = 99, p = 0.0095), comprising 12 percent of his activity budget in 2019 and not being observed at all in the 2020 condition. The SAF’s inactivity level changed significantly (U = 42, p = 0.01), more than doubling between the conditions to a total of 56 percent of her activity budget in 2020.There were also downward trends in foraging behavior observed in five of the six gorillas in the troop, with as much as a 27.8% decrease in the behavior between the conditions (Figure 2). Conversely, the AM’s foraging behavior increased from 28.86% of his activity budget in 2019 to 67.12% of his 2020 activity budget.Other noted trends in the activity budgets included an increase in inactivity during the 2020 condition for five of the gorillas, with a simultaneous increase in locomotor behavior shown by four of the gorillas. It is important to note that although these changes trended similarly for a majority of the subjects, these changes did not show statistical significance.All six gorillas had downward trends in autogrooming between the two conditions. Three of four gorillas who were observed plucking in 2019 did not pluck in 2020, however, it is important to note that this behavior occurred infrequently, with each gorilla plucking less than five times in the 2019 condition. The SAF plucked at a much higher rate in 2020, increasing from 0.45% in 2019 to 2.67% of the total activity budget in 2020.Regurgitation and reingestion was observed in both adult female gorillas in each of the conditions. AF1 exhibited no change in the frequency of performing R/R, while for AF2 R/R comprised 23.40% of her total activity budget in 2020, up from 15.31% in the condition with visitor presence.Differences in social play were marginal and did not reflect a significant change in the behavior between the two conditions.4. DiscussionThe findings of this case report suggest no significant differences in the subjects’ activity budgets between the two conditions. Our findings were consistent with a number of studies that examined visitor effects on gorillas. While we observed some variation in whole-troop behavior, any behavioral changes that were significant at the individual level were not consistent throughout the troop.We observed slight overall differences in foraging, inactivity, and locomotor behaviors. Foraging behavior decreased in five of the six subjects when visitors were not in attendance; this is contradictory to several previous studies that observed a decrease in foraging when crowd sizes increased in zoo-housed gorillas [17] and chimpanzees [18], respectively. We also observed a general pattern of increased inactivity when visitors were absent, consistent with several other studies [19,21].It has been suggested that abnormal or undesirable behaviors occur less frequently in primates when crowd levels are low [15,23]. We observed similar trends in autogrooming behaviors, with modest declines of the behavior seen in five of six gorillas, and the AM ceasing to perform the behavior all together. Similar patterns have been observed in other studies [14]; however, the authors prefaced this by stating that any changes in abnormal behaviors were not consistent. We also found inconsistency in the occurrence of abnormal behaviors, most notably with regurgitation and reingestion. While AF1 did not change her rate of performance of this behavior, AF2 engaged in regurgitation and reingestion at nearly double the 2019 rate. Despite this large increase, the change was not statistically significant.Overall, our findings corroborate what the literature suggests: individual variability in gorilla response to visitor presence leads to inconsistent and unpredictable changes in activity budgets. While we observed generalized trends in behavior, each gorilla’s response to the absence of zoo guests was varied, and despite observing specific trends in the troop’s overall activity budgets, these trends can be exaggerated by even a single member of the troop that responded strongly—either positively or negatively—to the absence of guests.With only a single gorilla—the AM—showing statistically significant changes in one of the seven analyzed behaviors in the opposite direction of the rest of the troop, we did not find overwhelming evidence that zoo guests cause significant stress to gorillas in zoo-housed settings, in contrast to previous findings [27] that zoo visitors cause overt stress to troops. Our case report findings are consistent with the conclusions drawn from several other studies [14,24], that is, although there were some changes to behavioral patterns between the conditions, any findings cannot be generalized to suggest that zoo guests negatively or positively impact the welfare of gorillas in zoos. Our findings suggest that we must consider welfare at the individual level, as both intrinsic (sex, age, rearing status, and dominance rank in troop) and extrinsic (exhibit, weather, and visitor activity) factors uniquely influence each gorilla’s behavioral response. Demonstrating such patterns continues to be challenging, due to small sample sizes and inherent individual variation.Though we did not find consistent and significant differences in behavior, we note that this case study is based on only one group of gorillas with limited data collection during an unprecedented pandemic. Consequently, we must consider the limitations of our study. First, the gorillas were given access to their off-exhibit holding areas on certain days by the exhibit staff whilst the zoo was closed to the public. We were unable to collect behavioral data whilst the gorillas were in holding, which may have impacted our results. Second, we treated zoo visitors as a binary variable (visitors were either in attendance or not in attendance). The problem with this approach has been highlighted by previous authors [24,27]. To provide more detailed information on the impact of zoo guests, future visitor effect studies must better quantify guest activity levels and interactions with gorillas as both initiators of and responders to gorilla behavior. Finally, our sample size was small. Even if each individual gorilla is considered as an independent data point (rather than the group as a single data point), we lack the power with six individuals to draw firm conclusions. For example, although 5 out of 6 gorillas showed decreases in foraging, we would have needed a larger group size, with 7 of 8 gorillas showing changes in the same direction to achieve statistical significance. We note that the behavior of the AM was consistently and noticeably different than that of the rest of the troop. Had we not adjusted our analyses for multiple comparisons, we would have seen significant changes in four behaviors, with notable increases in foraging and locomotion, and decreases in inactivity and autogrooming.Other potential factors that could have influenced individual behavior include personality and/or exhibit design. These variables are both complex and difficult to measure. Gorillas’ personality types could have some potential impact on how they react to visitors [14]. For example, animals with more dominant personalities might respond differently to visitor changes than those with subordinate personalities. This could potentially be related to the unique behavior changes we saw in the AM, the most dominant gorilla in the troop. Exhibit design may also influence behavioral responses. The exhibit in our study was indoors with glass separating the gorillas from the visitors, allowing visitors to be in very close proximity to the gorillas which could have influenced their behavior. Each zoo has a different exhibit design, some include more space and vegetation to hide or a large moat offering significant spatial separation between gorillas and visitors. If our study were to be replicated at another zoo, these exhibit design factors would need to be taken into consideration.In November of 2020, Buffalo NY was once again designated as an “orange zone” due to a rise in COVID-19 cases. This designation imposed restrictions on all non-essential businesses, including the Buffalo Zoo. Restrictions kept all of the indoor exhibits at the zoo closed to the public for several months, during which our team continued to collect behavior data. In the future we hope to again analyze the data from the second closure period to get a more complete picture of the gorilla’s response to a lack of visitors.Further examination of the characteristics and behaviors of zoo visitors is likely to be an important area for future investigation. We would like to see if the visitors’ behavior can influence the gorillas’ behavior, as other taxa have been observed reacting according to specific visitor characteristics [28]. For example, some visitors passively observe the gorillas while others are loud and attempt to get the animals’ attention. We have observed visitors doing things such as knocking on the glass or beating their chest to provoke some sort of behavior change from the gorillas. By taking visitor behavior into account, we may be able to discover more specific details about visitor effect in gorillas.We recognize the importance of visitor effect studies for the improvement of species-specific welfare standards that continuously evolve as we learn more about how animals in managed care interact with humans. In order to best serve their animal collections, we believe that zoological institutions should strive to permit a greater degree of animal choice within exhibit spaces. Studies such as these, that explore the interaction between human and non-human animals in the zoo, may inform future exhibit design and husbandry practices.Considering the fact that all zoos offer unique environments, we encourage other zoos to conduct similar research if they are presented the opportunity to have exhibits closed to the public. A multi-institutional study could provide a more extensive look at how gorillas respond to visitors and possibly help us better understand what other factors such as crowd noise or behavior, exhibit design, or troop composition may influence gorilla responses.5. ConclusionsWe found that there were no consistent, significant changes in the gorilla’s behavior between 2019 and 2020. The small changes in behavior that we saw were not always consistent across the whole troop and few were statistically significant. We did see a slight overall decrease in autogrooming from all gorillas but it was not a significant difference. Of the other behaviors we focused on, there was no clear pattern of increase or decrease across the whole troop. For some individuals certain behaviors declined while the same behavior increased in other individuals. The most notable changes were in the behavior of the AM, who may have been less attentive to activities outside of the enclosure when visitors were absent. The findings of this case report highlight the importance of collecting routine data in order to facilitate comparison when unpredictable events occur. Such information could could shed light on the ways in which zoo guests may or may not impact the behavior and welfare of zoo collections.

animals : an open access journal from mdpi

10.3390/ani12070884

PMC8996952

Autochthonous sheep and goat breeds are at risk of extinction as they produce less milk and meat compared to other breeds, around 50% less for milk and 30% less for meat production, leading to a reduction in the number of animals bred. However, thanks to rural policies, sheep and goat biodiversity is recovering and livestock farms can recognize and sustain their added value to the territory, the landscape, the maintenance of biodiversity and to sustainability through the adoption of a local supply chain in the medium and long term. Financial support for farms is not enough, integrated policies are needed to focus on further training and education and to improve communication channels to increase consumer awareness of biodiversity and quality.

European agriculture and rural development policies have promoted the diversity and genetic types of autochthonous breeds to increase sheep and goat biodiversity. Agri-environmental measures to support livestock farmers, have been the main tools used by these policies over the last twenty years. The COLAUTOC, “Collection of a seed bank for native sheep and goat breeds and strategies to increase their numbers”, research project in Basilicata, Southern Italy, investigated sheep and goat farms with autochthonous breeds with results proving a reduction in the number of sheep and goat farms in general, particularly those with native breeds at risk of extinction, a clear indication of policy failure with a devastating loss of sheep and goat biodiversity. The COLAUTOC used a variety of methodological tools, including, desk analysis, focus groups, interviews, school workshops and a survey on sheep and goat meat consumption. The results indicated that a recovery in sheep and goat biodiversity could be achieved by supporting farm livestock strategies with a view to diversify production methods and activities, whilst, recognizing the value added to the territory, landscape, biodiversity and sustainability. The strategies identified by the COLAUTOC project require a local vision, using agri-environmental funds to invest in training on innovation for farms, provide tools to improve the quality of life in the rural areas, to increase communication channels to further knowledge and awareness of biodiversity. These strategies could contribute to meeting several 2030 Agenda goals.

1. IntroductionIncreasing the number of endangered native sheep and goats in Basilicata is crucial for the development of sustainable biodiversity from an ecological, economic and social perspective. The local agro-pastoral system [1] fits the adaptive cycles [2,3,4] of exploitation, conservation, collapse and reorganization, determined by a wide and articulated network of interactions between people and the surrounding environment. The resilient [5] characteristics of animal husbandry requires a reorganization of the ecological available resources to enhance marginal and inner areas, guaranteeing the production of consumer goods and services, while preserving public assets such as landscape, environmental and social biodiversity, animal welfare, quality of life, cultural traditions and heritage [6]. As a result, the sheep and goat sector provides “benefits” to the local economy [7,8,9].Sector comparison and literature analyses refer to “adaptive governance” as the most effective coordination tool, based on networks of individuals, agencies and institutions at various organizational levels, who are capable of undertaking progressive or new directional changes to improve structures and processes to create individual and collective actions [10]. The conservation of biodiversity is an essential part of ecosystems and it is necessary to introduce agricultural multifunctionality [11,12,13] to improve income opportunity.Recent research shows that various livestock farms consider agricultural multifunctionality as essential in the generation of short supply chains [14], direct relationships [15] and collective benefits. Agricultural multifunctionality also maximizes labor returns and boosts ecological capital, tightly tying corporate income to the territory and reducing its dependence on the global market [16].A new European agricultural model is emerging, centered on agronomically sound and sustainable agricultural systems, characterized by high-added-value farming and high-quality primary and processed products [17]. The new agricultural strategies are quality differentiation and the construction of new alternative markets where high quality products meet consumers needs [18].There are more than 70 million sheep and goats (85% sheep and 15% goats) in the EU, often raised in economically fragile areas such as mountainous regions [19]. Goat and sheep farming systems in the Mediterranean represent one of the most important agricultural activities, particularly in fragile inner areas with more prevalent pastoral systems, including low levels of mechanization and the production of typical products, mainly cheeses [20]. The sheep and goat supply chain in Italy has little economic relevance compared to national agricultural production with an Added Value (VA) of just over 1% of the national agriculture VA [21]. The Italian sheep and goat sector is mainly oriented towards dairy production while meat is a secondary product that generates a value equal to one third of that of sheep’s milk (163 million for meat and 442 million for milk) [21].The national sheep and goat population amounts to 6,179,121 sheep and 1,054,549 goats [22]; stable over the last five years. Sheep and goat meat consumption is concentrated almost exclusively in two periods of the year coinciding with the Easter and Christmas holidays, with a legacy of traditions that are still very much alive in society. In recent years, the meat sector has been facing a crisis linked to the effects of an increase in vegetarian and vegan diets. The closure of the HO.RE.CA channels, the prolonged absence of tourists and the restrictions due to the COVID-19 pandemic have compromised the lamb market.The EU rural development policy links a territorial approach to specific growth objectives through the integration of tools, resources and interventions [23]. Farm diversification and multifunctionality hold a place in the European rural development policy as they are considered a lever of socio-economic development that addresses the typical rural area problems such as an ageing population, declining employment, rural exodus, preservation of territory and its rural heritage, availability of services, new business activities, income sources, quality products and the environment [24]. The COLAUTOC project, “Collection of a seed bank for native sheep and goat breeds and strategies to increase their numbers”, was approved in 2017 and financed by the 2014–2020 Basilicata RDP—Sub-measure 10.2 “support for the conservation of genetic resources in agriculture and forestry”. The goals include the protection and enhancement of four endangered sheep breeds (Gentile di Puglia, Leccese, Altamurana and Trimeticcio di Segezia) and four native endangered goat breeds (Capra di Potenza, Garganica, Jonica, Rossa Mediterranea).The aim of this paper is the identification of possible development paths linked to the territory, landscape, biodiversity and sustainability that could be supported by public policy through the multifunctional approach.2. Materials and MethodsOver the last twenty years, the animal husbandry sector has seen the progressive replacement of both prestigious and specialized native and rustic breeds. With so many indigenous breeds at risk of extinction, serious socio-economic repercussions would impact the protection of the territory. Indeed, beyond their purely productive function, sheep and goat farms maintain a strategic role in the environmental landscape and cultural protection of the hilly mountainous areas, particularly those that are marginalized. Basilicata is rich in vegetal and animal agricultural biodiversity with close links to cultural and gastronomic traditions (Figure 1).The protection of biodiversity is fundamental in the maintenance of a natural balance. The data show 42,078 raised goats and 171,536 raised sheep in Basilicata [22] with the number of raised animals in continuous decline. The trend in the population of native sheep and goat breeds has reduced in the past 5 years (Figure 2), with the exception of the Capra di Potenza and the Rossa Mediterranea breeds which have increased.Sheep and goat production is 2.2% of the total Lucanian agriculture, including dairy products [25], in particular “Pecorino di Filiano” and “Canestrato di Moliterno”, two cheeses with a protected designation of origin (PDO), and a range of cheeses which are traditional agricultural products (PAT) [26], such as Cacioricotta, Caprino, Casieddo, Pedraccio, Pecorino and mixed Pecorino.In an attempt to promote its popularity in 2008, the “Agnello delle Dolomiti Lucane” brand [27] (product recognized in the P.A.T. list) was launched, it is the property of the Edere Lucanum Cooperative, which was created to promote local goat and sheep production using a local supply chain and ensuring consumer safety. The cooperative brings together livestock farms from the Lucanian hills and mountains dedicated to the breeding of sheep and goats and has launched an integrated supply chain project for the enhancement of lambs and kids born and raised in the mountain areas [27].The EU plays an active role in the protection of biodiversity and in October 2020, member states approved the 2030 targets proposed by the Commission to step up efforts to protect and restore the natural environment and ecosystems. At the international level, the EU contributes to ensuring compliance to its global commitments to protect nature and biodiversity, through multilateral conventions such as the Convention on Biological Diversity and the Convention on International Trade concerning endangered species of wild fauna and flora. These strategies have been applied through community planning for agriculture and rural development, in the provision of specific measures of the RDPs aimed at biodiversity, dedicating funding to preserve, restore and enhance ecosystems. The 2007–2013 Basilicata RDP [28] has already considered measures to protect the environment from the promotion of organic agriculture to the conservation of endangered breeds and the provision of indemnities linked to the presence of farms in sites of particular environmental value [29] (Natura 2000 sites) and in areas subject to natural constraints. In particular, Measure 214 dedicated to the subject matter of animal biodiversity safety with: Action 3—Sub-Action B “Protect and conserve local animal breeds in danger of extinction”. This measure protected the endangered breeds through actions supporting conservation interventions, mainly in situ, of native breeds. Action 5 “Agrobiodiversity—Integrated action projects” to encourage the implementation, recovery and improvement of agrobiodiversity. It should be noted that these actions have not received much attention, with only 53 farms benefiting from Action 3 [30] and eleven projects approved for Action 5 [31]. The reasons could be due to the lack of information and assistance to farmers on the opportunities of the tenders, and in the low amounts of aid which are not particularly desirable incentives.The Basilicata region [32] has allocated 43% of financial resources to biodiversity and environmental sustainability objectives from the 2014–2020 agricultural programme, using further actions to promote the collection, characterization, use, conservation and enhancement of animal genetic resources, particularly through two sub-measures: 10.1.3 “Breeders and Growers Custodians” and 10.2 “Conservation and Sustainable Use of Genetic Resources in Agriculture”. The first sub-measure supports the protection of animal breeds and vegetal genetic diversity registered in the Regional Directory for the protection of autochthonous vegetal and animal genetic resources of agricultural interest. The Directory was established by Regional Law no.26 of 2008 [33] with the aim of maintaining and increasing consistency through on-site breeding, favoring a livestock production regime capable of guaranteeing high-quality production and recognized its added value [34,35,36]. Participation of farms in the sub-measure during this period is low, despite the presence of a network of custodian farmers and germplasm banks that house many autochthonous breed seeds which are of agricultural interest and are at risk of extinction.The second sub-measure funded native breed farms in partnership with the Research Body Institute; eleven projects including 149 partners were funded [37]. Increasing the number of native breeds is, without a doubt, the best strategy for conserving biodiversity, and is also useful in the response to environmental sustainability and the evolution of consumer demand, as well as contributing to the security of farms in the area [20,38].Various research tools were adopted including field surveys and desk analysis, the latter focusing on national and regional data and the results were shared with regional experts from the sheep and goat sector (production, research and other institutions) at two focus groups.The Regional Breeders Association (A.R.A., project partner) gave us the 34 Lucanian biodiversity farms with local breeds of interest, that is all the goat and sheep regional biodiversity farms. Authors interviewed the 34 farms over the telephone between May and June 2020, based on the following:☞F The figure of the farmer and the role of his family on the farm;☞Type and characteristics of sheep and goat breeding;☞Production and marketing of the products;☞Quality of production and/or presence of a farm brand;☞The use of technology;☞Farm land infrastructure;☞Collaboration in business activities with third parties.A second survey was carried out online with over 650 responses on the daily purchasing and consumption habits of Lucanian sheep and goat meat in Italy also using official Ministry of Agricultural Policy channels and social platforms.In addition, one study visit was carried out by classes from Year Four, from two schools (an Agrarian Institute and an Arts High School), called “rural walks”, in order observe the biodiversity farm. Students met farmers on their land who provided them with first-hand experience of their sheep and goat biodiversity farms. Workshops were carried out in both schools over the following months. 3. ResultsAnalysis of local native sheep and goat farms in Lucania (Figure 3) found that 46% raise the “Capra di Potenza”, 38% the “Garganica” goat, with the “Rossa Mediterranea” goat and the “Gentile di Puglia” sheep taking up the remaining 16%. Direct business management is the most widely used method adopted by family-run businesses, with sheep and goats raised in extensive systems, mainly fed on Lucanian pastures. Product quality is the main strength of food production in these areas and should be recognized as adding value.The autochthonous breeds of the COLAUTOC project compared to other breeds raised in Basilicata are characterized by lower yields of milk and meat. The difference is significant, about 50% less for milk production and 30% less for meat production. The low productivity of biodiversity breeds has, over time, led to a reduction in the number of animals bred [34,39,40].In total, 44% of farmers sell the raw material (meat and/or milk) to wholesalers or processing cooperatives in regional and national territories (Figure 4). Only 9% of breeders support the “Agnello delle Dolomiti Lucane” brand which was created with the aim of ensuring a better position on the market and to recognize the quality linked to the territory. The marketing methods are often not efficient or effective.The use of technological devices to carry out farm activities was detected in 79% of farms; 38% of the interviewees used a company computer and 41% used a smartphone.Farmers found that municipal and/or provincial road infrastructures were difficult to use due to their state of repair, even more complicated by the restrictive access routes to farms, classified as “bad” by 47% of farmers; some farmers are forced to reach farms exclusively on foot in adverse weather conditions. In addition, biodiversity farms have been heavily affected by the COVID-19 pandemic and the economic crisis of the last two years. In fact, the farms interviewed highlighted the need for training and consultancy services capable of supporting breeding activities, products and marketing methods.The second survey identified consumer purchasing tendencies, providing a comprehensive review of the future prospects of the sector and potential solutions to reverse the disappearance trend, particularly in indigenous breeding.The survey highlighted a growing sensitivity to issues such as production sustainability and animal welfare that influences eating habits and purchasing choices. Although 49% of the sample stated that they have reduced meat consumption in recent years as they are convinced that it does not bring health benefits, the remainder often cited environmental and ethical aspects as a reason for a reduction in meat consumption (Figure 5).In total, 33% of the interviewees said they would still be willing to buy more meat if it was from local agro-zootechnical farms (short chain) or if they were sure of the traceability of the product. Improving the quality of products and the environment, through less use of preservatives, buying seasonal produce, reducing pollution from transport, etc. and the exploitation of the features of the territory are considered essential in the promotion of the growth of the local economy.The third activity was aimed at informing young people of the importance of local goat and sheep breeds and their related products, the landscape, biodiversity and sustainability and the imminent threat to local traditions handed down from generation to generation. Two high schools were chosen to take part in school study visits of the farms and then attended workshops, the Agrarian Institute and the Artistic Institute (Arts, Music and Dance). Students interviewed a farmer and his family and gathered photographic material and during the workshops, developed a business plan and a brochure with a logo and painted scenes depicting their experience. The farm was representative of the local mountains, types of pastures and animals and the “physical” differences between varieties and breeds. The results from desk analyses, focus groups and exchanges with students highlighted that the sector would benefit from an in-depth knowledge of the product quality, production methods and ethical aspects linked to social and environmental sustainability which are little known to the public. 4. DiscussionThe Lucanian sheep and goat sector has two different facets; on the one hand, biodiversity farms with a few hundred native breeds can integrate and enhance the resources from the surrounding area, yet remain less productive, while intensive breeding of highly productive breeds is much more lucrative. Increased interest from the European Union and the consumer in environmental sustainability and biodiversity means that Lucanian breeders of native breeds need to supplement their income by making use of territorial resources, creating business diversifications which are all necessary conditions to help curb the abandonment of the territory. The importance of the presence of autochthonous breeds has also been highlighted by the survey which revealed the potential of this type of breeding, whilst also promoting cultural heritage to the younger generation. The knowledge system is needed to inform, educate and enable farms to improve their production methods, valorization and economic viability [41,42,43]. As such, some measures of the Basilicata RDP [44,45], such as information and consultancy services, must be made available to the agro-zootechnical system.In European countries in the Mediterranean, sheep and goat farming is mainly practiced in marginal agricultural areas, these pastures very often represent the only form of productive use of the land and play an important role in preserving social, cultural and environmental values [46]. The INCIPIT project, “Start-up program for a conservation plan for the Altamurana sheep population”, assessed the economic sustainability of this kind of breeding which in the long-term resulted as economically unsustainable, as opposed to the Comisana allochthonous breed. The SHEEP UP project, funded by the Veneto region in 2019, aimed to “define an innovative model of integrated economic enhancement of sheep farming for indigenous breeds (Brogna, Foza, Lamon and Alpagota) in marginal areas of the Veneto mountains” [47].The Lucanian biodiversity breeds are indigenous and rustic [20,48]; however, their product utilization is not capitalized as most of them are dual-purpose [48,49,50]. In order to maintain these breeds for the conservation of biodiversity it is essential to compensate lower income due to low productivity with adequate aid policies.The role and importance of multifunctionality should not be underestimated, it can be achieved with complementary activities such as involving the entire family unit, allocating functions other than those specifically agricultural to better enhance products and their value [51,52]. Public policies are investing in actions capable of achieving the objectives of relaunching the sectors and seeking forms of support and improvement. Tools such as traceability, transparency, labeling, strengthening of the supply chain relationships, support for business diversification processes, promotion of short supply chains and direct sales of products processed on the farm, tourist incentives and the opening of rural areas and farms are among the clearest examples of new business models. Additional enabling factors must be considered to improve competitiveness, maintain biodiversity and to protect the territory and cultural heritage, particularly if located in marginal areas.The protection of biodiversity is, and will increasingly be in the near future, vital to the consolidation of the productivity of any ecosystem. In order for this protection to become common practice, it is also necessary to provide adequate support for the protection of both vegetal and animal breed biodiversity, both in economic terms, and in terms of knowledge and services. The 2023–2037 Common Agricultural Policy (C.A.P.) [53] programming has paid due attention to the issue, providing financial instruments which play an important role in the knowledge system and support Lucanian farmers in the preservation of endangered breeds.5. ConclusionsThere is an increasing focus on the quality of agri-food products and as a result biodiversity breeding in Basilicata has an important and invaluable role in the livestock supply chain. Breeding stocks are deteriorating and support from the rural development policy is urgently needed. Over the years, regional development policies have contributed to protecting and increasing the number of breeds and in some cases have been successful in reaching their goals (Capra di Potenza and Rossa Mediterranea). Opportunities must be created to increase farmers’ income through the diversification of agricultural activities and improve their skills, knowledge and ability to introduce innovations and communicate the value and quality of their products. The surveys highlighted the need for consultancy services, improved infrastructure and new networks between farmers. Further lines of investigation could include the study of the profitability of these farms, for example high-quality brand, in marginal and inner areas [54,55].

animals : an open access journal from mdpi

10.3390/ani13061028

PMC10044566

Between 2018 and 2022, we surveyed small mammals at 23 sites in Lithuania—meadows, commercial orchards and berry farms, kitchen gardens, homesteads and farms—with the aim to assess the proportion of shrews in the community and their diet using stable isotope analysis. We found that in these natural, agricultural, and commensal habitats, common (Sorex araneus) and pygmy (Sorex minutus) shrews were under-represented—having a proportion of 3.1%, less than a half that of the long-term average in the country. The diet of these two species was similar in both agricultural and commensal habitats. On farms and in orchards with intensive farming, there were no catches of shrews. Contamination by plant protection products and a lack of invertebrates, which are the main food of shrews, may be factors limiting their numbers in the agriculturally managed habitats. Two species of water shrews, Neomys fodiens and Neomys anomalus, were found for the first time in homesteads, including in outbuildings, and their diet requires further investigation.

Shrews are a less studied group of small mammals than rodents. Between 2018 and 2022, we surveyed 23 sites in Lithuania, including natural and anthropogenic habitats, with the aim to assess the proportion of Soricidae in small mammal communities and their diet based on stable isotope analysis. The average representation of Soricidae was 3.1%, about half the long-term average in other habitats in the country. The highest proportions were in meadows and farmsteads, at 4.9% and 5.0% respectively. Shrews were not trapped on farms or in young orchards, and their relative abundance was very low in intensively managed orchards (0.006 individuals per 100 trap days). Neomys fodiens and N. anomalus were unexpectedly found in homesteads, including in outbuildings. Sorex araneus and S. minutus had similar diets. The trophic carbon/nitrogen discrimination factor between invertebrates and shrew hair was 2.74‰/3.98‰ for S. araneus, 1.90‰/3.78‰ for S. minutus in the orchards. The diet of N. fodiens and N. anomalus at the homesteads requires further investigation. We propose that the under-abundance of shrews may be due to contamination by plant protection products and a lack of invertebrates under intensive agricultural practices.

1. IntroductionThe family Soricidae is divided into two subfamilies, Soricinae and Crocidurinae [1], the latter not currently found in Lithuania. With more than 370 species, the family Soricidae is a very diverse family of mammals [2], exhibiting genetic diversity [3,4]. The family Soricidae originated in Eurasia [5] and is still very abundant in boreal forests [6]. Moisture is thought to be a key factor in the evolution of the Soricidae [5] and is of great importance for the ecology of shrews [7,8]. According to Sheftel and Hanska [6], wet and vegetation-rich habitats in the Eurasian boreal forests are characterized by the highest abundance and species richness of shrew communities.Four shrew species occur in Lithuania: the common shrew (Sorex araneus), the pygmy shrew (S. minutus), the water shrew (Neomys fodiens) [9], and the Mediterranean water shrew (N. anomalus), the last one identified only in 2012 [10]. As for the habitat preferences, S. araneus is more common in meadows, the banks of water reservoirs, reedbeds, old gardens, parks, forest edges, swamps, wet leaf litter, and forests, but is less common in dry forests. S. minutus is mostly found in wetland biotopes (marshy meadows, reedbeds, raised bogs, fens), but also in meadows, forest edges and forests. N. fodiens lives at the edges of various water bodies, covered by trees, shrubs, and dense grasses, and is encountered in biotopes adjacent to water bodies (reedbeds, meadows) and occasionally in forests and meadows farther from water [11]. Until 2020, all N. anomalus in Lithuania had been trapped near water, in reedbeds, or in sedge habitats [10]. This is consistent with the habitats investigated by other authors, notably overgrown water banks, marshes, and peat bogs [12,13,14,15].Data on the relative abundances of shrews and the proportions of their species in small mammal communities are heterogeneous. In the Carpathian Mountains, S. araneus accounted for 0.8%, and all insectivores for only 1.3%, of the total number of small mammal individuals trapped [16]. This contrasted with the work of Benedek et al. [17], who reported that the proportion of all insectivorous species in the Carpathians was 27.4%, while S. araneus alone was 23.6%. This is also much higher than the 8–15% reported by Bryja and Rehak [18]. According to Baláz and Ambros [19], various shrew species differ in their preferences of forest type and altitude in the Carpathian Mountains.In Finland, the relative density of S. araneus was 0–5 individuals per 100 trap nights for most of a 20-year period, occasionally exceeding 10 ind. per 100 trap nights in autumn during some years [20]. Similar densities of 0.6–3.7 ind. per 100 trap nights for S. araneus and much lower densities of 0.03–1.6 ind. per 100 trap nights for S. minutus were recorded in forests of Fennoscandia two decades later [21]. In other habitats, the abundance of shrews may differ, e.g., S. minutus can dominate in peatbogs. In Poland, this species accounted for 26.4% of all trapped individuals, while all Soricidae species as a whole accounted for 43.1% [22].In Lithuania, S. araneus has been recorded as dominant in the tawny owl (Strix aluco) diet: in 1999, in three forests in the central part of the country, shrews accounted for 40.7%, 38.3%, and 46.4%, respectively, while species proportion in these forests in 2000 was 26.7% [23]. A figure of 26.7% was also recorded in a protected wetland site in the north of the country in 2000.Long-term data on the shrew proportion in small mammal communities are also available from China [24] and several European countries [25,26,27]. A decline in S. araneus abundance was recorded in the Czech Republic between 1991 and 2015, with S. araneus and S. minutus accounting for an average of 8.1% and 1.3% of small mammals, with a maximum 23.0% and 3.7%, respectively, in young spruce stands [26]. Over the last 60 years, significant changes in the shrew communities have also been recorded in the Republic of Moldova [25]: S. araneus remains the most abundant species, while Neomys has declined considerably, and Crocidura has increased. Between 1975 and 2022, the average proportion of S. araneus in Lithuania was 10.0%, while that of S. minutus was 3.3%. Both species were declining [27]. On average, the proportion of Soricidae in the diet of S. aluco between 1999 and 2005 was similar, with S. araneus accounting for 7.2%, S. minutus 4.5%, and N. fodiens 0.1% of the total number of small mammals hunted [28].Agricultural and anthropogenic habitats may have different compositions of small mammals [29,30,31]. In an Italian agricultural zone (maize, corn, and sunflower fields), S. araneus was one of the two main species of prey of barn owls (Tyto alba), accounting for about 10% of all small mammals preyed upon, with no habitat preference. Crocidura shrews (21%), however, were associated with open and cultivated habitats, while Neomys shrews were preyed upon along canals and ditches [32]. Elsewhere, the Greater white-toothed shrew (Crocidura russula) accounted for 16.1% of the small mammals recorded in olive groves in Portugal [33], whereas in Serbia, in a mosaic of clover, maize, wheat, and soybean fields, all Soricidae accounted for 23.6% of the prey of owls, of which S. araneus accounted for 5.19% and S. minutus for 0.86% [34]. In the suburbs of Warsaw (Poland), S. araneus accounted for 4.9% and S. minutus for 0.2% of the prey of S. aluco [35].Diet composition and resource overlap among species is key to understanding ecological communities, including the adaptation to different environments [36]. Differences in the diet of S. araneus and S. minutus in grassland habitats have been shown to reduce competition between these species, as shown by studies of the digestive tract contents [37]. In contrast, an almost complete overlap of trophic niches between these species was found in a mountainous area with limited food supply, suggesting competition [38]. In marshland in the Bialowieza Forest in eastern Poland, abundant invertebrate prey allowed four species of Soricidae to coexist. In this case, the diets of S. araneus, S. minutus, N. fodiens, and N. anomalus overlapped considerably, despite signs of prey selectivity [39]. Using isotopic analysis, an overlapping diet was also found between S. araneus and S. minutus in a resource-rich floodplain meadow in western Lithuania [40]. The importance of abundant invertebrates as key micro-environmental features for shrews [41,42] was thus confirmed.Insectivores are not listed as pests; therefore, they are rarely monitored [43]. As with other mammals, climate change [44] is an important factor influencing the distribution and biology of shrews [45,46]. In agricultural habitats, shrews are very important as model species, as they are more likely to be contaminated by various compounds used as insecticides, plant protection products, etc., and, consequently, they show higher contamination levels [47]. Shrews also respond positively to habitat protection measures [17,48] and can therefore demonstrate the effectiveness of the measures.The aim of this study was to assess the proportions of Soricidae in small mammal communities in meadows, commercial fruit and berry farms, and commensal habitats (homesteads, kitchen gardens, and farms) of Lithuania, as well as their trophic niche, as determined by stable isotope analyses of their hair.2. Materials and Methods2.1. Study SitesBetween 2018 and 2022, small mammals were surveyed at 23 sites in various regions of Lithuania: 10 commercial apple orchards (AO), two plum orchards (PO), three raspberry plantations (RP), three currant plantations (CP), one highbush blueberry plantation (B), two homesteads (H), one kitchen garden (KG), and two farms (F) (Figure 1). The average size of apple orchards was 63.7 ha, of the plum orchards was 0.81 ha, of the currant plantations was 22.0 ha, of the raspberry plantations was 2.3 ha, and of the blueberry plantation was 3.80 ha.Agricultural study sites were old fruit orchards (AO2–9), middle-aged fruit orchards, and berry plantations (AO10; PO2; CP1–3; RP1, 2; B1), young orchards, and plantations (AO1, 6; PO1; RP3). The intensity of on-site agricultural measures was also different, from high (AO1, 2, 4, 6, 8, 10; RP2, 3; B1), to medium (AO3; PO1; CP3; RP2), and to low (AO5, 7, 9; PO2; CP1, 2; RP1). We characterized three levels of intensity depending on soil scarification, grass mowing, mulching of the plant interlines, and the usage of rodenticides and plant protection agents. Sites with only grass mowing once or several times per season were classified as low intensity, while usage of two measures from those listed above, once or twice per season, was defined as medium intensity. The application of several measures or frequent application of two measures per season was defined as high intensity. The nearest non-agricultural habitat was used as a control in every study site, these being mowed or unmowed meadows, or forest (Table A1).The kitchen garden was characterized by a limited number of buildings and a limited period of availability of human food. Both homesteads had a variety of buildings, some of which contained human food that was readily available for most of the year. The farms had the most complex building structures and unlimited access to human food and fodder for livestock, poultry, or rabbits throughout the year.2.2. Small Mammal TrappingIn orchards and plantations, small mammals were snap-trapped using a standard method—rows of 25 traps at 5 m spacing, set for three days and checked once per day in the morning. Depending on the size of the orchard, two to four trapping lines were set, while in the meadows, the number of lines was one or two.Inside the building structures of the kitchen garden, homesteadss and farms, small mammals were trapped opportunistically using 5 to 20 traps. Opportunistic trapping was also performed around all accessible buildings. Thus, the term “commensal habitats” was used sensu lato. More details of trapping are given in [49,50].In 2018–2021, we trapped small mammals in the orchards and their control meadows twice a year: in June and September–October. In 2022, small mammals were trapped in the orchards only in autumn. Trapping effort in 2018–2022 amounted to 36,978 trap days. Details regarding the trapping effort in orchards and plantations are given in Table A2. Differences in trapping efforts were inevitable due to differences in habitat availability, but, as shown in our previous publications [49,50,51], they did not affect the trappability of the more abundant species and the diversity of the small mammal community. This was tested using species accumulation curves (Figure A1) under different trapping efforts. Using long-term data, it was shown S. araneus was the fourth-most abundant species in Lithuania [27]. The species accumulation curves show that it should be trapped with the minimum trapping effort, or a very low number of trapped individuals (Figure A1); therefore the absence of this species cannot be related to under-trapping.Trapping on the homesteads, farms, and kitchen gardens took place throughout the year, with at least several trapping sessions each season. The trapping efforts on farm F1 amounted to 1530 trapping days and on homestead H2, to 210 trapping days. On homestead H1, the trapping effort in 2019–2020 was 1480 trapping days. In the other commensal habitats, the precise trapping effort was not calculated due to the use of the opportunistic trapping method.Therefore, the relative abundance of small mammals, including shrews, expressed as the number of individuals per 100 trap days, was not always available for habitats other than orchards.Trapped shrews were identified by external features, such as the base of the tail and the teeth in the Sorex species, using an identification key [9], and the hairy tail keel in the Neomys species [10].2.3. Stable Isotope AnalysisIn addition to stomach analysis, pellet analysis and laboratory feeding trials were used earlier to investigate shrew diets [37,38,39], and stable carbon and nitrogen isotope signatures in the hair may be used as a diet proxy [40]. Isotopic niches are substitutes for trophic or dietary niches, with a higher ratio of nitrogen (15N/14N) indicating consumption of animal foods [51].We analyzed carbon and nitrogen isotopic ratios in the hair of shrews, using an elemental analyzer (Flash EA1112) coupled to an isotope ratio mass spectrometer (Thermo Delta V Advantage) via a ConFlo III interface. About 5 mm of hair was clipped from the back of individuals between the shoulders. Before analysis, dirty hair samples were washed in deionized water and methanol, then desiccated. Dry samples were weighted (0.5–1 mg) into tin capsules and stored in the sample plate. The stable isotope (13C/12C and 15N/14N) ratios were expressed relative to international standards, Vienna Pee Dee Belemnite, and atmospheric air, respectively. More details of sample preparation and analyses are given in [40,51].Prior to preparation and analysis, samples (n = 10) of small mollusks and arthropods from orchards were stored in a freezer at below –20 °C. After drying in an oven at 60 °C to a constant weight for 24–48 h, invertebrate samples were homogenized to a fine powder, using mortar and pestle and a Retsch mixer mill MM 400 [51].2.4. Data AnalysesWe estimated the proportion of Soricidae and the two most abundant species, S. araneus and S. minutus, in the total number of small mammals trapped (Table 1), presenting it as mean and 95% CI for each habitat type. Differences in proportions were assessed using the G test from an online calculator [52].The δ13C and δ15N values of the hair samples were expressed as the arithmetic mean ± 1 SE and the range, from the minimum to maximum observed value. The isotopic values of species and trophic groups, including those with sample size n < 5, were visualized in isotopic biplots.We used ANOVA to determine the influence of the habitat, the age of the orchard, and the intensity of the agricultural measures on the dependent parameters: the relative abundance and the hair δ15N and δ13C values in S. araneus and S. minutus. Differences between groups were evaluated with the post-hoc Tukey’s test, and pairwise comparisons were completed using the Student’s t test. The normality of the distributions of the hair δ15N and δ13C values were tested using the Kolmogorov–Smirnov’s D test online [53]. Homogeneity of variances of the hair δ15N and δ13C values in S. araneus and S. minutus was assessed using the Levene test (Table A3).The minimum confidence level was set as p < 0.05. However, the small sample size of two Neomys species may result in a low power for statistical analysis. Calculations were performed in Statistica for Windows, version 6.0 (StatSoft, Inc., Tulsa, OK, USA); biplots were drawn in SigmaPlot ver. 12.5 (Systat Software Inc., San Jose, CA, USA).3. Results3.1. Shrew Proportions in Different Small Mammal CommunitiesDuring the study, 3141 small mammal individuals representing 14 species were trapped. Of these, 96 were shrews: 65 S. araneus, 26 S. minutus, three N. fodiens, and two N. anomalus. The proportions of Soricidae varied between habitats (G = 37.14, p < 0.001), with the highest proportion in the control habitats and homesteads. Shrews were not trapped on farms (Table 1). Of all Soricidae species, the highest number of shrews were trapped in the control habitats, 47.7% (CI = 36.8–58.3%), with lower numbers in apple orchards, 15.5% (CI = 7.3–25.8%), then raspberry plantations, 13.8% (CI = 6.1–23.7%), and currant plantations, 10.3% (CI = 3.9–19.3%).The relative abundance of all species was low, with S. araneus being most abundant in meadows, followed by berry plantations. S. minutus was most abundant in meadows (Table 1). The highest relative abundance of S. araneus and S. minutus was recorded in the control meadows, at 2.67 and 4.00 ind. per 100 trap days, respectively.Agricultural treatment intensity had no effect on the relative abundance of S. minutus (ANOVA, F2,322 = 0.92, p = 0.43) and a weak effect on the relative abundance of S. araneus (F2,322 = 3.03, p < 0.05). The relative abundance of the latter species was 0.006 ind. per 100 trap days in intensively managed crops, 0.09 in moderately managed crops, 0.19 in low-managed crops, and 0.28 ind. per 100 trap days in control habitats, with a difference of more than ten times.The relative abundance of S. araneus and S. minutus was not affected by the age of the orchard or plantation (F = 0.96, p = 0.46); however, in the young crops, shrews were not trapped at all.3.2. Stable Isotope Ratios of ShrewsStatistics for the stable isotope ratios of the insectivorous species in agricultural and commensal habitats are given in Table 2.In the commercial orchards, the trophic niche of S. araneus was wider than that of S. minutus, with a range of δ13C values of 1.9-fold, and δ15N values of 1.5-fold. The two species were fully separated according δ13C and did not differ according δ15N (Figure 2). The δ15N value of N. fodiens was about 2.4 times higher than those of Sorex shrews.We calculated the trophic discrimination factor, TDF, of invertebrates (the most likely food for shrews) and the shrew hairs (Figure 2). The TDF of carbon and nitrogen was 2.74‰/3.98‰ for S. araneus, 1.90‰/3.78‰ for S. minutus, and 3.03‰/13.50‰ for the single N. fodiens individual in the orchards. The TDF of nitrogen in the latter species indicates that N. fodiens can use not only invertebrates, but also other food sources of animal origin.In the commensal habitats, namely the kitchen garden and the homestead, the four Soricidae species did not exhibit a distinct separation in dietary space (Table 2), but the number of trapped individuals was minimal. Lower δ15N values were observed for both Neomys species compared to those of S. araneus and S. minutus; however, a larger sample is required for statistical analysis. In Lithuania, N. fodiens and N. anomalus were trapped for the first time in commensal habitats such as outbuildings.Compared with invertebrates, the TDF of carbon and nitrogen in shrews from commensal habitats was similar to those from commercial orchards: for S. araneus (2.73‰/3.70‰) and for S. minutus (1.63‰/3.88‰). In N. fodiens, the TDF was 2.16‰/2.09‰, and in N. fodiens, it was almost the same, 2.42‰/2.09‰.3.3. Trophic Position of Insectivores in Relation to Other Groups of Small MammalsIn commercial orchards, the positions of the small mammal trophic groups were well separated (Figure 3a) in terms of both δ13C (F = 184.3, p < 0.001) and δ15N (F = 28.8, p < 0.001).Four herbivore species, the common vole (Microtus arvalis), short-tailed vole (M. agrestis), root vole (M. oeconomus), and water vole (Arvicola amphibius), had the lowest δ13C and δ15N values, which were different from all other trophic groups (HSD, p < 0.001). The three granivore species, the striped field mouse (Apodemus agrarius), yellow-necked mouse (A. flavicollis), and harvest mouse (M. minutus), showed similar δ13C values, with their mean different from other trophic groups at p < 0.001. In terms of mean δ15N, they exhibited higher values than did herbivores (p < 0.001) and lower than did the insectivores (p < 0.05). Insectivores and omnivores, these being the bank vole (Clethrionomys glareolus), house mouse (Mus musculus) and Northern birch mouse (S. betulina), did not differ in terms of either δ13C or δ15N means (Figure 3a).The separation of small mammal trophic groups by δ13C (F = 16.7, p < 0.001) and δ15N (F = 34.1, p < 0.001) was also significant in commensal habitats (Figure 3b). However, intergroup differences were not as pronounced as those in orchards. In commensal habitats, insectivores had higher mean δ13C values than herbivores (HSD, p < 0.01), but did not differ from those of granivores and omnivores.The mean δ15N value of insectivores was only higher than that of granivores (HSD, p < 0.005). Other small mammal trophic groups were heterogeneous, with herbivorous M. arvalis and omnivorous C. glareolus having higher δ15N values than Neomys shrews. The granivore A. agrarius was at the same level as both Sorex species, while the omnivore M. musculus was characterized by the highest δ15N (Figure 3b).4. DiscussionThe results of the 2018–2022 small mammal trapping indicate that shrews were an under-represented group in the agricultural and commensal habitats of Lithuania, and their proportion was approximately half the long-term average proportion of Soricidae in other habitats [27]. However, two Neomys species were unexpectedly trapped in commensal habitats, including outbuildings far from the nearest water source.Despite some data on shrews in agricultural and anthropogenic habitats [29,30,31,32,33,34,35], agro-ecological studies are mostly limited to rodent communities [54,55,56,57,58,59,60]. Studies on insectivores in anthropogenic habitats are mostly limited to birds and bats [61,62,63,64,65,66], and the same applies to agricultural areas [67,68,69,70]. As such, the problem is that in some cases, there is no reference point against which to compare recent data [71]. However, insectivores may be important as vectors of pathogens [72,73,74], including Yersinia enterocolitica recently identified in S. araneus from Great Britain [75]. Therefore, our study is of value, as we provide long-term primary data on insectivores in the country for comparison purposes [27], although knowledge of the pathogen situation is still limited [76].As for shrews living in commensal habitats, this group can be expected to be under-represented, as this is a general problem of simplified diversity, especially in urban areas [77]. The hostility of anthropogenic environments, such as urban habitats, is compensated by changes in Soricidae behavior, such as increased boldness and high individual variation in aggression [78]. The presence of suitable habitats is of primary importance in the anthropization gradient, but these habitats in urbanized areas create opportunities for contact and interaction between humans or domestic animals with wild animals [79]. Shrews can be found in very unusual habitats, including airport fields: at Chisinau airport in Moldova, one shrew species was recorded from three species found in the adjacent area [80].We propose the hypothesis that one of the reasons for the low numbers of shrews in agricultural habitats is the biomagnification of pollutants. Shrews have a higher contamination rate than mice [47], and these authors argue that the effectiveness of so-called organic or ecological farming to avoid insectivore pollution is limited. This is partly contradicted by Pelosi et al. [81], who found higher concentrations of pesticide residues in the soil and earthworms of recently treated areas. The higher contamination of shrews than rodents is based on their position in the food chain, and even new “safe” insecticides concentrate in shrews [82]. This is consistent with general patterns of terrestrial vertebrate exposure to pollutants [83]. We will not speculate further on this point, but in our study, in the orchards with the most intensive agricultural treatments (including the use of plant protection products), shrews were not trapped. The susceptibility of shrews to pesticide exposure, “that can be oral via direct consumption and watering or grooming, trophic transfer, inhalation, and/or dermal contact” [84], justified the suitability of shrews as a focal species for risk assessment of plant protection products.Another reason for their low numbers may be the limited trophic resources available to shrews, these absent due to agricultural treatment in orchards, berry plantations, and homesteads. Such agricultural activity was not present in the other commensal habitats. The diet of shrews is based exclusively on invertebrates [37,39,41,42]. S. araneus mainly consumes coleopterans, insect larvae, araneids, opilionids, and isopods [85], whereas araneids, lumbricids, and coleopterans are also common foods for S. minutus [38]. These prey groups should be affected in orchards, but are unlikely to be affected in commensal habitats. Recent observations of S. minutus in commensal habitats (roof cavities) have been attributed to possible avoidance of harsher climates, resulting in a change in diet [86]. In our study, S. araneus and S. minutus did not differ in stable isotope values between commensal and agro-habitats (see Figure 2).With regard to the diet of Neomys shrews, there are differences in the prey related to the hunting method, as only N. fodiens can hunt underwater [87]. As all of our trappings for both N. fodiens and N. anomalus were in atypical agricultural or commensal habitats, the dietary characteristics are unknown. Two possible reasons for the high δ15N value in N. fodiens from orchards can be suggested as the influence of fertilization or preying/scavenging on vertebrate food. Occasional vertebrates in the diet of N. fodiens have been previously recorded [88]. An increase in δ15N values in the hair of small mammals was detected under the influence of guano from a colony of great cormorants, Phalacrocorax carbo [89]. Therefore, both factors are possible. It is also known that pesticide use can alter the diets of shrews and rodents [90].The dispersal of shrews between control areas and orchards is possible, as the migration distance of S. minutus ranges from 475 to 2570 m [91]. The migration distance of S. araneus is unknown, but it has a very limited home range of about 500 sq. m and an activity radius of 13 m [92]. Therefore, the diet of the shrews in our study probably corresponded to the habitat in which they were trapped.Finally, we suggest that there was no possible influence of removal trapping on shrew presence and abundance sensu Sullivan et al. [93]. Long-term trapping of similar intensity has been used in several studies in Lithuania and has not shown any resultant significant changes in the proportions of any small mammal species, including shrews [27,40,94,95]. Therefore, the reasons given for the low abundance or absence of shrews in intensively managed orchards must be correct and not influenced by trapping.5. ConclusionsBased on the results of the small mammal survey in agricultural and commensal habitats, it can be concluded that Soricidae (S. araneus and S. minutus) were an under-represented group of small mammals in orchards and homesteads, their proportion being less than the average in the other habitats. The diets of these two species in both habitat groups were similar, as determined by stable isotope analysis. We hypothesize that the main reasons for their limited abundance are intensive agricultural practices, contamination with plant protection products, and a lack of invertebrates, which are the main food of shrews. The presence of N. fodiens and N. anomalus was not expected in the homesteads, and their diet requires further investigation.

animals : an open access journal from mdpi

10.3390/ani11113151

PMC8614257

Endometrosis is a serious problem mainly affecting older mares’ fertility. Despite the importance of this disease, its etiology and pathogenesis are not fully known. Thus, no effective treatment exists to cease or restore degenerative processes and fibrogenesis in the mares’ endometria. The nuclear factor kappaB (NF-κB) is an important factor regulating cell metabolism. Nevertheless, it is also known to promote inflammation and fibrosis in various tissues and species, as well as in the mares’ endometria. The main goal was to bring new knowledge regarding endometrosis pathogenesis, which could allow for therapy development. Endometrial samples, collected postmortem from cyclic mares in estrus or diestrus, were classified histologically and used for gene expression assessment. Gene transcription of NF-κB subunits (subunit RelA—RelA; subunit 1—NF-κB1; subunit 2—NF-κB2), pro-inflammatory molecules (monocyte chemoattractant protein-1—MCP-1; interleukin-6—IL-6), and hyaluronan synthases (hyaluronan synthase 1—HAS 1; hyaluronan synthase 2—HAS 2; hyaluronan synthase 3—HAS 3) were compared among endometrosis types (active, non-active, destructive, non-destructive), according to the classification of Hoffmann and co-authors. These results suggest that activation of the NF-κB canonical pathway is involved especially in destructive endometrosis, the type when endometrial glands are damaged. These data give substantial information for further evaluations and treatment development.

Endometrosis is an important mares’ disease which considerably decreases their fertility. As classic endometrial classification methods might be insufficient for tissue pathological evaluation, further categorization into active/inactive and destructive/non-destructive types was developed by Hoffmann and others. This study aimed to compare NF-κB pathway genes transcription among histopathological types of endometrosis, following Hoffmann and co-authors’ classification. Endometrial samples, collected postmortem from cyclic mares (n = 100) in estrus or diestrus, were classified histologically and used for gene transcription assessment. Gene transcription of NF-κB subunits (RelA, NF-κB1, NF-κB2), pro-inflammatory molecules (MCP-1, IL-6), and hyaluronan synthases (HAS 1, HAS 2, HAS 3) was compared among endometrosis types (active, non-active, destructive, non-destructive). Most individual mRNA samples showed high expression of RelA, NF-κB1, and MCP-1 gene transcripts and the destructive type of endometrosis, simultaneously. The expression of RelA and NF-κB1 genes was higher in active destructive group than in the other groups only in the follicular phase, as well as being higher in the inactive destructive group than in the others, only in the mid-luteal phase. The increase in gene transcription of the NF-κB canonical activation pathway in destructive endometrosis may suggest the highest changes in extracellular matrix deposition. Moreover, the estrous cycle phase might influence fibrosis pathogenesis.

1. IntroductionDegenerative Endometrial Fibrosis, also referred to as endometrosis, is a major problem in equine reproduction, negatively affecting mares’ fertility. As its occurrence is widespread, endometrosis is an important cause of financial losses in the equine breeding industry. Main paramount features of this disease encompass periglandular fibrosis of the endometrium and degenerative changes of endometrial glands associated with dysfunction of affected glandular epithelial cells [1,2]. Since adequate endometrial gland response is crucial in the nutrition of the embryo, as the severity of endometrosis increases, the risk of embryonic death increases [1,3]. The basic classification method, introduced by Kenney and Doig (1986), focuses on the percentage of affected glands and layers of periglandular fibrosis. This classification allocates uterine biopsy samples into categories I, IIA, IIB, and III [3]. However, more recent studies have assessed the damage of glandular epithelial cells and the metabolic activity of periglandular stromal cells as the basis for the development of an additional endometrosis classification system into four histopathological types [1,2,4]. This classification divides uterine biopsy samples using the terms destructive or nondestructive endometrosis for the description of the damaging glands, and active or inactive endometrosis for the characterization of the metabolic activity of the stroma. Following Schöniger and Schoon’s opinion, these two classifications complement each other, and their combination allows for a better description of the affected mare’s endometrium [2]. It should be kept in mind that the better the description of the current state of the endometrium, the more adequate the assessment of the severity of the endometrial fibrosis, and hence a more accurate prognosis of the future fertility of the mare [1,2,4].Even though these two classification methods were developed, the etiology and pathogenesis of endometrosis still require further clarification. Some investigation has been carried out on cellular differentiation and periglandular myofibroblast transformation [1,5,6,7], the cycle of asynchronous differentiation [1,4,8,9], failure of innate immunity [10,11,12], extracellular matrix (ECM) composition [13,14,15], and the role of proinflammatory molecules and neutrophil extracellular traps action in stroma fibrosis remodeling [16,17,18,19]. Therefore, numerous biological indicators of endometrial pathophysiology are in the field of interest as biomarkers that can be objectively evaluated during prognostic or diagnostic protocols and treatment responses. Among such biomarkers of endometrosis, the proteins calponin, vimentin, desmin, and smooth muscle actin [1,5,6,7] have been proposed for the assessment of myofibroblast transformation. In addition, estrogen and progesterone receptors, uteroglobin, uterocalin, calbindin, and glycogen [1,4,8,9] have been used as indicators of cycle asynchronous differentiation. Likewise, ß-defensin and indoleamine 2,3-dioxygenase 1 have been suggested as indicators of innate immunity failure in the cytoplasm of endometrosis affected glands [10,11,12]. Moreover, hyaluronan synthases (HASs) have been suggested as the indicators of the production of ECM components with diverse biological functions [13,14,15], as well as monocyte chemoattractant protein-1 (MCP-1), interleukin-6 (IL-6), and tumor necrosis factor α (TNFα) as the proinflammatory molecules involved in the activation of fibrogenesis pathway by acting on cells residing in ECM [16,17,18,19,20]. Given the need to evaluate regular and abnormal cellular function within the mares’ endometria, some of these biomarkers should be considered to have prognostic value for breeding success or for the response to treatment of fibrosis [2,21].It is worth noting that in humans, the inhibition of the nuclear factor kappaB (NF-κB) pathway is one of the most popular research approaches for the prevention and treatment of fibrosis-related diseases [22,23,24,25,26]. The NF-κB is a pleiotropic transcriptional regulator of the transcription of genes that are responsible for immunity and inflammatory functions [27]. The NF-κB family of proteins consists of c-Rel, RelA (p65), RelB, NF-κB1 (p50/p105), and NF-κB2 (p52/p100) [27,28], which are systematized into two activation pathways—canonical (RelA, NF-κB1) and non-canonical (RelB, NF-κB2) [28,29]. Both pathways lead to the degradation of the inhibitory protein IκBα or C-end of p100, respectively [28,29]. This proteasome-dependent degradation is responsible for the release of NF-κB to the nucleus, causing numerous gene expression initialization, e.g., MCP-1, IL-6, and HAS [23,27,28]. Interestingly, in our recent research, the endometria of healthy mares were mostly devoid of NF-κB pathway gene expression, whereas those with endometrosis frequently showed high expression of RelA in category III endometria, high expression of NF-κB1 in categories IIA, IIB, and III, as well as high expression of NF-κB2 in categories IIA and III. These differences have been predominantly shown in the follicular phase of the estrous cycle [20]. Since delivered results provided interesting insights for NF-κB signaling pathway involvement in endometrosis pathogenesis regarding basic Kenney and Doig classification [20], further studies are required for better understanding the relationship between histopathological features of affected mares’ endometria and the NF-κB signaling pathway, using the endometrosis classification of Hoffmann and co-authors [1]. Thus, this study aimed to evaluate the expression of genes involved in the NF-κB signaling pathway in mares’ endometria in relation to Hoffmann et al. [1] four histopathological types of equine endometrosis. Specifically, gene transcription of NF-κB subunits (RelA; NF-κB1; NF-κB2), pro-inflammatory molecules (MCP-1; IL-6) and hyaluronan synthases (HAS 1; HAS 2; HAS 3) was compared among four endometrosis types (active, non-active, destructive, non-destructive) at different phases of the estrous cycle (estrus, diestrus). In the future, it is expected, as an ultimate goal, to further evaluate the potential use of NF-κB inhibitors in prophylaxis and treatment of equine endometrosis.2. Materials and Methods2.1. Sample CollectionBiological material for this study consisted of equine internal genitalia and blood collected from 100 Polish warmblood mares (aged from 4 to 25 years). At a commercial slaughterhouse in Poland, samples were collected postmortem in the reproductive season (from April to September), following the European (Council Regulation (EC) No 1099/2009) and Polish (Regulation (MARD) Dz.U. 2004 205 poz. 2102) welfare mandates. No permission from the Ethical Committee following the National Legal Regulation (Act of 15 January 2015 on the Protection of Animals Used for Scientific or Educational Purposes, Dz.U. 2015 poz. 266) was needed for sample collection after slaughter.Blood samples, each with a volume of 10 mL, were collected during exsanguination into dry tubes for hormone concentration analyses (BD Vacutainer®, Plymouth, UK). Blood samples were transported to the laboratory at +4 °C, and centrifuged (2000× g, 5 min), for serum retrieval, kept at −20 °C until further hormonal analysis. Serum progesterone (P4) concentration was determined using a commercial radioimmunoassay with the sensitivity of 0.15 ng/mL (KIP 1458; DIAsource ImmunoAssays SA, Ottignies-Louvain-la-Neuve, Belgium; intra-assay coefficient of variation <5.6%; inter-assay coefficient of variation <8.8%). The sample dilution recommended by the manufacturer’s protocol was used. The absorbance was measured by Multiscan Reader (Labsystem, Helsinki, Finland) using Genesis V 3.00 software.Ovaries were collected into containers with cold saline (0.9% NaCl, Polfa S.A., Lublin, Poland) for macroscopic examination, transported at +4 °C to the laboratory, and sectioned. The presence of follicles and/or corpus luteum was noted, and their diameter was measured.From each animal, two endometrial samples from the uterine body were collected immediately after evisceration, no longer than 5 min after the mare’s death by exsanguination. One sample was inserted into containers with the 10% neutral phosphate-buffered formalin (Sigma-Aldrich, Poznan, Poland) for histological examination, and the second one into RNase-free Eppendorf tubes (Eppendorf AG, Hamburg, Germany), snap-frozen in liquid nitrogen, and stored at −80 °C for gene transcription analyses. Endometrial samples for histopathological examination were fixed in formalin for 24 h and then moved to 70% ethanol (Sigma-Aldrich, Poznan, Poland) for one week, and then processed for paraffin-embedded blocks.2.2. Phases of Estrous Cycle DeterminationThe phases of the estrous cycle were determined based on the P4 concentration, and on the macroscopic examination of mares’ ovaries, following Roberto da Costa et al. protocol [30]. Mares were included in the mid-luteal phase group (MLP) when serum P4 concentration was >1 ng/mL and on both ovaries, none of the follicles were >35 mm in diameter, and at least one corpus luteum was demonstrated. Mares were assigned into the follicular phase group (FLP) when serum P4 concentration was <1 ng/mL, and there was at least one follicle >35 mm in diameter in any of the ovaries, and no corpus luteum present. Each group, MLP and FLP, consisted of 50 mares (total n = 100). None of the mares were excluded due to the failure of inclusion into any one of the two phases of the estrous cycle.2.3. Histopathological Types of EndometrosisFixed endometrial samples were embedded in paraffin equivalent for standard histological staining procedures. The paraffin blocks were cut in 9 μm sections on rotation microtome Leica RM2255 (Kawa-Ska, Zalesie Gorne, Polska) and mounted on glass slides. Then, slides were deparaffinized and rehydrated in a series of immersions in xylene and decreasing concentrations of ethanol (Sigma-Aldrich, Poznan, Poland). Samples were stained using standard hematoxylin-eosin (HE) protocol (hematoxylin, 3801520E, Leica, Buffalo Grove, IL, United States; eosin, HT1103128; SigmaAldrich, Poznan, Poland) and mounted under Canadian balsam resin for histological evaluation (Sigma-Aldrich, Poznan, Poland).The HE-stained slides were evaluated under a light microscope (Olympus BX43, Warsaw, Poland, magnification 40×–1000×) to assess the presence of inflammation and the appearance or severity of pathological degenerative changes. Endometrial samples that were chosen for RNA isolation did not appear actively inflamed in the macroscopic examination and did not reveal any inflammatory cell infiltration in the histopathological examination. Endometrosis was recognized when the microscopic hallmark, the concentric arrangement of stromal cells and/or collagen fibers around affected glands, was observed [2,31]. Endometrial samples were classified as belonging to histopathological types inactive nondestructive (IN), inactive destructive (ID), active nondestructive (AN), and active destructive (AD), according to specific pathological features [1,2]. In the nondestructive type, glandular epithelial cells were intact, whereas in the destructive type of endometrosis, degenerative lesions and necrosis were observed. The features of periglandular stromal cells’ metabolic activity allowed for the inclusion into the active or inactive type. In the active type, active stromal cells characterized by an oval shape, pale cytoplasm, and ovoid hypochromatic nuclei were noted, whereas in the inactive type, inactive stromal cells with spindle-shaped elongated hyperchromatic nuclei were observed [2].The mares with healthy endometrial tissue were included in the control group (C; n = 20). In addition, the remaining 80 mares were assigned to each of the four endometrosis histopathological types, as follows: (i) inactive nondestructive endometrosis (E IN; n = 20), (ii) inactive destructive endometrosis (E ID; n = 20), (iii) active nondestructive endometrosis (E AN; n = 20), and (iv) active destructive endometrosis (E AD; n = 20) (Figure 1). In all groups, half of the samples were collected from mares in FLP (C, n = 10; E IN, n = 10; E ID, n = 10; E AN, n = 10; E AD, n = 10) and the other half in MLP (C, n = 10; E IN, n = 10; E ID, n = 10; E AN, n = 10; E AD, n = 10). Part of the results on the transcription of selected genes involved in the NF-kB signaling pathway in endometria were classified in Kenney and Doig’s (1986) categories, I, IIA, IIB, III, as previously documented [20].2.4. Gene Transcription EvaluationFrozen endometrial samples were mechanically disrupted in a liquid nitrogen environment. Afterwards, 50 mg of each sample were homogenized in Lysing Matrix D tubes (MP Biomedicals, Irvine, CA, United States), and total RNA was extracted using High Pure RNA Tissue Kit (Roche, Rotkreuz, Switzerland). The extraction protocol recommended by the manufacturers was used. Then, a DNase treatment was performed. The RNA concentration was determined using DS-11 FX spectrophotometer (DeNovix, Wilmington, DE, United States) with absorbance ratios A260/280 and A260/230 of approximately 2.0. The further analysis inclusion criterion was RNA concentration above 100 ng. None of the samples were excluded due to insufficient RNA concentration.Real-time PCR (qPCR) amplification was performed using a TaqMan™ RNAto-CT™ 1-Step Kit (No 4392938, ThermoFisher, Swedesboro, NJ, United States) and a Quant-Studio™ 6 Flex Real-Time PCR System (Applied Biosystems, Wilmington, DE, United States). The commercially available equine-specific TaqMan Gene Expression Assays (No 4448892 and 4441114, ThermoFisher, Swedesboro, NJ, United States) were used. The list of primers and 6-carboxyfluorescein (6-FAM) and 6-carboxytetramethylrhodamine (TAMRA)-labeled TaqMan probes used for the qPCR analysis was presented in Domino et al. [20]. Real-time PCR reaction had a 10 mL volume and included 15 ng of total RNA, 5 mL of TaqMan® RTPCR Mix (2×), 0.25 mL of TaqMan® RT Enzyme Mix (40×), 0.5 mL of TaqMan probe, and both PCR primers (ThermoFisher, Swedesboro, NJ, USA) for each gene of interest. The PCR protocol included four steps as follow reverse transcription (15 min at 48 °C), enzyme activation (10 min at 95 °C), 40 cycles of denaturation (15 s at 95 °C) and annealing/extension (1 min at 60 °C). Each sample was run in triplicate.2.5. Data AnalysisEach endometrial sample was double categorized using estrous cycle criterion and endometrosis criterion. The estrous cycle determination data were listed as MLP or FLP for each endometrial sample. Independently, healthy endometria (C), and histopathological types of endometrosis data were allocated to each one of the following groups: C, E IN, E ID, E AN, or E AD. In each endometrial sample, transcription of the following eight genes was assessed by qPCR: RelA, NF-κB1, NF-κB2, MCP-1, IL-6, HAS 1, HAS 2, and HAS 3. Raw data of genes transcription were normalized using the geometric mean of mRNA detected from two independent endogenous reference genes (GAPDH, HPRT1). The semi-quantitation of the target gene expression was performed in a comparative CT method (ΔΔCT method), where the target gene expression in the samples of category C was considered as ΔCt Control Value.2.6. Statistical AnalysisUnivariate marginal distributions of Expression Fold Change (2−ΔΔCt) of the qPCR data were tested independently for each endometrial samples category and each target gene using a univariate Kolmogorov–Smirnov test. The comparison between histopathological types was assessed by Kruskal–Wallis test, followed by Dunn’s multiple comparisons test. The comparison between phases of estrous cycle was performed Unpaired t-test with Welch’s correction for normally distributed data pairs or Mann–Whitney test for non-Gaussian data pairs. Numerical data were reported on the box plots using minimum and maximum values, lower and upper quartiles, as well as median. The control group level was estimated as a maximal value of gene expression in a control group and marked on plots using a dashed line. The destructive group level was introduced when only destructive type samples were above the marked level. The destructive group level was marked on selected plots using a dashed line. The percentages of samples in each histopathological type with the value above the level of control group and destructive type were also calculated. All statistical analysis was performed using GraphPad Prism6 software (GraphPad Software Inc., San Diego, CA, USA), where the significance level was established as p < 0.05.3. ResultsThe heterogeneity of the distribution of transcripts of target genes in the control and endometrosis groups was observed (Figure 2, Figure 3 and Figure 4), hence the intra-group variability was high. Therefore, the individual samples distribution was visualized including individuals of each endometrosis histopathological type and healthy endometrium.Individual sample distribution of mRNA levels of NF-κB subunits (Figure 2), proinflammatory proteins (Figure 3), and hyaluronan synthases (Figure 4) differed in relation to the control group. Most individual transcript samples above the level of the control group represented NF-κB1 gene (60% E ID, 50% E AD, and 10% E IN), then RelA gene (50% E ID, 25% E AD, 15% E ID, and 5% E IN), MCP-1 gene (60% E ID and 10% E IN), and NF-κB2 gene (15% E AD, 10% E ID, and 5% E IN). For these four target genes, the individual distribution of transcripts of the destructive type dominated over the non-destructive type.Transcription levels of all the genes under study were calculated for the destructive type. Most individual mRNA samples above the level of the destructive type corresponded to MCP-1 gene transcripts (60% E ID), NF-κB1 gene transcripts (40% E ID and 30% E AD), and RelA transcripts (25% E ID and 20% E AD). However, transcription levels of NF-κB2 and HAS 3 above the level of the destructive type of endometrium only occurred in 10% of E AD for NF-κB2 and 10% of E ID for HAS 3 endometria. The remaining target genes transcripts did not meet the destructive type-level criterion.The transcription of the genes under study in the histopathological types of equine endometrosis differed in relation to the control group for NF-κB subunits of canonical (RelA, NF-κB1), but not for the non-canonical (NF-κB2) pathway (Figure 5), for MCP-1 and IL-6 (Figure 6), as well as for HAS 2, but not for HAS 1 and HAS 3 (Figure 7).The transcript expression of RelA gene was higher in E AD (p = 0.033) than in the other groups in the follicular phase, but not in the mid-luteal phase of the estrous cycle, as well as in E ID (p = 0.018) in mid-luteal phase, but not in the follicular phase. Moreover, RelA gene transcription also differed between follicular and mid-luteal phases in the E AD (p = 0.006; RelA higher in FLP), but not in E ID (p = 0.114) histopathological types (Figure 5A). Similarly, the transcription of NF-κB1 gene increased more in E AD (p = 0.044) than in the other groups in the follicular phase, but not in the mid-luteal phase. In addition, similarly to RelA, the transcription of NF-κB1 gene was higher in E ID (p = 0.023) than in the other groups in mid-luteal phase, but not in the follicular phase. The NF-κB1 gene transcription was raised in the follicular phase, only in the E AD (p = 0.004) histopathological type (Figure 5B). No differences in the expression of NF-κB2 gene were found between either the histopathological type or phases of the estrous cycle (Figure 5C).The transcription of the MCP-1 gene in the endometrium was higher in E ID (p = 0.030), than in the other groups, but only in the mid-luteal phase. In addition, MCP-1 gene transcript was higher in the mid-luteal than in the follicular phases in the E ID histopathological type (p = 0.047) (Figure 6A). On the contrary, the transcription of IL-6 gene was increased in E AD (p = 0.028) than in the other groups, but just in the follicular phase. However, no differences in the transcript level of IL-6 were found between phases of estrous cycle in E AD (p = 0.343), and in any other histopathological groups (Figure 6B).No differences in the transcription of HAS 1 (Figure 7A) and HAS 3 (Figure 7C) genes were found between either histopathological type of endometrosis or phases of estrous cycle. However, the transcript levels of HAS 2 gene were higher in E AD (p = 0.044) than in the other groups in the follicular phase, but not in the mid-luteal phase. Additionally, in all endometrosis groups, no differences in the mRNA levels of HAS 2 were found between estrous cycle phases (Figure 7B).4. DiscussionAn interesting finding about the present results is the heterogeneity of gene transcription within a group. This can possibly indicate the complexity of endometrosis pathology and the involvement of some other pathways in cooperation with NF-κB. Additionally, despite showing similar histopathological features, some mares could have been in a different stage of the disease. Thus, allocation of endometrial samples to the specific classification groups of Hoffmann and co-authors (2009) could be somehow inaccurate. In a single endometrial biopsy, usually endometrial glands may show different types of cells. Therefore, a biopsy assignment to Hoffmann et al.’s classification groups has been made based on the state of most glands [1].In a previous study, we evaluated the transcription of NF-κB pathway genes regarding Kenney and Doig’s endometrial categories, but not the histopathological types of endometrosis [20]. Interestingly, in the present study, the destructive type of endometrosis showed the highest differences in the transcription of several genes. In other studies, this type of endometrosis depicted a larger modification of ECM, especially the increase in proteoglycans, fibronectin and laminin expression [1,3]. Our results suggest that in this specific type of endometrosis, severe changes in ECM may be associated with the NF-κB pathway (Figure 8), which may regulate the production of connective tissue fibers. Since the destructive endometrosis is thought to decrease fertility more significantly than the non-destructive endometrosis, the NF-κB pathway might be involved in endometrial changes, which might impair pregnancy success [3].Regarding Kenney and Doig´s categories, our previous study showed an increase in NF-κB gene transcripts in the canonical pathway activation (NF-κB1, RelA) and in the non-canonical pathway (NF-κB2), observed only in FLP along with the increase in endometrosis severity [20]. However, considering the various histopathological types of mare endometrosis, the main differences regard the canonical pathway of activation, since RelA, NF-κB1, and MCP-1 genes transcripts increased in over than half of the endometria. It has been discovered that this pathway is coactivated by TNFα, another cytokine taking part in endometrosis pathogenesis, inducing fibroblasts’ transformation into myofibroblasts [21,32]. The known increased presence of TNFα in the endometrium may be responsible for the increase in NF-κB pathway proteins expression. The abovementioned activation may result in the promotion of MCP-1 expression, a potent factor increasing monocyte infiltration. Interestingly, these cells are the main producers of TNFα [32], thus this mechanism may act in a virtuous cycle.Another explanation may be the fact that destructive endometrosis affects glands and glandular epithelium far more than nondestructive endometrosis [1,3]. The obtained results may show the influence of NF-κB on the glandular epithelium, causing its degeneration, alteration in secretion composition and basal lamina degradation, which altogether decreases fertility [8]. However, there is no direct evidence proving NF-κB involvement in basal lamina degradation, which needs to be further studied.An increase in RelA transcription was noted in E AD in FLP and in E ID samples in MLP, similarly to NF-κB1, associated with the canonical pathway. In addition, MCP-1 increased in E ID in MLP, whereas IL-6 raised in E AD in FLP, thus showing that the transcription of those genes depended not only on the type of endometrosis, but also on the phase of the estrous cycle. In contrast, when considering Kenney and Doig’s endometrial classification, IL-6 decreased significantly in FLP in samples with endometrosis [20]. These findings imply that endometrial changes induced by the NF-κB pathway are estrous cycle-dependent. Moreover, this may suggest that the metabolic activity of fibroblasts in the endometria of mares may depend on estrous cycle regulation and variability in ovarian steroid hormones levels. However, further studies, including estrogen and progesterone receptors evaluation, are needed to confirm this theory, considering the expression of proven receptors during endometrosis [1,2,3].Among the hyaluronan synthases, only the transcription of HAS 2 showed significant changes in the endometrosis samples evaluated in the present study. An increase in HAS 2 mRNA levels in E AD tissue retrieved in the FLP, similarly to RelA transcript data, suggests a connection between these proteins in endometrosis pathogenesis. On the contrary, when considering Kenney and Doig´s categories, HAS 3 gene transcriptions increased instead, in endometrosis tissues obtained in FLP [20]. Ohkawa et al. found that hyaluronan synthesis by fibroblasts is mediated by RelA, after stimulation by TNFα [33]. The difference found in gene transcription only in the follicular phase gives another evidence, that endometrosis pathogenesis might be somehow connected with the estrous cycle, as previously suggested [19].The NF-κB is known for stimulating ECM deposition in various tissues [22,23,27,29,32]. A quantitative assessment of endometrosis can be carried out based on RelA, NF-κB1, and MCP-1 gene transcription levels, as previously described for the use of uterocalin, uteroferrin, uteroglobin, and calbindin [8]. The achieved results may be helpful in the classification of endometrosis, as well as for the prognosis of disease development in clinical cases. However, further studies comparing NF-κB canonical pathway proteins and epithelial cell degeneration are necessary for confirmation of this assumption. This study has shown the importance of the NF-κB pathway on the pathogenesis of endometrosis. Since NF-κB inhibitors have been successfully studied in suppressing ECM deposition in various tissues [34,35,36], this approach should be further studied as a therapeutic means for endometrosis, allowing for stopping or even reversing fibrosis.The main limitation of this study is that the obtained data only pertain to gene transcription, and the assessment of the end products of genes in the endometrium is necessary. The lack of immunohistochemistry for protein localization can be considered as a part of the explanation for the differing results of previous studies. Therefore, further studies encompass the use of immunohistochemistry for localization of proteins, and comparison among the various endometrosis types, which is crucial for further assumptions.Summing up, activation of the NF-κB canonical pathway may be associated with degeneration and necrosis of glandular epithelial cells, as results showed significant changes in gene transcription in destructive endometrosis. Moreover, steroid hormones possibly modulate the NF-κB canonical pathway. Additionally, the activation of proinflammatory molecules, promoted by NF-κB, may play a role in gland deformation and damage, acting on residual inflammatory cells located in ECM, but also promoting infiltration of further leukocytes. In this study, RelA, NF-κB2, and IL-6 transcription was increased in comparison with the control group in FLP in the active nondestructive type of endometrosis, whereas in our previous study, RelA, NF-κB1, NF-κB2, HAS 1, and HAS 3 transcription similarly increased in FLP in the respective types of Kenney and Doig´s categories of endometrial classification when endometrosis was present [20]. Both findings suggest active remodeling of ECM in this phase of the cycle. Minor changes in the luteal phase may suggest that another set of chemokines present in the endometrium might be necessary to activate fibroblasts and myofibroblasts.5. ConclusionsThe NF-κB pathway is important in the regulation of fibrosis during endometrosis, regarding the different types of this condition, based on histopathological lesions. The NF-κB canonical pathway is upregulated especially in destructive fibrosis, indicating the highest intensity of changes in ECM deposition. The MCP-1 gene transcription increased in the follicular phase in the inactive destructive type of endometrosis, whereas IL-6 transcript levels raised in the mid-luteal phase in the active destructive endometrosis. Nevertheless, for further conclusions future studies are necessary, comprising more endometrial samples and additional research approaches.

animals : an open access journal from mdpi

10.3390/ani13050910

PMC10000096

A long birthing process is not only stressful for both the sow and her piglets, it also decreases the chances of survival of piglets during birth or in the first days of life. Oxygen supply from the mother to the fetus via placenta and umbilical cord is crucial. This oxygen supply may be impaired by successive uterine contractions, partly or completely blocking placental and/or umbilical blood flow. Providing the mother with the right amount of energy and other nutrients needed for the birthing process could reduce its duration and, as a consequence, increase peri-partum piglet survival. In addition, nutrients that enhance blood flow (and therefore oxygen flow) to piglets during birth may also impact piglet survival.

The birth process is a crucial event for piglet survival. Along with increasing litter sizes, not only has the duration of parturition increased, but placental blood flow per piglet has reduced and placental area per piglet has become smaller, making these piglets more susceptible for hypoxia. Diminishing the risk of piglet hypoxia by either reducing the total duration of parturition or increasing fetal oxygenation may reduce the incidence of stillbirth and early post-partum mortality. This review discusses options to do so by nutritionally supporting the sow in the final pre-partum period, after discussing the role of uterine contractions and placental blood flow. Providing sufficient energy seems to be a logical first step, but also other nutrients needed for uterine contractions, such as calcium, or enhancing uterine blood flow by using nitrate seem promising. These nutrient requirements may depend on litter size.

1. IntroductionThe parturition process is challenging for both the sow and her piglets. For the sow, parturition is an energy demanding, stressful, and painful event [1]. For piglets, it is also a stressful event, and the odds of dying are highest during parturition and the first days of life [2]. The parturition process in sows has been studied mainly from a behavioral or endocrine point of view [3,4]. Only a few studies, however, have investigated peri-partum uterine contractions and placental or umbilical blood flow and the changing metabolic status and nutritional requirements of the sow during the peri-partum period [5]. Along with increases in litter size, the challenges to the perinatal piglet have increased, which are either related to in utero circumstances such as a decrease in uterine blood flow per piglet [6], a decrease in piglet [7,8,9] and placental size [2,10,11], or an increase in farrowing duration [12]. The survival rate for piglets mainly depends on fetal oxygenation, which in turn is related to farrowing duration [13,14], the duration and intensity of uterine contractions [15], and placental blood flow and therefore oxygen flow [16]. The maternal diet needs to provide the nutrients for uterine contractions and for sufficient placental blood flow, and its role has been investigated in recent studies [17,18,19,20,21,22]. This review focuses on possible interventions in the maternal diet that may facilitate the parturition process, affecting uterine contractions and/or placental blood flow in the perinatal period. 2. Uterine Contractions The parturition process in the sow can be divided into three stages: (1) increase in myometrial activity and dilation of the cervix (approximately 6–12 h), (2) expulsion of the piglets with the sow lying down and in abdominal straining (approximately 5–8 h), and (3) expulsion of the placenta (approximately 4 h, which may already start during stage 2) [12,23]. Several reviews discuss the complex endocrine changes in the peripartum period [24,25,26,27], so here, we only highlight the major changes. The increase in myometrial contractions in stage 1 results from a cascade of endocrine events. Fetal cortisol induces a release of endometrial PGF2α, which induces luteal regression and thereby results in a decline in progesterone. PGF2α also stimulates the release of relaxin by the corpora lutea, producing oxytocin and uterine smooth muscle contractions [28]. Exogenous prostaglandin injection to induce luteal regression and thereby induce parturition does not influence the parturition process itself. It is mainly a tool to optimize parturition management when given, at most, two days before parturition [29]. The placenta produces estrogens, and the changed progesterone/estrogen ratio increases the expression of oxytocin receptors on the myometrium, causing an increase in number as the ratio between progesterone and estrogen changes [27,30]. The changed progesterone/estrogen ratio stimulates myometrial contractions starting at 4–9 h before the expulsion of the first piglet. These contractions last for 2–3 min each and occur at regular intervals [31]. Contractions keep increasing in frequency and amplitude, and straining efforts of the sows start to appear the last few hours before expulsion of the first piglet [31]. As soon as the first fetus enters the cervix, stage 1 of parturition is considered to be completed [32]. Then, the Ferguson reflex is activated, releasing oxytocin from the pituitary. The increased oxytocin levels stimulate abdominal muscle straining to expel fetuses [33]. The frequency of uterine contractions is highest when piglets and the placentae are being expelled [34], but large variations occur among sows in frequency, duration, and amplitude and for an individual sow from one hour to the next of the expulsion phase [35]. During this phase, on average, uterine contractions last for 1–2 min and occur at a frequency of 18 per hour [34]. Maffeo et al. [15] gained insight into the frequency and amplitude of contractions during different timepoints of the parturition process using two implanted strain gauges (one in each horn) during spontaneous births. The frequency, amplitude, and duration of contractions 12, 5, and 1 h before the birth of the first piglet, during piglet expulsions, and during placenta expulsion are shown in Figure 1. Exogenous oxytocin injections can maintain and reinforce spontaneous parturition [36] by increasing the frequency of contractions 13-fold and their intensity 2-fold when compared to spontaneous contractions. This can result in a reduced duration of farrowing, but could also impair the normal physiology of contractions [37], which may result in a reduction in piglet vitality at birth and an increase in incidence of stillbirth [38]. This is likely due to damage of the umbilical cord [39] or a decrease in placental blood flow [40]. During the expulsion phase, tubo-cervical contractions move the fetuses towards the cervix. In addition, cervico-tubal contractions occur, which are likely meant to shorten the uterine horns and to prevent accumulation of fetuses at the caudal ends of the uterine horns [31] and/or to keep fetuses at a fixed place to keep the umbilical cord functional before expulsion [34]. Contractions are initiated at the two ends of the horns and convey (either as a tubo-cervical or cervico-tubal contraction) to the proximal end of the horns [34], but may rebound in the opposite direction when reaching the end of the horn [31]. Empty parts of the horn also contract [31]. Cervico-tubal contractions end when the horn is empty of piglets, indicating that the presence of piglets close to the cervix initiate these contractions [31]. It is estimated that four to five uterine contractions, with an average duration of 11.5 s and an intensity of 9.4 mm Hg, are needed to expel one fetus [38,41,42]. As soon as the horns are completely empty, contractions are only tubo-cervical and appear very frequent and regular for placentae expulsion [15,34]. It is unknown whether or not there is synchrony in the timing of contractions between the two horns, but this seems likely, since muscle fibers fuse at the common uterine body [43], and the birth order of fetuses from both uterine horns appears to happen fully at random from one horn or the other [34]. It is also unclear whether crowding of piglets occurs during contractions. It might be that crowding does occur when fetuses are stuck or when a stillborn piglet causes a delay in the birth process. The birth interval after which a stillborn piglet is born is approximately twice as long as that of a liveborn piglet (28 vs. 15 min) [14,44]. It is unknown whether the increase in birth interval is a cause or consequence of the increased birth interval [45].3. Uterine Blood FlowMost studies evaluating the duration of farrowing in sows only consider stage 2 of parturition, the time during which fetuses are expelled [12,14,17,22,46,47,48], as this stage determines the level of asphyxiation of piglets and can easily be observed. Asphyxiation mostly occurs due to strong uterine contractions combined with placental space limitation and/or reduced placental–uterine connection, which together reduce or obstruct placental blood flow [32]. As an initial response to reduced blood oxygen levels, fetal heartrate drops [49] and fetal movements increase, which in turn promote myometrial contractions, making a positive feedback system to reduce the duration of farrowing [32]. In fetuses with a prolonged inadequate oxygen supply, blood CO2 concentrations will rise, and hypoxia starts to occur. To reduce fetal oxygen consumption, not only will fetal limb and body movements reduce [50], but also fetal heartrate falls (bradycardia) [51,52] and metabolic rate reduces [52]. When fetal blood O2 concentration drops below a certain threshold level, adenosine triphosphate (ATP) production shifts to anaerobic glycolysis, and fetal lactate levels increase [51]. This anaerobe metabolism is faster than aerobe metabolism, but can only provide energy for a short period of time (up to 2 min) [53]. Lactate also lowers blood pH, which can affect functioning of the central nervous or cardiovascular system [54]. Lactate levels at birth have been related with chances of dying during lactation. For example, English and Wilkinson [55] showed that piglets that died pre-weaning had higher blood lactate concentrations at birth than survivors (383 vs. 303 μg lactate/mL blood; p < 0.01, respectively). Furthermore, Langendijk et al. [56] found a higher pre-weaning mortality when blood lactate concentrations in umbilical cord blood was increased (8.5% and 10.9% for 4.45–6.40 mmol/L and >6.40 mmol/L, respectively). Thus, the level of asphyxia at birth appears to be related to the chances for pre-weaning survival.It is not known whether the number, duration, and amplitude of contractions, or the duration of stage 1 of parturition, is related to litter size. It is also not known whether the durations of stage 1 and 2 of parturition are related. It is known that the duration of stage 2 of parturition is related to litter size; it indeed takes more time to deliver more piglets [12]. Combining the data of 15 studies that measured the duration of stage 2 of parturition in the last 18 years [12,14,17,46,47,57,58,59,60,61,62,63,64,65,66] shows an estimated increase of 27 min in duration of stage 2 of parturition per extra piglet (Figure 2, averages per study). The deviation from the predicted line for farrowing duration based on litter size is sometimes quite large, which may be caused by differences in e.g., breed, housing, or management (i.e., use of birth assistance and exogenous hormones). Summarizing, the total duration of parturition (stage 1, 2, and 3), in which a sow experiences frequent and powerful uterine contractions, can take up to 24 h in the hyper-prolific sow [26]. Research on duration of parturition mainly focuses on phase 2 of parturition, i.e., the period during which the piglets are born, since this phase is the most easy to observe. It is unknown what the impact is of phase 1 on the sow, her piglets, and how related phase 1 and 2 of parturition are with each other. Most of the studies investigating the intensity, number, and duration of uterine contractions for the different phases of parturition in the sow were done three to four decades ago [15,31,34]. It is unknown whether the intensity, number, and duration of uterine contractions relate to the current litter sizes and other aspects of the current highly prolific sow. 4. Placental and Umbilical Cord FunctionalityThe placenta is responsible for nutrient and oxygen exchange between the sow and her fetuses. The fetus has a diffuse placenta in which many closely spaced chorionic villi are distributed over the entire outer surface of the chorion [32], which ensures transport and diffusion of nutrients from the maternal to the fetal blood. Additionally, specific structures called areolae on the placenta absorb the products secreted by the endometrial glands (e.g., growth hormones, hormones, transport proteins lymphokines, cytokines) [32]. The surface area of the chorio-allantoic membrane mainly increases in size between day 35 to 70 of gestation, with little change between day 70 to 100 of gestation [10]. Vascularization of the allantoic membrane starts at approximately day 15 post-insemination, i.e., 2 days after contact between the trophoblast and maternal epithelium [67], and increases until mid-gestation, after which vascularity remain relatively constant [68,69]. By that time, blood vessels occupy about 3–4% of the chorio-allantoic membrane, but with large variation among individual fetuses, among litters, and between breeds [68,69]. Blood capillaries from the chorionic villi merge and eventually form larger vessels that enter the umbilical cord [32]. In addition to vascularization, nutrient supply to the fetus is also affected by uterine blood flow, which increases as gestation progresses [6]. Although it seems likely, it is not known, whether vascularization of the placenta and placental blood flow are related. Blood flow [70] and placental area [2] per piglet both seem negatively correlated with litter size, which likely explains why average piglet birth weight decreases as litter size increases [7,9,71]. No studies were found showing a clear relationship between litter size and placental vascularization. Wilson et al. [69] found differences between breeds in placental vascularization at the fetal–maternal interface. Vascular density was higher in Meishan placentas compared to Yorkshire placentas, although placental size was larger in Yorkshire sows. Whether placental blood flow differs between breeds has not been evaluated. Placental characteristics and the incidence of pre-weaning mortality appear to be related, although these relationships might be confounded with piglet birth weight. Both placental surface (−20.4%) and placental weight (−14.8%) were lower in piglets that died before weaning compared to surviving conspecifics, which was most likely caused by a lower birth weight of piglets that died before weaning [2]. Baxter et al. [72] found no difference in vascularization score of placentas of piglets that survived or died before weaning.The umbilical cord connects the fetus to the placenta, and it contains one vein that carries oxygen and nutrient-rich blood to the fetus and two smaller arteries that transport deoxygenated blood from the fetus back to the placenta [73]. These vessels are surrounded by Wharton’s jelly, a gelatinous connective tissue consisting mainly of hyaluronic acid, in which collagenous and reticular fibers form a loose meshwork [74]. An intact and functional umbilical cord is of crucial importance for fetal oxygen and nutrient supply. Umbilical cord length of piglets was found to be 35 cm on average (ranging between 17 to 50 cm) and was positively correlated with piglet weight [9]. Umbilical cord length is not correlated to the position of a piglet in the uterus, but its elasticity (up to 37.5% of its length) allows it to stretch as a piglet is transported through the uterine horn at parturition, making it possible for piglets at the end of the uterus to be born with intact umbilical cords. The tension required to break an umbilical cord varies from 545 to 2000 g [75,76]. The percentage of piglets born with a broken umbilical cord lies between 21 to 71% [9,56], and Rootwelt et al. [9] showed that broken umbilical cords occur most in the second and last third of piglets born (2.3 times more often compared to the first third of piglets born). When or where an umbilical cord breaks has, to our knowledge, not been studied in pigs. It can be hypothesized that the umbilical cord breaks at a weak spot or occurs randomly over the full length of the umbilical cord, potentially caused by a weak spot in the Wharton’s jelly or the first place where umbilical cord blood flow has stopped. It is also unclear which placental or other sow and/or piglet characteristics might be related with umbilical cord length, thickness, strength, or breaking point. It seems likely that larger piglets, which have a larger placenta [2,9], also have a thicker umbilical cord that may also be less prone to breaking. Curtis et al. [77] suggested that stillborn piglets (that weighed less than live-born litter mates) have a smaller umbilical cord that is more likely to break, suggesting a relationship with piglet birth weight and umbilical cord thickness/strength. However, Langendijk and Plush [49] found a similar weight distribution in live and stillborn piglets, suggesting that the hypothesis of Curtis et al. [77] might not be true. Piglets born alive but with a broken umbilical cord (as observed at the moment of birth) showed a lower vitality score and had an higher risk for post-partum death compared to piglets born with an intact umbilical cord [2]. A recent review estimated the association between incidence of stillbirth and a broken umbilical cord before expulsion to be 50% or more [49]. In addition, even when the cord does not break, extensive stretching might lead to vasoconstriction and limited blood flow, increasing the risk for stillbirth [39]. In summary, in larger litters, placental blood flow per piglet is reduced [70] and placental area per piglet is smaller [2], which likely explains why average piglet birth weight is lower [7,9,71] and partially explains why incidence of pre-weaning mortality increases. An intact and functional umbilical cord is key for fetal oxygen and nutrient supply and therefore survival. Studies on how, where, or when an umbilical cord breaks and which sow and/or piglet characteristics are related to its breaking are limited. A better understanding of the complex interactions between placental/umbilical cord blood flow, contractions, breaking of the umbilical cord, and other characteristics of the modern sow might provide insights in how perinatal piglet losses can be reduced. In conclusion, placental and/or umbilical cord blood flow might be under pressure in large litters, which might be related to stillbirth and pre-weaning mortality. 5. The Potential of Maternal Nutrition to Reduce Farrowing DurationRelationships between placental characteristics, uterine contractions, placental and umbilical cord blood flow, and piglet losses are summarized in Figure 3. Additionally, in this figure, the potential effects of maternal nutrients on these events are included. Providing the right nutrients to the sow and her fetuses may not only enhance placental development and fetal growth but could also affect uterine blood flow in the perinatal period and/or affect the duration of farrowing. Studies evaluating nutritional solutions aiming to reduce farrowing duration by enhancing uterine contractions or affect placental characteristics and therefore affecting piglet losses during or shortly after parturition will be discussed in the next paragraph.Nutritional interventions in the perinatal period aiming to decrease stillbirth and to increase piglet vitality right after birth should stimulate uterine contractions (frequency or intensity), increase placental nutrient and/or oxygen supply to the fetus, and/or provide the sow with the energy to prevent fatigue. To prevent constipation [78] and metritis, mastitis and agalactia (MMA) [79] feed allowance in some European countries is lowered to 2.0–3.0 kg/sow/day, beginning 2–3 days before the expected farrowing date. It can be questioned whether this lower energy and nutrient intake and the type of nutrients supplied sufficiently facilitates energy and nutrient requirements during parturition. Consequently, feeding strategies in the perinatal period might need to be reconsidered. 5.1. EnergyThe total duration of farrowing (stage 1, 2, and 3) can take up to 24 h in the hyper-prolific sow [26] and is positively related to litter size [80]. When we expect our sows to give birth to larger litters, we should provide them with the right nutrients to perform this activity. Focus on energy requirements seems to be a logical first step, since farrowing is likely a highly energy-demanding activity [61,81]. It seems likely that modern sows do experience a limitation in available energy around farrowing. Van Kempen et al. [82] suggested that sow exhaustion during farrowing caused by energy depletion could impair the number and intensity of uterine contractions, thereby increasing the duration of farrowing and consequently increasing stillbirth rate. That sow exhaustion occurs was also suggested by Mosnier et al. [5], who found a higher sow plasma lactate concentration at day 1 post-partum (approximately 1.4 mmol/L) compared to day 4 (approximately 0.9 mmol/L). The higher concentration of lactate in sow blood is likely due to increased metabolic activity and uterine contractions of sows during farrowing (also seen by an increase in body temperature [83]). A recent study, in which lactate levels were determined more frequently around parturition (every 6 h pre-farrowing and every 3 h post-partum), showed that sow blood lactate levels were indeed increased during parturition, but were already increased at 9 and 3 h before the expulsion of the first piglet [65], which is likely related to higher activity during nest-building behavior and by increased uterine contractions during phase 1 of parturition. Three hours after farrowing, lactate levels started to decrease again [65], indicating that sows shifted back to their aerobe metabolism. The energy requirement for the farrowing process is expected to be comparable to moderate to heavy exercise [61]. Recent estimates of energy requirements estimated during the transition period (10 days pre-farrowing to 10 days post-farrowing) included maintenance, heat loss, mammary growth, fetal growth, and colostrum/milk production, but not requirements for the farrowing process itself, resulting in the lowest estimated energy requirements at the day of farrowing [59]. Other recent evaluations of amino acid and energy requirements also did not include parturition requirements [84]. Feyera et al. [81] were the first to give an estimate of energy requirements of farrowing, which was based on an evaluation of different feed amounts and therefore daily energy intake around farrowing, aiming for the shortest farrowing duration and lowest number of interventions during farrowing. The estimated energy requirement for farrowing was 16 MJ ME (approximately 30% of the total energy requirements on the day of farrowing). In Figure 4, the calculated energy requirements of sows provided by Theil et al. and Feyera et al. are combined for the last day of gestation, the day of farrowing, and day 1 and 2 of lactation. These estimates included energy requirements for maintenance purposes, heat loss due to reproduction costs and diet induced thermogenesis [85,86], colostrum/milk production, fetal growth, mammary growth, growth of uterine tissue, nest-building behavior, and energy requirements for farrowing.In addition to the study by Feyera et al. [81], Che et al. [87] evaluated effects of energy intake on the day of farrowing on farrowing duration. Strategies of how the energy intake was increased differed between these two studies. Che et al. [87] increased energy level of the diet by increasing fat levels (soybean oil) and increasing daily feed supply with 0.2 kg/sow/day (from day 90 of gestation until farrowing), while Feyera et al. [76] increased feeding levels (from 1.8 to 5.0 kg/sow/day from day 108 of gestation until 24 h after farrowing). Both studies also differed in average litter size and average farrowing duration. Because farrowing duration increases with increasing litter size (Figure 2), it can be assumed that energy requirements for farrowing also increase with litter size. For estimating the energy requirements per piglet born or per 60 min of farrowing, it was assumed that the energy requirements were met when farrowing duration was shortest. For the treatments with the shortest farrowing duration, number of piglets born and average farrowing durations are shown in Table 1. Calculations on average energy requirement per piglet and per 60 min for both studies turned out to be quite close. Calculations suggest that optimal daily energy intake on the day of farrowing depends on the average litter (2.44 MJ ME/piglet born) and/or farrowing duration (8.66 MJ ME/hour of farrowing duration). Since only two studies were available, the findings should be confirmed in additional experiments. 5.2. Glucose as a Source of Energy during FarrowingATP (adenosine triphosphate), derived primarily from glucose by glucogenesis, is the main energy source for uterine contractions [88]. Blood glucose levels rise during farrowing, which can be explained by the increased glucolysis under the influence of adrenalin and cortisol [19,89]. Sow blood glucose levels originate from carbohydrates in the diet and/or glycogen reserves. A negative correlation was found between sow arterial glucose level, measured 1 h after the birth of the first piglet and farrowing duration, suggesting that a low energy status of the sow indeed increased farrowing duration. Feyera et al. [17] showed that farrowing duration linearly increased with time when the last meal was more than 3.13 ± 0.34 h before the onset of farrowing (defined as the birth of the first piglet). Theil et al. [37] showed that sows lack glucogenic energy on the day of farrowing and start using the glycerol part of triglicerides as an energy source (rather than nonesterified fatty acids in a normal catabolic state), making triglycerides and glucose the only two energy sources for the uterus during farrowing [37,90]. Although duration of farrowing has been related to the energy status of the sow, other dietary factors (e.g., type of energy, mineral levels, other supplements) might play a role as well. These factors will be discussed below.5.3. Other CarbohydratesThe role of dietary fibers in sow nutrition around farrowing are mostly studied in relation to the prevention of constipation and therefore easy passage of piglets through the birth canal [18,58]. Effects of dietary fibers on the duration of farrowing have been reviewed extensively [25] and will thus not be discussed here. No information is available on possible effects of dietary fibers on uterine contractions. However, dietary fibers can be a source of energy from the gastrointestinal tract up to several hours after a meal [91]. The type of carbohydrates consumed appears to be an important factor for exercise performance in athletes [92]. The glycemic index (GI) of carbohydrates is a tool to predict blood glucose, insulin, and therefore energy supply of diets [93] High GI foods (e.g., sugars and starches) provide a high and relatively short peak in blood glucose. Low GI feed ingredients (e.g., pectins) could provide lower but longer levels of blood glucose. Combining different types of carbohydrates, providing fast-, medium-, and slow-release glucose, might increase the period after feeding in which sufficient glucose is available to supply the energy needed for the farrowing process. It can be concluded that a better understanding of perinatal energy requirements (and the composition of these energy sources) is needed to optimize the farrowing process and consequently reduce piglet losses.5.4. Calcium and MagnesiumCalcium is an essential mineral for muscle contractions [94,95] and therefore also essential for myometrial contractions during farrowing (Figure 3). Studies evaluating calcium requirements in the peri-partum period are limited. Geisenhauser et al. [96] evaluated effects of a single-dose calcium supplementation on top of feed (400 mmol Ca, source calcium lactate) on the day of farrowing and found a significant reduction (−34% on sow level) in the incidence of dystocia (defined as birth interval > 60 min) and decreased time for placenta expulsion (4–19 min faster). Le Cozler et al. [19] evaluated plasma calcium levels in gilts before, during, and after farrowing and observed no change in plasma calcium levels during parturition (measuring 2 h before the birth of the first piglet to 7 h after). This is in contrast to a recent study of Nielsen et al. [65], who also evaluated plasma calcium profiles around parturition (measuring 33 h before the birth of the first piglet until 24 h after farrowing) and found a drop in calcium levels 9 and 3 h before the expulsion of the first piglet, but no changes in sow blood calcium levels during the first 24 h post-farrowing. These ambiguous results in perinatal plasma calcium profiles might be related with differences in litter size (12.2 vs. 24.6 total born for Le Cozler et al. [97] and Nielsen et al. [65], respectively) and/or farrowing duration (175 vs. 486 min for Le Cozler et al. [97] and Nielsen et al. [65], respectively), suggesting that hyper-prolific sows might have higher calcium requirements in the perinatal period. The benefits of calcium supplementation to sows before farrowing on incidence of stillbirth and piglet vitality remain unclear but might be related to the plane of feeding in this period and the calcium source and concentration in the diet. Magnesium promotes the relaxation of smooth muscle cells and inhibits contractions of the uterine myometrium. Magnesium sulphate is used in human medicine to prevent pre-term labor and pre-term birth [98], which suggests that magnesium supplementation before farrowing might have a negative effect on myometrial contractions. However, Le Cozler et al. [19] observed a drop in magnesium levels in sow blood 1 h after the first piglet was born, which was likely due to the role of magnesium in dephosphorylation of ATP to provide energy for muscle contractions, as ATP must be bound to a magnesium ion to be biologically active. The synergistic and antagonistic role of magnesium with calcium might explain why calcium levels were constant and magnesium levels dropped during parturition [97]. In pig husbandry, magnesium is used in sow diets as an effective laxative to prevent constipation [99]. However, as with calcium, research on magnesium supplementation for sows in the perinatal period is limited. Plush et al. [99] showed an increase in stillbirth incidence (+0.3 stillborn piglets/litter, p = 0.01) when sows were supplemented with magnesium sulphate (2.85 kg/mton feed, receiving 2.5 kg of feed/sow/day) from 5 days pre-farrowing until 3 days post-farrowing. It can be speculated that magnesium induced relaxation of the myometrium, which consequently increased the duration of farrowing, but this was not evaluated.Vitamin D is essential for intestinal calcium absorption, plays a central role in calcium homeostasis, and directly impacts muscle contractions [100,101]. Vitamin D is usually added to sow diets in the form of cholecalciferol (vitamin D3), which is transported to the liver and hydroxylated to 25-hydroxycholecalciferol [25(OH)D3], or by directly feeding the 25(OH)D3 [102]. Although requirements for sows are known for gestation and lactation [103], vitamin D requirements specifically during parturition are not. Some studies evaluated maternal vitamin D supplementation on offspring status in muscle fibers and therefore lean development and growth performance in later life [104,105,106,107]. However, we found no studies that evaluated maternal vitamin D supplementation and the effects on parturition characteristics and piglet losses. It can be concluded that although both calcium and magnesium play an important role in myometrial contractions, research on supplementing sows with one or both of these minerals in the perinatal period is very limited. Consequently, calcium and magnesium requirements of the sow in the perinatal period and potential factors influencing these requirements (e.g., litter size) are currently unknown.5.5. Vasoactive ComponentsDietary arginine (as recently reviewed by [108,109]) and nitrate supplementation [61,110] to the sow both aim to influence placental vascularization and/or placental–fetal blood flow. Although converted differently, both arginine (oxidized in a reaction catalyzed by the NO synthase family [111]) and nitrate (non-enzymatically converted via the NO3-NO2-NO pathway [112]) are precursors for nitric oxide (NO). NO is an endothelium-derived relaxing factor, causing vascular vasodilation [113,114], which plays an important role in regulating placental–fetal blood flow and consequently nutrient and oxygen transfer from mother to fetuses [37,115]. This higher blood, and therefore nutrient and oxygen, flow may lead to an increased piglet birth weight and/or oxygenation during parturition, which is hypothesized to lead to a lower incidence of stillbirth, increased vitality, and therefore a decreased incidence of pre-weaning mortality. Arginine is mostly supplemented in the first stage of gestation to increase placental angiogenesis [116], with several studies showing a beneficial effect on embryo survival, fetal development, placental weight, piglet weight, and number born alive (as reviewed by [108,117]). Fewer studies have used arginine supplementation up to or close to the moment of parturition. Neither placental weight (when supplementing 1% of L-arginine from day 22 until day 114 of gestation [118]) nor piglet birth weight and stillbirth rate (when supplementing 25.5 g/d from day 77 of gestation until term [119]) were affected in these studies. Van den Bosch et al. [61,110] evaluated effects of dietary nitrate supplementation starting 7 days before farrowing and found a linear dosage effect on piglet vitality (by scoring individual piglet vitality [72]) and piglet birth weights and a tendency for a lower pre-weaning mortality rate, which may have been driven by an increased placenta size and/or vasodilation. The use of NO precursors to enhance either placental vascularization and/or blood flow may benefit piglet vitality and survival.6. ConclusionsParturition is not only a stressful, painful, and energy-demanding event for sows, but it also affects the perinatal survival of her offspring. Along with increases in litter size, farrowing duration has increased and uterine blood flow per piglet, placental development, and piglet weight have decreased, which has increased the challenges to the perinatal piglet. Potential maternal nutritional factors that stimulate uterine contractions and/or increase uterine blood flow (by providing adequate energy and/or minerals or by the use of NO precursors) may reduce the duration of parturition and/or increase perinatal piglet survival. However, knowledge on the exact nutritional requirements before, during, and after parturition and the impact that meeting these requirements may have on piglet characteristics and perinatal survival is limited. Current feeding strategies in the perinatal period might not support the modern hyper-prolific sow adequately in energy and other nutritional requirements.

animals : an open access journal from mdpi

10.3390/ani13050891

PMC10000034

Hepatitis E virus (HEV) is a zoonotic pathogen, with an increasing number of cases worldwide. In Asia, including Mongolia, infections are associated with the zoonotic HEV-3 and HEV-4 genotypes, and pigs, deer, and wild boars are the main reservoirs. Recent studies have revealed that sheep are hosts of the virus in several countries. The aim of our study is to diagnose HEV RNA in feces and liver samples of sheep in Mongolia and clarify the origin of the virus and characterize its chain of infection. From our results, we found HEV genotype 4 in sheep and it was closely related to pig HEV genotype 4 in the same region. On Mongolian pig farms, pigs are fed with the raw internal organs of sheep for fattening the pigs as a free resource of protein. There is a concern that the spread of HEV could affect livestock feeding.

Hepatitis E is a viral infectious disease in pigs, wild boars, cows, deer, rabbits, camels, and humans as hosts caused by Paslahepevirus. Recently, it has been detected in a wide variety of animals including domestic small ruminants. Mongolia is a land of nomadic people living with livestock such as sheep, goats, and cattle. Due to how Mongolian lifestyles have changed, pork has become popular and swine diseases have emerged. Among them, Hepatitis E disease has become a zoonotic infectious disease that needs to be addressed. The HEV problem in pigs is that infected pigs excrete the virus without showing clinical symptoms and it spreads into the environment. We attempted to detect HEV RNA in sheep which had been raised in Mongolia for a long time, and those animals living together with pigs in the same region currently. We also conducted a longitudinal analysis of HEV infection in pigs in the same area and found that they were infected with HEV of the same genotype and cluster. In this study, we examined 400 feces and 120 livers (pigs and sheep) by RT-PCR in Töv Province, Mongolia. HEV detection in fecal samples was 2% (4/200) in sheep and 15% (30/200) in pigs. The results of ORF2 sequence analysis of the HEV RT-PCR-positive pigs and sheep confirmed genotype 4 in both animals. The results suggest that HEV infection is widespread in both pigs and sheep and that urgent measures to prevent infection are needed. This case study points to the changing nature of infectious diseases associated with livestock farming. It will be necessary to reconsider livestock husbandry and public health issues based on these cases.

1. IntroductionThe hepatitis E virus (family Hepeviridae; subfamily Orthohepevirinae; genus Paslahepevirus) is one of the main viral causes of acute human hepatitis worldwide [1]. Out of eight different genotypes, HEV-3 and HEV-4 are confirmed as zoonotic. Suidae are generally recognized as the main reservoirs of these genotypes. HEV-4 has been identified in China in goats [2], cows [3], cow’s milk [4], and sheep [5]. The disease is endemic in Asia, Africa, and Latin America [6]. The presence of anti-HEV antibodies in sheep and goats has been reported worldwide [7,8]. High similarity between human and ruminant HEV sequences has also been confirmed [9,10]. This raises concerns about the zoonotic transmission of HEV from these animal species [10,11]. In connection with this, it has been suggested that the consumption of contaminated milk, meat, and/or dairy products from sheep could be a source of HEV infection in humans [12,13]. However, information about the relationship between other livestock and sheep is not well understood. Mongolia is a country of animal husbandry with 30 million sheep [14]. It also has the third-largest population of sheep in the world [15]. Sheep meat is considered a staple food for Mongolians. Due to recent dietary changes, pig farming is being promoted in Mongolia. Most of the pig farms are located in Töv Province. Currently, ham, bacon, liver paste, and sausages are common foods for Mongolians that are prepared from pork at pig farms in Töv Province. This situation must be considered an important public health issue for food safety. There have been several studies on the molecular characterization of HEV in both pigs and human patients in Mongolia [16,17,18,19,20]. In a report on Mongolians, HEV prevalence was 12%, which is higher than in Russia (1.2%) and Japan (3%), despite lower pork consumption in Mongolia compared to the other countries. Although there have been reports of HEV infection in sheep from China, there is no information regarding HEV transmission from sheep in Mongolia. The purpose of this study was to determine the risk of HEV infection in native sheep associated with the introduction of pigs into livestock in Mongolia.2. Materials and Methods2.1. SamplingsThis was a cross-sectional study conducted in Töv Province and Ulaanbaatar city (Figure 1) between 2020 and 2022. Considering a 95% confidence interval (95% CI) and the desired precision of ±5%, the sample size was calculated as 384. We sampled 400 feces (200 sheep feces and 200 pig feces), and 120 livers (60 sheep livers and 60 pig livers). Sheep fecal and liver samples were equally collected in the Bayanchandmani, Batsumber, Undurshireet, and Zaamar soums (districts) in Töv Province. The liver and fecal samples were transported to the laboratory on ice. The Bayanchandmani and Batsumber soums are close to Emeelt slaughterhouse, which is the main slaughterhouse in the central region of Mongolia. The study area for the sheep was set near Ulaanbaatar city, where pig farming is most prevalent in Mongolia. Sheep and free-roaming livestock were raised in the same region as the pig farms. The fecal samples were collected from 200 pigs and the liver samples were collected from 60 pigs slaughtered in Ulaanbaatar city. The ORF2 region of HEV RNA was a target sequence for viral detection [21]. In addition, the farmers were asked questions that included general knowledge about HEVs and possible contact between livestock to analyze risk factors.2.2. RNA Extraction and Detection of HEV RNA by RT-PCRFecal samples from sheep and pigs were suspended 10% (w.p.v.) in sterile PBS. The fecal and liver samples were homogenized with zirconium beads in a tissue lyser (QIAGEN, Hilden, Germany). Next, the homogenized samples were centrifuged at 8000 rpm for 5 min, and the supernatants were collected in sterile tubes. RNA from pig livers, experimentally infected with HEV, was used as a positive control, and non-HEV-infected healthy pigs were used as a negative control [22]. Viral RNA was extracted from fecal supernatants using a QIAamp Viral RNA extraction kit (QIAGEN, Hilden, Germany) according to the kit’s protocol, and 50 mg from each liver tissue was lysed with 1 mL Trizol (Invitrogen, Carlsbad, CA, USA). RNA was precipitated with 0.5 mL isopropanol and washed with 1 mL of 75% ethanol. The RNA was solubilized in 20 µL RNase-free water [21]. A one-step RT-PCR kit (QIAGEN, Hilden, Germany) was used to amplify the ORF2 region of the HEV (primer sequences used: ORF2-F1; (5772-5794) 5′-AATTATGCYCAGTAYCGRGTTG-3′, ORF2-R1; (6416-6439) 5′-CCCTTRTCYTGCTGMGCATTCTC-3′). The reaction conditions were reverse transcription at 45 °C for 60 min and 95 °C for 15 min as a PCR cycle, denaturation at 94 °C for 30 s, annealing at 55 °C for 30 s, extension at 72 °C for 75 s for 35 cycles, and final extension at 72 °C for 7 min [21]. Nested PCR: the RT-PCR products were then diluted and amplified again using Takara Ex-Taq (Takara Bio. Inc., Shiga, Japan) to check the PCR amplification of the F1-R1 region (primer sequences used [23]: ORF2-F2 (5953-5975) 5′-GTWATGCTYTGCATWCATGGCT-3′, ORF2-R2 (6341-6363) 5′-AGCCGACGAAATCAATTCTGTC-3′) under the following conditions: 95 °C for 2 min (1 cycle); 94 °C for 30 s, 55 °C for 30 s, 72 °C for 30 s (35 cycles); and 72 °C for 5 min (1 cycle). The nested PCR products were electrophoresed on a 1.5% agarose gel, and amplified RNA bands were detected using a transilluminator (Toyobo-FAS-III, Toyobo Co., Ltd., Osaka, Japan) followed by ethidium bromide staining. The expected amplified HEV RNA band (348 bp) was sequenced after purification [21].2.3. DNA SequencingThe PCR products from each DNA sample were purified using a FastGene Gel/PCR Extraction Kit (NIPPON Genetics Co., Ltd., Tokyo, Japan) and sequenced using an ABI Prism Big Dye Terminator v3.1 Cycle Sequencing Kit (Applied Biosciences, Foster City, CA, USA). The sequences were analyzed using a 3500 Genetic Analyzer (Life Technologies, Carlsbad, CA, USA) [21]. 2.4. Phylogenetic AnalysisThe ORF2 gene sequences were then compared with those in the NCBI GenBank database using the multiple alignment function of the online ClustalW tool. Phylogenetic trees were constructed using the neighbor-joining method in MEGA (version X). A total of 28 sequences were submitted to GenBank through an online submission system and accession numbers were obtained and compared with previously reported sequences [24]. The same data were utilized to generate an ML phylogenetic tree, which was initially used to conduct the Bayesian maximum clade credibility (MCC) host a discrete traits tree by using the software BEAST v1.8.4 (http://tree.bio.ed.ac.uk/software/beast/, accessed on 12 June 2022). The strict clock and the best fit GTR + G + I nucleotide substitution model with a constant population size coalescent tree prior were used. The MCMC was run at 50,000,000 generations and sampled at every 5000 generations. The effective sample size (ESSs) of the analysis was checked by using the software Tracer v1.6 (http://tree.bio.ed.ac.uk/software/tracer/, accessed on 12 June 2022). The MCC host discrete traits output tree was generated by using TreeAnnotator v1.10.4 (http://tree.bio.ed.ac.uk/software/beast/, accessed on 12 June 2022) afterburn 10% of the first trees. The host phylogenetic tree was reconstructed by using the software FigTree v.1.4.3 (http://tree.bio.ed.ac.uk/, accessed on 12 June 2022) [21]. 2.5. Anti-HEV Antibody Detection from Sheep and PigsSerum samples from sheep (n = 42) and pigs (n = 45) collected in the study area were investigated for antibodies to HEV. In-house ELISA was used in this study as previously described [21]. Anti-HEV antibodies were detected by enzyme-linked immunosorbent assay (ELISA) using VLPs derived from HEV genotype 3 as antigens. For the detection of antigen-bound IgG, an anti-pig IgG antibody-HRP conjugate and an anti-sheep IgG antibody-HRP conjugate (Bethyl Laboratories Inc., Montgomery, TX, USA) were applied as a secondary antibody according to the method described in a previous report [21]. The serum samples were diluted 1:100 with PBS containing 0.05% Tween 20, and 10% of Block Ace (DS Pharma Promo Co., Ltd., Osaka, Japan) and incubated for 1 h at room temperature. After the secondary antibody reactions, 50 μL of TMB (3,3′,5,5′-tetramethylbenzidine) (Kirkegaard & Perry Laboratories Inc., Baltimore, MD, USA) was added, and after 10-min incubation at room temperature, 50 μL of 2 M sulfuric acid was added to stop the reactions. The optical density (OD) value at 450 nm was measured by a microplate spectrophotometer (Thermo Fisher Scientific Inc., Waltham, MA, USA). Positive control sera were obtained from pigs experimentally infected with HEV, and negative sera were obtained from non-infected, healthy pigs. The cut-off value was set at 0.295 by calculating the average value + 2 standard deviation of the negative samples (n = 5). The sensitivity and specificity of this ELISA test in 20 experimentally infected and 24 negative pigs were 90.0% (95% CI: 68.30–98.77) and 91.67% (95% CI: 73.0–98.97), respectively.2.6. Statistical AnalysisThe age of the pigs was categorized as piglet less than 6 months; older than 6 months; over 1 year old. All epidemiological data were triple-entered and cleaned in Microsoft Office Excel 2010 (Microsoft Co., Redmond, WA, USA). We performed univariable and multivariable analysis using IBM SPSS Statistics 26 (IBM, Chicago, IL, USA) and Fisher’s exact test calculation [25]. Categorical variables were compared using Fisher’s exact test and χ2 test. A statistically significant difference was considered when the p-values were <0.05. The adjusted odds ratio (OR) and the 95% confidence interval (CI) for HEV risk factors were analyzed using univariate and multivariable logistic regression. 3. Results3.1. Prevalence of HEV RNA in Sheep in Töv Province, MongoliaAll of the fecal and liver samples were examined for the HEV ORF2 region using nested RT-PCR. The HEV ORF2 region was detected in sheep liver and fecal samples (Table 1). A total of 3/60 (5%) positive sheep livers were found near farms, which is in Bayanchandmani soum, Töv Province. Three RT-PCR-positive sheep liver samples were sequenced, and these sequences belonged to genotype 4 (accession numbers LC752752, LC752753, and LC702420). The sequence was highly homologous (98%) to those detected from pig samples (PF121, PF101, and PF175, which is located in the Songino Khairkhan district of Ulaanbaatar city; accession numbers LC413554, LC413553, and LC413557) (Table 1). The HEV prevalence in the feces samples was 2% (4/200) in sheep. 3.2. Prevalence of HEV RNA among Pigs in Töv Province, MongoliaPig feces (PF) and pig livers (PL) were collected from a farm (sample numbers: MGL-PF 1-200, MGL-PL 1-60). A total of 200 pig feces samples and 60 pig liver samples were examined for the HEV ORF2 region using nested RT-PCR, and the HEV ORF2 regions were detected in fecal samples (30/200, 15%) and liver samples (4/60, 6.6%) using nested RT-PCR (Table 2). 3.3. Genetic Analysis of the HEV from Pigs and Sheep The nucleotide sequence analysis of the HEV ORF2 region detected in pigs identified genotypes 3 and 4. The pig-derived HEVs belonging to genotype 3 were divided into two clusters. The viruses belonging to genotype 4 were divided into two clusters, one of which showed high homology to the sheep sequence (Supplementary Tables). The accession numbers of the HEV-3 type were LC413551, LC413555, LC413556, LC413558, and LC413573. The accession numbers of the HEV-4 type were LC413550, LC413552, LC413553, LC413554, LC413557, and LC413574. The HEV detection rate for those older than 6 months was 12.3%, with 75% of the positive samples belonging to genotype 3 and 25% belonging to genotype 4. Pigs over 1 year of age were slaughtered and RT-PCR for HEV in the liver was performed. The results showed that 6.6% (4/60) of the samples were positive and that the positive samples belonged to genotypes 3 and 4, with two animals in each (accession numbers LC413570, LC413569, LC413550, and LC413574) (Table 2, Supplementary Tables). Phylogenetic analysis showed that 19 out of the 25 sequences belonged to genotype 3 (Figure 2 and Figure 3).The sequence analysis of the ORF2 region of three of the HEV-detected sheep belonged to genotype 4 (accession numbers: LC702420, LC752752, and LC752753). The sequence was identical to the genotypes (LC413553, LC413554, and LC413557) detected in pigs from the same region. Their amino sequences were also 99% identical to a previously reported sequence (ABO93596). Homology comparison of the sequences of HEV genotype 4 identified in Mongolian sheep and China (KU904269) showed a homology of 86%. Therefore, the HEV genotype 4 identified in Mongolian sheep was different from the HEV genotype 4 in China.The Bayesian MCC hosts a discrete traits tree for 302 bp HEV ORF2 nucleotide sequences (nt position 6022–6324) of a total of 40 Mongolian strains, including 28 sequences from our study and reference strains (six swine HEV-ORF2 sequences, genotype 3 [16], four human HEV-ORF2 sequences [19], and two Bactrian camel HEV-ORF2 (BcHEV1, BcHEV2) sequences [26]) obtained from the GenBank database (Figure 3). The phylogenetic host tree indicated transmission between HEV hosts, the host at the node indicated the ancestor of the sub-group, and the number at the node indicated the posterior probability. Genetic analysis of HEV determined in Mongolian people suggested that it was derived from genotype 4 of HEV in pigs and sheep in that country. In addition, six swine HEV-ORF2 sequences, three sheep HEV-ORF2 from our study, and four human HEV-ORF2 sequences from a previous study [19] belong to genotype 4 of HEV. The other 25 pig HEV-ORF2 sequences belong to genotype 3, including 19 sequences from our study and 6 sequences from F. Lorenzo’s study [16].3.4. Anti-HEV Antibody Detection from Sheep and PigsSerum samples from sheep (n = 42) and pigs (n = 45) collected in the study area were investigated for anti-HEV IgG. HEV-3 genotype VLP protein was used as the ELISA antigen. The HEV seropositive animals were 11.9% (5/42) of the sheep and 35.5% (16/45) of the pigs. 3.5. Statistical ResultsUnivariable analysis was performed (Supplementary Table S3). Categorical variables were compared using Fisher’s exact test and χ2 test. Age and foreign introduction of pigs are statistically significant (p < 0.05), while gender and feeding source is not statistically significant. A risk assessment was performed for each variable (Table 3). The odds ratio between the age ranges was calculated when the age of 1 year old is the reference, and the highest was a less than 6 months old piglet, OR = 3.42. The odds ratio between the feeding source was calculated when mill offal is the reference; the highest OR equals 2.6 in the small ruminant offal. The odds ratio between whether a foreign introduction is introduced or not is the reference, the highest OR equals 3.06 in the foreign introduction. These factors led to the higher risk for HEV cases in Mongolia. These three risk factors were statistically significant (p < 0.02–0.003, 95% CI). Gender was not statistically significant.4. DiscussionHEV has been detected in pigs and other animals all over the world including in Asia [6]. The HEV genotypes predominantly detected in Asia are genotypes 3 and 4, which are known to cause zoonoses. In this study, we used molecular methods to demonstrate the prevalence of HEV in Mongolian pigs and sheep. HEV genotype 3 has been detected in Mongolia previously [16]; however, the sequences of the viruses obtained in this study were different from those previously reported. Moreover, our results have shown the efficient detection of HEV RNA in feces. Virus shedding is an important factor in virus transmission among pigs on farms [27]. Thus, these results imply that sanitary control on pig farms is an important issue for healthy livestock management in the country. Since meat is the staple diet of the Mongolian people, there is concern that meat contaminated with HEV could cause adverse situations, especially for pregnant women and other hepatitis virus carriers. The prevalence of HEV in the swine liver requires caution for handlers at slaughterhouses and in meat processing factories. The phylogenetic tree analysis showed that genotypes 3 and 4 were detected in pigs. The HEV gene sequence detected in pigs in Mongolia is highly homologous to previously reported cases, which may be due to the establishment of the virus based on the importation of live pigs to Mongolia. The history of live piglet imports from China suggests that Mongolian HEVs originate from two different sources. HEV genotypes 3 and 4 are similar to Asian isolates, including those from Japan and China [28]. One HEV-4 genotype cluster was also identified in sheep-derived HEVs, suggesting that HEVs can be transmitted between pigs and sheep. From the results of the phylogenetic analysis, 19 sequences belonged to cluster HEV-3 and six sequences belonged to cluster HEV-4. The HEV strain may be transmitted between farms via human and pig movement. Pigs are usually fed on the by-products of slaughterhouse-derived sheep’s internal organs. The pigs had no restrictions on their movement. Phylogenetic analysis of the ORF2 region revealed that the HEV genotypes in the infected pigs in the vicinity of Ulaanbaatar city are homologous to those in sheep. This could be because sheep co-graze near pig farms in the same area. In comparison with HEVs from sheep detected in Töv Province, phylogenetic analysis showed genetic similarity with PF121, PF101, and PF175 from pigs. The geographical distance between Bayanchandmani soum (the sheep sample area) and Songino Khairkhan district intersects with livestock movement and feed distribution. Therefore, it is quite possible that HEV of livestock origin between the same regions could be transmitted via various routes. Mongolian sheep and goats usually co-graze in pasturelands together with other livestock, including cattle. We assume that sheep are not HEV reservoirs but are spillover infected animals. Sheep are most likely spillover infected animals due to HEV infection from pigs. The maximum clade credibility tree analysis in this study suggests that sheep HEV is of porcine origin. Chinese and Italian researchers have detected HEV in fecal and milk samples from goats and sheep [2,9,29]. HEV isolates have been detected in the southern and eastern parts of China, including Yunnan Province, Tai’an region, and Shandong Province [2,10,30]. With regard to HEV detection efficiency and the age of the tested pigs, the detection rate was low in subjects aged approximately 1 year old but high in younger populations (less than 6 months old) [27]. Fecal shedding of the virus is frequently detected in young pigs under 6 months of age and this age is a risk factor for environmental shedding of the virus. On the other hand, since the virus is detected in the liver even at the age of over 6 months, there is a risk of HEV infection in the livers of pigs shipped to the market. The main concern is that the virus shed from infected pigs is transmitted to other pigs. Each year, 10–20% of pigs are replaced by foreign introductions, mainly from China. Fattened pigs are slaughtered for meat, and piglets are fattened on farms. The statistical analysis indicated that the foreign introduction of pigs was a strong factor and that another possible cause of infection could be the feeding source. Except for piglets, all of the pigs were fed with mill offal as a fattening food. According to a cross-sectional study, there was a statistically significant difference in terms of the use of small ruminant offal and mill offal. Free-range pigs can contaminate the water or hay resources of sheep that graze near pig farms. Gender was not found to be associated with the risk estimation analysis. Improvements are needed to reduce the opportunities for contact with other livestock in the future. In our study, HEV-antibody-positive sheep were detected (positivity at about 11.9% (5/42) in sheep and 35.5% (16/45) in pigs), and previous reports of HEV infection epidemiology around the world also suggest that ruminant livestock is at risk of HEV infection [7,31,32,33,34,35,36]. If there is the contamination of HEV from the internal organs of livestock, the possible transmission risk of the virus in Mongolia will be high. Mongolian people’s main source of food is meat. HEV-contaminated food can lead to bad situations, especially in pregnant women and other hepatitis virus carriers. HEV has been reported to have been detected in sheep from neighboring China. Anti-HEV antibodies were detected in 35.2% of sera and 57.7% of slaughter meat samples from Xinjiang, which borders the Mongolian west border. All of the isolates belonged to genotype 4 [5]. Moreover, various types of livestock are mixed-reared in China, and it has been pointed out that HEVs are transmitted between livestock [10]. To date, sheep infected with HEV-4 have been reported from China as follows: KU904269 [30] sheep HEV. In our study, the MCC analysis suggested that the pig and sheep HEV detected in this study could be transmitted to humans, presenting a public health issue. A detailed investigation is required for virus transmission among livestock, and investigation and management of HEV-infected animals are also important for public health. The risk verification of the use of pig feed from Mongolian livestock would be a further research point from the perspective of zoonotic diseases. 5. ConclusionsThe present results suggest that the spread of HEV in Mongolian livestock is a transmission risk owing to the management of introductions among pigs. Furthermore, HEV of the same genotype was shared among pigs and sheep reared in the same area. As sheep offal is fed to pigs, livestock husbandry management to interrupt HEV transmission and prevent its spread appears necessary. The virus was also confirmed in the livers of slaughtered pigs and sheep, suggesting the need for a call to attention with regard to food hygiene. These data show that measures are needed to improve prevention and control strategies for food safety.

animals : an open access journal from mdpi

10.3390/ani13071181

PMC10093658

The molecular mechanisms of skin pigmentation in Andrias davidianus are not clear. In this study, we identified two albino individuals and found that Andrias davidianus had distinct regulatory mechanisms of skin pigmentation that differed from other vertebrates. The key signaling pathway and transcription factors related to melanin synthesis in other vertebrates did not play a significant role in Andrias davidianus. MITF mRNA in Andrias davidianus had a unique splicing form that was not reported in other vertebrates and a unique mutation existed in the SLC24A5 gene in albino Andrias davidianus. The results contributed to understanding the molecular mechanism of skin pigmentation in Andrias davidianus and accelerating the acquisition process of Andrias davidianus species with specific body colors by genetic means, which will help to enrich the aquaculture market of Andrias davidianus.

The Chinese giant salamander (Andrias davidianus) has been increasingly popular in the aquaculture market in China in recent years. In the breeding process of Andrias davidianus, we found that some albino individuals were extremely rare and could not be inherited stably, which severely limits their commercialization in the aquaculture market. In this study, we performed transcriptome and small RNA (sRNA) sequencing analyses in the skin samples of wild-type (WT) and albino (AL) Andrias davidianus. In total, among 5517 differentially expressed genes (DEGs), 2911 DEGs were down-regulated in AL, including almost all the key genes involved in melanin formation. A total of 25 miRNAs were differentially expressed in AL compared to WT, of which 17 were up-regulated. Through the integrated analysis, no intersection was found between the target genes of the differentially expressed miRNAs and the key genes for melanin formation. Gene Ontology (GO) and KEGG pathway analyses on DEGs showed that these genes involved multiple processes relevant to melanin synthesis and the key signal pathway MAPK. Interestingly, the transcription factors SOX10 and PAX3 and the Wnt signaling pathway that play a key role in other species were not included, while the other two transcription factors in the SOX family, SOX21 and SOX7, were included. After analyzing the key genes for melanin formation, it was interesting to note an alternative splicing form of the MITF in WT and a critical mutation of the SLC24A5 gene in AL, which might be the main reason for the skin color change of Andrias davidianus. The results contributed to understanding the molecular mechanism of skin pigmentation in Andrias davidianus and accelerating the acquisition process of individuals with specific body colors by genetic means.

1. IntroductionThe Chinese giant salamander (Andrias davidianus), which currently represents the largest amphibian worldwide, is of great scientific, ecological, and economic importance [1]. With the gradual sophistication of artificial propagation and breeding technology and the increasing enlargement of the breeding scale, the artificial breeding of Andrias davidianus in China has gradually emerged as a new variety with an extremely promising market. The Andrias davidianus mainly has brown or black skin, with very few individuals showing other colors. However, body color serves as an important economic indicator in aquaculture, and its diversity will certainly lead to significant economic benefits. We identified several albino individuals (complete or incomplete) during the breeding of Andrias davidianus, and giant salamanders with this special body color are particularly popular among consumers in the market as an ornamental animal. However, such albino individuals are quite scarce, and their body color cannot be stably inherited, which significantly limits their commercialization process in the aquaculture market.The animal’s body color is an important phenotypic feature that is primarily determined by various pigments synthesized by chromatophores or pigment cells [2]. Up to now, six types of pigment cells have been reported in vertebrates that determine the animal’s body color by regulating its cell number and the production and release of pigments [3,4,5]. The skin color of Andrias davidianus (black or brown) is mainly determined by melanocytes. The synthesis of melanin mainly involves the tyrosine metabolic pathway, of which MITF is considered to be the master regulator that is able to phosphorylate the key enzymes involved in melanin synthesis, including tyrosinase (TYR), tyrosinase-related protein 1 (TYRP1), dopachrome tautomerase (DCT), and SILV (Silver Locus Protein Homolog), to activate their transcription [6,7]. The regulation of melanin synthesis is principally achieved through MITF. Transcription factors such as paired box 3 (PAX3), sex-determining region Y box 10 (SOX10), and lymphoid enhancer binding factor 1 (LEF1) can bind to the MITF upstream promoter to affect the formation of melanin by mediating MITF expression [8,9,10]. MAPK and Wnt signaling pathways are two major pathways involved in melanin formation that can affect melanin synthesis by regulating the activity or expression of MITF [11,12]. Other genes such as OCA2, SLC24A5, and SLC45A2-encoded ion-exchange proteins can influence melanin formation by regulating the concentrations of positive ions such as Na+, K+, and H+ in melanosomes [13,14,15]. Additionally, several noncoding RNAs, such as miR-206, miR-429, miR-137, and miR-330-5p, have been demonstrated to regulate MITF or to directly regulate key genes in the tyrosine metabolic pathway [16,17,18,19]. Conclusively, the melanin synthesis pathways have been extensively studied, and the results have revealed that these signaling pathways seem to be conserved in vertebrates [7,20,21]. However, the regulation of body color in Andrias davidianus has not yet been reported, and whether the regulation of body color in Andrias davidianus is specific or the same as in humans, mice, or other reported vertebrates remains unknown.In the present study, through combined transcriptome and sRNA sequencing analyses, we found that the vast majority of key genes involved in melanin synthesis were differentially expressed between wild-type and albino Andrias davidianus with two different body colors, which is similar to the findings in other vertebrates. However, specificity was also identified in the regulation of body color in Andrias davidianus, mainly manifested as follows: (1) there were few differentially expressed miRNAs between the two types of Andrias davidianus with different body colors and almost no intersection between the target genes of those differentially expressed miRNAs and the key regulators of melanin synthesis, suggesting that miRNAs do not play a critical role in the body color regulation in Andrias davidianus; (2) by analyzing MAPK and Wnt signaling pathways, which are key pathways in other vertebrates, we found that the Wnt signaling pathway seems to have no function in body color regulation in Andrias davidianus; (3) PAX3 and SOX10, two key transcription factors for melanin synthesis, were not differentially expressed between the wild-type and albino Andrias davidianus, but we identified two other differentially expressed transcription factors of the SOX family (SOX7 and SOX21), showing consistency in their expression and regulation patterns (transcriptional activation or repression); (4) we identified a novel alternatively spliced form of MITF in wild-type Andrias davidianus that is not present in other vertebrates; (5) a critical mutation on the SLC24A5 gene identified in albino Andrias davidianus, which has not been reported in other animals, could directly affect pigmentation. With these findings in mind, as one of the few studies exploring the regulation of body color in Andrias davidianus, we investigated the molecular mechanisms underlying the regulation of body color in Andrias davidianus and, in particular, found that the regulation of body color in Andrias davidianus differed from those in other vertebrates, which will certainly advance the application of molecular biology in the propagation and breeding of Andrias davidianus with specific body color and the ability to stably inherit to enrich the aquaculture market.2. Materials and Methods2.1. Experimental AnimalsWild-type and albino Andrias davidianus (two individuals, respectively) were purchased from a Andrias davidianus breeding base in Luoyang, Henan Province. All animal experiments were conducted in accordance with the guidelines and approval of the Animal Care and Welfare Committee of Luoyang Normal University (Approval code is 0020080A).2.2. mRNA and sRNA SequencingThe degradation and contamination of RNA, particularly DNA contamination, were monitored using 1.5% agarose gels. The NanoDrop 2000 Spectrophotometer (Thermo Fisher Scientific, Wilmington, DE, USA) was employed to determine the concentration and purity of RNA. The integrity of RNA was evaluated using the RNA Nano 6000 Assay Kit on the Agilent Bioanalyzer 2100 System (Agilent Technologies, Santa Clara, CA, USA).For mRNA sequencing, a total amount of 1.5 μg of RNA per sample was used as input material for rRNA removal using the Ribo-Zero rRNA Removal Kit (Epicentre, Madison, WI, USA). The NEBNextR UltraTM Directional RNA Library Prep Kit for IlluminaR (NEB, Ipswich, MA, USA) was utilized according to the manufacturer’s instructions to produce sequencing libraries, with index codes added to assign sequences to individual samples. The protocol involved fragmenting the RNA with divalent cations at high temperature in the NEBNext First-Strand Synthesis Reaction Buffer (5×), followed by synthesis of the first-strand cDNA using random hexamer primers and Reverse Transcriptase. Subsequently, DNA Polymerase I and RNase H were used for second-strand cDNA synthesis, and exonuclease/polymerase activities were employed to convert any remaining overhangs into blunt ends. Adenylation of the 3′ ends of DNA fragments was performed, and NEBNext Adaptors with hairpin loop structures were ligated in preparation for hybridization. To ensure the selection of insert fragments ranging from 150 to 200 bp in length, the library fragments underwent purification using AMPure XP Beads from Beckman Coulter, located in Beverly, MA, USA. The cDNA that had been size-selected and adaptor-ligated was treated with 3 μL of USER Enzyme (NEB, Ipswich, MA, USA) at 37 °C for 15 min prior to PCR. The PCR was carried out using Phusion High-Fidelity DNA polymerase, along with Universal PCR primers and Index Primer. Lastly, PCR products were purified (AMPure XP system) and library quality was assessed on the Agilent Bioanalyzer 2100 and qPCR. To conduct sRNA sequencing, 2.5 ng of RNA was utilized as input material for RNA sample preparations. The NEBNextR UltraTM small RNA Sample Library Prep Kit for IlluminaR (NEB, Ipswich, MA, USA) was employed to generate sequencing libraries, following the manufacturer’s guidelines. Index codes were assigned to associate sequences with each sample. The process involved ligating the 3′ SR Adaptor as the first step. To prepare for ligation of the 3′ SR Adaptor for Illumina sequencing, a mixture of the adaptor, RNA, and nuclease-free water was incubated at 70 °C for 2 min in a preheated thermal cycler. The tube containing the mixture was transferred to ice, and 3′ Ligation Reaction Buffer (2×) and 3′ Ligation Enzyme Mix were added to facilitate the ligation of the 3′ SR Adaptor. The reaction was incubated at 25 °C for 1 h in a thermal cycler. In order to prevent the formation of adaptor-dimers, the excess of 3′ SR Adaptor (that remains free after the 3′ ligation reaction) was hybridized with the SR RT Primer, which transformed the single-stranded DNA adaptor into a double-stranded DNA molecule. Ligation-mediated processes do not support dsDNAs as substrates. The second step involves ligating the 5′ SR Adaptor, followed by the synthesis of the first chain through reverse transcription. Subsequently, PCR amplification and size selection were carried out. For fragment screening purposes, PAGE gel electrophoresis was performed, and small RNA libraries were created by recycling rubber-cutting pieces. Finally, the PCR products were purified using the AMPure XP system, and the quality of the library was evaluated on the Agilent Bioanalyzer 2100 system.2.3. De Novo Assembly and Sequence AnnotationThe clean reads were assembled using the Trinity software (https://sourceforge.net/projects/trinityrnaseq/files/ (accessed on 1 July 2021), trinityrnaseq-2.1.1). As there is no reference genome, in order to identify genes with low expression or genes with only partial fragments that are measured, the combined assembly of all samples of the same species can make the assembly results more comprehensive and facilitate subsequent analyses. The transcript reads were first broken into kmer (kmer is a continuous DNA sequence; assuming a read length L and kmer size K, then the Kmer number for each read is L − K + 1). The high-frequency kmer is then used as the seed to build a contig based on overlap relationships and cluster contigs of alternative spliced or other parallel genes. A separate de Bruijn graph was constructed for each contigs set and then the reads were aligned back to remove the sequencing error path and use the dynamic programming scoring algorithm to obtain the final transcripts, while selecting the longest transcripts in each locus as UniGene for subsequent analysis.2.4. Analysis of Differentially Expressed GenesThe alignment results were calculated with eXpress software (https://pachterlab.github.io/eXpress/, express-1.5.1-linux_x86_64 (accessed on 10 July 2021)) to obtain the read count number of each sample to each UniGene, convert them to FPKM (Fragments Per Kilobase Million), and then analyze the expression level of genes. Differential expression analysis was performed based on the UniGene expression abundance values of 2 samples, and the main analysis software was DESeq2 (https://bioconductor.org/packages/release/bioc/html/DESeq2.html (accessed on 20 July 2021), version 1.14.0). Genes with |logFoldChange| ≥1 and adjusted p-value (padj) ≤ 0.05 were considered as the differentially expressed genes for further analysis.2.5. Functional Annotation, Pathway Enrichment and Network ConstructionTo annotate gene function, the following databases were employed: Nr (NCBI non-redundant protein sequence database), Nt (NCBI nucleotide sequences database), SWISS-PROT (a non-redundant protein sequence database that is manually curated), GO (Gene Ontology database), COG (Clusters of Orthologous Groups of proteins database), KOG (Clusters of Protein homology database), and KEGG (Kyoto Encyclopedia of Genes and Genomes database).The clusterProfiler R package was employed to carry out Gene Ontology (GO) Enrichment Analysis of differentially expressed genes (DEGs), applying hypergeometric testing to identify significantly enriched GO entries in comparison to the entire genome background. The database KEGG (http://www.genome.jp/kegg/ (accessed on 21 July 2021)) was used for pathway analysis. The clusterProfiler R packages were utilized to identify KEGG pathways that were significantly enriched relative to the whole genome background. The DEG sequences were blasted (blastx) against the genome of a related species (which had protein–protein interaction data available on the STRING database, http://string-db.org/) to obtain the predicted protein–protein interaction (PPI) of the DEGs. Finally, Cytoscape was utilized to display the PPI network of these DEGs.2.6. Reverse Transcription-Quantitative Polymerase Chain Reaction (RT-qPCR)To verify the mRNA expression levels of selected genes, a quantitative RT-PCR (qRT-PCR) test was conducted. Reverse transcription was carried out in a 20 µL reaction mixture, which included 2 µL of total RNA, 1 µL of 5 µM RT primer, 1 µL of 10 mM dNTP, 4 µL of 5X PrimeScript Buffer, and 200 units of PrimeScript RTase (TaKaRa, Kusatsu, Shiga, Japan). The reaction was incubated at 42 °C for one hour and terminated by heating at 85 °C for 5 min. qPCR was performed with SYBR Green PCR Master Mix (TaKaRa, Kusatsu, Shiga, Japan) and all reactions were performed in triplicate. GAPDH was employed as the internal reference control.2.7. Dendrogram Construction of MITFThe dendrogram of the relationships among the MITF of Andrias davidianus and other vertebrates were generated by SHOOT.bio (https://www.shoot.bio/, accessed on 5 October 2022). All the sequences were downloaded, and the sequence similarity was then calculated using the MEGA (https://www.megasoftware.net/, version 11) program to generate a branching pattern.2.8. Protein Model ConstructionA bHLH-Zip model of the MITF protein was constructed using SWISS-MODEL (online program accessed at https://swissmodel.expasy.org/, accessed on 1 September 2022)3. Results3.1. Overview of mRNA and miRNA Sequencing DataTo investigate the molecular mechanism of skin color variation in Andrias davidianus, we performed mRNA and small RNA sequencing of skin tissues from the wild-type (WT) and albino (AL) Andrias davidianus (Figure 1). The analysis of the mRNA sequencing data showed that 24,946,613 and 24,281,191 total sequences were obtained from two biological replicates in the AL group, and 25,071,096 and 24,314,935 total sequences were obtained from the WT group. After combined assembly of all samples of each group, 239,881 and 209,055 transcripts with N50 values of 1269 and 1257 were obtained in the AL and WT groups, respectively (Table 1). The average GC content was 48% and47%, respectively, and the percentages of Q30 bases were more than 94% for all the samples, suggesting high sequencing quality. Four databases including the SWISS-PROT database, Nr database, Nt database, and GO and KEGG database were used for gene prediction. The results showed that 29,407, 41,840, 21,058, 23,157 and 17,989 transcripts had significant hits against SWISS-PROT, Nr, Nt, GO, and KEGG, respectively (Table 1). Cumulatively, a total of 46,221 unique genes were predicted, allowing at least one significant hit against at least one of the three databases (Table 1).Four small RNA libraries were constructed by deep sequencing. As shown in Table 2, 11.46 million (M) and 11.43 M total reads were obtained in the AL group. Subsequently to removing the low-quality and adaptor sequences, a total of 11.35 and 11.16 M clean reads were ultimately obtained. All identical sequence reads were then classified as groups, and 1.75 and 1.67 M unique reads were obtained. In these unique reads, 560,498 and 475,608 reads were annotated, respectively. In the WT group, 11.15 M and 11.05 M total reads were obtained, corresponding to 10.76 M and 10.52 M clean reads, and 1.08 and 1.06 M unique reads, respectively. In these unique reads, 481,482 and 447,731 reads were annotated, respectively. The novel and known miRNAs from each group are shown in Table 2 and Supplementary File S1.3.2. Enrichment and Pathway Analyses of Differentially Expressed Genes (DEGs)Through analyzing all unique genes, 5517 unique genes were found to be differentially expressed between the wild-type and albino Andrias davidianus with |logFC| ≥1 and padj value ≤0.05 set as the criteria (Figure 2A). Among these DEGs, 2606 genes were down-regulated and 2911 genes were up-regulated in the wild-type Andrias davidianus compared with those in albino Andrias davidianus (Figure 2A and Supplementary File S2). Gene set enrichment analysis and pathway analysis were performed on all DEGs to analyze the functional and regulation differences underlying the phenotypic variations. According to the GO terms, the DEGs were classified into three major functional categories, including 4743 DEGs in biological process (BP), 2046 DEGs in cellular component (CC), and 1861 DEGs in molecular function (MF) categories (Supplementary File S3). The majority of the GO terms related to pigmentation and melanogenesis including pigment cell differentiation, melanin biosynthetic and metabolic processes, developmental pigmentation, melanocyte differentiation, melanosome and pigment granules, and melanosome membranes were significantly enriched and encompassed most of the key genes regulating pigmentation (Table 3). However, among these genes, we did not detect the presence of transcription factors PAX3 and SOX10, which may affect pigmentation by regulating MITF in other vertebrates (these two transcription factors were poorly expressed in the skin of two types of Andrias davidianus with different body colors and there was no statistical difference between the two). Intriguingly, we found two SOX family transcription factors, SOX21 and SOX7, in the DEGs (Supplementary File S4). As a transcription activator, SOX7 was highly expressed in wild-type Andrias davidianus (2.46-fold change), while the transcription inhibitor SOX21 was less expressed in wild-type Andrias davidianus (2.93-fold change), which was consistent with the high expression pattern of pigment regulatory genes in wild-type Andrias davidianus.In the KEGG pathway analysis, the DEGs were involved in 279 pathways and the top 25 enriched pathways are shown in Figure 2B. In these significantly enriched pathways, we found two melanin-associated signaling pathways, tyrosine metabolism and the MAPK signaling pathway (Figure 2B, Table 4 and Supplementary File S3). However, the Wnt signaling pathway, another important melanin-related pathway in other vertebrates, was not identified, and the majority of the Wnt-signaling-pathway-related genes were unchanged in Andrias davidianus with different body colors (Table 4). It was suggested that the Wnt signaling pathway might not exert the same critical role in Andrias davidianus as it does in other vertebrates. Interestingly, we found a spliceosome in the significantly enriched pathways (Figure 2B), suggesting a key role of gene splicing in regulating pigmentation in Andrias davidianus. We indeed found a novel splicing form of the MITF gene in wild-type Andrias davidianus that is absent in other animals (see Section 3.4). This unique splicing pattern might directly affect the skin pigmentation in Andrias davidianus. A regulatory network was constructed using the DEGs related to pigmentation in Andrias davidianus, in which all of the genes are divided into three parts: MAPK signaling pathway-related genes, melanin synthesis and metabolism-related genes, and transcription factors (Figure 2C). MITF is the ‘hub gene’ that plays a critical role in pigmentation of Andrias davidianus. To confirm the reliability of the RNA-seq, 10 DEGs were chosen for validation by RT-qPCR. The expression levels of all 10 DEGs determined by RT-qPCR were concordant with their mRNA sequencing data (Figure 2D), indicating a strong association between mRNA profiling and the RT-qPCR data.3.3. Integrated Analysis of Differentially Expressed miRNAs and mRNAsTo identify miRNAs that may be involved in melanin synthesis, we comprehensively analyzed the differentially expressed miRNAs and mRNAs. A total of 25 differentially expressed miRNAs were identified based on the criteria of |logFC| ≥ 1 and padj value ≤ 0.05 (Figure 3). Among these miRNAs, 17 downregulated and 8 upregulated miRNAs were found in the wild-type Andrias davidianus compared to the albino Andrias davidianus (Figure 3). We then obtained 5000 putative target genes of these differentially expressed miRNAs (Supplementary File S5). As previously mentioned, there were 2606 downregulated genes and 2911 upregulated genes in the wild-type Andrias davidianus relative to the albino Andrias davidianus. Then, we intersected the target genes of 17 downregulated miRNAs with 2911 upregulated genes, yielding 99 intersection genes (Supplementary File S5). Next, the target genes of 8 upregulated miRNAs were intersected with 2606 downregulated genes, from which 55 intersection genes were acquired (Supplementary File S5). Subsequent analysis of these intersection genes showed the absence of the key genes that could regulate melanin synthesis yet the presence of only a few MAPK signal pathway-related genes. These findings suggest that miRNAs do not play a key role in the regulation of body color in Andrias davidianus.3.4. Splicing of MITF and Mutation of SLC24A5As mentioned above, we found the spliceosome to be the significantly enriched pathway (Figure 2B). To screen the genes affected by DEGs related to the spliceosome, we scanned the sequences of all mRNAs that might be involved in the regulation of pigmentation in Andrias davidianus. The results showed a unique splicing form of the MITF gene in the wild-type Andrias davidianus, which has not been reported in other animals (Figure 4, Figure 5 and Figure 6). More precisely, through the sequence alignment of MITF among 328 different animals (Supplementary File S6), we identified deletion in the 3rd and 5th exons of MITF in Andrias davidianus (either wild-type or albino, Figure 4). More importantly, compared with the albino Andrias davidianus, a form of MITF mRNA in the wild-type Andrias davidianus was characterized as the insertion of 90 nucleotides between the 7th and 8th exons (30 extra amino acid residues of the protein) (Figure 4 and Figure 5A). The phylogenetic tree depicted the distance between the MITF in Andrias davidianus and their orthologs from vertebrates, which indicated that the MITF in Andrias davidianus belonged to a member of the MIiF-TEF family and belonged to the same branch of the evolutionary tree as the MITF protein of Xenopus laevis (Figure 5B). Interestingly, the MITF in Andrias davidianus was more closely related to that in terrestrial animals than in aquatic animals (Figure 5B). By analyzing this uniquely spliced form of the MITF protein in wild-type Andrias davidianus, we found 30 extra amino acid residues located in the loop of the basic/helix-loop-helix/leucine zipper (bHLH-Zip) structure of the MITF protein (Figure 5A). bHLH-Zip transcription factors usually function as dimers. A bHLH-Zip model of the MITF protein was constructed using SWISS-MODEL (Figure 5C), which revealed a longer loop structure for the wild-type MITF protein that might allow this protein to form a dimer with another helix through more flexible folding and packaging, or the DNA-binding domain of the dimer to have a more flexible and open spatial structure, thereby exerting regulatory effects on its downstream target genes and affecting the pigmentation process in Andrias davidianus.After scanning all mRNA sequences that might be involved in the regulation of pigmentation in Andrias davidianus, we identified an interesting mutation in SLC24A5 in the skin of albino Andrias davidianus (Figure 7). The mutation was presented in a cysteine-rich region (CCTCC) on the intracellular loop of the transmembrane protein NCKX5 encoded by SLC24A5, named the 4C region (Figure 7). Similar cysteine-rich regions also exist in other animals (Figure 7). This mutation was manifested as a loss of three amino acid residues of CCT in the 4C region in the albino Andrias davidianus (Figure 7). Cysteine residues play a crucial role in many proteins, particularly in enzyme reactions and intermolecular/intramolecular interactions [22]. In Xenopus laevis, the 4C mutant in NCKX5 could not rescue the reduction in pigmentation caused by NCKX5 knockdown [23], indicating a critical role of 4C in the functionality of NCKX5. The mutation in the 4C region of the NCKX5 protein in Andrias davidianus might be another major cause of skin albinism in addition to the alternative splicing of MITF.4. Discussion4.1. Key Genes Regulating Pigmentation in Andrias davidianusMelanin is an amino-acid-derived biological pigment synthesized from melanocytes and is a polyphenolic polymer that can be mainly classified into two types: brown/black eumelanin and red/yellow pheomelanin [24]. Existing studies have shown that the pigmentation of melanin consists of four stages: melanosome formation, melanosome maturation, melanin synthesis, and final transfer and deposition of melanosomes containing a great deal of melanin in the skin keratinocytes, through which the skin color is presented [25]. Therefore, melanin pigmentation in the skin is closely related to melanosome formation, melanin synthesis, melanosome transportation, and the transcription activation of related genes at various stages. In this study, we conducted GO analysis on the DEGs between wild-type and albino Andrias davidianus and found that these DEGs involved multiple processes related to pigmentation, including pigment cell differentiation, melanin biosynthetic and metabolic processes, developmental pigmentation, melanocyte differentiation, melanosome and pigment granules, and melanosome membranes. After detailed analysis, these DEGs were demonstrated to participate in almost all of the processes of melanin pigmentation consisting of four stages (Figure 8). (1) Melanosome formation. Melanosome is a specialized membrane-bound organelle with striatal structures formed by amyloid fibers in melanocytes [26]. A pigment-cell-specific protein, the premelanosome protein (PMEL)—also known as SILV—plays a key role in the formation of melanosomes [27,28]. SILV is one of the molecules essential for the formation of melanosome fibers, which can independently assemble and form the striatal structure of melanosomes and maintain the environmental balance in melanosomes [27]. Studies have illustrated that SILV gathers on the intraluminal vesicles (ILVs) of melanosomes and gradually forms amyloid fibers with the elongation of ILVs [28,29]. A large number of amyloid fibers are packed into sheets and eventually form oval melanosomes [29]. (2) Melanosome maturation. The ion-exchange proteins encoded by the OCA2, SLC24A5, and SLC45A2 genes play a key role in maintaining the internal environment stability of the melanosomes during melanosome maturation. They can regulate the concentration of positive ions such as Na+, K+, and H+ in melanosomes and jointly maintain the acid–base balance in the environment of melanosomes [13,30,31]. Their absence causes an abnormal morphology of melanosomes and amyloid fiber formation disorder, leading to a reduction in the melanin content [32]. In addition, RAB38 can control the transfer of melanin synthase (TYR, DCT, and TYRP1) to melanosomes, thus regulating the maturation of melanosomes [33,34]. (3) Melanin synthesis. The synthesis of melanin, especially the eumelanin, which determines the animal skin color (black or brown), is a continuous enzymatic reaction process. Melanin production is initiated from the TYR-catalyzed synthesis of dopa-quinone from L-tyrosine. Specifically, tyrosine is catalyzed by TYR and transformed into dopa (i.e., L-3,4-dihydroxyphenylalanine, L-DOPA), and L-DOPA is further oxidized to L-dopaquinone in the presence of TYR. In the absence of cysteine, L-dopaquinone undergoes cyclization to generate dopachrome, which in turn forms the intermediates 5,6-dihydroxyindole (DHI) and 5,6-dihydroxyindole-2-carboxylic acid (DHICA) through carboxylation and decarboxylation under the action of DCT. Then, DHI and DHICA form the two intermediates 5,6-indolequinone (IQ) and indole-5,6-quinone-2-carboxylic acid (IQCA) under the action of TYR and TYRP1. Finally, the heteropolymer formed through the binding of IQ to IQCA is regarded as eumelanin [4,35]. During this process, TYR, DCT, and TYRP1 are key rate-limiting enzymes and the expression of all three enzymes is regulated by the transcription factor MITF [32]. 4) Melanosome transportation. The transfer of melanosomes from melanocytes to keratinocytes is a key process to maintain skin pigmentation [36]. Rab GTPases play a role in the transfer of melanosomes, in which RAB11A knockout significantly affects the transfer of melanosomes [36,37]. In summary, the difference in skin color between wild-type and albino Andrias davidianus correlates with the gene expression changes during the whole formation to the transportation process of melanosomes. However, the differential expression of genes involved in the whole process does not seem to be regulated by miRNA. In fact, when we used these DEGs to construct a regulatory network, we found that MITF was a hub gene (Figure 2C), suggesting that MITF was one of the key genes for the skin color difference between two different types of Andrias davidianus.4.2. Signaling Pathway Regulating Pigmentation in Andrias davidianusStudies have illustrated that in most vertebrates, the Wnt/β-catenin and MAPK signaling pathways regulate pigmentation by modulating the transcription factor MITF [8,38]. In the MAPK signaling pathway, activated downstream ERK signaling molecules can inhibit MITF phosphorylation in an indirect or direct manner, thereby blocking the binding of MITF to downstream targets; moreover, the phosphorylation of MAPKs/ERK can increase the degradation of MITF, block the binding site (MITF M-box) on the tyrosinase promoter, and effectively inhibit the reduction in melanin synthesis caused by MITF transcription [38,39]. In the Wnt/β-catenin signaling pathway, accumulated β-catenin in melanocytes enters the nucleus and binds to LEF1, thereby acting on the promoter region of MITF and affecting its transcription level [8]. However, in this study, the analysis of the above two signaling-related genes found that the vast majority of the Wnt-signaling-related genes did not change in the skin of the wild-type and albino Andrias davidianus (Table 4), which suggests that the Wnt signaling pathway does not play a key role in the process of skin pigmentation in Andrias davidianus as it does in other vertebrates.4.3. SOX Family and Pigmentation RegulationThe SOX family includes approximately 20 transcription factors, which contain a high-mobility-group (HMG) domain that binds to downstream DNA sequences [40]. In mammals, members of the SOX family can be subclassified into nine groups, while the HMG domain is highly conservative in the SOX family, with a ≥90% identity in the amino acid sequences of SOX proteins in the same group but approximately a 60% identity in the amino acid sequences of SOX proteins among different groups [41]. In vitro experiments have demonstrated that the HMG domains of SRY, SOX5, SOX9, and SOX17 have the same core binding sequence (5′-AACAAT-3′) [42,43,44,45]. In melanocytes, MITF, DCT, and TYR are common target genes of SOX10 and SOX9 [46], while SOX5, another member of the SOX family, can competitively bind to SOX10 target genes such as MITF and DCT, thus restraining the melanin synthesis [47]. These results indicate that SOX family members have overlapping target genes, and the role of several SOX family members may be substantiated or suppressed by other members.Studies have unveiled that SOX10 plays a crucial role in melanin synthesis in the majority of vertebrates. Its role is manifested in two aspects: (1) SOX10 and PAX3 co-activate the expression of MITF [48], which affects the synthesis of melanin by regulating the expression of TYR, DCT, TYRP1, etc. [10,48,49]. (2) SOX10 can bind to the regulatory genes of melanin synthesis, such as TYR [50], DCT [51], and TYRP1 [52], synergistically with MITF to activate the expression of these genes [51], while MITF cannot induce their expression individually [50,53]. However, the role of SOX10 in melanin synthesis is also challenged. It is believed that once the expression of MITF is established in melanocytes, other members of the SOX family substitute for the role of SOX10 in later development [54]. It has been demonstrated that after SOX10 mutation in zebrafish, MITF can induce the expression of TYR independent of SOX10 and completely rescue pigmentation [50]. In this study, we found that the expression of SOX10 and its partner transcription factor PAX3 was very low in the skin of both the wild-type and albino Andrias davidianus (especially PAX3, which was almost not expressed) and exhibited no difference between the two different types of Andrias davidianus. In melanocytes, SOX10 and PAX3 form a complex to achieve the transcriptional regulation function, while SOX10 alone cannot activate the expression of downstream genes such as MITF [48], indicating that SOX10 and its partner transcription factor PAX3 have an extremely limited role in the pigmentation process of the skin of Andrias davidianus. Interestingly, we analyzed other members of the SOX family and found that the other two members, SOX7 and SOX21, were highly expressed in the skin of wild-type and albino Andrias davidianus and were differentially expressed between the two. SOX7, belonging to group F of the SOX family, usually contributes to the transcriptional activation of target genes, while SOX21, belonging to group B, acts as a transcriptional inhibitor [55]. The expression level of SOX7 was 2.46-fold upregulated in WT skin compared with AL, while the SOX21 expression in the AL skin was 2.93-fold upregulated compared with WT (Supplementary File S3), suggesting that SOX7 and SOX21 could act as transcriptional activators and inhibitors in the pigmentation of the skin of Andrias davidianus. Whether SOX7 acts as a substitute for SOX10 to activate the expression of MITF and genes related to melanin synthesis, such as TRY, DCT, and TYRP1, or whether SOX21 acts as an inhibitor to inhibit the expression of these genes remains to be further proved.4.4. MITF SplicingAs a transcriptional activator, MITF can regulate the expression of multiple genes related to pigmentation, including melanosome assembly and melanin synthesis [32]. MITF consists of at least four isomers, MITF-A, MITF-C, MITF-H, and MITF-M [56,57], which show differences in the amino-terminal but share the entire carboxyl portion encoded by exons 2 to 9 of the MITF gene [58]. Among them, MITF-M is only expressed in melanocytes and melanoma cells, which is a lineage-specific isoform in melanocytes [56,57]. There are two major isoforms of MITF-M with and without six amino acids (6a, ACIFPT) inserted in exon 6, named (+) MITF and (−) MITF, respectively [56,57,59]. In this study, we found that the MITF in Andrias davidianus showed great uniqueness compared with other vertebrates. By comparing the MITF protein sequences of Andrias davidianus and 328 other species, we found its uniqueness mainly presented in the following three aspects: (1) Exon 3 and exon 5 of MITF were deleted simultaneously in Andrias davidianus (Figure 4 and Supplementary File S6). Compared with common MITF protein sequences, the deletion of exon 3 also exists in amphibians such as Xenopus tropicalis, Protopterus annectens, Bufo bufo, Nanorana parkeri, and Rana temporaria (Figure 6A). However, exon 5 deletion was not presented, particularly the deletion of both exon 3 and exon 5 in Andrias davidianus. (2) MITF in most animals either contains 6a or does not contain 6a, while the sequence at 6a of MITF was altered to DATVYY in Andrias davidianus (Figure 6B). Sequence changes at 6a of MITF also exist in other animals such as Microcaecilia unicolor, Bufo gargarizans, Geotrypetes seraphini, and Amblyraja radiata, but these sequences have no consistency with each other (Figure 6B). (3) An insertion of 30 amino acids between exons 7 and 8 of MITF existed in the wild-type Andrias davidianus but not in albino Andrias davidianus (Figure 5A and Figure 6C). Insertions between exons 7 and 8 are rare in other animals, and the insertion sequences found only in Rana temporaria, Myotis lucifugus, Pogona vitticeps, and Apteryx rowi are completely different from those in the wild-type Andrias davidianus (Figure 6C). Therefore, it could be concluded that the splicing process of the MITF in Andrias davidianus is more complicated than those in other vertebrates. Interestingly, through the analysis of the DEGs in wild-type and albino Andrias davidianus, we found that these DEGs were enriched in the spliceosome pathway (Figure 2B). These spliceosome-related DEGs might be the main reason why MITF showed different splicing forms in two types of Andrias davidianus with different skin colors. As previously mentioned, the insertion of 30 amino acids changed the bHLH-Zip structure (Figure 5A,C), thereby affecting the expression of the MITF downstream genes, which might be one of the major reasons for the differential expression of key melanin-related genes (such as TYR, DCT, TYRP1) in the skin of wild-type and albino Andrias davidianus.4.5. SLC24A5 MutationThe SLC24A5 gene, the fifth member of the solute carrier family 24, encodes a potassium-ion-dependent cation exchange protein (NCKX5) located on the melanosome membrane. NCKX5 can transport calcium ions into the melanosome and pumps sodium ions out of the organelle, which plays a crucial role in maintaining the concentration gradient of hydrogen ions and calcium ions inside and outside the melanosome [60]. The formation of an ion gradient is involved in the synthesis of melanin in the melanosome [60]. The mutation of the SLC24A5 gene regulates the PH in melanocytes and affects the maturation and catalytic activity of tyrosinase, resulting in different color phenotypes [13]. Additionally, intracellular calcium can affect the production of melanin by activating the PMEL protein [61].The transmembrane protein NCKX5 contains a cytoplasmic loop, which divides the transmembrane structure of the NCKX5 protein into two transmembrane segments, TMS1 and TMS2 [62]. By comparing the cytoplasmic loop sequences of 470 different vertebrates (Supplementary File S7), we found a cysteine-rich region (4C or 3C) that was conserved in all animals (Figure 7). Cysteine residues play an important role in many proteins, especially in enzyme reactions and intermolecular/intramolecular interactions [22]. The 4C or 3C region in the cytoplasmic loop of NCKX5 is believed to participate in the transition metal binding and S-acylation, thus affecting the localization of the NCKX5 protein on the membrane [63,64]. It was also indicated in another study that the mutation of a specific cysteine resulted in a significant reduction in the expression of NCKX2, another member of the NCKX family [65]. It was found in Xenopus laevis that the 4C mutant in NCKX5 could not rescue the reduction in pigmentation triggered by NCKX5 knockdown, indicating that 4C exerted a very important role in the function of NCKX5 [23]. In this study, we revealed that the mutation in the 4C region (CCTCC) of the albino Andrias davidianus caused the deletion of three amino acid residues of CCT (Figure 7). This mutation might be another major cause of skin albinism in addition to the alternative splicing of MITF.5. ConclusionsIn this study, through mRNA and small RNA sequencing analyses of wild-type and albino Andrias davidianus, we identified the genes and signaling pathways involved in melanin synthesis, pigmentation, etc., and found that Andrias davidianus has distinct regulatory mechanisms that differed from other vertebrates as follows. (1) The difference in body color between the wild-type and albino Andrias davidianus did not seem to be significantly influenced by miRNAs. (2) The Wnt signaling pathway, the key signaling pathway related to melanin synthesis in other vertebrates, does not play a significant role in Andrias davidianus (Figure 7). (3) MITF acts as the core transcription factor that influences body color in Andrias davidianus; however, its regulator, SOX10, as well as PAX3—the partner transcription factor of SOX10—do not play a role in the pigmentation process in Andrias davidianus. The expression of two other members of the SOX family, SOX7 and SOX21, significantly differs between the two types of Andrias davidianus (Figure 7). (4) MITF in Andrias davidianus has a unique splicing form, and specifically, the insertion of 30 amino acids in the bHLH-Zip structure might directly affect the functionality of MITF. (5) A unique mutation exists in the SLC24A5 gene in Andrias davidianus, resulting in the deletion of the 4C region of the protein, thereby affecting the protein activity. The identification of the molecular mechanisms underlying the regulation of body color in Andrias davidianus will advance the utilization of molecular biology in the breeding of Andrias davidianus species with a specific body color that can be stably inherited to enrich the aquaculture market.

animals : an open access journal from mdpi

10.3390/ani12050605

PMC8909261

The objective of this study was to determine postmortem measurements for predicting carcass traits in growing rabbits. A total of 50 New Zealand White × Californian male rabbits were used. Data recorded at slaughtering included carcass and noncarcass components (viscera and offal). Our results indicated that the use of carcass measurements could accurately and precisely (r = ≥0.76 and ≤0.84) be used as alternatives to predict the carcass weights and carcass tissues in growing rabbits.

The objective of this study was to determine post-mortem measurements for predicting carcass traits in growing rabbits. A total of 50 clinically healthy New Zealand White × Californian male rabbits with a body weight (BW) of 1351 ± 347 g between 60 to 80 days of age were used. Body weight was recorded 12 h before slaughtering. Data recorded at slaughtering included carcass weights (HCW). After cooling at 4 °C for 24 h, carcasses were weighed (CCW) and then were carefully split longitudinally with a band saw to obtain left and right halves. In the right half carcass, the following measurements were recorded using a tape measure: dorsal length (DL), thoracic depth (TD), thigh length (TL), carcass length (CL), lumbar circumference (LC). The compactness index (CCI) was calculated as the CCW divided by the CL. Thereafter, the right half carcass was weighed and manually deboned to record weights of muscle (TCM), and bone (TCB). The CCI explained of 93% of variation for TCM (R2 = 0.93 and a CV = 9.30%). In addition, the DL was the best predictor (p < 0.001) for TCB (R2 = 0.60 and a CV = 18.9%). Our results indicated that the use of carcass measurements could accurately and precisely (R2 = ≥ 0.60 and ≤0.95) be used as alternatives to predict the carcass tissues composition in growing rabbits.

1. IntroductionIn Mexico, per capita consumption of rabbit meat varies between 30 and 134 g per person per year, and in recent years there has been a growing interest in the production of rabbits [1,2]. Rabbit production is a favourable activity for small and medium producers, due to the versatility of this species, the little investment required, and the possibility of generating income throughout the year [3]. However, rabbit production systems in Mexico have been hampered by certain factors, such as the lower economic importance of this species compared to livestock and poultry production and limited information on their management, reproduction, and productivity, including carcass yield and carcass characteristics [4].In Mexico, the main rabbit breeds for meat production are New Zealand and California [5]. From an economic point of view, in rabbit production, carcass yield is the most important trait [6]. The evaluation of the carcass allows them to be classified according to carcass weight and carcass tissue composition and distribution [5]. Therefore, in rabbit production, knowing carcass traits should be feasible and practical to assess [7]. This information could help to improve genetics more quickly without the need for lengthy progeny testing to determine the merit of carcass traits [8].In this sense, a professional’s measurements could be a means of describing body size and conformation, which are important traits in meat animals. On the other hand, it is important to know the relationship between certain carcass traits and carcass measurements in rabbits [7,9]. Knowledge of the carcass tissue composition and its distribution in rabbits is valuable information because one of the main challenges in the market of meat, along with ensuring food safety, is the commercialization of meat and meat products. For that, is necessary to generate information on carcass tissue composition to provide valuable information to improve the economic viability of rabbit production systems [2]. Until now, information on the use of post mortem measurements for predicting carcass traits of growing rabbits raised in tropical conditions is scarce. Therefore, the objective of the present study was to determine post mortem measurements for predicting carcass traits in growing rabbits.2. Materials and Methods2.1. Experimental Site and AnimalsAll animals were managed in compliance with the guidelines and regulations for ethical animal experimentation of the División Académica de Ciencias Agropecuarias, Universidad Juárez Autónoma de Tabasco (ID project PFI: UJAT-DACA-2015-IA-02). The climate (Am) of the region is tropical humid with rains in the summer, altitude is 9 m above sea level, with an average annual rainfall of 1958 mm, a relative humidity close to 75%, and an average annual temperature of 27 °C.In this study, 50 clinically healthy New Zealand White × Californian fattening male rabbits with body weight (BW) of 1351 ± 347 g and between 60 to 80 days of age were used. All animals were obtained from a commercial farm and were fed a standard commercial diet (17% crude protein, 11% crude fibre, 2% fat, and 11% ash). Feed and water were provided ad libitum. Rabbits were housed in individual raised-slatted floor cages (45 × 30 × 40 cm), having a photoperiod of 10 h and natural ventilation.2.2. Slaughter of Animals and Carcass MeasurementsFeed and water were withdrawn 12 h before slaughtering, and BW was recorded. Animals were slaughtered according to the Mexican Official Standard NOM-033-SAG/ZOO-2014 for the humane slaughtering of animals. After slaughtering, hot carcass weight (HCW) was recorded. After cooling at 4 °C for 24 h, carcasses were again weighed (CCW) and then were carefully split longitudinally with a band saw to obtain left and right halves. In the right half carcass, the following measurements were recorded: dorsal length (DL) was considered as the interval between the first cervical vertebra and the seventh lumbar vertebra; thoracic depth (TD) was considered between the fifth and seventh thoracic vertebra and longitudinally surrounding the ribs until ending at the sternum; the thigh length (TL) was the interval between the seventh lumbar vertebra and the distal part of the ischium; the carcass length (CL) was calculated as the sum of the dorsal length and thigh length data; the lumbar circumference (LC) was the circumference of the carcass at the level of the seventh lumbar vertebra. Measurements were performed using a tape measure [10]. With this information, the compactness index (CCI) was calculated as the CCW divided by the carcass length. Thereafter, the right half carcass was weighed and manually deboned to record weights of muscle (TCM), and bone (TCB) [11]. Dissected tissues of the right half carcass were adjusted as whole carcasses. Carcass management was carried out at the Meat and Meat Products Technology Laboratory from Universidad Juárez Autónoma de Tabasco.2.3. Data AnalysesFor the statistical analysis and internal validation of the model, the data were read in the Python environment as follows: descriptive statistics were obtained using the description function of the “pandas” package [12]. The relationship between carcass traits and carcass measurements was determined by linear regression equations using the “lmfit” package [13]. The models and their residuals were plotted with the “matplotlib” package [14]. The goodness-of-fit of the regression models was evaluated using the Akaike Information Criterion (AIC), the Bayesian Information Criterion (BIC), the coefficient of determination (R2), the mean square error (MSE), and the root of MSE (RMSE). The last three parameters were obtained using the “scikit-learn” package [15]. The predictive capacity of the three models was evaluated by cross-validating k-folds (k = 10). This approach was undertaken by randomly dividing the set of observation values into nonoverlapping k-folds of approximately the same size. The first fold is treated as a validation set, and the model fits the remaining k − 1 folds (training data). The ability of the fitted model to predict the actual observed value was evaluated using the mean square error of prediction (MSEP), the root mean square error of prediction (RMSEP), and the R2 mean absolute error (MAE). The MSEP was calculated as the squared distance between the predicted value and the true value. The RMSEP was calculated by summing all squared prediction errors during cross-validation and is an indicator of the reliability and predictive ability of the model. The lower the RMSEP value, the higher its predictive ability for the model.The MAE was calculated by taking the summation of the absolute difference between the actual and calculated values of each observation over the entire array and then dividing the sum obtained by the number of observations in the array. Lower values of root RMSEP and MAE indicate a better fit. The k-folds cross-validation was performed using the “scikit-learn” package [15], which allowed a comparison of numerous multivariate calibration models.3. ResultsThe descriptive statistics of body weight, carcass traits, and postmortem measurements are presented in Table 1. Body weight ranged from 718 to 2491 g, while HCW and CCW ranged from 297 to 1390 g and 280 to 1334 g, respectively. With regard to postmortem measurements, mean values of DL, TD, CL, TL, and LC were 26.55 ± 2.83, 9.71 ± 1.38, 31.78 ± 3.18, 5.27 ± 0.97, and 13.98 ± 2.02 cm, respectively. The mean value for ICC was 21.63 ± 5.15 g/cm.The correlation coefficients (r) between body weight, carcass traits and post mortem measurements are presented in Table 2. Except relationship between TCB and TL (p > 0.05), significant positive correlations were obtained between body weight and carcass traits with all post mortem measurements. The correlation coefficient (r) between the variables ranged from moderate to high (r = 0.47 to 0.97). The BW was highly correlated with CCI (r = 0.96), LC (r = 0.87), and TD (r = 0.86). On the other hand, both HCW and CCW had high correlations with CCI (r = 0.97 and r = 0.97, respectively) and LC (r = 0.91 and r = 0.90, respectively). Finally, TCM strongly correlated with CCI (r = 0.96) and LC (r = 0.90), while TCB showed moderate correlations with DL (r = 0.77) and CL (r = 0.75).Significant predictors, determination coefficient (R2), mean square error (MSE) and p-value for predicting the carcass tissue composition are shown in Table 3. For the prediction of TCM, the CCI was again the most significant variable (R2 = 0.93 and CV = 9.30%, p < 0.001), but the precision showed a slight increase at R2 = 0.95 when the CL was added to the prediction model. On the other hand, the most significant predictor variable for total TCB was DL with R2 = 0.60 and CV = 18.9%.The goodness-of-fit of the equations was calculated by the k-folds cross-validation technique is shown in Table 4. The proposed models showed adequate goodness-of-fit based on internal validation. The equations showed good performance according to the goodness-of-fit evaluation and internal validation (Table 4). With exception of the model for predicting TCB, all models had an R2 ≥ 0.93; however, the values of RMPE indicated a good performance of the fitted model for predicting carcass tissue composition. These models may be used both in experimental and commercial farms to predict carcass traits in growing rabbits (Figure 1).4. DiscussionIn general, mean values and ranges of variation for HCW and CCW were lower than those reported by Ortiz-Hernández and Rubio-Lozano [16] in different rabbit breeds. Such differences are due to the different mean values of rabbits’ body weights used in that study (~2000 g) versus this study (1329 g). Paci et al. [17] have previously reported that slaughter weight, age, and genotype are important factors that affect variability in the performance and composition of rabbit carcass. The observed carcass yield was consistent with that reported by Montes-Vergara et al. [6] in New Zealand White (NZ)-breed rabbits, however, in the present study animals were New Zealand White × Californian rabbits.In the study, the highest correlation was observed between hot carcass weight and cold carcass weight with the carcass compactness index. Previously, Venturini et al. [18] showed in lambs that the higher the cold carcass weight, the higher the observed carcass compactness index with a correlation coefficient of r = 0.95, consistent with the results of this study. The carcass compactness index was highly correlated with the total carcass muscle, while the dorsal length correlated with the total carcass bone. These relationships could be because the carcass compactness index is a strong indicator of carcass conformation, as it evaluates the amount of muscle tissue deposited in the carcass in a unit of length. This is important in economic terms since the market for meat has a preference for more compact carcasses and more muscle tissue [19]. Michalik et al. [7] reported in French Lop rabbits that the total meat weight was correlated with hips circumference (r = 0.69), thigh circumference (r = 0.68), and pelvis width (r = 0.66), while the total bone weight was correlated with the pelvis width (r = 0.73) and thigh length (r = 0.65).The high coefficients of determination of the regression equations obtained in the present study indicated that carcass compactness index could be used as the only variable to accurately predict body weight and the carcass traits. However, the precision could increase when considering the carcass length in the prediction models. Earlier, Blasco et al. [20] reported that total carcass muscle in California rabbits could be correctly predicted using only carcass weight or slaughter weight as a predictive variable, showing correlation coefficients of r = 0.88 and r = 0.84, respectively; therefore, it was not essential to add more predictor variables to the models. Later, Hernández et al. [10] showed that the composition of the carcass in rabbits can be clearly defined using the weight of the carcass, the meat/bone ratio of the hind leg, and the weight of the perirenal fat deposit. Recently, Michalik et al. [7] reported that the meat weight in the whole carcass in French Lop rabbits can be predicted (r = 98) using pelvic width, chest girth, thigh circumference, and carcass weight. These equations can be applied in selection works aimed at improving the meatiness of the carcass of the species.The cross-validation of equations obtained in the present study, indicated a good performance according to the goodness-of-fit evaluation based on cross-validation because the values of RMSEP indicated a good performance of the fitted model when predicting carcass traits.5. ConclusionsOur results indicated that the use of carcass measurements could accurately and precisely (R2 = ≥0.60 and ≤0.95) be used as alternatives to predict carcass weights and carcass tissues from growing rabbits. The equations showed good performance according to the goodness-of-fit evaluation based on cross-validation. With exception of the model for predicting TCB, all models had an R2 ≥ 0.93, in addition to the values of RMSEP indicating the good performance of the fitted model in predicting the carcass tissue composition.

animals : an open access journal from mdpi

10.3390/ani13071249

PMC10093597

In addition to the historical indicator of mammary gland health, milk somatic cell count (SCC), the differential SCC (DSCC) has been introduced to improve the accuracy of mastitis detection. No studies have yet explored DSCC variability in local breeds used for milk production such as Burlina and Alpine Grey. Although local cattle breeds show greater rusticity and resistance to disease compared to cosmopolitan specialized dairy breeds, udder health status needs to be monitored for reasons related to profitability, management improvement, and animal welfare. In the present study, we aimed at investigating the factors affecting SCC and DSCC in Italian local breeds. Finally, by combining both SCC and DSCC, we attempted to estimate the effect of the udder health status on milk yield and composition traits.

Milk differential somatic cell count (DSCC) represents the percentage of polymorphonuclear neutrophils and lymphocytes out of the total somatic cell count (SCC) and has been proposed in recent years as a proxy for udder health in dairy cows. We investigated phenotypic factors affecting SCC and DSCC using 3978 records of 212 Alpine Grey and 426 Burlina cows farmed in Northern Italy. The linear mixed model accounted for the fixed effects of breed, parity, lactation stage, sampling season, and first-order interactions of breed with the other effects. Cow, herd-test-date nested within breed were random. Subsequently, four udder health status groups (UHS) were created by combining SCC and DSCC to assess the UHS impact on milk yield and quality. DSCC was greater in Alpine Grey (66.2 ± 0.8%) than Burlina cows (63.2 ± 0.6%) and, similarly to SCC, it increased with days in milk and parity regardless of breed. Milk yield and composition were affected by UHS in both breeds. These results suggest that also udder health of local breeds can be monitored on a large scale through SCC and DSCC for reduction in biodiversity loss and increased farm profitability. However, in addition to milk data, the introduction of mastitis recording and monitoring plans is advisable.

1. IntroductionIn Italy, local cattle breeds are farmed for their contribution to a multifunctional and sustainable development and maintenance of confined areas. Representative examples of local breeds are the Burlina and the Alpine Grey, which are both well adapted to marginal environments where farming conditions can often be challenging [1,2]. Thanks to their good grazing aptitude, these dual-purpose breeds have been reared for both meat and milk production in extensive and semi-extensive systems. In this regard, the adaptability and the ability to transform poor pastures into valorized animal products make Burlina and Alpine Grey important genetic resources to preserve [1,2,3].Generally, local cattle breeds show greater rusticity and resistance to disease compared to cosmopolitan genotypes specialized for dairy like Holstein [4]. Nevertheless, milk quality and udder health status need to be monitored in local as in cosmopolitan breeds for reasons related to profitability, management improvement, and animal welfare. Although enormous efforts have been put at different levels to improve udder health, mastitis is still the most impacting disease in cows [5]. In addition to the milk somatic cell count (SCC, cells/mL), historical indicator of mammary gland health, the differential somatic cell count (DSCC, %) has been recently introduced. The DSCC has been proposed as a promising novel trait to improve mastitis detection accuracy to be used as a proxy for management purpose (e.g., selective dry-cow therapy) [6,7,8]. In bovine milk the epithelial exfoliated cells account for approximately 10% of total SCC, with polymorphonuclear neutrophils, macrophages, and lymphocytes being the main components of SCC [9]. The DSCC is the fraction of SCC that includes polymorphonuclear neutrophils and lymphocytes [6,7,8]. Considering that the composition of SCC changes in the presence of inflammation, monitoring traits like DSCC in parallel with SCC sounds meaningful. In fact, SCC and DSCC observed during inflammation may differ from those observed after the onset of inflammation. For example, polymorphonuclear neutrophils are the predominant cell type in the early stages of inflammation and they decrease in chronically affected quarters [10,11]. Cows with one or more quarters suffering from a chronic mastitis are generally defined as ‘chronic’: these are expected to have a dampened immune response, particularly in presence of certain pathogens [12]. In fact, while the immune response activation occurs normally in healthy animals, a tolerance status can be observed in presence of chronic infections, i.e., attenuation of the inflammatory response, reduction in the concentration of inflammation biomarkers in both plasma and milk, and less evident or non-evident signs [12].Few milk laboratories carrying out official milk analyses are equipped with devices able to record DSCC in Italy. Data are thereby routinely registered only in certain Italian regions. This explains why, to date, milk DSCC of Burlina and Alpine Grey has never been explored.In the era of precision livestock farming, however, farmers are expected to improve detection accuracy of quarters/cows with inflammation(s) to be monitored or treated [13], particularly in local endangered populations. The recent European limitations on the antimicrobial use, in addition, have changed the routine of dairy farmers, making the analysis of SCC trend within lactation indispensable for a smart and correct application of selective dry-cow therapy protocol.The DSCC phenotypic variability and relationship with SCC have never been investigated in Italian local breeds. Therefore, in the present study we investigated the effect of season, parity and stage of lactation on SCC and DSCC in Burlina and Alpine Grey. Subsequently, we evaluated if and how the udder health status identified with different combinations of SCC and DSCC can affect milk yield and composition.2. Materials and Methods2.1. Data EditingInformation on individual milk samples was retrieved from the official routine milk testing database of the Breeders Association of the Veneto Region (ARAV, Vicenza, Italy). Routine milk testing is carried out every 4–5 weeks and include registration of daily milk yield (MY, kg/d). The sampling period covered 24 months, from January 2019 to December 2020 and included all year-round calving herds. The infrared-predicted traits included, protein, casein, and lactose content (%), and urea (mg/dL) and beta-hydroxybutyrate concentration (BHB, mmol/L) determined using the Combifoss 7 DC (Foss, Hillerød, Denmark). SCC and DSCC were obtained by flow cytometry as described in [6].The casein index was calculated as the ratio between casein and protein. To achieve a normal distribution of the data, SCC was transformed to SCS through the formula of Ali and Shook [14]: SCS = 3 + log2(SCC/100,000), and score of DSCC, i.e., DSCS, was calculated using the same formula starting from DSCC expressed in cells/mL, which was obtained as: DSCC (cells/mL) = SCC (cells/mL) × DSCC (%). Finally, following Benedet et al. [15], the BHB was log10-transformed. According to Schwarz et al. [7], the good separation index (GOSE) of Combifoss 7 DC provides information about reliability of the measured SCC and DSCC. For this reason, 168 test-day records lacking good separation (GOSE = 0) were discarded. Moreover, only samples with SCC ≥ 10,000 and ≤ 5,000,000 cells/mL analyzed within 5 d from sampling were considered. This ensured the presence of highly reliable SCC and DSCC [6]. Records not belonging to cows between 5 and 305 days in milk (DIM) or which were of a parity greater than 9 were discarded. Similarly, cows with less than 3 test-day records within each lactation and herd-test-dates with less than 3 cows were also discarded. Values of MY and fat, protein, and lactose content deviating more than 3 standard deviations from the mean were considered as missing.The final dataset included 3978 test-day records from 212 Alpine Grey and 426 Burlina cows located in 20 and 16 herds, respectively. Following Schwarz et al. [7], test-day records were categorized based on SCC and DSCC into four udder health status (UHS) groups (Figure 1). Briefly, we considered cows with SCC ≤ 200,000 cells/mL and DSCC ≤ 65% as healthy (UHS1), and cows with SCC ≤ 200,000 cells/mL and DSCC > 65% suspicious of mastitis (UHS2). SCC > 200,000 cells/mL and DSCC > 65% were the inclusion criteria for the UHS3 group. Based on the most accredited interpretation, finally, SCC > 200,000 cells/mL and DSCC ≤ 65% (UHS4) identified animals with chronic intramammary infection(s).2.2. Statistical AnalysisData manipulation, editing, and analysis were carried out in R software v. 4.1.2 [16]. In the first step, the analysis of variance was performed for SCS, DSCC (%) and DSCS as dependent variables through the following mixed linear model: yijklmnop = µ + Bi + Sj + Pk + Dl + Tm + (B × S)ij + (B × P)ik + (B × D)il + Cn + Ho(Bi) + eijklmnop(1) where yijklmnop is the dependent variable; µ is the overall mean; Bi is the fixed effect of the ith breed (i = Alpine Grey and Burlina); Sj is the fixed effect of the jth season of sampling (j = 4 season: December to February, March to May, June to August, and September to November); Pk is the fixed effect of the kth parity (k = 1, 2, 3, 4, and ≥5, with the last containing data up to parity 9); Dl is the fixed effect of the lth stage of lactation (l = 6 classes of 50 d each); Tm is the day of analysis calculated as the difference between the sampling date and the milk analysis date (m = 0 to 5 days); (B × S)ij is the fixed interaction effect between breed and season of sampling; (B × P)ik is the fixed interaction effect between breed and parity; (B × D)il is the fixed interaction effect between breed and stage of lactation; Cn is the random effect of the nth cow (n = 691) ~ N(0, σ2C), where σ2C is the cow variance; Ho(Bi) is the random effect of the oth herd-test-date (o = 344) nested within breed ~N(0, σ2H(B)), where σ2H is the herd-test-date variance; and eijklmnop is the random error ~N(0, σ2e), where σ2e is the residual variance. Model diagnostics were checked through analysis of distribution, variance homogeneity, and independence of residuals.Subsequently, four combinations of SCC and DSCC were used to include UHS as independent variable in a further mixed linear model and estimate its impact on MY and major milk composition traits:yijklmnopq = µ + Bi + Sj + Pk + Dl + Um + Tn + (B × S)ij + (B × P)ik + (B × D)il + (B × U)im + Co + Hp(Bi) + eijklmnop(2) where all the effects, except for UHS group (Um), were the same included in Equation (1). The UHS group (Figure 1) was included as main effect and in interaction with breed. Multiple comparisons of least squares means were performed using the Bonferroni adjustment with significance at p ≤ 0.05, unless otherwise stated.3. Results3.1. SCS and DSCC VariabilityThe raw means of daily production and gross composition traits of the two breeds were similar, while the average SCS, DSCC, and DSCS were greater for the Alpine Grey than for the Burlina (Table 1). The coefficient of variation (CV), calculated as the ratio of the standard deviation to the mean, was 27% for urea in Alpine Grey and 30% in Burlina; BHB was more variable in the former (CV = 34%) than in the latter breed (CV = 28%). Negative values of BHB refer to samples with low concentrations before log-transformation.As regards Pearson’s correlations (Table 2), SCS was strongly positively correlated with both DSCC (0.66) and DSCS (0.99) and inversely with MY, lactose, and casein-index, similarly to DSCS. DSCC presented not significant coefficients with protein and casein and, in general, urea concentration was not significantly correlated with the udder health traits studied with. In both breeds most of the test-day records presented SCC < 200,000 cells/mL (Figure 1). 36 and 45% of the data of Alpine Grey and Burlina belonged to UHS1, and 31 and 26% to UHS2 (Figure 1). The udder health group with the lowest frequency (<4%) was UHS4 (Figure 1).Both least squares means of SCS and DSCS were similar in the two breeds (Table S1); however, the estimated DSCC was greater in Alpine Grey (66.2 ± 0.8%) than Burlina (63.2 ± 0.6%). Although lactation stage was significant for both SCS and DSCS (Figure 2), the interaction between lactation stage and breed was not (Table S1).The lowest SCS and DSCS were observed in early lactation, whereas the greatest at the end. The two breeds showed different patterns of DSCC (p < 0.05) throughout DIM (Figure 3), with Alpine Grey having the minimum in early lactation (62.5 ± 1.1%), a peak corresponding with the MY lactation peak (Figure 3; 66.2 ± 1.1%) and a subsequent drop between 106 and 155 DIM followed by a second increase and stabilization. On the contrary, least squares means of DSCC were similar across lactation stages in Burlina cows, with a mild peak in correspondence of the MY lactation peak (Figure 3).SCS, DSCC, and DSCS were significantly affected by parity and its interaction with breed (Table S1). SCS steadily increased with cow’s parity (Table 3) and the last parity class presented the greatest DSCS in both breeds (Table 3).Season of sampling—but not its interaction with the breed—affected the SCS, DSCC, and DSCS variability (Table S1, Figure 4). Least squares means were generally the greatest in summer (SCS = 3.34 ± 0.09; DSCS = 2.71 ± 0.10), except for DSCC whose seasonal trend looked different, being characterized by a single peak in summer (66.6 ± 0.6%) and a drop in autumn (63.3 ± 0.7%).3.2. Effect of Udder HealthIn both breeds, the UHS significantly affected daily MY and composition. The interaction between UHS and breed was in fact significant for protein and casein, BHB, and urea, whereas there was only a tendency for the casein-index (Table S2). Least squares means of Burlina MY (16.10 ± 0.24 kg/d) was greater compared to Alpine Grey (15.30 ± 0.32 kg/d) and MY differed according to the UHS group (Figure 5).The greatest daily MY was in fact observed in UHS1 (17.10 ± 0.21 kg/d), i.e., when SCC ≤ 200,000 cells/mL and DSCC ≤ 65%. This estimate, however, was similar to that of UHS2. Overall, cows presenting SCC > 200,000 cells/mL had a sharp drop in MY, in fact, the lowest production (13.40 ± 0.41 kg/d) was observed in the UHS4 group.UHS significantly affected part of the composition traits (Figure 5). The greatest lactose content was observed in groups with SCC ≤ 200,000 cell/mL, i.e., UHS1 (4.78 ± 0.01%) and UHS2 (4.77 ± 0.01%), while the lowest content belonged to UHS4 records (4.58 ± 0.02%). The lowest and the greatest fat content was observed in UHS2 (3.67 ± 0.03%) and UHS4 (3.98 ± 0.06%), respectively. The interaction between UHS and breed was significant for protein and casein content and casein index (Table 4).In both Alpine Grey and Burlina, UHS1 and UHS2 presented the lowest protein content, whereas the greatest was observed in UHS4 (Alpine Grey, 3.53 ± 0.04%; Burlina 3.60 ± 0.03%). When comparing the two extreme classes, UHS4 and UHS1, the relative reduction in protein was greater in Burlina than in the other (−0.21 and −0.12%, respectively). The same was observed for casein content of Burlina milk (Table 4). In Alpine Grey breed the numerically greatest estimate of casein was found in UHS3, which was not significantly different from UHS1 and UHS2. The lowest casein-index was observed for UHS4. The greatest casein-index estimate was calculated for groups with SCC < 200,000 cells/mL, i.e., UHS1 and UHS2.As regards the milk BHB (Table 4), indicator of negative energy balance, all breeds had the greatest estimates in UHS4, with the score estimated at −1.22 ± 0.06 and −1.16 ± 0.04 for Alpine Grey and Burlina, respectively. In Burlina cows, milk BHB of UHS3 was similar to that of UHS4. In general, the lowest BHB was observed for records with SCC ≤ 200,000 cell/mL, i.e., UHS1 and UHS2, and the relative increase in BHB was greater in Alpine Grey than Burlina. Finally, the effect of UHS on the urea concentration was different in the two breeds: in Alpine Grey it ranged from 23.40 to 24.00 mg/dL with no differences among UHS groups, whereas in Burlina the lowest estimate was found in UHS3 whereas the highest in UHS4 and UHS1.4. DiscussionCompared to the endangered Burlina breed, the Alpine Grey is present in a greater geographical area of the Northern Italian Alps. At the present, Italian Alpine Grey population consists in 17,548 officially registered heads in 1784 farms [2]. The main breeding goal in this breed is to improve the aptitude for milk and meat production in parallel, maintaining the typical morphological characteristics [1]. On the other hand, Burlina is only present in 39 farms, and only in the mountain areas of the Veneto region involved in a specific conservation plan. In 2020 this breed accounted for 1060 heads [2]. From a genetic standpoint, the interest is to preserve Burlina in purity, focusing on the inbreeding level for motivations related to biodiversity [3]. Local breeds are often characterized by robustness, optimal fertility, and longevity. Indeed, Burlina and Alpine Grey have a calving-conception interval average of 91 and 99 days, respectively, which are significantly lower than that reported for Holstein Friesian (127 days) [17]. Moreover, the frequency of abortions is very low (≤0.2%) and the frequency of twin calving is greater in Alpine Grey (3.11%) than Burlina (0.26%) [17]. The age at first calving of these local breeds is higher than Holstein Friesian; however, the average number of lactations is greater (3.34 for Alpine Grey and 3.31 for Burlina) [17]. Despite the type of breeding of Alpine Grey and Burlina, calvings are distributed through the year, with slightly higher percentages in autumn [17].4.1. SCS and DSCC VariabilityBeyond being influenced by breed, udder health is first and foremost affected by management. Local breeds are generally confined to mountain areas where the traditional rearing system is the tie-stall barn with summer grazing. Different studies [18,19] have demonstrated that SCS of Alpine Grey, Holstein Friesian, and Simmental are similar and greater than SCS of Brown Swiss. Gottardo et al. [18] and Visentin et al. [19] reported estimates of 2.59 and 2.61 for SCS in Alpine Grey cows farmed in South Tyrol. Regarding Burlina breed, Penasa et al. [20] reported an average SCS of 3.56. In the same breed, Niero et al. [21] observed an average of 3.13. SCS tends to be greater in Burlina than Holstein Friesian cows [22]. In the present study average SCS was numerically greater in Alpine Grey than Burlina. However, DSCC was significantly greater in Alpine Grey. To our knowledge, no studies have explored DSCC variability in local breeds such as Burlina and Alpine Grey so far. In terms of raw means, DSCC of Burlina and Alpine Grey was greater if compared with the results of Bobbo et al. [23] that found a raw means of 61.8% in Holstein Friesian, 60.8% in Brown Swiss and 61.1% in Simmental cows. If compared to the local Rendena breed (DSCC = 66.6%), Burlina DSCC is lower [23].In lactating cows, the number of mammary gland secretory cells increases with parity/age, resulting in a progressive lifetime increase in MY. At the same time, however, this exposes cows to major stress, compromised immune response and elevated sensitivity to intramammary infection [7,24]. This is in line with results in Table 3, which suggested that udder health is generally worse in older cows. Our results showed that SCS and DSCS had similar trends across DIM, with the lowest SCS in early lactation stages, likely due to a dilution effect. After the MY peak, SCS steadily increased until the end of lactation. On the contrary, DSCC, like SCC, peaked together with MY between 56 and 105 DIM, but was high even in late lactation. This demonstrates that the fractions of SCC changes along DIM, with the amount of polymorphonuclear neutrophils increasing towards the end of lactation [25,26]. According by Schwarz et al. [7], SCC and SCS increased in the warm season due to exposure to heat, reduced feed intake and greater susceptibility to diseases; in addition, higher temperatures and greater humidity in the summer are optimal conditions for proliferation of environmental (bedding) bacteria [27].4.2. Udder Health Status Group and Daily Production and CompositionMY of Alpine Grey and Burlina were similar and, in accordance with Schwarz et al. [8], were influenced by UHS. When SCC increases, the daily MY decreases due to intramammary infections. Mastitis pathogens and agents released during immune response are responsible for damages of udder tissue, which is particularly evident when mastitis is subclinical or recurrent [28,29]. This might explain the lower MY observed for UHS4 (‘chronic’; [7]) compared to the others. Cows with high SCC show lower MY followed by a concentration of certain solids, namely fat and protein. Despite this, mastitis is known to cause unfavorable changes in specific detailed fractions, e.g., casein content reduction and lipolysis occurrence. Intramammary infection causes an influx of leukocytes from the blood, with alteration of the osmotic equilibrium in the alveoli and thus modification of milk composition [5].In the study of Bobbo et al. [23], the concentration effect was responsible for the great fat content found in cows with chronic mastitis (here UHS4). In the same study, cows suspicious of intramammary infection (here UHS2) were characterized by the lowest milk fat content probably due to the lipolytic activity stimulated by neutrophils recruitment [30]. The proteolytic activity impairs the casein fraction, and with SCC/mastitis the proteolytic activity becomes elevated, thereby reducing the favorable proteins that are synthesized within the mammary gland (α-casein, β-casein, α-lactalbumin, and β-lactoglobulin). This creates grounds for increased transfer of protein fractions from blood such as serum albumin and immunoglobulins [31]. The greatest protein and casein contents were found in group UHS4, where MY was the lowest leading to a concentration of some solids. For the reason explained above, the casein index was expected to be the lowest in UHS4. This agrees with Mariani et al. [32] who observed the lowest casein index in cows with simultaneously high SCC and low DSCC.As regards lactose, results are in agreement with Costa et al. [29]. Overall, cows with high SCC presented lower lactose content due to leakage caused by the compromised alveolar epithelial integrity. Mastitis creates damages at the epithelial level in the alveolar structures; all the substances released during and after mammary gland tissues inflammation, such as bacteriostatic and bactericidal factors, disrupt tight junctions of the basal membrane that separates blood and milk [29,33,34]. Even if deeper investigations are needed, lactose losses are generally expected to be greater in cows/quarters suffering from a condition of chronic mastitis.The association between cow’s udder health and infrared-predicted milk BHB has only been marginally investigated in the literature. The present study showed that milk BHB was maximum in the UHS4 group. Moyes et al. [35] observed that udder inflammation was associated with an increase in milk BHB, indicating that animals with consistently high SCC throughout their lifetime could be more susceptible to metabolic disorders and severe negative energy balance.Milk urea concentration was the lowest in UHS3 group. This is, at least partly, in line with Mariani et al. [32] who observed a lower concentration of urea in milk of Holstein-Friesian and Simmental cows that presented high values for both DSCC and SCC. Pegolo et al. [36] did not report significant associations between milk urea and SCC at different levels of DSCC in Holstein Friesians. Milk urea is usually adopted to monitor protein utilization and nitrogen efficiency at herd level for management purpose (bulk milk). A large concentration of urea in individual milk is instead indicative of inefficient nitrogen utilization/protein metabolism due to either unbalanced protein content of feed (e.g., temporary excess) or cow-related physiological factors. In fact, urea in milk is not influenced by breed but rather by the interaction of multiple environmental factors.4.3. DSCC and SCC in PractiseThe UHS of cows influences the economic profit of the farmer both directly and indirectly. Findings of this study demonstrate that cows with high SCC (UHS3 and UHS4) tend to produce less [37] and to present a deteriorated milk quality. Within the supply chain, high SCC in bulk milk leads to penalties for the farmers, particularly in Italy where some milk components important for cheesemaking are considered in the milk payment system [38]. Even when the milk is processed directly at the farm, the consequences of UHS3 and UHS4 negatively affect the milk and cheese yield (e.g., lower casein index). In addition to this, costs for any veterinary treatment during the lactation and/or the dry period must be accounted for while also an increased culling rate could arise [5]. Ideally, we will need further validation data to confirm whether DSCC I) significantly improves diagnostic accuracy of mastitis or II) is useful for the identification of cows needing treatment at dry-off. This would lead to a decrease in antibiotic use and costs at farm-level.5. ConclusionsIn the present study we demonstrated how the different milk somatic cell fractions were differently affected bybreed (Apline Grey and Burlina), stage of lactation, parity, and season of sampling. Primiparous showed lower SCC and DSCC compared to pluriparous and udder health seemed in general worse in the summer period. Moreover, findings releval that udder health status can affect MY and milk composition in local cattle breeds. This is the first study investigating proxies for udder health in Italian local breed. Our results highlight that udder health of less cosmopolitan/autochthonous cows can be monitored through official milk analyses exploiting various proxies measurable in milk. To improve udder health, the introduction of mastitis recording and monitoring plans is advisable in local as in cosmopolitan breeds and may allow the set-up of dedicated genetic strategies. The low productivity of dual purpose/local breeds compared to the highly selected populations must be compensated through maximization of their resistance to disease, longevity, functionality, and efficiency, with a positive indirect reduction in biodiversity loss.

animals : an open access journal from mdpi

10.3390/ani11113039

PMC8614431

Salinity is a stress factor for benthic invertebrates. Based on a 2-year study of 9 coastal lakes along the southern Baltic Sea, representing freshwater, transitional, and brackish ecosystems, we have shown that benthic fauna was structured by sea water intrusion (=fluctuation of salinity). The increase in salinity gradient resulted in a decreasing trend in the richness and abundance of benthic species, while the diversity showed a slightly positive trend, but below statistical significance (p < 0.05). The abundance of benthic organisms was the highest in brackish costal lakes, where the marine component of fauna was identified. Due to the greatest instability of environmental conditions in lakes periodically linked with the sea (transitional), we found the lowest species number, α-diversity, and abundance, regardless of the season. Salinity appears as a key factor in controlling the functioning of the ecotone (coastal lakes, lagoons) between the marine and terrestrial environments. Salinity is a prerequisite for the proper assessment of the ecological status of coastal lakes and the development of effective methods of protecting coastal lakes.

Benthic communities were studied in nine Polish coastal lakes of the Baltic Sea; representing three levels of hydrological connection with the sea (isolated, periodically connected, and permanently connected), with resultant differences in salinity (freshwater, transitional, and brackish). The lakes classified in this way allowed us to investigate biodiversity in relation to the degree of environmental pressure. Stress intensity in coastal water bodies, resulting from contrasting marine and terrestrial influences, varied from mild to severe. Spatial variation in environmental predictors affected species richness more strongly than seasonal fluctuations. The broader the spatial salinity gradient, the smaller the species number recorded. Differences in the intensity of natural instability only slightly affected species number and α-diversity. In Baltic coastal lakes, characterized by low salinity (max. 7.5 PSU), benthic faunal communities were dominated by large populations of opportunistic species. This applied primarily to closed systems and those periodically influenced by seawater intrusion. The marine component of fauna played a more important role in increasing the diversity of benthos in permanently open water bodies (brackish). The highest density of benthic fauna was recorded in them, whereas low values were associated with the strongest instability, observed in lakes periodically linked with the sea (transitional).

1. IntroductionCoastal water bodies (lakes, drowned river valleys, and smaller intermittently open and closed lagoons) are sites of contact between marine and terrestrial environments, associated with mixing of variable amounts of fresh water from tributaries and seawater caused by the intrusion. As a result, their biotope is characterized by complex environmental gradients, comprising salinity, water exchange, morphometric features, nutrients, turbidity, and sediment properties [1,2]. Simultaneously, contributions of individual physicochemical predictors depend on the main hydrodynamic energy source [3,4,5]. In the coastal lakes with major tributaries (rivers), environmental gradients are determined by the amount of fresh water. By contrast, in the water bodies permanently connected with the sea, the gradients are affected mostly by the degree of seawater intrusion. In this way, two alternative stable states develop, with contrasting levels of water salinity [6,7]. According to the theory of alternative stable states, the system can remain in one of the possible states defined by a specific composition of biocenoses and habitat properties in ecologically significant time scales [8,9]. Specific cases are transitional lakes, with a periodic variable dominance of river water (freshwater-brackish) or seawater (brackish-freshwater), where fluctuations in environmental conditions are particularly high [10,11]. This can be also observed in river deltas [12,13]. This leads to continuous structural and functional changes within individual biocenoses colonizing the lakes, according to the assumptions of adaptive cycles [14,15].The Baltic Sea is not a typical, salty sea. The mean salinity of its surface waters in the southern zone is only 7 PSU (range 2–20 PSU), i.e., 1/5 as high as in oceans. This is due to the dominance of the supply of fresh water (from rivers and precipitation) over the saltwater intrusion from Kattegat. As a result, its coastal lakes are characterized by a relatively small gradient of salinity, from <0.5 PSU to 7.5 PSU [16]. However, studies have shown that these ecosystems are highly unstable, leading to a remarkable variation in animal diversity [17,18]. The repeated pattern of species richness, decreasing from the places affected by seawater intrusion to internal parts of lakes, is well-documented [17,19,20,21]. Moreover, species diversity strongly depends on the possibility of migration [22] and varies in relation to increasing environmental instability caused by frequent changes in proportions between fresh and brackish waters [23]. In coastal ecosystems with limited seawater intrusion, some specialized animal species can tolerate environmental extremes and potentially develop large populations within a broad range of salinity levels [24]. Bamber et al. [25] found that eurytopic (opportunistic) species colonizing such ecosystems are better adapted to environmental variation, most probably because of the level of genetic plasticity, but under extreme salinity conditions, species richness is expected to decline [26]. It is necessary to investigate and compare the spatial and spatiotemporal patterns of fauna and flora in coastal lakes with various levels of natural stress to distinguish and quantify the major factors that structure communities, i.e., critical extremes and instability level as a predictor of species distribution [27].Previous studies on benthic invertebrates inhabiting the Baltic coastal lakes have provided only some of the necessary information, as they were based only on information from selected lakes [17,21]. This study attempts to identify relations between the level of variation in abiotic conditions and fluctuations in the structure of invertebrate fauna of all southern Baltic coastal lakes along a gradient of salinity. The analyses concerned spatial and seasonal variation in benthic invertebrate communities (zoobenthos) in three types of coastal lakes: brackish (>3.0 PSU), transitional (0.5–3.0 PSU), and freshwater (<0.5 PSU). In this respect, it is the first comprehensive study of the taxonomic diversity of the benthic fauna of the lakes along the coast of the southern Baltic Sea.2. Materials and MethodsSamples were collected from 9 Baltic coastal ecosystems (Figure 1), varying in the level of hydrological connectivity, which was reflected in their salinity gradient. In the present study, all major lakes of the southern Baltic coast were sampled. This supplemented the information on the taxonomic composition of the benthic fauna obtained in the 2014–2015 study [17,21].Lakes Łebsko, Resko, and Ptasi Raj are permanently connected with the sea (brackish), but Łebsko is the largest water body on the southern coast of the Baltic (71 ha), and Ptasi Raj is the smallest (<0.5 ha). Łebsko is fed by the Łeba River, while Resko by the Błotnica, both of which later flow into the sea. Lake Ptasi Raj is in fact a bay separated from the sea by a levee with a system of floodgates, which allow seawater intrusion. Lakes Liwia Łuża, Kopań, and Gardno (transitional: freshwater-brackish) are like estuaries, as they are mostly fed with fresh water but periodically are exposed to seawater intrusion. Lakes of this type are supplied with fresh water from an extensive system of drainage ditches (Liwia and Kopań) or a large river (Gardno). They also have a permanent connection with the sea (lakes Liwia and Gardno) or only a periodical one (Kopań). Freshwater lakes are represented by Wicko, Dołgie, and Sarbsko, devoid of hydrological connection with the sea. All the lakes receive pollution loads from the catchment, which accelerate their eutrophication [16,28]. Samples were collected primarily from soft lake sediments in 2019 and 2020 seasonally: in spring, summer, and autumn. Numbers of sampling sites depended on lake size: 4 in Ptasi Raj, 5 in Liwia Łuża, Resko, Kopań, Sarbsko, Gardno, and Dołgie, 8 in Wicko, and 11 in Łebsko. In each season at each site, 3 replicates of benthos samples were taken using an Ekman bottom dredge (0.03 m2 of the bottom each). The samples were sieved through a 0.5-mm mesh and next preserved with 6% formalin. In the laboratory, the bottom macroinvertebrates were sorted, identified to species level (if possible), and counted. We divided the organisms into 3 groups: opportunistic, euryhaline, and marine, as suggested by Reizopoulou et al. [23]. Opportunistic species are characterized by a low level of specialization and adapt to changes easily, while euryhaline species tolerate various levels of salinity. Identification and classification was based on available keys and information extracted from online databases, [29,30]. On the basis of biological data, α-diversity was assessed (Shannon index, H’).Simultaneously with the biological sample collection, we measured physicochemical parameters at the same sites (in situ): salinity, dissolved oxygen (%DO), chlorophyll a concentration (Chl-a), NO3−, NO2−, and NH4+ with a calibrated AP-7000 Aquaprobe (AquaRead, UK). To determine total inorganic nitrogen (TIN), we summed up values of NO3−, NO2−, and NH4+ [31]. We also took water samples for laboratory analyses, including total phosphorus (TP). Laboratory analyses followed the Standard Methods [32]. Conductivity values (µS cm−1) were related to salinity values (PSU) as reported in Wagner et al. [33].Differences between the three lake types in environmental parameters were assessed using principal component analysis (PCA). Spearman rank correlations (r) between biotic and abiotic parameters were calculated. Community structure was described using multidimensional scaling (MDS) based on a similarity matrix constructed using Bray–Curtis similarity index. Before the analysis, data for seasons from 2 years prior were averaged and log-transformed (y = log (x + 1)). Differences between variables for lake types were tested by analysis of variance (ANOVA) with the Kruskal–Wallis test by ranks (p < 0.05). ANOSIM test (R) was used for matrices describing the zoobenthos (numbers of opportunistic, euryhaline, and marine species), testing the null hypothesis that they did not differ significantly between the study lakes and seasons. Statistical analyses were performed using PRIMER v7 software.3. ResultsEnvironmental parameters varied widely between the lakes and seasons of sample collection (Table S1). Generally, brackish lakes had high temporal ranges of abiotic variables in spring and autumn, whereas freshwater ones had them in summer. Salinity gradients in brackish lakes were strongly sloping spatially, while transitional lakes more clearly varied seasonally.Regardless of the season, the most important physicochemical parameters differentiating the abiotic conditions in the investigated coastal lakes were: salinity, conductivity, oxygen saturation and ammonium concentrations (one-way ANOVA, p < 0.0001). In addition, statistically significant differences in total phosphorus and total inorganic nitrogen were found in the seasons. Among the analyzed parameters, only the concentrations of chlorophyll remained similar (did not differ significantly statistically) in the studied lakes regardless of the season (Table 1).Differences in variation of abiotic parameters in the study areas are probably due mostly to the level of primary productivity, aerobic conditions, and hydrological connection with the sea. The variation between the nine lakes (representing three lake types) in environmental parameters is illustrated by results of the PCA (Figure 2A). The PC1 axis (explains 35% of total variance) is associated with increasing salinity (and EC) gradient from low salinity in freshwater Lake Sarbsko in the right-hand part of the plot to sites with high salinity in the left-hand part of the plot (Lakes Ptasi Raj and Łebsko). Interestigly, it shows summer and autum effects of marine water intrusion to the lakes. The PC2 axis (31.35% of total variance) correlates positively with the aeration conditions in the lakes, while negatively with mineral nitrogen and phosphorus.Multidimensional scaling based on qualitative-quantitative analysis of benthic fauna allowed us to identify benthic communities corresponding to individual types of coastal lakes. Seasonal variation was more conspicuous in brackish lakes, whereas in freshwater ones (with negligible temporal variation in salinity) community structure varied only slightly (Figure 2B). Species matrices for the whole data set can be classified into three major groups: euryhaline (tolerating a broad range of salinity levels and temperatures), opportunistic, and marine species. Among the analyzed physico-chemical parameters of water, the strongest correlations (Spearman’s ranks) with the abundance of benthic fauna in coastal lakes were found in concentrations of nitrates (r = 0.91), oxygen saturation (r = 0.77), and salinity (r = 0.66). The number of identified species (r = 0.93 and r = 0.76, respectively) and their α-diversity (r = 0.88 and r = 0.67, respectively) were clearly related to the concentration of TIN and NO2-. Benthic diversity was strongly associated with TP concentrations (r = 0.83).In total, we identified 48 taxa of benthic invertebrates (of 10 taxonomic groups), including 26 opportunistic, 13 euryhaline, 7 marine, and 2 unclassified ones (Table 2). Because of the low salinity of the investigated lakes, their invertebrate community structure is unique, dominated by opportunistic species (= eurybionts) accompanied by small numbers of marine and euryhaline species. The level of hydrological connectivity between the sea and coastal lakes increased the taxonomic composition of benthic fauna groups. The species denisty in individual lakes is presented in Table S2. The ANOSIM test of the species composition matrix showed that the lake types differed significantly at p < 0.001 except for transitional vs. freshwater, where significance level reached only 0.1. The greatest significance of differences was recorded for transitional vs. brackish lakes (R = 0.14, p < 0.001). The qualitative structure of benthic fauna did not differ significantly between seasons within lake types, but differences between lake types were noticeable. The strongest seasonal influence was observed between brackish and transitional lakes: in summer (R = 0.16, p < 0.01), spring (R = 0.16, p < 0.02), and autumn (R = 0.1, p < 0.05). Additionally, community structure in autumn differed between brackish and freshwater lakes (R = 0.12, p = 0.01). In lakes with higher salinity, communities “moved” between opportunistic and tolerant euryhaline species, depending on seasonality of changes in environmental parameters and intensity of seawater intrusion and eutrophication. However, opportunistic species were the major group of benthic fauna in the study lakes, accounting for 62% of the total number of identified taxa. The most common members of this group were Chironomus f.l. plumosus and Polypedilum nubeculosum. In brackish lakes with greater volume and undisturbed intrusion of seawater, marine and euryhaline species were more numerous (Figure 3). Overall, in brackish lakes, a greater total number of species was recorded and the resultant higher diversity was linked with seawater intrusion, reflected in the presence of marine species. The other lakes, with significant or total loss of hydrological connection with the sea, were characterized by lower diversity. In this context, transitional lakes were distinct, as in spring their benthic species diversity was the lowest.In lakes of all types, the abundance of opportunistic invertebrates was to a large extent shaped by C. plumosus, which in spring reached up to 40% of the total catch. Their abundance was similar in brackish and transitional lakes, where they accounted for about 35% of the total benthic fauna. Euryhaline species reached the highest densities in brackish lakes in spring, in transitional ones in summer, and in freshwater ones in autumn. This group was represented most abundantly by Gammarus duebeni in transitional and brackish lakes, while by Bezzia nobilis in freshwater ones. Densities of marine organisms were high only in brackish lakes in summer. In lakes permanently connected with the sea, the most abundant marine species was Hydrobia ulvae, whereas in periodically connected ones, Gammarus oceanicus. Benthic fauna reached the highest density in a brackish lake (Resko) and was the lowest in a transitional one (Kopań) (Figure 4). The abundance of benthic organisms was the highest in brackish costal lakes (x¯ = 760 indiv. m−2), where the marine component of fauna were identified. Due to the greatest instability of environmental conditions, the lowest abundance (x¯ = 300 indiv. m−2) was found in lakes periodically linked with the sea (transitional). Significant differences in density were found between brackish and transitional lakes (R = 0.060, p = 0.027) and freshwater and transitional lakes (R = 0.053, p = 0.029). Opposing salinity regimes (freshwater vs. brackish water) resulted in similar benthic fauna density values. In individual lake types, significant seasonal differences in the abundance of benthic fauna were observed only between spring and autumn, within all groups: brackish (R = 0.070, p = 0.024), transitional (R = 0.11, p = 0.003), and freshwater (R = 0.12, p = 0.003). Opportunistic species reached the highest densities in brackish lakes in autumn, in transitional lakes in spring, and in freshwater lakes in summer.The overall data analysis (Figure 5) confirmed a high significance of the patterns associated with the level of environmental changes mean species richness (R = 0.37, p = 0.0001) and mean density (R = 0.10, p = 0.001) was negatively correlated with the variation in salinity, expressed as standard deviation of salinity values within the study lakes for each sample. In the case of α-diversity, salinity gradient also strongly affected the variation in benthic animal communities (R = 0.55, p = 0.0001). However, correlations between other benthos descriptors and mean salinity values were not statistically significant.4. DiscussionContributions of various predictors to shaping the natural gradients of water conditions in coastal water bodies depend on the major source of hydrodynamic energy in the system [1] and the intensity of intrusion of seawater [34]. Chemical and physical gradients affect animal and plant communities in many ways [35,36,37], so species richness in coastal lakes depends not only on salinity but on a complex of factors reflecting the degree of their isolation (time needed to restore marine conditions) [20,38]. When the time is longer, a stronger stimulus is needed to change the regime [17]. In coastal water bodies, the salinity gradient is the major environmental factor shaping species distribution [21,39,40,41,42,43]. It is particularly significant in the case of lakes connected with the Baltic Sea, which are brackish (~7 PSU). Coastal ecosystems with spatial variation in values of predictors were treated as ecotones and a two-ecocline model was proposed (physical transitional zone), which in the case of freshwater species is combined with gradients caused by the influx of fresh water (rivers, canals) to mid-estuary, and in the case of marine species, from the connection with the sea to mid-estuary [34,40]. In coastal water bodies, the environmental gradient is shaped mostly by seawater intrusion, strongly determined by the opposition of marine and terrestrial factors, and species richness following the one-scale pattern. In this study, the influence of seawater was the main factor determining the complexity of benthic fauna structure in the ecosystems being studied, while the degree of hydrological connectivity of each water body was optimally reflected in salinity and dissolved oxygen levels (Table 1). This concept has recently been the subject of numerous studies and has been well explained [42]. Differences in natural stress were also reflected in the presence of various invertebrate groups. In the communities being studied, opportunistic species formed the major group, including common species of shallow eutrophic lowland lakes (e.g., C. plumosus). They are highly tolerant to osmotic stress, which allows them to colonize abundantly coastal lakes [24]. They are accompanied by euryhaline species, treated as characteristic of habitats of this type. Marine species were limited to brackish lakes (Łebsko, Resko, and Ptasi Raj), where they increased the species richness of the ecosystem (see Table 2). An increasing number of reports emphasize the ecological significance of the dependence between seawater intrusion within systems and biotic zonation [21,23,44,45]. In the Baltic coastal lakes under study, we observed a positive linear relationship between species richness (S) and the slope of the salinity gradient in each water body, which indicates their dependence on seawater intrusion. The higher the salinity gradient, the lower the mean number of species in individual ecosystems. Benthic fauna abundance (S) and diversity (H’) showed a similar pattern, but the correlation with salinity variance was feeble. It seems that the abundance is more strongly linked with food availability and seasonal biological cycles, while species richness is mostly connected with the intensity of environmental stress. In a similar study of three Mediterranean lagoons with a broader salinity gradient (0–12 PSU), stronger positive correlations of species richness and diversity with salinity and weaker correlations in the case of abundance were observed [23]. Seasons seem to less strongly affect the structure of brackish communities, which was confirmed by MDS results (Figure 2B). Similar results were reported by Obolewski et al. [17] who stated that temporal changes in species richness and community structure often prove to be non-significant in coastal water bodies, irrespective of their hydroecological type. According to Nicolaidou [46], the lack of seasonality is attributed mostly to continuous reproduction of some abundant species and both trophic and other interactions between species. It is worth noting that only our most recent studies provide sufficient information on the level of taxonomic diversity of benthic invertebrates. Therefore, the presented results can only be treated as fully representative for the coastal lakes of the southern Baltic Sea. Compared to the results obtained from our research from 2014–2015, we managed to identify a much larger number of species. In these studies, 48 taxa were found, while only 28 were found in the earlier one [17].The high trophic state of Baltic coastal lakes was reported many times [28,47,48]. Taking into account the specificity of saline conditions, the coastal habitats with a small number of species and low diversity should be carefully assessed as severely degraded. In particular, in the case of transitional coastal lakes, low biodiversity rates are due to natural stress caused by: (i) unstable salinity conditions that benthic species must tolerate to survive and (ii) variable environmental parameters due to seawater intrusion. Our observations showed that there is a specific dispersion of benthic fauna in the gradient zone between sea and land along which the intensity of salinity stress varies from mild to severe. It is worth noting that salinity, controlled by the hydrodynamics of water at the interface between the lake and sea, influences the variability of the physicochemical conditions of the coastal environments. In light of the above, our study points to the role of brackish sea water intrusion as a factor in improving oxygenation in the bottom zones of lakes. It is worth emphasizing that in the case of coastal lakes, diffusion and photosynthesis, as well as the inflow of oxygenated sea waters are responsible for the oxygenation of the waters [18,49].The measures of diversity and the associated biotic indices proved to be less effective for coastal water bodies with a narrow salinity gradient. This was mostly due to the dominance of species tolerant of natural stress factors [17,18,27,49,50]. This situation was described as a “paradox of estuary quality” and can lead to false classifications based on the level of hydrological connectivity [51,52]. The occurrence of intrusion of brackish seawater causes “refreshment” of the biotope but destabilizes environmental conditions [16,53]. Moreover, hydrological connectivity also constitutes a “window of opportunity” for migration of alien marine and euryhaline species, and their expansion from the Baltic Sea to lakes [54,55].5. ConclusionsSalinity is a key determinant of species diversity in coastal waters. Species richness and diversity of benthic communities in coastal waters are strongly linked with environmental gradients determined by the level of hydrological connection with the sea. Various indices used to assess the ecological potential of water bodies, including diversity indices, should take into account the level of natural instability evoked by marine water intrusions. This aspect concerns both the potential chances of migration and adaptation of organisms, but also leads to permanent changes in habitat conditions and dominance of opportunistic species. Assessment of biodiversity along salinity gradients in coastal lakes is the only and optimal way to recognize natural or anthropogenic stress factors. Salinity should be a prerequisite for the proper assessment of ecological status and the development of effective methods of protecting coastal lakes.

animals : an open access journal from mdpi

10.3390/ani11113071

PMC8614502

The social complexity hypothesis (SCH) for communication predicts that species with complex social systems exhibit complex communication systems. Testing the SHC in a broad range of species can contribute to a better understanding of human evolution because a co-evolutionary runaway process between social and vocal complexity may have shaped human language. Here we compare patterns of vocal complexity between the two species of African elephants: the savanna elephant exhibiting a complex social organization and the forest elephant exhibiting a simpler social organization. We review the existing literature and present novel insights into the vocal communication system of the elusive forest elephant, along with a first direct comparison with savanna elephants. Our findings suggest that the African elephants may contradict the SCH, as well as other factors potentially shaping patterns of vocal complexity across species. A better understanding of vocal complexity in the two species of African elephants will depend on continuing advancements in remote data collection technologies to overcome the challenges of observing forest elephants in their dense rainforest habitat, as well as the availability of comparable data quantifying both structural and contextual variability in the vocal production of both species of African elephants.

The social complexity hypothesis (SCH) for communication states that the range and frequency of social interactions drive the evolution of complex communication systems. Surprisingly, few studies have empirically tested the SHC for vocal communication systems. Filling this gap is important because a co-evolutionary runaway process between social and vocal complexity may have shaped the most intricate communication system, human language. We here propose the African elephant Loxodonta spec. as an excellent study system to investigate the relationships between social and vocal complexity. We review how the distinct differences in social complexity between the two species of African elephants, the forest elephant L. cyclotis and the savanna elephant L. africana, relate to repertoire size and structure, as well as complex communication skills in the two species, such as call combination or intentional formant modulation including the trunk. Our findings suggest that Loxodonta may contradict the SCH, as well as other factors put forth to explain patterns of vocal complexity across species. We propose that life history traits, a factor that has gained little attention as a driver of vocal complexity, and the extensive parental care associated with a uniquely low and slow reproductive rate, may have led to the emergence of pronounced vocal complexity in the forest elephant despite their less complex social system compared to the savanna elephant. Conclusions must be drawn cautiously, however. A better understanding of vocal complexity in the genus Loxodonta will depend on continuing advancements in remote data collection technologies to overcome the challenges of observing forest elephants in their dense rainforest habitat, as well as the availability of directly comparable data and methods, quantifying both structural and contextual variability in the production of rumbles and other vocalizations in both species of African elephants.

1. IntroductionThe various forms of animal social organization constitute adaptive strategies that enable individuals to maximize their fitness in the face of ecological constraints imposed on their survival and reproduction [1]. Vocal communication is often a crucial component of social behavior, enhancing the benefits and mitigating the costs associated with a species’ social organization as it serves to coordinate the interactions and, as such, maintain the relationships between individuals cooperating and competing within and between social groups [2]. The social complexity hypothesis for communication states that complex social organization drives the evolution of complex communication systems [3]. The underlying notion is that animals living in highly variable and complex social environments (e.g., individuals interacting frequently and in diverse ways), need to convey a broader range of information to coordinate their social interactions. Such increased expressiveness can be achieved via various mechanisms, including an increase in vocal repertoire size and acoustic variation within and between call types, as well as the combination of call types into larger utterances. A vocal mediation of social relationships, in turn, may further facilitate the formation of complex social organizations [4], particularly when the animals’ ability to interact directly is limited [5], giving way to a co-evolutionary feedback mechanism ratcheting up social and vocal complexity. Even though already mentioned by Darwin [6], surprisingly few studies have empirically tested the social complexity hypothesis for vocal communication systems (e.g., [4,7,8,9]). Some of those have offered mixed results and leave many aspects of social and vocal complexity unexplored [10]. Filling this gap is particularly important because a co-evolutionary runaway process between social and vocal complexity may have been a crucial driving force shaping the most intricate communication system, human language [5], the evolutionary origin of which remains unresolved. The open-ended generativity of human language broadly arises from two underlying mechanisms of speech production: the intentional modification of the vocal tract to vary the energy distribution across frequencies (the so-called ‘formant structure’) to produce vowels [11], and the flexible combination of acoustic units into more complex utterances, generally referred to as syntax [12]. How the occurrence of analogs of syntax and formant modulation relates to variation in social complexity across non-human species remains little understood. In the absence of a fossil record, comparative studies of the vocal behavior of closely related species showing distinct differences in social complexity are a powerful tool to better understand the evolution of complex communication skills, including key features of human language. Fission-fusion societies in which groups regularly split into smaller subgroups are excellent systems to pursue such studies. Such plasticity in social cohesion may create unique challenges to the maintenance of social relationships; as such, distinctive selective pressures act on underlying communicative abilities [13], for instance in order to keep contact, cooperate and re-enforce social bonds or dominance hierarchies. Human language may have evolved to facilitate the maintenance of long-term social relationships when frequent direct interactions became impossible in the fission-fusion societies of our hunter-gatherer ancestors [14,15]. Previous studies related to language evolution focused largely on primate vocal communication, which was long considered fundamentally different from human language. With a strict division between language and primate vocal communication increasingly dissolving [16], our understanding of the evolutionary origins of human language will benefit from broadening investigations to evolutionarily more distant taxa, but with similar sociality. We here aim to highlight the African elephant (Loxodonta spec.) as a highly suitable system in which to explore evolutionary pathways leading to complex communication skills. The two species, the forest elephant (L. cyclotis) and the savanna elephant (L. africana), exhibit marked differences in the level of complexity in their respective fission-fusion societies. How these observed differences in social complexity relate to the two species’ vocal behavior remains poorly understood due to difficulties studying forest elephants in their dense forest habitat. A climatic transition to more arid conditions resulting in the reduction of forests [17] likely drove the divergence of savanna elephants from a forest elephant-like animal that preferred forest habitats between 2.6 and 5.6 million years ago [18,19,20]. While extant savanna elephants may inhabit a variety of habitats, including deserts, savannas, subtropical and temperate lowland and montane forests, forest elephants almost exclusively inhabit the dense tropical rainforests of Central Africa. Adult females of the primarily herbivorous savanna elephants exhibit complex multi-tiered fission-fusion societies, in which family units, consisting of a matriarch, her offspring and her close adult female relatives and their offspring, as well as more extended family “bond groups” may split and rejoin on a regular basis [21,22,23] while maintaining long-term social relationships within extensive social networks [24]. It is assumed that savanna elephants exhibit such strong social bonds because they benefit from cooperative defense against large pack-hunting predators (including humans), increased opportunities for social learning (allomothering, leadership) and sharing of knowledge about resource distribution [25,26,27,28]. In contrast, females of the more frugivorous forest elephants are found in smaller groups consisting of only one adult female with her dependent offspring likely due to elevated competition for food and the absence of non-human predators [29,30,31]. While multiple family groups exhibit stable and differentiated associations with one another, similar to the fission-fusion sociality of savanna elephants [32,33,34,35], the social networks of forest elephants appear smaller and lack multi-tiered structuring [36]. In both species, the coordination of social interactions within and between groups relies on characteristic rumble vocalizations. Rumbles are tonal calls with a rich harmonic structure and a low fundamental frequency that extends into the infrasonic range [37,38]. Both species habitually combine rumbles with broadband call types (laryngeal roars, cries, barks, here broadly referred to as roars, and sometimes with trumpets and snorts) into more complex combinatorial call types (Figure 1). In the following, we present the current state of knowledge on forest elephant vocal communication in comparison to savanna elephants, with direct reference to evidence for the social complexity hypothesis from other species. We present findings based on already published studies, as well as results based on new data used to conduct a first direct comparison between savanna and forest elephant vocal behavior. We discuss if our current assessment of differences in the vocal communication system supports the social complexity hypothesis and conclude with suggestions for future research.2. MethodsTo collect information on patterns of vocal complexity in the two species of African elephants we followed two approaches. First, we undertook a detailed literature review. These findings are indicated with the appropriate citation. Second, we collected new data on forest elephants, and re-analyzed already available data for savanna elephants, as described in the following. Results based on these new data are indicated under new findings in separate paragraphs.2.1. Forest ElephantsData collection on call contexts took place at Dzanga Bai, a forest clearing in Dzanga-Ndoki National Park in the southwestern Central African Republic (2.963° N, 16.365° E). The clearing is approximately 10 hectares in size, characterized by a sandy pan intersected by a permanent stream. Elephants enter the clearing for several reasons, primary among them being access to mineral rich water in small monopolizable depressions or pits that the elephants dig themselves [31].We conducted behavioral observations on the contexts of calls produced mainly by unidentified individuals from September 2018 to April 2019 from an 8 m high observation platform at the edge of the clearing. We used opportunistic sampling paired with simultaneous audio-recording using an Earthworks omnidirectional microphone, capable of accurately recording the very low frequency calls of elephants, attached to a Sound Devices MixPre3 Audio recorder, and a 48 kHz sampling rate. For each audible call for which we were able to identify the caller and context with a high level of confidence, detailed notes on the behavioral contexts were taken. These notes were examined and collated into distinct context categories with the goal to compile the first ethogram of call contexts in forest elephants. Spectrograms of sound recordings were generated in Raven Pro Sound Analysis Software® (version 1.5) using a Hann window with a frequency and time resolution of 0.98 Hz and 0.0255 s. Based on visual inspection of these spectrograms, we categorized calls into rumbles (N = 304), roar only (N = 88) and combination types: rumble then roar (RU-RO; N = 25), roar then rumble (RO-RU; N = 67), rumble, roar, rumble (RU-RO-RU; N = 38) and roar, rumble, roar (RO-RU-RO; N = 2). To ascertain that these call types can be assigned reliably, DH and a second observer independently scored a subsample of N = 75 calls. The percent agreement between the observers was 97%.A subset of rumbles recorded with sufficient quality (high signal-to-noise ratio, no concurrent environmental noises) was used to investigate contextual differences in the structure of rumbles based on acoustic measurements conducted following the protocols described in Hedwig et al. [39]. We used discriminant function analysis and linear regression analysis to investigate whether rumbles can be assigned to the context categories based on their acoustic structure, focusing on the most frequently observed contexts and excluding the rare calls we recorded from adult males and infant calves (N = 246; see Table S1 in Supplementary Material for detailed sample sizes by context and age-sex class).2.2. Savanna ElephantsWe recorded elephants primarily in Amboseli, Kenya between 1986 and 1990, 1998 and 2006, and in 2020, from a population of known-aged individuals studied since 1972. To obtain suitable sample sizes of calls from individuals, we focused on one family of 27 elephants in 1999 and 2000. In 1998 we also recorded elephants in Laikipia and Maasai Mara, Kenya, and from semi-captive orphan elephants in Tsavo, Kenya. The vast majority of data included in this paper are from individually known callers from Amboseli. Through 1990 we used a Nagra IVSJ recorder; between 1998 and 2003 an HHB PDR 1000 DAT recorder and after 2003 a factory modified Nagra Ares BB. Most recordings were made with an Earthworks QTC1 omni-directional microphone. All our equipment was capable of accurately recording the very low frequency calls of elephants (down to at least 10 Hz; see Poole 2011 for more detail).We made observations from a vehicle (except the Tsavo orphans). When a suitable group was found we parked near an individual or sub-group that provided good visibility (5–20 m to the nearest elephant). Once the elephants moved greater than about 25 m away, we moved the vehicle again. Although many elephant calls persist over long distances, the best quality calls and field data are typically from the closest individuals; accordingly, we varied the nearest elephant. We noted the specific location, group size and type and the individuals present. When we heard a call, we recorded the call type, call context-type, caller, distance to the source and any contextual or other comments about the situation or the behavior of the calling individual. We use the term call type to refer to the broad, structurally differentiated categories of sounds (e.g., rumble, roar, trumpet, etc.), and the term context type to refer to a priori subtypes initially differentiated from a combination of sound quality and social context (e.g., begging-rumble, musth-rumble, let’s-go-rumble, greeting-rumble). Many of these have since also been structurally differentiated [40]. We noted caller, call type and context-type with a level of confidence (A: certain, B: fairly confident, C: educated guess, D: no idea). A call context-type assigned confidence “A” required that both the behavioral context and sound quality matched the context-type designation. Elephant calls were systematically logged from field notes into a custom-designed FileMaker Database (N = 6642). Our long term studies of savanna elephants resulted in the recent release of The Elephant Ethogram [41], a detailed library of the communication and behavior of African savanna elephants, which provides a foundation for comparative studies focused on the vocal communication systems of the two species of African elephants. Building on from Poole and Granli [42], The Elephant Ethogram suggests and defines 23 behavioral contexts (https://www.elephantvoices.org/elephant-ethogram/ethogram-table.html, accessed on 23 October 2021), at least 20 of which are associated with vocalizations. To conduct a first direct comparison of the contextual use of rumbles between the two species of elephants, we have aligned the savanna elephant behavioral contexts (N = 15; and, within these, the specific call sub-types) that we believe best match the broader contexts currently described for forest elephants. Future studies will need to harmonize the contexts for a more accurate comparison.To compare the behavioral contexts of rumbles, roars and combinations between the two species, we selected all SE rumbles that had been placed in behavioral contexts and had been assigned a context-type with an accuracy of A or B (N = 3006), and likewise, all SE roars and rumble-roar combinations with a context-type accuracy of A or B (roar only: N = 90; RU-RO: N = 12; RO-RU: N = 33; RU-RO-RU: N = 22). Pardo et al. [43] demonstrated that these call types can be assigned reliably with a mean percentage of agreement between observers of 90%. For a subset of rumbles, we used Signal 4.0 to take acoustic measurements. Due to the high frequency of overlapping calls, measurement was not automated, rather it was taken in the spectrograph view using the cursor. We used linear regression analysis to investigate contextual differences in the duration of rumbles, for which acoustic measurements and information on the age of the caller were available (N = 1177 rumbles recorded from 53 known individuals (median number of calls per individual = 5 [interquartile range 1–32]); see Table S2 in Supplementary Material for detailed sample sizes by context and age-sex class). 3. Results3.1. Rumble Repertoire Structure, Repertoire Size and Contextual UseSocial and vocal complexity can be described through diverse attributes [44]. Among the most used attributes of vocal complexity is the size of a species’ vocal repertoire. Across avian and mammalian taxa, vocal repertoire sizes relate to various aspects of social complexity, including group size [4,7,8,45,46,47], social and mating system [9,48,49,50], level of gregariousness [48], time spent grooming [4] and the number of social roles in a group [7]. However, exceptions in some taxa of frogs, birds and primates highlight that factors other than social complexity, or at least other aspects of it, may also shape patterns of vocal complexity across species [51,52]. Even though assumed to be a driving force behind the evolution of human language, how the degree of complexity of the hierarchical structure characteristic for fission-fusion societies relates to patterns of vocal complexity has not been investigated in a comparative perspective. Freeberg et al. [3], however, suggest that communicative requirements may be affected by the fact that relationships across the different tiers of the social system may have different qualities. Moreover, an aspect of vocal complexity even more overlooked is the level of structural variation within and between call types. Within-species comparisons of acoustic variation demonstrate that call types involved in coordinating often socially more complex close-range interactions exhibit particularly high variation compared to those used for long-distance communication [53,54,55,56], suggesting social complexity also plays a role in shaping the degree of gradation within vocal repertoires. The cornerstone of any research aiming to utilize the genus Loxodonta to elucidate relationships between social and vocal complexity is detailed quantitative information on the acoustic structure of their vocal repertoire, as well as its contextual use. Classification schemes and estimates of the size of the rumble repertoire of savanna elephants vary widely and remain unclear. Studies were either carried out in captive settings on a small number of individuals with a limited range of social contexts or on wild elephants, yet, often with small sample sizes per individual. Defining repertoire sizes is an inherently difficult endeavor often influenced by bias due to human perception or classification approaches (i.e., based on context with subsequent quantification of the acoustic structure of resulting context types versus based solely on quantifying acoustic structure), as well as the statistical tools used. Based solely on the acoustic structure, savanna elephant rumbles can be categorized into three to six highly intergraded types [57,58]. Others argue that savanna elephant rumbles constitute a single call type with graded variation [59]. Rumble repertoire assessments that also take into account the contextual use and vocal production mechanism of rumbles have proposed up to 19 rumble types [40,60,61]. Addressing the problem of small sample sizes in wild elephants, one study focused on a family of 27 individuals [40] and, using step-wise discriminant function analyses, classified 14 of these apparent rumble types given in specific contexts well above that expected by chance (57–79%) (54–81% with cross validation) [40]. These results suggest that, despite the highly graded acoustic structure of savanna elephant rumbles, there appear to be rumble sub-types given within specific narrow contexts. Behavioral responses to playback experiments (e.g., [62]) add further support to this assertion.A quantitative assessment of the acoustic structure of forest elephant rumbles recorded at four sites across Central Africa using passive acoustic monitoring suggests that, compared to savanna elephants, the rumbles of forest elephants may exhibit a higher, but less modulated fundamental frequency, which reaches its maximum frequency earlier in the call and also exhibits higher formants [39]. Based solely on the acoustic structure, the results also indicate a possibly larger, but similarly highly graded, repertoire of rumbles. Using cluster analysis, forest elephant rumbles can best be classified into five to eight rumble types. While these rumble types were structurally highly graded, discriminant function analysis indicated they are predictable with a classification accuracy between 75% and 81% [39]. Interestingly, forest elephants used a larger set of seven rumble types when in a forest clearing compared to only four when in the forest, with four types observed exclusively in the clearing and one exclusively in the forest [39]. As passive acoustic recordings are entirely blind to the context in which recorded calls occurred, it remains to be ascertained how these categories reflect differences in age and sex of the callers or behavioral context.3.2. New FindingsOur new data on the contextual use of rumbles by forest elephants collected at Dzanga Bai indicate that forest elephants produce rumbles in seven broad distinct contextual categories when in the clearing (Table 1). Similar to savanna elephants, forest elephant rumbles can, to some degree, be assigned to these different contexts based on their acoustic structure. Discriminant function analysis assigned 56% of forest elephant calls to the correct context, with call duration explaining most variation between contexts. Linear regression analysis, controlling for a positive correlation between caller age and call duration, indicated context had a significant effect on rumble duration (likelihood ratio test; x2 = 84.39, df = 4, p < 0.001, N = 246 forest elephant rumbles). While rumbles given in a logistical context were on average the longest, they were not significantly longer than those produced during affiliation and separation. Rumbles produced by calves during nursing were on average the shortest rumbles, with no significant difference from rumbles given during competition, but both competition and nursing rumbles exhibited significantly shorter durations than those produced during affiliation, separation and logistics contexts (Figure 2, see Table S3 in Supplementary Material for detailed model output).Our analysis suggests that both species use rumbles in similar broad contexts, but at different rates. Savanna elephants in Amboseli appear to produce a higher proportion of rumbles in affiliative, logistics and sexual contexts, in contrast to the high proportion of rumbles emitted in separation and competition contexts by forest elephants at Dzanga Bai (Figure 3). Part of this discrepancy though may be due to the categorization of contact-rumbles under logistical context in savanna elephants, while calls given in a similar context may have been categorized under a separation or an unspecific context in forest elephants. Exactly equivalent events and calling patterns involving contact-rumbling in savanna elephants have not been identified in forest elephants. In savanna elephants, contact-rumbles are unlike the separated-rumbles made by calves who are lost and looking for their mothers. Since the separation context defined for forest elephants is largely composed of calls by calves who are separated from their mothers, we chose to compare these with separated-rumbles and retain the contact-rumbles in the logistics context for the savanna elephants. Rumbles used by both species in the same broad contexts appear to be structurally similar. In both forest and savannah elephants, rumbles associated with nursing were significantly shorter than other rumbles, while those associated with logistics were longer than those produced in other contexts (Figure 2, see Table S4 in Supplementary Material for model output).3.3. Formant ModulationMammalian vocal production follows principles similar to the production of human speech, conceptualized in the source-filter theory which decomposes the acoustic structure of vocalizations according to their mode of production [63,64]. A vocalization’s fundamental frequency reflects the frequency of the vibrations of the vocal folds (i.e., the source). As the sound subsequently travels through the vocal tract (i.e., the filter), the length and shape of the tract defines how energy is distributed, resulting in characteristic patterns of frequencies with particularly high energy, the so-called resonant frequencies or formants of the vocalization. Across mammals, including humans, body size correlates with fundamental frequency, formant frequencies and formant spacing [65,66,67,68,69], and fundamental frequency and its modulation vary with the motivational state of the caller, formalized by Morton (1977) as motivation-structural rules [70]. A hallmark of human speech is the intentional modification of the vocal tract independently of source-related components to produce vowels with distinct formant structure, enabling speakers to encode information about external entities independent of motivational state and body size [11,71]. Non-human vocal tracts were long considered uniform tubes allowing little variation in formant structure [72]. While more recent studies suggest that the vocal tract of monkeys can produce human-like vowels [16,73,74,75], and that variation in formant structure in monkey vocalizations can encode information about external contexts [76,77,78], it remains poorly understood if non-human mammals alter formant structure independently of fundamental frequency and motivational state. For instance, in Diana monkeys, formant structure varies independently of the fundamental frequency [78,79], whereas in baboons variation in formant structure is linked to fundamental frequency [75]. Moreover, it has been argued that even if correlated with fundamental frequency, formant modulation may not be a consequence of the motivational state alone [16]. A comprehensive simultaneous investigation of variation in formant structure and fundamental frequency in relation to body size, motivational state and the behavioral context in species exhibiting unusual flexibility in the size and shape of their vocal tract, such as elephants, is particularly suitable to further elucidate formant modulation in non-human mammals and the evolutionary origins of the mechanisms underlying speech production in humans.Elephants exhibit unique morphological adaptations of the hyoid apparatus and pharynx, which allows for exceptional flexibility in the shape of their unusually elongated and plastic vocal tract in form of the trunk [80,81]. Such flexibility does not only enable savanna elephants to produce idiosyncratic, novel and imitated sounds [40,82,83] but it may be critical to the formation of the species’ complex social organization. Savanna elephants critically rely on their low-frequency rumble vocalizations to coordinate interactions and stay in contact with other family groups over large distances [37,40,84]. Savanna elephants may intentionally facilitate such long-distance vocal communication by actively including the trunk in vocal production thereby lowering the formant structure of rumbles, which likely renders them less prone to attenuation during sound propagation [60]. However, the functions of formant modulation in savanna elephants are not completely understood, as the energy distribution of rumbles also varies with other contextual factors (as can be seen in [40]) including information about nearby threats [85]. Passive acoustic monitoring recordings used to quantify the acoustic structure of forest elephant rumbles indicate that similar to savanna elephants [60,84,85,86], forest elephant rumbles also exhibit two formants below 250 Hz [39]. In particular, the second formant showed a distinct bimodal frequency distribution, suggesting a formant modulation (possibly involving the trunk) as found in savanna elephants [60,84]. The results also suggest an intentional modulation of the second formant, because its center frequency varied independently of variation in the fundamental frequency, which is usually related to motivational state [39].3.4. Call CombinationHuman languages construct meaningful expressions through the flexible combination of acoustic units into more complex utterances, generally referred to as syntax. Phonological syntax (or phonology) constructs words out of acoustic units that are not themselves meaningful. Lexical syntax generates compositional messages (i.e., sentences) to which each unit contributes its own meaning [12,87,88]. Two types of syntax, analogous to phonological and lexical syntax in human language, were recognized by Marler [87]. Phonological syntax is found among various species ranging from birds to gibbons and was traditionally viewed as the simpler and evolutionarily older form [89], however, others suggest that lexical syntax, even though more restricted to non-human primates, may have evolved first [90]. Call combinations have rarely been investigated in detail in terrestrial mammals other than primates (but see [91,92]), and here, most studies have focused on predator alarm calls (e.g., [93]). Given the inherently social nature of human language, studying call combinations produced in social contexts seems critical. Investigating the potential for lexical and phonological syntax in the combinatorial calls of the two African elephant species, at different levels of social complexity, may shed light on the evolutionary sequence producing the two forms of syntax in human language.Both species of African elephants habitually combine their characteristic low-frequency rumble vocalizations with broadband calls (here loosely referred to as roars, Figure 1). Using forest elephant recordings conducted at two forest clearings using passive acoustic recordings, as well as recordings collected at several savanna elephant sites, Pardo et al. [43] compared the structure of these combinatorial calls between the two species. Forest elephants and savanna elephants appear to exhibit a similar repertoire of rumble-roar combinations, but forest elephants produced a higher proportion of combination calls than savanna elephants (forest elephants: 4–8% of calls, savanna elephant: 1–4%) and the proportion of different combination types differed significantly between the two species, the cause of which appears to be socio-ecological rather than phylogenetic [43]. In savanna elephants, combination calls were most often produced by immature individuals and least often by adult males and in diverse contexts, including agonistic, affiliative and sexual interactions, as well as related to nursing, separation and disturbance [43]. 3.5. New FindingsOur new data on the use of combinatorial calls by forest elephants at Dzanga Bai and additional data on savanna elephants from Amboseli reveal that, in accordance with findings by Pardo et al. [43], the use of call combinations, as opposed to stand alone roars, appears to be more prevalent across age/sex classes in the forest as compared to the savanna elephant (combination calls made up 60% of forest elephant roars and only 43% of savanna elephant roars; Figure 4). Our first comparison of the contextual use of roars and combination calls produced by adults of the two species suggests that roars, as well as combination calls of adult male elephants of both species are produced predominantly in competition contexts; however, adult male forest elephants appear to produce many more combination calls than adult male savanna elephants (Figure 5). Adult females of both species appear to produce roars and combination calls in a broader range of contexts than do adult males and more combination calls than stand-alone roars. As expected for syntactic systems, in females of both species combining a roar with a rumble appeared to modify the contexts in which roars were being used. However, contextual use of roars and combinations differed between the two species. In female forest elephants, the combination with a rumble appeared to increase the number of contexts in which a roar was produced. While roars were observed only in competition contexts, combinations of rumbles and roars were associated with five contexts, including competition as well as sexual, affiliative, separation and logistical contexts. In Amboseli adult female savanna elephants, roars and combination calls were predominately observed in a sexual context (78%, N = 32), but a more detailed look at the specific behavior reveals that combination calls also appear to modify the context. The majority of roars or combination calls given in a sexual context were by an estrous female being chased by a male (N = 18). Of these, 11 were roars. The remaining calls given in a sexual context were all combination calls and occurred in more social settings, following a mating (mating-pandemonium) or when females were greeting a musth male (female-chorus). Likewise, combination calls were also given in affiliative contexts and when a family was mobbing predators, while the remaining stand-alone roars were observed in a competitive context (Figure 5). 3.6. Environmental Constraints on Vocal Mediation of InteractionsVocal mediation of social interactions may facilitate the formation of complex social organizations, particularly when individuals cannot engage directly [4]. Estimating the distance over which vocalizations can be detected and interpreted is key to our understanding of the extent of such vocal coordination, but, effective detection distances have been determined for only a few species [94,95,96,97,98]. Detection distances are limited as sound experiences reverberation and absorption during propagation, leading to the attenuation and distortion of the acoustic signal. The strength of such degrading effects depends on frequency-related and temporal features of the acoustic signal in relation to environmental characteristics. For example, in forest habitats with dense vegetation, high pitched signals attenuate faster than they would in open habitats, such as savannas [99]. Savanna elephants appear to be able to recognize each other from the harmonic structure of some rumbles up to a distance of 2.5 km, enabling them to coordinate interactions over long distances and to form extensive vocal recognition networks [24,84]. Simulation models suggest that when conditions are optimal, savanna elephants may be able to detect rumbles over distances of up to 10 km [100]. Models of the attenuation of forest elephant rumbles based on amplitude measurements of rumbles recorded in a Central African rainforest suggest that forest elephant rumbles attenuate faster than savanna elephant rumbles [98]. Under optimal conditions when ambient sound is lowest, forest elephants may be able to detect a rumble of average fundamental frequency and source pressure level up to 3.2 km. However, under average ambient conditions, an average rumble would be completely masked by background noise at only 800 m, with the harmonic structure of the majority of rumbles attenuating after only 100 m. Such short detection distances suggest that the ability to coordinate interactions over long distances is limited in forest elephants which may severely constrain the maintenance of social complexity compared to levels documented in savanna elephants. Hedwig et al.’s [98] estimation of detection distance assumed a noise perception of forest elephants similar to that of other vertebrates. However, detailed information on hearing sensitivity and auditory filters is lacking for any species of elephants, and for frequencies below 100 Hz for terrestrial vertebrates in general, but critically needed to better understand the functioning of low-frequency rumble vocalizations within the elephant social systems.4. DiscussionThe social complexity hypothesis for communication states that the range and frequency of social interactions drive the evolution of complex communication skills enabling individuals to flexibly convey a wide range of information to maintain their social relationships (e.g., [3]). Intriguingly, the first insights into the vocal communication system of the elusive forest elephant suggest that African elephants may contradict the social complexity hypothesis. The socially less complex forest elephant appears to exhibit communication skills that are at least as sophisticated as savanna elephants, a species with one of the most complex social systems observed among mammals. Comparison based solely on the acoustic structure of rumbles of both species indicates a seemingly larger but equally highly graded repertoire of rumble types in forest elephants as compared to savanna elephants [39,57]. In addition, both species appear to exhibit communication skills that may increase the generativity of their vocal repertoires: the modulation of formant structure through the intentional inclusion of the trunk into vocal production [39,60,84] and the combination of roars with rumbles into contextually more complex call combinations [43,101].These preliminary findings suggest that the complex communication skills observed in savanna elephants may be ancestral traits that evolved in the context of a forest-dwelling last common ancestor, instead of constituting socio-cognitive adaptations to the environmental conditions of a savanna habitat. Our study modelling the attenuation of forest elephant rumbles suggests that sound transmission conditions in dense rainforests may inhibit long distance communication and, thus, vocal coordination of social relationships. As such, the potentially sophisticated communication skills of extant forest elephants, and likely those of their forest-dwelling ancestors, permit the transmission of exceptionally complex information, but only within the context of their small groups. Increased predation pressure by large pack-hunting mammals and seasonal abundance of food resources in the savanna may have driven the formation of larger social groups. The exceptionally low frequencies of rumble vocalizations and the power with which these sounds can be produced [37] are likely a byproduct of the elephants’ large body size. Powerful, very low frequency calls paired with reduced attenuation in the open savanna, may have allowed savanna elephants a larger spatial range over which information can be transmitted and interactions between groups mediated.Our first insights into the patterns of the vocal complexity in Loxodonta appear to challenge the social complexity hypothesis and offer an exciting field for future comparative research. The emergence of complex communication skills may be driven by other factors than social complexity alone [3], none of which, however, appear to be accountable for the complex communication skills in forest elephants. First, the decreased visibility in the rainforest would predict a less graded rumble repertoire in the forest elephants compared to the savanna elephant to reduce ambiguity in signal interpretation, but this is not evident [39]. Second, predation pressure may bring about an increase in repertoire size through the evolution of predator specific alarm call types [102] or call combinations [103]. Despite humans currently and historically being key predators of both species of African elephants, savanna elephants are assumed to experience higher predation pressure due to the presence of large pack hunting predators, which are absent in the rainforest environment. Recent research documents that variation in the acoustic structure of savanna elephant rumbles encodes context specific information about the presence of external threats [61,85]. Whether forest elephant rumbles encode similar information currently remains unknown. Third, a high overlap of a species’ ecological or acoustic niche with other species may drive signal diversity to enhance species recognition [104]. However, such overlap should be minimal, as elephants occupy an acoustic niche reaching into the infrasound range, largely inaccessible to the vocal production of most other sympatric species. Lastly, communicative complexity may emerge through neutral evolutionary processes and constitute the result of mutation and recombination independently of any social or ecological factors [105]. Phylogenetic analysis can be used to test for neutral evolution as closely related species are expected to exhibit similar levels of complexity compared to evolutionarily more distant species. The elephantid family consists of only three extant species, including the Asian elephant Elephas maximus. Results regarding the repertoire of combinatorial calls in the three species, however, do not indicate that neutral evolutionary processes have played a role as all three elephant species exhibit a similar repertoire size of call combinations [43]. Factors that remain underexplored as potential drivers of vocal complexity are life history traits and associated levels of parental care and social learning. Among terrestrial mammals, the forest elephant displays an exceptionally slow and low reproductive rate with a late median age of primiparity of 23 years and long median interbirth intervals of 5.6 years [106]. While reproductive rates generally correlate negatively with body size [107], forest elephants exhibit substantially lower reproductive rates and slower life histories compared to savanna elephants, despite their smaller body size [106]. As suggested for the long slow life histories characteristic for primates [108], these low reproductive rates may be driven by challenges associated with foraging in a highly variable forest environment on food sources, such as fruit with seasonally limited availability and patchy distribution, or foliage often high in toxins [106]. Slow life histories go hand in hand with extended parental care and a potentially high degree of social learning. The complex communication system observed in forest elephants may reflect the strong bonds between female forest elephants and their offspring which facilitate social learning needed to maximize survival in a highly complex environment. Similarly, strong social bonds have been put forth to explain complex cognitive abilities in some monogamous pair-bonded bird species [109].In our first comparison, the frequency of rumbling in different contexts showed a marked discrepancy between the two species of Loxodonta. Here, we speculate whether our observations represent a species- or rather site-specific distinction, shaped by environmental features particular to the sites. Observations of forest elephants took place at Dzanga Bai, a social arena in a dense forest where elephants gather in large numbers due to the distinct availability of minerals through monopolizable pits, whereas observations of savanna elephants took place largely in Amboseli’s open grasslands, with its saline soils and numerous swamps. These site-specific differences likely account for the large discrepancy between the frequency of rumbles given in a competitive context as competitive interactions constitute the most frequently observed social interaction between forest elephants at Dzanga Bai (unpublished data DH). Another large difference occurs in the context of separation. In both species, calves were responsible for the majority of calling in a separation context. It is possible that forest elephant calves spend more time further from their mothers or siblings than do savanna elephants because their survival is not threatened by large pack-hunting predators. While savanna elephant calves also habitually wander further from their mothers to socialize, Dzanga Baimay provide forest elephant calves with a perhaps unusual opportunity for social interaction and exploration. However, the large differences between the frequency of rumbles given in affiliative, logistical, and even sexual, nursing and anti-predatory contexts point more to species-specific differences. Given that savanna elephants have larger families and more frequent fission-fusion events [21,22,23], we would expect to see more frequent greetings and bonding events, and more calling related to the coordination of members’ movements and their location relative to one another in savanna relative to forest elephants. Yet, since forest clearings are assumed to constitute important social arenas [35], the degree of difference we observed between the two species is surprising. In addition, forest elephant females exhibit an older age of first reproduction compared to savanna elephant females (forest elephant: 23 years [106], savanna elephant: 14 years [110]), along with longer interbirth intervals (forest elephant: 5.6 years [106], savanna elephant:~4.5 years [110]) and generation lengths (forest elephant: 31 years, savanna elephant: 23 years [111]). As such, estrous behavior, mating and even nursing and weaning events would be expected to occur less frequently in forest elephants than among savanna elephants. Furthermore, the difference between the frequency of rumbles that occurred during anti-predatory events can likely be explained by the lack of large predators in the rain forest. In summary, we suggest that differences between the two species in the degree of fission-fusion sociality and life history parameters may account for more of the observed discrepancies in the frequency of calling by context than do site-specific differences.Conclusions are drawn regarding differences in vocal complexity exhibited by the two species of African elephants, however, they suffer from insufficient information on forest elephants and a lack of directly comparable data and methods for both species. As we are only beginning to understand forest elephant behavior, any conclusions regarding the level of vocal complexity in this species must be drawn with caution. Yet, the first insights presented here can serve as hypotheses guiding much needed future studies on the structure and contextual use of vocalizations of these elusive animals. Direct observations of forest elephants are difficult. The dense vegetation in the Central African rainforest poses extreme limitations in visibility and it is not possible to observe them from vehicles as is habitually done in savanna elephant studies. Forest clearings, however, such as Dzanga Bai where elephants venture out in the open and aggregate in large numbers, provide unique opportunities to observe forest elephant behavior from observation platforms [32]. Our results show that the Bai studies can provide comprehensive insights into the contextual use and structure of the vocal repertoire of forest elephants. However, our results are based on calls that we were able to hear and localize with sufficient confidence to define context, and, as such, low amplitude rumbles and those produced in subtle contexts currently remain unexplored. The development of advanced acoustic recording methods, such as localization of calls using acoustic arrays consisting of time-synchronized recording units, paired with simultaneous high resolution video recording will be imperative to advance our understanding of forest elephant rumbles. Not surprisingly, our work also demonstrates that not all aspects of forest elephant vocal communication can be observed at forest clearings. Bai aggregations represent highly social and unique contexts, where forest elephants spend only a fraction of time. A better understanding of forest elephant social and vocal communication critically relies on the development of innovative methods to study them in their forest environment. Passive acoustic recording in combination with camera traps as well as acoustic recorders on radio collars are promising, yet challenging, methodological avenues that require further development.If comprehensive studies of forest elephants are a challenge, so are conducting meaningful comparisons with savanna elephants. The recently released The Elephant Ethogram, an extensive repository of savanna elephant behavior [41], provides an excellent reference point for forest elephant observational studies and the groundwork for future comparative work on the contextual use of rumbles in both species. Our comparison of the contextual use of rumbles and rumble-roar combinations in the two species, however, highlights how site- or population-specific features could lead to erroneous conclusions regarding species-level differences. In addition, despite the considerable number of insightful studies dedicated to savanna elephants, various aspects regarding their vocal communication require more research. Conclusions drawn about the importance of rumble vocalizations in the maintenance of relationships in a fission-fusion setting are largely based on playback experiments simulating optimal sound transmission conditions [24,84,112] and focused on specific rumble types, such as contact and estrous rumbles. Attenuation models or playback experiments considering the natural range of conditions under which vocal communication may take place in savanna elephants are needed to get a more realistic view of the role of long-distance communication in facilitating their complex social organization [98].5. ConclusionsFirst insights into the vocal communication system of the elusive forest elephant suggest complex communication skills. These findings contradict the social complexity hypothesis, which predicts a less complex communication system in the forest elephant compared to the socially highly complex savanna elephants. We propose the exceptionally slow and low reproductive rate and associated high levels of parental care in forest elephants as drivers of vocal complexity in this species. However, conclusions regarding differences in vocal complexity between the two species need to be drawn cautiously. A better understanding of vocal complexity in the genus Loxodonta will depend on continuing advancements in remote data collection technologies, such as passive acoustic recording and camera trapping, to overcome the challenges of observing forest elephants in their dense rainforest habitat. Finally, inconsistent methodologies currently strongly inhibit direct comparison between forest and savanna elephant vocal behavior. Studies based on directly comparable data and methods, quantifying both structural and contextual variability in the production of rumbles in both species are critically needed to understand patterns of vocal complexity within the genus Loxodonta, and beyond.

animals : an open access journal from mdpi

10.3390/ani13061056

PMC10044621

The paper describes a study to assess the stability of the genomes of bovine embryo transfer recipients following in vitro fertilization using cytogenetic tests and to analyze the effects of selected vitamins and micro- and macroelements on genome integrity. Genome stability was analyzed using the sister chromatid exchange, fragile site, and comet assays. The effects of selected micro- and macroelements and vitamins on the levels of chromosomal instabilities generated in the cows were analyzed.

Genome instability can lead to a wide variety of diseases. Many endogenous and exogenous factors influence the level of damage to genetic material. Genome integrity depends on factors such as the fidelity of DNA replication, normal DNA organization in the chromosomes, and repair mechanisms. Genome stability influences fertility, embryonic development, and the maintenance of pregnancy. In the case of in vitro fertilization, it can be an important factor determining the success of the procedure. The aim of the study was to assess the stability of the genomes of recipient cows following in vitro fertilization using cytogenetic tests and to analyze the effects of selected vitamins and micro- and macroelements on genome integrity. Genome stability was analyzed using the sister chromatid exchange, fragile site, and comet assays. The material for analysis was peripheral blood from 20 Holstein-Friesian heifers that were embryo transfer recipients. The effect of selected micro- and macroelements and vitamins on the genome stability of the cows was analyzed. Folic acid was shown to significantly influence the level of damage identified using the SCE, FS, and SCGE assays, while iron affected SCE and SCGE results, and zinc affected FS.

1. IntroductionChromosome instability is a form of genome instability indicating increased chromosome damage [1]. Genome integrity is continually subject to disturbances by exogenous and endogenous factors, which can cause various changes in DNA, leading to chromosome damage. Maintenance of genome integrity is crucial in every species for the normal functioning and survival of the organism and the complete transfer of genetic potential to the next generation [2]. Genome stability depends in part on normal DNA organization in the chromosomes, the fidelity of DNA replication, mechanisms responsible for controlling stability, and DNA damage repair pathways. Chromosome instabilities are caused by disturbances in the pre-mitotic phase of the cell cycle, but are also the consequence of the damage and rearrangement of chromosomes resulting from an abnormal cell response to DNA damage or abnormal DNA synthesis [3,4]. Difficulties in analyzing chromosome instability stem from its unclear molecular background. It may result from the effects of non-genetic factors such as physiological processes, e.g., ageing, hormone balance, inflammation, and metabolism, as well as various environmental factors, such as diet.Vitamins and macro- and microelements help to maintain genome stability and integrity and are an important element of genetic and anti-carcinogenic prevention, because they influence gene expression and the DNA structure [5,6,7]. This is why the quantity and quality of nutrients that influence gene expression and DNA synthesis and repair are so important. Micro- and macroelements and vitamins are cofactors of enzymes catalyzing DNA replication, methylation, and repair [8]. In dairy cattle, they have a major influence on reproduction and productivity [9]. Both deficiencies and surpluses negatively affect balance in the body, disturbing regulatory functions in metabolic processes [10]. Calcium (Ca) is the mineral present in the highest quantities in vertebrates. It is an essential element regulating numerous physiological processes. It activates enzyme activity and has anti-tumor, antiproliferative, proapoptotic, and antimutagenic properties [11]. It takes part in cell division and specialization and in sperm activity, plays an essential role in the fertilization of the ovum, and inhibits chromosome breakage [7,11,12]. Iron (Fe) is another essential microelement for cell function, including the storage and transport of oxygen and numerous enzymes. It supports immune processes in the body and the production of secondary metabolites [13]. It takes part in catabolism and the synthesis of numerous compounds, including nucleic acids; prevents the oxidation of nitrogen bases and the breakage of DNA strands; and regulates the life cycles of cells and the expression of certain genes [14,15]. It is a cofactor of enzymes that break down reactive oxygen species (oxidase, peroxidase, and catalase), protecting cells against oxidative stress, which disturbs the progress of replication and leads to the formation of DNA breaks [16]. Zinc (Zn) influences numerous physiological and metabolic processes and reproduction in animals [9,17]. It takes part in DNA replication, the synthesis of DNA, RNA and proteins, and gene expression [18,19,20]. It is also an essential element for cell proliferation and differentiation, preventing cancer and other diseases [9,20,21,22]. Moreover, it protects the body against the adverse effects of free radicals, preventing DNA lesions and breaks [23]. Vitamins are also very important functional substances, which are essential to maintaining health and normal productive, reproductive, and growth parameters, and, in pregnant cows, they prevent embryo death and developmental defects [20]. Vitamins B9 (folic acid) and B12 (cobalamin or cyanocobalamin) prevent DNA strand breaks and the oxidation of bases and are involved in the synthesis of nitrogen bases and in DNA transcription and repair. Anomalies in these biological functions result in an increased number of lesions in genetic material and a reduction in DNA methylation, which can lead to developmental defects and carcinogenesis. Folic acid has a strong influence on genome stability. A deficiency of folic acid in the diet leads to errors in DNA synthesis, changes in the degree of methylation, and chromosome breaks. This is why a suitably balanced diet for pregnant cows is so important [24,25,26,27,28]. Sites in chromosomes with CGG repeats are particularly sensitive to folic acid deficiencies, which can lead to the defective segregation of chromosomes in these sequences and even cause instability of the entire chromosome [29,30]. A B9 deficiency leads to a number of disorders of the nervous and digestive systems, inhibition of cell growth and regeneration, reduced milk yields in cows, chromosomal disorders, the transfer of abnormal amounts of DNA, and fetal defects [25,26,27,28]. Vitamin B12 is closely correlated with folic acid [31]. It plays an important role in cell growth and development by participating in numerous reactions and processes in the body. In combination with folates, it influences hematopoietic processes, DNA synthesis, and the methylation of DNA and RNA. It also has anti-tumor effects and additionally takes part in erythropoiesis; the metabolism of carbohydrates, fats, and nucleic acids; and neuron function, regulating mental processes [32,33]. Vitamin B12 deficiency leads to neurological disorders, apathy, loss of appetite, anemia, reduced body condition and productivity, and DNA damage [34]. Excessive intake of concentrate feed increases the concentration of propionic acid in the rumen, which prevents the production of adequate amounts of cobalamin. Micro- and macroelements and vitamins are important regulators of DNA synthesis and repair. This is why a suitably balanced diet with the most important minerals and vitamins ensuring genome stability is so important. Cytogenetic tests are sensitive methods for detecting chromosome damage, providing information about genome stability. They include the sister chromatid exchange assay, fragile site assay, and comet assay. They also provide information about the potential for the repair of DNA damage, the control of the entire cell cycle, and the effects of malfunctioning cellular mechanisms responsible for maintaining stability. The sister chromatid exchange assay is used to quantify genetic material exchanged between sister chromatids during mitosis. The test makes it possible to detect DNA damage in the form of single- and double-strand breaks induced by mutagenic and genotoxic factors [35]. A sister chromatid exchange (SCE) is associated with malfunctioning repair pathways [36], leading to the fragmentation of sister chromatids as a result of DNA strand breakage and resealing. This rearrangement is accompanied by the exchange of regions of the parent strands in the duplicated chromosomes between sister chromatids, which is possible owing to cohesion between chromatids [37,38]. Fragile sites (FS) in chromosomes are sites that are especially sensitive to breaks, gaps, and constrictions under replication stress [39]. They are the consequence of malfunctioning mechanisms of repair of disturbances in the progression of replication forks during replication and transcription [36]. The lack of complete replication and repair of DNA damage at FS results in the transfer of genome damage and instability between generations of cells [39]. FS are regarded as hotspots of chromosome instability and rearrangement in cancer [24]. Depending on their frequency in the population and means of induction, rare fragile sites (RFS) and common fragile sites (CFS) are distinguished [40,41]. The former, which occur in up to 5% of the population, are inherited according to the laws of Mendel and are classified based on their sensitivity to a lack of folic acid (folate-sensitive vs. non-folate-sensitive) [41,42,43]. They most often take the form of numerous trinucleotide repeats, or, less often, di- or tetranucleotide repeats. For folate-sensitive fragile sites, they are CCG trinucleotide repeats, while, in the case of non-folate-sensitive FS, they are AT-rich repeated sequences. The high elasticity of repeated sequences affects the dynamics of replication, reduces the effectiveness of links between nucleosomes, and leads to the decondensation of genetic material [41,43]. Repeated nucleotide sequences are able to form secondary and tetrahelical structures that block replication forks, resulting in delayed replication [42,43,44]. CFSs are an integral element of the chromosome structure. They occur in the genome spontaneously and affect large genomic regions consisting of hundreds or even thousands of kilobases [40,45,46]. They include AT-rich nucleotide sequences without repeats, do not show a tendency to expand, and are arranged in the form of islands, which increases the elasticity of the sequences [42,47]. As in the case of RFSs, in CFSs, secondary structures also cause disturbances in replication and higher-order chromatin organization [43,46]. The comet assay, i.e., single-cell gel electrophoresis (SCGE), is a rapid and sensitive technique for the identification of DNA degradation in individual cells. An advantage of this method is that it can detect various types of errors, e.g., DNA single- and double-strand breaks, alkali-labile sites, DNA single-strand breaks associated with incomplete excision repair sites, and apurinic/apyrimidinic sites. The comet assay therefore identifies many types of DNA damage leading to instability and mutations induced by genotoxic and mutagenic stress factors, such as ionizing radiation [48,49,50,51,52]. A major advantage of this test is that a great many individual cells can be analyzed, which enables a more precise analysis of instabilities in the genetic material [53].Eating disorders deregulate genetic, epigenetic, and epigenomic mechanisms. Improper intake of minerals, vitamins, and, importantly, folates is correlated with abnormalities in fetal programming, an increased risk of complications during fetal development, and the occurrence of diseases in adult life [54]. Disproportions in nutrients and deficiencies of vitamins and minerals can lead to functional disorders in the adult animal and developmental disorders in the fetus obtained from in vitro fertilization. Assisted reproductive techniques are used not only in cases of infertility, but also to increase reproductive performance and accelerate breeding progress. This is particularly important in cattle, due to the relatively long generational interval in comparison with other livestock species. Therefore, in vitro fertilization is a very important issue in breeding. It makes it possible to produce embryos of high genetic value, combat reproductive disorders, and predict fertility. The OPU/IVP (ovum pick-up/in vitro production) method consists of numerous stages: the acquisition of male and female gametes, bringing them to fertilization capacity; the fusion of the oocyte with the spermatozoon via IVF (in vitro fertilization) or microinjection of the spermatozoon into the cytoplasm of the oocyte; the in vitro culture of the embryo; the evaluation of the embryo up to the morula and blastocyst form; and the transfer of the embryos into the recipient [55,56]. An increasing number of breeders are using modern reproductive techniques in their herds (e.g., sexed semen, embryo transfer, or in vitro fertilization). For the process to be successful, each step is controlled regarding the transfer and deposition of the embryos in the reproductive tract of the cow [56].Genetic and nutritional factors have a major impact on processes. In recipients, these factors affect the maintenance of the pregnancy and the unassisted birth of healthy calves [57]. The choice of recipients with a stable genome and optimal levels of micro- and macroelements and vitamins in the body vastly improves the chance of obtaining healthy calves and future adult animals [10,58]. The analysis of genome stability using cytogenetic techniques and the assessment of levels of micro- and macroelements and B vitamins makes it possible to monitor the health potential of recipient cows and their calves, and thus the cows’ diet is an important element in the preparation of cows for fertilization. The aim of the study was to assess the genome stability of bovine embryo transfer recipients following in vitro fertilization using cytogenetic tests and to analyze the influence of selected vitamins and micro- and macroelements on genome integrity.2. Materials and Methods2.1. AnimalsAll experiments were conducted in accordance with the recommendations in Directive 63/2010/EU and the Journal of Laws of the Republic of Poland of 2015 on the protection of animals used for scientific or educational purposes. The study was approved by the Polish Laboratory Animal Science Association (nos. 3235/2015 and 4466/2017).The research material was peripheral blood drawn from the tail veins of 20 Holstein-Friesian cows at the age of 18–20 months. The blood was obtained from bovine embryo transfer recipients following in vitro fertilization, in roughly the third month of gestation. All cows were from the same herd.2.2. Cell Culture Peripheral blood lymphocytes were cultured in vitro in Lymphogrow growth medium for 72 h at 38.5 °C (5% CO, with stable humidity). At 69 h of culture, colchicine was added (2.5 µg mL−1). At 24 h, 5-bromodeoxyuridine (BrdU) was added to the cultures intended for SCE assays (10 µg mL−1), and at 65 h, BrdU was added to the cultures for the FS test (5 µg mL−1). Potassium chloride (0.65% KCl) was used as a hypotonic solution. The cells were fixed with Carnoy fixative (3:1 methanol–acetic acid).2.3. Sister Chromatid Exchange AssayThe FPG technique [59] was used to detect sister chromatid exchanges in the following steps: digestion with 0.01% RNase, incubation in a solution of 0.5 × SSC (sodium chloride + sodium citrate; pH = 7.0) with Hoechst 33258 solution, UV irradiation twice, overnight incubation at 4 °C, incubation at 58 °C, and 4% Giemsa staining. Stained sister chromatid exchanges were counted in 20 metaphases from each individual.2.4. Fragile Site AssayMicroscope slides for the identification of fragile sites were prepared using the technique of differential staining chromosomes in the following steps: incubation in Hoechst 33258 solution (1 mg/100 mL), UV irradiation, incubation in 2 × SSC, and 4% Giemsa staining. Twenty metaphases were examined from each individual. Chromatid breaks, chromatid gaps, and chromosome breaks were identified.2.5. Comet AssayThe single-cell gel electrophoresis (SCGE) assay (comet assay) was performed on microscope slides [60]. Lymphocytes were isolated with Histopaque-1077. Slides coated with a layer of 0.5% normal melting point (NMP) agarose gel were spotted with lymphocytes mixed with 0.5% low melting point (LMP) agarose gel and then embedded in LMP agarose. Samples prepared in this manner were subjected to alkaline lysis (2.5 M NaCl, 100 mM Na2EDTA, 0.4 M Tris-HCl, 1% sodium N-lauroylsarcosinate, 10% Triton X-100, 1% DMSO, pH = 10) to release DNA from the cell and remove proteins. This was followed by alkaline denaturation in electrophoresis solution, neutralization with Tris-HCl, and staining with ethidium bromide. DNA integrity was determined on the basis of the percentage content of DNA in the tail (%T DNA) of the comet. Fifty cells were analyzed for each animal. Changes observed in cells were classified according to Gedik’s scale: N—no DNA damage or less than 5% damage in the comet tail; L—low level of damage (5–25%); M—moderate damage (25–40%); H—high level of damage (40–95%), and T—over 95% DNA damage [61].2.6. Analysis An Olympus BX50 microscope was used for microscopic analysis. The MultiScan image analysis software from Computer Scanning Systems was used to analyze chromosome damage identified in the form of sister chromatid exchanges and fragile sites. The CASP 1.2.2 software [62] was used to analyze degraded DNA of lymphocytes identified by the comet assay. Levels of micro- and macroelements (Ca, Fe, and Zn) and vitamins (B9 and B12) in the blood serum were tested by a commercial veterinary laboratory. The results were presented in a table. The reference values for the minerals and vitamins were Ca 2.3–2.8 mmol/L; Fe 25–35 µmol/L; Zn 10.7–19.9 µmol/L; B9 17–24 ng/mL; B12 150–200 pg/mL [63].Statistical analysis of the results was performed using STATISTICA 12.5 MR1 PL software. The effect of the level of vitamins (B9 and B12) and minerals (Ca, Fe, and Zn) on the frequency of chromosomal instabilities (SCE, FS, and SCGE) was assessed. Means between individuals within groups were compared by one-way ANOVA. Significance of differences between means for a given type of instability within factors was assessed by Tukey’s test (p < 0.05). In addition, the relationships between the content of vitamins and minerals and the level of damage were tested by correlation analysis. Simple regression equations were built for significant relationships. All calculations were performed for p < 0.05.3. ResultsThe study evaluated the genome stability of cows following in vitro fertilization. Figure 1 present images of metaphase chromosomes tested by the SCE and FS assays and nuclei of lymphocytes tested by the comet assay. In addition, the blood serum of the cows was analyzed for the content of Ca, Fe, Zn, and vitamins B9 and B12, and the effect of the micro- and macroelements on the integrity of the genetic material was assessed. The average frequency of SCEs in the cows was 5.0 ± 7.9 SCEs/cell. Differences were shown between cows in the average frequency of SCEs, including statistically significant differences between the cows with numbers 6, 7, and 17 and those with numbers 2, 3, 8, 9, 11, and 16. The most SCEs were noted in cow no. 16, and the fewest in no. 17 (Table 1). The average frequency of FS was 3.2 ± 1.1 FS/cell. Differences in FS frequency were shown between cows, but were statistically significant only between no. 16 and all other cows. The frequency of this type of damage was highest in cow no. 16 and lowest in nos. 1 and 17 (Table 1). The average %T DNA in the cows was 3.8 ± 1.4. Differences were found in the frequency of damage identified by the comet test, including statistically significant differences, e.g., between no. 1 and nos. 3, 11, and 16. The most damage was observed in cow no. 16, and the least in no. 17 (Table 1). The low frequency of damage to both DNA and chromosomes in the cattle suggests that the animals’ genome was stable. Based on an additional criterion, i.e., Gedik’s scale, the animals were classified as N (14 cows) or L (6 cows), with a low level of DNA damage. N indicates no DNA damage or less than 5% damage in the comet tail, while L represents a low level of damage (5–25%).Table 2 presents analyses of minerals and vitamins. Serum levels of Ca in all cows were within the reference range, averaging 2.5 mmol/L. The average serum concentration of Fe was 34.8 µmol/L. The Fe level was elevated in eight cows (nos. 2, 4, 8, 9, 10, 11, 13, and 20), but within the reference range in the others. The average Zn level was 14 µmol/L. The cows with nos. 11, 13, 16, and 18 had low Zn values, below the reference range, while cows 1, 3, and 5 had elevated values. The average content of vitamin B9 in the blood of the cows was 12.4 ng/mL. Vitamin B9 levels were outside the reference range in most of the individuals; only cow no. 17 had a correct concentration. Four cows (nos. 1, 2, 6, and 7) had elevated levels of vitamin B9, while its level was low in the others. The average vitamin B12 level in the blood of the cows was 151.1 pg/mL. Only cows no. 8 and 9 had correct concentrations of this vitamin, while, in all the others, it was low. Analysis of variance showed that the number of SCE, FS, and SCGE instabilities depends on the concentration of vitamin B9 in the blood. The highest levels of SCE, FS, and SCGE instability were noted when the B9 level was very low, while the least damage was detected when its content was within reference values. Analysis of the correlation between the vitamin B9 concentration and the number of lesions showed a significant negative correlation (Table 3). As the vitamin B9 concentration in the blood increased, the number of instabilities decreased. A 1 ng/mL increase in the vitamin B9 level caused a decrease in the level of damage by 0.06 for SCE, 0.038 for FS, and 0.206% for SCGE (Table 4).The iron level in the blood did not significantly influence the level of SCE and FS instability, but significantly influenced the level of damage detected by SCGE. The number of lesions detected in SCGE was highest when the iron level was higher than the reference values and lowest when it was below the reference values. Analysis of the correlations between the iron content in the blood and the level of instability showed a positive correlation between the concentration of this microelement and SCE and SCGE damage (Table 3). A 1 µmol/L increase in the iron level was associated with a 0.046 increase in SCEs and a 0.21% increase in the SCGE result (Table 4). In the case of zinc, analysis of variance showed a significant relationship between the number of FS and the content of this element in the blood. Deviations from the reference values (above or below) were associated with an increase in the level of chromosome damage. Zinc levels in the blood did not significantly influence the number of SCE and SCGE instabilities. Analysis of the correlation between the zinc level in the blood and the level of damage to genetic material showed no significant relationships (Table 3). Analysis of variance and correlations showed no significant relationships between concentrations of vitamin B12 and calcium in the blood and the level of SCE, FS, and SCGE damage. 4. DiscussionCattle are the most widespread species of livestock. Intensive evaluation of the use value and breeding value has resulted in a vast increase in milk performance. Selection for high yields has unfortunately been carried out at the cost of health and reproductive parameters [64]. A milestone in the process of improvement of cattle was the development of biotechnological breeding methods (e.g., cryopreservation of semen, artificial insemination, in vitro fertilization, and embryo transfer), resulting in healthier animals with high milk yields [65]. A helpful tool for achieving this goal is in vitro fertilization, which makes it possible to select donor cows with the desired traits [57,66,67]. Maintenance of pregnancy and the birth of a healthy calf are associated not only with the recipient cow’s physical condition, but also determined by the integrity of the genetic material. Studies assessing genome stability in cows used in assisted reproduction, e.g., in vitro fertilization, can make it possible to evaluate and select the best cows, i.e., those with high genome stability. Studies analyzing chromosomal instabilities in cattle most often focus on numerical and structural mutations in chromosomes [3,68,69,70]. There are fewer studies on chromosomal instabilities resulting from errors in replication, transcription, or malfunctioning repair mechanisms and control points tasked with catching these errors. They can be identified using cytogenetic tests, such as the sister chromatid exchange, fragile site, and comet assays. These are extremely valuable tools for evaluating animals, because they are highly sensitive and provide information about malfunctions in a number of important cellular processes responsible for maintaining genome stability and integrity [71]. According to Danielak-Czech and Słota [72], Nino-Soto and King [73], and Danielak-Czech et al. [74], instabilities negatively affect reproduction in cattle by extending the calving interval, reducing the effectiveness of artificial insemination, or causing the loss of embryos. Luna et al. [75] also found that a high number of chromatid breaks and gaps causes reproductive problems in cows. Many mutagenic, genotoxic, and carcinogenic factors negatively affect animal health [76,77]. Abnormalities occurring during cell division can generate various forms of damage to genetic material, resulting in abnormally developed embryos, which die in the early stages of embryonic development [78,79]. Abortion results in new, delayed estrus, prolonging the calving interval and thus generating further economic losses for the breeder [69]. The group of cows analyzed in the present study was homogeneous in terms of age, breed, and location. According to researchers, these factors significantly influence the frequency of instabilities [36,80,81,82,83,84,85]. Therefore, the results obtained are a reliable indicator of the level of genome stability in the recipient cows, following the exclusion of the above-mentioned factors that negatively affect genome integrity. The level of damage observed in our study was low (SCE 5.0, FS 3.2, and %T DNA 3.8). According to Azimi [86], the average frequency of SCEs for healthy cattle ranges from 5 to 14. Deviations from this standard indicate pathological changes in the body [87]. According to Di Meo et al. [88,89], Peretti et al. [90], and Wójcik et al. [91], the incidence of SCEs is characterized by species conservatism. Our analysis of various studies revealed that the frequency of spontaneous SCEs in cows ranges from 3.2 to 8.3 [80,82,85,86,92]. This wide range of averages is influenced by the breed of cow. Lower SCE values have been observed in indigenous cow populations, while the frequency of these instabilities in Holstein-Friesian cows was 5.1, 6.8, 7.1, 7.1, and 8.3 [80,85,86]. The mean SCE values obtained in our study are lower than in those cited above, which indicates high genome stability in the recipient cows. Unfortunately, there are no published reports of the results of the identification of SCE, FS, and DNA analyzed by the comet test in cows impregnated following in vitro fertilization. Fragile sites have been identified in cattle chromosomes by Peretti et al. [71], Wójcik and Szostek [85], Danielak-Czech and Słota [93], Di Meo et al. [94], and Genualdo et al. [95]. The frequency of spontaneous FS ranged from 0.21 to 3.0/cell. The average FS frequency of 3.2 obtained in our study is similar to the results reported by Wójcik and Szostek [85] (3.5), Danielak-Czech and Słota [93] (3.0), and Rodriguez et al. [96] (2.5). There is also very little information on the use of the comet test in cattle. It has been used in genetic toxicology monitoring, e.g., of the effect of ivermectin, copper, or lead on the DNA structure [97,98,99,100], and to identify damage to genetic material induced by oxidative stress in cow embryos [101] and in cows with Bovine papillomavirus [102]. Tharwat et al. [103] also used SCGE to investigate apoptosis in the peripheral blood cells of dairy cows three weeks before expected parturition, during the week of parturition, and after three weeks. They observed a higher frequency of DNA damage in the week of parturition and three weeks after parturition than during pregnancy. According to the authors, these findings are explained by endocrine changes, immunosuppression in the peripartum period, changes in diet, metabolic and immune disorders, and stress factors associated with the change of location in the cowshed.Genome stability is determined by many traits, as well as by exogenous factors such as diet [84]. Pregnant cows should have a suitably balanced diet, which is a key factor influencing the levels of micro- and macroelements and vitamins in cattle. Supplementation reduces the risk of stillbirths and disease in the mothers [104]. Failure to monitor the composition of the feed and blood biochemical parameters when correcting deficiencies, or the use of feed additives that are not adjusted to the animals’ needs, can disturb homeostasis and may also cause toxicosis during pregnancy, leading to abortion or stillbirths [10,105]. Deficiencies of vitamins and minerals, especially Zn, are especially dangerous. Embryo transfer to heifers with inadequate concentrations of these elements, or of total protein, urea nitrogen, albumins, and beta-carotene, accompanied by an elevated total bilirubin concentration and excessive nitrogen intake, is associated with problems with embryonic development and implantation. Disorders of fat metabolism reduce the secretion of progesterone, which is responsible for maintaining pregnancy [105]. The major organs in the fetus are formed in the first few months of pregnancy. This is why it is so important to provide the mother with a suitably balanced diet, to avoid deficiencies that could adversely affect the stability of the genetic material of the mother and fetus [105,106,107]. Minerals such as calcium, iron, and zinc are important elements influencing physiological functions, genetic resistance, and reproductive functions [108]. In the present study, all cows had normal serum levels of calcium. A correct Ca level has a positive effect on genetic resistance and productivity, but its quantity is also correlated with the bioavailability of zinc [11,109]. In our study, four recipient cows had low Zn levels, and much higher frequencies of SCEs were shown in these cows in comparison to the others. According to Gressley [9], Seyrek et al. [18], and Omur et al. [20], Zn is an essential element preventing oxidative stress, which adversely affects the progress of replication forks and causes DNA single- and double-strand breaks. The SCE assay proved to be a highly sensitive diagnostic tool in detecting the negative effect of Zn deficiency on the cows’ genetic material. According to Mirowski [19] and Meglia et al. [21], Zn deficiencies can be a consequence of increased stress but also of an unbalanced diet and colostrum production. Excess zinc can lead to tumor formation and reduce the bioavailability of calcium, iron, copper, and phosphorus [110]. In our study, cows with elevated Zn had slightly lower levels of Ca and Fe than other cows, but the values were within the reference ranges. No negative effect of the elevated Zn level on the genetic material was observed. Some cows had elevated Fe levels in the blood. As the Fe level increased, the frequency of damage detected by the SCE and comet assays increased as well. Sanders et al. [100] also used the comet assay and other molecular techniques to determine that excess Fe contributes to oxidative stress, cytotoxicity, and genotoxicity in cells and identified an increase in DNA damage. A low level of Fe, according to Regmi and Dhakal [108], causes reproductive failure, e.g., embryonic death.Group B vitamins are crucial to preserving genome stability. In the present study, the level of vitamin B9 was normal in only one cow; it was elevated in four and low in the others. In the case of B12 as well, only two cows had normal concentrations of this vitamin, while in the others, it was below the norm. Folic acid is an essential nutrient that plays a crucial role in immune processes and prevents disease in pregnant animals. In the present study, as the vitamin B9 concentration increased, the frequency of chromosomal instabilities identified by the SCE, FS, and comet assays decreased. Folic acid plays an important role in the expression of genes, including those responsible for immunity [28]. It takes part in DNA synthesis, transcription, and repair. The consequences of disturbances in these biological functions include an increased frequency of damage to genetic material and a reduced level of DNA methylation [7,31]. Cobalamin is closely associated with folic acid. Girard and Matte [31,111] reported that cows with low serum levels of B12 did not react positively to supplementation with folates. According to Mirowski [112], low levels of this vitamin adversely affect reproduction, metabolism, and health status in cows. Khan et al. [27] also claim that folic acid and vitamin B12 are essential nutrients that influence the level of metabolic and immune stress during pregnancy and the peripartum period. According to Mirowski [19] and Kincaid and Socha [113], levels of vitamins and minerals in advanced pregnancy are varied, e.g., the level of zinc in the blood falls as pregnancy advances, possibly due to impending parturition and lactation. Therefore, it is essential to monitor micro- and macroelements and vitamins in pregnant cows at various stages of advancement of pregnancy.5. ConclusionsThe analyses showed which of the vitamins and minerals affect the stability of genetic material. The level of chromosome instability detected by the SCE, FS, and SCGE assays significantly depends on the level of vitamin B9 in the blood. As its content increases, the level of damage decreases. SCE and SCGE instability also depends on the iron level in the blood. The number of FS significantly depends on the zinc concentration in the blood; deviations from reference values of zinc cause an increase in the level of this type of damage. Higher content of this element increases the number of these lesions. Zinc content did not significantly increase the number of SCE and SCGE instabilities. No relationship was found between the level of SCE, FS, or SCGE chromosome instabilities and the concentration of calcium or vitamin B12 in the blood.

animals : an open access journal from mdpi

10.3390/ani11041176

PMC8074599

The continuous development of innovative technologies and the large-scale implementation of these solutions on farms are dynamically influencing the so-called precision livestock farming—PLF. Pig producers striving to increase the profitability of production, food safety, and food itself are increasingly willing to invest in rationalisation systems that raise the technological standards of pig breeding. The use of modern systems in livestock management and animal welfare is based on the use of non-invasive monitoring devices such as cameras, microphones, or detectors and information technology-based data archiving and management systems that support farmers/breeders in the daily running of the farm. Precision farming technologies, which are beneficial for animal welfare as well as for the profit of the livestock producer, help to solve the problems of large-scale animal production and satisfy the expectations of food regulators and consumers themselves. The aim of the paper was to gather contemporary knowledge on innovative technologies applied on pig farms. The paper presents and compares methods of controlling herd behavioural parameters with the use of various monitoring systems and their purpose. The paper also includes a review of potential limitations that may occur in the daily use of the above-mentioned devices. The review presents results on the effectiveness of their use.

In recent years, there have been very dynamic changes in both pork production and pig breeding technology around the world. The general trend of increasing the efficiency of pig production, with reduced employment, requires optimisation and a comprehensive approach to herd management. One of the most important elements on the way to achieving this goal is to maintain animal welfare and health. The health of the pigs on the farm is also a key aspect in production economics. The need to maintain a high health status of pig herds by eliminating the frequency of different disease units and reducing the need for antimicrobial substances is part of a broadly understood high potential herd management strategy. Thanks to the use of sensors (cameras, microphones, accelerometers, or radio-frequency identification transponders), the images, sounds, movements, and vital signs of animals are combined through algorithms and analysed for non-invasive monitoring of animals, which allows for early detection of diseases, improves their welfare, and increases the productivity of breeding. Automated, innovative early warning systems based on continuous monitoring of specific physiological (e.g., body temperature) and behavioural parameters can provide an alternative to direct diagnosis and visual assessment by the veterinarian or the herd keeper.

1. IntroductionIn recent years, the world has seen rapid changes in the dynamics and efficiency of pig production. The general trend of increasing production, with reduced employment, requires optimisation and a comprehensive approach to herd management [1]. Modern pig production should therefore be based not only on a modern infrastructure and a precisely designed feeding program, but also on the use of modern technologies for monitoring health and welfare of the entire herd [2,3,4].Herd health programs for swine include biosecurity, routine health control, and other preventive procedures allowing maintenance of a high health status of pig herds [5]. The health of pigs on the farm directly translates into the economics of production. Diseases lead to higher morbidity and mortality in different age groups and higher veterinary costs for the purchase of medicines and vaccines and more frequent veterinary visits [6,7]. Herds with low health status are also characterised by low productivity, reduced growth, and higher feed consumption. As a consequence, the fattening period is extended and the production efficiency is decreased [8].Productive performance of pigs is a reliable indicator of the efficiency of production under different housing conditions [9,10]. The criteria for assessing animal welfare cover even more characteristics, including indicators of health and ethological parameters [11]. In practice, it is difficult to identify one basic and easy-to-use measure, which demonstrates the imperfections of each indicator and, on the other hand, the complexity of the concept of welfare [12].In recent years, the market of equipment and systems for continuous, automatic health and behaviour monitoring in pig herds has been enriched by innovative technologies. Modern pig production systems based on intelligent technologies allow for planned, efficient, and thus more cost-effective production [5]. Considering the used methodology and the scope of application, three categories can be distinguished among the available devices. The first category devices are only aimed at detecting specific animal behaviour by means of special sensors. An example of such a solution is the system automatically measuring the frequency of pig visits to the feeder and the time taken to feed by means of radio frequency identification technology—RFID [13,14]. Another example is the use of real-time video visualisation using conventional (2D) monochromatic or colour cameras or 3D cameras to depict activity level, area occupancy, aggression, gait scores, resource use, and posture [15,16,17,18]. The second category devices allow for detection and recording of specific behaviours, such as drinking [19], feeding [20], or spatial distribution [21], which are further processed into numeric data and presented, e.g., in the form of a graph on a mobile phone monitor. This type of device allows for identification of changes in animal behaviour but requires farm workers to interpret the data. The last category involves intelligent production systems, automatically analysing the recorded changes in the physiological and behavioural parameters. These systems are based on optimal settings of farm environment, have the ability to extract deviations from theses settings, and automatically make decisions to adapt the production environment to optimal production conditions [22,23]. The goal of this paper was to gather knowledge on novel technologies applied on pig farms in order to promote their health, productivity, and welfare. The article should be in the interest of pig farmers, pork retailers, and researchers working in the field of meat science.2. Challenges of Pig FarmingThere were about 677.6 million pigs worldwide as of January 2020 [24]. Pork is the second-most consumed meat in the world, with the consumption reaching 23.0 kg/capita [25]. Population growth increases demand for meat. The statistics on the projected pork consumption indicate a global increase, by about 17%, predicted for the period from 2021 to 2029 [26].Modern pig production is characterised by intensification and specialisation of production. These two factors lead to an increase in animal productivity and thus contribute to higher economic efficiency of production. On the other hand, they cause serious ecological problems as well as problems related to animal welfare, herd health, and food safety [27]. A high level of welfare is a guarantee of good health of the animal as well as the elimination of antibiotics or other drugs [28].A series of programs were launched in European Union in order to evaluate and improve the welfare of farmed animals, with the Welfare Quality® program resulting in the development of welfare protocols for the animal species under large-scale production [29,30,31,32]. Welfare Quality® protocols contain major welfare principles targeted at the needs of animals under intensive production and based on the Five Freedoms of animal welfare [33]. The protocols are designed in a manner that allows for their species-specific adaptation. According to the available research results, the protocol dedicated for pigs is a useful and reliable tool for identification of farms keeping pigs at poor level of welfare [34].In order to prevent economic losses due to diseases occurring in the herd, pig farmers should acquire at least basic skills to diagnose and deal with the appearance of the disease unit in their shed. [35]Changes in animal behaviour preceding or accompanying subclinical and clinical signs may be of significant diagnostic value. They are often referred to as sickness behaviour, including changes in eating habits, social behaviour, mobility, and posture. By definition, subclinical disease is latent, and thus direct monitoring based on staff observation is ineffective. This is due to the fact that it is time-consuming, inaccurate, and impractical in terms of work organisation [22,36].Automated early warning systems, based on continuous monitoring of specific physiological (e.g., body temperature) and behavioural parameters, can provide an alternative to direct observation of animals [23,37]. A good example are methods based on artificial intelligence. These methods employ computer tools able to track animal behaviour [38,39,40,41,42] and distinguish individuals from each other [43].Today, commercial pigs are exposed to a great number of stress factors, including stocking densities, high concentration of animals in a limited area, limited possibilities of movement and motivated behaviour expression, and frequent regrouping of animals. The microclimate in pig buildings also has a huge impact on pig welfare and production results. It affects animal health, reproduction parameters, and feed intake [37]. Harmful consequences of stress depend on the sensitivity of the animals to stressors as well as their severity and duration of action. Among farm animals, pigs are characterised as having the lowest tolerance to high environmental temperatures. This is due, among other things, to low adaptability of the thermoregulatory medium in the brain, low number of sweat glands, presence of the subcutaneous fat layer, and intensive metabolism. Exceeding the body’s ability to adapt to high temperatures (hyperthermia) is a threat to health and life [44,45]. Hyperthermia in pigs leads to poor condition of the animals, decreased daily gains, and longer fattening period. Moreover, it negatively affects the quality of pork resulting from the interaction between the muscle pH value and the temperature in the process of post-mortem denaturation of muscle proteins [46].Among the consequences of stress, one can name reduced appetite, reproductive disorders, and reduced immunity. In the aftermath, the pig farmers observe lower daily gains, poor reproductive results, and the appearance of infectious diseases and high mortality [47].3. Welfare Monitoring Systems for PigsThe market of systems designed for permanent, automatic monitoring of farm animal welfare is constantly evolving. Automated innovative early warning systems (PLF—precision livestock farming), based on continuous monitoring of specific behavioural and physiological parameters, are an alternative to direct visual assessment by staff or veterinarian. Fast and accurate acquisition of real-time data on animal movement and feed intake frequency enables early detection of diseases and facilitates further management of the herd. Thanks to sensors (cameras, microphones, accelerometers, RFID sensors, and temperature sensors), behavioural patterns of animals are gathered and combined through algorithms. The data derived from PLF technologies can be used to derive warnings and trigger notifications and alarms [48]. With the development of the Internet of Things (IoT, i.e., the interconnection between computing devices via the Internet), decision making can be better informed by connecting PLF information with other data streams, and components of farm management can be automated or even controlled remotely [15,49,50]. This allows for the ability to detect problems early enough to prevent potential, negative effects on productive performance of animals [51]. There are many benefits of precision livestock production, including increased productivity and profitability, increased safety and quality of animal products, and improved animal welfare, as well as reduced environmental impact and combating climate change. The use of precision livestock production in animal nutrition has been shown to reduce feed costs by up to 25% [52,53]. In 2016, the total turnover in the precision agricultural technology market was estimated at USD 4.8 billion. Current forecasts put the market turnover in this area at USD 12.6 billion by 2025 [54]. The adoption of these technologies varies considerably. RFID and accelerometer technologies are well integrated, but other technologies still have to achieve a viable market share [48].3.1. Vision-Based SystemsAmong the adopted PLF methods, video monitoring seems to be the most commonly implemented. It provides non-invasive and efficient tools to be able to record not only the behaviour of a group of animals but the behaviour of each individual [35]. By means of image analysis, the results are converted to detailed data on animal distribution (location and proximity) [18,55] and activity (position and movement) [56,57]. Imaging is also used in pigs to measure body weight [58,59,60] and to detect lameness [61], aggressive behaviour [62], and heat [63].Over recent decades, two-dimensional (2D) monochrome and colour have been widely used in computer vision due to its low cost and high efficiency. Many researchers have proposed different systems to extract livestock characteristics, such as body size or body condition, on the basis of 2D images [64,65]. For example 2D image analysis allows for monitoring and estimation of pig growth rates to an accuracy of 1 kg [58]. In turn, the number of cameras (video sets) intended for animal observation depends on the monitored area and the height at which the camera is placed. The quality of monitoring is also influenced by the number of animals per square meter [22]. As many studies show, simple dome cameras are sufficient to monitor the behaviour of the inmates [41,65,66,67]. For example, it can be a CCTV camera with IP67 waterproof rating and 2MP (1080) resolution, with f/2.8/F1.6 minimum aperture and with built-in infrared heater [67]. Monochrome cameras typically have greater light sensitivity and are thus more ideal for recording under lower light conditions than colour cameras [15].In the work of Chen et al. [65], a behaviour identification and monitoring study was conducted on eight pigs in pens of approximately 4 square meters, with the camera positioned at a height of 2.4 m. This study used the neural modelling technique using deep learning algorithms. It should be noted that the authors used a more difficult modelling technique in relation to convolutional networks and searched for their own indexes to describe the image. They obtained 98.5% accuracy in terms of behaviour identification through connection of a relatively simple camera with neural modelling technology. The results of research conducted by Chen et al. [65] allow for high efficiency of algorithms used in systems for monitoring animal behaviour [65]. Reikert et al. [67] applied the deep learning system in combination with 2D cameras in order to detect the position and posture of pigs. The authors obtained slightly lower precision results compared to the previously described study (87.4 and 80.2%), but the area of pens and the stocking of animals were much larger [67].Despite continuous development, 2D imaging technology still has some limitations. It requires appropriate ambient lighting; provides only a flat projection of the animal [68]; is influenced by distance, wavelength, and applied filters [69]; and also requires a contrasting background, e.g., a bright pig against a dark pen wall [15]. Additionally, data extraction from images taken in various environmental conditions leads to inaccurate operation of computer tools for image processing and analysis [70,71].Three-dimensional (3D) (RGBD) cameras equipped with high-resolution lenses, infrared sensors, or depth sensors with time of flight (ToF) technology give greater possibilities compared to cheaper two-dimensional (2D) ones [35]. ToF technology sends a pulse of infrared light from LED several times per second and records the delay between the pulse and its return. The 3D cameras can operate regardless of the visual light environment, including in total darkness; are unaffected by changing light conditions including changes in contrast and shadow; and are less prone to errors due to occlusion [15,17]. Three-dimensional technology opens the possibility to reconstruct the geometry of animals’ bodies and to link abnormal morphological changes to behavioural changes [7,72]. Cameras equipped with ToF technology (kinect cameras) are extremely useful in precision animal husbandry due to their relatively low cost, ability to handle large databases, low power requirements, and ability to adapt to changing light and background conditions [35,73]. However, these types of equipment have a limited distance range (i.e., up to 4.5 m), and the accuracy of the depth data measured by such devices decreases squarely with increasing distance [74].Despite these limitations, the ability of kinect cameras to detect the movement of individual animals is satisfactory. This was proven in the study of Kim et al. [74], which shows that one Kinect set installed at a height of 3.8 m is sufficient for accurate (94.47% sensitivity) monitoring of an area measuring 2.4 by 2.7 m.Although video-based recognition of pig behaviour has made significant progress, there are still some unsolved problems [71]. Vision data may require considerable processing and there have been studies on the trade-off between the video image quality and computational processing requirements [75]. Software challenges include detecting individual pigs on the basis of selected features by means of feature selection algorithms [76]. In addition, cameras are susceptible to dust and damage from ammonia, being part of the pig farms’ environment, although this can potentially be negated through ingress protection enclosures and maintenance [77].To ensure accurate and continuous monitoring of individual animals on a modern livestock farm, farmers today need reliable and inexpensive technology [3]. Such systems already exist in cattle breeding and include GEA CowView system, or Lely Qwes. For pigs, the RO-MAIN Smart Cam and eYeNamicTM system is currently the best-known system used to identify and track the pattern of activity in a group of pigs during the growing period. Other newer solutions are still in the realm of research [48].3.2. Sound-Based SystemsReal-time monitoring can be carried out not only by camera and image analysis but also by microphone and sound analysis [78]. Audio recordings combined with voice analysis and machine learning algorithms are used to detect heat stress and conditions of illness or suffering in animals [5]. Respiratory diseases and/or discomfort associated with poor air quality can cause changes in vocal characteristics and some acoustic signs, such as coughing and sneezing [79]. Monitoring coughing is particularly useful, as it can be easily distinguished from other sounds [80]. Specialised microphones or groups of microphones (microphone arrays) give the ability to distinguish infectious cough from coughs caused by accumulated ammonia or dust, and allow for automated sound source location [81]. Currently available sound analysis systems are so accurate that they can detect and locate respiratory disease outbreaks between individual pens [82]. Several studies have taken up the subject of animal coughing sounds analysis, under laboratory and farm conditions [82,83,84]. The first study on cough detection in pigs was conducted by Van Hirtum et al. [85] and was followed by additional research on refining algorithms for pigs’ cough detection [82,84]. In the study of Ferrari et al. [86], the authors used cough-sound analysis to identify respiratory tract infections in pigs. They found significant differences in several major acoustic parameters, including peak frequency, duration, and time occurring between consecutive coughs in healthy and infected pigs. In turn Exadaktylos et al. [84] proposed a method to identify sick pigs in real time by analysing the sound of coughing, with a recognition accuracy of 85%. Research results on the application of cough algorithms allowed for the development of a commercial tool, the respiratory distress monitor, able to detect infected pigs 2-12 days before the farmer or veterinarian [87]. Van Hirtum and Berckmans [88] suggested that cough sound recognition could be used as a biomarker of air pollution. This thesis was confirmed in a recent study of Wang et al. [79]. Unfortunately sound detection and analysis on pig farms is impeded by the noisy environment of the farm [80].3.3. Temperature-Based SystemsTemperature meters typically use thermometers embedded in a data logger or a sensor installed in the ear tag or subcutaneous transponder [89,90,91]. However, as Hartinger et al. [91] reported, this method is characterised by a high degree of variability, which makes it moderately reliable. It has been reported [92] that subcutaneous implanted transponders show a temperature around 1 °C lower compared to rectal measures. Explanation of this discrepancy lies in the position of the transponder, the amount of adipose tissue in the region of measurement, behavioural factors, environmental changes, heat radiation, or blood perfusion in the connective tissue in the implant area. Lohse et al. [92] have shown in their studies that transponders introduced into skeletal muscles show a better correlation with rectal temperature than transponders introduced subcutaneously. An alternative to invasive body temperature measurement with a transponder is to measure the temperature distribution on the body surface by using thermal imaging. Thermography, also known as thermovision, is a method of remote and non-contact assessment of body surface temperature distribution. This technique allows for visualisation of infrared radiation, and thus can obtain information about physiological and pathological processes taking place in the body of humans and animals [93]. The application of thermovision is absolutely non-invasive and has no risk of spreading infections [94,95]. In the diagnosis of farm animals, thermovision is used to investigate injuries and inflammation of the locomotor system, detect infectious diseases, diagnose heat and pregnancy, and monitor welfare and stress levels [93]. Modern thermovision methods make it possible to determine temperature changes both in terms of values and spatial distribution, both in static and dynamic terms. Thermal imaging cameras can produce high resolution images with a temperature accuracy of up to 0.08 °C [96]. Temperature readings depend on the animal’s temperature, environmental conditions, and thermoregulation of the peripheral circulatory system. At higher ambient temperatures, thermoregulation results in increased blood flow to the skin tissue, causing an increase of surface temperature [97]. In adult pigs, the temperature measured on the body surface is lower compared to younger animals due to the insulating effect of subcutaneous fat [97]. Skin surfaces behind the ears or near the sternum are hairless and lacking in fat insulation, and therefore better reflect adult body temperature [94].3.4. Activity-Based SystemsAccelerometers are among the most promising technologies for monitoring livestock behaviour [35]. These instruments are primarily used to measure linear or angular acceleration, and allow for very accurate monitoring and analysis of animal activity: posture and walking patterns, the length of time it spends standing up, delays in lifting, or even antepartum activity in pens, making it possible to detect the onset of labour in sows [98]. Triaxial accelerometers allow for the possibility of collecting three-dimensional information and measure the earth’s force by determining the angle of a device (e.g., wireless acceleration sensor nodes placed on the back to record the three-axis movement of pigs) and by measuring the acceleration forces [35]. Several studies have described automatic detection by accelerometers of standing and walking behaviour in pigs [99,100,101]. Studies of accelerometer readings installed on ear tags have shown that although the ear is virtually independent of the animal’s locomotor system, the range of data provided by the device is sufficient to reliably detect early lameness in pigs [102]. Other research indicate that a combination of data from accelerators with data from body temperature sensors allows for automatic detection of infections 1–3 days before using specific diagnostic methods [23]. Thus far, high accuracies have been found for movement and resting behaviours in cows and pigs, while the development of algorithms for analysing feeding and drinking behaviours in pigs is far behind these developed for cattle [103].Disease, welfare, and productivity problems can have an impact on the feeding patterns of pigs, and may lead to a reduced feeding time or longer intervals between feed intakes [104,105]. RFID at feeding and drinking areas has been used to measure occurrence and duration feeding and drinking behaviour of individual pigs’ [20,106,107]. An RFID system requires an RFID transponder (ear tag) and an RFID antenna or receiver (located at the feeder or drinker) [22]. The device is implanted primarily in the ear tags and stores information such as the animal’s unique identification number and farm identification number. These data can be used immediately to identify individuals or can be stored and analysed later [35]. Low-frequency RFID is used, for example, in electronic feeders, and makes it possible to dose individually adjusted feed rations [108]. At the same time, data from RFID readers are also used to analyse the frequency of visits to the feeders and the time taken to feed, which allows for early detection of behavioural signs of health problems [107]. Nevertheless, low frequency RFID has two main disadvantages: low reading range (<1 m) and the impossibility to identify more than one animal at a time within the reader’s range [104,109]. There is research on the application of high-frequency UHF readers to track multiple animals simultaneously and at longer ranges (3–10 m) [110,111]. Such systems often include anti-collision algorithms to avoid data loss when multiple tags are within the reading range [112,113]. Thanks to its high sensitivity (88.58%) and specificity (98.34%), the HF RFID system performs well in recording feeding visits of pigs [105]. An example of a commercial system used in pig farming, based on UHF-RFID technology, is the SLIDE® system (Simplum Gliwice, Poland). One of the key elements of this system, which distinguishes it from other similar solutions, is the long reading range of approximately 4–5 m, allowing for the automation of the data acquisition process. The system allows not only for monitoring of animals, but also offers the possibility of a very detailed analysis of the individual indicators of each tagged pig, taking into account the factors influencing them and the relationships between individuals [114].RFID solutions are eagerly used in various sectors, e.g., in factories and warehouses. However, it is important to be aware that the conditions in pig stables, which are mostly based on concrete and reinforced structures, may disrupt the transmission of waves and data. Other disadvantages of RFID-based technology include frequent loss or failure of tags, pain and stress for the animal during tagging, and the need to remove the tag prior to slaughter [35]. An idea worthy of attention is the use of beacons—microcontrollers equipped with BLE (Bluetooth Low Energy) transmitters in pig farming as devices used to identify behaviour and physiological condition. Studies on these systems have been successfully carried on cattle, although the difference in the daily behaviour of cows and pigs is important [115]. Table 1 summarises the advantages and disadvantages of equipment used in precision pig farming.4. Automatic Health and Welfare Monitoring Systems for Pigs—Farmer and Consumer PerspectiveAutomatic systems for health problem detection in pigs are practical from the scientific point of view and are undoubtedly a common topic in research on detection of health problems on commercial pig farms. The automatic health and behaviour measurement seem to perfectly fit management of big stables; it allows for recording and storing of valuable data, and thus carrying out continuous observations on large numbers of pigs. However, the major problem remains unsolved: How do we convince pig farmers to adopt novel solutions? Are these solutions economically profitable? The economic aspect of health monitoring systems for pigs is still undefined, as no research has been made to compare the outputs and the costs of all inputs used. Moreover, the popularity and possibility of implementation of automatic systems for pig health monitoring is affected by a group of additional factors: effectiveness and reliability of measures, the awareness and technical knowledge of pig farmers, and the housing system and herd size used [116].Recent years have seen a series of major leaps forward in the technologies and methods of automated animal observation and monitoring, notably under the generic terminology of PLF whose general aim is to increase the efficiency of livestock farming systems while reducing the workload [2].Commercial pig farms are an aggregation of technical solutions that allow for increased production while limiting the labour. In times of sustainable agriculture and high welfare farming, the commercial pig farms have limited opportunities to follow these trends and to compete with the good reputation of organic pig farms. The modern pig production should meet the public requirements of animal welfare, and the use of automatic systems for welfare monitoring might be a chance of fulfilling these requirements. PLF technologies have the potential to monitor animal health and behaviour in ways that go beyond those of conventional welfare monitoring and observation. PLF allows for the establishment of welfare indicators that are not dependent solely upon periodic human observation and measurement [2]. In addition, PLF provides the opportunity to observe animal behaviour without interference.Problems that need to be resolved in the near future include inter alia, technical, and scientific issues related to the definition of welfare. In addition, it is necessary to establish effective welfare indicators, improve the reliability of data collected using new observation technologies, develop welfare automation technology dedicated to extensive farming, regulate matters related to the ownership of data generated by PLF technologies in order to effectively manage these data, and further the possibility of exchanging such information between participants in the food chain. To date, however, this potential is both underdeveloped and under-studied [48].Balzani and Hanlon [117] underline that animal welfare links a variety of perspectives: animal science, veterinary science, public opinion, and the perspective of the farmer, which is often disregarded. Therefore, implementation of automatic welfare monitoring systems should be preceded by research that relate science with farm practice and social reaction. The practical aspects such as economic profitability and consumer feedback should be carefully analysed as this kind of research is lacking. With growing concern for animal welfare, the pressure on the implementation of PLF technology in pig farms will also increase, both from food chain operators and consumers [48,118]. The question is how will consumers feel about the use of novel technologies to assist the farmer with the monitoring of the welfare of pigs. Are they willing to pay higher price for pork produced with this extra supervision? How much educational input is required to create social attitudes that favour novel technologies? The automatic health and welfare monitoring systems in pig stables should be promoted as one of the paths in sustainable animal production, and one of systems that allow for production at high welfare standards. A pork production chain that employs use of systems supporting health and welfare of pigs should be traceable for consumers and promoted by the authorities of each country.The farmer’s attitude to pig welfare monitoring systems is even more important than the social opinion. Because the EU directive (Council Directive 98/58/EC) defining the rules of pig farming charges farmers and stock-people with the responsibility to inspect animals at regular intervals (usually, at least once a day) to verify their wellbeing, farmers’ knowledge of the biological and behavioural needs of pigs is key to bringing about changes that promote welfare automation in the pig industry [119,120]. If the farmer is aware that the stable requires changes to produce pigs at high welfare standards, the decision on implementation of novel technological solutions will depend on their economic profitability.5. ConclusionsThis review concluded that automatic health monitoring systems should be widely implemented into the pig industry in order to increase the effectiveness of healthy pig production. The monitoring systems are developing together with the knowledge on effective animal production, requirements considering the level of welfare, and the developments in available technologies. Implementation of novel technologies for health monitoring may be an answer to the demands of society and animal welfare organisations. The research in the field of swine industry deliver a number of practical solutions. The solutions that are already commonly used are automated weight measurement, electronic identification of pigs, automated measurement of feed and water intake, accelerometers (in ear tags) measuring activity, and systems to monitor and manage the microclimate (humidity, temperature, ventilation). The automatic welfare monitoring systems give much more data on the pig herd and are much more developed that these commonly used technologies. However, without proving their economic profitability and defining the reliable possibilities of application, automatic health/welfare monitoring systems will not gain popularity. Research is lacking in this field. Though the social pressure may be the “drive motor” of changes in animal production, the economic aspects of pig heath monitoring systems will decide on the scale of their implementation. Acquisition of data defining the health on pigs in real-time is the key to early disease recognition and disease prevention. The expectations considering the monitoring systems gradually increase, and with time we can observe new technologies that allow us to trace individuals and monitor pigs without stressing the animals.Summing up the issues discussed in this review, one last thing is still lacking—a system that will allow us to link the health and welfare measures of an individual pig with the data on the quality attributes of obtained pork. Only the careful analysis of this relation would allow for a reliable assessment of the role of pigs’ health and welfare in the economical effectiveness of the swine industry, aimed at the production of high-quality meat.

animals : an open access journal from mdpi

10.3390/ani11061702

PMC8226439

Canine degenerative myelopathy (DM) is a chronic, progressive, and fatal neurodegenerative disease. Although degenerative changes in dogs with DM are observed not only in the spinal cord white matter but also the dorsal root ganglion (DRG) neurons, these changes are undetectable on conventional magnetic resonance imaging (MRI). Therefore, we investigated the ability of water-excitation MRI to visualize the DRG in dogs, and whether volumetry of DRG has a premortem diagnostic value for DM. Using water-excitation MRI, DRG could be depicted in all dogs. To normalize the volumes of DRG, body surface area was the most suitable denominator. The normalized DRG volume in dogs with DM was significantly lower than those in control dogs and dogs with intervertebral disc herniation. The results of this study revealed that widespread atrophy of DRG was likely to occur in DM. Moreover, volume reductions of DRG were observed in dogs with DM in both the early disease stage and late disease stage. Our research suggests that the DRG volume obtained by the water-excitation technique could be used as a clinical biomarker for DM.

Canine degenerative myelopathy (DM) is a progressive and fatal neurodegenerative disease. However, a definitive diagnosis of DM can only be achieved by postmortem histopathological examination of the spinal cord. The purpose of this study was to investigate whether the volumetry of DRG using the ability of water-excitation magnetic resonance imaging (MRI) to visualize the DRG in dogs has premortem diagnostic value for DM. Eight dogs with DM, twenty-four dogs with intervertebral disc herniation (IVDH), and eight control dogs were scanned using a 3.0-tesla MRI system, and water-excitation images were obtained to visualize and measure the volume of DRG, normalized by body surface area. The normalized mean DRG volume between each spinal cord segment and mean volume of all DRG between T8 and L2 in the DM group was significantly lower than that in the control and the IVDH groups (P = 0.011, P = 0.002, respectively). There were no correlations within the normalized mean DRG volume between DM stage 1 and stage 4 (rs = 0.312, P = 0.128, respectively). In conclusion, DRG volumetry by the water-excitation MRI provides a non-invasive and quantitative assessment of neurodegeneration in DRG and may have diagnostic potential for DM.

1. IntroductionCanine degenerative myelopathy (DM) is a fatal neurodegenerative spinal cord disorder that develops in several breeds including German Shepherds, Boxers, and Pembroke Welsh Corgis (PWC) [1,2]. The etiology of DM has not yet been fully elucidated; however, a previous study reported that DM-affected dogs were homogeneous for the A allele of a superoxide dismutase 1 (SOD1) missense mutation, SOD1: c.118G > A, which predicts a p.E40K amino acid substitution [3]. Age at the onset of clinical signs was 8 years or older in most cases [4]. The clinical signs of DM initially appear in the pelvic limbs as spastic upper motor neuron paresis and general proprioceptive ataxia, which progresses to flaccid tetraplegia and eventually dyspnea [2,5].Currently, the clinical diagnosis of DM is based on the following criteria: confirmation of the progression of clinical signs, identification of reported SOD1 mutations, and exclusion of other progressive spinal cord disorders that clinically mimic DM [2,4]. However, a definitive diagnosis of DM can only be achieved by postmortem histopathological examination of the spinal cord [2]. Therefore, novel diagnostic tools with higher specificity to DM are needed in order to make a faster and more accurate premortem diagnosis.The pathological changes in the spinal cord in DM are characterized by axonal degeneration [3,6,7], axonal loss [3,6,7], demyelination of the white matter in the spinal cord [7,8], loss of thoracic sensory root axons [9,10], and degenerative changes in the dorsal root ganglion (DRG) neurons [10]. Although these marked histopathological changes occur at every part of the spinal cord [7], conventional magnetic resonance imaging (MRI) techniques do not depict DM lesions [7,11]. Currently, technological advances have led to the development of high-field MRI for clear visualization of the spinal cord and the peripheral nerve tissue. The water-excitation technique has been demonstrated to provide better fat suppression and overall better image quality compared with conventional T1-weighted fat saturation [12]. In humans, magnetic resonance neurography with water-excitation was introduced as a modified method for visualizing the peripheral nervous system [13,14]. In addition, a previous study reported that nerve root volume could be measured using three-dimensional fast field echo water-excitation [15].The first aim of the current study was to establish the normalization of DRG volume in dogs using the water-excitation technique. The associations between DRG volume or spinal cord cross-sectional area and body weight, body surface area, or vertebral body length were evaluated. The second aim was to investigate the diagnostic ability of the water-excitation technique for DM. Dogs with intervertebral disc herniation (IVDH) were included in this study because IVDH is the most common cause of hindlimb paralysis that needs to be differentiated from DM. We hypothesized that the water-excitation technique provides clear depiction of DRG and the volume of DRG is reduced in DM dogs because of widespread degenerative changes and loss of nerve root axons compared to control dogs and dogs with IVDH.2. Materials and Methods2.1. AnimalsThis study was conducted as a retrospective cross-sectional study. All dogs underwent MRI at the Animal Medical Center of Gifu University between August 2019 and January 2021. All owners signed an informed consent form (approved by the Animal Medical Center of Gifu University and Use Committee, protocol #E20005, #2020-230). First, in order to establish the normalization of DRG volume in dogs, this study included control dogs that had no clinical or imaging evidence of vertebral or spinal cord disorders. Second, this study included dogs with DM, dogs with IVDH, and control dogs in order to compare DRG volume. Control dogs in the first study and the second study were the same population. In the DM group, all dogs were diagnosed with DM according to the following criteria: clinical signs consistent with DM (adult onset, slowly progressive, and non-painful paraparesis progressing to tetraplegia) [2,16], unremarkable findings on spinal cord imaging with conventional MRI sequences (Achieva dStream, Philips, Amsterdam, The Netherlands), and genetic testing confirmed homozygosity for the SOD1 c.118G > A missense mutation (A/A) [17]. All DM-affected dogs had progressive clinical signs for at least a year at the time of manuscript preparation. We also included dogs in the DM group that underwent MRI within 24 h after death, prior to necropsy. The number of dogs that received postmortem MRI is stated in the results section. The owners of the dogs were instructed to store the dogs in a cool condition and place refrigerants over the entire spine in order to minimize postmortem changes until they brought the dogs to us. The disease stage of DM was classified into four clinical stages as previously described [2,7]. The clinical stages were characterized as follows: stage 1, general proprioceptive ataxia and upper motor neuron paraparesis; stage 2, non-ambulatory paraparesis to paraplegia; stage 3, lower motor neuron paraplegia to thoracic limb weakness; and stage 4, lower motor neuron tetraplegia and brainstem signs. All dogs in the IVDH group had thoracolumbar spinal cord compression by herniated intervertebral discs, which were confirmed by MRI with or without subsequent gross confirmation at surgery. Thoracolumbar IVDH cases were graded as previously described [18,19]. The clinical grading of thoracolumbar IVDH was as follows: grade 1, thoracolumbar pain only; grade 2, ambulatory paraparesis; grade 3, non-ambulatory paraparesis; grade 4, paraplegia with positive deep pain sensation; and grade 5, paraplegia with a loss of deep pain sensation. Imaging analyses were performed to rule out the presence of concurrent diseases that may contribute to the neurological status of each dog. Exclusion criteria of this study for the DM or IVDH group were as follows: dogs with an incomplete diagnosis, intracranial disorders, vertebral/spinal cord tumors, and intramedullary or intradural extramedullary lesions that can be detected by conventional MRI.2.2. MRI SequencesAll MRI sequences were acquired using a 3.0-Tesla MRI system with an 8-channel coil as an RF coil and field of view adapted to the size of the animal (Figure A1). For MRI procedures, general anesthesia was induced with intravenous propofol (PROPOFOL injection, Fuji Pharma Co. Ltd., Toyama, Japan) and maintained with a mixture of isoflurane (Isoflurane, Pfizer Inc., New York, NY, USA) in oxygen and room air. In the control group, the protocol consisted of a sagittal and transverse T1-weighted sequence (repetition time (TR)/echo time (TE) 570/13.8 ms; slice thickness 1.5 mm) and T2-weighted sequence (TR/TE 3113/90 ms; slice thickness 1.5 mm). In the DM and IVDH groups, the protocol consisted of a sagittal and transverse T1-weighted sequence (TR/TE 570/13.8 ms; slice thickness 1.5 mm), T2-weighted sequence (TR/TE 3113/90 ms; slice thickness 1.5 mm), and contrast enhanced T1-weighted sequence after intravenous injection of 0.1 mmol/kg of gadodiamide hydrate (OMNISCAN, Daiichi-Sankyo, Tokyo, Japan). Water-excitation imaging parameters were as follows: water-excitation time: 13 msec, repetition time: 10.12–10.24 msec, invention time: 150–170 msec, slice thickness of transverse image: 0.375–0.500 mm, slice thickness of coronal and sagittal images: 1.1–1.2 mm, and sequence flip angle: 30°.2.3. Image AnalysisImage data were analyzed using OsiriX MD version 4.1.2 (OsiriX Pixmeo, Geneva, Switzerland). All images included in the present study were anonymized by H.K. and measurements were performed by E.N. E.N. is a practicing veterinarian who received training in veterinary radiology and neurology for seven years. Using water-excitation images, a stack of sequential image slices that cross-sectioned the DRG were selected for quantification. Water-excitation images included the thoracolumbar spinal cord between the T8 and L2 intervertebral disc levels. Volumetry of DRG was performed as previously described with minor modifications (Figure 1) [20]. Water-excitation images were a continuous image of the nerve roots from the intervertebral foramen to the entry of the spinal cord, including the full volume of the DRG on both sides. The transverse images of the DRG were manually segmented by tracing the borders using software that calculated the cross-sectional area of the DRG based on the number of pixels contained within the traced contour. To overcome the inadequate definition between the DRG and spinal cord profiles at the point where the nerve root began to enter the spinal cord, the boundary between the spinal cord and the DRG was defined by tracing the contour of the spinal cord. DRG volume was calculated by summing the cross-sectional areas (ai) of each DRG image, multiplying it by the slice thickness (0.375–0.5 mm) (ti), and expressing it in the following formula, according to Cavalieri’s principle:DRG volume = ∑aitiEach segment of DRG volume was averaged on the left and right sides. The DRG volume was measured in duplicate and the average value was adopted. To normalize the volumes of DRG, the ratios of body weight, body surface area, and L2 vertebral body length to DRG volume were calculated. Vertebral body length was measured on a sagittal water-excitation image. The cross-sectional area of the spinal cord was measured on a transverse water-excitation image at the center of each vertebral body. To evaluate the diagnostic utility of the DRG volume for DM, normalized DRG were compared with a normalized cross-sectional area of the spinal cord.2.4. Statistical AnalysesStatistical analyses were performed using Easy R software [21]. In the control group, the correlation coefficient (rs) was calculated by evaluating the correlation between the DRG volumes from T8 through L2 and body weight, body surface area, and vertebral body length from T8 through L2 by Spearman’s rank correlation coefficient. The normalized DRG volumes were compared among the DM, IVDH, and control groups using the Kruskal–Wallis test. Post-hoc comparisons employed the Mann–Whitney U-test with Bonferroni correction. Bilateral differences of DRG volumes were calculated from the absolute value of the left-right DRG ratio. In all analyses, a P value of < 0.05 was considered significant.3. Results3.1. Sample PopulationThe characteristics of all dogs are shown in Table 1. The control group consisted of laboratory animals at Gifu University (n = 4) and client-owned dogs (n = 4). The client-owned dogs in the control group had transient limb ataxia but no structural lesions in the central nervous system (n = 2), intracranial neoplasia (n = 1), or idiopathic epilepsy (n = 1). Laboratory dogs in the control group had no structural lesions in the central nervous system (n = 3) or idiopathic epilepsy (n = 1). Breeds in the control group included Beagle (n = 4), Boston terrier (n = 1), mixed-breed (n = 1), French bulldog (n = 1), and PWC (n = 1). Five dogs were spayed females and three dogs were castrated males. The median body weight, median body surface area, L2 vertebral body length, and median age of the dogs in control group were 11.4 kg (range, 6.6–14.0 kg), 0.49 m2 (range, 0.33–0.58 m2), 16.9 mm (range, 13.4–18.8 mm), and 6.0 years (range, 2.8–10.0 years), respectively. We included eight DM-affected Pembroke Welsh Corgis in the DM group. The clinical stages of DM were as follows: stage 1 (n = 4) and stage 4 (n = 4). Four PWCs, which were categorized as stage 4, underwent MRI within 24 h of death (Dog #9, 10, 11, and 12). These dogs were diagnosed with DM based on histopathological examination of the spinal cord. The other four PWCs were diagnosed with DM according to the inclusion criteria. Median body weight, median body surface area, L2 vertebral body length, and median age of the dogs in DM group were 12.3 kg (range, 10.1–16.8 kg), 0.52 m2 (range, 0.46–0.63 m2), 17.2 mm (range, 16.3–18.0 mm), and 13.2 years (range, 10.8–15.9 years), respectively. There were two spayed females, one intact female, and five castrated males. In the IVDH group, we included 24 dogs diagnosed with thoracolumbar IVDH. The neurological grades were as follows: grade 1 (n = 2), grade 2 (n = 5), grade 3 (n = 4), grade 4 (n = 8), and grade 5 (n = 5). The locations of IVDHs were T12-T13 (n = 12), T13-L1 (n = 9), L1-L2 (n = 6), T11-T12 (n = 4), L2-L3 (n = 4), L4-L5 (n = 2), and T9-T10, T10-T11, L3-L4, L5-L6, and L6-L7 (n = 1). The number of disc herniations in each dog was one (n = 15), two (n = 4), three (n = 1), four (n = 3), and five (n = 1). Surgical treatment was performed in 15 dogs, and non-surgical treatment was selected in nine dogs. The median body weight, median body surface area, median L2 vertebral body length, and median age of the dogs in the IVDH group were 6.7 kg (range, 3.0–14.1 kg), 0.33 m2 (range, 0.20–0.58 m2), 14.4 mm (range, 10.0–16.9 mm), and 11.7 years (range, 2.6–15.7 years), respectively. There were three intact females, six spayed females, nine intact males, and six castrated males. Breeds in the IVDH group included: Miniature dachshund (n = 11), Toy poodle (n = 4), French Bulldog (n = 2), Pug (n = 2), Border Collie (n = 1), Chihuahua (n = 1), mixed-breed (n = 1), Miniature Schnauzer (n = 1), and Pekingese (n = 1). The control dogs were significantly younger than the dogs with DM (P = 0.009) and IVDH (P = 0.012). There was no significant difference in age between the DM and IVDH groups. The dogs with IVDH had significantly lower body weight, body surface area, and L2 vertebral body length than the control dogs (P = 0.003, 0.005, 0.009, respectively) and dogs with DM (P < 0.001). There were no significant differences in body weight, body surface area, or L2 vertebral body length between the control and DM groups.3.2. Normalization of the DRG Volumes in Control DogsThere were no significant differences in DRG volume among spinal cord segments. Therefore, we used the mean DRG volume of all spinal cord segments between T8 and L2 in the correlation analyses. The strongest correlation was found between body surface area and mean DRG volume (rs = 0.792, P = 0.024) (Table A1). Body weight was found to have a moderate correlation with DRG volume (rs = 0.691, P = 0.037), but L2 vertebral body length did not have a significant correlation with DRG volume (rs = 0.612, P = 0.176). Therefore, comparisons of DRG volume among the three groups were carried out using body surface area as a denominator for normalization. There were no correlations between the mean cross-sectional spinal cord area of the spinal cord segment and body weight (rs = −0.048, P = 0.911), body surface area (rs = 0.124, P = 0.812), and L2 vertebral body length (rs = 0.571, P = 0.151).3.3. Normalized DRG Volumes between the DM, IVDH, and Control GroupsAt each spinal cord segment, normalized DRG volumes were significantly lower in the DM group than in the control group at T9 (P = 0.038), T10 (P = 0.042), and L2 (P = 0.045) (Figure 2 and Table 2). Normalized DRG volumes were also significantly lower in the DM group than the IVDH group at T8 (P = 0.009), T9 (P = 0.003), T10 (P = 0.003), T11 (P = 0.010), T12 (P = 0.035) T13 (P = 0.031), L1 (P = 0.041), and L2 (P = 0.007) (Figure 2 and Table 2). The normalized mean DRG volume of all spinal cord segments between T8 and L2 was significantly lower in the DM group than in the control group and the IVDH group (P = 0.011, P = 0.002, respectively; Figure 3 and Table 2).3.4. DRG Volumes in DM Dogs with Different StagesThere was no correlation in the mean normalized DRG volume between DM stage 1 and stage 4 (rs = 0.312, P = 0.128). At each segment, there was also no significant difference in the mean normalized DRG volume between DM stage 1 and stage 4.3.5. Laterality of DRG Size ChangeThe bilateral difference of mean DRG volume of all spinal cord segments between T8 and L2 was 9.8% (standard deviation [SD] 6.0) in the control group, 15.3% (SD 8.2) in the DM group, and 19.8% (SD 13.2) in the IVDH group, and no significant difference was observed among the three groups (Table A2). In the IVDH group, the bilateral difference of DRG volumes at the lesion (25.3%; SD 14.7) was higher than that at the non-lesion site (15.9%; SD 9.5).3.6. Spinal Cord Cross-Sectional AreaThere were no significant differences in mean cross-sectional area of the spinal cord among the three groups. At each spinal cord segment, there was also no significant difference in the cross-sectional area of the spinal cord among the three groups.4. DiscussionThe present study demonstrated that the water-excitation images depicted DRG and the nerve roots of dogs. The water-excitation sequence is a fat suppression sequence, which is a selective excitation technique to suppress signals from fat tissues by exploiting the difference between water and fat resonance frequencies. This sequence visualizes DRG clearly due to its high spatial resolution and high signal-to-noise ratio [22]. Slice thickness is thinner with water-excitation than with short tau inversion recovery, and reduced slice thickness improves spatial resolution and better visualization of anatomical details [23]. The water-excitation technique produces thinner slice images and provides a clearer depiction of the DRG and nerve roots [24]. As DRG in dogs is not visualized by conventional MRI sequences, water-excitation MRI has potential to be used as a non-invasive diagnostic test for diseases affecting DRG.The ratio of DRG volume to body surface area showed a strong positive correlation, which can be used to normalize DRG volume across dogs with different sizes. In a previous study of chronic inflammatory demyelinating polyneuropathy (CIDP) in humans, DRG normalized by body surface area was useful for the diagnosis and assessment of the severity of CIDP [15]. We found that the normalized DRG volume was significantly reduced in the DM group compared with the control and the IVDH group. This suggests that the DRG volume obtained by the water-excitation technique could be used as a clinical biomarker for DM.In an early study, Wallerian degeneration of the dorsal nerve roots and central chromatolysis of the DRG neurons was reported in dogs with DM [9]. More recently, a decreased number of axons in the T8 dorsal nerve root and degenerative changes of DRG neurons in DM-affected dogs have been reported [10]. In mouse models of amyotrophic lateral sclerosis and diabetic neuropathy, axonal degeneration caused impaired axonal transport of proteins and metabolites, resulting in neuronal cell death [25,26]. Therefore, it is considered that nerve cell death due to axonal degeneration occurs in the DRG of DM-affected dogs, leading to decreased DRG volume. Although the histopathological findings of T9-L2 DRG in DM-affected dogs remain unknown, development of concurrent degeneration of DRG neurons in other regions is more likely, given the widespread axonal loss and demyelination in the white matter not only in the caudal thoracic spinal cord but also in the cervical and lumbar spinal cords in DM dogs [3,6,7]. In particular, since degenerative lesions are located in the dorsal and lateral funiculus of the spinal cord through which the axons of DRG neurons pass, widespread atrophy of DRG is likely to occur in DM. There was no significant difference in DRG volume between the early and late disease stages in this study. This finding was in contrast with the observation that the C7 dorsal roots of dogs with DM gradually decreased in number with disease progression [27]. A previous study showed that hyporeflexia of the patellar reflex was described in dogs with DM at early disease stage, which was accounted for by the degenerative change of dorsal nerve root and central chromatolysis of DRG neurons [9]. Therefore, DRG volumes may decrease even at the early disease stage. This finding favors an early diagnosis of DM with this non-invasive MRI technique. On the other hand, a decrease in DRG volume may not correlate with lesion load of the spinal cord and therefore may not be suitable for longitudinal monitoring of pathological progression.The initial clinical signs of DM share similarities with other progressive spinal cord disorders. IVDH is the most common spinal cord disorder in dogs that are also predisposed to DM; therefore, we included dogs with IVDH as a “disease control” in this study. Our study revealed that DRG volume measurement using water-excitation MRI was capable of distinguishing DM from IVDH. Normalized DRG volumes in all spinal cord segments and mean DRG volume were significantly lower in the DM group than in the IVDH group. The reduction of DRG volumes in DM occurred in multiple spinal segments that parallel the diffuse degeneration of the spinal cord, whereas the DRG volume changes were focal in the IVDH group, decreasing in the proximity of the lesion site. In human and rodent studies, ipsilateral DRG at the site of spinal cord injury was atrophied due to demyelination and Wallerian degeneration of the axons of DRG, resulting in dying back degeneration and death of sensory neurons [20,28]. The duration and severity of the disease also had an impact on DRG size as atrophied DRG recovered its size over time after injury [29,30,31]. The wide range of DRG sizes in the IVDH group may be attributed to disease duration, location, and severity of injury in this study.Several limitations of the present study should be considered. Measurements of DRG were performed and analyzed by a single observer. Although measurements were performed in duplicate, further study is needed to evaluate intra- and inter-observer errors. The sample size was small, especially the number of dogs in the control and the DM groups. In the DM group, MRI data for four dogs were obtained postmortem in order to compare the difference of DRG volumes between the early stage and late stage. This comparison was only possible by using postmortem MRI as dogs with DM in the late stage suffers from respiratory disfunction that hinders diagnostics requiring general anesthesia. Postmortem MRI was performed within 24 h of death in an attempt to minimize any postmortem changes; however, postmortem changes in DRG must be investigated in terms of their effects on histopathological changes and MRI data. In the central nervous system, the previous study showed that comparing premortem and postmortem MRIs for cerebral microbleeds yielded comparable imaging performance [32]. We stored these dogs in a cool condition immediately after death in order to minimize potential postmortem changes. We considered that the obtained MRI data were as close to the premortem state as possible. The other four dogs were tentatively diagnosed with DM without histopathological confirmation. These four dogs were still alive at the time of manuscript preparation; therefore, this study could not compare the histopathological findings of DM with MRI. All dogs in the DM group were PWCs in the present study. In Japan, DM is most common in Corgis, and the number of other breeds that are prone to develop DM is small. The fact that dogs in the DM group only included a single breed was one of the limitations of this study. Dogs in the control group were significantly younger than those of the other two groups. Although a previous study reported no difference in the volume of DRG with age in humans [33], the relationship between DRG volume and age in dogs warrants further investigation. 5. ConclusionsWater-excitation was a useful technique for DRG volumetric analysis in dogs with DM. Volumetry of normalized DRG by the water-excitation technique provided a non-invasive and quantitative assessment of neurodegeneration in DRG and may have diagnostic potential for DM.

animals : an open access journal from mdpi

10.3390/ani12010089

PMC8749849

Food-responsive enteropathy is the most common diagnosis given for dogs with chronic enteropathy, and there are no tests that can replace treatment trials. Furthermore, there is a lack of information on the specific nutritional status of these patients regarding the lipid profile that could relate them to the state of health/disease. This study evaluated differences in short-chain fatty acids and the total fatty acid profile of faeces and plasma as possible indicators of food-responsive enteropathy (FRE), as well as its relationship with body condition and the chronic enteropathy activity index. Changes in the long-chain fatty acid of plasma, and short-chain, branched and odd-chain fatty acids of faeces were detected in sick dogs, and high correlations were observed between some of these compounds and the existing calculated indices.

The aim of this study was to evaluate differences in short-chain fatty acids (SCFAs) and the total fatty acid profile of faeces or plasma as possible indicators of FRE in comparison with healthy dogs. FRE dogs had a lower concentration (p = 0.026) of plasma α-tocopherol as an indicator of the oxidative status of the animal, and lower C20:5n-3 (p = 0.033), C22:5n-3 (p = 0.005), polyunsaturated fatty acids (PUFA) (p = 0.021) and n-6 (p = 0.041) when compared with the control dogs; furthermore, sick dogs had higher proportions of plasma C20:3n-6 (p = 0.0056). The dogs with FRE showed a decrease in the production of faecal levels of SCFAs, mainly propionic acid (C3) (p = 0.0001) and isovaleric acid (iC5) (p = 0.014). FRE dogs also had a lower proportion of C15:0 (p = 0.0003), C16:1n-9 (p = 0.0095), C16:1n-7 (p = 0.0001), C20:5n-3 (p = 0.0034) and monounsaturated fatty acids (p = 0.0315), and tended to have lower n-3 (p = 0.058) and a reduced desaturase activity index in the stool when compared with the control group. However, the dogs with chronic enteropathy tended to have greater C20:4n-6 (p = 0.065) in their faeces as signs of damage at the intestinal level. The faecal parameters were better predictors than plasma. The highest correlations between faecal odd-chain, medium- or long-chain fatty acids and SCFAs were observed for C15:0 that correlated positively with faecal acetic acid (C2) (r = 0.72, p = 0.004), propionic acid (r = 0.95, p = 0.0001), isobutyric acid (iC4) (r = 0.59, p = 0.027) and isovaleric acid (r = 0.64, p = 0.0136), as well as with total SCFAs (r = 0.61, p = 0.02). Conversely, faecal C20:4n-6 showed a high inverse correlation (r = −0.83, p = 0.0002) with C2 and C3 (r = −0.59, p = 0.027). Canine inflammatory bowel disease (IBD) activity (CIBDAI) index correlated negatively mainly with faecal measurements, such as C3 (r = −0.869, p = 0.0005) and C15:0 (r = −0.825, p = 0.0018), followed by C16:1/C16:0 (r = −0.66, p= 0.0374) and iC5 (r = −0.648, p = 0.0310), which would indicate that these fatty acids could be good non-invasive indicators of the chronic inflammatory status, specifically FRE.

1. IntroductionChronic enteropathy (CE) is a very common diagnosis given for dogs with chronic digestive signs, with an estimated prevalence of 70% in cases with chronic diarrhoea [1]. CE can be further subdivided retrospectively by the response to treatment into food-responsive enteropathy (FRE), antibiotic-responsive enteropathy (ARE), immunosuppressant-responsive enteropathy (IRE) and non-responsive enteropathy (NRE) [2]. Several retrospective studies suggested that FRE is probably the most common CE in dogs, with a prevalence greater than 60–70% of cases [1,3]. The significance of the effect of diet in different subtypes of CE is increasing. In fact, it was recently described that dogs with protein-losing enteropathy with a previous non-response to a combination of dietary therapies, glucocorticoids and immunosuppressive medications can achieve remission following a dietary change [4,5,6].During recent years, the scientific focus has been directed to identifying biomarkers of interest in prognosis and treatment. However, to date, there are no tests that can replace treatment trials [7,8,9,10].Short-chain fatty acids (SCFAs) are major end products of dietary fibre formed by bacteria [11]. Their role in health and disease was previously evaluated, taking into account their relationship with digestive microbiota and their effects on the immune system and gastrointestinal motility [12,13,14]. Previous studies in human medicine have shown a decrease in faecal concentrations of SCFAs in different chronic digestive diseases, such as inflammatory bowel disease [15]. Similar results were found in human populations with a high risk of colon cancer [16]. In veterinary sciences, the effect of dietary change or intervention on SCFAs was widely evaluated in healthy dogs [17,18,19], but information about faecal SCFAs in different canine digestive diseases is limited. To the best of our knowledge, systematic evaluation of faecal SCFAs has only been performed in dogs with acute diarrhoea [20] and CE [21].In addition, some studies revealed the potential interest of other fatty acids present in faeces as diagnostic indicators of certain chronic intestinal diseases. This is due to the important functions that lipids have in the body, from being part of cellular structures to being a source of energy, participating in metabolic regulation or as precursors of certain substances [22]. Thus, DePreter et al. [23] found that faecal medium-chain fatty acids were decreased in human patients with inflammatory intestinal diseases and considered hexanoate levels as a good tool for gut disease prediction. Song et al. [24] observed a certain relationship between humans with colorectal cancer and the amount of monounsaturated or polyunsaturated fatty acids in the stool, although there were contradictory results when associating certain long-chain fatty acids with the appearance of disease. Furthermore, some of these fatty acids, such as odd-chain fatty acids (OCFA), were described as coming mainly from gut-derived propionic acid synthesised endogenously in the body [25]. Hence, disorders of propionate were detected through plasma total odd-chain fatty acids determination [26]. OCFA was also used as a potential indicator of rumen function, as well as bacterial matter [27]; however, in non-ruminant species, OCFA are present in small proportions [28]. Recent studies carried out in humans show that plasma and tissue OCFA may be associated not only with lipid status in the organism [28] but also with the gut microbiota [29]. However, there is a lack of information on the diagnostic utility of the fatty acid profile (SCFAs, OCFAs or long-chain fatty acids) in the stool or plasma in chronic intestinal diseases in dogs. In addition, some studies reported that long-chain fatty acids could be involved in the development and treatment of some chronic inflammatory diseases [30]; therefore, the complete fatty acid profile in faeces and plasma as non-invasive procedures deserve more attention. Thus, the aim of this study was to evaluate differences in SCFAs and the total fatty acid profile of faeces and plasma as possible indicators of inflammatory chronic disease in the dog, specifically food-responsive enteropathy, and to study the possible correlation between SCFAs as indicators of gut homeostasis and the fatty acid profile in order to determine the most adequate fatty acid for intestinal disease prediction.2. Materials and Methods2.1. Animals and Sample CollectionHealthy dogs included in the study did not have any clinical signs, including digestive signs, within the past 4 months before the sample collection. Physical examinations and routine bloodwork of these dogs had to be normal to be enrolled in the study. Asymptomatic dogs with chronic diseases were excluded from the study.The criteria for the inclusion of sick dogs in the study included the persistence of clinical signs of chronic digestive disease (vomiting, diarrhoea, weight loss or anorexia/hyporexia) for at least 3 weeks. All dogs included in the study had a favourable response to an elimination diet (hydrolised protein or novel protein diet) after one month. Based on the response to dietary therapy, the disease of these dogs was classified as FRE. No dog included in the study had protein-losing enteropathy. Signalment, including age, sex, breed, sexual status, body weight and body condition score, was collected for every dog. Information about specific clinical signs related to chronic digestive disease was obtained in order to calculate the chronic enteropathy activity index (CIBDAI), as previously described [31].Faecal samples were collected by the owners after spontaneous defecation and received at the clinic in less than 3 h, where they were immediately frozen at −20 °C until analysis. Blood (2 mL) extracted using jugular or cephalic venipuncture were collected in heparine tubes. Plasma obtained after subsequent centrifugation was stored at −80 °C. Faecal and blood samples of dogs with FRE were collected before starting the dietary treatment. The pre-experimental diet for all dogs was mainly based on cereals, animal proteins and vegetable/animal fats (averaged percentages according to the manufacturer’s composition: humidity, 9.5 ± 0.0; crude protein, 26.8 ± 3.4; crude fat, 11.7 ± 4.4; ash, 5.9 ± 1.7; crude fibre, 1.8 ± 0.5; soluble fibre, 6.0 ± 0.7; nitrogen-free extractives, 38.3 ± 12.5; Ca, 0.9 ± 0.1; p, 0.7 ± 0.1; C18:2, 2.9 ± 1.1; ∑n-6, 2.7 ± 1.2; ∑n-3, 0.7 ± 0.1; mg/kg vitamin E: 619.2 ± 245.5; metabolic energy/100 g: 3332.3 ± 645.2. No vitamins, minerals, energy or any other supplements were administered at least 4 days before sample collection. Their participation in the study was always carried out through the informed consent of the owners. All procedures and protocols were approved by the Animal Research Committee of the Veterinary Medicine Teaching Hospital, Complutense University of Madrid (reference number 11/2021). 2.2. Laboratory Analysis2.2.1. Concentration of Vitamin E in Plasma SamplesThe concentration of vitamin E in the plasma samples was quantified as described elsewhere [32]. Vitamin E (α-tocopherol) was extracted directly without saponification. Duplicate plasma aliquots were mixed with a dibasic sodium phosphate buffer (0.054 M) adjusted to pH 7.0. Tocopherol was extracted via centrifugation (600× g for 10 min at 4 °C) after the addition of hexane to the mixture. After the evaporation of the upper layer, the remaining residue was dissolved in ethanol. Tocopherol was analysed using reverse-phase HPLC (HP 1100, equipped with a diode array detector) (Agilent Technologies, Waldbronn, Germany) [32]. Identification was carried out using the pure compound (Sigma-Aldrich, Alcobendas, Madrid) and quantification (µg of α-tocopherol per mL of plasma) was carried out by means of a standard curve built with the pure compound.2.2.2. Analysis of Short-Chain Fatty Acids in Faecal SamplesDetermination of short-chain fatty acids in the faecal samples was carried out as previously described [33]. Frozen dried stool samples were accurately weighed in a 2 mL safe-lock micro test tube. Two glass balls (2 mm Ø) and 1.0 mL distilled water were added. After being tightly capped, the tubes were placed on the adapters and homogenised for 5 min at 30 Hz in a Mixer Mill MM400 (Retsch technology, Haan, Germany). The final system was allowed to separate via centrifugation (10 min, 10,000 rpm). The extraction was repeated three times. Then, the faecal suspension was transferred into a vial and the internal standard (20 mM 4-methylvaleric acid solution) was spiked and the pH was adjusted to 2–3 by adding 25% phosphoric acid. Finally, this solution was placed in vials for gas chromatography injection. Chromatographic analysis was carried out using an Agilent 6850N GC system equipped with a flame ionisation detector (FID) (Agilent Technologies, Waldbronn, Germany). A fused-silica capillary column with a free fatty acid phase (DB-FFAP 125-3237, J&W Scientific, Agilent Technologies Inc., Santa Clara, CA, USA) of 30 m × 0.53 mm i.d. coated with a 0.50 µm thickness film was used. Nitrogen was used as the carrier gas at a constant pressure of 15 psi. The initial oven temperature was 100 °C maintained for 0.5 min, raised to 180 °C at 8 °C/min and held for 1.0 min, then increased to 200 °C at 20 °C/min and finally held at 200 °C for 5 min. The temperatures of the FID and the injection port were 240 °C and 200 °C, respectively. The flow rates of hydrogen, air and nitrogen as makeup gases were 40, 300 and 30 mL/min, respectively. Data handling was carried out with HP ChemStation Plus software (Agilent Technologies, Waldbronn, Germany). Identification and quantification were carried out using pure standards (Sigma-Aldrich, Alcobendas, Spain). An aqueous stock standard solution was prepared for each acid with a concentration of 400 mM for acetic acid, propionic acid and n-butyric acid; 200 mM for n-valeric acid and i-valeric acid; 100 mM for i-butyric acid; 50 mM for n-caproic acid; and 15 mM for n-heptanoic acid.2.2.3. Extraction of Total Fat and Fatty Acid Profile of Plasma and Faecal SamplesPlasmatic and faecal total lipids were extracted and then analysed for fatty acid profile determination. A solvent mixture of dichloromethane-methanol 8:2 was added to lyophilised weighted samples (Lyoquest, Telstar, Tarrasa, Spain) and after homogenisation in a mixer mill (MM400, Retsch technology, Haan, Germany) and centrifugation (8 min at 10,000 rpm), the upper layer containing lipids were collected. The lipid content was quantified gravimetrically after evaporation of the solvent in a nitrogen stream [34]. Fatty acid methyl esters (FAMEs) were obtained by heating the lipids (80 °C for 1 h) in the presence of methanol:toluene:H2SO4 (88:10:2 by volume), as described elsewhere [35]. After esterification, FAMEs were extracted with hexane and separated in a gas chromatograph (HP 6890 Series GC System; Hewlett Packard, Avondale, PA, USA) after direct injection of the sample. The gas chromatograph was provided with an automatic injector (hold at 170 °C), a flame ionisation detector (hold at 250 °C) and a capillary column (HP-Innowax polyethylene glycol, 30 m × 0.316 mm × 0.25 µm). After injection, the oven temperature was increased to 210 °C at a rate of 3.5 °C/min, then to 250 °C at a rate of 7 °C/min [35]. Identification and quantification of the FAMEs were made by comparing the retention times with those of authentic standards (Sigma–Aldrich, Alcobendas, Spain). Results were expressed as grams per 100 grams of quantified fatty acids.Different indices were measured to estimate the desaturase or elongase activities.The Δ9 desaturase index was calculated as the ratio of C18:0 to C18:1n-9 and as the ratio of C16:0 to C16:1.The elongase index was calculated as the ratio of C18:0 to C16:0 and as the ratio of C20:5 to C22:5.2.3. Statistical AnalysisData were analysed following a completely randomised design using the general linear model (GLM) procedure contained in SAS (version 9; SAS Inst. Inc., Cary, NC, USA).Data were presented as the mean of each group and the standard error of the mean (SEM), together with significance levels (p-values). Tukey’s test was used to separate the treatment means. The differences between means were considered statistically significant at p < 0.05. Pearson correlations (among SCFAs and plasma or faecal fatty acids, or CIBDAI index and the other variables) were calculated using the Statgraphics-18 program. A linear adjustment between variables was carried out by means of the Statgraphics-18 program (Statgraphics Centurion XVIII, version 18.1.12).3. Results and Discussion3.1. Signalment of DogsData regarding the age, sex, sexual status, body weight, body condition score (BCS) and canine inflammatory bowel disease activity index (CIBDAI) are shown in Table 1. The breeds of dogs with FRE (n = 9) were three mongrel dogs and one each of labrador retriever, cocker spaniel, miniature schnauzer, Maltese, short-haired dachshund and chihuahua. The breeds of the healthy dogs (n = 6) were 5 mongrel dogs and one Gordon setter. Differences concerning age, body weight or body condition score between the FRE and control groups were not statistically affected. The breed or reproductive status was not evaluated because of the insufficient numbers of individuals to study these effects. Previous studies indicated a higher prevalence of chronic enteropathy in purebreds, such as German shepherd, rottweiler, Weimaraner, border collie, or boxer when compared with mixed-breed dogs [36]. However, based on the study of these factors on the microbiome in dogs with IBD, other authors did not observe changes due to breed or reproductive status, although they did observe changes due to the disease [37].3.2. Oxidative Status and Lipid Plasma Profile of the DogsThe oxidative status, total fat and fatty acid profile of the plasma are presented in Table 2. The FRE dogs had a lower concentration (p = 0.026) of α-tocopherol as an indicator of the oxidative status of the animal, whereas no changes were observed in the proportion of plasma fat. Other authors reported reduced plasma antioxidant concentrations (mainly vitamin E and A) in human patients with inflammatory chronic disease [38]. The pathogenesis studies of inflammatory bowel disease (IBD) revealed that human patients with IBD had an excessive amount of oxidised molecules compared with healthy controls [39,40]. According to Rezaie et al. [39], in order to counteract this state, the organism responds with higher antioxidant production, which reduces the deposits and the capacity of the organism’s response to oxidative stress. Yuksel et al. [41] also reported that total antioxidant status in humans could be a good predictor of IBD. Hence, the use of substances based on the assessment of the redox status was recently proposed as a therapeutic alternative [42,43]. In addition, in rats, a relationship was found between different regions in the gastrointestinal tract and the oxidative status according to the microbiota since some microorganisms are able to produce endogenous antioxidants [40]. It is interesting to highlight that in the present study, the oxidative status, measured as the plasma vitamin E concentration in the alpha-tocopherol form, was also an indicator of greater C3 (r = 0.56, p = 0.044) in faeces, and a positive trend was observed for IC4 (r = 0.51, p = 0.072) and total SCFAs (r = 0.53, p = 0.064). Therefore, the higher the concentration of vitamin E in the plasma, the higher the content of C3 in faeces. There is no previous information on this result in dogs with chronic diseases, but some studies carried out in mice point to a direct connection between SCFAs and the inhibition of oxidative stress and inflammation [44].Concerning the lipid plasma profile, previous investigations did not find any changes in plasma triglyceride content in human patients with IBD [45]. In the present study, the specific fatty acid profile of the plasma (Table 2) revealed that the FRE dogs had higher C20:3n-6 (p = 0.0056) and tended to have higher C18:1n-9 (p = 0.072) and C20:2n-6 (p = 0.062), although the differences were not statistically significant; meanwhile, these FRE dogs had lower C20:0 (p = 0.003), C20:5n-3 (p = 0.033), C22:5n-3 (p = 0.005), polyunsaturated fatty acids (PUFA) (p = 0.021) and n-6 (p = 0.041), and tended to have lower proportions of C18:2n-6 (p = 0.051) and n-3 (p = 0.056), although these last two were not statistically different when compared with the control group. Other authors [46,47,48] reported decreased polyunsaturated fatty acids in different intestinal chronic diseases. In a detailed study carried out in humans, Esteve-Comas et al. [48] found a relationship between the severity of the disease (ulcerative colitis and Crohn’s disease) and the degree of decrease in n-3 and n-6 fatty acids. These authors also observed an increase in monounsaturated fatty acids in both diseases corresponding to the status severity. Similarly, Kuroki et al. [47] found negative correlations between the Crohn’s disease activity index and serum polyunsaturated fatty acids. The main essential polyunsaturated fatty acids come mainly from the diet, while monounsaturated fatty acids can be synthesised endogenously in the body. A lower proportion of polyunsaturated fatty acids could be related to malabsorption processes, or to a higher lipolytic activity of these fatty acids that are used preferentially for energy supply [49] as structural parts of cell membranes or as precursors of inflammation-regulating substances [50] in a high-requirement status, such as chronic digestive disease [47], consequently resulting in a higher proportion of other plasma fatty acids. In addition, a predominance of monounsaturated fatty acids due to desaturation phenomena to obtain energy in metabolic states in which there was a significant decrease in polyunsaturated fatty acids was described [22,49]. Conversely, in humans with ulcerative colitis, Bazarganipour et al. [51] reported increases of EPA and DHA (20:5n-3 and C22:6n-3) in the blood of patients with a more severe stage of the disease since these fatty acids are precursors of resolvins and maresins that are synthesised in order to repair the barrier disruption and were attributed to anti-inflammatory properties; however, dietary supply of these essential fatty acids should have been considered. As indicated by Hengstermann et al. [52], discrepancies in results between studies could be attributed to the malnutrition status and dietary therapeutic alternatives used to counteract the disease state. Furthermore, in the present research, it is interesting to highlight the greater proportions of some n-6 fatty acids (C20:3n-6 and C20:2n-6) observed in plasma from FRE dogs when compared with the control dogs, contrary to the decrease in n-6 fatty acids observed by other authors [47]. It was described that C20:3n-6 is the immediate precursor of PGE1 and C20:4n-6 (the main component of the phospholipids membranes) [22], and C20:3n-6 can be obtained directly from C20:2n-6 via Δ8-desaturase in a direct alternative route that is mainly activated with high eicosanoid requirements, such as in inflammation [53]. This is the first study in which the plasma fatty acid profile of dogs with inflammatory digestive chronic diseases was studied, specifically in dogs with food-responsive CE. The specific increase in these long-chain n-6 fatty acids (C20:2 and C20:3) in the blood could indicate their diagnostic potential as indicators of inflammation.3.3. Short-Chain Fatty Acid (SCFA) Profile in Faecal SamplesIn the present research, the short-chain fatty acid profile of faecal samples was also quantified (Table 3). Dogs with FRE showed a decrease in faecal levels of SCFAs. Similar results were found in dogs with different acute and chronic digestive diseases [20,21]. It was suggested that decreased SCFAs might contribute to the inflammatory status of these disorders [21]. Intestinal microbiota of dogs with CE showed a decrease in the phylum Firmicutes, especially in Clostridium XIVa and IV, which are significant producers of SCFAs [54,55].When analysing the specific content of SCFAs in the present study, the propionic acid concentration decreased in the dogs with FRE. A similar decrease in the concentrations of faecal propionic acid was also found in dogs with acute diarrhoea [20] and in dogs with CE [21]. These similar results were not unexpected, taking into account the fact that the previous study performed in dogs with CE [21] included some dogs with FRE, but also dogs with other types of CE. The significance of the difference in faecal propionate concentrations found in our study when comparing healthy dogs and dogs with FRE was the most prominent among all SCFAs, similarly to what was previously found in dogs with CE [21]. Propionate can play a role in the pathogenesis of chronic intestinal inflammation in dogs [21]. Its role in the immune system and intestinal inflammation has been widely evaluated, especially in vivo and in rodents. Among these functions, propionate is able to regulate the size and function of the colonic Treg cells that express the transcription factor Foxp3 and protect against colitis in mice [56]. The decreased levels of propionate in dogs with different CE could be potentially significant, taking into account the fact that dogs with IBD have decreased numbers of Foxp3-positive Treg cells in the duodenal mucosa [57,58].Concerning other SCFAs, information in the literature about faecal branched-chain fatty acids (BCFAs) in dogs is very limited. The relative concentration of isovalerate was associated with increased colitis and the IL-1β concentration of the intestinal mucosa in experimental models of IBD [59]. Our study showed that dogs with FRE also had lower concentrations of faecal isovaleric acid (p = 0.014), similar to what was found in rodent models of colitis [60]. Conversely, Guard et al. [20] reported that dogs with acute diarrhoea have similar levels of BCFA to healthy dogs. In contrast, in the present research, faecal butyric acid in dogs with FRE was not altered in comparison with healthy dogs. Similar results were found in dogs with chronic intestinal disease [21], while dogs with acute diarrhoea had higher levels of faecal butyric acid [20]. It was hypothesised that these results could be due to a reduction in the utilisation of butyrate by epithelial cells or a loss into the intestinal lumen in dogs with chronic intestinal inflammation [21].3.4. Total Fatty Acid Profile in Faecal SamplesThe total fatty acid profile of faeces from dogs affected with intestinal chronic disease, specifically FRE, or the control group is presented in Table 4. The FRE dogs had a lower proportion of C15:0 (p = 0.0003), C16:1n-9 (p = 0.0095), C16:1n-7 (p = 0.0001), C20:5n-3 (p = 0.0034) and monounsaturated fatty acids (MUFA) (p = 0.0315), and tended to have lower ∑n-3 (p = 0.058) when compared with the control group. However, the FRE dogs had a greater proportion of C18:0 (p = 0.017) and tended to have greater C20:4n-6 (p = 0.065) in faeces. This fatty acid profile was different from that found in plasma samples; however, some results were connected with those observed in blood, such as the proportion of ∑n-3 fatty acids. It is also interesting to highlight the high proportion of arachidonic acid (C20:4n-6), which could be the result of excessive membrane destruction in sick dogs or to greater production of this fatty acid to repair cellular damage at the intestinal level. This fact coincided with the higher proportions of C20:3n-6 and C20:2n-6 in blood as a faster alternative route for the synthesis of C20:4 [53], which could point to these fatty acids as possible indicators of inflammatory processes.The lower presence of some fatty acids, such as C15:0, in faecal samples could be in part associated with the different microbiome activity in the digestive system in these sick dogs. It was observed that odd-chain fatty acids (OCFA), such as C15:0 and C17:0, can be endogenously synthesised in the body from short-chain fatty acids, such as propionic acid (C3:0), which mainly come from processes of microbial fermentation in the gut [25]. Sick dogs also had lower levels of MUFA, such as C16:1n-9 and C16:1n-7, in the faeces, which could be explained by the higher metabolic use of these fatty acids, although no significant differences were observed in the blood levels. Other authors reported the high predisposition of C16:1n-7 to undergo β-oxidation [47,61], as well as the preferential use of monounsaturated fatty acids to obtain energy after polyunsaturated fatty acids [35,47]. The poorer utilisation of nutrients and malnutrition associated with animals with inflammatory digestive disease could induce this greater metabolic utilisation rate of certain fatty acids in the present study. Furthermore, the lower presence of these monounsaturated fatty acids and high level of C18:0 in dogs with inflammatory disease could also be explained by a possible lower desaturase activity at the level of the enterocyte membrane. This was confirmed by the lower C16:1/C16:0 and C18:1/C18:0 indices as indicators of the desaturase activity, which were observed in the faecal samples from the sick dogs. Garg et al. [62] reported that although the activity of desaturases in the intestine was lower than in the liver, it had an important effect on the properties of the enterocyte membrane. Therefore, according to the results of the present study, a greater alteration of the intestinal cell membrane could affect such a desaturation capacity. However, it is very interesting to observe in the present study how the elongase capacity found in the intestinal sample was higher in sick dogs. This was more marked in the case of the elongase activity of monounsaturated fatty acids C16:0/C18:0 than in the elongase activity of polyunsaturated fatty acids C22:5/C20:5. Oreshko et al. [63] reported a tendency towards increased serum elongase activity in human patients with celiac disease when compared with healthy controls. The diseased animals probably adapted their metabolic pattern to achieve a greater synthesis of saturated and long-chain polyunsaturated fatty acids as precursors of other energy-supplier fatty acids and main constituents of the membrane structure [63], as well as the synthesis of compounds with anti-inflammatory characteristics.3.5. Correlations between Fatty Acids and SCFAsThere is no previous information on the global diagnostic possibilities of SCFAs, plasma or faeces fatty acid profile for FRE in dogs; therefore, this is the first study in which these compounds were evaluated in healthy dogs and dogs with food-responsive chronic enteropathy. Since some of these measurements are considered of interest in humans, we looked for possible relationships between the different compounds. In the faecal SCFAs, the greater correlations were detected for propionic acid (C3), isobutyric acid (IC4) and isovaleric acid (IC5), followed by acetic acid (C2) and valeric acid (C5) (Table 5). The faecal fatty acids that presented the greatest correlations were C15:0, C16:1n-7 and C20:5n-3, followed by C18:0, MUFA and C20:4n-6. The highest correlations between faecal OCFA, medium- or long-chain fatty acids and SCFAs were observed for C15:0, which correlated positively with C2 (r = 0.72, p = 0.004), C3 (r = 0.95, p = 0.0001), IC4 (r = 0.59, p = 0.027) and IC5 (r = 0.64, p = 0.0136), as well as with total SCFAs (r = 0.61, p = 0.02). Conversely, C20:4n-6 showed a high inverse correlations with C2 (r = −0.83, p = 0.0002) and C3 (r = −0.59, p = 0.027). C16:1n-7 correlated positively with C3 (r = 0.85, p = 0.0001), IC4 (r = 0.66, p = 0.0102) and IC5 (r = 0.72, p = 0.0037); meanwhile, C20:5 correlated to a lesser extent with C3 (r = 0.62, p = 0.017), IC4 (r = 0.55; p= 0.042) and IC5 (r = 0.68, p = 0.0072). These fatty acids could therefore be considered as indicators of the intestinal health status and mucosa integrity.On the other hand, faeces moisture and fat were negatively correlated with SCFA (Table 5). A higher number of correlations were observed for faeces moisture than for fat. Hence, faeces moisture correlated negatively with C3 (r = −0.68, p = 0.0077), IC4 (r = −0.55, p = 0.040) and IC5 (r = −0.56, p = 0.035), and showed a greater correlation for total SCFAs (r = −0.80, p = 0.0007) than the other variables. Other authors found a greater SCFA proportion and faecal moisture in the colons of healthy mice [64]; however, in the present research sick dogs were affected with diarrhoea and higher moisture in the stool could be associated with a clinical sign of disease. Therefore, according to the present results and as stated before, C3 could be a good indicator of chronic diarrhoea, but more research is needed in order to know if this compound could be affected between different CE.The correlations between SCFA and plasma fatty acids, plasma fat and tocopherol concentration were also determined (Table 6). A lower number of significant correlations were observed in the plasma than in the faeces. IC4 and IC5, followed by C3, were the SCFAs that had the greatest number of correlations with the other plasma variables. IC4 correlated negatively with C18:0 (r = −0.61, p = 0.025) and positively with C18:3n-3 (r = 0.64, p = 0.018), C20:1n-9 (r = 0.57, p = 0.042), C20:5n-3 (r = 0.60, p = 0.031), C22:4n-6 (r = 0.68, p = 0.010) and total n-3 (r = 0.61, p = 0.025). In the same way, IC5 correlated positively with C20:0 (r = 0.56, p = 0.046), C20:5n-3 (r = 0.68, p = 0.010), C22:5n-6 (r = 0.68, p = 0.011), total PUFA (r = 0.56, p = 0.048) and n-3 (r = 0.59, p = 0.013). According to these results, plasma n-3 fatty acids levels could be interesting predictors of intestinal health, probably in connection with the nutritional status of the animal.Finally, the correlation between BCS and CIBDAI scores and the other parameters of plasma and faeces were also evaluated (Table 7). The significant correlations were mainly detected in faecal parameters. Moreover, the CIBDAI score presented a higher number of significant correlations than BCS. Hence, the CIBDAI score correlated negatively mainly with faecal measurements, such as C3 (r = −0.869, p = 0.0005) and C15:0 (r = −0.825, p = 0.0018), followed by C16:1/C16:0 (r = −0.66, p = 0.0374) and iC5 (r = −0.648, p = 0.0310), as well as the sum of C2 + C3 (r = −0.659, p= 0.0273) and the sum of C2 + C3 + C4 (r = −0.632, p = 0.0369). In addition, the CIBDAI score was also positively correlated with faeces moisture (r = 0.790, p= 0.0039). This index is considered a reliable measure of inflammatory activity in canine IBD [31]; therefore, according to the results of the present study, the CIBDAI score would be a good indicator of chronic inflammatory status. Its quantification, together with C3, other or total SCFAs, C15:0 or desaturase capacity in faecal samples would reinforce this non-invasive diagnosis technique in dogs with chronic inflammatory diseases.4. ConclusionsIn conclusion, the dogs with FRE had a lower oxidative status and higher plasma proportions of C20:2 and C20:3 as indicators of chronic inflammation, as well as lower propionic acid and branched-chain fatty acids, such as isovaleric acid, in their stools. The short-chain fatty acids correlated better with the total fatty acid profile of the faeces. The high correlations observed between most of the SCFAs and OCFA, such as C15:0, in the faeces indicates the diagnostic potential of this compound. Sick dogs also showed signs of damage at the intestinal level with a greater presence of arachidonic acid (C20:4), as well as a reduced desaturase activity in the stool. Further studies would be warranted in order to elucidate whether specific profiles of faecal SCFAs, OCFA or long-chain fatty acids could be found in dogs with different CEs, such as FRE, ARE and IRE.

animals : an open access journal from mdpi

10.3390/ani13111870

PMC10251909

Grass carp reovirus genotype Ⅱ (GCRV Ⅱ) is the leading cause of death in grass carp. To investigate the involved molecular responses against the GCRV Ⅱ infection, we performed comparative transcriptomic analysis in the spleen and liver of rare minnow injected with virulent and attenuated strains. Results showed that the virulent strain infection especially induced tissue-specific alteration and caused severe suppression of hemorrhage related pathways in spleen. Our finding provides new insights on the interactions between host and GCRV Ⅱ.

Grass carp reovirus genotype Ⅱ (GCRV Ⅱ) causes a variety of fish hemorrhagic disease, which seriously affects the sustainable development of grass carp aquaculture in China. Rare minnow (Gobiocypris rarus) is an ideal model fish to study the pathogenesis of GCRV Ⅱ. To investigate the involved molecular responses against the GCRV Ⅱ infection, we performed comparative transcriptomic analysis in the spleen and liver of rare minnow injected with virulent strain DY197 and attenuated strain QJ205. Results showed that the virulent DY197 strain induced more differently expressed genes (DEGs) than the attenuated QJ205 strain, and tissue-specific responses were induced. In the spleen, the attenuated and virulent strains induced different DEGs; the attenuated QJ205 infection activated steroid synthesis pathway that involved in membrane formation; however, virulent DY197 infection activated innate immunity and apoptosis related pathways while suppressing cell proliferation and migration related pathways that are important for damage tissue repair, as well as hemorrhage related pathways. In the liver, the attenuated and virulent strains infection induced similar DEGs; both strains infection activated immunity and apoptosis related pathways but suppressed metabolism-related pathways; virulent DY197 infection especially activated protein digestion and absorption-related pathways and suppressed steroid synthesis pathway. To conclude, virulent strain infection especially induced tissue-specific alterations and caused severe suppression of hemorrhage-related pathways in spleen. Our findings will contribute to better understanding of the interactions between host and GCRV II.

1. IntroductionGrass carp reovirus (GCRV) is a double-stranded RNA (dsRNA) virus belonging to the genus Aquareovirus of the family Reoviridae [1]. GCRV infects a variety of fish and caused serious hemorrhage disease, resulting in huge economic losses to the aquaculture industry in China [2,3]. According to the VP6 protein sequence of GCRV, the known GCRV isolates were classified into three genotypes (Ⅰ–Ⅲ), and the sequence similarity between different genotypes was less than 20% [4,5]. A preliminary epidemiological analysis and detection of grass carp hemorrhagic disease fish collected from 2015 to 2017 in China showed that the positive rate of GCRV Ⅱ was as high as 89.8% [6], indicating that GCRV Ⅱ was the most common etiological agent of grass carp hemorrhagic disease. The mortality rates of grass carp caused by different GCRV Ⅱ strains were different. For example, the mortality rates of grass carp infected with HZ08, 109, and HuNan1307 strains were 30%, 80%, and 100%, respectively [7,8]. Investigating the pathogenic mechanism of GCRV Ⅱ is of great significance to improve the prevention and control of grass carp hemorrhagic diseases.The rare minnow (Gobiocypris rarus) is a small cyprinid species endemic to China, with a total length of 3–6 cm and a short reproductive cycle. It is widely used in studies of ecotoxicology fields as a model fish [9]. In addition, rare minnow has been demonstrated to be sensitive to GCRV Ⅱ, making it an ideal model fish for research of GCRV Ⅱ pathogenesis [10,11]. In teleosts, the spleen is a primary hematopoietic and peripheral lymphoid organ [12] and is mainly responsible for microorganism defense, antigen presentation, and the start of adaptive immune responses [13,14]. The liver of teleosts serves as an important immune organ by housing numerous immune cell populations in addition to its functions in metabolism and the storing of nutrients [15]. Moreover, previous studies have shown that the liver and spleen are among the most severely infected tissues of grass carp and rare minnow [11,16,17,18].Transcriptomic analysis can provide a comprehensive understanding of the intricate biological processes of fish response against infection at the transcriptome level [19,20]. With the rapid development of next-generation sequencing technology, a growing number of fish infectious diseases studies have been conducted using transcriptomics technology. For example, studies of Atlantic salmon infected with viruses [21], crucian carp and tilapia infected with bacteria [22,23], and large yellow croaker infected with parasites [24] have been reported. There are also several transcriptome studies conducted on GCRV-infected grass carp and rare minnow. He et al. [3] analyzed the kidney transcriptome of grass carp infected with GCRV Ⅰ and GCRV Ⅱ and found that mRNA expression of metabolism-related genes was downregulated and mRNA expression of immune-related genes was upregulated on the 5th day after GCRV Ⅱ infection, and the complement and coagulation cascade was the most enriched pathway. Chen et al. [25] studied the transcriptome of grass carp kidney cells (Ctenopharyngodon idellus kidney, CIK) infected with GCRV. It was found that there were three stages of infection: in the early stage (0–8 h), differentially expressed genes (DEGs) were mainly related to viral adhesion; in the middle stage (8–24 h), DEGs were mainly related to viral phagocytosis and transmission; in the late stage (24–72 h), DEGs were mainly concentrated in steroid metabolism that is important for membrane formation and apoptosis that involved in cell lysis. Lin et al. [26] studied the transcriptome of rare minnow infected with genotype Ⅱ virus GCRV-HZ08; when they compared it with the transcriptome of grass carp infected with GCRV-HZ08, they found that the responses of the two species were similar. Based on the above research results, GCRV Ⅱ infection may lead to host innate immune activation, metabolic dysfunction, and coagulation system disorders. However, the transcriptomic responses to virulent and attenuated GCRV Ⅱ infection have not been reported, as well as the difference in response between tissues has not been paid much attention in previous studies.In the previous study, the experiments of mortality statistics, viral load measurement, and histological examination were conducted on rare minnow after attenuated GCRV Ⅱ isolate QJ205 and virulent GCRV Ⅱ isolate DY197 infection. The infection of QJ205 caused slightly muscular hemorrhage symptoms and 5% mortality in rare minnow, associated with low virus copy numbers and no obvious pathological changes in the spleen and liver. In contrast, DY197 infection led to severe muscular hemorrhage symptoms and 95% mortality in rare minnow, as well as approximately 100-fold virus copy numbers of those infected with attenuated QJ205 and severe cell necrosis in the spleen and liver [27]. To further dissect the virulence-specific and tissue-specific molecular mechanism, in the present study, we performed comparative transcriptome analysis in the spleen and liver of rare minnow after virulent and attenuated isolate infection. These results would undoubtedly help us to better understand the pathogenesis and host–pathogen interaction of GCRV Ⅱ infection.2. Materials and Methods2.1. Experimental FishApproximately 1500 healthy rare minnows weighing 1–1.5 g and measuring 4–4.5 cm were acquired from the Institute of Hydrobiology, Chinese Academy of Sciences (Wuhan, China). The fish were raised in a 500 L tank with a flow-through system and plenty of aeration at 28 °C prior to the experiment. The water was replaced every day, and the fish were fed twice daily. After ten days, if there were no abnormal symptoms, the virus challenge experiment was carried out.2.2. VirusThe attenuated strain (GCRV-QJ205) and the virulent strain (GCRV-DY197) of GCRV Ⅱ were isolated from diseased grass carp that had been collected from the cities of Qianjiang in Hubei province and Deyang in Sichuan province, respectively. The spleens and livers of diseased grass carp were homogenized with a 5-fold amount of phosphate-buffered saline (PBS), and three freeze–thaw cycles at −80 °C were performed to isolate the virus. The tissue homogenate was then centrifuged for 30 min at 2880× g. The filtrate from the supernatant was diluted to a titer of 1 × 106 RNA copies/L for the subsequent viral challenge experiment after being filtered through a 0.22 μm Millipore filter (Millipore, Billerica, MA, USA). 2.3. Virus Infection and Sample Collecting Rare minnows were divided into three groups at random and given intraperitoneal injections of PBS (as a control group), QJ205, and DY197 at doses of 10 μL (1 × 106 RNA copies/μL) for each fish. The samples (livers and spleens) of 480 and 9 fish from each group were collected at 5 days post-infection (dpi) and split into three parts as biological replicates for transcriptome sequencing and quantitative real-time PCR (qPCR) validation, respectively. The spleens and livers of the control, QJ205, and DY197 groups were designated as control-spleen (C-S), attenuated-spleen (A-S), virulent-spleen (V-S), control-liver (C-L), attenuated-liver (A-L), and virulent-liver (V-L), respectively.2.4. RNA Extraction, cDNA Library Construction, and SequencingUsing the Trizol reagent kit (Invitrogen, Carlsbad, CA, USA), the total RNAs were extracted from the spleen and liver samples in accordance with the manufacturer’s instructions. After the total RNA was isolated, Oligo(dT) beads were used to enrich mRNA. The short fragments created by using fragmentation buffer to break up the enriched mRNA were then reverse transcribed into complementary DNA (cDNA) using random primers. The second-strand cDNA fragments were then repaired at the terminal, A base was added and connected to the Illumina sequencing adapters. Agarose gel electrophoresis was used to size-select the ligation products, followed by PCR amplification and Illumina NovaSeq 6000 sequencing.2.5. De Novo Assembly and AnnotationTo get high-quality clean reads, the raw reads were further filtered by fastp (version 0.18.0). The parameters were as follows:(1)removing reads containing adapters;(2)removing reads containing more than 10% of unknown nucleotides (N);(3)removing low-quality reads containing more than 50% of low-quality (Q-value ≤ 20) bases.Subsequently, using the default settings in Trinity software [28], clean reads were de novo assembled and then were mapped back to the National Center for Biotechnology Information (NCBI) to remove GCRV Ⅱ contamination using the program Blast. Each cluster’s longest RNA was designated as a unigene. To annotate the unigenes, we used BLASTx software (http://www.ncbi.nlm.nih.gov/BLAST/, accessed on 1 June 2022) with an E-value threshold of 1 × 10−5 to NCBI non-redundant protein (Nr) database (http://www.ncbi.nlm.nih.gov, accessed on 1 June 2022), the COG/KOG database (http://www.ncbi.nlm.nih.gov/COG, accessed on 1 June 2022), the Kyoto Encyclopedia of Genes and Genomes (KEGG) database (http://www.genome.jp/kegg, accessed on 1 June 2022), and the Swiss-Prot protein database (http://www.expasy.ch/sprot, accessed on 1 June 2022). 2.6. Gene Expression Analysis and Enrichment Analysis Gene expression levels were firstly estimated by mapping clean reads from each library back to the transcriptome assembly using Bowtie2 software [29] and then calculated read counts and normalized as FPKM (Fragments Per Kilobase of transcript per Millions mapped reads) values for each sample using RSEM software [30]. The DESeq R package [31] was used to carry out the differential analysis, and p-values were modified using Benjamini-Hochberg’s method. Finally, DEGs were designated as genes with an absolute value of log2 (Fold change) greater than 1 and an adjusted p-value less than 0.05. Then DEGs were enriched and analyzed with Gene Ontology (GO) term and KEGG pathway.2.7. Validation of DEGs by RT-qPCREight DEGs that were significantly differentially expressed in at least one isolate-infected group in each tissue were selected for the quantitative reverse transcript PCR (RT-qPCR) validation. As mentioned above, total RNA was extracted from spleen and liver tissue samples. The manufacturer’s instructions were followed to produce cDNA from the total RNA using a PrimeScript RT reagent Perfect Real Time Kit (TaKaRa, Dalian, China). Then, the reaction of qPCR was performed and analysed using a Rotor-Gene Q Series Software 1.7 supplied with the instrument (QIAGEN, Hilden, German). An amount of 10 μL of TB Green Premix Ex Taq Ⅱ (TaKaRa, Dalian, China), 2 μL of the cDNA sample, 0.8 μL (10 μM) of each primer, and ddH2O in a total volume of 20 μL made up the reaction mixtures. The reactions were amplified for 30 s at 95 °C, followed by 40 cycles of 95 °C for 10 s, 60 °C for 15 s, and 72 °C for 20 s.Primer sequences of eight DEGs were listed in Table 1. For normalization of gene expression, β-actin gene was used as an internal control. Primers had a Tm of roughly 60 °C, and PCR products ranged in length from 100 to 200 bp. qPCR was conducted three times for each sample as technique replicates.3. Results3.1. Transcriptome Sequencing, De Novo Assembly, and AnnotationTranscriptomic sequencing generated totally 783 million raw reads from 18 libraries, which were deposited in the Sequence Read Archive (SRA) at the NCBI repository (accession number: PRJNA954066). After quality-filter analysis, 780 million clean reads were produced and de novo assembled into a total of 62,638 unigenes with an N50 length of 2385 bp (Table S1). All libraries gave Q20 ≥ 97%, Q30 ≥ 92%, and mapped percent ≥ 85% (Table S2). All unigenes were functionally annotated using four public databases, including Nr, Swiss-Prot, GO, and KEGG, for the assembled reference transcriptome. The results showed that 48.27% of unigenes were annotated by at least one of public database (Table S3). The Nr annotation demonstrated that 51.67% of unigenes in rare minnow liver and spleen could be annotated in the database of Pimephales promelas and Anabarilius grahami (Figure S1).3.2. Identification and Enrichment of Differentially Expressed GenesThe PCA score plots showed good repeatability of the data, as the liver and spleen data sets were separated, while the same data set was clustered together (Figure 1A). These results demonstrated the sequencing data had high quality and was suitable for further investigation. To determine DEGs involved in response to GCRV Ⅱ infection in liver and spleen tissues of rare minnow, pairwise comparison for differential expression analysis was performed. In the spleen, compared with the control group, 145 DEGs (135 upregulated and 10 downregulated) were identified in the attenuated QJ205 infection group and 1461 DEGs (614 upregulated and 847 downregulated) in the virulent DY197 infection group (Figure 1B). In the liver, 227 DEGs (134 upregulated and 93 downregulated) were identified in the QJ205 infection group and 1461 DEGs (976 upregulated and 249 downregulated) in the DY197 infection group compared with the control group (Figure 1B). In both tissues, the virulent DY197 induced more DEGs than the attenuated QJ205, indicating the response to the infection of rare minnow was positively correlated with the virulence of GCRV Ⅱ. In the spleen, only six upregulated genes and seven downregulated genes were shared between the virulent and attenuated groups, indicating that the virulent and attenuated strain infection induced different responses (Figure 1C). In the liver, a total of 103 upregulated genes and 46 downregulated genes were shared in the virulent and attenuated strain infection groups, indicating that the virulent and attenuated strain infection induced similar responses in the liver (Figure 1C).3.3. Enrichment Analysis of Differentially Expressed Genes in SpleenIn the spleen, KEGG enrichment analysis (Figure 2) showed different pathways were induced in both infected groups. Among the upregulated pathways, innate immunity-related pathways, such as RIG-Ⅰ, TOLL, Nod-like receptor signaling pathways, JAK-STAT signaling pathway, lysosome, phagosome, and apoptosis, were significantly enriched in the virulent DY197 infection group. Lipid metabolic pathways, such as steroid metabolism, biosynthesis of unsaturated fatty acid, and fatty acid metabolism, were significantly enriched in the attenuated QJ205 infection group. In the downregulated pathways, cell migration and proliferation-related pathways such as focal adhesion, extracellular matrix receptor interaction (ECM-receptor interaction), regulation of actin cytoskeleton pathways, adaptive immunity-related pathways such as hematopoietic cell lineage, T cell receptor signaling pathway, and hemorrhage-related pathways such as malaria and platelet activation were significantly enriched in the DY197 infection group. In addition, as shown in Table 2, the expression levels of DEGs in these pathways changed more significantly in the virulent DY197 infection group.3.4. Enrichment Analysis of Differentially Expressed Genes in LiverIn the liver, KEGG enrichment analysis (Figure 3) showed that similar pathways were induced in both infected groups. Immunity pathways and protein digestion and absorption pathway were significantly activated after both strains of infection; the proteasome pathway was only significantly enriched in the virulent DY197 infection group; while metabolic pathways were inhibited, and the steroid synthesis pathway was only enriched in the virulent DY197 infection group. As shown in Table 3, virulent DY197 infection not only induced more DEGs but also induced greater changes in the expression level of DEGs.3.5. Validation of Differentially Expressed Genes by qPCREight DEGs involved in the immune-related pathways, lysosome pathway, and hemorrhage-related pathways were selected for qPCR validation. These eight DEGs included MHC class I antigen (MHCI), interferon regulatory factor 3 (IRF3), C-X-C motif chemokine 8 (CXCL8), signal transducer and activator of transcription 1b (STAT1B), cathepsin B (CTSB), urokinase plasminogen activator surface receptor (PLAUR), hemoglobin subunit alpha (HBA), and platelet glycoprotein Ib beta chain (GP1BB). As shown in Figure 4, qPCR expression trends of these eight DEGs were consistent with transcriptome results, which confirmed the accuracy and reliability of RNA-seq results.4. DiscussionGCRV Ⅱ causes severe hemorrhagic disease in grass carp and affects the aquaculture industry in China. Previous studies have demonstrated that GCRV Ⅱ infection induced innate immunity activation, metabolic dysfunction, and coagulation disorder. However, the underlying virulence-specific and tissue-specific pathogenesis of GCRV Ⅱ infection remains to be further studied. For this reason, we performed a comparative transcriptomic analysis in the spleen and liver of rare minnow injected with virulent isolate DY197 and attenuated isolate QJ205 to investigate the possible involved molecular responses against the GCRV Ⅱ infection. The results showed the number of DEGs was positively correlated with the virulence of GCRV Ⅱ. In the spleen, compared with attenuated QJ205 infection, virulent DY197 infection activated innate immunity and apoptosis-related pathways but suppressed adaptive immunity, cell proliferation and migration, and hemorrhage-related pathways. In the liver, except innate immunity and apoptosis-related pathways, virulent DY197 infection especially activated protein digestion and absorption-related pathways, both innate and adaptive immunity-related pathways, and cell migration and proliferation-related pathways and caused slight suppression of hemorrhage-related pathways. The different regulatory mechanisms in the spleen and liver after GCRV Ⅱ infection were shown in Figure 5.4.1. Immune ResponseAs pattern recognition receptors (PRRs), RIG-I, Toll, and Nod-like receptors can recognize the unique pathogen-associated molecular pattern (PAMPs) or damage-associated molecular pattern (DAMPs) components of the organism and initiate downstream inflammatory responses in response to infection when the host is infected with a pathogen [32]. Extracellular cytokines can be recognized by corresponding receptors, stimulate the JAK-STAT pathway, regulate transmembrane receptor communication to the nucleus, and promote the expression of related antiviral genes [33]. However, the activation of STAT also causes tissue damage and leads to hemorrhage [34,35]. Members of the cytokine signal transduction inhibitors (SOCS) family are key regulators of immune balance [36,37]. In this study, in the spleen of virulent DY197-infected group, the mRNA expression levels of Toll-like receptor 3 (TLR3) and Toll-like receptor 8 (TLR8) in the Toll-like receptor signaling pathway; interferon regulatory factor 3 (IRF3), interferon regulatory factor 7 (IRF7), interferon (IFN-α and IFN-β), Retinoic acid-inducible protein I (RIG-I) in RIG-I receptor pathway; melanoma differentiation associated gene 5 (MDA5), DEXH-box polypeptide 58 (LGP2), JAK1 and STAT1B in JAK-STAT signaling pathway were significantly upregulated, enhancing inflammation to restrict viral infection, while mRNA expression levels of SOCS1, SOCS3 and SOCS6 were upregulated, controlling inflammation and avoid excessive damage. This regulatory mode was also observed in the liver of the virulent DY197 infection group. In comparison, the mRNA expression levels of related genes were slightly upregulated or even downregulated in the spleen and liver of the attenuated QJ205-infected group.Phagocytosis of macrophages and degradation of lysosomes are the last step of phagocytosis and elimination of microorganisms [38]. For example, as a protease of lysosomes, cathepsin L (CTSL) is involved in a variety of immune responses, including apoptosis, antigen presentation, and inflammation [39,40]. In this study, as important effecting genes and antigen presentation genes in the lysosome and phagosome pathways, mRNA expression levels of cathepsins (CTSL, CTSS, CTSZ, CTSA, CTSK, and CTSB) and major histocompatibility complex class Ⅰ antigen (MHC Ⅰ) were significantly upregulated in both virulent DY197 infected liver and spleen and slightly upregulated or even downregulated in attenuated QJ205 infected liver and spleen, indicating that the virulent DY197 infection activated phagocytosis of macrophages. Similar to our study, the activation of phagosome and lysosome pathways was also found in the spleen and kidney of GCRV Ⅱ-infected grass carp [3,41].However, in terms of adaptive immunity, the B cell receptor signaling pathway was activated in both liver and spleen, while the T cell receptor signaling pathway behaved differently in the liver and spleen. CD8 cell surface receptors CD8A and CD3Z genes were downregulated in the spleen, while CD3Z genes were upregulated in the liver. Previous studies in human viruses, such as herpes simplex virus (HSV) and SARS coronavirus 2 (SAR-COV-2), have demonstrated that activation of the kynurenine pathway of tryptophan metabolism can enhance antioxidant to alleviate inflammation in innate immune cells, but depletion of tryptophan will lead to suppression of T cell [42,43]. In this study, kynurenine-oxoglutarate transaminase 1 (KYAT1), the kynurenine pathway gene of tryptophan metabolism, was significantly upregulated in both spleen and liver after the virulent DY197 infection (Table 2 and Table 3). T cell was suppressed in the spleen and activated in the liver, which may be related to a sufficient supply of tryptophan because of liver-specific protein synthesis and metabolism function as discussed below. Tissue-specific inhibition of T cells provides us new insight into further investigation of the pathogenesis of GCRV Ⅱ infection.4.2. Apoptosis and Necroptosis-Related PathwaysApoptosis and necroptosis can be activated through stimulation of IFN and tumor necrosis factor (TNF), DNA damage, depletion of cellular NAD+, production of iron-dependent ROS, mitochondrial permeability change, etc. [44]. IFN transcription activates RNA reactive protein kinase, triggering necrosis through JAK1–STAT1-dependent transcription [45]. TNF stimulates the production of caspase-7/8, which plays an important role in the pathogen clearance and apoptosis of damaged cells [46]. DNA damage causes mitochondrial apoptosis induced by BAX (Bcl-2-associated X protein) [47]. Transcriptomic studies of GCRV-infected CIK [25] showed that necroptosis and apoptosis pathways were activated, and the steroid synthesis pathway that involved cell membrane formation [48] was suppressed in the later stage of infection. In this study, after virulent DY197 infection, the mRNA expression levels of tumor necrosis factor receptor superfamily member 1A (TNFRSF1A), tumor necrosis factor superfamily member 10 (TNFSF10), caspase-7, caspase-8, BAX genes were significantly upregulated in the liver, and the mRNA expression levels of caspase7 and BAX genes were significantly upregulated in the spleen; while the mRNA expression levels of these genes were slightly upregulated after attenuated QJ205 infection in both liver and spleen. In addition, the steroid synthesis pathway associated with membrane formation was suppressed in both spleen and liver after the virulent DY197 infection, which was consistent with the virulent DY197 infection that caused severe cell necrosis and tissue damage observed by histological examination [27]. Interestingly, the steroid synthesis pathway was activated in the spleen of attenuated QJ205 infected rare minnow, which may indicate that the spleen had passed the peak of antiviral response and began to synthesize steroids to restore membrane formation at 5 dpi. Previous studies have shown that hemorrhage symptoms gradually appeared on the 5th to 7th day in the head kidney of grass carp after a virulent strain of GCRV Ⅱ infection, and the upregulation of immune gene expression and downregulation of metabolic gene expression caused by viral infection would be reversed on the 7th day [3]. This reversal may be a regulatory mechanism for recovery after viral infection. Another study on viral load of GCRV Ⅱ-infected grass carp showed spleen and kidney were the tissues with the first increase in viral load and reached the peak of viral load on the 5th day, which may be due to the accumulation of virus caused by immune, hematopoietic, or glomerular filtration in these two tissues [16,17]. In studies of rare minnow infected with a virulent isolate of GCRV Ⅱ, the spleen was also the tissue that reached the viral load peak relatively quickly [11]. Considering GCRV Ⅱ tends to replicate faster and cause higher mortality in rare minnow than in grass carp after GCRV Ⅱ infection [11,16], it is possible that the spleen has passed the peak of disease and started to recover in rare minnow on the 5th day after attenuated QJ205 infection.4.3. Protein Digestion and AbsorptionThe protein digestion and absorption pathway was activated only in the liver after both strain infections. Solute carrier family 15 member 1 (SLC15A1) chiefly mediates di/tripeptides absorption from protein digestion [49]. Meprin alpha, a zinc metalloprotease, was previously reported to be capable of cleaving a variety of substrates (e.g., protein kinases, basement membrane proteins, cytokines), and participate in the regulation of fibroblast activation and production of extracellular matrix [50]. In this study, the mRNA expression of meprin alpha subunit A/B (MEP1A, MEP1B) and SLC15A1 genes was significantly upregulated after both strain infections in the liver. The proteasome can degrade a large number of damaged and misfolded proteins, and then the production can be used to synthesize new proteins required by the organism. Thus, the proteasome controls many biological processes, including cell cycle, cell survival, and apoptosis [51,52]. In this study, in the liver, the proteasome pathway was the most significantly different pathway between the virulent DY197 and attenuated QJ205 infection group. In contrast, in the spleen, only two proteasome genes were significantly upregulated after virulent DY197 infection, and no related genes were upregulated after attenuated QJ205 infection. The activation of protein digestion and absorption and proteasome pathways induced by GCRV Ⅱ infection may lead to an increase in oligopeptides and amino acids of proteolytic products, which was consistent with protein synthesis and metabolism for liver-specific function. The tissue-specific activation of protein digestion and absorption and proteasome pathways may be responsible for T cell activation in the liver but suppression in the spleen after virulent DY197 infection as described above. In addition, the lack of protein digestion and absorption and proteasome activation may result in the inhibition of many biological processes controlled by these two pathways in the spleen after virulent DY197 infection, including cell cycle, cell survival, and apoptosis [51,52].4.4. Cell Proliferation and MigrationFocal adhesion is a subcellular structure that regulates the adhesion response of cells to the extracellular matrix [53]. Focal adhesion kinase is the core of the focal adhesion pathway; it can communicate with integrin, vascular endothelial growth factor receptor (VEGFR), platelet-derived growth factor receptor (PDGFR), insulin-like growth factor receptor (IGFR) and actin-related proteins that interact with each other to regulate cell proliferation, migration, and survival [54,55]. The regulation of the actin cytoskeleton pathway is downstream of the focal adhesion pathway and is involved in the regulation of cell movement. Previous studies have shown that focal adhesion kinase can interact with phosphoproteins of rabies virus and participate in viral infection [56]; during infection of shrimp, white spot syndrome virus was also found to interact with integrin proteins of the focal adhesion pathway [57]. Studies of GCRV-infected CIK, kidney, and spleen of grass carp showed that the focal adhesion and regulation of actin cytoskeleton pathways were activated at the initial stage of infection, suggesting these two pathways may be involved in the binding of the virus to the receptor; while these two pathways were suppressed at the later stage of infection, suggesting that the host translation mechanism was hijacked or shut down to promote virus replication and transmission [3,25,41]. In this study, after virulent DY197 infection, the mRNA expression of platelet-derived growth factor C (VEGFC), platelet-derived growth factor receptor subunit B (PDGFB), integrin alpha-2 (ITGA2), myosin regulatory light polypeptide 9b (MYL9), myosin light chain kinase (MYLK) genes involved in focal adhesion and regulation of actin cytoskeleton pathways were significantly downregulated in the spleen, which may cause cell proliferation and migration disorders that are important for repairing of tissue damage. The damage to vascular endothelial cells can lead to hemorrhage. In comparison, attenuated QJ205 infection induced only slight downregulation of mRNA expression of related genes in the spleen. Our spleen results were similar to previous transcriptome studies on the spleen of grass carp infected with GCRV Ⅱ [41]. However, our data suggested that the focal adhesion and regulation of actin cytoskeleton pathways were activated in the liver after GCRV Ⅱ infection, which had not been reported. Such tissue-specific regulatory mode of these two pathways may be controlled by metabolite abundance, as activation of protein digestion and absorption and proteasome pathways was liver-specific function.4.5. Hemorrhage-Related PathwaysThe complement and coagulation cascade pathway was activated in the spleen and liver after the virulent DY197 infection. In comparison, attenuated QJ205 infection induced only slight changes in the expression of genes involved in this pathway. The complement and coagulation cascade system has been reported to play an important role in innate immunity [58,59]. Overactivity of the complement cascade, however, can lead to endothelial damage, platelet activation and aggregation, hemolysis, and thrombosis [60,61]. Thus, significant activation of the complement and coagulation cascade pathway may account for the hemorrhagic symptoms after virulent DY197 infection. Malaria is an infectious disease caused by plasmodium, which causes a decrease in hemoglobin in human blood [62]. In this study, the mRNA expression of hemoglobin subunit alpha (HBA), hemoglobin subunit beta (HBB), thrombospondin-1 (THBS1), platelet glycoprotein Ib beta chain (GP1BB) genes involved in malaria and platelet activation pathways were significantly downregulated in the spleen after the virulent DY197 infection. In comparison, the mRNA expression of these genes was relatively slightly downregulated in the liver after the virulent DY197 infection. Obviously, although GCRV Ⅱ infection in the liver activated the complement and coagulation cascade system, due to the activation of proteasome, focal adhesion, and regulation of actin cytoskeleton pathways, vascular endothelial cells in the liver may be actively repaired after damage, alleviating hemorrhage symptoms. 5. ConclusionsIn this study, a comparative transcriptomic analysis in the spleen and liver of rare minnow injected with virulent strain DY197 and attenuated strain QJ205 was conducted to investigate the possible involved molecular responses against the GCRV Ⅱ infection. The results showed the virulent DY197 strain induced more DEGs than the attenuated QJ205 strain, and tissue-specific responses were induced. In the spleen, virulent DY197 infection activated innate immunity and apoptosis-related pathways but suppressed adaptive immunity, cell migration and proliferation, and hemorrhage-related pathways. In the liver, except innate immunity and apoptosis-related pathways, virulent DY197 infection especially activated protein digestion and absorption-related pathways, both innate and adaptive immunity-related pathways, cell proliferation and migration-related pathways and caused slight suppression of hemorrhage-related pathways. These results would help us to better understand the interactions between the host and GCRV II.

animals : an open access journal from mdpi

10.3390/ani11051400

PMC8157221

Gas concentration is a relevant parameter for the estimation of emissions in dairy farms, but few studies have investigated the influence of cow behavior and barn management on gas concentrations in open buildings. In this study, concentrations of ammonia, methane, and carbon dioxide were investigated in an open dairy barn in a hot Mediterranean climate. Since hot climate conditions cause heat stress to the cows, gas concentrations were statistically analyzed to assess whether variation of environmental and animal-related parameters produced significant effects on the level of gas concentrations in the barn environment. In this study, it was statistically proved that daily gas concentrations were influenced by both the effect of micro-climate conditions, connected with the barn typology, and of barn management on the animals. Therefore, the mitigation strategies for the reduction of these gases could be pursued through the improvement of the barn management aimed at modifying cow behavior and through the control of climatic conditions in relation to the building features.

Measurement of gas concentrations constitutes basic knowledge for the computation of emissions from livestock buildings. Although it is well known that hot climate conditions increase gas emissions, in the literature the relation between gas concentrations from open barns and animal-related parameters has not been investigated yet. This study aimed at filling this gap by evaluating daily gas concentrations within an open-sided barn in hot Mediterranean climate. The influence of microclimatic parameters (MC) and cow behavior and barn management (CBBM) were evaluated for ammonia (NH3), methane (CH4), and carbon dioxide (CO2) concentrations. Results showed that both MC and CBBM affected concentrations of NH3 (p < 0.02), CH4 (p < 0.001), and CO2 (p < 0.001). Higher values of NH3 concentration were detected during the cleaning of the floor by a tractor with scraper, whereas the lowest NH3 concentrations were recorded during animal lying behavior. Measured values of CO2 and CH4 were highly correlated (C = 0.87–0.89) due to the same sources of production (i.e., digestion and respiration). The different management of the cooling systems during the two observation periods reduced significantly CH4 concentrations in the barn when the cooling system in the feeding area was switched off. Based on methodological choices due to the specific barn typology, parameters related to animals can provide information on the variation of gas concentrations in the barn environment in hot climate conditions.

1. IntroductionAgriculture and livestock farming are known to be activities with a great environmental impact. Among the main gases emitted from dairy farming, methane (CH4) and carbon dioxide (CO2), produced during enteric fermentation and manure management, have relevant impacts that contribute to global warming [1]. Another atmospheric pollutant, though it is not considered a greenhouse gas (GHG), is ammonia (NH3), which is emitted during manure management and produces environmental impact, such us eutrophication, soil acidification, and nutrient-N enrichment of ecosystems [2,3,4,5]. The evaluation of the application of mitigation strategies and technologies for emission reduction requires a reliable quantification of gas emissions. This quantification is based in turn on gas concentrations and the ventilation rate of livestock buildings. This latter parameter depends on the gas concentration difference between indoor and outdoor when applying the CO2 mass balance method [6] for estimating emissions from naturally ventilated (NV) dairy houses. Therefore, the knowledge of the variation of gas concentration in relation to the main parameters is of utmost importance. In this field of investigation, the barn structure, the housing system, the barn management, and the climatic conditions are the main influencing factors of emissions [3,4,7,8,9] and specific techniques or their combination can reduce emissions. However, in the literature there is a lack in the investigation on gas concentrations in open structures with partial or whole absence of perimeter walls. These structures are typical in a hot summer Mediterranean climate (Csa in Koppen classification) where the natural ventilation is generally integrated by a cooling system (e.g., fans and sprinklers) to reduce heat stress of the cows [10,11]. Consequently, the indoor microclimatic conditions are both influenced by the outdoor climatic conditions and the management of the barn (e.g., switching on/off of the cooling system, setpoints for climatic parameters, and number of cooling sessions). Microclimatic parameters (MC) of the barn represents one of the main factors that affect animal behavior, physiology, and productivity, as well as emissions of gaseous pollutants [12], especially during the warm seasons. Based on the use of the temperature humidity index (THI) to evaluate the risk of heat stress in cows, Hoffmann et al. [13] synthesized knowledge about activity and lying behavior as non-invasive animal-related parameters. In detail, they described recent outcomes in the literature on how heat stress affected the degree of physical activity. In another study, Porto et al. [10] studied the influence of the alternation of different cooling systems on lying, standing, and feeding behavior under heat stress conditions. They found that the management of the two cooling systems affected the analyzed behaviors. However, in the literature the influence of animal-related parameters (e.g., THI, cow activity/lying/feeding index) on gas concentrations has not been thoroughly investigated. Attention has been focused mainly on the relation between concentration and ventilation rate or air exchange rate [14,15,16,17], emissions and climatic variables [8,18,19,20,21,22,23], emissions and barn management or animal activity [23,24,25], and barn management and animal behavior [10,26,27,28]. For instance, Saha et al. [21] analyzed the influence of external wind speed and direction on sample point concentrations. They found that the inflow, i.e., speed and direction of the incoming wind, strongly affects the spatial distribution of NH3 and CH4 concentrations. The distribution of airflow was investigated by Fiedler et al. [14] within an NV dairy barn. A linear correlation (r = −0.7) showed lower CO2 concentrations for higher wind speeds during two weeks of measurements. On this basis, this study aimed at increasing knowledge on gas concentrations connections with environmental and animal-related parameters. The hypothesis to be proved was the dependence of gas concentration levels on MC and cow behavior and barn management (CBBM) in hot climate conditions. The main objectives of this study were to: (1) study gas concentration distribution in the open barn equipped with a cubicle loose housing system; (2) evaluate the effect of climatic parameters on gas concentrations; and (3) assess the relations between non-invasive animal-related parameters and gas concentrations.2. Materials and Methods2.1. Building and Site DescriptionMeasurements were carried out in a dairy barn equipped with a cubicle loose housing system, located in Pettineo/Pozzilli district (37°01′ N, 14°32′ E) in the province of Ragusa (Italy), at an altitude of 234 m a.s.l.The dairy house building, about 55.50 m long and 20.80 m wide, has three completely open sides, i.e., the SE, NE, and NW sides, without perimeter walls; the SW side is closed by a continuous wall with small openings; in the NE side there is a row of trees; and the roof is symmetric with a central ridge vent. The barn has solid floor and includes three pens for lactating cows, each composed of a resting area, a feeding area, and service alleys (Figure 1). In detail, the resting areas of the three pens are equipped with 64 head-to-head cubicles made of concrete kerbs filled with sand. The building is equipped with two cooling systems (Figure 2): a fogging system with fans located in the resting area; and a sprinkler system with fans located in the feeding alley.2.2. Data AcquisitionData analyses were carried out in 2016 during spring and summer in two observation periods. These latter were composed of the week from 15/06 to 21/06, named “week 1” (W1) hereafter, and the week from 01/07 to 07/07, named “week 2” (W2) hereafter. In these two weeks, the gas concentrations, climatic and MC, and CBBM were continuously monitored by specific devices and procedures described in the following subsections.2.2.1. Measurement of Gas ConcentrationsConcentrations of CO2, NH3, and CH4 were continuously measured by an INNOVA photo-acoustic analyzer composed of a Multigas Monitor mod 1412 i and a multipoint sampler 1409/12 (Lumasense Technology A/S, Ballerup, Denmark). The sampler system was made of AISI-316 stainless steel and PTFE (polytetrafluoroethylene) tubing to minimize adsorption of samples [29]. The system had 12 inlet channels. An air-filter was attached to the end of each sampling tube to keep the sampler free of particles. The detection limits, declared by the manufacturer, are the following: 0.2 ppm for NH3, 0.4 ppm for CH4, and 1.5 ppm for CO2.Continuous measurements were carried out at twelve sampling locations (SLs) with a sampling frequency of 15 min. SLs were located within the functional areas of the barn where cow urine and feces are released most, i.e., in the feeding area along the manger and in the service alley at the east side of the barn (Figure 1).These SLs were located at a height of 20 cm from the barn floor in the animal-occupied zone in order to analyze the gas concentrations where they are most significant for animal presence [30]. The outdoor SL was located at point 7 of Figure 1, at a height of about 3 m above the floor outside the barn and upwind at 2 m from the front of the barn, to acquire background concentrations. The INNOVA system was calibrated by the manufacturer two weeks before the experiment started, and it was operated to acquire data for the experiment.2.2.2. Climatic and Microclimatic Data MeasurementsMeasurements of climatic and microclimatic data were carried out by sensors installed inside and outside the barn. Air temperature and relative humidity sensors (Rotronic Italy s.r.l., Milano, Italy) were located in pen 1 and pen 2 at a height of about 2.0 m above the floor and outside the building at the ridge vent above the roof of the barn. The air temperature sensors were platinum thermo-resistances (Pt 100 ohm 0 °C) with a measurement range between −40 and +60 °C and a precision of ±0.2 °C (at 20 °C). The hygrometer was a transducer with a sensitivity of ±0.04%RH/°C and a precision of ±2% (at 20 °C). The position of these two combined sensors was inside a shelter in order to reduce possible inaccuracies due to direct radiation on the sensors. Sensors for the measurement of indoor airflow velocity and direction were located inside the building in pen 2 at a height of about 2.0 m above the floor, and wind speed and direction sensors were placed outside the building at the ridge vent above the roof of the barn. The anemometers were two-dimensional sonic sensors (WindSonic, Gill instruments Ltd., Lymington, UK) characterized by: a velocity measuring interval of 0 ÷ 60 m s−1, with a precision of ±2% (at 12 m s−1), a resolution of 0.01 m s−1, and a threshold of 0.01 m s−1; and a direction measuring interval of 0 ÷ 359°, with a precision of ±3% (at 12 m s−1), and a resolution of 1°.The measured values of wind and air temperature and relative humidity, airflow velocity and direction, wind speed and direction, were recorded at intervals of five seconds by a data-logger CR10X (Campbell, City, UK) that every five minutes computed the average values and stored them in memory locations. 2.2.3. Barn Management and Cow Daily Routine RecordingsSixty-four Friesian cows were housed in the barn, with primiparous cows mainly located in pen 2 (Figure 1). The daily routine of the cows showed different phases influenced by milking, feed delivery, cleaning, and operation of the cooling systems, which determined cows’ motor activity (e.g., feeding, standing, or walking) or else lying (e.g., resting, ruminating, or sleeping). The cleaning was done once a day at about 07:30 a.m. by a mechanical tractor with scraper. In the scraper, a hard rubber was applied to the blade to ensure a better cleaning effect. During the cleaning, the manure was moved to the manure storage area, south of the barn. The milking session was carried out twice a day at about 5:00 a.m. and 5:00 p.m. The feed was delivered every day after cleaning and it was moved into the manger before the first and the second milking sessions. Moreover, cows had ad libitum access to a mixed ratio that was not modified during the two weeks of observation.Both the cooling systems were manually switched off during the milking sessions and the cleaning of the feeding alley. Fans were automatically switched on when the air temperature exceeded 22 °C, whereas the sprinkler and fogging systems were operated when the air temperature was greater than 27 °C. The forced ventilation was automatically switched off during wetting to avoid the scattering of water. Different operating conditions of the cooling systems were established during data acquisition. Specifically, the sprinkler system in the feeding alley was switched off during W2 in pen 2 in a contextual experiment described by D’Emilio et al. [28].2.2.4. Behavioral Activity RecordingsBehavioral activity was monitored by a 24-h video-recording system [31], which was composed of ten cameras (Kon.Li.Cor, Perugia, Italy), located at a height of 4 m above the pen floor in the first and in the second pen (Figure 1).The analysis of cows’ behaviors on the recorded images was carried out by a skilled operator using the scan sampling method [32,33]. The visual assessment was based on the count of the number of cows in activity (e.g., feeding, standing, and walking) and in lying with a frequency of 15 min. These parameters were used in the computation of cow behavioral indices, i.e., cow lying index (CLI), cow standing index (CSI), and cow feeding index (CFI), according to Bava et al. [26]. 2.3. Data Analysis and Statistical ModellingData collected during the observation periods were organized in a dataset to carry out statistical analyses on gas concentrations values performed by using Microsoft® Excel and R free software environment.2.3.1. Gas Concentration DistributionVariability of gas concentrations at the different sample locations was studied for all the values recorded in the two weeks. Specifically, a one-way analysis of variance (ANOVA) was conducted to assess the gas distribution at different positions of the SLs. Gas concentrations at three groups of SLs were analyzed: central SLs (SL03-SL04-SL05-SL06); perimeter SLs (SL09-SL10-SL11-SL12); corner SLs (SL01-SL02-SL08). On this basis, the groups were separated by Tukey′s honestly significant difference at p < 0.05 (post hoc test).2.3.2. Influence of Micro-Climate Parameters on gas ConcentrationsThe influence of MC on gas concentrations was analyzed by identifying different ranges of similar climatic conditions. To this aim, wind, and airflow direction data were divided into eight different sectors of 45° (from 0–45° to 315–360°) as it was done in previous studies [14,17]. Figure 1 shows how each sector is related to the orientation of the building. The angles used in the representation gives the direction from which the wind is blowing (e.g., 180° indicates that wind is blowing from 180°). The study of the frequency of data values in those ranges was conducted to obtain the prevailing wind and airflow directions. The influence of airflow velocity on gas concentrations was assessed selecting gas concentrations at the prevalent indoor direction. Then, gas concentrations were divided into two groups of indoor airflow velocity (v ≤ 0.5 m/s; v > 0.5 m/s), based on the study carried out by Schrade et al. [19]. Then, for evaluating the equality of their mean values, the one-way ANOVA test was applied.In the post hoc analyses the mean values were separated by Tukey′s honestly significant difference at p < 0.05. 2.3.3. Influence of Animal-Related ParametersThe influence of animal-related parameters was assessed through two different statistical analyses, based on data grouped by THI and CBBM. In the first analyses, the effect of THI on gas concentration expressed the combined effects of temperature and relative humidity on animal stress under specific conditions [10]. The THI was computed by using the following relation [34], suggested by Bohmanova et al. [35] for hot climate conditions and approved by the Italian Ministry for Agricultural, Food and Forestry Policies [36]:THI = (1.8 × Tdb + 32) − (0.55 − 0.55 × RH/100) × (1.8 × Tdb − 26)(1) where Tdb is the dry bulb air temperature (°C) and RH is the air relative humidity (%).The categories of THI for heat stress in dairy cattle were assigned based on the study by Zimbelman and Collier [37] and Hempel et al. [38] and adapted from Armstrong [39] as follows: The first group was related to values of THI < 68 and corresponded to no stress conditions for cows; the second one was related to values 68 ≤ THI ≤ 72 and identified the stress threshold; the third one corresponded to the condition 72 < THI ≤ 78 and described a low risk of thermal stress for cows; and the fourth one was related to the interval 78 < THI < 84 and indicated that cows were in thermal stress. Conditions of emergency for cows (THI ≥ 84) were not recorded in this study. In the second statistical analyses, the CBBM conditions were considered as broad categories: They were subdivided into three groups, i.e., cow activity, cow lying, and barn cleaning, and were identified in the video recordings. The first group (cow activity) included those activities that facilitated the mixing between urea and feces: (1) When cows were moved in groups from the barn to the milking parlour (cow transfer for milking); (2) the permanence of the cows in the feeding alley when they ate (feeding); (3) when cows were in standing position or walked in the alleys (standing/walking). The second group (cow lying) included the period that cows spent in cubicles resting, rumination, or sleeping. The third group (barn cleaning) included the cleaning of the feeding and service alley. In detail, the mixing of urine and feces and their removal, produced by the tractor with a scraper, was considered different from the mixing performed by the cows.The influence of THI and CBBM on gas concentrations was assessed by using a one-way ANOVA with p-value (level of significance) lower than 0.05. If the test was significant (p < 0.05), the post hoc test applied was the Tukey test which identified differences between groups. The results related to the two weeks were compared to analyze whether statistical significances were recurrent in both periods. Further investigations were carried out to compute the correlation between (i) NH3 and CH4 and (ii) NH3 and CO2 and (iii) CO2 and CH4 following the Pearson correlation coefficient application [21,23]. 3. Results3.1. Gas Concentrations DistributionGas concentration profiles for CO2, NH3, and CH4 changed in time and with SLs during the day inside the barn and showed recurrent peaks during each day. Indoor gas concentrations showed a different pattern compared to the outdoor one. Figure 3 shows the variation of NH3 concentrations in all SLs during an average day, which was representative of the variation in the two weeks. Gas concentrations were higher in the central SLs than in the perimeter ones. There were two peaks during the day, whereas gas concentrations decreased in the central hours between 9 a.m. and 5 p.m. An example of gas distribution of NH3 is described in Figure 3. Gas distribution was uneven inside the barn where the concentration level decreased from the inside to the outside.A decrease in gas concentration data acquired at the centre of the barn was observed along the longitudinal axis of the building in the NW-SE direction of the airflow. Although symmetrically located in the corner of the barn, NH3 concentrations at SL1 were higher than those at SL8. The one-way ANOVA highlighted that the four groups of SLs (e.g., central SLs, perimeter SLs, corner SLs, outdoor SL) had a significant difference among them (p < 0.001). Mean values of gas concentrations, expressed in ppm, with the related standard deviation for each group statistically analyzed for both W1 and W2 are shown in Table 1. In both weeks, the central SLs (SL03 to SL06) detected the highest gas concentrations, and they were statistically different from gas concentrations in the other groups of SLs. Results showed a significant difference (p < 0.05) in gas concentrations between indoor SLs and the outdoor SL07. On average, it was found that outside concentrations of NH3 measured about 18.33% of the indoor concentrations in W1 and 17.21% in W2, i.e., mean indoor NH3 concentration was about 5.5 ÷ 6 times the outdoor one. The CO2 outdoor concentrations were on average about 82.65% of the indoor concentrations in W1 and 82.30% in W2, i.e., mean indoor CO2 concentration was about 1.20 times the outdoor one. Outdoor concentrations of CH4 were, on average, about 50.45% of indoor concentrations in W1 and 42.64% in W2, i.e., mean indoor CH4 concentration was about 2.00–2.40 times the outdoor one. 3.2. Effect of Climatic Parameters and Micro-Climate Conditions on Gas ConcentrationsStatistical measures of climatic parameters in W1 and W2, reported in Table 2, showed no significant differences (p < 0.001) for air temperature, relative humidity, and velocity between W1 and W2. Since the prevailing wind and airflow direction moved from NE to SW towards the manure storage area (>85%), W1 and W2 were considered replicates for MC conditions and, thus, the influence of airflow velocity on gas concentrations was assessed.The group of locations that exhibited the highest values of gas concentrations were selected to perform further analyses and specific datasets were created by filtering them in relation to different MC and CBBM variables. In detail, Table 3 showed the results of the analyses carried out on gas concentrations in relation to the selected indoor airflow velocity ranges in the two weeks. The ANOVA showed significant differences for CO2, NH3, and CH4 values of concentrations in air at changing of the airflow velocity. In particular, the results reported in Table 3 show that, when airflow velocity is lower than 0.5 m s−1, gas concentration mean values are statistically different from those when airflow velocity is higher than 0.5 m s−1. Therefore, gas concentrations are generally high when airflow velocity is low and vice versa. The results of this statistical analysis pointed out that increasing indoor airflow velocity at least above 0.5 m s−1 is effective in reducing gas concentration within the breeding environment. Based on these results, the daily trend of each gas (Figure 4) was analyzed for airflow velocities lower than 0.5 m s−1. NH3 showed two peaks in both weeks due to cleaning and cows’ feeding activity. The first peak occurred in the morning during cleaning between 7 a.m. and 8 a.m., whereas the second peak occurred at 8 p.m. after about one hour from the end of the milking session, when cows were in feeding. Concerning CO2 and CH4, the correlation coefficients (C) between these gases were equal to 0.87 in W1 and 0.89 in W2, whereas no correlations were found between NH3 and CH4 (C = 0.54 in W1 and C = 0.50 in W2) and NH3 and CO2 (C = 0.41 in W1 and C = 0.43 in W2). The results show that in both weeks the lowest gas concentration values of CO2 and CH4 were recorded at night when cows were mainly in lying (Figure 5b), whereas higher gas concentrations were found during the afternoon. Moreover, the daily trend of gas concentrations showed that there were two peaks in CH4 concentration at 5 p.m. and at 7 p.m. at low airflow velocities. As it was reported in Figure 4, the dataset selection (values related to v ≤ 0.5 m s−1) excluded the measurements during two intervals (9 a.m.–3 p.m. and 8 p.m.–10 p.m.) when cows were resting (Figure 5b). In these intervals, gas concentrations always decreased for all gases due to the effect of gas removal by the airflow. With the aim of reducing this effect on data, the groups of gas concentrations at low airflow velocities (v ≤ 0.5 m s−1) were analyzed in the following investigations in order to better identify the effect of THI and CBBM on gas concentrations.3.3. The Effect of THI on Gas ConcentrationsThe analysis of gas concentrations at different THI ranges showed that there was generally a significant relation between THI and gas concentrations in both weeks at an air speed lower than 0.5 m s−1. Table 4 reveals that CO2 and CH4 concentrations were significantly different at the different THI ranges (p < 0.001). In detail, the mean values of CO2 and CH4 were the highest when THI was higher than 78, whereas they were the lowest when THI was under 68. However, CO2 and CH4 gas concentrations at THI < 68 were not always different from those at 68 ≤ THI ≤ 72, whereas gas concentrations at THI lower than 72 where always different from those at THI higher than 72.The statistical analyses confirmed that NH3 was influenced by THI (p < 0.005) in both weeks. The results of the Tukey test post hoc test showed that NH3 concentrations when 72 < THI ≤ 78 were significantly different from those when THI ≤ 68. In detail, the highest values of NH3 concentrations occurred when 72 < THI ≤ 78. 3.4. Effect of CBBM on Gas ConcentrationsDuring W2, the deactivation of the sprinkler system in the feeding alley of pen 2 changed cow behavior in that area. Figure 5 shows that CFI in pen 2 was generally lower than in pen 1.The area under the CFI curve in pen 1 was bigger (A = 6.50 CFI day−1) than that under the CFI curve in pen 2 (A = 6.17 CFI day−1) with a reduction of the time spent at feeding for cows in pen 2. On the contrary, the area under the CLI curve in pen 2 is bigger (A = 11.61 CLI day−1) than that under the CLI curve in pen 1 (A = 11.13 CLI day−1), increasing time spent in lying. Moreover, a statistical reduction (p < 0.006) of CH4 concentration was observed from W1 (15 ppm) to W2 (12 ppm).The application of one-way ANOVA and post hoc test to the gas concentration data acquired during periods at low airflow velocities allowed to statistically prove the influence of CBBM, i.e., activity, lying, and cleaning, on the level of gas concentrations. As it was reported in Table 5, results showed a significant influence of CBBM on gas concentrations with p < 0.001 in both weeks. Specifically, gas concentrations of CO2 and CH4 during cow activity were always statistically different from those during cow lying, whereas NH3 concentrations during barn cleaning, cow lying, and cow activity were all significantly different. In detail, gas concentrations during cleaning were the highest and those during lying were the lowest.4. Discussion4.1. Gas Concentration DistributionGas distribution of NH3, CH4, and CO2 was uneven inside the barn where the concentration level decreased from inside to outside (Figure 3), similarly to what was observed by Wang et al. [5]. In open structures the effect of boundary conditions produced significantly differences between gas concentrations at central SLs and perimeter or corner SLs. This could be attributed to the absence of the perimeter walls. Moreover, the presence of the feeding alley in the central area of the barn increased the animal activity there. Consequently, the gas concentrations were higher in the central area. The decrease of gas concentrations along the longitudinal axis of the building was to be ascribed to the prevalent airflow direction in the NW-SE direction, composed by the air flux of the fans and the wind direction. In the housing system and the layout of the barn, functional areas influenced the gas concentration distribution. Consequently, multi-location measurements have a relevant role to understand and analyze the heterogeneous distribution of the gas, in agreement with results reported by other authors [17,40,41].4.2. Methodological ConsiderationsIn the barn under study, the environmental conditions related to the activation of axial fans and MC conditions significantly influenced gas concentrations due to the open building structure. In order to reduce the effect of gas removal by the airflow on the analyzed data, two main methodological considerations were put forward and the consequent choices were made in this study. The first regarded the selection of gas concentrations in the central SLs and the second involved the selection of gas concentrations at low airflow velocities (v ≤ 0.5 m s−1). The observed reduction of gas concentrations from central to perimeter SLs (Table 1) and when operating the cooling systems (Table 3) revealed that airflow velocity increased dilution and flushing. Moreover, the exchange of ventilation air in the building removed polluted air from the barn due to the influence of air movement as it was found by Angrecka and Herbut [9] in a NV dairy barn with open curtains during summer. In fact, the performance of ventilation systems was affected by the power and direction of wind in these open structures [42]. Therefore, these methodological choices allowed us to assess the influence of animal-related parameters on gas concentrations when the environmental parameters had the minimum effect.4.3. Effect of THI on Gas ConcentrationsThe highest values of CH4 and CO2 production occurred mainly during the day, when cows were subjected to thermal stress and increased their breathing activity, as it was described by Hoffmann et al. [13]. The statistical analyses (Table 4) delivered the similar results for CO2 and CH4, in the post hoc tests, due to the strong correlation between CO2 and CH4. These results are in line with other studies [20,24] and highly depend on the fact that CO2 and CH4 have the same source of production due to digestion and respiration [43]. Although Zimbelman and Collier [37] found a threshold of THI equal to 68 for cows’ heat stress, in this study the results showed that there was not a significant difference between gas concentrations at THI ≤ 68 and gas concentrations at 68 ≤ THI ≤ 72 for CO2 and CH4.The lowest values of gas concentration corresponded to conditions at THI < 68 recorded at night hours when cows are mainly at sleeping. Lower mean values of NH3 were found when cows were in thermal stress (78 < THI < 84) because this condition limited their activities as it is further explained in the following Section 4.4. Furthermore, the activation of the sprinkler system during the hottest hours of day (i.e., at high THI values) increased the water on the floor, thus diluting the urines in the puddles, and consequently reducing NH3 concentrations in air, similarly to what was described in other studies [4]. The highest values of NH3 concentrations occurred when 72 < THI ≤ 78 in the period of cow activity. The discussion of this point is reported in the following subsection.4.4. Effect of CBBM on Gas ConcentrationsDuring W2, cows in pen 2 reduced time spent at feeding to respond to heat stress. Although there is evidence that cows spend more time standing than lying under stress conditions to increase heat dissipation [13,27], in the barn under study, the activation of the fogging system in the resting area increased cow comfort during the hottest hours of the day and, thus, time spent lying increased [10,11].Moreover, the presence of sand in the cubicles increased the cooling effect of the fogging system located above the cubicles, as well as ensuring a fresh bed and safe hygienic-sanitary conditions [27,44].According to other authors [13,26,45], hot climate conditions cause a depressive effect on dry matter intake because the reduction of time spent at feeding reduces the dry matter intake. Moreover, Zetouni et al. [46] proved how there is a high correlation between CH4 production and dry matter intake; consequently, the observed decrease in CH4 production between W1 and W2 in this study could be ascribed to a reduction of dry matter intake due to hot climate conditions. The results of the statistical analyses reported in Table 5 highlighted that barn management strategies (e.g., the operation of cooling systems) are capable of producing an effect on gas concentration levels because cows respond to the management inputs with a different behavior (e.g., cows increased lying when the sprinkler system at the feeding alley was not operated, to maximize their welfare). In detail, the peaks in the daily trend of NH3 (Figure 4) are related to cleaning and cow activity. The first peak was the results of the cows’ activity (e.g., feeding and milking) and the consequent cleaning in the morning, whereas the second peak was due to the animal activity (e.g., feeding and milking) in the afternoon at 8 p.m. The cleaning interval had the highest concentration of NH3 (mean values are 10.70 ppm in W1 and 10.40 ppm in W2) due to both the mixing of urine and feces and the accumulation of NH3 during the day [7,30]. In fact, chemical processes for NH3 production are triggered by the effects of the mechanical cleaning by the tractor and the urine–feces mixing due to the activity of the cows besides other effects such as the phases of urine excretion. In the barn under study, during the cleaning of feeding and service alleys, cows were moved by the farmer away from the alleys where the tractor was going to operate. Animals were confined in the other part of the pen that was not yet under cleaning, where cows were in activity (e.g., standing, feeding, and walking) and enhanced the mixing of urine and feces on the floor. Therefore, the cleaning operation was the operation with the highest NH3 concentrations because it combined the effect of the tractor and the animal activity on the gas production. During the second peak of NH3, gas concentrations had high values after about one hour from the end of the milking session. This could be explained by the fact that after the milking sessions cows were in feeding. In fact, the farmer switched on the cooling system located in the feeding alley to encourage cows moving towards the feeding area. This action prevented cows from returning to lying immediately after milking; in this case, sand could enter inside the udder sphincter, causing poor hygienic conditions and increasing the risk of mastitis [47]. In the barn under study, after about 30 min from the end of each milking sessions, the sprinkler system in the feeding alley was switched off and the one in the resting area was turned on to make cows leave the feeding area and go to the stalls, in order to encourage cow lying. Concerning CO2 and CH4, findings seem to be in contrast with the main source of gas production, i.e., rumination during lying [48]; however, this is to be attributed to the influence of environmental conditions when cows are often in lying (Figure 5). In the conditions of the barn considered in the experiments, the management of the cooling system modified the trend of these gases by lowering it during the central hours of the day. The daily trend of gas concentrations at low airflow velocities reported in Figure 4 shows that there are two peaks in CH4 concentration at 5 p.m. and at 7 p.m. The first peak is seen as being determined by the switching off of the fans, at the beginning of the milking session, which reduced gas dilution. The second peak could be due to the enteric fermentation that occurs approximately 4 h after feeding [48]. Regarding CO2, another influencing factor was the passage of the tractor during the scraping of the floor or feed delivering, which contributed to the peaks of gas concentrations in Figure 4c. 5. ConclusionsThis study improved knowledge on the influence of environmental and animal-related parameters on gas concentrations in open barns with cubicle housing system. Based on the barn structure, the management of the barn and the response of animals produced effects on the variation of gas concentrations in the barn environment. Knowledge on how gas concentrations are affected by MC and CBBM can be applied to improve the application of mitigations strategies in the daily management of the barn. The control of specific operations (e.g., frequency of manure removal, number of milking, cooling sessions, time of application of urea inhibitors, mechanical efficiency of tractors) could provide precise information to reduce gas concentrations. The analyses performed in this study laid the ground to further study aimed at investigating how the variation of gas concentrations could affect the estimation of emissions in open structures. However, this latter point is very challenging for researchers due to the difficulties in the estimation of the ventilation rate by using internal tracer methods (i.e., CO2 balance, heat balance, and moisture balance). Further improvements should assess whether it is possible to extend current protocols for measuring gas emissions from NV dairy barn for open structures without the presence of perimeter walls.

animals : an open access journal from mdpi

10.3390/ani11061594

PMC8228038

Various methods such as in situ, gas production and enzymatic methods are exercised to estimate the in vivo fermentable organic matter (FOM). However, each of these methods has its limitations. The in vivo method with fistulated animals for FOM determination is expensive, laborious and negatively affects animal welfare. Similarly, the in situ method also requires rumen fluid and is costly. However, enzymatic methods eliminate the need for fistulated animals and are comparatively simple, cheaper, faster, have greater repeatability, and also ensure the standardization of the process. Additionally, in situ technique can be disregarded as a standard method to test the accuracy of other techniques in cases where in vivo testing is not feasible. Therefore, in the current study, we compared the in situ nylon bag technique with the in vitro neutral detergent cellulase method and chemical composition to estimate in vivo FOM of roughages.

In Vivo fermentable organic matter (FOM) reflects the energy production and the potential of rumen’s microbial protein synthesis. However, the in vivo method with fistulated animals for FOM measurement compromises animal welfare and is laborious as well as expensive. Although the alternative in situ nylon bag technique has been widely used, it is also costly and requires rumen liquor. Therefore, the present study was performed to compare the in situ nylon bag technique with the in vitro neutral detergent cellulase (NDC) method or chemical composition to estimate in vivo FOM of roughages. For this purpose, we selected 12 roughages, including six each from forages and crop residues. Our results have shown the strong correlation equations between FOMin situ and FOMNDC of forages (n = 6; R2 = 0.79), crop residues (n = 6; R2 = 0.80), and roughages (n = 12; R2 = 0.84), respectively. Moreover, there were also strong correlations between the chemical composition of roughages and FOMin situ (n = 12; R2 = 0.84–0.93) or FOMNDC (n = 12; R2 = 0.79–0.89). In conclusion, the in vitro NDC method and chemical composition were alternatives to in situ nylon bag technique for predicting in vivo FOM of roughages in the current experiment.

1. IntroductionIn Vivo fermentable organic matter (FOM) of roughages reflects the rumen’s energy production and the potential of microbial protein synthesis [1]. The determination of in vivo FOM of forages with fistulated animals is costly, time-consuming, and negatively affects animal welfare [2]. On the other hand, in the French small intestine digestible protein system, FOM is calculated from organic matter total tract digestibility (OMD) [3,4].Previous studies have shown that the methods of in situ nylon bag, gas production, pepsin–cellulase (PC), Tilley and Terry, and neutral detergent cellulase (NDC) are well correlated with in vivo OMD [5,6,7,8,9]. Although the in situ nylon bag technique has been widely used, this method requires fistulated animals and is also expensive [5]. Other existing in vitro methods, such as Tilley and Terry [5] and gas production [7], also need rumen liquor. Compared to rumen liquor-based methods, enzymatic methods eliminate the need for fistulated animals, are simpler, cheaper, faster, have greater repeatability, and ensure the standardization of the process [1,9]. Moreover, the PC method requires regular evaluation to ensure accurate results, while the NDC method has not been updated [6] and is faster than the pretreatment of samples with pepsin [10]. Therefore, it seems that NDC method may be an attractive alternative for predicting in vivo FOM. Moreover, it was shown that in vitro digestibility determined by rumen fluid or enzymes was superior to chemical characteristics [5,11]; however, the chemical composition method is considered the simpler, faster, and cheaper method [12]. Thus, the chemical composition method has also attempted to predict in vivo FOM in the current study. Kitessa et al. [3] reported that the in situ nylon bag technique could be disregarded as a standard method to test the accuracy of other techniques in cases where in vivo testing is not feasible. Additionally, the in situ nylon bag technique with the Bang-Bang (BB) apparatus was inexpensive, portable, and easy to operate to measure the rumen degradation characteristics of feedstuffs than traditional steel chain or flexible plastic tubes for binding the bags used in the in situ nylon bag technique [13]. Therefore, in the current study, we considered the in situ nylon bag technique with the BB apparatus as the reference method.To our knowledge, no study has performed an in vitro NDC method to predict in vivo FOM, and the correlation of chemical composition to predict in vivo FOM is also controversial and unclear. This study aimed to determine the possibility of predicting in vivo FOM of roughages using the in vitro NDC method or chemical composition. Thus, we hypothesized that the in vitro NDC method and chemical composition are suitable methods for an alternative in vivo FOM of roughages.2. Materials and Methods2.1. Sample CollectionTwelve roughage samples, including six each from forages and crop residues, were collected from five main beef cattle breeding areas in China. Specifically, the samples were langsdorff small reed (Hulunbuir City, Inner Mongolia), alfalfa, avena nuda straw, corn straw (Zuoyun County, Shanxi Province), mixed forage, oat straw, hulless barley straw, rapeseed straw (Hezuo City, Gansu Province), lolium perenne (Xiahe County, Gansu Province), poa annua (Nagqu Prefecture, Tibet Autonomous Region), alpine kobresia (Maizhokunggar County, Tibet Autonomous Region), and barley straw (Haiyan County, Qinghai Province). Mixed forage was collected from the natural meadow. Three samples were selected from each forage and crop residue for further analysis.2.2. Chemical AnalysisThe samples were oven-dried at 55 °C for 48 h and ground to a 2-mm mesh-screen size using a feed mill (DF-20, Wenling Linda Machinery Co. Ltd., Wenling, Zhejiang, China). Dry matter (DM; method 930.15) and ash (method 942.05) were analyzed by AOAC methods (2000) [14]. Nitrogen was determined using a protein analyzer (Rapid N III, Elementar Inc., Germany), and CP was calculated as the percentage of nitrogen × 6.25. Further, crude fiber (CF), neutral detergent fiber (NDF), and acid detergent fiber (ADF) were determined using a fiber analyzer (ANKOM A220, ANKOM Technology Corp., Macedon, NY, USA). Ether extract (EE) was extracted using an automatic extractor (ANKOM XT101, ANKOM Technology Corp., Macedon, NY, USA). Nitrogen-free extract (NFE) was calculated using the formula: w(NFE) = w(DM) − w(CP) – w(EE) – w(CF) – w(Ash). The chemical compositions and digestibility of each sample were calculated in triplicate.2.3. In Situ Nylon Bag TechniqueThree Angus steers fitted with a permanent rumen cannula (450 ± 15 kg) were utilized to determine the effective rumen degradability of the organic matter of roughages with the nylon bag technique within the BB apparatus [13]. Animals were fed a total mixed ration at 8:00 h and 16:00 h according to NRC 2000 to meet the ME requirement for 1.3 × maintenance [15], and ad libitum access to water and a mineral block was provided. This animal experiment was approved by the China Agricultural University Animal Care and Use Committee (AW28059102-1, Beijing, China). The sample was weighed (4.00 ± 0.01 g) in nylon bags (80 × 140 mm) with a pore size of 37 µm, and then suspended in the rumen before morning feeding. Bags were removed after 6, 24, 48, 72, 96, 120, and 144 h of incubation. After removal, the bags were immediately immersed in cold water to stop fermentation, washed six times (1 min/rinse) in a washing machine until the water was clean, then dried at 65 °C for 48 h. Rumen degradation kinetics was determined using the following equations. Rumen dynamic degradation rate: y = a + b (1 − e−ct)(1) Effective degradability: ED = a + (b × c) / (c + k) (2)Here, y is the rumen degradation rate at time t, a, b, c, t, and k stand for the rapidly degraded fraction (%), the slowly degraded fraction (%), the degradation rate constant at which b is degraded (%/h), incubation time, and rumen passage rate at 0.0253%/h, respectively. The effective degradability (ED) of organic matter was regarded as the rumen fermentable organic matter (FOMin situ) [16].2.4. In Vitro Neutral Detergent-Cellulase Plus Amylase MethodThe in vitro organic matter digestibility was determined by NDC plus amylase method using small nylon bags (37 µm, 25 × 60 mm). Samples (0.50 ± 0.001 g) were transferred to small nylon bags, sealed (FR-300B, Blueberry, Shanghai, China), boiled in neutral detergent solution with heat-stable α-amylase for 75 min (ANKOM A220, ANKOM Technology Corp., Macedon, NY, USA), then oven-dried at 65 °C for 48 h. For the cellulase buffer solution preparation, we incubated 20 g cellulase [17] in a 1 L acetic acid buffer (pH 4.8) for 1 h at 40 °C. Two small nylon bags were randomly placed in 100-mL culture tubes and pre-warmed overnight at 39 °C. The next morning, cellulase buffer solution (80 mL) was added into each preheated 100-mL culture tube using an automatic pump and then incubated for 24 h in a 40 ± 2 °C water at 40–60 rpm (DSHZ-300A, Jiangsu, China). Following incubation, the bags were immersed in cold water to stop fermentation, washed as in situ nylon bag technique, and oven-dried at 65 °C for 48 h.2.5. Statistical AnalysisWe analyzed the chemical composition and in situ organic matter degradation characteristics of samples using a two-tailed Student’s t-test [18]. Then we compared the FOMin situ and FOMNDC of samples using the general linear model (GLM) procedure of SAS version 9.0 (SAS Institute Inc., Cary, NC, USA): yij = μ + ai + eij, where yij is jth observation value at the ith level of factor a, μ is the population mean, ai is the treatment effect at level i of factor a, and eij is the individual random residual error. The data of the prediction equations were analyzed using the linear regression procedure (REG) of SAS version 9.0 (SAS Institute Inc., Cary, NC, USA): y = b0 + b1x1 + b2x2 + ⋯ + bmxm, where y is the dependent variable and x1, x2, x3, ⋯ xmrepresent m independent variables. Statistical significance was set at p < 0.05.3. Results3.1. Chemical Compositions of RoughagesThe data regarding chemical analysis for contents (%) of DM, OM, EE, CP, CF, NDF, ADF, Ash, and NFE are presented in Table 1. Compared with crop residues, EE, CP and NFE of forages were higher (p < 0.01), but CF, NDF, and ADF were lower (p < 0.01).3.2. In Situ Organic Matter DegradationIn Situ organic matter degradation characteristics of roughages are shown in Table 2. Compared with crop residues, a (p < 0.01), b (p < 0.01), c (p = 0.01), and FOMin situ (p < 0.01) were higher in the forages.3.3. In Situ Organic Matter Disappearance RateIn Situ organic matter disappearance rate (OMDin situ) of forages (a) and crop residues (b) are demonstrated in Figure 1. In brief, our data show that at 144 h, OMDin situ of forages from high to low were mixed forage (84.82), poa annua (82.92%), alpine kobresia (77.55%), lolium perenne (64.24%), alfalfa (57.21%), and langsdorff small reed (56.23%). Consequently, OMDin situ of crop residues were 71.27% in barley straw, 65.88% in corn straw, 63.77% in oat straw, 57.91% in hulless barley straw, 46.72% in avena nuda straw, and 42.08% in rapeseed straw.3.4. Prediction Equations of FOMin situ Based on Chemical CompositionThe regression equations between FOMin situ and chemical composition (EE, CP, CF, NDF, ADF, Ash, and NFE) of roughages (n = 12) are presented in Table 3. From equations 1 to 5 achieved higher reliability (p < 0.01), which contain CF (R2 = 0.84), ADF (R2 = 0.87), EE and CF (R2 = 0.92), EE, CP and ADF (R2 = 0.92), EE, CP, and CF (R2 = 0.93).3.5. Comparison of FOMNDC and FOMin situThe FOMNDC and FOMin situ were presented in Table 4. For forages and crop residues, we only obtained a difference in alfalfa (p = 0.02).3.6. Correlation Analysis between FOMNDC and FOMin situ of Forages, Crop Residues, and RoughagesThe correlation analysis between FOMNDC and FOMin situ of forages (a), crop residues (b), and roughages (c) is presented in Figure 2. Our results show the strong correlation equations between FOMin situ and FOMNDC of forages (n = 6; R2 = 0.79), crop residues (n = 6; R2 = 0.80), and roughages (n = 12; R2 = 0.84), respectively.3.7. Prediction Equations of FOMNDC Based on Chemical CompositionThe regression equations generated between FOMNDC and chemical composition (EE, CP, CF, NDF, ADF, Ash, and NFE) of roughages are presented in Table 5. Based on our findings, it was shown that the R2 increased in equations 1 through 8 (p < 0.01), which contains a chemical composition of CF (R2 = 0.79), EE and NDF (R2 = 0.83), ADF (R2 = 0.84), EE, CP and NDF (R2 = 0.84), EE and ADF (R2 = 0.84), EE, CP and ADF (R2 = 0.88), EE, CP, CF (R2 = 89), EE and CF (R2 = 0.89), respectively.4. DiscussionIn Vivo FOM depends on rumen dynamic processes, while in vivo OMD depends on the digestion of OM in the total digestive tract, and a reduction in the rumen can be compensated with the enhancement of the fermentation of the hindgut. Therefore, in vivo FOM’s determination is more difficult than that of in vivo OMD; the measurement of in vivo FOM is also less precise than in vivo OMD. Consistently, the differences between alternative methods will probably be more pronounced when correlated with in vivo FOM than with in vivo OMD [1].It was shown in a previous report that the in vitro method is the most inaccurate in predicting the digestibility of some low-quality feeds, such as hay and grain by-products [3]. The low-quality feed has higher contents of insoluble material, which up to some extent, determines the digestibility of a feed. When the roughage is treated with neutral detergent solution, the soluble carbohydrates, pectin, proteins, and other soluble components dissolve, leaving insoluble cell walls, which can be degraded by cellulase [10]. Therefore, in our study, we selected the NDC method. Consequently, to combine the characteristics of beef roughage, we used low-quality roughages as experimental material, which is in line with previous findings [19].Although studies have suggested that the feedstuff’s chemical composition cannot be used to predict OMD satisfactorily [3,20,21]. On the contrary, a good relationship between chemical composition and FOMin situ or FOMNDC of roughages was observed in the current study. These findings suggest that it is possible to predict in vivo FOM by the chemical compositions of a sample. Moreover, research has shown that chemical composition is related to DMI, DMI is negatively related to NDF, and NDF associated with gastrointestinal filling [22], and a strong relationship between NDF and ADF is well established, which can explain that equations based on ADF and NDF are accurate enough to predict FOMin situ or FMNDC of roughages. Additionally, the prediction of EE, CP, and CF in the same equation was more accurate than that of CF or ADF alone, suggesting that using chemical composition to predict in vivo FOM may be more correlated with fiber components. In the future, with increasing the number of samples, more accurate prediction equations are expected to be obtained.The findings of our study showed a strong correlation between FOMNDC and FOMin situ of roughages (R2 = 0.84). Consistently, Mary et al. [10] had documented the correlation between NDC and in vivo values and seemed to be consistent with those obtained by Tilley and Terry techniques. Moreover, that relationship was increased with the number of samples. Givens et al. [23] also reported a higher R2 value between in vivo OMD and in vitro OMD in the spring forage than in the autumn forage. Future samples collected only once to construct the equation will not be adequate; the collection of more samples at different times of the year is suggested.It may be noted that enzyme-based FOM measurements did not take into account possible interactions between microbial species present in the rumen. Although the nylon bag technique feed samples are digested in the actual rumen environment, many of the procedures involved in this method have not been standardized, and the range of variability within laboratories may be greater than all other in vitro methods [3]. Therefore, different methods have their advantages and disadvantages, but as long as the correlation between methods is strong, they might be replaced by each other.5. ConclusionsAltogether, it was concluded that there is a strong relationship between FOM values obtained with the in situ nylon bag technique and the in vitro NDC method, which FOMin situ = 0.77 FOMNDC + 9.90 (forages, R2 = 0.79), FOMin situ = 0.88 FOMNDC + 5.57 (crop residues, R2 = 0.80), FOMin situ = 0.81 FOMNDC + 7.92 (roughages, R2 = 0.84). Moreover, the FOM predicted by chemical composition correlates very well with the in situ nylon bag technique (R2 = 0.84–0.93) and in vitro NDC method (R2 = 0.79–0.89), respectively. Therefore, the NDC method and chemical composition seem adequate to develop equations to predict the in vivo FOM. However, more research is warranted to develop local equations considering specific pasture types, environmental conditions such as harvest season, and phenological stages, and to corroborate such predictions with in vivo data where conditions permit.

animals : an open access journal from mdpi

10.3390/ani12010019

PMC8749751

The study of age and growth patterns in skates and rays can be conducted by analyzing mineral deposition patterns inside the vertebrae as biological features may influence age estimation. For the giant electric ray (Narcine entemedor), age was estimated by analyzing the vertebrae and an annual deposition pattern was found. After considering additional biological features such as birth date and date of capture, a more precise description of growth pattern was made. We concluded that this species is a moderate body size elasmobranch with moderate longevity and fast growth. Our results provide useful information for the future management of this exploited species.

The age and growth rate of the giant electric ray, Narcine entemedor, was estimated using growth bands deposited in the vertebral centra of 245 specimens. Differences in size and age distribution were found between the sexes, a pattern that suggests the annual deposition of band pairs, possibly occurring in April. Multimodel inference and back-calculation were performed to three age data sets of females considering their reproductive cycle and time of capture, among which the von Bertalanffy growth function was found to be the most appropriate (L∞ = 81.87 cm TL, k = 0.17 year−1). Our research supports the idea that age can be determined via biological features such as birth date and growth band periodicity. We concluded that N. entemedor is of a moderate body size, moderate longevity and is a fast-growing elasmobranch species.

1. IntroductionTraditionally, it has been recognized that elasmobranchs, due to their biological characteristics (late maturation, low fecundity, slow growth), are especially vulnerable to overfishing [1,2,3,4]. Nevertheless, evidence has proven that life-history traits of this group may vary between K and r strategies, allowing some species to respond differently to fishing pressure [5,6,7]. In this sense, it is important to understand the life-history parameters of the species, particularly those that are related to the degree of vulnerability and risk to fishing pressure, providing basic information for demographic models [8,9,10]. Among the most important parameters for this purpose are the ones related to the age and growth of the species [11].The number of age and growth studies in elasmobranchs has increased significantly in recent years [12]; however, the techniques and structures used for these purposes have remained constant, based mainly on the identification and count of opaque and translucent banding patterns present in hard anatomical structures such as vertebral centra, denticles and dorsal spines [13,14].Many studies on the age and growth of elasmobranchs have been encouraged by the increasing exploitation of this group, which has been documented by numerous researchers, reporting the important effects of fishing mortality on batoid populations [15,16,17,18]. In Mexican elasmobranch fisheries, the main species of batoids captured are benthic, such as Pseudobatos productus, Zapteryx exasperate and Hypanus dipterurus [19,20,21,22], although some pelagic species such as Pteroplatytrygon violacea and Myliobatis californica have been found frequently in the catch [22,23]. In all these study cases, Narcine entemedor (Jordan and Starks, 1895) have been reported with low abundances; nevertheless, Villavicencio-Garayzar [24] and Márquez-Farías [25] reported that they are commonly captured in artisanal fisheries of Mexico, especially during spring and summer. In Bahía de La Paz, an artisanal fishery on batoids captures around 14 species, among which N. entemedor ranks third in captures [26].The giant electric ray N. entemedor is an endemic batoid of the eastern tropical Pacific Ocean distributed from southern Baja California and the Gulf of California to Peru. While the diet and feeding ecology [27] and reproductive biology [24,28] of the giant electric ray have been investigated, there is little information on the age and growth of this species. Moreover, information on the species is limited to the Pacific coast of Mexico, while life-history traits could vary in Central and South America. The objective of this study was to estimate age and growth parameters for N. entemedor in the southern Gulf of California using a multimodel inference approach, and to consider the influence of biological features in the estimates. We conclude that using data such as birth date may result in a more precise description of individual growth.2. Materials and Methods2.1. Collection of SamplesThe specimens were captured in collaboration with a fisherman who has a commercial fishing permit under Mexican fishing regulations and laws (CONAPESCA-103053993316-1); therefore, the data in this study are fishery-dependent. Monthly samplings were made from October 2013 through December 2015 in the south of Bahía de La Paz, located in the southern portion of the Gulf of California (24°25′ N, 110°18′ W). The organisms were captured using monofilament gill nets (200–300 m long, 1.5 m high, 20–25 cm stretch mesh) set in the afternoon at depths between 10 and 30 m over sandy bottoms and recovered the next morning. Individuals were measured for total length (TL in cm), and the sex was determined by the presence of copulatory organs in males (claspers; [28]). Vertebrae were collected from the abdominal region of each specimen and placed in a freezer.2.2. Vertebral PreparationVertebrae were thawed, excess tissue manually removed and individual centra were separated using a scalpel. Cleaned vertebrae centra were dried at room temperature. A qualitative analysis to test the effectiveness of diverse treatments to enhance the visibility of growth bands was evaluated. Three cutting thicknesses (0.3, 0.4 and 0.5 mm) and two dyes, Alizarin Red S (0.01 g/250 mL water) and Bismarck Brown Y (0.01 g/250 mL alcohol 95%), were tested to enhance the visibility of the centra growth bands. Staining was performed at different times of exposure, from 1 min to saturation. However, none of the dyes tested presented an improvement in the clarity of growth bands; thus, vertebrae were instead prepared as follows. Centra were fixed on wooden structures using a cyanoacrylate-based adhesive. Sagittal sections of 0.4 mm thick were made using double saws fitted with diamond-impregnated blades, ensuring that the focus of the centrum was included. Subsequently, sections were cleaned with scalpel and water and then dried at room temperature.2.3. Reading of Growth BandsThin sections were observed under a stereo microscope (Olympus SZX9) and digitized using a video camera (Sony CCD-IRIS-RGB). The sections were illuminated using reflected light on a dark background and submerged in a thin layer of water to improve the observation. The birth band was defined as the angle change on the centrum side [29]. Additionally, a centrum of a 12.4 cm TL near-term embryo [28] was polished and compared with a thin section of an adult. The size of the embryo’s centra and the birth band coincided; furthermore, no pre-birth bands were observed. The radius of each vertebra was measured on the corpus calcareum long a straight line through the focus of each vertebra with SigmaScan Pro 5.0.0 Software (Systat Software, Palo Alto, CA, US). The vertebral radius (VR) was plotted against TL and tested for a linear relationship to determine if these vertebrae provided a suitable structure for age determination and for back-calculated estimation of length at previous ages. The influence of sex in the VR-TL relationship was evaluated with a test of slopes and elevations [30].A training exercise counting the bands of a subsample (n = 50) was performed by two readers to refine the identification and growth band counts criteria. The following criteria were established: (1) identification of the presence of pairs of growth bands (one translucent and one opaque; [31]), (2) identification of a birth band through a change in the angle of the corpus calcareum in the place closest to the focus of the vertebra [29] and (3) counting of translucent bands (Figure 1). The two readers (reader 1 was most experienced) then did a simultaneous and independent band count without knowing the sex or size of the specimens. Readings of bands were performed on the corpus calcareum due to the poor visibility in the intermedialia zone. This procedure was repeated twice. Any vertebra yielding an age estimate that differed between counts was re-examined by both readers jointly; if no consensus was reached, the sample was discarded.2.4. Precision and AccuracyAccording to Campana [31], aging errors can be expressed as follows: (a) discrepancies in the reproducibility of repeated measurements on a given structure (precision), and (b) differences between the closeness of the age estimate to the true value (accuracy). Thus, count reproducibility, as indicated by reader variability, was determined by calculating the percent of agreement (PA) by ±1 mark [32], average percent error (APE; [33]) and coefficient of variation, which is an alternative precision analysis that uses the standard deviation rather than the absolute deviation (CV; [34]). Each method was applied to the total of the sample. Additionally, to assess whether there are systematic differences between the readings made by the readers, age-bias plots of band counts [35] and Bowker’s test of symmetry were performed [36].2.5. Periodicity of Band FormationThe periodicity of band pair formation was evaluated using two methods. The “centrum edge analysis” considers whether the last deposited band was translucent or opaque and relates it to the month of capture [37]. The “marginal increment analysis” was undertaken by measuring the distance from the last band to the edge of the centrum (marginal width) as a proportion of the distance between the last and the penultimate band pair (previous band pair width; [38]). (1)MIR=MWP=VR−RnRn−Rn−1 where MIR = the marginal increment ratio, MW = marginal width, P = previous band pair width, VR = the vertebral radius, Rn = the distance from the focus to the last complete growth mark band and Rn−1 = the distance from the centrum origin to the penultimate complete growth mark(Figure 1). Those distances were measured using SigmaScan Pro 5.0.0 Software (SPSS Inc., Chicago, IL, USA). Individuals that presented only one translucent band (referred to as the birth band) were not considered for MIR analysis. Mean MIRs were plotted against months to examine trends in band formation. A Kruskal–Wallis test was used to examine for differences among months, followed by a nonparametric multiple comparison test to find the months among these differences were presented [30]. In addition, a Kolmogorov–Smirnov distribution test was used to examine for differences in age structures between sexes [39].2.6. Age AdjustmentFor the individual age estimation, three data sets were analyzed, one using the growth band counts information (unadjusted) and two more performing an adjustment to age, considering reproductive cycle (Adjustment 1) and time of capture (Adjustment 2). For the unadjusted analysis, it was considered that the birth band formed shortly after parturition, irrespective of reproductive seasonality. Adjustment 1 was adjusted to the period between birth and first band formation. In the study area, N. entemedor has two birth peaks per year, a major one during August and a minor one during January [28]; the month of band formation is April (see Section 3). We considered the birth month to be August; thus, the time between birth and formation of the first band was assumed to be seven months (0.58 years). Adjustment 2, the second age adjustment integrated the capture date; therefore, age was adjusted with the time between the month of band formation and capture month.2.7. Back-CalculationDue to the small sample size of juvenile giant electric rays, back-calculated estimates of length at previous ages were calculated for each of the three data sets. Back-calculated lengths were calculated using the proportion-based back-calculation equation proposed by Francis [40], modified from Hile [41]. The equation used to back-calculate the lengths at presumed ages was:(2)Li=−(ab)+(Lc+ab)(VRiVRc) where Li is the TL at time i, a and b are parameters obtained from the linear relation between total length and vertebral radius, Lc is the TL at capture; VRc is the VR at capture and VRi is the VR at age i.2.8. Growth EstimationA multimodel inference approach was used to determine the most appropriate candidate growth model [42]. The candidate set of models consisted of the traditional 3-parameter von Bertalanffy growth model (VBG-3; [43]); a 2-parameter modified form of the VBG forced through the length-at-birth (L0) (VBG–2; [44]), where L0 was estimated using the largest near-term embryo (i.e., 14.5 cm TL) reported by Burgos-Vázquez et al. [28]; the 3-parameter Gompertz growth model (GG-3) and the logistic model with three parameters (LG-3; [45]).The four growth models were fitted to a combination of back-calculated lengths and sample data for the three data sets previously described (unadjusted and adjusted ages), and the resulting parameters were estimated and compared. The parameters in the candidate growth models were estimated when the negative log-likelihood was maximized with a nonlinear fit using the generalized reduced gradient method, assuming a multiplicative error in the residuals [46,47]. The objective function is expressed as follows:(3)−logL(θi|data)=∑n[−12ln(2π)]−[−12(σ2)−(lnTLO−lnTLE)22σ2] where n is number of data, i indicates the number of parameters for each candidate growth model selected (VBG-3, VBG-2, GG-3, and LG-3), TLO is the total length observed, and TLE is the total length estimated. For σ, the following analytical solution [48] is proposed:(4)σ=1n∑t=1n(lnTLO−lnTLE)22.9. Confidence IntervalsTo estimate the confidence intervals (CI) of the θi parameters in the candidate growth models, two approaches were used: (1) the likelihood profile method [48] for the parameter t0 because there is no correlation between parameters and (2) the likelihood contour method when there is a correlation between parameters [49,50,51], which is observed in the parameters L∞ and k. For the likelihood profile method, a chi-square distribution with 1 degree of freedom (df) was used, and therefore, values equal to or less than 3.84 were accepted within the CI. For the likelihood contour method, a chi-square distribution with 2 df was used, and values equal to or less than 5.99 were accepted within the CI [30]. The CIs were estimated based on Haddon [49].2.10. Model SelectionModel performance was evaluated using Akaike’s Information Criterion (AIC), where the best model was the one with the lowest AICc value. For model comparisons, the delta AIC (ΔAIC) and Akaike weights (wi) were calculated [42]. The ΔAIC is a measure of each model relative to the best model and is calculated as ΔAIC = AICi − minAIC, where AICi is the AIC value for model i and minAIC is the AIC value of the best model. Models with ΔAIC of 0–2 had substantial support, while models with ΔAIC of 4–7 had considerably less support, and models with ΔAIC > 10 had essentially no support. Akaike weights (wi) represent the probability of choosing the correct model from the set of R-candidate models and was calculated as:(5)wi=e−0.5ΔAIC∑i=1Re−0.5ΔAIC3. Results3.1. Collection of SamplesA total of 305 specimens (260 females and 45 males) were initially used for the aging study. Of the processed vertebrae, 245 (80%) were readable from 209 females ranging in size from 49 to 84 cm TL and 36 males ranging from 41.5 to 58.8 cm TL, with females being significantly larger than males (D = 0.84, p < 0.001) and having a well-represented sample for each month of the year (Table 1). Growth bands were poorly visible in the intermedialia zone (Figure 2); therefore, reading was performed on the corpus calcareum. Significant linear relationships between VR and TL (p = 0.001) were found for both sexes (females: TL = 0.08VR−0.65, r2 = 0.81; males: TL = 0.07VR−0.2, r2 = 0.67), verifying that these vertebrae were suitable structures for age determination. The mean radius of the observed birth band was 0.79 ± 0.08 mm (mean and S.E.). Similarly, the mean VR of the near-term embryo was 0.69 ± 0.02 mm TL, indicating that the birth band was identified correctly.3.2. Precision and AccuracyAge estimates agreed closely between readers. Age band counts resulted in an APE between readers of 3.3% and CV of 4.7%, with a PA of 69%, PA ± by one band of 95% and PA ± by two bands of 100%. Both age-bias plots (Figure 3) and Bowker’s test of symmetry (X2 = 14.5, df = 9, p = 0.89) indicated no systematic differences between readers. These precision and accuracy values indicate a high level of reproducibility.3.3. Periodicity of Band FormationThe categorization of growth bands at the edges of the vertebrae as opaque or translucent was possible. There were no differences in monthly marginal increments (Kruskal–Wallis: H11, 246 = 16.7, p = 0.11), although a pattern was observed in both marginal increment analysis and edge analysis (Figure 4). The mean MIR and the translucent edge percentage were highest during April and lowest during June, suggesting that a single band pair is formed annually on the vertebral centra of N. entemedor during April. Assuming the formation of a pair of bands each year, 14 age groups were identified for N. entemedor (Table 2 and Table 3), as well as the formation of a birth band right after birth. The fourth and fifth age groups were predominant, whereas the 14–15 year groups were poorly represented (Figure 5a,c,e). The one-year age group was represented only by males (n = 2), and in the 2-year age group, males were more frequent than females. For the 3–6 year age groups, females were increasingly more frequently than males, which were absent in the remaining age groups. The age structure was different between sexes (D = 0.66, p < 0.001).3.4. Growth Estimation and Model SelectionDue to a low sample size of males, growth models were only adjusted to the observed and back calculated age-length data of females. Growth models fitted to the data are shown in Table 4, with CIs for growth parameters L∞ and k for each one of the data sets analyzed. Their likelihood contours are shown in Figure 5b,d,f. Based on AIC values, the VBG-3 presented the best fit to the data to describe the growth of N. entemedor females for every data set analyzed (unadjusted and adjusted ages; Table 4 and Figure 5a,c,e). Furthermore, the VBG-3 was the only fitted model with empirical support (Δi = 0) for every data set analyzed, while the rest of the models had no support (Δi > 10). In each dataset, VBG-3 obtained the maximum −log-likelihood values. The VBG-3 unadjusted-age data set showed the maximum −log-likelihood value among the three, while the rest of the parameters varied slightly between data sets, L∞ ranged from 81.5 to 82.1 cm TL, and the estimates were close to the observed maximum length (TL = 84.0 cm); k remained constant at 0.17 cm year−1.4. DiscussionWe found sexual dimorphism by size between sexes, with females (TLmax = 84 cm) being larger than males (TLmax = 58 cm). Similar differences in sizes between sexes were described for N. entemedor by Villavicencio-Garayzar [24] off the west coast of Baja California Sur (Bahía Magdalena) and for other closely-related species, such as N. brasiliensis [52], Torpedo californica [53], T. marmorata [54] and T. nobiliana [55]. This suggests a selective advantage for larger-sized females; as has been mentioned, the larger size in female elasmobranchs facilitates the accommodation and nourishment of embryos [56]. Similarly, the differences in estimated maximum ages between sexes (6 years for males, 14 years for females) suggests both sexes are not equally long-lived. Furthermore, elasmobranch sexual segregation has been documented related to sex differences in body size, which possibly confers differences in attributes such as predation risk and nutritional requirements [57]. Therefore, the absence of males TL > 59 cm (and >6 years old) in our sample could be explained by differential mortality (or differential longevity) or by sexual segregation. In addition, gear selectivity can be ruled out as a factor for the observed differences because the same gear was used during the full study; thus, differences in size and age by sex seem to be characteristic of the species.In recent decades, the need to perform age validation has been stressed [31,58]. Villavicencio-Garayzar [24] verified the annual deposition through marginal increments for N. entemedor off the west coast of Baja California, concluding that annual band formation occurs during June. Moreover, annual deposition has been verified through marginal increment analysis for another Torpediniformes species, Torpedo marmorata [54], as well as for other batoid species such as Dipturus trachyderma [59], Bathyraja parmifera [60] and Urotrygon rogersi [7]. Accounting for this, annual deposition of a pair of bands was assumed in our study; however, we suggest being cautious since MIR did not show monthly significant differences. A pattern was observed both in MIR and edge analyses which suggests that annual band deposition concludes in April.Although the VBG-3 model in the unadjusted age data set showed the maximum −log-likelihood value among the three data sets, the length at birth described by this model, as estimated from the intersection of the growth curve with the length axis (L0 = 23.0 cm TL), was the farthest from the length of birth described for the species in the southern Gulf of California (14.5 cm LT; [28]). Considering that the rest of the parameters varied slightly between data sets, according to statistical results (–log-likelihood, AIC and Wi) and biological interpretation (L∞, k and L0 values), we considered the VBG-3 based on the adjusted age to date of capture (age Adjustment 2) to be the growth model that best describes individual growth of N. entemedor. The multimodel inference approach allows the analysis of an alternative growth hypothesis for N. entemedor and avoids the risk of using an inappropriate model a priori [61].The estimated ages for females in our study, 2–14 years, were similar to ages, 1–15 years, estimated for females by Villavicencio-Garayzar [24] off the west coast of Peninsula Baja California, Mexico. Nonetheless, the ages found for males in our study were 1–6 years (LT = 41–59 cm), which differed from males aged 1–11 years (LT = 24–67 cm) obtained by Villavicencio-Garayzar [24]. This could be due to differences in longevity between study areas related to differences in environmental conditions (e.g., temperature; [62]) or differences in the spatial distribution of older males since sexual segregation among elasmobranchs have been documented related to sex differences in body size [57].The L∞ estimated for females in our study (TL = 81.87 cm) was similar to L∞ = 82.6 cm TL estimated for females by Villavicencio-Garayzar [24] and close to the maximum observed length (TL = 84 cm). On the other hand, the growth coefficient estimated for females in our study, k = 0.17 year−1, was lower than k = 0.30 year−1 obtained for females by Villavicencio-Garayzar [24]. In this regard, it has been documented that a limited representation of the sizes in the sample, particularly of small and/or large individuals, can bias parameter estimates using the VBG [63]. However, in our case, the back-calculated lengths at previous ages provided a good representation of younger organisms, and this was observed in the closeness of our L0 estimation to the observed birth length for N. entemedor. Compared with closely-related ray species, our estimation was similar to k = 0.18 year−1 found for combined sexes of T. marmorata [54] but proved higher than k = 0.07 year−1 obtained for T. californica females [53].5. ConclusionsAcknowledging the difficulty of sampling young individuals, we suggest the use of back-calculation estimations where information associated with early stages is limited. Furthermore, we conclude that age adjustment is a useful practice. Although age has traditionally been analyzed on a yearly basis (as a discrete variable), our study indicated that adjusting age to biological features, such as birth date, catch date and the periodicity of growth band deposition, may result in a more precise description of individual growth. Finally, we conclude that N. entemedor is a moderate body size elasmobranch species with moderate longevity and fast growth, which is a life history pattern typical of species that grow quickly to overcome mortality in the early life stages.

animals : an open access journal from mdpi

10.3390/ani12050537

PMC8908859

Cutaneous wound healing is a complex and tightly regulated biological process to restore physiological and anatomic function. Current knowledge of cutaneous wound healing is mostly based on studies in laboratory animals and humans. The histological and immunological features of skin, for example, cutaneous thickness, cellular components, and immune response, are not identical among animal species, and these differences may lead to substantial effects in cutaneous wound healing. In field observation, large cutaneous wounds in cetaceans could heal without medical treatments. However, little is known about the underlying mechanisms, and there is no histological study on full-thickness wound healing in cetaceans. The current study characterizes the macroscopic and histological features of large full-thickness wound healing in Fraser’s dolphins (Lagenodelphis hosei). The differences of wound healing between cetaceans and terrestrial mammals were shown from the histological aspect, including rete and dermal ridge appearance, repigmentation, and adipose tissue regeneration. Better understanding of the mechanism of full-thickness wound healing in cetaceans will shed light on veterinary and human regenerative medicine, leading to novel therapies.

Cetaceans are tight-skinned mammals that exhibit an extraordinary capacity to heal deep soft tissue injuries. However, essential information of large full-thickness wound healing in cetaceans is still lacking. Here, the stages of full-thickness wound healing were characterized in Fraser’s dolphins (Lagenodelphis hosei). The skin samples were collected from normal skin and full-thickness cookiecutter shark (Isistius brasiliensis)-bite wounds of stranded carcasses. We defined five stages of wound healing according to macroscopic and histopathological examinations. Wounds in Stage 1 and 2 were characterized by intercellular and intracellular edema in the epidermal cells near the wound edge, mixed inflammatory cell infiltration, and degradation of collagen fibers. In Stage 3 wounds, melanocytes, melanin granules, rete and dermal ridges were noticed in the neo-epidermis, and the adipose tissue in adjacent blubber was replaced by cells and fibers. Wounds in Stage 4 and 5 were characterized by gradual restoration of the normal skin architecture including rete and dermal ridges, collagen bundles, and adipose tissue. These phenomena were quite different from previous studies in terrestrial tight-skinned mammals, and therefore, further in-depth research into the mechanisms of dolphin wound healing would be needed to gain new insights into veterinary and human regenerative medicine.

1. IntroductionIn humans and most mammals, cutaneous wound healing is a complex biological process which has been divided into three continuous and overlapping phases: inflammation, proliferation, and remodeling [1]. The inflammation phase occurs first and directly after skin injury. A fibrin clot forms immediately to re-establish hemostasis and prevent invasion of microorganisms as well as providing a scaffold for inflammatory and other cells to crawl into the wounded area [2]. Inflammatory cells, such as neutrophils and macrophages, are recruited to the wounded area for clearing invading bacteria and cellular debris. As the clearing process approaches an end, a change of local microenvironmental signals promotes macrophage phenotype switching from a pro-inflammatory phenotype to an anti-inflammatory phenotype, contributing to the progression of wound healing [3,4]. The second phase of wound healing is the proliferation phase, which includes angiogenesis, granulation tissue formation, and re-epithelialization. Granulation tissue is composed of primarily new blood vessels, immune cells, fibroblasts, and an abundance of extracellular matrix (ECM) produced by the fibroblasts [5]. Re-epithelialization, which involves the proliferation, differentiation, and migration of keratinocytes, is an essential process for wound closure [6]. Furthermore, wound contraction promoted by myofibroblasts aids wound closure [7]. In the last phase of wound healing, most of the cells in the granulation tissue undergo apoptosis or emigrate from the wounded area, leaving a scar tissue containing few cells with an excess of abnormal ECM [2]. ECM composition changes during wound healing processes are under the regulation of cytokines, growth factors, and matrix metalloproteinase (MMPs). These are secreted by fibroblasts, macrophages, and endothelial cells [8]. ECM remodeling enhances the strength of repaired tissue; however, the repaired tissue does not return to a completely normal status in humans and most tight-skinned mammals [9].Although the basic mechanisms of wound healing are preserved in most mammals, heterogeneity in wound healing has been found in different species, individuals, and anatomical sites [10,11,12,13]. For example, wound closure in tight-skinned species, such as humans and pigs, is primarily by re-epithelialization, whereas wound closure in loose-skinned animals, such as rats, mice, and rabbits, is primarily caused by contraction of the panniculus carnosus muscle (reviewed in [12]). Another example is the rate of granulation tissue formation and wound contraction in cats being rather slow compared to dogs, even though both dogs and cats are closely related in the order Carnivora. This difference could be related to variability in cutaneous blood supply during wound healing [10]. Heterogeneity in wound healing is not a phenomenon restricted to between species but also exists within the same species. Horses and ponies, both belonging to Equus ferus caballus, show considerable differences in second-intention wound healing [13]. The authors noted that the inflammatory response in ponies was strong but short, leading to a better quality second-intention healing, while horses had a weak and chronic inflammatory response, resulting in the production of poorly vascularized exuberant granulation tissue. Previous studies showed that the formation of hypertrophic scars after wounding can be observed in red Duroc pigs and Mexican hairless dogs but not in other breeds of pigs and dogs [14,15,16]. Genetic variations leading to heterogeneity in wound healing also occur in humans. The prevalence of keloid scarring in certain ethnic populations, such as African American, Asian, Mediterranean, and Hispanic, is higher than in Caucasian populations [11,17]. Although the heterogeneity of wound healing in several terrestrial mammals has been studied, little is known about the timing, sequence, and mechanism of wound healing in mammals that live in a significantly different environment: the marine habitat. Cetaceans, which inhabit water for the entirety of their lives, have been reported to possess great healing capacity [18,19,20]. Cetaceans possess a unique cutaneous anatomy, which is supposed to be an adaptation to the aquatic environment [21]. Cetacean skin is characterized by an exceptionally smooth, rubbery texture without hairs or other skin appendages (excepting newborn odontocetes that possess hairs on the rostrum and mysticetes that have vibrissae on the rostrum and mandible) [22,23]. The epidermis consists of a stratum externum, a stratum spinosum, and a stratum basale; the stratum granulosum and stratum lucidum are absent [22]. Melanin granules are distributed in all epidermal layers, including the stratum externum [24]. Most of the cells in the stratum externum retain flattened nuclei, which is referred to as parakeratosis [22]. A thick epidermis with elongated rete ridges interdigitating with dermal ridges (dermal papillae) is one of the extraordinary characteristics of cetacean skin. The serrated interface between epidermis and dermis increases the ratio of germinative to superficial cells, consequently contributing to a greater proliferative capacity in cetacean skin [25]. Moreover, the well-developed rete ridges could function to enhance skin adherence against hydrodynamic friction, which prevents the epidermis being torn off from the dermis during high-speed swimming [26]. Blood vessels and nerve fibers are present in the dermis and dermal ridges, providing nutrition and sensory perception [27]. Beneath the dermis is blubber, a thick layer of specialized subcutaneous adipose tissue found only in marine mammals [28]. Blubber contains numerous adipocytes, intermingled with blood vessels, nerves, collagen fibers, and elastin fibers [27,29]. The literature shows that blubber plays an important role in thermoregulation, metabolic energy storage, buoyancy, body streamlining, and locomotion [30].Cetaceans commonly sustain traumatic injuries such as abrasions, conspecific biting or shark biting, anthropogenic trauma caused by fishing entanglement or propeller strike in free-ranging individuals, and thermal burns in stranded cetaceans [18,31,32,33,34,35]. Interestingly, observations report deep cutaneous wounds in wild cetaceans can completely heal without medical treatment [20]. Some antimicrobial compounds, for example organohalogens and isovaleric acid, were found in the blubber layer [36,37] and could provide a natural protection against infection contributing to wound healing [20]. Furthermore, the regenerative ability in cetacean skin is associated with the adipose-derived stem cells existing in the blubber layer [38]. To the best of our knowledge, there are only two studies describing the histological features of cutaneous wound healing in cetaceans [39,40] that focused on superficial wound healing in bottlenose dolphins and beluga whales, respectively. The studies showed that both species shared a similar sequence of cellular and vascular change during the healing process with humans and laboratory animals. The only remarkable difference between cetaceans and terrestrial mammals was no solid fibrin clot or scab formation during wound healing in cetaceans. Instead, a layer of degenerative cells mixed with vesicles covered the wound surface in cetaceans. The authors suggested that the formation of this degenerative layer might relate to the change of cellular osmolarity in cells when exposed to seawater. This could provide a similar function to a solid fibrin clot or scab in terrestrial mammals to be a protective barrier between the external environment and the underlying tissue.Although the aforementioned studies on superficial wound healing in captive dolphins and whales described the sequence and the timing of wound healing, the essential information of large full-thickness wound healing in cetaceans is still lacking. The aim of the current study was to describe the macroscopic and histological features of full-thickness cutaneous wound healing in Fraser’s dolphins (Lagenodelphis hosei). It would not be surprising if there are significant differences in large full-thickness wound healing between cetaceans and other mammals. Understanding the differences and underlying mechanisms may contribute to the development of novel therapies to treat severe trauma patients (both non-human and human) and make progress in the field of skin wound healing research.2. Materials and Methods2.1. Sample CollectionThe skin samples used in the current study were collected from four dead stranded Fraser’s dolphins. The samples included normal dolphin skin and full-thickness wounds caused by cookiecutter shark (Isistius brasiliensis) bites. These shark bite wounds were approximately 5–8 cm in diameter and 2–3 cm in depth, and mainly present on the dorsolateral and ventral parts of the body. Wounds in different healing states, including recently created, healing, and healed, were collected for subsequent analysis. Samples in poor condition (e.g., sloughing epidermis) were ruled out from the current study. Body condition and carcass condition were assessed according to previous studies [41,42]. Freshly dead animals were classified into carcass Code 2; animals presenting moderate decomposition were classified into carcass Code 3. The sample condition and the details of each animal are listed in Table 1.2.2. Tissue Preparation and Histochemical StainingThe skin samples taken from the stranded dolphins were fixed in 10% neutral buffered formalin for 3 days and then embedded in paraffin. For hematoxylin and eosin (H&E) staining and Fontana-Masson staining, the tissues were cut into 4 μm sections; for Masson’s trichrome staining, the tissues were cut into 5 μm sections. Slides were deparaffinized in xylene and rehydrated in graded ethanol, and then subjected to H&E staining, Fontana-Masson staining, and Masson’s trichrome staining according to accepted protocol. Images were recorded with a Whited WM100 microscopy (Whited, Taipei, Taiwan).3. Results3.1. Normal SkinThe epidermis consisted of a stratum externum, stratum spinosum, and stratum basale. No hair follicles or other skin appendages were observed. The outermost layer of the epidermis was composed of flattened keratinocytes with pyknotic nuclei, defined as parakeratosis. Some of the spinous cells showed clear nuclear haloes. Melanin granules appeared in all layers of the epidermis. Prominent epidermal rete ridges extended downward into the dermis to construct an interdigitated interface between epidermis and dermis (Figure 1). The dermis consisted of a complex network of collagen fibers. Blood vessels and nerve fibers were clearly observed in the reticular dermis. The blubber layer was rich in adipocytes, intermingled with collagen fibers, elastin fibers, nerve fibers, and blood vessels. Collagen fibers presented in a basket-weave orientation. A higher quantity of blood vessels was observed in the deep layer of blubber than in the superficial layer.3.2. WoundsFive stages were classified in the wound healing progress in Fraser’s dolphins (Table 2 and Figure 2): Stage 1, new wound; Stage 2, initially healing wound without granulation tissue; Stage 3, healing wound with granulation; Stage 4, healed wound with cellular and vascular blubber; Stage 5, healed wound without cellular and vascular blubber.3.2.1. Recently Created or Immature Wound; Stage 1Grossly, the wounds showed sharp edges with little or no bleeding. Underlying tissues were exposed to the environment with no necrotic tissue covering. Histologically, intercellular and intracellular edema were noted in the stratum spinosum near the edge of the wound (Figure 3). In the edematous region, the epidermis was pale and swollen. Some keratinocytes presented with karyolytic, pyknotic, or karyorrhectic nuclei while some presented without nuclei (Figure 3B). There was no obvious change in the dermis and blubber. Only a few collagen fibers at the wound edge were necrotic. This stage of cetacean wound healing was classified as Stage 1.3.2.2. Initiation of Wound Healing in Wound; Stage 2The macroscopic appearance of the wounds was characterized by a raised and puckered epidermal edge with a layer of yellow-white substance covering the surface of the wounds. Histologically, the epidermal cells near the wound edge were edematous and the region of cellular edema extended to the adjacent area, approximately 2.4–7.9 mm from the wound edge (Figure 4B). Exfoliation of degenerating epidermal cells was noted. The yellow-white substance covering on the wound surface consisted of necrotic collagen and necrotic adipose tissue, approximately 0.3–4.2 mm in width (Figure 4C). Next to the necrotic tissue was an inflammatory cell infiltration zone, approximately 0.7–6.3 mm in width (Figure 4D). The predominant inflammatory cells were granulocytes. Adjacent to the inflammatory cell infiltration zone, hyperemia and hemorrhage were noted in the blubber, predominantly in the middle and lowest layers of blubber, extending up approximately 10 mm on either side of the wound edge (Figure 4E). Degradation of collagen fibers in the dermis and blubber close to the wound edge was noted (Figure 5A,B). Adipose tissue in the blubber was replaced by a loose fibrin network composed of fibrin, erythrocytes, and inflammatory cells (Figure 5C,D). This stage of wound healing was classified as Stage 2.3.2.3. Mature Open Healing Wound; Stage 3Under gross view, neo-epidermis showed in the wound margin. In the center of wound, the void was filled with a reddish tissue. Histologically, rete and dermal ridges were noticed in the neo-epidermis (Figure 6B). Melanocytes and melanin granules were also observed in the neo-epidermis (Figure 6C). Part of the thick collagen fibers broke into pieces and part of the adipose tissue was replaced by numerous cells and thin fibers (Figure 6D). In the wound center, reddish granulation tissue consisted of cells, extracellular matrix, and numerous microscopic blood vessels (Figure 6E). Trichrome staining showed that thin collagen fibers and blood vessels appeared in the papillary dermis that connected to the neo-epidermis (Figure 6F,G). Keratinocytes in the neo-epidermis and uninjured adjacent skin were enlarged and edematous, approximately extending 7.8 mm away from the wound edge. In the uninjured area adjacent to the wound, the degenerating epidermal cells had exfoliated, resulting in the loss of the stratum externum and part of the stratum spinosum. This stage of wound healing was classified as Stage 3.3.2.4. Immature Healed Wound; Stage 4Grossly, the surface of wound was fully epithelialized. Histologically, no cellular edema was observed in the epidermis. Melanin granules appeared in all the layers of neo-epidermis. Rete and dermal ridges were present in the neo-epidermis. According to the histological features, such as collagen thickness, cell density, and blood vessel density, this category of healed wounds was subdivided into two stages. The dermis and blubber layer were filled with numerous thin collagen fibers which oriented parallel to the skin surface. Numerous blood vessels, fibrocytes, fibroblasts, and other cells existed among the collagen fibers (Figure 7B). Thick collagen fibers were present in the lower part of the blubber (Figure 7C). Scattered rounded spaces were noticed beside blood vessels. Nerve fibers were rarely observed. A wound with the features mentioned above was classified as Stage 4.3.2.5. Mature Healed Wound; Stage 5In macroscopic view, the mature healed wound was closed, showed little to no contraction lines, and was evenly pigmented similar to the surrounding unwounded skin. In the more “mature” wounds, which were classified as Stage 5, the dermis and blubber layers were less cellular and vascular (Figure 8). The presence of rete and dermal ridges was observed (Figure 8B). It was clear that adipocytes appeared in the perivascular region (Figure 8C), but the amount of adipose tissue was varied among different samples. Most of the wounds in this stage comprised thin collagen fibers, while a few contained thick collagen fibers similar to the collagen bundles in the normal skin. Few nerve fibers could be observed in the dermis. The architecture of Stage 5 healed wounds was significantly similar to unwounded skin.4. DiscussionIn the current study, the process of full-thickness wound healing in Fraser’s dolphins was characterized. The normal skin structure of Fraser’s dolphins is quite similar to previously described cetacean species [22,27]. The skin characteristics of these cetacean species share several similarities, for example, parakeratosis in the outermost layer of the epidermis, absence of hair follicles and other skin appendages, relatively simplified cellular strata in the epidermis, prominent epidermal rete and dermal ridges, and the distribution of blood vessels in the dermis and blubber. Compared to terrestrial tight-skinned mammals, humans and pigs, the thickness of normal cetacean epidermis is much greater. Increased thickness of the epidermis happens in humans, but only during abnormal conditions, for example, injury or autoimmune diseases like psoriasis [43]. A thickened epidermis in cetaceans could provide better protection against environmental insults and maintain homeostasis in the aquatic environment and is considered one adaptation to the aquatic life [21,25,44]. The current study showed the presence of rete and dermal ridges after full-thickness wounding indicating the importance of rete ridges in dolphin skin. These structures do not appear after full-thickness wound healing in humans and pigs, and only limited rete and dermal ridge regeneration was observed in partial-thickness wounds in pigs [14]. The interdigitated rete and dermal ridges create an epidermis firmly connected to the dermis, preventing detachment from the underlying tissue during high-speed swimming [26]. It was surprising to observe rete and dermal ridges in the neo-epidermis in Stage 3 wounds in the current study, which might provide better adhesion during the wound closure process. Moreover, the convoluted basement membrane is accompanied by a higher ratio of basal cells to superficial cells compared to terrestrial mammals, consequently increasing the proliferative capacity and potentially contributing to wound closure during skin wound healing [39,45].The epidermis layer in Stage 1 and Stage 2 was edematous without a solid fibrin clot or scab, similar to reports for superficial wounds in bottlenose dolphins and beluga whales [39,40]. A previous field study showed that the surface of shark-inflicted wounds was covered with blubber coming from the wound adjacent area within the first day after injury [20]. However, our findings were not consistent with this. The macroscopic appearance of the wounds in Fraser’s dolphins was a layer of yellow-white substance covering the surface of the wounds in Stage 2, and this substance was necrotic collagen and adipose tissue in the microscopic observation. A layer of necrotic tissue might serve as a mechanical barrier to maintain homeostasis and protect underlying tissues from further damage and enable wound healing. Of note, regions of adipose tissue in the blubber were replaced by a loose fibrin network that could provide a scaffold for the migration of fibroblasts, inflammatory cells, and other cell types to the wounded area [9]. Recent studies have shown that mouse adipocytes can undergo cellular reprogramming by dedifferentiation into preadipocytes and mesenchymal stem cells, or by transdifferentiation into myofibroblasts, indicating that adipocytes have a high cellular plasticity (reviewed in [46]). The disappearance of adipocytes during wound healing in cetaceans might result from apoptosis, or dedifferentiation or transdifferentiation into other cells. Cetaceans possess a thick layer of subcutaneous adipose tissue. This raises the question of if the adipose tissue around the full-thickness wound in cetaceans supplies essential fibroblasts and endothelial cells utilized for the formation of granulation tissue during wound healing. Granulation tissue consists of primarily new blood vessels, immune cells, fibroblasts, and an abundance of extracellular matrix (ECM) [5]. In full-thickness wounds, the void of the wound needs to be filled with granulation tissue before re-epithelialization [7]. A full-thickness cookiecutter shark wound in a human required a significantly long time to heal, even though a skin graft had been applied [47]. In contrast, a similar wound on a stranded cetacean was filled with granulation tissue within days, followed by epithelial migration, and completely closed in the next two months (H.-V.W., personal communication). Previous studies showed the origin of fibroblasts forming the granulation tissue in full-thickness wounds could arise from multiple populations of cells, for example, perivascular sheaths and pericytes (shown in rabbits), hematopoietic cells with mesenchymal characteristics (fibrocytes) (shown in mice), and stromal elements in the adipose layer (shown in rabbits and pigs) (reviewed in [7]). If adipose tissue in cetacean skin is an essential element to supply fibroblasts and endothelial cells for the formation of granulation tissue, adipose tissue regeneration would be necessary in order to prepare required materials for future wounds. Further studies are required to understand the mechanism of the rapid formation of granulation tissue in cetacean skin wounds.Interestingly, small, rounded spaces with obvious membrane structures were noticed next to blood vessels in Stage 4 and Stage 5. We hypothesized that these small, rounded spaces could be neo-adipocytes and that this is the location of adipocyte regeneration after full-thickness wounding in cetaceans. Adipocyte regeneration has been studied in laboratory animals but is yet to be fully elucidated. In mouse studies, it was reported that adipogenic progenitors reside in the perivascular niche of adipose tissue, suggesting adipose progenitors might come from endothelial cells or pericytes (reviewed in [48,49,50]). Morphological and genetic evidence showed white and brown fat depots originated from cells which displayed endothelial characteristics [51]. The authors further suggested the possibility of cellular reprogramming with the interconversion between adipocytes and endothelial cells, contributing to the maintenance of homeostatic equilibrium during adipose tissue expansion and reduction. It would be interesting to investigate the cellular reprogramming in the possible coordination between angiogenesis in Stage 3 and adipogenesis in Stage 4 in wound healing process in cetaceans.Wound healing with less fibrosis has been reported in humans under certain circumstances, for example, adult oral mucosa wounds and early gestation fetal skin wounds. These wounds exhibit less inflammatory cell infiltration during wound healing, which is considered one of the critical factors for scarless wound healing [52,53]. Of note, inflammatory response occurred in a comparatively smaller area during the healing process in large full-thickness wounds of Fraser’s dolphins. It needs to be emphasized that the cookiecutter shark bite wounds collected in this study were relatively large, 5–8 cm in diameter and 2–3 cm in depth. Similar wounding in humans requires medical intervention and more than six months to repair [47]. However, the infiltration of inflammatory cells in Fraser’s dolphin skin only extended approximately 4–7 mm away from the edge of a Stage 2 wound. One of the factors contributing to the limited inflammatory response might be certain special compounds in cetacean skin. It was reported that blubber contains organohalogens and a high proportion of isovaleric acid, which exhibit antimicrobial properties [36,37,54]. The presence of these antimicrobial compounds in blubber could provide a synergistic effect to defend the body against infection and restrict pathogen distribution to a relatively small area. Another possible explanation for this phenomenon is that the inflammatory response in cetaceans might be able to eradicate pathogens more efficiently than other animals. Although the inflammatory phase is generally believed to be vital for wound healing to proceed, there is evidence to suggest otherwise [8]. Skin wounds in PU.1 null mice, which lacked macrophages and functioning neutrophils, could heal within the normal time course [55]. Moreover, wounds in these macrophage-removed mice healed without scar, similar to what is reported for fetuses. This study demonstrated that inflammatory cells might not be essential in wound healing as long as microbial infection is controlled. However, it has been reported that the metabolic regulation of innate immune cell phenotypes is significant during skin regeneration, regeneration-competent versus -incompetent mice differ in neutrophil ability, and macrophages are required for skin regeneration in African spiny mice [56,57,58]. There is a lack of information on the immune response during full-thickness wound healing in cetacean skin. Further studies on the relationship between immune modulation and the enhanced healing ability of cetacean skin is essential. Moreover, it could be hypothesized that bacterial infection during wound healing in cetaceans may hinder skin regeneration and lead to impaired wound healing and excessive scarring.Melanin in the skin is synthesized by melanocytes and is suggested to provide protection of epidermal cells from ultraviolet radiation (UVR)-induced DNA damage [59]. A previous study showed that whales with more pigmentation possessed fewer UVR-induced skin lesions, indicating the importance of a photoprotective function for melanin in cetaceans [60]. Melanin was observed in all layers of the epidermis in cetaceans and the wide distribution of melanin was suggested to be a unique photoprotective strategy for the adaptation to aquatic life [24]. In addition to the photoprotective function, many studies showed the antimicrobial and immunomodulatory properties of melanin (reviewed in [59]). In the current study, melanocytes and melanin were observed in the migrating epithelial tongue in Fraser’s dolphins. This phenomenon was quite different from a previous study in pigs, which did not find melanin in the neo-epidermis of full-thickness wounds until they were fully re-epithelialized [61]. Studies on human melanocytes showed that they are not only a source for melanin but also participate in skin immunity through cytokine production, antigen recognition, and antigen presentation [59,62,63]. It remains unknown what the dynamic change of melanocytes during wound healing is, the relationship between intact and healed skin color, and the number of melanocytes in cetaceans. Furthermore, it is important to study the immune functions of melanin and melanocytes in cetacean skin and compare the findings with those in terrestrial mammals.Differences of collagen bundle thickness, spacing, and orientation were observed at different healing stages in the current study. Thin collagen fibers in a parallel arrangement mixing with numerous fibroblasts, fibrocytes, and blood vessels were noticed in the dermis and blubber in Stage 4, while the collagen fibers were comparatively thicker, and the number of cells decreased in Stage 5. Previous studies showed that collagen thickness is related to the composition [64]. In normal human skin, type I and type III collagen are the two major types of collagens [8]. Type I collagen, which is relatively thicker, helps maintain skin structure and tissue integrity, while type III, which is relatively thin, provides tensility, flexibility, and softness [8,64]. It has been reported that normal human skin contains 80–90% type I collagen and 10–20% type III collagen [65]. During wound healing, type III collagen appears earlier than type I collagen, and the percentage of type III collagen may increase up to 30% in granulation tissue [8,66]. In the remodeling phase, type III collagen undergoes degradation, and the proportion of the two collagen subtypes gradually returns to approximately normal ratios in the mature scar [8,66]. Cetaceans can dive several hundreds to thousands of meters deep [67]. Collagens in cetacean skin are essential to maintain skin architecture and body profile during diving. Cetaceans’ diving capacity could be compromised if healed wounds did not function similar to normal skin, especially for a cetacean with numerous healed wounds. However, little is known about the composition of collagen content in cetacean skin, and it would be interesting to understand if cetacean healed wounds equip a normal composition of collagen. Further studies on the quantification of different collagen subtypes in cetacean skin is needed. We surmise that the composition of collagens in cetacean skin, and the timing of ECM remodeling, is different from humans due to the exposure to an extreme marine environment and high water pressure during diving.5. ConclusionsTo the best of our knowledge, this is the first study to characterize histological features of full-thickness wound healing in cetaceans, which demonstrate this adaptation to the aquatic environment. The most significant findings in cetacean full-thickness wound healing included (1) the early appearance of melanocytes and melanin during wound healing; (2) the presence of adipose tissue and rete and dermal ridges in the completely healed wound. These phenomena are quite different from those in terrestrial tight-skinned mammals. It shows that the full-thickness wounds in cetaceans heal in a regenerative manner rather than repair. We hypothesize that the thick blubber layer and limited inflammatory response are critical factors contributing to the enhanced healing ability of cetacean skin. Further studies to elucidate the mechanisms of immune modulation, angiogenesis, adipocyte regeneration, and collagen reconstruction during full-thickness wound healing in cetaceans may shed light on veterinary and human regenerative medicine, leading to novel therapies.

animals : an open access journal from mdpi

10.3390/ani13061063

PMC10044279

Reproductive activity in mares shows a seasonal pattern that is associated with an increasing photoperiod with a pronounced incidence of ovulations during spring and summer. The photoperiodic control of reproductive activity is mediated by melatonin, secreted by the pineal gland, which plays an inhibitory role in ovulatory activity via the hypothalamic-pituitary axis. Considering that there is little information on the direct effect of melatonin on the development of equine ovarian follicles, we studied the seasonal variations in mRNA expression of gonadotropin receptors (Fshr and Lhr), melatonin receptors (Mt1 and Mt2), melatonin-synthetizing enzymes (Asmt and Aanat) and melatonin concentration in developing follicles (small—<20 mm, medium—20 to 35 mm and large—>35 mm) from five mares raised in natural photoperiods. There was an increased mRNA expression of gonadotropin receptors and melatonin-related genes and an increase of melatonin levels in developing follicles during the spring/summer seasons. The total number of large follicles (potential ovulatory follicles) was significantly higher during the spring/summer seasons. Our results demonstrate that melatonin upregulates the mRNA expression of melatonin receptors and melatonin-forming enzymes in mare developing follicles during reproductive seasons.

This study investigated the seasonal variations in mRNA expression of FSH (Fshr), LH (Lhr) receptors, melatonin (Mt1 and Mt2) receptors, melatonin-synthetizing enzymes (Asmt and Aanat) and melatonin concentration in developing follicles from mares raised in natural photoperiods. For one year, ultrasonographic follicular aspiration procedures were performed monthly, and small (<20 mm), medium (20 to 35 mm) and large (>35 mm) follicles were recovered from five mares. One day before monthly sample collections, an exploratory ultrasonography conducted to record the number and the size of all follicles larger than 15 mm. The total number of large follicles were higher during the spring/summer (8.2 ± 1.9) than during autumn/winter (3.0 ± 0.5). Compared to autumn/winter seasons, there was an increase of Fshr and Aanat mRNA expressions in small, medium and large follicles, an increase of Lhr and Asmt mRNA expressions in medium and large follicles and an increase of Mt1 and Mt2 mRNA expressions in small and large follicles during spring/summer. The melatonin levels in follicular fluid were also higher during the spring/summer seasons. The present data show that melatonin locally upregulates the mRNA expression of Mt1 and Mt2 receptors and melatonin-forming enzymes in mare developing follicles during reproductive seasons.

1. IntroductionThe incidences of ovulatory activity in mares are dependent on the time of year. During the winter, the incidence of ovulation is minimal or absent, increasing during the spring, it becomes maximum in summer, and decreases during the autumn [1]. Although the incidence of ovulations presents a seasonal pattern, estrus behavior in mares can be expressed even during the anovulatory period [1,2]. The seasonal pattern of reproductive activity comes from an endogenous circannual rhythm, which is determined by several environmental factors including the photoperiod [3]. The beginning of the reproductive activity in mares is related to the increase in daylight hours, and an additional lighting exposure during short-day seasons can anticipate the start of the breeding season [3,4]. Several studies have shown that the neuroendocrine control of seasonal reproduction in mares is mediated by melatonin secreted by pineal gland [5,6]. The administration of melatonin in subcutaneous implants to pony mares during the summer produces a significant reduction of hypothalamic GnRH [7] and alters the secretion of LH [8] according to the ovarian status (intact or ovariectomized). In addition, exposing mares to constant light decreases the concentration plasma melatonin and increases hypothalamic GnRH [9], suggesting a regulatory role for melatonin at the central level.In addition to the effects of melatonin on reproduction being mediated mainly through its action in hypothalamic and pituitary regions [10,11], numerous studies have shown that melatonin may act directly on the regulation of ovarian function [12,13,14,15]. During the ovarian follicle development, melatonin may act in the estrogen and progesterone productions [16,17,18] by regulating the steroidogenic gene expressions [16,19] including the mRNA expression of LH receptor [17,20] through the MT1 and MT2 receptors present in granulosa-luteal cells [17,21,22]. Melatonin also protects the oocyte from oxidative stress [23] improving oocyte’s quality whose effects could be attributed to its free radicals scavenging action [24]. The concentration levels of melatonin in follicular fluid [25], probably synthetized by the ovary [26,27,28], varied according to the human [14] and porcine [29] follicle sizes, and circadian and seasonal changes in melatonin concentration were also detected in human preovulatory follicular fluid [30]. In general, melatonin plays a positive effect on oocyte maturation, fertilization, and embryo development as well in the direct regulation of steroidogenesis [12,13,14,15,16,18,19,20,21,22,23,27,28,29] during the follicle development. Because of these physiological characteristics, melatonin plays an important role in human assisted reproductive technologies (ART) by improving the clinical outcomes of IVF-embryo transfer [13,31]. Like in humans, ART procedures were also used in horses and some similarities in these technologies have been shown between women and mares [32].Considering that there is evidence of a direct effect of melatonin on the equine ovary showing no photoperiodic differences in mRNA expression of Mt1 receptors in developing follicles [17], we studied the seasonal variations in mRNA expression of FSH (Fshr), LH (Lhr), melatonin (Mt1 and Mt2) receptors, melatonin-synthetizing enzymes (Asmt and Aanat) and melatonin concentration in developing follicles from mares raised in natural photoperiods.2. Materials and Methods2.1. MaresFive fertile crossbred mares from the Laboratory of Theriogenology Dr. O. J. Ginther of Faculty of Engineering and Animal Science, University of São Paulo (aged 6 to 14 years and weighing 300–400 kg) were used in this study. All animals were clinically healthy with no evidence of diseases or reproductive disorders. Mares were kept under natural photoperiods in Southeast Brazil (21°59 Southern latitude and 47°26 Western longitude from Greenwich and 634 m altitude). The mares were kept exclusively on pasture of Mombaça grass (Panicum maximum Jacq. cv. Mombaça), with access to water and trace-mineralized salt ad libitum. They also received individual and daily protein-energy maintenance supplementation.2.2. Aspiration Procedure and Follicular Cells RecoveryThe aspiration procedures were carried out every month for one year. One day before the follicular aspiration, an exploratory ultrasonography was performed for ovarian assessment when the number and the size of all follicles larger than 15 mm and luteal development were recorded. Immediately before the aspiration procedures, the mares were sedated with Butorphanol (0.01 mg/kg) and Romifidine (0.04 mg/kg). An ultrasound machine with a multifrequency linear transducer (5–8 MHz) was used for follicular aspiration procedures [33]. Each follicle was aspirated with a 12-gauge double lumen needle (WTA®, Cravinhos, SP, Brazil) attached to a vacuum pump whose pressure varied between 150 mmHg and 250 mmHg according to the size of the follicles. The probe protected with a sanitary plastic condom was inserted transvaginally for follicular aspiration, and the ovaries were manipulated transrectally and placed next to the ultrasound probe for visualization of the follicles. Data were collected during the light phase of the day, between 8:00 a.m. and 11:00 a.m.The aspirated follicles were collected in tubes containing Tissue Culture Medium 199 with HEPES (Gibco®/12350-039, Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA) [20]. To avoid any individual differences, the follicular (granulosa and theca cells) cells were isolated from a pool of follicles from different sizes which were categorized as small (<20 mm), medium (20 to 35 mm) and large (>35 mm). After the collection, the separated samples were put into 1000 µL of lysis solution (TRIzol®, 15596-018, Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA) for RNA isolation and stored at −80 °C. After centrifugation at 4 °C for 10 min, the follicular fluid samples from different sizes of follicles were also stored at −80 °C.2.3. mRNA Extraction and qRT-PCRFor RNA extraction, the TRIzol® RNA isolation protocol was used [22]. The aqueous phase of the thawed sample, previously diluted in TRIzol® reagent, was separated with 200 µL of chloroform. The RNA precipitation and the washing of RNA pellet were carried out using 500 µL of isopropanol alcohol and 1000 µL of 75% ethanol, respectively. Then, the RNA pellet was dissolved in DEPC treated water. The RNA quantification was conducted at 260 mm on NanodropTM 1000 equipment (Thermo Fisher Scientific, Waltham, MA, USA).The first-strand cDNA was produced from 1 µg of total RNA pellet diluted in RNase-free water using reverse transcriptase (Superscript III, 18080-044, Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA) and random primers (48190-011, Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA). The Quantitative Reverse Transcriptase Polymerase Chain Reaction (qRT-PCR) was performed on QuantStudio 6 Flex Real-Time PCR equipment (Applied Biosystems, Inc., Foster City, CA, USA) using the relative quantification analyses (2−ΔΔCT method) [34] and reported as arbitrary units. The PCR efficiency of the target and reference genes was 95%. All samples were analyzed in duplicate, and Gapdh was used as a reference gene [35]. The accession number of the genes and the primer sequences are listed in Table 1.2.4. Melatonin Concentration in Follicular FluidThe concentrations of melatonin (pg/mL) in follicular fluid samples were collected during the light phase of day and measured using a commercial melatonin ELISA kit (IBL International, Hamburg, Germany) according to the manufacturer’s instructions. The samples were assayed in duplicate, and the sensitivity of the assay was 0.5 pg/mL.2.5. Statistical AnalysisData were expressed as mean ± SEM, calculated from at least five replications. Seasonal (spring/summer vs. autumn/winter) effects on the number of follicles and the mRNA expression in each class of follicles were analyzed using the Welch’s t-test and the non-parametric Mann–Whitney test, respectively. Seasonal effects on melatonin concentrations in follicular fluid (FF) were analyzed using the two-way ANOVA followed by Bonferroni’s post-test. A correlation analysis between melatonin levels in FF and mRNA expression of melatonin-related genes was also performed. All analyses were performed using GraphPad Prism (GraphPad Software version 9.40; San Diego, CA, USA).3. Results3.1. Annual Distribution of the Number of Developing Ovarian Follicles and Seasonal Variation of mRNA Expression of FSH (Fshr) and LH (Lhr) Receptors in Ovarian Follicles of Different SizesThe ovaries of five mares were monitored monthly by ultrasonography for one year. The total number of large follicles were significantly higher during spring/summer than during autumn/winter (Table 2). Considering the mRNA expression of Fshr and Lhr in follicles from different sizes (Figure 1), the small, medium and large follicles presented an increase of Fshr mRNA expression during the spring/summer seasons. The Lhr mRNA expression in medium and large follicles was also increased during spring/summer seasons.3.2. Seasonal Variation of mRNA Expression of Melatonin-Related Genes in Ovarian Follicles of Different SizesThe mRNA expression of melatonin receptors (Mt1 and Mt2) in ovarian follicles is presented in Figure 2. The expression of both receptors in small and large follicles was higher in spring/summer seasons than in autumn/winter seasons. No seasonal variation of Mt1 and Mt2 mRNA expressions was observed in medium follicles. Figure 3 shows the mRNA expression of melatonin synthetizing enzymes (Amst and Aanat). A significant increase of Asmt and Aanat mRNA expressions was observed in medium and large follicles during the spring/summer. In small follicles, the mRNA expression of Aanat was also increased during spring/summer seasons, but no seasonal difference of Asmt mRNA expression was observed.3.3. Seasonal Variation on Melatonin Concentration in Follicular Fluid from Developing FolliclesConsidering seasonal melatonin concentration differences in developing follicles (Figure 4), melatonin levels in medium and large follicles were significantly higher during the spring/summer seasons. During the autumn/winter seasons, no differences in melatonin concentration were observed in small follicles. The correlation analysis (Table 3) revealed a significant positive correlation between seasonal melatonin levels in follicular fluid and the seasonal mRNA expression of melatonin forming enzymes (Asmt and Aanat) in medium and large follicles. In large follicles, seasonal melatonin concentrations in follicular fluid were also positively correlated with seasonal variation in mRNA expression of melatonin receptors (Mt1 and Mt2). No significant correlation was observed in small follicles.4. DiscussionThe present study investigated the seasonal variation of melatonin concentration and mRNA expression of melatonin-related genes in developing follicles of mares living under natural photoperiods in Southeast Brazil. First, we verified the annual follicular activity by monitoring the ovaries one day before the monthly collection of the samples. The ovarian follicles were classified according to the follicular dynamics that are characterized by the development of an ovulatory and anovulatory major waves and anovulatory minor waves that are mainly regulated by circulating gonadotropin levels [36]. In mares, the emergence of a follicular wave occurs in response to FSH surge which reach the peak when a cohort of follicles attains approximately 13 mm in diameter (small follicles). The declining FSH levels lead to the selection of one largest follicle with 21 to 23 mm in diameter (medium follicles) which deviates in diameter from the smaller follicles (subordinate follicles) that go into regression. After the deviation, the largest follicle continuously grows, becoming a dominant follicle (large follicles) with approximately 35–45 mm in diameter which may ovulate or stop growing and regress, depending on the occurrence of the LH surge [36].Our research revealed that the number of large follicles was significantly higher during the spring/summer seasons confirming the seasonal follicular activity whose annual changes in photoperiod have been the main environmental factor that synchronizes this seasonality in temperate, subtropical and tropical environments [3,4,5,37]. It has been reported that the mare follicular activity decreases during the winter, and the follicular waves during the non-breeding season are characterized by the absence of large follicles and unaltered presence of small and medium follicles [38]. We also analyzed the mRNA expression of FSH and LH receptors, which reflects the action of these gonadotropins in developing follicles [39]. The present study revealed that, during spring/summer seasons, there was a significant increase of Fshr mRNA expression in small, medium and large follicles and an increase of Lhr mRNA expression in medium and large follicles. The development of follicular waves has been associated with FSH surge for major and minor waves. In ovulatory, transitional and anovulatory seasons, FSH concentration has been more important for follicular growth before deviation while LH plays an important role during deviation and in stimulating the development of dominant follicles [36,38]. Moreover, it has been shown that no seasonal differences were observed in circulating FSH associated with follicular activity, and significant LH requirements are determinant for the growth of large follicles during the ovulatory and anovulatory seasons [38]. However, large follicles have shown a higher responsiveness to LH during the ovulatory season (spring/summer) than during the anovulatory season (autumn/winter) as indicated by high levels of Lhr mRNA expression in large follicles during the ovulatory season [40]. Our results suggest that, during the spring and summer seasons, small, medium and large follicles have an increased responsiveness to circulating FSH, and medium and large follicles have an increased responsiveness to LH levels.Our findings also showed that the mRNA expression of melatonin-related genes in developing follicles and melatonin concentration in follicular fluid varied seasonally. The mRNA expression of Mt1 and Mt2 receptors in small and large follicles was significantly elevated during the spring/summer seasons. In the same way, we also observed an increase of mRNA expression of Asmt and Aanat and an increase of melatonin levels in developing follicles during the spring/summer seasons. These results suggest a putative direct effect of local producing melatonin on the ovarian follicle development independent of the seasonal effects of melatonin on the photoperiodic control of reproductive activity via the hypothalamic-pituitary axis. As previously mentioned, melatonin is an important signal in the seasonal control of reproductive activity in mares [6,7,8,9,10]. The breeding season period is associated with increasing daylength and takes place during the spring and summer months when the duration of the nocturnal melatonin secretion reflects the length of scotophase [11,41]. In fact, it has been demonstrated that pinealectomy and melatonin treatment at the timing of onset of nocturnal melatonin secretion blocked the ability of mares in responding to stimulatory long photoperiods [6,7,9,42]. On the contrary, our results showed an increase of the mRNA expression of the melatonin related genes (Mt1, Mt2, Asmt and Aanat) and an increase of melatonin concentration in developing follicles during the spring and summer months, suggesting a direct effect of local synthetized melatonin on follicle development in the same period of ovulatory activity when melatonin signal is reduced. In addition to confirming the previously reported direct effect of melatonin on ovarian function in mares [17], the present findings also suggest that melatonin is acting locally in all steps of follicular wave development. Apparently, melatonin action has been more pronounced in dominant follicles which have the potential to ovulate. The increase of mRNA expression of melatonin-synthetizing enzymes and melatonin levels in large follicles revealed a local and beneficial role of melatonin in the final follicular growth during the ovulatory season. One of the most important effects of melatonin on ovarian follicle growth is to protect the oocyte and follicular cells from intrafollicular oxidative stress by reducing reactive oxygen species (ROS) and acting as a potent free radical scavenger during the ovulation process [14,15,23] since ovulation is like a local inflammatory response whose ROS are generated [43]. Our results are compatible with the premise that melatonin in follicular fluid was locally secreted by oocytes and/or follicular cells [26,27,28], and the elevated melatonin levels during the reproductive season play a beneficial role in follicular growth and protection. It has been demonstrated that high concentrations of melatonin in follicular fluid were positively correlated with the quantity of good-quality oocytes, oocytes fertilized and good-quality embryos [44,45], critical conditions for the success of assisted reproductive technologies [13].The present research shows that melatonin has the potential to be used in equine ART programs [46] by acting as an antioxidant agent for in vitro maturation cultures [47]. Indeed, the role of melatonin as an antioxidant in human ART programs has recently been well documented [48]. Melatonin could be used as a supplement in culture media improving oocyte maturation and fertilization and embryonic development [13,23,31,49]. Oral administration of melatonin to patients in human ART programs was able to improve fertilization rates and the quality of oocytes and embryos [23,50]. Additionally, these results could be also considered important as a basis for further studies which focus on human ART programs since there are similarities in the dynamics of follicular growth between the two species [32,51].5. ConclusionsThe current findings demonstrate that melatonin, probably from peripheral ovarian melatonin synthesis, upregulates the mRNA expression of melatonin receptors and melatonin-forming enzymes in mare developing follicles during the reproductive season.

animals : an open access journal from mdpi

10.3390/ani12010070

PMC8749698

Ice cream may be used as a carrier to deliver probiotics and prebiotics. In this study, we decided to investigate the possibility of using sheep milk from the Olkuska breed for ice cream manufacture and evaluate the viability of Lactobacillus and Lacticaseibacillus strains and the chemical, physical and organoleptic properties of dairy ice cream during storage. The obtained results contribute to a more practical application of different probiotic strains for the fermentation of ice cream mixes and the possibility of using apple fiber in ice cream production. Moreover, the study’s findings showed that symbiotic ice cream with acceptable physicochemical and organoleptic characteristics might be produced from sheep milk of the Olkuska breed.

The aim of this study was to determine the possibility of using Olkuska sheep milk for the production of ice cream with probiotics and prebiotics. The study examined the effect of the storage and type of bacteria used for the fermentation of ice cream mixes and partial replacement of inulin with apple fiber on the physicochemical properties, viability of probiotic cultures and organoleptic properties of sheep’s milk ice cream stored at −22 °C for 21 days. The addition of apple fiber reduced the pH value of ice cream mixes before fermentation. In ice cream mixes and ice cream with apple fiber, the lactic acid content was higher by 0.1–0.2 g L−1 than in their equivalents with inulin only. These differences persisted during the storage of the ice cream. After fermentation of the ice mixes, the bacterial cell count ranged from 10.62 log cfu g−1 to 12.25 log cfu g−1. The freezing process reduced the population of probiotic bacteria cells in ice cream with inulin from 0.8 log cfu g−1 in ice cream with Lactobacillus acidophilus, 1.0 log cfu g−1 in ice cream with Lacticaseibacillus paracasei and 1.1 log cfu g−1 in ice cream with Lacticaseibacillus casei. Freezing the varieties with apple fiber also resulted in a reduction of viable bacterial cells from 0.8 log cfu g−1 in ice cream with L. paracasei and Lb. acidophilus to 1 log cfu g−1 in ice cream with L. casei, compared to the results after fermentation. The highest percentage overrun was determined in ice cream with L. paracasei and Lb. acidophilus. Ice cream with L. casei was characterized by significantly lower overrun on the 7th and 21st days of storage. Although L. paracasei ice cream had the highest overrun, it did not cause a significant reduction in the probiotic population during storage. After seven days of storage, the first drop differed significantly depending on the type of bacteria used for fermentation of the mixture and the addition of apple fiber. L. casei ice cream had a longer first drop time than L. paracasei and Lb. acidophilus ice cream. Partial replacement of inulin with apple fiber resulted in a significant darkening of the color of ice cream mixes. Depending on the type of bacteria used for fermentation, the addition of apple fiber decreased the value of the L* parameter. Ice cream mixes and ice cream with inulin and apple fiber were characterized by a high proportion of yellow. Partial replacement of inulin with apple fiber reduced the hardness of ice cream compared to inulin-only ice cream. Moreover, the panelists found that ice cream with inulin was characterized by a sweeter taste than ice cream with apple fiber. Moreover, the addition of apple fiber favorably increased the flavor and aroma perception of the mango-passion fruit. Therefore, the milk of Olkuska sheep could be successfully used for the production of symbiotic dairy ice cream.

1. IntroductionThe parameters of sheep milk are influenced by various factors including genetic, physiological and environmental. Polish sheep breeds, such as Olkuska, are well adapted to local environmental conditions. This breed is characterized by resistance to diseases and demanding environmental conditions, moreover good milk yield [1].Sheep milk is characterized by a higher content of total solids compared to cow and goat milk. Moreover, sheep milk is characterized by a high content of micro- and macroelements, vitamins, protein and fat [2].The protein content of raw milk differs among the species and the sheep raw milk has the higher protein content (5.5%). According to Balthazar et al. [3] the total casein content in sheep raw milk is 85% of total proteins, where αS1, αS2, β and κ-casein represent 6.7%, 22.8%, 61.6% and 8.9%, respectively. The remaining 15% of milk proteins includes major whey proteins β-lactoglobulin and α-lactalbumin as well as other protein constituents [4].Milk proteins have a wide range of functional properties such as emulsifying, thickening, gelling and foaming. Milk proteins promote the formation and stabilization of oil droplets in emulsions or air bubbles in foams in food formulations [5]. Moreover, the higher viscosity of sheep milk may also be attributable to an increased water binding capacity in the milk proteins [6]. These functional properties of milk proteins are used to produce dairy products such as ice cream.Raw milk from different species remains an element of human nutrition. Therefore, the free and total amino acid profile of milk from different species plays a crucial role for both milk producers and processors as well as consumers to achieve innovative new product design, complexity, flavor and functionality [4]. The characteristic profiles of free amino acid content vary among species. According to Landi et al. [4], the free amino acid found in the highest amounts in raw cow, sheep and goat milk is glutamic acid (9.07 mg per 100 g), tyrosine (4.72 mg per 100 g) and glycine (4.54 mg per 100 g). In contrast, raw goat milk is a rich source of taurine (14.92 mg per 100 g), which is found in small amounts in raw cow milk (1.38 mg per 100 g) and sheep milk (2.10 mg per 100 g).Sheep milk could be considered a substitute for cow’s milk for allergy sufferers. Specific antibodies in milk-allergic individuals (IgE) poorly recognize the αS1-casein, αS2-casein and β-casein protein fractions from goat and sheep milk, which is not observed with cow milk [3,7]. Nevertheless, protein polymorphism is described as playing an essential role in the induction of different degrees of an allergic reaction [3,8].Sheep’s milk ice cream can be an excellent carrier of bioactive compounds, including probiotic cultures and prebiotic ingredients. The high protein level and fat in sheep’s milk allows for the production of ice cream with a higher density, which translates into better protection of probiotic cells against oxygen access during ice cream storage and during passage through the gastrointestinal tract [9].Probiotics added to food benefits by restoring the intestinal microflora balance [10,11,12] with a minimum number of live probiotic cells of 106–109 cfu g−1 is required for a therapeutic effect [13,14,15]. Probiotic bacteria used in ice cream production should be freeze-resistant and survive for a particular time at low temperatures [16,17,18]. Lactobacillus and Bifidobacterium are the most commonly used probiotic strains in food production [19,20]. Few studies show that probiotics in milk ice cream have better survival in the presence of inulin [21,22]. Inulin can be used as a replacement for fat and sugar, improving the viscosity of ice cream, thus affecting better overrun, resistance to melting and maintaining shape by stabilizing the foam [23] due to the ability to bind water and create a gel network, giving a feeling of smoothness and creaminess [24,25] in addition, it reduces the hardness of reduced-fat ice cream [26].Recent studies indicate that prebiotic compounds may also increase the bioavailability of milk protein [27]. Simultaneous consumption of prebiotic ingredients that ferment into acetate, propionate, and butyrate stimulates the growth and development of probiotic bacteria [28]. Fruit fibers are gaining more and more recognition among consumers and are considered natural additives with a low degree of processing [29]. This group of prebiotics includes apple fiber, a natural fruit product containing protein, carbohydrates, polyphenols and pectins [30]. The addition of fiber to dairy products fits well with the “clean label” trend in the natural food market segment.Sheep milk ice cream with the addition of prebiotics such as inulin and apple fiber, apart from the health benefits and properties stimulating the growth of probiotic bacteria, is an innovative product with functional properties. There are few publications on the production of ice cream from sheep’s milk with probiotic strains and prebiotics in the scientific literature. Therefore, the study aimed to determine the possibility of using Olkuska sheep milk for the production of ice cream with probiotics and prebiotics.2. Materials and Methods2.1. MaterialsRaw morning and cooled (4 °C) sheep’s milk for the production of ice cream was purchased from the farm “Owcza Zagroda” (Wyżne, Podkarpacie, Poland) in June 2021. A flock of 35 Olkuska breed sheep were kept on the farm. Green fodder and hay with the addition of cereals (oats, barley and maize in a quantity of 0.5 kg/sheep/day) from the breeder’s farm were used for feeding animals. Milking was conducted by hand. Before ice cream manufacture, milk was filtrated to remove dirt and foreign particles.The chemical composition of milk was determined in the chemical composition analyzer of milk and dairy products Bentley B-150 (Bentley, MN, USA).The following materials were used to prepare the ice cream mixture: inulin (carbohydrate 97 g/100 g, including sugars 7 g/100 g, fiber 90 g/100 g, fat 0 g/100 g and protein 0 g/100 g; Orafti HP, Oreye, Belgium); apple fiber (carbohydrate 87 g/100 g, including sugars 27 g/100 g, fiber 51 g/100 g, fat 3.3 g/100 g and protein 5.1 g/100 g; Aura Herbals Jarosław Paul, Sopot, Poland) composed of 100% micronized apple fiber; white sugar (Polish sugar, Toruń, Poland); mango-passion fruit flavor essence (Browin, Łódź, Poland) with the composition: natural and identical to raw mango and passion fruit flavors, citric acid E330 and mango juice. Probiotic bacteria (Chr. Hansen, Hoersholm, Denmark) were used to produce the inoculum: Lacticaseibacillus paracassei L-26, Lacticaseibacillus casei 431 and Lactobacillus acidophilus LA-5.2.2. Manufacture of Ice Cream MixesSheep’s milk (84.9%), sugar (11%) and flavor essence (0.1%) were mixed and then divided into two parts. Inulin (4%) was added to the first part of the blend and divided into three groups: CP, CC and CA. Inulin (2.5%) and apple fiber (1.5%) were added to the second part and analogously divided into three groups: CPF, CCF and CAF. The milk with additives was mixed and homogenized with a homogenizer (Nuoni GJJ-0.06/40, Zhejiang, China) at 60 °C with a pressure of 20 MPa, and then pasteurized at 85 °C, 1 min. After heat treatment, it was cooled to 37 °C.The CP and CPF groups were inoculated with a monoculture Lacticaseibacillus paracasei L-26, the CC and CCF groups were inoculated with Lacticaseibacillus casei 431. The CA and CAF groups were inoculated with Lactobacillus acidophilus LA-5. Each starter probiotic culture was previously inoculated in sheep’s milk at 40 °C for five hours. After five hours, 5% (w/w) of inoculum containing 9 log CFU of g−1 bacteria was added to the ice mixes. The fermentation process of the ice cream mixes was carried out in an incubator (Cooled Incubator ILW 115, POL-EKO-Aparatura, Wodzisław Śląski, Poland) at 37 °C for 10 h. After cooling to 5 °C, it was conditioned at the specified temperature for 12 h in an incubator and then froze in a DeLux 48816 freezer (UNOLD AG, Hockeheim, Germany) for 40–50 min with a freezing temperature down to −22 °C. The prepared ice cream was packaged in 100 mL plastic cups and stored at −22 °C for twenty-one days.The experiment was repeated on three occasions.2.3. Physicochemical AnalysisThe chemical composition of the ice cream mixes was determined using a Bentley B-150 Milk and Milk Product Analyzer (Bentley, MN, USA). The determination of the pH value was performed with a FiveEasy digital pH meter (Mettler Toledo, Greifensee, Switzerland) with an electrode InLab®Solids Pro-ISM (Mettler Toledo, Switzerland) with an integrated temperature sensor. The content of lactic acid was determined (g of lactic acid L−1) by titration of samples of ice cream mixes and dissolved ice cream with 0.1 N NaOH (Chempur, Piekary Śląskie, Poland) according to the method of Jemaa et al. [31]. The melting rate, first dropping time and total melting time was determined at an ambient temperature of 22 °C. Ice cream samples were placed on a wire mesh grid (95 mm diameter, holes 5 × 5 mm, wire thickness 0.5 mm). Then the time until the ice cream wholly dissolved was recorded [32]. Ice cream overrun was estimated as the air volume ratio in frozen ice cream to the importance of melted ice cream expressed in % [33]. Five samples were tested for each ice cream variant, and it was repeated was repeated in triplicate.2.4. Microbiological AnalysisThe number of probiotic strains (Lacticaseibacillus paracasei L-26, Lacticaseibacillus casei 431, Lactobacillus acidophilus LA-5) was determined in the ice mix, then immediately after freezing and on the 7th and 21st days of storage at −22 °C. 10 g of each sample was diluted in 90 mL of sterile peptone water solution (0.1%) (BTL Sp. z o.o., Łódź, Poland). Serial dilutions from 1 log CFU g−1 to 8 log CFU g−1 were made. The inoculation was performed by the plate-deep method using MRS agar (Biocorp, Warszawa, Poland) and incubated anaerobically in a vacuum desiccator at 37 °C for 72 h using the GENbox anaer (Biomerieux, Warszawa, Poland). The cultured probiotic colonies were counted with a colony counter (TYPE J-3, Chemland, Stargard Szczeciński, Poland). The result was expressed as log CFU g−1. Five samples were tested for each ice cream variant, and it was repeated was repeated in triplicate.2.5. Color of Ice CreamThe color of the ice cream was measured with a precise colorimeter (model No. 145, Shenzhen, China) using the CIElab system. The following parameters were measured: L*—brightness of ice cream (0—black, 100—white), a* (− a*—shades of green, + a*—shades of red) and b* (− b*—shades of blue, + b*—shades of yellow), C—color saturation and purity and h° as a hue of color. Before analysis, the device was calibrated on a white and black reference standard [34,35]. Five samples were tested for each ice cream variant, and it was repeated was repeated in triplicate.2.6. Organoleptic AnalysisOrganoleptic assessment for six groups of ice cream was carried out by a trained team of 15. The samples of ice cream encoded (with a random three-digit code) were analyzed on a nine-point linear scale with marginal, structured markings. The left end of the scale indicated the following features: not very characteristic (appearance); soft (hardness); sandy (smooth); immediate (spreadability); dark (color); hardly perceptible (taste, smell) [36]. The right side of the scale defined the features: very distinctive (appearance), hard (hardness), very smooth (smooth); delayed (spreadability); light (color); very intense (distinguishing features of taste and smell). After analyzing the tested ice cream sample, the panelists were asked to rinse their mouths with water to avoid a cold transfer effect [37].2.7. Statistical AnalysisThe mean and standard deviation were calculated using Statistica v. 13.1 (StatSoft, Tulsa, OK, USA). One, two and three-way ANOVA was performed. The significance of differences between the mean values was verified with the Turkey test (p < 0.05).3. ResultsThe chemical composition of sheep milk was: fat 6.56 ± 0.5%, protein 4.60 ± 0.3% and lactose 5.04 ± 0.10%, and was similar to the results obtained by Musial et al. [2]. Olkuska sheep milk was only characterized by a higher lactose content.3.1. Chemical Composition of Ice Cream MixesThe chemical composition of the ice cream mixes fermented by various strains of probiotic bacteria is presented in Table 1.The ice cream mixes contained 3.68–3.75% protein, 6.36–6.46% fat and 18.43–18.50% carbohydrates. The ice cream mixes did not differ significantly in terms of protein, fat and carbohydrate content. Partial replacement of inulin with apple fiber and fermentation of ice cream mixes with different probiotic strains do not significantly differentiate their chemical composition. Similar content of protein (3.39%) and carbohydrates (18.39%) in the control milk ice cream was demonstrated by Ismail et al. [38]. Moreover, Balthazar et al. [39] developed and tested sheep’s milk ice cream with a higher fat content (10.03%) and lower protein content (3.2%).Table 2 shows the moisture results of ice cream after 7 and 21 days of ice cream storage. There were no significant differences in moisture between individual ice cream groups and during storage, which was confirmed by the 3-factor ANOVA (Table 3). These results are in line with the studies by Abdelazez et al. [40] and Ranadheera et al. [41], who showed that the moisture of frozen products does not change significantly during freezer storage.Table 2 shows the pH values determined before and after the fermentation of the ice mixes. As expected, the 1.5% addition of apple fiber lowered the pH value of ice cream mixes by about 0.2 units before fermentation. The optimal pH for Lactobacillus and Lacticaseibacillus growth is 5.5–6.0 [42]. The addition of apple fiber favorably reduced the pH value to 6.1, creating conditions similar to the optimal ones for fermentation than in mixtures with inulin only. Reducing the pH value of ice cream with apple fiber might be due to the presence of organic acids. Apple fiber obtained from apple pomace contains organic acids (0.6–0.9%) such as malic, quinic, citric and shikimic acids as well as sugars (7%), fiber (5–6%), trace amounts of protein as well as microelements: calcium, magnesium, iron and potassium [43,44].As in our study, in the study by Favaro-Trindade et al. [45], the addition of acerola pulp reduced the pH value. In our research, after fermentation, the lower pH value of mixtures with apple fiber (CPF, CCF and CAF) was maintained compared to their counterparts only with inulin (CP, CC and CA). It was shown that using the same fermentation conditions for all mixes, the pH value decreased the most in the fermentation in CP mixtures fermented with L. paracasei. Most likely, it results from the specificity of L. paracasei because this strain, unlike L. casei, ferments lactose to produce L (+) lactic acid, and about 50% of cells also can ferment inulin [46].Significantly higher pH values were obtained in Lb-fermented CC mixtures. L. casei and CA from Lb. acidophilus (Table 2). The decrease in the pH value of the mixtures results from the metabolic activity of probiotics in the ice cream mix and the inclusion of prebiotic preparations, i.e., apple fiber. A 3-factor analysis of variance showed that the type of probiotic bacteria significantly affected the pH value and lactic acid content in ice cream (Table 3). In the studies of Soukoulis et al. [47], the pH value of fermented and non-fermented ice cream was between 4.5 and 6.3. In studies by Akca and Akpinar [48], probiotic milk ice cream with the addition of Lacticaseibacillus rhamnosus and Bifidobacterium animalis ssp. lactis Bb-12 was characterized by a pH value of 5.18–5.54 during 90 days of storage. The authors noted the lowest pH value in ice cream with powdered grape seed pulp with prebiotic properties.In our research, an inverse relationship with the pH value was found for lactic acid content in ice cream mixes and ice cream. In ice cream mixes and ice cream with apple fiber, the lactic acid content was higher by 0.1–0.2 g L−1 than in their equivalents with inulin only. These differences persisted during the storage of the ice cream. The lowest content of lactic acid after fermentation was determined in the CA ice cream mix fermented by Lb. acidophilus.The highest amount of lactic acid was found in the mixture of CPF with apple fiber fermented by L. paracasei. The analysis of variance shows that lactic acid content is significantly influenced by single factors (bacterial type, storage time or fiber) and interactions between bacterial type and storage time. However, the combined effect of these three factors on the lactic acid content turned out to be insignificant.Similar results were obtained by Ismail et al. [38] in ice cream enriched with pomegranate peel powder and Abd El-Rashid and Hassan [49], who used Doum palm fruit to make ice cream. According to Farias et al. [50], ice cream’s pH value and acidity do not change during the storage period, regardless of whether they have been fermented or not. As in the studies by Farias et al. [50], our results indicate that extending the storage time from 7 to 21 days does not increase the lactic acid content.The pH value and acidity of probiotic products can significantly affect the survival of probiotic bacteria cells in fermented ice cream. The addition of growth promoters and prebiotics such as inulin has been shown in many studies to significantly improve the viability of probiotic organisms [51]. Akin et al. [52] studied ice cream containing probiotic bacteria (Lb. acidophilus and Bifidobacterium lactis), and their results suggest that the addition of inulin stimulates growth Lb. acidophilus and Bifidobacterium lactis, which improved their survival. Therefore, partial replacement of inulin with apple fiber in our research might also contribute to the differentiation of growth and survival of probiotic bacteria cells.3.2. Microbiological Analysis of Ice CreamThe number of bacterial cells in the mixes and ice cream depending on the type of bacteria, storage time and the addition of apple fiber is presented in Table 4.After fermentation of the ice cream mixes, bacteria cells ranged from 10.62 log cfu g−1 to 12.25 log cfu g−1. The addition of apple fiber did not significantly affect the growth of L. paracasei in the CPF ice cream mix compared to CP. In turn, this addition contributed to an increase in the number of Lb. acidophilus cells by approximately one log cfu g−1 in CAF mixtures with apple fiber compared to the CAF mixture with inulin only. It should be mentioned that Lb. acidophilus, especially, has a high cytoplasmic buffering capacity (pH 3.72–7.74), which allows it to resist cytoplasmic pH changes and obtain stability under acidic conditions [53,54]. According to Talwalkar and Kailasapathy [55] and Haynes and Playne [56], and Takahashi et al. [57], this stability is influenced by the enzyme H + -ATPase.In this study, the highest number of bacterial cells was determined in the CC-fermented mixture with L. casei with inulin. However, the addition of apple fiber resulted in a reduction in the number of cells by about 1 log CFU g−1 in the CCF mixture. These studies indicate that a vital problem is selecting probiotic bacteria for the fermentation of sheep’s milk mixtures because partial replacement of inulin with apple fiber changed the fermentation conditions (e.g., pH value) and differentiated the number of probiotic cells after fermentation.The resistance of probiotics to freezing damage varies among probiotic strains. Microorganisms that show a better ability to survive under freezing conditions can dehydrate without rupturing the cytoplasmic membranes. Such cells can reduce the number and growth of intracellular ice crystals and thus reduce the heat transfer of their cells; both factors minimize microbial cell damage [58].In our research, the freezing process reduced the population of probiotic bacteria cells in all ice cream groups. After freezing, significant cell count reductions were observed by 0.8 log cfu g−1 in CA ice cream, 1.0 log cfu g−1 in CP ice cream and 1.1 log cfu g-1 in CC ice cream with inulin. This means that the survival rate of bacteria when freezing sheep’s milk ice cream mixes depends on the probiotic strain. Lb. acidophilus showed the best survival and the lowest reduction of the population after freezing. However, it should be added that this strain multiplied the least when fermenting the CA mix, which resulted in the lowest number of Lb. acidophilus cells in CA after fermentation. This is essential information for ice cream producers because fermentation should be extended in industrial production or the starter dose of bacteria increased to obtain a more significant number of Lb. acidophilus cells. These treatments are needed to obtain a comparable number of bacterial cells to those obtained in Lb mixtures: L. paracasei and L. casei.Freezing mixtures with the addition of apple fiber also resulted in a reduction of viable bacterial cells from 0.8 log CFU g−1 in CPF and CAF to 1 log cfu g−1 in CCF, compared to the results after fermentation. This means that the reduction in the probiotic population in apple fiber ice cream due to freezing is similar to their inulin counterparts in CCF and CAF ice cream and less only in CPF ice cream. Additionally to the damage caused by probiotic cell freezing, the inclusion of oxygen in the mixture (aeration process) could result in an additional reduction in the number of viable probiotic bacteria cells since the oxygen content, and the redox potential (which is directly proportional to the amount of oxygen) is essential. Most of the probiotic Lactobacillus and Lacticaseibacillus strains are organisms derived from the intestines with microaerophilic or anaerobic metabolism. Therefore, molecular oxygen and high redox potential values are critical factors for these bacteria [53,59,60].After seven days of freezing storage at −22 °C, the viability of the probiotics in all ice cream groups decreased, and the population was reduced by another 0.8–1.2 log cfu g−1 compared to the number of bacterial cells after freezing. The most intense reduction in the log cycle (1.2 log cfu g−1) was found in CC and CCF ice cream with L. casei compared to the number of cells determined after freezing. The highest survival after seven days of ice cream storage was characterized by L. paracasei, where the reduction in the number of bacterial cells was only 0.8 log cfu g−1 in CP ice cream and 0.9 log cfu g−1 in CPF ice cream with apple fiber, compared to the number of cells determined after freezing.In our study, extending the storage time of ice cream from 7 to 21 days did not significantly affect the number of bacterial cells in all ice cream groups. After seven days and 21 days of ice cream storage, the lowest number of bacterial cells was determined in CA and CAF ice cream. After 21 days, the CAF ice cream showed a population reduction of only 0.1 log CFU g−1 compared to the 7th day of storage. In contrast, probiotic ice cream stored at −20 °C for 90 days, tested by Turgut and Cakmakci [61], showed a reduction in the number of cells of Lb. acidophilus with 0.38 log CFU g−1 at this time. Furthermore, in the study by Salem et al. [62], the number of live probiotics in ice cream decreased by 2.23, 1.68, 1.54, 1.23 and 1.77 log CFU g−1, respectively, for Lb. acidophilus, B. bifidum, Lb. reuteri, Lb. gasseri and Lb. rhamnosus within 12 weeks of frozen storage (−26 °C).The highest number of bacterial cells after 21 days of ice cream storage was determined in CP and CPF ice cream (Table 4). L. paracasei was characterized by the best survival rate, which predisposes it to be used to produce probiotic ice cream from sheep’s milk. The conducted 3-factor ANOVA confirms that the survival and the number of probiotic bacteria cells are significantly influenced by all tested factors (type of bacteria, storage time and fiber) and their interactions.After 21 days of storing ice cream, the number of bacterial cells in each group of ice cream exceeded 8 log cfu g−1, which means that the ice cream can be classified as a probiotic food [63,64]. According to Nezhad et al. [65], and at a concentration of 6–7 log cfu g−1, the daily therapeutic dose of probiotics is about 8–9 log cfu g−1 to compensate for losses during the digestive process. In our research, all ice cream groups meet these recommendations for 21 days of storage.3.3. Physical Analysis of Ice CreamDetailed analysis of the influence of the bacteria used for fermentation on the aeration of ice cream (Table 5) shows the highest percentage of overrun in the ice cream with L. paracasei and Lb. acidophilus. L. casei ice cream was characterized by significantly lower overrun on the 7th and 21st days of storage. Although the L. paracasei ice cream had the greatest overrun, it did not cause a significant reduction in the probiotic population during storage.The literature reports that grape, peach and apple pomace is valuable source of pectin, especially pectin with many methoxylated groups, which have favorable gelling properties [66]. This type of pectin also improves the aeration ability, which is also confirmed by our research results.According to Feizi et al. [67], aeration decreases with the increasing addition of chia seed lyophilisate. The authors attribute less aeration of ice cream with a higher chia lyophilisate to the rheological properties of the mixes. Increasing the viscosity of the mixtures reduces the speed of whipping, although a certain level of density is necessary for optimal whipping and air retention. In a similar study, the aeration of ice cream for samples containing basil lyophilisate (0.1 and 0.2% w/w) was 46.5% and 42.5%, respectively [68].According to Soukoulis et al. [69], prebiotics affects the incorporation of air and stabilize the foam by increasing the viscosity of the water phase (increasing the concentration of solute and gelling) surrounding the surface between air cells, raising the physical barrier against destabilization of air cells. Numerous ice cream studies have shown that inulin, oligofructose or resistant starch significantly improve air incorporation (aeration) and related properties such as melt resistance and shape retention [23,70]. The melting rate is influenced by many factors, including the chemical composition, the amount of air introduced, the size of the ice crystals, and the structure of the fatty globules formed during freezing [71].Table 5 shows the first dropping time and total melting rate ice cream tests after 7 and 21 days of storage. It is generally known that the melting rate of ice cream correlates with its aeration [72]. Therefore, the CC ice cream with the lowest overrun had the longest first drop and total melting times. The high melting rate of ice cream with high overrun was probably due to poor air cell stability, air cell size distribution and a network of fat globules formed during freezing. Air, fat globules and ice are the main microstructural components of ice cream and significantly influence the melting or dripping behavior [73,74].It should be added that the time of the first drop after seven days of storage in the freezer differed significantly depending on the type of bacteria used to ferment the mixture and the addition of apple fiber. The ice cream fermented with L. casei had a longer first drop time than that of L. paracasei and Lb. acidophilus. Extending the storage time from 7 to 21 days significantly reduced the first drop time in all ice cream groups by 57 s in CP, 93 s in CPF, 131 s in CC, 28 s in CCF and 68 s in CA 70 s in CAF. Furthermore, in a study by Bahram-Parvar et al. [71], the melting rate of ice cream increased with increasing shelf life, which may be due to an increase in ice crystal size due to ice recrystallization. Guven et al. [75] found that the addition of various combinations of selected hydrocolloids, such as salep, firewood flour, guar gum and sodium alginate, significantly changes the first drop’s time.The conducted research shows that the addition of apple fiber significantly shortens the total melting time. It is most likely related to the properties of apple fiber resulting from a lower water retention capacity than inulin. Inulin is a hygroscopic substance; it reacts with water molecules limiting their free movement and stabilizing the ice mixture, reducing meltability [76]. According to Syed et al. [76], the higher content of water-soluble compounds, i.e., inulin and apple fiber, resulted in the enrichment of the liquid phase. Thus, the freezing point was moderately lowered, and the percentage of water in frozen form also decreased, resulting in a lower melting point.Several researchers have reported conflicting results about the effects of inulin on the melting properties of ice cream. Akbari et al. [26] and Akalin et al. [77] found that low-fat ice cream with different levels of inulin (up to 4%) showed significantly worse melting parameters compared to the control ice cream (no inulin with 10% fat content). In contrast, El-Nagar et al. [78] observed that incorporating inulin into yogurt ice cream mixes resulted in a reduction of the melting time. Akalin and Erisir [79] also indicated that using both inulin and oligofructose in ice cream improves the melting properties of ice cream with a 4% fat content. Akin et al. [52] confirmed that adding inulin delays the melting of ice cream because inulin can act as a stabilizer by binding water molecules.However, the presence of pectin in apple fiber may also contribute to the cryopreservation of ice cream by controlling the mobility of water molecules in the non-frozen aqueous phase due to its thermodynamic incompatibility with the current separation phase proteins [25]. Goh et al. [80] report that protein–protein and protein–polysaccharide at the interface with fat globule/air cell and dispersed components in the serum phase can also influence melting properties [67].In our study, the difference in total melting time was most significant in the L. casei ice cream, where the melting time was 1206 s shorter in the CCF ice cream with apple fiber compared to the CCF ice cream with inulin. On the other hand, CP and CPF ice cream fermented with L. paracasei had a significantly lower total melting time. In these ice cream, the addition of apple fiber reduced the total melting time by 488 s compared to CP ice cream with inulin only. Extending the shelf life from 7 to 21 days significantly reduced the total melting time only in L. casei-fermented ice cream (CC and CCF) and Lb. acidophilus (CA and CAF). However, in ice cream with L. paracasei, the storage time did not significantly affect the total melting time.3.4. Color Parameters of Ice CreamThe results describing the color parameters of ice cream and ice cream mixes are presented in Table 6.The brightest was the CP mixes fermented with L. paracasei, while the slightly darker CC mixes fermented with L. casei and CA with Lb. acidophilus. Partial replacement of inulin with 1.5% of apple fiber resulted in a significant darkening of the color of ice cream mixes. Depending on the type of bacterial mixtures used for fermentation, the addition of apple fiber increased the value of the L* parameter from 13.2 to 16.6.After seven days of freezing storage in CPF and CAF ice cream, the L* brightness of the ice cream increased compared to the blends. The remaining ice cream (CP, CC, CA and CCF) also tended to increase the brightness of the ice cream, but the differences were not significant. This color change in ice cream samples was due to ice crystals and air bubbles inside the ice cream formed during whipping during freezing [81].According to Nozière et al. [82], inulin molecules interact with casein micelles, which together with fat globules are responsible for light scattering and, consequently, for a high L* value. Extending the storage time to 21 days also increased the L* parameter in all ice cream groups. Higher L* values indicate a brighter, more white color of the ice cream.The a* (+ a—redness, − a—green) parameter in CP, CC and CA ice cream mixes and ice cream with inulin only took negative values, while CPF, CCF and CAF mixes and ice cream with apple fiber-positive values. This means that in ice cream mixes and ice cream with inulin only (CP, CC and CA), a greater proportion of green color was observed, while in mixes and ice cream with apple fiber (CPF, CCF and CAF), the red color was dominant. After the 7th day of storage, the proportion of green color in ice cream with inulin (CP, CC and CA) increased compared to the a* parameter determined in ice cream mixes (CP, CC and CA). Moreover, in the ice cream with apple fiber, an intensification of the red color was observed compared to the a* value determined in the mixes (CPF, CCF and CAF).Extending the storage time of ice cream with apple fiber from 7 to 21 days increased the proportion of red color. The analyzed ice cream mixes and ice cream with inulin and apple fiber were characterized by a high ratio of yellow (+ b*). The minor balance of yellow was found in the CA ice cream mix fermented by Lb. acidophilus. On the other hand, the most yellow CP mixes and ice cream fermented by L. paracasei. This effect is confirmed by the studies of Acevedo-Martinez et al. [83], which inform that the type of probiotic bacteria and the 5% addition of fructo-oligosaccharides may significantly impact the color of the analyzed products.A 1.5% addition of apple fiber resulted in the intensification of the yellow color in CPF, CCF and CAF mixtures. Moreover, after 7 and 21 days of freezing storage, the yellow color was found. These results are confirmed by the analysis of variance, which shows that the type of bacteria, storage time and addition of apple fiber and the interactions of these factors significantly influenced the intensity of yellow color. Furthermore, when analyzing the results of the C color saturation and the h° hue of ice cream mixes and ice cream, a significant effect of apple fiber was found.Apple fiber is characterized by a beige color, which is formed during the drying of apple pomace. During drying, the enzyme polyphenol oxidase causes oxidation of polyphenols resulting in browning of the powder. The same effect is obtained from the non-enzymatic browning reaction, i.e., the reaction of sugars with amino acids, occurring at high speed at an elevated temperature and with a water content of about 30% [84,85]. The color components of dried apple fiber, determined by Rząca and Witrowa-Rajchert [30], indicate a large proportion of red and yellow, which explains the role of these colors in forming the color of ice cream in our research.3.5. Organoleptic AnalysisThe results of the organoleptic evaluation of ice cream on days 7 and 21 of freezer storage are presented in Table 7.The most characteristic appearance of milk ice cream was that of ice cream with CA and CP inulin, both after 7 and 21 days of freezing storage. This is probably because the use of apple fiber caused a significant reduction in L* brightness, and the fiber ice cream was darker and redder than inulin only ice cream. The ANOVA, also, confirms that the addition of fiber had a significant effect on the appearance of sheep’s milk ice cream.In the Akalin et al. [77] study, ice cream with apple fiber also scored lower for appearance due to its darker color. Crizel et al. [86], also, observed that the orange peel fiber used lowered the acceptance of ice cream among consumers.Ice cream hardness depends on the aeration and size of the ice crystals. The hardness is the greater the larger the ice crystals, while with the increase in overrun, the hardness decreases [87,88]. Moreover, in our research, the highest hardness was found in the least aerated CC and CCF ice cream. On the other hand, CPF and CAF ice cream, with the highest percentage of overrun, turned out to be the softest.According to Franck [89], the added inulin of ice cream improves its texture due to its ability to bind water molecules and form a gel network of molecules. In our research, ice cream only with inulin (CP, CC and CA) was also characterized by higher hardness than their counterparts with the addition of apple fiber. Additionally, El-Nagar et al. [78] found in yogurt ice cream that the addition of inulin increased the hardness of the ice cream. Moreover, in the studies of Tiwari et al. [90], the milk ice cream with 4% inulin was harder than the control ice cream. Akalin and Erisir [79] and Di Criscio et al. [22] also demonstrated the effect of inulin addition on ice cream hardness. In ice cream tested by Hashemi et al. [91], an increase in hardness was also observed in ice cream where hydrogenated vegetable oil was replaced with inulin.The addition of apple fiber in our study reduced the hardness of ice cream compared to ice cream with inulin only, but these differences were not significant. According to Bahram-Parvar et al. [71], hardness is a parameter that indicates the smoothness and wateriness of ice cream samples [92]. In our research, the smoothness of ice cream was significantly influenced by the addition of apple fiber, which reduced the feeling of smoothness and creaminess during consumption, increasing their sandiness at both times. The significant effect of the addition of apple fiber is confirmed by the ANOVA (Table 3).According to Abdullah et al. [93] and Soukoulis and Tzia [94], the most important parameter determining the acceptance of frozen desserts is their taste. In our research, the sweetest ice cream turned out to be CA ice cream from Lb. acidophilus on both dates. This is most likely due to the low lactic acid content of 0.50–0.51 g L−1 in CA ice cream compared to other ice cream groups. Moreover, the panelists, analyzing the taste of sheep’s milk ice cream (Table 7), found that ice cream with 4% inulin was characterized by a sweeter taste than ice cream with apple fiber. The least sweet were the CAF, CPF and CCF ice cream with 1.5% apple fiber at both storage times. This is probably related to the acids in apple fiber and the low pH and high lactic acid content of the ice cream. The panelists indicated that the addition of apple fiber positively increased the taste and aroma of the mango-passion fruit, but the differences in the notes were not significant.As in the studies by Turgut and Cakmaci [61] also in our studies, fermentation guided by L. paracasei, L. casei, Lb. acidophilus, the addition of inulin and apple fiber did not introduce any foreign taste and smell to the ice cream. Furthermore, according to Salem et al. [62], no foreign taste was found in the milk ice cream with probiotic strains during storage. The conducted ANOVA (Table 3) shows that the storage time and type of bacteria used for fermentation and their interactions do not significantly affect the organoleptic characteristics of probiotic ice cream from sheep’s milk. Only the addition of fiber significantly influenced the appearance and smoothness of the ice cream.4. ConclusionsSymbiotic ice cream with acceptable physicochemical and organoleptic characteristics may be produced from sheep milk of the Olkuska breed. Moreover, partial replacement of inulin with apple fiber contributed to increased overrun in the ice cream and to a shorter run-off time of the first drop and the total melting time. The addition of apple fiber stimulated the growth of L. paracasei and Lb. acidophilus. The best survival rate was found for L. paracasei. Results demonstrated the effects of using apple fiber and various types of bacteria in milk ice cream, mainly probiotics that have not yet been studied and used in sheep’s milk ice cream. Partial replacement of inulin with apple fiber is, also, a way to reduce production costs.

animals : an open access journal from mdpi

10.3390/ani13101648

PMC10215587

INGA FOOD, S.A. initiated a crossbreeding program involving two Iberian pig varieties: Retinto and Entrepelado. The primary objective of this program is to produce an F1 hybrid sow that exhibits enhanced reproductive performance. In a previous investigation, variations in the reproductive performance of sows, specifically litter size, were observed among the reciprocal crosses. These variations indicate the presence of genomic imprinting effects. To assess the influence of genetic origin, we developed a multivariate gametic model to estimate the gametic correlations between paternal and maternal effects. Gametic correlations lower than one could potentially explain the performance differences observed across the reciprocal crosses. Despite having limited data, the study’s findings suggest that the gametic correlation estimate between paternal and maternal effects on litter size is lower in the Entrepelado population compared to the Retinto population.

INGA FOOD, S.A. initiated a crossbreeding program between two Iberian pig varieties, Retinto (R) and Entrepelado (E), with the goal of producing a hybrid sow (F1). Several studies have been conducted to evaluate its productive performance, and these studies have revealed differences in litter size between the two reciprocal crosses, suggesting the presence of genomic imprinting effects. To further investigate these effects, this study introduces a multivariate gametic model designed to estimate gametic correlations between paternal and maternal effects originating from both genetic backgrounds involved in the reciprocal crosses. The dataset consisted of 1258 records (the total number born—TNB and the number born alive—NBA) from 203 crossbred dams for the Entrepelado (sire) × Retinto (dam) cross and 700 records from 125 crossbred dams for the Retinto (sire) × Entrepelado (dam) cross. All animals were genotyped using the GeneSeek® GPP Porcine 70 K HDchip (Illumina Inc., San Diego, CA, USA). The results indicated that the posterior distribution of the gametic correlation between paternal and maternal effects was distinctly different between the two populations. Specifically, in the Retinto population, the gametic correlation showed a positive skew with posterior probabilities of 0.78 for the TNB and 0.80 for the NBA. On the other hand, the Entrepelado population showed a posterior probability of a positive gametic correlation between paternal and maternal effects of approximately 0.50. The differences in the shape of the posterior distribution of the gametic correlations between paternal and maternal effects observed in the two varieties may account for the distinct performance outcomes observed in the reciprocal crosses.

1. IntroductionThe Iberian breed is widely renowned for its ability to produce some of the highest-quality pork [1]. This breed is particularly well-adapted to the “Dehesa” environment in southwestern Spain, which is characterized by a savannah landscape and is composed of grass, cork, and holm oaks with seasonal production. Traditionally, Iberian pig production was dominated by purebred varieties and extensive management practices. However, in recent decades, there has been a shift toward more intensive farming practices that incorporate crossbreeding with Duroc boars to improve growth and efficiency at commercial stages [2].The regulatory norms for Iberian pig production allow crossbreeding, as long as the sow is of purebred Iberian stock. The reproductive performance of the Iberian sows is lower than that of white pig populations [3], which is a major limitation of its use in intensive farms. Therefore, improvement in the reproductive efficiency of Iberian sows is crucial for their economic sustainability. Several studies have identified genetic variability for prolificacy within and between varieties of Iberian pig [4,5]. To take advantage of this variability, the INGA FOOD, S.A. company has developed a crossbreeding scheme between two Iberian varieties (Retinto and Entrepelado) to generate an F1 hybrid sow, which exhibits significant heterosis for litter size [6]. However, this study also found differences in the reproductive performance between the two reciprocal crosses (Entrepelado × Retinto, ER, vs. Retinto × Entrepelado, RE), suggesting that these differences may be attributed to parental imprinting [7] (i.e., the effects from alleles may differ whether they are transmitted by paternal or maternal gametes). In fact, there is increasing evidence of the importance of imprinting in placenta development [8], and certain imprinted genes have been proposed as candidates for pig litter size [9].In recent years, some algorithms have been proposed to develop a genomic analysis of imprinting [10] from the genomic information provided by commercial genotyping devices. However, knowledge of the parental haplotype phase of the SNP markers is required to differentiate the paternal or maternal gametic effects. Some approaches have been developed to reconstruct haplotype phases [11].Phenotypic information from reciprocal crosses offers the opportunity to compare the paternal and maternal effects of each parental population. In the absence of imprinting, the correlation between the paternal and maternal effects from the same population should be one. Imprinting, on the other hand, results in a lower correlation. Accordingly, the goal of this study was to apply the multivariate gametic model developed in a previous study [12] that utilizes genomic information and is capable of estimating the paternal and maternal gametic contributions of Retinto and Entrepelado varieties in the ER and RE crosses, along with their correlations.2. Materials and MethodsPhenotypic and Genomic Data. The phenotypic data used in this study consisted of the total number born, TNB, and the number of piglets born alive, NBA, in 203 ER and 125 RE sows. The ER sows were the offspring of 38 purebred Entrepelado boars and 139 Retinto dams, whereas the RE sows were generated from 38 Retinto boars and 92 Entrepelado dams. A summary of the data is presented in Table 1.Genotyping was performed with the GeneSeek® GPP Porcine 70 K HDchip (Illumina Inc., San Diego, CA, USA) on all ER and RE crossbred sows, as well as on 341 Retinto and 350 Entrepelado purebred individuals. Due to shared purebred ancestors, there was some degree of relationship between a subset of the ER and RE crossbred sows and the purebred individuals, although not all of them were genotyped. The original genotype data consisted of 60,224 autosomal SNPs, which were filtered by excluding SNP markers with a call rate below 0.90 and a minor allele frequency lower than 0.05 in each population. Among these, 4212 were discarded due to a call rate lower than 0.90, 11,234 were found to be monomorphic, and 9876 and 11,516 had a minor allele frequency lower than 0.05 in the Entrepelado and Retinto populations, respectively. Finally, a total of 23,386 SNPs were retained.Haplotype Phasing. AlphaPhase software [11] was used for each chromosome separately, utilizing genotypes of both crossbred and purebred individuals, as well as a pedigree of 1601 individuals. AlphaPhase was executed with a tolerance of 1% of genotype errors and 1% disagreement between genotypes and haplotypes. The number of surrogates and percentage of surrogate disagreement was set to 10. Nine different scenarios were applied with core lengths of 75, 100, and 125 SNPs and tail lengths of 100, 150, and 200 SNPs (see Table 2). The scenarios were evaluated for concordance, and haplotype assignments that coincided in seven or more scenarios were retained for subsequent analysis.Statistical Model. Once the haplotype phases were calculated, data were analyzed with the model proposed by Shiri et al. [12]:yER=XERbER+BERsER+ZERpE+WERmR+eERyRE=XREbRE+BREsRE+ZREpR+WREmE+eREIn this equation, yER and yRE refer to the vectors of phenotypic records (TNB or NBA) for the ER and RE crosses, respectively. The terms bER and bRE correspond to systematic effects, and sER and sRE represent the permanent sow environmental effects. Paternal effects for the Entrepelado (E) and Retinto (R) populations are denoted by pE and pR, respectively. Maternal effects for the Entrepelado (E) and Retinto (R) are represented by mE and mR. Additionally, eER and eRE are the residual effects for the ER and RE crosses, respectively. The systematic effects vectors included the order of parity with five levels (first, second, third, fourth, and fifth or more) and herd–year–season with thirty-four levels. Further,XER,XRE,BER,BRE,ZER,ZRE,WER, and WRE are the corresponding incidence matrices.Following [12], the prior distribution of the permanent sow environmental effects was:sERsRE~N00,I⊗S where:S=σsER200σsRE2 where σsER2 and σsRE2 are the variances of the permanent sow environmental effects for ER and RE, respectively. The prior distributions of the gametic effects for the Entrepelado (E) and Retinto (R) populations are:pEmE~N00,GE⊗VE pRmR~N00,GR⊗VR where:VE=σpE2σpmEσpmEσmE2 and:VR=σpR2σpmRσpmRσmR2 where σpE2, σmE2, and σpmE refer to the variances of the paternal and maternal gametic effects and the covariance between them, respectively, for the Entrepelado population. Similarly, σpR2, σmR2, and σpmR represent the variances of the paternal and maternal gametic effects and the covariance between them, respectively, for the Retinto population. Additionally, GE and GR are the gametic relationship matrices of the Entrepelado or Retinto gametes, respectively, regardless of whether they are transmitted as paternal or maternal gametes. These matrices describe the relationships among the gametes from Entrepelado and Retinto origins, and they are calculated using the algorithm proposed by Nishio and Satoh [10]:GE=MEME′∑iNSNPqEi1−qEi GR=MRMR′∑iNSNPqRi1−qRi where ME and MR are the matrices of the number of genotyped individuals (n) × the number of SNP (NSNP), whose elements MEi,j (or MRi,j take the value qEj (or qRj) or −1−qEj (or −1−qRj), depending on whether the jth allele of the gametes transmitted for the ith individual is A1 or A2 and of Entrepelado (or Retinto) origin. Additionally, qEj and qRj represent the allelic frequencies of the A2 allele in the Entrepelado (E) and Retinto (R) populations, respectively. The prior distributions for the (co) variance components and the systematic effects were assumed to be flat. The analysis was performed using Bayesian inference with the Gibbs sampler [13] and implemented with Gibbsf90 software [14]. The analysis was performed using 10 million iterations after discarding the first million.At each iteration of the Gibbs sampler, the (co) variances components samples were utilized to compute the samples from the marginal posterior distribution of the correlations between the paternal and maternal gametic effects for Entrepelado (rpmE) and Retinto (rpmR):rpmE=σpmEσpE2σjE2 and rpmR=σpmRσpR2σjR23. Results and DiscussionHaplotype Phasing. The results of comparing haplotype phasing using nine combinations of core length and core tail parameters using Alphaphase software are presented in Figure 1.The average degree of similitude was 0.89, and it was consistently above 0.86. Specifically, the predicted haplotype phase was identical across all nine scenarios for only 78.74% of the analyses but had concordance in more than seven scenarios for 92.5% of SNPs. These findings indicated that the output of the phasing algorithm was highly dependent on the specific set of parameters used for its implementation when medium-density SNP chips were used.Calculation of Gametic Matrices. The diagonal values of the gametic matrices for the Entrepelado population ranged from 0.894 to 1.100, while for the Retinto population, they ranged from 0.901 to 1.179. Table 3 shows the distribution of the gametic relationships observed in the off-diagonal elements of the gametic matrices.The calculated gametic matrices yielded results consistent with the familiar relationships of the individuals, as gametic relationships around 0.50 indicated that the individuals shared sire (or dam), while gametic relationships around 0.25 suggested that the sires (or dams) of the individuals were fullsibs.Variance Components. The posterior mean and standard deviation estimate of the variance components are presented in Table 4.Furthermore, Figure 2 shows the posterior distributions of the ratios of gametic variances in the Entrepelado × Retinto (E × R) and Retinto × Entrepelado (R × E) crosses. The posterior mean estimates were similar, ranging between 0.034 for the Retinto maternal gametic effects in the ER cross and 0.043 for the Entrepelado paternal gametic effects in the RE cross.These results indicate that there are no relevant differences in the amount of genetic variance contributed by the paternal and maternal origins in either of the two reciprocal crosses, based on the available information.Gametic Correlations. The posterior distribution of the gametic correlations for the TNB and NBA in the Entrepelado and Retinto populations are presented in Figure 3 and Figure 4, respectively.The posterior distribution of the correlation between gametic effects in Retinto and Entrepelado showed notable differences in shape. Specifically, the posterior distributions of the gametic correlations in the Retinto population exhibited a higher degree of positive asymmetry compared to those in the Entrepelado population. In fact, the posterior probabilities of a positive gametic correlation in the Retinto population were 0.80 and 0.78 for the TNB and NBA, respectively. In contrast, the posterior probabilities of a positive gametic correlation in the Entrepelado population were 0.50 (TNB) and 0.54 (NBA).Although caution is needed in interpreting the results due to the limited amount of phenotypic and genotypic information, the shape of the posterior distribution of gametic correlations suggests a potential role of genomic imprinting. This is because a gametic correlation substantially lower than one indicates that the same combination of alleles in a gamete may produce different effects on offspring depending on whether they are transmitted by paternal or maternal gametes, which is consistent with the theory of genomic imprinting. Genomic imprinting is an epigenetic phenomenon that causes genes to be expressed depending on whether they are inherited from the father or mother [7].Several theories have been postulated to explain the evolutionary origin of genomic imprinting [15], and one of the most popular is the parental investment theory [16]. This theory argues that imprinting is the result of a conflict between the evolutionary success of paternally and maternally derived genes. In mammalian reproduction, the evolutionary success of paternally inherited genes is associated with the increase in fetal growth, while for maternally inherited genes, it is associated with the number of offspring. This theory is reinforced by the discovery of numerous imprinted genes known to regulate aspects of mammalian development [17], including growth, behavior, and placental function [18] and, furthermore, there is increasing evidence of imprinted genes in the pig genome [9,19,20].From a practical perspective, a low or null gametic correlation between paternal and maternal gametes within the same population indicates that a selection program to improve the performance of the crossbreeding individuals needs to be specifically designed, especially in the Entrepelado population. This is because the selection of purebred animals to increase the performance in the Entrepelado × Retinto cross may not have any noticeable consequences in the performance in the Retinto × Entrepelado cross. Furthermore, this result also may explain the differences in performance among the reciprocal crosses observed by Noguera et al. [6], who proposed using the Retinto variety as a boar and the Entrepelado as a sow, providing better performance than the opposite cross.4. ConclusionsThe bivariate model proposed in this study provides estimates of the gametic effects of each founder population as either paternal or maternal, as well as their correlation. In the absence of parental imprinting, a perfect correlation of one would be expected. However, our results detect a significant deviation from this ideal scenario, indicating possible differences in the performance of crossbred individuals depending on the paternal or maternal origin of the gametes. These findings provide evidence of the presence of imprinting effects in Iberian pig populations, which could have implications for the design of future breeding programs.

animals : an open access journal from mdpi

10.3390/ani11092606

PMC8470203

The slow-growing Korat chicken (KR) is economically attractive, as KR meat has a high selling price and has thus been used in Thailand to support smallholder farmers. However, low feed efficiency in KR stockbreeding makes the product less competitive and improving KR feed efficiency is central to increasing KR profitability. Using RNA sequencing, we compared the jejunal transcriptomic profiles of low- and high-feed conversion ratio (FCR) KR chickens, to identify FCR-related transcriptional variation and biological pathways. Gene Ontology and Kyoto Encyclopedia of Gene and Genome analysis revealed that the main pathways involved in KR FCR variation are related to immune response, glutathione metabolism, vitamin transport and metabolism, lipid metabolism, and neuronal and cardiac maturation, development, and growth. This is the first study to investigate, in the jejunum, the molecular genetic mechanisms affecting the FCR of slow-growing chickens. These findings will be useful in line-breeding programs to improve feed efficiency and profitability in slow-growing chicken stockbreeding.

Improving feed efficiency is an important breeding target for the poultry industry; to achieve this, it is necessary to understand the molecular basis of feed efficiency. We compared the jejunal transcriptomes of low- and high-feed conversion ratio (FCR) slow-growing Korat chickens (KRs). Using an original sample of 75 isolated 10-week-old KR males, we took jejunal samples from six individuals in two groups: those with extremely low FCR (n = 3; FCR = 1.93 ± 0.05) and those with extremely high FCR (n = 3; FCR = 3.29 ± 0.06). Jejunal transcriptome profiling via RNA sequencing revealed 56 genes that were differentially expressed (p < 0.01, FC > 2): 31 were upregulated, and 25 were downregulated, in the low-FCR group relative to the high-FCR group. Functional annotation revealed that these differentially expressed genes were enriched in biological processes related to immune response, glutathione metabolism, vitamin transport and metabolism, lipid metabolism, and neuronal and cardiac maturation, development, and growth, suggesting that these are important mechanisms governing jejunal feed conversion. These findings provide an important molecular basis for future breeding strategies to improve slow-growing chicken feed efficiency.

1. IntroductionIn poultry breeding, improving feed efficiency—the efficiency of converting energy and nutrients from feed into tissue—presents an important environmental and economic challenge [1]. Low feed efficiency raises production costs and reduces competitiveness, particularly when combined with unstable feed costs [2], and improving feed efficiency could increase profitability. Feed efficiency is commonly measured in poultry production using the feed conversion rate (FCR, the ratio of feed intake to body weight gain [3,4]), especially in the production of chickens for meat [5,6]. In male slow-growing broilers, selecting for higher FCR can effectively increase feed efficiency and improve the growth rate and market weight, without affecting carcass composition [7]. However, FCR is also highly associated with production traits; some studies show that selecting for lower FCR increases the body weight gain, but it can also increase the feed intake, increase the average daily gain, and reduce the meat quality [7,8]. It is thus difficult to improve FCR via traditional breeding. Therefore, understanding the molecular basis for FCR is necessary to improve poultry production.The slow-growing Korat chicken (KR) is produced from an indigenous breed that retains its ancestral behavioral and phenotypic traits [9]. The KR line—a crossbreed between a male of a Thai indigenous chicken line (Leung Hang Khao) and a female of a broiler line (Suranaree University of Technology)—was established to support smallholder farmers, to ensure food security in communities, and to contribute to preserving indigenous chicken breeds. KR meat has a unique taste—compared to broiler meat it is firmer and chewier, with lower fat and higher collagen content, giving it a higher selling price [10,11]. KR chickens have an average daily weight gain of 19.8–21.0 g/d, and FCR of 2.2–2.3 [12]; they are sent to the market at ca. 1.2–1.7 kg bodyweight, at 10 weeks of age.Digestive efficiency is important in determining feed efficiency, and is highly heritable [13]. The digestive system is essential for the conversion of ingested food into the nutrients required for growth, maintenance, and reproduction [14]. The conversion of energy and nutrients from feed into tissue depends partly on nutrient absorption [15]. The jejunum is an important small intestine section that ensures nutrient absorption [16,17].RNA sequencing (RNA-seq) has been used widely and successfully to examine economically important traits in many cultured species such as carp [18], sheep [19], cows [20], and chickens [21]. Many studies have been conducted on poultry feed efficiency using RNA-seq. However, most have focused on the selection on residual feed intake [21,22,23,24]; few data are available on FCR selection. A prior study in ducks reported that FCR selection is associated with significant gene expression variation [25]. Recent RNA-seq studies have been conducted on native chickens [23,24], dwarf chickens [21], divergent lines [26], and broiler lines [22,27]. These report that selection for a higher feed efficiency induces gene expression variation in the chicken muscle [22,24,27], duodenum [21,22,23], and jejunum [22], for which few data are available. Juanchich et al. [26] examined gene expression in the gizzard and jejunum of broiler chickens in a line divergently selected for their digestive efficiency. They did not find differentially expressed genes (DEGs).To the best of our knowledge, no data have yet been published on the effect of FCR selection on jejunal gene expression in slow-growing chicken. The objective of this study is to examine the effect of FCR selection on the jejunal gene expression in slow-growing chickens. Our ultimate goal is to find candidate genes that can improve FCR. Here, using RNA-seq to compare the jejunal transcriptome profiles of low- and high-FCR KR groups, we examined the biological pathways connecting FCR and feed efficiency. We identified 56 genes related to the FCR that are enriched in biological processes related to immune response; the metabolism of glutathione, vitamins, and lipids; and neuronal and cardiac maturation, development, and growth. These genes are potentially central to governing jejunal feed conversion. These findings will improve understanding of the mechanisms underlying feed efficiency in slow-growing chickens and will improve breeding selection programs aimed at producing slow-growing chickens while minimizing feed costs.2. Materials and Methods2.1. Ethics StatementThe animal handling and maintenance procedures used in the present study were approved by the Ethics Committee on Animal Use of Suranaree University of Technology, Nakhon Ratchasima, Thailand (permit number: U1-02631-2559).2.2. Experimental Animal and Tissue CollectionWe used 75 one-day-old male slow-growing KR chickens. They were raised in individual cages under a 16L:8D light regimen and fed ad libitum three types of commercial feed (CPF Co., Ltd., Nakhon Ratchasima, Thailand): a starter diet (21% protein), a grower diet (19% protein), and a finisher diet (17% protein) at 0–3, 4–6, and 7–10 weeks of age, respectively. An automatic nipple watering system was installed individually in each cage, and water was freely available. The FCR was calculated as previously described [7], using the following equation:(1)FCR=FIBWG where FI represents the total feed intake from 1 week to 10 weeks (g), and BWG is the body weight at 10 weeks of age (g) minus the body weight at 1 week (g).The chickens were then ranked by FCR at 10 weeks of age. Six individuals with extreme FCR values were chosen: three with extremely low FCR (1.83–1.99), and three with extremely high FCR (3.18−3.36). After 8 h of fasting, these six males were stunned using chloroform for knockout and were sacrificed by neck cutting for bleeding. The jejunum was dissected immediately from the carcass, cut into 1 cm segments, snap-frozen in liquid nitrogen, and subsequently stored at −80 °C until further processing. The significance of differences in growth performance between the FCR groups was determined using Student’s t-test. The statistical significance threshold was set at p < 0.05.2.3. RNA ExtractionTotal RNA was extracted from each sample using TRIzol reagent (Thermo Fisher Scientific, Carlsbad, CA, USA). Jejunal tissue from each bird was lysed and homogenized in TRIzol reagent. After centrifugation, the supernatants were transferred to new tubes and incubated with chloroform for 5 min. Samples were then centrifuged for 10 min at 12,000× g (Thermo Fisher Scientific, Langenselbold, Germany). Pellets were precipitated using isopropanol, washed with 75% ethanol, and dried at 25 °C for 5 min. RNA pellets were resuspended in 20 µL of nuclease-free water. The quantity and quality of the extracted RNAs were monitored using an Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA), a NanoDrop (Thermo Fisher Scientific, CA, USA) device, and a 1% agarose gel. One microgram of total RNA with RIN > 7.5 was used for library preparation.2.4. RNA Library Preparation and SequencingRNA-seq library preparation, sequencing, and analysis were carried out by Vishuo Biomedical Ltd. (Bangkok, Thailand), following the procedure of [28]. For RNA-seq preparation, poly (A) mRNA isolation was performed using the NEBNext® Poly(A) mRNA Magnetic Isolation Module (New England BioLabs, Ipswich, MA, USA). mRNA fragmentation and priming were performed using NEBNext® First Strand Synthesis Reaction Buffer and Random Primers (New England BioLabs, Ipswich, MA, USA). First-strand cDNA was synthesized using ProtoScript® II Reverse Transcriptase (New England BioLabs). Second-strand cDNA was synthesized using the Second Strand Synthesis Enzyme Mix (New England BioLabs). Bead-purified double-stranded cDNA was treated with NEBNext End Prep Enzyme Mix (New England BioLabs) to repair both ends and to add a dA-tail in one reaction, followed by T–A ligation to add adaptors to both ends. The size selection of adaptor-ligated DNA was then performed using beads, and fragments of ~420 bp (insert size of ~300 bp) were recovered. Libraries were amplified by polymerase chain reaction (PCR) for 13 cycles. The PCR products were purified using beads, and their quality and quantity were checked using a Qsep100 (BIOptic Inc., Taiwan, China), and a Qubit 3.0 Fluorometer (Invitrogen, Carlsbad, CA, USA). The libraries were sequenced using paired-end configurations with a read length of 2 × 150 bp on an Illumina HiSeq X instrument (Illumina, San Diego, CA, USA). Image analysis and base calling were conducted using the HiSeq Control Software (HCS) + OLB + G A Pipeline-1.6 (Illumina).2.5. Gene Expression and Differential Expression AnalysisTo ensure high-quality data, low-quality reads and those containing adapter contamination were removed using Cutadapt v. 1.9.1 [29]. Read bases with a phred quality score less than 20 (Q20), sequencing adapters, and reads containing poly-N were filtered out to generate clean data. The guanine cytosine (GC) content of the clean reads was then calculated. All downstream analyses were based on high-quality, clean data. The reads were mapped to the chicken genome (GRCg6a, GenBank: GCA_000002315.5) using HISAT2 v. 2.0.1 [30]. Cufflinks v. 2.2.1 [31] was used to assemble the mapped reads. Alternative splicing events were extracted, quantified, and compared using ASprofile v. 1.0 [32]. Gene-level read counts were enumerated using HTSeq v. 0.6.1 [33]. Differential gene expression between high- and low-FCR samples was analyzed using the DESeq2 R package [34]. p-values were adjusted using a Benjamini–Hochberg correction [35] to control the false discovery rate. The criteria for identifying DEGs were FC > 2 (or |log2 FC| > 1), p < 0.01, and a q-value < 0.27.The visualization of the differences and similarities between high- and low-FCR samples was performed using a principal component analysis (PCA) of the transcripts and DEGs, using the ropls R package [36]. A volcano plot (created using EnhancedVolcano in R [37]) was used to visualize the jejunal transcriptome and DEGs. A heatmap (generated using pheatmap in R [38]) was used to illustrate the DEG expression profile for each sample.2.6. RNA-Seq Validation via RT-qPCRTo confirm the differential expression results, eight genes (i.e., LY6E, PLAC8, LOC771880, MLKL, ADV, IFI6, PLA2G4B, and LBFABP) were randomly selected and their expression was measured by quantitative reverse transcription PCR (RT-qPCR). Total RNA was converted into first-strand cDNA using a High Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific, Carlsbad, CA, USA). The primers (Table S1) for each gene were designed using NCBI Primer BLAST (https://www.ncbi.nlm.nih.gov/tools/primer-blast/, accessed on 28 October 2021) and the Ensembl genome browser (https://ensembl.org/, accessed on 28 October 2021). The amplification efficiencies for each primer pair were calculated prior to RT-qPCR validation, and the efficiency of reaction values from 90 to 110% were used for qPCR reactions. RT-qPCR reactions were conducted in triplicate on a LightCycler® 480 Real-Time PCR System (Roche Diagnostics GmbH, Mannheim, Germany) using SYBR® Green Chemistry. The thermocycling program consisted of an initial denaturation step at 95 °C for 10 s, followed by 45 cycles of 95 °C for 30 s, 60 °C for 30 s, and 72 °C for 1 min, with a final extension at 72 °C for 5 min. Relative gene expression was calculated using the 2−ΔΔCt method, using the housekeeping gene GAPDH as an internal control [31]. To validate the RNA-seq results, we conducted linear regression analysis of the log2 FC scores of the RNA-seq and RT-qPCR analyses using a regression analysis tool from the data analysis tool pack of Microsoft® Excel® 2016 (Microsoft Corp., Redmond, WA, USA). The correlation between log2 FC was calculated with the Pearson test using SPSS v. 24.0 for Windows software (SPSS Inc., Chicago, IL, USA). The statistical significance threshold was set at p ≤ 0.01.2.7. GO Term and KEGG Pathway Enrichment AnalysisFunctional annotation and Gene Ontology (GO) term enrichment were performed using the Ensembl and Entrez Gene (NCBI) databases for Gallus gallus, using ViSEAGO [39] in R. The dataset of the jejunum-expressed genes was used as a background for DEG GO term enrichment (Fisher’s exact test, p-value < 0.05). Multi-dimensional scaling (MDS) plots constructed from the semantic similarity distances between enriched GO terms were generated using ViSEAGO [39].Pathway enrichment analysis was based on Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway units. A hypergeometric test was used to identify the DEG pathways that were significantly enriched against the transcriptome background, using the following formula:(2)p=1−∑i=0m−1(Mi)(N−Mn−i)(Nn) where N is the number of genes with pathway annotations; n is the number of DEGs in N; M is the number of genes annotated for a particular pathway in all genes; and m is the number of DEGs annotated for this pathway.A scatter plot was used to graphically represent the KEGG enrichment analysis results. The degree of enrichment was assessed using the Rich factor (the ratio of the number of DEGs in the pathway to the total number of genes in the pathway), q-value < 0.05, and the number of genes enriched in each pathway.3. Results3.1. Feed Efficiency Associated with Low- and High-FCRAt 10 weeks of age, FCR was significantly lower in the low FCR group (1.93 ± 0.05) than in the high-FCR group (3.29 ± 0.06) (n = 3 per group; p < 0.05; Table 1). The low-FCR group had significantly lower feed intake and significantly higher body weight gain than the high-FCR group (p < 0.05).3.2. Genome Mapping StatisticRNA-seq of the six jejunum epithelial samples generated 264.9 million raw reads (Table 2). After filtration, 6.3 Gb on average of high-quality data (Q20 > 95%, Q30 > 90%) was retained for each sample. The filtered data were subsequently aligned with the reference genome. Over 83% of the clean reads per sample were mapped to the Gallus gallus genome assembly. Between 78.15% and 80.49% reads were uniquely mapped, whereas 4.76–7.84% reads were mapped more than once.Mapped reads were assigned to genomic features, exons, introns, and intergenic regions. Most of the sequences (59.09–70.04%) were mapped to exonic regions; 13.34–21.38% to intergenic regions; and 12.12–27.57% to intronic regions (Figure 1). This indicates that our sequences matched the reference genome mainly in coding regions and were therefore acceptable for further analysis.3.3. Differential Expression ProfilingIn total, 24,356 transcripts were identified in the jejunum samples. Based on the PCA (Figure 2), high-FCR samples were closely grouped together, whereas low-FCR samples were scattered, reflecting natural biological variation in gene expression in the low-FCR group. Differential gene expression analysis revealed 56 DEGs (p < 0.01, log2 FC > 1), of which 31 were upregulated and 25 downregulated in the low-FCR group relative to the high-FCR group (Figure 3). Those genes differentially expressed in response to differences in FCR are summarized in Table 3. Among the low-FCR individuals, individual L2 had a distinct pattern of DEG expression (Figure 4).We conducted PCA of the DEGs to examine the high level of natural variation in the low-FCR group. There was a distinct separation between the high- and low-FCR groups (Figure 5), providing evidence that these DEGs are appropriate for separating these groups, in spite of the natural variation in the low-FCR group.To validate these results, eight DEGs (i.e., LY6E, PLAC8, LOC771880, MLKL, ADV, IFI6, PLA2G4B, and LBFABP) were randomly selected for RT-qPCR assays using the same RNA samples used for RNA-seq. The linear regression of RNA-seq and RT-qPCR log2 FC scores (Figure 6) reveals that these selected DEGs showed concordant expression patterns using both methods, with a strong positive association (R2 = 0.9875). Moreover, Pearson’s correlation also showed that this association is significantly correlated (r = 0.960, p < 0.01). Thus, the RT-qPCR analysis validated the RNA-seq results.3.4. Functional Annotation and GO Term Enrichment AnalysisThe significantly enriched Ensembl-derived GO terms are shown in Figure 7. Table S2 shows the associated DEGs, and Table 4 shows the significantly enriched Entrez Gene-derived GO terms. GO term enrichment revealed 22 GO terms in the BP category, 6 in the MF category, and none in the CC category. The Ensembl-derived MDS plots, which arrange the GO terms into several main groups, reveal the most important functions associated with jejunal differences in FCR (Figure 7A). Based on the Ensembl-derived MDS plots and the Entrez Gene-derived GO terms, the main biological processes implicated are: immune response; neuronal and cardiac maturation, development and growth; and glutathione and vitamin metabolism.3.5. Pathway Enrichment AnalysisThe top 30 most significantly enriched KEGG pathways are shown in Figure 8, and Table S3 shows the associated DEGs. The pathways for vitamin digestion and absorption, and the primary immunodeficiency pathways, had the highest Rich factors, indicating their importance in jejunal differences in FCR.Consistent with the GO term analysis, KEGG pathway enrichment analysis revealed that the main biological pathways involved in jejunal differences in FCR were those related to immune response; neuronal and cardiac maturation, development and growth; glutathione and vitamin metabolism; and lipid metabolism.4. DiscussionThe jejunum is the primary site of nutrient absorption [16,17]. The jejunum transcriptome has been studied previously in broiler lines with differences in digestive efficiency [26] and ducks with differences in FCR [25], an indicator of feed conversion efficiency. Our study represents the first analysis of jejunal transcriptomic differences associated with FCR in slow-growing chickens. We investigated DEG–associated functional networks to elucidate feed conversion and feed efficiency in slow-growing chickens.For the KR chickens that we studied, FCR was 1.93 in the low-FCR group and 3.29 in the high-FCR group. In a KR population, the market-age FCR averages 2.2 [12], which is below that of commercial slow-growing meat-type chickens, at 3.22 [40], and Chinese yellow slow-growing chickens, at 3.15 [7,40]. We observed that, for KR chickens, higher body weight can be achieved with reduced feed intake, even at FCR values as low as 1.93. In Chinese slow-growing chickens, selecting for FCR can improve the market weight, even though FCR is negatively correlated with feed intake [7,21]. This suggests that the growth dynamics of KR chickens may be unique among slow-growing chickens.Our functional annotation of DEGs revealed an enrichment of biological processes related mainly to immune response, glutathione metabolism, vitamin transport and metabolism, lipid metabolism, and neuronal and cardiac maturation, development, and growth. This suggests that these might be important mechanisms governing jejunal feed conversion in KR chickens.4.1. Immune ResponseImmune defense is central to intestinal function, because the intestinal epithelium is in contact with the feed and microbiota [41]. Although we did not select for immune-related traits, immune response was the most important enriched pathway, representing various DEGs. Consistent with many research studies on poultry, in which selecting for higher feed efficiency was found to be associated with muscular and intestinal epithelial immune response [22,24,25,27], we found that FCR in KR chickens is associated with differences in jejunal immune response.Within the innate immune system, macrophages are essential for maintaining mucosal homeostasis and epithelial renewal [42]. Jejunal tissue is enriched in genes related to immune defense and immune response via macrophages [26], highlighting the importance of macrophages in the immune system of the chicken jejunum. Two DEGs, SPON2 (Spondin-2) and MMP10 (Matrix metalloproteinase 10, or stromelysin 2), with higher expression in the low-FCR group, are involved in macrophage activation and function. In contrast, in the muscle of high feed efficiency chicken, a decrease in macrophage activation has been predicted [22]. SPON2 encodes a protein that binds directly to bacterial lipopolysaccharide (LPS), functioning as an opsonin for macrophagic phagocytosis of bacteria [43]. In the jejunum of steers, genes related to bacterial LPS were more highly expressed in high feed efficiency (high gain–low intake) individuals than in low feed efficiency (low gain–high intake) individuals, suggesting that high feed efficiency is associated with higher macrophage bactericidal potential [44]. Therefore, higher SPON2 expression in the low-FCR group may indicate a greater ability to eliminate bacterial LPS via the macrophage pathway.MMP10 encodes a critical cell-autonomous mediator controlling macrophage activation [45]. Our results suggest that higher SPON2 and MMP10 expression might ensure the maintenance of a healthy environment in the low-FCR jejunum, with a greater potential to eliminate bacteria and other undesirable particles. Their lower expression in the high-FCR group highlights the importance of this pathway for feed efficiency. Therefore, higher SPON2 and MMP10 expression might be essential for improving feed efficiency.Several of the DEGs are related to T-cell activation (i.e., LAPTM4B, LOC771880, LAG3, and LY6E). T-cells are essential in the adaptive immune response, producing interferons and interleukins to coordinate an appropriate immune response [46]. Intestinal T-cells are critical in control and protection against pathogenic infections [47]. T-cell proliferation is associated with improved growth performance in animals treated with feed supplements and provides a protective immune response [48,49]. LAPTM4B, which encodes a protein that negatively regulates active transforming growth factor beta 1 (TGF-β1) production in human regulatory T-cells (Tregs) [50], was expressed more highly in the low-FCR group. The production of active TGF-β1 is one mechanism whereby human Tregs suppress immune responses [51]. Thus, higher LAPTM4B expression in the low-FCR jejunum may lead to T-cell activation, which would not occur to the same extent in the high-FCR group.LOC771880 (CD8A) and LAG3 were more highly expressed in the high-FCR group. CD8 encodes a cell-surface glycoprotein (antigen) found on most T-cells [52,53]. LAG3 encodes a protein that functions as a T-cell inhibitor [54,55,56]. CD8+ T-cells are precursors of cytotoxic T-cells, which are efficient immune effectors with an important role in eliminating virus-infected cells [57]. CD8+ T-cell proliferation is associated with higher growth performance in pigs with viral infection [48]. The CD8A expression profile in the high-FCR jejunum may thus indicate that its immune system is potentially more sensitive to viral infection. This is supported by our finding that LY6E, which promotes viral entry in human cell lines [58], was more highly expressed in the high-FCR group. Together, our findings reveal that the lower T-cell inhibitor expression in the low-FCR jejunum may promote T-cell activation, which is essential in the adaptive immune response. In contrast, in the muscle of native chickens selected on residual feed intake, genes relative to T-cell activation were downregulated in high feed efficiency chickens [24].Changes in the cell death process may lead to severe disorders, including inflammatory diseases; this crucial process is therefore finely controlled in the intestinal barrier [59]. Previous studies have reported that selection for higher feed efficiency in chickens involves variation in the expression of muscular genes related to cell death [24,27]. Apoptosis and necroptosis are programmed forms of cell death involved in intestinal barrier homeostasis and renewal [59]. Two of the DEGs were related to apoptosis and necroptosis pathways. PLAC8, which was more highly expressed in the high-FCR group, was first identified via a microarray analysis of the murine placenta [60]. Its function in apoptosis has also been demonstrated [61]. Huang et al. [62] reported that butyrate, a short-chain fatty acid produced by gut microbes, reduces PLAC8 expression and induces apoptosis in the human colon. A previous study in commercial broiler chicken showed that muscular genes related to cell death and survival were upregulated in a high feed efficiency group [27]. In contrast, a recent study in native chickens found an upregulation of muscular genes related to the apoptosis pathway in a low feed efficiency group [24]. We propose here that the higher expression of PLAC8 in the high-FCR jejunum may reduce apoptosis, thus compromising jejunal barrier homeostasis and renewal.In contrast to the PLAC8 expression profile, MLKL was more highly expressed in the low-FCR group. MLKL mediates TNF-induced necroptosis [63,64,65,66], and the RIPK3–MLKL pathway is suggested to be important for activating necroptosis in digestive organs [67]. The higher MLKL expression in the low-FCR jejunum may promote necroptosis, ensuring jejunum barrier homeostasis and renewal, in contrast to the effect of PLAC8 in the high-FCR jejunum. PLAC8 and MLKL expression thus appears to be important for regulating homeostasis and renewal of the KR jejunal epithelium, ensuring epithelial integrity, and thereby ensuring a suitable environment for feed absorption.4.2. Glutathione MetabolismHigher oxidative stress is associated with low feed efficiency in broilers [68], steers [44], and cattle [69]. The antioxidant system maintains a diverse microbiota in the luminal epithelia of the gastrointestinal tract [70]. Production of reactive oxygen species and reactive nitrogen species by gastrointestinal epithelial cells or enteric commensal bacteria causes intestinal inflammation and impairs absorption [70]. Our findings revealed two DEGs (MMACHC and CHAC1) associated with glutathione metabolism; they are closely involved, respectively, in vitamin metabolism and neuronal function. MMACHC, more highly expressed in the low-FCR group, encodes a protein that functions closely with vitamin B12 and its derivatives, using glutathione to generate cob(I)alamin (a vitamin B12 derivative) and glutathione thioether [71]. Given that the formation of a thioether derivative in the glutathione pathway serves to detoxify xenobiotic compounds [72], our findings suggest that the low-FCR jejunum may have greater cellular detoxification potential than the high-FCR jejunum. MMACHC functions as a glutathione S-transferase. A previous study in a native chicken showed an upregulation of several glutathione S-transferase in the muscle of high feed efficiency native chickens, suggesting that responses to oxidative stress in high feed efficiency chickens is elevated [27]. In agreement with this, the findings of two previous studies suggest that chickens with high residual feed intake (low feed efficiency) are more susceptible to oxidative stress, since an upregulation of several muscular and duodenal genes responsible for ROS production in low efficiency animals was found [21,24].CHAC1, also more highly expressed in the low-FCR group, encodes a protein that catalyzes glutathione cleavage into 5-oxo-l-proline and a Cys-Gly dipeptide, functioning as a glutathione-degrading enzyme [73,74]. Higher CHAC1 expression may thus sensitize low-FCR jejunal cells to oxidative injury; nevertheless, other pathways may balance redox homeostasis. Moreover, glutathione degradation by CHAC1 may represent an important novel pathway in neuronal development and pathogenesis [75].4.3. Vitamin Transport and MetabolismVitamin binding and metabolism pathways were enriched in the jejunal DEGs. In the beef steer jejunum, vitamin binding-related genes were significantly enriched in a high-feed efficiency group [44], indicating that this biological pathway related to vitamin binding may be important for feed efficiency. AVD, which was more highly expressed in the high-FCR group, encodes a protein that binds to biotin (water-soluble vitamin B8). AVD is localized in chicken intestinal goblet cells; given that its expression increases in response to bacterial LPS stimulation, it may serve as an antibacterial mucus layer in the intestinal epithelium [76]. Variation in the composition of the jejunal mucus layer may be highly important, considering that mucus layer properties are important for the absorptive function of the small intestine [77]. However, considering that the gene expression profiles of the high-FCR individuals might reduce their immune response, the higher AVD expression may ensure a minimal defense system to protect the epithelium. AVD and SPON2 (higher expressed in the low-FCR group) are related to the bacterial LPS degradation pathway [43,76]. Therefore, considering their contrasting expression profiles, we propose that the differences in FCR in KR chickens may be associated with different pathways responding to bacterial LPS stimulation.MMACHC, related to glutathione metabolism and more highly expressed in the low-FCR group, encodes a cytosolic chaperone responsible for the processing and intracellular trafficking of cobalamin (water-soluble vitamin B12) by participating in the conversion of vitamin B12 into adenosylcobalamin (AdoCbl) and methylcobalamin (MeCbl) [71]. AdoCbl is a cofactor of mitochondrial methylmalonyl CoA mutase, which breaks down certain protein building blocks (amino acids), fats (lipids), and cholesterol [78]. MeCbl is a cofactor of cytosolic methionine synthase, which converts homocysteine into methionine, which is used to produce proteins and other important compounds [78]. The higher MMACHC expression in the low-FCR jejunum may reflect a higher potential for vitamin B12 metabolism into AdoCbl and MeCbl, essential components for protein breakdown and protein synthesis, respectively. This potentially ensures jejunal epithelial renewal and the absorption and metabolism of protein from feed.Vitamin B12 interacts with superoxide at rates similar to those of superoxide dismutase; therefore, it may protect against chronic inflammation and modulate redox homeostasis [79]. The higher MMACHC expression in the low-FCR jejunum may thus represent a greater potential to modulate redox homeostasis by eliminating reactive oxygen species, thereby protecting the epithelium from inflammation. MMACHC is therefore an important candidate for improving the feed efficiency of slow-growing KRs.ADH1L (LOC10087280), highly expressed in the low-FCR group, participates in the metabolism of retinol (fat-soluble vitamin A) into retinoic acid, an important component that regulates gene transcription at specific DNA sites known as retinoic acid response elements (RAREs) [80]. In the murine liver, ADH1 may minimize toxicity by rapidly metabolizing retinol into retinoic acid, thus reducing retinol utilization by P450s [81]. Retinol may induce oxidative stress and modulate antioxidant enzyme activity [82], and it plays a critical role in enhancing immune function [83]. Therefore, higher ADH1L expression in the low-FCR jejunum indicates that retinol metabolism may be central to improving slow-growing KR feed efficiency.4.4. Lipid MetabolismOur functional analysis revealed the enrichment of pathways related to lipid metabolism. Processes related to lipids and fatty acid B-oxidation are highly enriched in the chicken jejunum [26], indicating the importance of this pathway with regard specifically to jejunal absorption. Many studies have shown that intestinal fatty acid degradation- and synthesis-, and fat transport-, metabolism-, and absorption-related genes are associated with feed efficiency in poultry [22,23,25]. PLAC8, a negative regulator of apoptosis and more highly expressed in the high-FCR group, also encodes a critical upstream protein that regulates brown fat differentiation and body weight, and controls thermoregulation [84]. Brown adipocytes oxidize fatty acids to produce heat in response to cold or excessive energy intake [84]. In mice, genetic inactivation of PLAC8 is associated with cold intolerance, late-onset obesity, abnormal morphology, and impaired brown adipocyte function [84]. Higher PLAC8 expression in the high-FCR jejunum may therefore indicate a higher potential for brown fat differentiation to promote body weight control and thermoregulation. This may reflect higher energy expenditure in the high-FCR than the low-FCR jejunum. The lower PLAC8 expression in the low-FCR group suggests that those individuals may be less able to adapt to changes in environmental temperature or feed intake, because of their reduced capacity for thermoregulation or body weight control.LBFABP, which was more highly expressed in the low-FCR group, is predominantly expressed in the chicken’s digestive tract [85], and encodes a protein belonging to the fatty acid binding protein family. Fatty acid binding proteins are abundant cytosolic lipid-binding proteins expressed in a tissue-specific pattern [86,87]. Their expression may facilitate intracellular fatty acid trafficking from uptake to storage or oxidation, or from lipid droplets for secretion [86,87,88]. In accordance with our results, a previous research study in duck showed that several fatty acid binding proteins were upregulated in the jejunum of a low-FCR group [25], revealing the importance of the peroxisome proliferator-activated receptor (PPAR) pathway in poultry feed efficiency [23,25]. The higher LBFABP expression in the low-FCR group is also consistent with the findings of Prakash et al. [89], who demonstrated that LBFABP is upregulated in high-feed efficiency broiler duodenums. It has been suggested that LBFABP plays a specific role in the liver in response to food intake, since its expression is higher in high-growth than in low-growth chickens [85]. The higher LBFABP expression in the low-FCR group suggests greater intracellular trafficking of lipids and consequently an increased capacity for lipid utilization (including storage and oxidation). We therefore suggest that LBFABP may be essential in controlling feed efficiency in slow-growing KR chickens.PLA2G4B, which was more highly expressed in the low-FCR jejunum encodes phospholipase A2 (in the cytosolic phospholipase A2 protein family), which hydrolyzes the sn-2 bond of phospholipids and releases lysophospholipids and fatty acids. Consistent with our results, a prior study in chickens (selected for residual feed intake) showed that PLA2G4A may be associated with feed efficiency, since it was upregulated in high feed efficiency duodenum [21]. Phospholipase A2 enzymes participate in membrane homeostasis by altering phospholipid composition. They also participate in energy production by supplying fatty acids for β-oxidation, in barrier-lipid generation, and in balancing saturated and unsaturated fatty acids [90]. They may also play an important role in metabolic disorders such as obesity, diabetes, hyperlipidemia, and fatty liver disease [91].PLA2G4B varies in its tissue expression pattern, regulatory mechanisms, and functions. Its role in regulating metabolism remains to be clarified [91]. However, it has been implicated in age-related changes in phospholipids and in reduced energy metabolism in monocytes [92]. Moreover, the phospholipase A2 family is responsible for arachidonic acid liberation from cellular membranes. Subsequent arachidonic acid metabolism leads to the production of prostaglandins and leukotrienes—key mediators of the gut inflammatory response [90]. PLA2G4B might be important in improving KR feed efficiency and warrants further study.4.5. Maturation, Development and GrowthSeveral of the identified pathways and biological processes in our study were related to neuronal and cardiac maturation, development and growth. This finding was also reported by Xiao et al. [23] and Zhou et al. [27] in chicken duodenum and muscle, respectively. In particular, C1QL1, CDK5R1, MYOC, and CHAC1, associated with neuronal function and development [93,94,95], were more highly expressed in the low-FCR group. The digestive system possesses a local nervous system (the enteric nervous system, ENS) [96,97,98], which regulates major enteric processes such as immune response, nutrient detection, microvascular circulation, intestinal barrier function, and the epithelial secretion of fluids, ions, and bioactive peptides [99]. It is therefore not surprising that jejunal genes involved in neuronal development were differentially expressed in our study in response to differences in FCR. To the best of our knowledge, the role of the ENS in feeding efficiency has not previously been examined.C1QL1 encodes a secreted protein proposed to regulate the number of excitatory synapses formed on hippocampal neurons [93]. CDK5R1 encodes a neuron-specific activator of CDK5 (cyclin-dependent kinase 5); CDK5R1/CDK5 has been suggested to play a critical role in neurite outgrowth and cortical lamination [94]. MYOC encodes a secreted glycoprotein that regulates the activation of various signaling pathways in adjacent cells, thereby controlling numerous processes such as cell adhesion, cell–matrix adhesion, cytoskeleton organization, and cell migration. Among its many roles, MYOC mediates myelination in the peripheral nervous system via ERBB2/ERBB3 signaling and participates in neurite outgrowth [95]. Interestingly, recent observations of Xiao et al. [23] suggest that the upregulation of several myosins (e.g., MYO1D, MYO1E, MYO1A) in duodenum from high feed efficiency chickens may be related to intestine digestion and absorption function. Moreover, Zhou et al. [27] reported an upregulation of the growth-related genes myogenin (MYOG) and myoferlin (MYOF) in high feed efficiency chicken muscle. Higher C1QL1, CK5K5R1, and MYOC expression in the low-FCR jejunum may function in ENS neuronal development, supporting the specific absorptive function of the jejunum and thus improving feed efficiency.CHAC1 is responsible for the cleavage of glutathione into 5-oxo-l-proline, also called l-pyroglutamic acid (PGA), an endogenous molecule formed by glutamate cyclization [72]. PGA has been studied in metabolic disease with glutathione synthetase deficiency [100] and in neurodegenerative disease [101]. Various neurotoxic actions have been attributed to PGA [100,101,102,103]. PGA binds to glutamate receptors [104] and inhibits glutamate uptake by synaptosomes [105]. Chronic glutamate-uptake inhibition can lead to slow neurotoxicity [106]. Considering these reported negative effects, we are concerned about the endogenous use of PGA by 5-oxoprolinase. This enzyme, which catalyzes the conversion of PGA into l-glutamate, has not yet been referenced in GRCg6a (GenBank: GCA_000002315.5); we were therefore unable to identify it in the KR jejunum. However, given that it has been detected in the digestive tract of other species including humans [107], we hypothesize that it occurs in the KR jejunum, and may participate in transforming the abundant PGA in glutamate. In pigs, glutamate is central in supporting maximum growth, development, and production performance [108]. Differences in CHAC1 expression in KR chickens might therefore affect ENS functions via the glutamate pathway, thereby crucially affecting feed efficiency.CHAC1 negatively regulates the Notch signaling pathway in embryonic neurogenesis [109]. We found that MESP1, which was more highly expressed in the low-FCR jejunum, was related to aspects of embryonic development such as cardiac conduction system development. MESP1 participates in the embryonic development of the murine heart, somites, and gut [110]. In muscle from pigs with differences in residual feed intake, functional analysis of DEGs comparing high and low feed efficiency groups revealed the enrichment of various biological processes related to growth, including cardiovascular system development and function [1]. In summary, this highlights the importance of genes related to embryonic development, such as MESP1 or CHAC1, in improving feed efficiency. In low-FCR KR chickens, high MESP1 and CHAC1 expression might be critical in establishing jejunum structure and function, both during and after embryonic development. The precise roles of MESP1 and CHAC1 in KR jejunum function and development remain to be elucidated.Several limitations of this study need to be acknowledged. Despite no evidence of area-specific gene expression in the jejunum, RNA has been isolated from the entire section of the jejunum. Therefore, it is not possible to determine which area of the jejunum is associated with significant gene expression variation. The major limitation of this study is its small sample size, which made it unsuitable for statistically powerful analysis. Moreover, our study reveals biological variation in one experimental group that can also correspond to a possible batch effect, although samples have been analyzed in the same conditions with quality control steps. A surrogate variable analysis may be a useful method to remove hidden variations should any exist. Thus, these aspects limit the power of the experiment to detect DEGs. Moreover, searching for differences in DEGs between animals that share the same genetic background can represent a limitation. Our list of DEGs was obtained considering a p-value < 0.01 and a q-value < 0.27, which represent a risk (27%) of a false positive. It has been previously accepted that q-value can be higher than 0.05; recent studies using RNA-seq have fixed the p-value at <0.05 or <0.01, while q-value was cut off at 0.1 [111] or higher than 0.1 [1,22,112,113] to increase DEG detection. One reason why we might optimize DEG detection is that a shortlist of DEGs reduces the significance of the enrichment analysis, limiting the conclusions that can be drawn from the results.Although the findings should be interpreted with caution, this study has several strengths. It is the first in-depth analysis of jejunal gene expression associated with feed efficiency in slow-growing chicken. Our RT-qPCR results confirmed the differential expression—as shown by RNA-seq—of several of the DEGs examined. Among the genes confirmed, several had a q-value between 0.05 and 0.26. Our list of enriched GO-terms and pathways were consistent with previous literature in poultry. Our study represents the first molecular portrait of the jejunal genes influencing feed efficiency in slow-growing chicken.It will be helpful for optimizing the method of detection of genes associated with feed efficiency. Future studies should perform the experiments described here with an increased sample size and should integrate different critical parameters (gene-specific mean counts and dispersion) revealed by the present study. Another possible area of future research would be to investigate and confirm the candidate genes highlighted in our study. This study opens up prospects for more in-depth investigations.5. ConclusionsThis is the first RNA-seq analysis of differential gene expression in the jejunal tissues of high- and low-FCR slow-growing chickens. The DEGs expressed more highly in the low-FCR group are associated mainly with the regulation of immune response activation, and specifically, of macrophages, T-cell, apoptosis, and necroptosis pathways; some are associated with the regulation of glutathione metabolism, the transport and processing of vitamins A, B8, and B12, and the metabolism of lipids, including the beta-oxidation of fatty acids. Others are associated with neuronal development and maturation. However, genes that promote FCR in KR chickens may negatively affect body weight control and thermoregulation, reducing their potential to adapt to changes in feed intake or environmental temperature. It would be interesting to assess the effects of FCR selection on body weight control and thermoregulation. Notwithstanding the relatively limited sample size, these findings provide an important molecular basis for future breeding strategies to improve slow-growing chicken feed efficiency.

animals : an open access journal from mdpi

10.3390/ani11051353

PMC8151493

Over the last decades, the dairy industry has primarily been focused on constantly increasing milk yields per cow. Consequently, the incidence of metabolic disorders is rising. In this study, we investigate the effect of hyperketonemia on the longitudinal progression of four metabolic biomarkers in dairy cows and possible predictive biomarkers for hyperketonemia. Our findings provide new insights into the metabolic challenges of dairy cows, and we propose novel approaches enabling an early onset diagnosis of hyperketonemia.

Currently about 30% to 50% of all dairy cows are affected by a metabolic or infectious disease during the transition period. A key factor for preventive actions is the ability to precisely predict metabolic diseases at an early stage. We report the longitudinal metabolic profile of non-esterified fatty acids, beta-hydroxybutyrate (BHB), total bilirubin, and aspartate aminotransferase in hyperketonemic dairy cows. Aiming for a novel measurement regime to improve metabolic health in dairy cows, we evaluated prognostic classifiers for hyperketonemia. In the observational longitudinal study, 99 healthy adult primiparous and multiparous Simmental dairy cows were included. Every cow was monitored weekly for 14 consecutive weeks, beginning two weeks prior to the expected day of parturition until peak lactation. Cows with serum concentrations of BHB > 0.8 mmol/L were considered hyperketonemic. Biomarker profiles were fitted by the maximum likelihood method using a mixed effects natural cubic spline model. In the hyperketonemic group, the BHB profile remained significantly higher than that of the control group until the end of the study period. As a prognostic classifier, the cut-off level of 0.54 mmol/L BHB measured on the 10th day post partum had the highest area under the curve. These results provide new longitudinal insights into the metabolic biomarker progression of dairy cows and enable an early onset diagnosis of hyperketonemia.

1. IntroductionIn recent decades, the dairy industry has been focused on constantly increasing milk yields. The high average milk yield per cow was mostly achieved by genetic selection in combination with optimized farm management and feeding strategies [1]. Moreover, Rauw et al. [2] claim that production traits such as milk yields are twice as heritable as metabolic traits (e.g., feed intake). High average milk yields and high metabolic rates contribute to an extended and more intense negative energy balance (NEB) around calving. NEB has been known to increase the risk for several metabolic diseases and infections [3,4,5,6]. LeBlanc [7] claims that 30% to 50% of dairy cows are affected by some form of metabolic or infectious disease during the transition period from dry-off to early lactation. One of the most important metabolic disorders in dairy cows is ketosis. Ketosis is defined by an elevated concentration of ketone bodies in blood. Ketone bodies are produced in the mitochondria of hepatocytes and are a part of the normal adaptive metabolic response. The three ketone bodies are beta-hydroxybutyrate (BHB), acetoacetate, and acetone. BHB is the most stable form in blood and is commonly used for diagnostics. Different cut-off levels for BHB have been published. Fürll [8] recommended a cut-off level for BHB of 0.62 mmol/L indicating hyperketonemia. BHB concentrations above 1.2 mmol/L have been defined as subclinical ketosis (SCK) [9]. SCK is associated with an increased risk of numerous diseases and infections such as displaced abomasum (DA), uterine infection, and mastitis [10,11,12]. Further, SCK is suspected to impair fertility and reduce milk yield [13,14]. Van Saun and Sniffen [15] estimate the median incidence risk of SCK at 53%, compiled over several studies. McArt, Nydam, and Overton [16] estimate the average total cost of one SCK case at USD 375 and USD 256 for primiparous and multiparous cows, respectively. BHB concentrations above 3 mmol/L are classified as clinical ketosis [17]. For clinical ketosis Kelton et al. [18] reported a 4.8% median incidence risk compiled over several studies. To reflect the effect of ketosis on energy metabolism, additional biomarkers need to be considered. The recommended biomarker panel for fat cow syndrome (FCS) or hepatic lipolysis consists of non-esterified fatty acids (NEFA), BHB, total bilirubin (tBIL), and aspartate aminotransferase (AST) [19]. FCS similar to ketosis develops due to an imbalance in the energy metabolism [13]. During FCS, the hepatic uptake of metabolized lipids exceeds their oxidation and lipid exportation from the liver. Consequently, excess lipids are accumulated as triacylglycerol in the liver, compromising its metabolic performance. NEFA is a metabolite during lipolysis and thereby an indicator of fat mobilization and NEB [20]. BHB as discussed above is a ketone body and a marker for energy metabolism. tBIL is a product of hemoglobin catabolism and biliary metabolized. An elevated concentration is related to a decreased bile flow and impaired liver function and is a marker for hepatocyte dysfunction and damage [21]. Furthermore, tBIL is positively correlated to NEFA due to their transport concurrence. AST activity serves as a marker for cell integrity. Increased serum activity is caused by damaged tissue and hepatic lesions [8,19,22].The aim of our study was to investigate and compare the long-term metabolic biomarker profile of cows with hyperketonemia and non-ketotic cows. Further, we wanted to identify novel prognostic classifiers for hyperketonemia and derive a cut-off level to avoid hyperketonemia. Our main hypothesis was that a persisting change in the biomarker course already occurs in mild forms of hyperketonemia. Further, we emphasize that the definition of cut-off points should rather be based on maintaining and nurturing health than on production yield and impairment of health. The ability to predict the risk and classify metabolic diseases in dairy cows early on is a key factor in taking effective preventive actions.2. Materials and Methods2.1. Animals, Study Design, Housing and DietsA longitudinal observational study design was chosen. The study was performed at the teaching and research dairy farm of the Vetmeduni Vienna (VetFarm Kremesberg, Pottenstein, Austria) between April 2016 and December 2017. In total, 104 healthy adult primiparous (number of samples (n) = 27) and multiparous (n = 74) Simmental dairy cows were enrolled in this study. During the study period, 2 cows died due to birth complications, 1 cow was euthanized due to toxic mastitis, 7 cows were sold during the study trial, and 1 cow was excluded due to repeated dangerous behavior during blood sampling. Cows with more than 10 observations were considered for the final data set (1256 observations from 99 cows). Lactating cows were fed a total mixed ration consisting of 19.23% hay, 16.67% grass-silage, 44.23% corn silage, 11.41% grain mix, and 8.46% protein supplement (RINDASTAR 39 XP; H. Wilhelm Schaumann GmbH & Co KG, Brunn am Gebirge, Austria) based on dry matter basis. Dry cows were fed with a dry-cow total mixed ration consisting of 24.25% hay, 9.33% grass silage, 46.08% corn silage, 12.13% barley straw, and 8.21% protein supplement (RINDASTAR 39 XP; H. Wilhelm Schaumann GmbH & Co KG, Brunn am Gebirge, Austria) based on dry matter basis. The mixed ration had a net energy lactation of 6.7 MJ/kg and a metabolizable energy of 10.4 MJ/kg. The composition of the grain mixture varied to a minor degree throughout the seasons. Feed was mixed and delivered by an automatic mixing and feeding system (Triomatic T15, Trioliet Feeding Technology, Oldenzaal, The Netherlands). Fresh feed was offered to lactating cows ad libitum eleven times per day (between 4:50 a.m. and 9:30 p.m.). Dry cows were offered fresh feed ad libitum twice per day (7:15 a.m. and 4.00 p.m.). All cows were housed in a free stall barn with straw bedding and had ad libitum access to water and mineral stones (Raiffeisenverband Salzburg reg. Gen.m.b.H, Salzburg, Austria) throughout the study. Twice a day (6:30 a.m. and 4:30 p.m.), the lactating cows were milked in a 4 × 4 tandem milking parlor (DeLaval GmbH Eugendorf, Austria). 2.2. Data CollectionEvery cow was monitored for 14 consecutive weeks. Sampling was performed once per week after morning milking approximately 2 to 3 h after first feeding. The first sample was taken 2 weeks prior to the expected day of birth.2.2.1. Blood SamplingThe blood was collected from the jugular vein by vacuum tube system (Vacuette®, Greiner Bio-one International, Kremsmünster, Austria) using 10 mL serum vacutainer tubes with coagulant (Vacuette® Z Serum Clot Activator, Greiner Bio-one International, Kremsmünster, Austria). The blood samples were kept at room temperature for 2 h to allow for clotting. The serum was separated by centrifugation at 3000× g for 15 min. Samples were stored at −80 °C until analysis, which was performed within a maximum of 8 weeks.2.2.2. Herd MonitoringHerd monitoring data was provided by VetFarm Kremesberg. Routine point of care BHB measurements were performed 7 days p.p., wherein coccygeal venous blood samples were analyzed by an electronic hand-held device (Freestyle Precision, Abbott Ges.m.b.H., Vienna, Austria). BHB concentrations above 1 mmol/L were considered as positive ketosis samples, and affected cows were treated with monopropylene glycol (PG) and glycerin (PropyLac®, Garant Tiernahrung Ges.m.b.H, Pöchlarn, Austria) as food supplement for a week. Treatment was started after the sample for the study was withdrawn. In total, 16 out of 1256 samples were drawn after a weekly treatment.2.3. Serum AnalysisIn all serum samples, tBIL, BHB, NEFA, and AST activity were analyzed with an autoanalyzer for clinical chemistry (Cobas 6000/c501; Roche Diagnostics GmbH, Vienna, Austria) using standardized colorimetric enzymatic assays. NEFA was analyzed with the ASC-ACOD method (Wako Chemicals, Richmond, VA, USA, inter-day coefficient of variability (CV) < 0.75%, inter-day CV at 0.55 mmol/L = 0.75%, inter-day CV at 1.08 mmol/L = 4.91%). BHB was determined using the Ranbut method (Randox Laboratories Ltd., London, UK, inter-day CV = 0.57%, intra-day CV = 0.99%). tBIL was measured with the Bilirubin Total DPD Gen.2 Kit (Roche Diagnostics GmbH, Vienna, Austria, inter-day CV = 1.6%, intra-day CV = 2.6%). AST activity was analyzed by kinetic measurement of the enzyme activity with pyridoxal phosphate activation recommended by the International Federation of Clinical Chemistry (Roche Diagnostics GmbH, Vienna, Austria, inter-day CV = 0.6%, intra-day CV = 0.8%). All measurements were performed at the Clinical Pathology Platform of the Vetmeduni Vienna.Samples with BHB concentrations > 0.8 mmol/l were classified as hyperketonemic. The cut-off was set in between the tolerance cut-off for individuals (0.62 mmol/L) recommended by Fürll et al. [23] and the cut-off used by the herd monitoring team (1.00 mmol/L). Further, Fürll [8] associates a BHB level > 0.85 mmol/L on the 3rd day p.p. in combination with other parameters as an indicator for increased fat mobilization and FCS. Cows with samples of BHB concentrations ≤ 0.8 mmol/L were selected as control group. SCK was defined as a BHB concentration > 1.2 mmol/L [24,25] and cows with samples of BHB concentrations ≤ 1.2 mmol/L were considered as control group. 2.4. Statistical AnalysisAll data were analyzed with R (Version 3.0.4). A first distribution of hyperketonemia and SCK over the study period was determined by an empirical cumulative distribution function. Overall hyperketonemia and SCK incidence were calculated by dividing the number of cows with hyperketonemia or SCK by the total number of cows tested. Further, the time span in days between last sampling ante partum (a.p.) to partum was calculated for each individual. The maximum calculated time span marks the smallest possible time interval for further classification analysis (e.g., every other day or weekly measurements). Using the maximum time span in classification models assures that at least one measurement point of each tested cow lies within the chosen time interval. The normality of all biomarkers (response variables) was tested with the Shapiro–Wilk test, with all variables requiring Log-transformation. A mixed effects natural cubic spline model was chosen to fit the course of hyperketonemia, SCK, and control cases over lactation stages. The model was fit to the data between 27 days a.p. to 80 days p.p. A family of splines with a degree of 8 (n = 8) was defined, and the optimal number of splines for the final fit was chosen based on the Akaike information criterion (AIC). The resulting spline function was smoothly joined at a fixed number of knots. The number of knots was defined by K = n − 1 (K = 7). The positions of the knots (20 days a.p., 7 days a.p., day of parturition, 7 days p.p., 14 days p.p., 25 days p.p. and 50 days p.p.) were chosen at time points, where structural changes were expected, while maintaining a uniform distribution of all knots throughout the fitted time span. Data obtained from the same cow were considered as repeated measurements. The final model was built in 3 stages. In the first stage a mixed effects model with solely a random intercept was fitted by maximum likelihood. In the second stage, hyperketonemia was added as a fixed effect to the spline coefficient to look for an overall effect of hyperketonemia. Finally, in the third stage hyperketonemia was additionally included as a random effect to the spline coefficient to evaluate a possible interaction of hyperketonemia and individuals. As described by Durrleman and Simon [26], spline functions are linear in the regression coefficients. Hence, the significance of the introduced covariates was evaluated by comparing all 3 models using a one-way ANOVA. To test if the biomarker concentration between hyperketonemic, SCK, and control cows differed significantly during the study period, one-sample t-tests were performed. The study period was subdivided into intervals of 6 days, starting at 15 days a.p. until 62 days p.p. A t-test was performed for all time intervals, t0, and t1 for each biomarker. The t-test was performed as a descriptive analysis; therefore, we did not correct for multiple measurements. As potential classifiers for hyperketonemia we considered the following features: (I) the median biomarker concentration a.p. (baseline), (II) the maximum biomarker value a.p., (III) biomarker concentration at partum (t0), (IV) daily biomarker concentration on the first day until the 10th day p.p. (t1), (V) biomarker increase from baseline to t1, (VI) biomarker increase from partum to t1, and (VII) biomarker concentration within different lactation. If measurements were not conducted on the exact time points, corresponding biomarker concentrations were linear interpolated using their nearest neighbor. All classifiers were tested by row-wise calculation of receiver operating characteristic (ROC) curves including their corresponding area under the curve (AUC). Single classifiers and random decision trees were evaluated. As criteria for the random decision trees, a minimum of 10 observations at each tree node and 5 cross validations were selected and analyzed using the R package “rpart”. Cut-off values were calculated by the criterion based on the Youden’s index using the R package “OptimalCutpoints”. The cost value of false negative (CFN) cases was set to 4 to reflect the impact of a false negative versus a false positive case. A false negative and thereby potentially missed ketosis case would cost more and has a higher impact than supplementing a false positive cow with PG and glycerin. The classifier achieving the highest AUC was presented. Additional data are shown in Appendix A.3. ResultsIn this study, 52% of the cows had at least one BHB test result higher than 0.8 mmol/L during the study and were classified as hyperketonemic. The cumulative distribution function of hyperketonemia cases in relation to the day of parturition is presented in Figure 1A. On the day of parturition, 2% of cows were hyperketonemic. On the 6th day p.p., the highest leap of cases (from 29% to 53%) was observed. Before the 10th day p.p. the case number elevated to 63%. A second leap, from 77% to 92% of cases, was observed between 30 to 50 days p.p. Furthermore, this means that 47% (measurement at 6th day) and 37% (measurement at 10th day) of cases progressing to hyperketonemia were not detected with a single-day measuring scheme. In the present study the overall SCK incidence was 20%. The cumulative distribution function of SCK cases in relation to the day of parturition is shown in Figure 1B. The final natural cubic spline fits of BHB (Figure 2A), NEFA (Figure 2B), tBIL (Figure 2C), and AST (Figure 2D) of hyperketonemia are shown. The spline fits represent a BHB course for an individual over time. Hyperketonemia had a significant effect on the time course of all tested biomarkers. For BHB the log likelihood ratio of the final model was χ2(17) = 48.06, p < 0.001. Two peaks of BHB concentration were observed throughout the fitted period. The first peak occurred directly after parturition, whereas the second occurred between 20 to 40 days p.p. Furthermore, the BHB concentration in the hyperketonemic group remained higher compared to the control group until 80 days p.p. For NEFA the log likelihood ratio of the final model was χ2(17) = 115.63, p < 0.001. In the hyperketonemic group, one peak of NEFA concentration was observed directly after parturition followed by a continuous decline until the end of the fitted period. In the control group, two peaks occurred, wherein the first was seen after parturition and the second between 20 to 40 days p.p. For tBIL the log likelihood ratio of the final model was χ2(17) = 86.58, p < 0.001. In both groups one peak of tBIL concentration was observed directly after parturition. For AST the log likelihood ratio of the final model was χ2(17) = 166.62, p < 0.001. In both groups one peak of AST concentration was observed directly after parturition. Subsequently, AST levels declined in both groups. After 30 days p.p., AST concentration steadily increased in the remaining study period. The BHB concentration was significantly higher in hyperketonemic cows than in the control group (p < 0.5) from 9 days a.p. to 62 days p.p. Detailed results of time intervals showing significantly higher biomarker concentrations are presented in Appendix A.The final natural cubic spline fits of BHB (Figure 3A), NEFA (Figure 3B), tBIL (Figure 3C), and AST (Figure 3D) of SCK are shown. The spline fits represent a BHB course for an individual over time. SCK had a significant effect on the time course of all tested biomarkers. For BHB the log likelihood ratio of the final model was χ2(17) = 47.55, p < 0.001. Two peaks of BHB concentration were observed throughout the fitted period. The first peak occurred directly after parturition, whereas the second occurred between 20 to 40 days p.p. Furthermore, the BHB concentration in the SCK group remained higher compared to the control group until 80 days p.p. For NEFA the log likelihood ratio of the final model was χ2(17) = 108.80, p < 0.001. In the SCK group, one peak of NEFA concentration was observed directly after parturition followed by a second peak around 40 days p.p. In the control group, one peak was observed after parturition and followed by a continuous decline until the end of the study period. For tBIL the log likelihood ratio of the final model was χ2(17) = 85.53, p < 0.001. In both groups one peak of tBIL concentration was observed directly after parturition. For AST the log likelihood ratio of the final model was χ2(17) = 166.79, p < 0.001. In both groups one peak of AST concentration was observed directly after parturition. Subsequently, AST levels declined in both groups. After 30 days p.p., AST concentration of the control group steadily increased in the remaining study period. Compared to the control group, in SCK cows the BHB and tBIL concentrations were significantly higher (p < 0.5) from 9 days a.p. until 62 days p.p. and from 2 days p.p. until 62 days p.p., respectively. Detailed results of time intervals showing significantly higher biomarker concentrations are presented in Appendix A.The classifier achieving the highest AUC (AUC = 0.915) for hyperketonemia was the BHB concentration on the 10th day p.p. (t1). A boxplot of BHB concentration at t1 for the hyperketonemic and control groups is shown in Figure 4A. The corresponding ROC curve is presented in Figure 4B. The BHB cut-off value for hyperketonemic classification was calculated to be 0.54 mmol/L. This criterion resulted in 73% specificity (Sp) and 92% sensitivity (Se). The classifier achieving the highest AUC (AUC = 0.914) for SCK was the BHB concentration on the 10th day p.p. (t1). A boxplot of BHB concentration at t1 for the SCK and control groups is shown in Figure 4C. The corresponding ROC curve is presented in Figure 4D. The BHB cut-off value for SCK classification was calculated to be 0.73 mmol/L. This criterion resulted in 82% Sp and 85% Se. A summary of the classification parameters is shown in Table 1. The random decision tree analysis for hyperketonemic and SCK showed consistent results compared to the single classifier analysis; the combination of the different features did not improve the classification.4. DiscussionThis study investigated the serum biomarker profile of hyperketonemia and SCK in Simmental dairy cows and prognostic classifiers for hyperketonemia and SCK. A long-term effect of hyperketonemia and SCK was observed on the concentration and time course of BHB. Additionally, a short-term effect of hyperketonemia and SCK after parturition was found in NEFA, tBIL and AST. To our knowledge, this study is the first to investigate the long-term effects of hyperketonemia and estimate a cut-off point of BHB concentration in serum to potentially prevent long-term effects of hyperketonemia in dairy cows. Several studies were conducted to evaluate a cut-off value for the diagnosis of SCK and its causal association with diseases and infections [9,10,25,27,28,29]. The authors found that a wide range of cut-off levels (>1.00 mmol/L up to >1.6 mmol/L) during the first 2 weeks p.p. were associated with decreased pregnancy rates, uterine infections and displaced abomasum. In our study, hyperketonemic and SCK cows showed higher biomarker concentrations after parturition in BHB, NEFA, tBIL, and AST. This effect might indicate increased metabolic stress during early lactation for ketotic cows [14]. Moreover, Djoković et al. [30] found higher BHB and NEFA concentrations in Simmental dairy cows during the first month of lactation. Increased levels of BHB and NEFA in early lactation might derive from a negative energy balance, leading to overstimulated fat mobilization [4,31,32]. Leblanc et al. [33] described an earlier NEFA increase with greater magnitude in cows experiencing metabolic diseases. However, data in our study showed a greater magnitude of increase, but the increase did not start earlier. Moreover, elevated AST and tBIL concentrations during the first weeks p.p. in hyperketonemic and SCK cows could indicate liver dysfunction [14,22,34,35,36].Long-term biomarker effects in ketotic Holstein dairy cows were shown previously with elevated BHB levels (BHB > 1.4 mmol/L) after 4 and 8 weeks p.p. [37]. Our results are in agreement with this finding. Furthermore, they revealed that once BHB concentration exceeded 0.8 mmol/L, the further BHB course differed significantly from the course of the control group. Moreover, the BHB concentration did not converge again with the level of the control group until the end of our study period. Hence, an altered BHB course is at least present until the peak lactation phase (80 days p.p.), which supports our main hypothesis. Further studies must be conducted to evaluate whether the changes in BHB concentration persist until the next lactation cycle, and if they are associated with undesirable long-term effects. Although this study was conducted on only a limited number of cows of a single breed with moderate milk yield, our results underline the importance of preventing hyperketonemia rather than managing or treating it for a short-term period. However, to our knowledge there are no breed-specific cut-off levels for the biomarkers used in this study. Benedet et al. [38] found small differences (i.e., BHB = 0.65 mmol/L in Holstein versus 0.63 mmol/L in Simmental dairy cows) in concentration, but the biomarker course followed the same biomarker profile over time. We are aware that the chosen cut-off values for this study were very conservative. However, we want to raise the questions: Why were the cut-off values for metabolic biomarkers increased over time? Did our cows change and adapt their metabolism to the increased metabolic stress, or did we push their limits until metabolic diseases arose? Hence, we are claiming that the current gold standard BHB cut-off values of 1.2 mmol/L and 1.4 mmol/L during the first 2 weeks p.p. are too high to prevent the long-term effects of hyperketonemia. As Suthar et al. [27] have already stated, disease-specific thresholds for BHB are widely discussed in research but are not feasible in practice. In our study, hyperketonemia was classified by a BHB concentration > 0.8 mmol/L. As Duffield et al. [10] have already noted, a decision for a cut-off level cannot be an arbitrary choice, but should be a reflection of production and health impairment. Moreover, by raising the cut-off from 0.62 to 0.8 mmol/L, we considered the increased metabolic stress of dairy cows during the transition period [12], as Fürll [8] associates a BHB level > 0.85 mmol/L on the third day p.p. in combination with other parameters as an indicator for increased fat mobilization and FCS. This is also supported by Pralle et al. [39], who found a mean BHB value of 0.8 mmol/L (SD = 0.02) in 1013 Holstein dairy cows. Therefore, it provides feasibility in practice and still reflects individual health status.The average incidence of SCK is about 40% [40,41]. In the present study the incidence of SCK was lower (20%) but within the expected range. Thirty percent of SCK cases were found within 10 days p.p., wherein most new cases were observed on the fifth day p.p. These results are in accordance with previous findings by Geishauser et al. [41]. Based on the used criteria, we found a hyperketonemia incidence of 52%, wherein 63% of cases were found until the 10th day p.p. These results are in accordance with Duffield et al. [42] and Oetzel [43]. Furthermore, this means that 47% (measurement on sixth day) and 37% (measurement on 10th day) of cases progressing to hyperketonemia are not detected at this stage and would not be detected by a single-day measurement regime, thus pointing out the need for a prognostic biomarker to enable effective preventative action early on. With our model of biomarker progression, such a prognostic classifier was defined. The prognostic classifier for hyperketonemia was 0.54 mmol/L serum BHB measured on the 10th day p.p. This suggests that in contrast to a fixed cut-off level measurable at different time points, a lower cut-off level at the right time point might be superior in the prognosis of hyperketonemia. Such a cut-off level is also supported by the physiological BHB levels for adult cows, published by Fürll [8], who suggested an upper control level of 0.53 mmol/L BHB. For cows exceeding the cut-off, we recommended anti-ketogenic food supplementary treatment such as PG and glycerin. The anti-ketogenic properties of PG and glycerin were shown in various studies, and they are recommended as prophylactic treatment for SCK [44,45,46]. These guidelines might allow increased success in transition management and improved individual health. 5. ConclusionsThe longitudinal biomarker profile of a mild form of hyperketonemia was investigated. Our results indicate that the progression of hyperketonemia is closely associated with the biomarker course of BHB, NEFA, tBIL, and AST. The most discriminative biomarker was BHB, which persisted at least until the peak lactation phase (80 days p.p.). The best prognostic classifier for hyperketonemia was identified as a cut-off level of 0.54 mmol/L on the 10th day p.p., achieved with a single measurement regime. This allows effective and early detection of hyperketonemia and is a promising approach for prevention, increasing the overall health of dairy cows.

animals : an open access journal from mdpi

10.3390/ani13091524

PMC10177084

Sentinels monitor their surroundings from vantage points for early detection of predators and rivals. The presence of multiple sentinels in a group may allow sentinels to relax their vigilance, especially if sentinels monitor different areas at the same time. We investigated sentinel behavior in groups of the Florida scrub jay (Aphelocoma caerulescens). Sentinels in this species turn their heads frequently to monitor different areas for potential threats. As predicted, we found that sentinels turned their heads less frequently in the presence of other sentinels. However, multiple sentinels tended to gaze in the same direction at the same time more often than predicted by chance alone. Gaze synchronization reduces the efficiency of collective detection by increasing the amount of time that some areas are not monitored by any sentinel. Despite the benefits of the presence of other sentinels, our results highlight the limits to collective detection when multiple individuals are vigilant at the same time.

Sentinels can detect predators and rivals early by monitoring their surroundings from vantage points. Multiple sentinels in a group may reduce the perceived predation risk by diluting the risk and increasing collective detection, especially if sentinels monitor different areas at the same time. We investigated sentinel behavior in groups of the Florida scrub jay (Aphelocoma caerulescens). Sentinels in this species turn their heads frequently to monitor different areas for threats. As predicted, we found that sentinels turned their heads less frequently in the presence of other sentinels. Multiple sentinels, however, tended to gaze in the same direction at the same time more often than predicted by chance alone. Gaze synchronization reduces the efficiency of collective detection by reducing visual coverage at any one time at the group level. Despite the benefits of the presence of other sentinels, our results highlight the limits to collective detection when multiple individuals are vigilant at the same time.

1. IntroductionLiving in groups has long been considered an adaptation against predation [1,2]. In particular, group members can dilute risk among themselves if predators can only target one individual at a time [3]. The many eyes and ears available in a group can also allow individuals to detect predators more quickly [4]. Predator detection by a few can be passed along rapidly to others in the group, which is a process known as collective detection [5]. Models of collective detection assume that each group member monitors the surroundings independently [6,7]. Independent monitoring ensures that detection ability at the group level is directly proportional to the size of the group. In groups where individuals interrupt foraging to initiate vigilance, effective collective detection can be achieved by initiating vigilance bouts regardless of whether other group members are already vigilant or not [8].Empirical studies on collective detection have often failed to document independent monitoring. Indeed, in many species, individuals are more likely to initiate vigilance bouts when others are already vigilant, which is a process known as synchronization [9,10,11,12,13,14,15]. Synchronization of vigilance may be costly because it increases the amount of time during which no one is vigilant at all. In addition, information about threats in the surroundings provided by several individuals that are all vigilant at the same time is more likely to be redundant thus reducing its value. Coordination of vigilance is the opposite process whereby individuals are less likely to initiate vigilance bouts when others are already vigilant [16]. Coordination, unlike synchronization, can increase the effectiveness of collective detection by decreasing the chances that no one is vigilant at all and by reducing temporal overlap in vigilance bouts at the group level. Nevertheless, coordination of vigilance is not common in animals and tends to occur in small groups where it is easier to monitor the vigilance of others, which is a prerequisite for effective coordination [10,17,18].Collective detection could also be less effective if vigilant group members are monitoring the same areas at the same time. Indeed, if all group members only monitored one specific area during vigilance, the group would be more vulnerable to threats coming from other directions. This is likely the case in species with eyes located frontally or laterally, which makes it impossible to monitor all surrounding areas simultaneously. Such animals must therefore make frequent head turns to monitor different areas including the blind spot behind their heads [19]. To ensure effective collective detection, such animals should not only initiate vigilance bouts independently of each other, but they should also monitor different areas independently by turning their heads in different directions. This would ensure a more even monitoring of all areas at the group level.There is limited information on collective detection in the context of acquiring information during vigilance bouts. In one study, birds in pairs synchronized their head turns suggesting that individuals paid attention to the vigilance of nearby companions [20]. However, the direction of their gaze was not examined. Other studies have examined the factors that affect the frequency of head turns but not in relation to collective detection. For instance, individuals in smaller groups or those with less experience of predators make more frequent head turns to increase visual coverage in riskier settings [21,22,23,24,25].Here, we examined the effectiveness of collective detection during vigilance bouts in a cooperative breeder, the Florida scrub jay (Aphelocoma caerulescens). In this species, members of a group perch on vantage points to monitor their surroundings for possible threats such as predators and intruding neighbors [26]. Known as sentinels, these group members can detect threats more easily and pass the information along rapidly to companions foraging nearby on the ground [27]. Because their eyes are located laterally, Florida scrub jays turn their heads frequently in different directions during sentinel bouts [25], providing visual coverage of all surroundings areas including the blind spot behind their heads [28]. Occasionally, two or more group members are present during sentinel bouts. This provides a unique opportunity to examine gazing strategies in a group during long, uninterrupted vigilance bouts aimed exclusively at distant threats.First, we aimed to determine whether sentinels benefit from the presence of other sentinels nearby. Nearby sentinels can dilute risk more effectively by reducing inter-individual distances [29]. Nearby sentinels can also monitor different areas at the same time, which improves collective detection. We predicted that the frequency of head turns in sentinels would be lower when other sentinels are present in response to the reduction in perceived predation risk. We then examined gazing strategies among multiple sentinels to gain insights into the benefits associated with collective detection. We predicted that multiple sentinels would gaze at different areas independently of one another to achieve better collective detection. The two possible alternatives to gaze independence are gaze synchronization or gaze coordination. Gaze synchronization was not expected because it implies that multiple sentinels are more likely to gaze in the same direction at the same time than predicted by chance alone thus reducing the effectiveness of collective detection. Although gaze coordination would increase collective detection benefits, it was not expected given that coordination of vigilance is not commonly reported in animals.2. Materials and Methods2.1. Study SiteThe study was conducted at Archbold Biological Station located in south-central Florida (USA). Small scrub oaks (Quercus spp.) are the dominant vegetation at the station. Florida scrub jays in the area live in all-purpose territories year round and are nearly all banded and monitored regularly to evaluate group size and breeding status [30]. Juveniles in this species remain in their natal territories after the breeding season and assist breeders in territory defense and predator detection. These juveniles can transition to helper status the following breeding season and remain with the family for one or more years. Observations were carried out in the non-breeding season from late February to early March in 2022 and during the last two weeks of January in 2023. At this time of year, the number of mapped territories was about 80 and groups ranged in size from two to nine. A typical group included two breeders and two non-breeders. At least one breeder was present in each territory accompanied by juveniles, if any, from the previous breeding season and by helpers, if any, from earlier breeding seasons.2.2. SamplingFlorida scrub jays were typically monitored from 7 to 11 am with a few exceptions in the late afternoon. During daily walks on different trails, one of us attempted to locate foraging groups with sentinels. Once a sentinel was detected, the observer used a video camera to record sentinel behavior. As Florida scrub jays at the station are accustomed to human presence, observations could be made at close range without obvious disturbances. A 60× zoom on the video camera also brought more distant sentinels into sharper focus, which allowed easy detection of head movements in all cases. Observations lasted for a scheduled 5 min unless the focal subject departed earlier. The observer noted how many other sentinels were present nearby and the total number of Florida scrub jays present including sentinels and foragers on the ground. Multiple sentinels in the same group were typically within a meter of one another and could all be fitted within the video shots. The identity and social status of the focal subjects were obtained later using information from the colored bands. We did not collect data during encounters between groups at territorial boundaries as sentinel behavior was rarely performed.2.3. Video AnalysisWe watched videos at low speed to determine the number of detectable head movements for each sentinel present in the video shots. The duration of a focal observation excluded all sequences during which the focal bird was not looking around. This included rare bouts of grooming or foraging or moments during which the focal bird was unintentionally outside the video shots.For the analysis of gazing strategies among multiple sentinels, we isolated video sequences with multiple sentinels from the set of focal observations. We excluded video sequences where one of the subjects could not be seen clearly. Most cases with multiple sentinels involved only two sentinels. In rare cases where three sentinels were present at the same time, we included only two of the three possible pairs chosen randomly to maintain independence among pairs. Overall, all pairs were unique. The duration of a focal observation with multiple sentinels excluded all sequences during which either sentinel was not looking around. We adopted the following procedure to assess gaze independence. In our study, most head movements occurred on the horizontal plane. Head orientation for one of the sentinels in a pair was obtained by projecting the position of the bill onto an imaginary circle in the horizontal plane centered on the head of this bird. The bill at the zero point on this circle seen from above is aligned with the long axis of the body from head to tail and represents the orientation of the head when the bill is pointing straight ahead (Figure 1). We then determined in which of four equal segments of 90° the bill of each sentinel in the pair was positioned on the circle during gazing. Congruent gazes occurred when the two bills were in the same quadrant at the same time. Non-congruent gazes implied that the two bills were positioned in different quadrants at the same time with at least 90° between them. More congruent gazes than expected by chance alone would be compatible with gaze synchronization. Fewer congruent gazes than expected would be compatible with gaze coordination. This scoring system is admittedly coarse but realistic given the field observations. Watching the videos at slow speed, we determined the percentage of time in the focal observation during which the two bills pointed in the same general direction (within 90° of one another).2.4. Statistical AnalysisIn the first analysis, we wanted to determine the effect of the presence of other sentinels on the frequency of head turns for focal individuals. We used a linear mixed model to examine the association between the frequency of head turns per minute and the following fixed effects: total group size, status of the focal sentinel (breeder v. non-breeder), and type of observation (other sentinels present or not). Focal subject id was used as a random effect to account for multiple observations of the same subjects. Group id was also considered a random effect to account for multiple observations of different birds within the same group. We fitted the following model: frequency of head turns = group size + status + observation type + group size*status + group size*observation type + (1|focal subject id) + (1|group id of the focal subject).The second analysis focused on gazing strategies among multiple sentinels. To obtain descriptive statistics for the percentage of time during which gazes were congruent among pairs of sentinels, we weighted each percentage by focal observation duration to give more weight to longer, more informative focal observations. We then computed the 95% confidence intervals around the weighted mean and determined whether it included the expected value or not.We used the following procedure to establish the expected value. The expected percentage of time during which gazes are congruent was based on observations of lone sentinels from the 2022 field season. For those sentinels, the percentage of time that the bill was positioned in each quadrant was obtained for each subject. Based on 50 focal observations, the bill was positioned on average 50.3% of the time in the front quadrant, 19.8% and 21.3% in the two side quadrants, and 8.6% in the back quadrant. At any given time, the bill of a sentinel is thus oriented in any one of these four quadrants. The orientation of the bill over a series of discrete time steps can be viewed as a multinomial distribution with four possible, mutually exclusive outcomes (i.e., quadrants) at each time with probabilities proportional to the above four percentages. Assuming that the two sentinels of a pair monitor the surroundings independently, we can calculate the chances that the two sentinels orient their heads in the same direction using the multiplication rule of two independent random variables [31]. For instance, the chances (expressed as a percentage) that the head of each sentinel is oriented in the front quadrant is given by 50.3% times 50.3%. These chances are calculated in a similar way for the other quadrants. We limited calculations of these joint occurrences to congruent gazes, namely, when the two sentinels orient their heads in the same direction.One difficulty in practice is that the bodies of the two sentinels may not be oriented in the same direction when their bills are pointing straight ahead. Therefore, the quadrants for each sentinel are not necessarily aligned. For instance, the front quadrants are oriented in different directions if the two sentinels face one another. To simplify calculations, we considered two extreme body orientations: parallel and antiparallel. In the parallel orientation, the bodies of the two sentinels are oriented in exactly the same direction so that all their quadrants are aligned. In the antiparallel orientation, the bodies of the two sentinels are oriented in fully opposite directions. With the antiparallel orientation, the front quadrants of each sentinel, for instance, point in the opposite direction. With these considerations in mind, we used the multiplication rule to calculate expected values for the two extreme body orientations. For congruent gazes in the parallel orientation, the expected value is given by: 50.3%*50.3% + 19.8%*19.8% + 21.3%*21.3% + 8.6%*8.6% = 34%. For the antiparallel orientation, the expected value is given by: 2*50.3%*8.6% + 2*19.8%*21.3% = 17%. Across all possible body orientations, the expected value should thus lie between 17% and 34%, which are the two extreme limits.3. ResultsFor head turns, we obtained a total of 213 focal observations over the two non-breeding seasons, including 48 cases with multiple sentinels. Multiple sentinels typically involved two birds and occasionally three (n = 3). Focal observations lasted for a median of 95 s (range: 13 to 366 s). Groups ranged in size from two to nine with a mode of four. Sentinels included breeders (n = 149, approximately 70%) as well as non-breeders (juveniles or helpers, n = 64, approximately 30%). Sentinels turned their heads on average 43.5 times per minute ranging from 15.6 to 73.8. The head-turning rate was higher in non-breeders than in breeders (F1,94.5 = 14.9, p = 0.0002). Overall, the rate of head turning decreased with group size (β (SE) = −2.4 (0.5), F1,170.3 = 22.1, p < 0.0001; Figure 2) and was lower when other sentinels were present rather than absent (β (SE) = −5.0 (1.5), F1,191.7 = 10.7, p = 0.001; Figure 2). Using model estimates, the presence of a nearby sentinel reduced the head-turning rate more than the addition of another group member on the ground did for single sentinels (5.0 units compared to 2.4 units). There was no significant interaction between status and group size (F1,157.7 = 1.4, p = 0.24) or presence of other sentinels and group size (F1,204.2 = 0.84, p = 0.36), and both interactions were removed for the final model.We assessed gazing strategies for 25 unique pairs of sentinels. Focal observations of pairs of sentinels lasted for a median of 66 s (range: 15 to 292 s). Groups with multiple sentinels ranged in size from two to seven. We did not record any obvious interactions between multiple sentinels. Adjusting for focal observation duration, the mean percentage of time during which gazes were congruent between sentinels in a pair was 43.1% (Figure 3). The 95% confidence interval for this percentage fell above the range of expected values based on independent gazing.4. DiscussionAs overall group size increased, Florida scrub jays reduced the frequency of head turns during sentinel bouts. This was true for breeders and non-breeders alike although the head-turning rate was higher in non-breeders than in breeders. In addition to the overall effect of group size, the presence of other sentinels also reduced the head-turning rate. The magnitude of this effect was greater than the addition of another group member on the ground for single sentinels. Sentinels in pairs synchronized rather than coordinated gazing. Indeed, gazes in pairs of sentinels were more likely to occur in the same broad direction at the same time than expected by chance alone.Breeding status and overall group size influenced the frequency of head turns in sentinels [25]. More experienced breeders probably have a better assessment of local predation risk than juveniles or helpers. Indeed, in many species, juveniles are more likely to give alarm calls [32], suggesting that they tend to overestimate predation risk. In larger groups, sentinels can benefit from the dilution provided by other group members foraging on the ground around them and perhaps from the ability of ground foragers to detect predators. The lower perceived predation risk in larger groups can thus translate into a lower rate of head turning during sentinel bouts.In addition to breeding status and overall group size, the presence of other sentinels during sentinel bouts also influenced the frequency of head turns. The presence of an additional sentinel can be beneficial by increasing the dilution effect. Dilution is more effective at closer range [33,34]. The results show that the presence of a nearby sentinel had more impact on the frequency of head turns than the addition of another forager on the ground for single sentinels. Nevertheless, increased dilution alone is unlikely to explain the reduction in head-turning rate. If sentinels relied solely on the dilution effect provided by nearby sentinels, there would be no need to make head turns to detect threats. The occurrence of head turns in bouts with multiple sentinels suggests that a sentinel relied to some extent on the ability of other sentinels to detect threats. As sentinels cannot detect threats coming directly from behind, the addition of another sentinel can potentially increase the area monitored by at least one bird at any one time, which can explain to some extent why the presence of other sentinels reduced their head-turning rate.Sentinels in pairs synchronized rather than coordinated gazing. Gaze synchronization comes at the cost of having longer periods during which no sentinel is monitoring blind spots. However, gazes were not congruent all the time, suggesting that collective detection, albeit imperfect, was not negligible. Gaze synchronization during sentinel bouts could actually bring benefits as long as congruent gazes are not too common. For instance, possible threats coming from one direction could be assessed by more than one sentinel at the same time meaning that misidentification is less likely [35]. This might be especially relevant when pairs of sentinels include breeders and younger, less experienced individuals. In addition, if predators tend to target laggards, there might be selection pressure to synchronize gaze direction [16]. This assumes that individuals detecting threats through their own eyes can flee sooner than those looking elsewhere, which can respond only after a delay to the flight responses of detectors. Future work is needed to assess these ideas.Little empirical work has focused on gazing strategies. One study found that individuals in pairs of captive birds synchronized their head turns but it did not examine gaze direction [20]. Gaze synchronization could arise from gaze following as one individual follows the gaze of another to look at the same thing [36]. Florida scrub jays have lateral eyes and probably a wide field of view [28]. For this reason, it is not clear what exactly individuals were looking at during sentinel bouts. As we could only determine head orientation, we cannot be certain that sentinels in pairs looked at the same thing during congruent gazes. Nevertheless, from the point of view of collective detection, congruent gazes between sentinels imply that their field of view is temporarily similar and that the blind spot behind their heads is not monitored. Non-congruent gazes, by contrast, allow for a fuller visual coverage of all areas surrounding sentinels including blind spots.Why did sentinels in the Florida scrub jay not coordinate gazing? Gaze coordination would ensure maximum collective detection. Models suggest that coordination should be more common in smaller groups [37,38], which is the case for multiple sentinels in the Florida scrub jay. However, gaze coordination requires monitoring neighbors to assess where they are looking. It is perhaps the case that the extra benefits from more efficient collective detection might not be sufficient to cover monitoring costs. A similar explanation was proposed to explain why coordination of vigilance was rare in animals that alternate between foraging and vigilance bouts [39].Overall, the presence of other sentinels was beneficial because it decreased perceived predation risk. However, the prevalence of sentinel bouts with multiple sentinels was low in this study. Sentinels must balance the need for better vigilance from vantage points against the need to forage on the ground to accumulate resources. As a consequence, sentinel bouts for a given individual are typically not very long in the Florida scrub jay [26]. The need to resume foraging might thus set a limit on the frequency of sentinel bouts with multiple sentinels. Multiple sentinels have not been reported in Arabian babblers (Turdoides squamiceps) [40] but have been documented in various other species of birds such as American crows (Corvus brachyrhynchos) [41], white-browed sparrow-weavers (Plocepasser mahali) [42], jungle babblers (Turdoides striatus) [43], and white-winged choughs (Corcorax melanorhamphos) [44]. Future tests of gazing strategies can thus be performed in other species with sentinels. More broadly, tests could be performed in any situation where multiple individuals are vigilant at the same time.5. ConclusionsPast research has shown that collective detection is not always effective in animals that alternate between foraging and vigilance bouts [5,45,46]. Our results show that this is also the case when considering where individuals gaze during vigilance bouts. Testing the limits of collective detection by also considering gaze direction will increase our understanding of the anti-predator function of group living in birds.

animals : an open access journal from mdpi

10.3390/ani11113322

PMC8614550

Mitochondrial DNA (mtDNA) analysis is a tool in assessing the maternal origin, phylogeny, and population structure of domestic animals. The Ecuadorian Pillareño Creole pig is a creole breed that comes from the descendants of Iberian pig introduced to Ecuador by the Spanish conquerors. This creole population represents important reservoirs of genetic diversity that are very essential to preserve; however, the introduction of new breeds of pigs has displaced this creole pig from Ecuador to the background. The objective of the mitochondrial DNA analysis was to identify the Ecuadorian Pillareño Creole pig populations, their origins, and their maternal lines. For this study, DNA samples from 34 Ecuadorian Pillareño Creole pigs were used, with the animals belonging to seven rural regions of Ecuador. The haplogroup network suggested that the Pillareño creole pig population can be grouped into a single haplotype and that it belongs to the European pig clades. The genetic relationship between the Ecuadorian Pillareño Creole and the European pigs, particularly the Iberian pigs from Spain, can be used to establish of an official breeding program for the conservation and valuation of these creole populations, with this genetic mitochondrial analysis potentially providing a better approach for the rescue of the Ecuadorian Pillareño Creole pig populations.

Domestic pig breeds reached America on the second Columbus trip; from this date, Iberian pig genetic resources were disseminated throughout the continent, forming diverse creole breeds. These Ecuadorian Creole pigs are important for food production but have been genetically eroded since the introduction of transboundary breeds. In this study, we sought to characterize this erosion more thoroughly through mitochondrial DNA D-Loop analysis of Ecuadorian Pillareño Creole pigs from seven regions of Ecuador. To allow comparison, we also included in our analysis sequences from wild species, commercial lines, and domestic pigs, which were obtained from the NCBI GenBank database. Creole pigs’ population showed overall moderate Hd values and low π values, and a negative value of Tajima’s D was observed. The greatest differentiation from the Ecuadorian Pillareño Creole pigs was observed between Asian wild and Asian domestic pigs. The haplotype analysis revealed three different phylogenetic clades (A, E I, and E II) and 65 haplotypes. Ecuadorian Creole populations were grouped into nine haplotypes for Clade E I and E II, which have not previously been reported for Creole Pillareño populations. Our analysis indicates that in the establishment of Creole Pillareño pigs, individuals most likely separated from the Asian pig population and appear to be genetically influenced by European and Iberian populations raised in Spain.

1. IntroductionDomestic pig colonization of the American continent started with the second trip of Columbus; in this expedition, several domestic species were carried to the Caribbean islands. Among them, pigs had a special role because the meat was used to feed the population and to prevent vitamin C deficiency [1]. From the Caribbean archipelago, the pig resources multiplied and were distributed in three main directions. The first was to North America, reaching the north of Mexico and the present southwestern United States. The second was to the south of Peru, reaching the Patagonia region. The third was to Central America through the Atlantic ports of Panama and Cartagena de Indias, Colombia, reaching the south of Mexico and the northern countries of South America, including Ecuador. This migration resulted in the local domestic breeds present in the different territories; these pig breeds are currently known as “Creole pigs”. Some feral populations also developed, which remain in several countries such as Argentina, Brazil, and Uruguay, among others.Today, different populations of these Creole pigs exist in America, for instance, in Colombia (San Pedreño and Zungo pig), Mexico (Pelon mexicano pig), Uruguay (Pampa Rocha pig), Argentina (Chancho Cimarrón pig), Cuba (Pinareño Creole pig, Cuban Creole pig), Venezuela (Apure Creole pig), Peru (Tumbes Creole pig), Guatemala (Chorti Creole pig), Brazil (Piau pig, Porco monteiro), and the United States (Ossabaw, Choctaw, Mulefoot, and Red Waddler pig), among others [2,3,4,5], but many more remain unknown, and studies characterizing these populations are needed. In Ecuador, the Pillareño Creole pig populations persist; these animals are normally under poor management conditions in marginal areas, generally involving family backyard production [6]. For more than 500 years, the Ecuadorian Pillareño Creole pigs have adapted to very different conditions in Ecuador and demonstrate climatic resistance [7], with a great adaptability to different ecosystems (Andinian, Coastal, and Amazonian), especially to extremely adverse conditions and to a diet with a low nutritional level [8]. Alongside the important social role of the Ecuadorian Pillareño Creole pigs, these animals have great potentiality for profitable and sustainable production due to their putative descendance from the Iberian pig, and probably carry its exceptional meat quality characteristics. Unfortunately, it was verified that in Ecuador, breeds of Iberian origin have tended to disappear due to the aggressive introduction of commercial breeds from northern European countries, which puts at risk a genetic heritage worthy of being conserved in order to take advantage of these capabilities, such as resistance to diseases, rusticity, and ability adaptive capacity to different environments [9].Historical documents illustrate that the Spanish conquerors released different lineages of the Iberian pig (Smooth Black, Hairy Black, Red Dish, Dark Brown, and Andalusian Blonde) [10] in America. Phylogeographical studies of domestic animals are based on finding genetic variations in mitochondrial DNA (mtDNA) because its variability is five times higher than for other types of markers [11,12]. The mtDNA structure shows nonrecombining patterns in pigs, forming a closed circular double helix DNA sizing around 16,500 bp encoding 13 hydrophobic polypeptides, 22 tRNAs, and 2 rRNAs [13,14].Generally, the research of genetic distances between breeds due to huge mutations [15] based on genetic variants in mtDNA has been centered on the D-loop region. This information is also available to characterize breeds and individuals in phylogenetic studies [16]. However, whole mtDNA sequences are needed to estimate the genetic relationships among breeds, characterize breed specificity, and identify individuals.Different American, European, and Asian pig breeds have been studied at the molecular level using diverse nuclear DNA marker systems [17,18], including microsatellites and single nucleotide polymorphisms (SNPs), but the relationships among Creole pigs have not been extensively evaluated using the mitochondrial DNA (mtDNA) D-loop control region. In terms of the possibilities of this molecular tool [13], mtDNA assessment can explain and provide additional support for the evaluation of distinctions between the Ecuadorian Pillareño Creole, European, Iberian, Asian, and commercial pig breeds on the basis of their relatedness [19,20].In the present paper, we investigated the Ecuadorian Pillareño local pig in terms of its possible Iberian origin and recent genetic erosion, understanding it as a loss of biodiversity due to crossbreeding with transboundary breeds. This was achieved by analyzing the mitochondrial diversity in Ecuadorian Pillareño Creole pig and testing its matrilineal relations with European domestic lines, Iberian pigs raised in Spain, Asian pig lines, commercial pigs, and wild pig breeds to evaluate its origin and inter- and intraspecific global connections with these other pig populations.2. Materials and Methods2.1. Sample CollectionAccording to the livestock census, as well as the creole pig Pillareño morphological and phenotypic measures, we selected backyard pigs raised by families in several locations throughout Chimborazo. A total of 34 blood samples of Creole Pillareño pigs were collected and preserved in 5 mL tubes containing ethylenediaminetetraacetic acid (EDTA). In order to avoid a high relationship between the animals, we collected the samples in seven different regions in Ecuador. For each region (Pungalá, Tunshi, Molobog Licto, Licto Pungala, Guamote, Pulinguí, and Penipe), five animals were selected, apart from Penipe, where there were only four animals. After the samples were obtained, they were preserved and stored at −18 °C for DNA extraction in the Animal Breeding Consulting Laboratory located at University of Córdoba, Spain, until subsequent use.2.2. Ethics StatementThis type of project does not fall under the legislation for the protection of animals used for scientific purposes, Organic Law for the Defense of Animal Rights. Data were collected during the application of regular zootechnical procedures without injuring the animals.2.3. DNA Extraction and AmplificationDNA was extracted from blood samples by using a Chelex-100 resin® analytical grade, 50–100 mesh, sodium form (BioRad, Madrid, Spain) under the protocol used by [1]. The mtDNA D-loop sequence was obtained from GenBank accession number AJ002189 [2] and primers (F: 5′-CGCCATCAGCACCCAAAGCT-3′ and R: 5′-TGGGCGATTTTAGGTGAGATGGT-3′) [21,22,23] to amplify a 637 bp product from [3,19,20,22,23] the mtDNA region. The reactions were performed following the steps by Canales [4]. The amplicon quality of the product of PCR were assessed in a 1.5% agarose gel stained with ethidium bromide (low EEO/multifunctional/molecular biology grade) using size 100 bp DNA ladder® (Invitrogen, Waltham, MA, USA) as a banding marker. After verifying the PCR product, the reactions were incubated using 10 µL with one unit of FastaP Thermosensitive Alkaline Phosphatase (Thermo Fisher Scientific, Waltham, MA, USA) and 10 units of Exonuclease enzyme (Thermo Fisher Scientific, Waltham, MA, USA) incubated by one cycle at 37 °C for 15 min and 80 °C for 15 min. The sequencing reactions were performed in both directions using PCR oligos by the dideoxy technique, using a commercial service at Macrogen Inc. services (Madrid, Spain). In addition, we included sequences from wild species (Sus celebensis indonesia, papuensis vanuatu, barbatus, and wild Spanish), many commercial lines, and domestic pigs (China, Indonesia, Papua New Guinea, Germany, Italy, Malaysia, France, Iberian, Black Jabugo, Large White, Duroc, and Pietrain), obtained from the NCBI GenBank database, resulting in a total of 134 sequences. More details about the country of origin and the number of samples for each breed are given in the electronic supplementary material (Table S1).2.4. Molecular D-Loop Analysis2.4.1. Mitochondrial Genetic Diversity and DifferentiationThe sequence editing, alignment, and construction of data matrices were performed using Mega v5 [5] and Gblocks 0.91b [6,7]. All new sequences were deposited in GenBank. The number of haplotypes (H) and polymorphic sites (S), amount of nucleotide diversity (π) and haplotype (Hd) diversity estimates, and the calculation of Tajima’s D-values Fu’s and Fs statistics for the Ecuadorian Creole pig populations were calculated using DnaSP v5 [7]. In addition, FST and coancestry coefficient (permuting haplotypes among population among groups) values from pairwise comparisons were computed with 5000 permutations using ARLEQUIN v3.1 [8]. A neighbor-joining (NJ) tree was constructed on the basis of the genetic distance matrix using Splitstree v4.14.6 software [9]. Analysis of molecular variance (AMOVA) [10] was used to calculate genetic variation and differentiation between populations by performing 10,000 permutations. We included the following external population sequences from the NCBI GenBank database: the Iberian-like population (Torbiscal line, Black Hairless, Red, Black Hairy, and Blond); wild species from Asia (Sus celebensis indonesia, papuensis vanuatu, and barbatus); wild Spanish pig, domestic pigs from Asia (China, Indonesia, Papua New Guinea, and Malaysia); European pigs from Germany, Italy, and France; and different commercial lines from Europe (Black jabugo, Large White, Duroc, and Pietrain).2.4.2. Genealogical Relationships between HaplotypesWith the software NETWORK v4.6.0.0 [9], we constructed a haplotype network to establish genealogical relationships between haplotypes and their frequencies using the median-joining method under the default parameters. We analyzed the relationship between haplotypes and sequence variation using phylogenetic inference. The matrices included haplotypes that were identified in this study and haplotypes for each population that were available from the NCBI GenBank database. The network consisted of 134 frequencies and included wild species (Sus celebensis indonesia, papuensis vanuatu, barbatus, and wild Spanish), many widely distributed commercial lines, local domestic pig breeds (China, Indonesia, Papua New Guinea, Germany, Italy, Malaysia, France, Iberian, Black Jabugo, Duroc, and Pietrain), and the Ecuadorian Creole pig (Table S1).3. Results3.1. Sequence Analysis, Genetic Diversity, and DifferentiationAfter we amplified the 637 bp product from [3] the mtDNA region, 34 sequences were edited and aligned, and 550 bp of the mtDNA D-loop was obtained from DNA samples of Pillareño pigs from Ecuador collected for this study. These sequences were registered in GenBank (accession numbers: MT317953–MT317986). D-loop sequences were aligned to a reference sequence from GenBank (accession number AJ002189); nine haplotypes with 25 polymorphic sites were identified in the population of Pillareño. The dominant haplotype was H_3 with n = 21 pigs (Table 1). All the populations showed overall moderate Hd values and low π values, with a negative value of Tajima’s D [11], which indicates an excess number of alleles from a recent population or genetic hitchhiking, with the Fu’s Fs tests showing positive values. All the results are shown in Table 2.In addition, we analyzed and constructed one genetic differentiation table, Table 3, which shows that the main divergence of Pillareño was observed between Asia domestic and Asia wild, and the lowest rates of genetic divergence were found between Pillareño and Iberic, Spanish wild, and commercial European. To confirm our results, using the neighbor-joining method (Figure 1), we estimated the genetic distances between populations from mitochondrial sequences.The coancestry coefficients [12] were calculated, and the greatest coefficients were observed with Pillareño–Asian domestic pigs; by contrast, the lowest genetic coancestry coefficients differentiation values were found between Pillareño and Iberic pigs (Table 4)The total amount of genetic variation detected was partitioned by AMOVA (p < 0.01), according to the following groups: Pillareño vs. European, Iberic, and commercial. According to the previous components divided by region and populations, the analysis results in Table 5 show that the genetic variability within population components was 69.42% of the total variance. A significant amount of within-group variation was also observed (28.36%), and a smaller but still significant result of difference among groups (2.22%) was found, with the high fixation index (FSC, FST and FCT) indicating that populations were well differentiated.3.2. Haplotype NetworkWe constructed a haplotype network to visualize the relationships between haplotypes and their frequencies, and the study showed that they are distinguished from each other by a moderate number of mutations. The network (Figure 2) showed and separated very clearly three different phylogenetic clades (A, E I, and E II) and 65 haplotypes. In the clade A, all the Asian domestic and wild pig haplotypes were grouped; in this clade, it was possible to observe four unique haplotypes for the Ecuadorian Creole pig.The other haplotypes conformed to the E I phylogenetic clades that corresponded to the European and E II that conformed to wild pig from Europe [13,14]. In these two clades, it was possible to observe that the majority of the sequences were of Pillareño; the network (Figure 2) displayed four principal haplotypes (H_1, H_2, H_3, and H_4). H_1 was the main haplotype and was composed of 23 individuals and three haplogroups. The dominant haplogroup corresponded to the Pillareño Creole pig samples, and the other two haplogroups corresponded to Iberian pigs and Spanish wild pigs. In the periphery of these haplotypes, we observed that they were surrounded by unique sequences of European domestic pigs and the haplotype H_2. H_2 is related to H_1 and was formed by the Iberian domestic pig haplogroup. The H_3 haplotypes contained European domestic pig samples. Haplotype H_4 comprised two haplogroups, which consisted of three individuals belonging to Pillareño Creole pig and two European individuals (Iberian and Duroc). H_2, H_3, and H_4 presented a star-like profile, consistent with the pattern of population expansion in the past.4. DiscussionThere is a new perspective regarding the use of adapted local breeds in programs of rural development that involves important concepts of sustainability, food security, and food sovereignty; even so, the Creole pigs are in extreme danger of extinction, owing to a severely reduced population. Ecuador is a good example in that this country has developed important policies for the economic growth of the poorest regions with Afro-American and indigenous populations. These recent programs have involved chickens [15], goats [16], and pigs [17].In this study, we present the first results to understand the evolution of the population of Pillareño Creole pig; the analysis suggests a moderate level of haplotype diversity (0.615) that represents the probability that two randomly sampled alleles are different, while the low nucleotide diversity (0.00968) represents the average number of nucleotide differences per site according to the analysis of generated sequences [18,19]. These two results are Hd and π, indicators that the Pillareño Creole pig population originated from a small number of founders [20], showing that the populations had in their evolution the effect of a bottleneck, producing genetic drift or changes in genetic sequences from the ancestors in Europe. These results differed from those reported for Alves [21], wherein pig populations of Iberian and maternal lineages preserved in the Torbiscal line were characterized and showed six mtDNA and 12 mtDNA haplotypes, respectively, but similar results reported in Mexican Pelon Creole pig [22] showed nine haplotypes. The negative value of Tajima’s D indicated an excess of rare haplotypes over what would be expected under neutrality and a signature indicating recent population expansion. Hence, the positive Fu’s Fs test results signified low levels and high frequency polymorphisms, indicating a recent decrease in population size and balancing selection [23]. These findings support the origin of the Pillareño Creole pig in those animals brought by the Spaniards in the conquest of Ecuador, but the sequential bottleneck produced a differentiated mitochondrial DNA profile.In the colonization of America by domestic animals, pigs were incorporated early because these animals were carried on the boats to prevent scurvy due to vitamin C deficiency through use of pig meat as a protein supply of animal origin [24].In the Table 3, is possible to observe that the Pillareño Creole and all the Asian populations showed genetic differences and variation with the European population, and this was possible because the Iberian populations contributed to the development of the Pillareño Creole [25]. It is also possible to observe the low mitochondrial genetic differences, assuming the complete influence of these populations in the Pillareño Creole pigs [26,27]; at the same time, the Spanish wild population showed a low-to-moderate distance from the Pillareño Creole population, likely because they mated with the European wild boar. Phoenician pigs are mentioned in historical reports as having been brought to southern Europe and the Iberian Peninsula by the Romans, and continued attempts to improve the husbandry and breeding of the native pigs contributed to the rise of the modern Iberian pig [28]. The coancestry coefficients (Table 4) are useful because they show how much the genomes of two individuals are expected to resemble each other [12], and the FST results for Pillareño Creole pig showed this population was preserved and was not influenced by commercial populations. This information was inferred from the neighbor-net results obtained (Figure 1). This is evidence for the purity of this breed in spite of the pressure from commercial lines of foreign breeds, and therefore this pressure has produced, at least at the maternal level, a reduction in the Pillareño population but not an indiscriminate crossbreeding with exotic breeds.In the genetic differentiation and the distribution of genetic variation by the AMOVA analysis, genetic differences were observed within populations, indicating that the populations of European, Pillareño Creole, and Iberian pigs shared common haplotypes and a maternal lineage, and it was also observed that the variation of mtDNA between populations and the proportion of unshared haplotypes in each group was significant and variable [29,30]. This supports the historical theories about the primary origin of the Latin American Creole pig populations.Very few studies of mtDNA have been performed in Creole pig populations; it is worth mentioning that we were able to find a distinctive Iberian genetic signature in all the Ecuadorian Creole pig haplotypes. This finding might be explained by the fact that the Iberian populations had a strong phylogeographic structure at the time of American colonization [31]. The population of this study was detected in the clade E I and E II, corresponding to the Iberian clades [21], and with clear evidence of separation from the Asian populations, with the exception of five individuals that presented a haplotype in Clade A.Accordingly, the presence of this individual in the Asian clade could be a possible explanation for the introgression of common Asian commercial lineages associated with Clade A or the historical influence of Philippine pigs [27] introduced more recently from their colonies in Asia by the Spaniards, shipped in the so-called “Navío de Manila”, which navigated the Pacific Ocean from the Philippines to the Pacific coast of America. The majority of Ecuadorian Creole individuals were of the principal haplotype. They shared haplogroups with wild Spanish individual and Iberian pigs, having a matrilineal relationship, because the Iberian pigs are currently genetically interacting with the Spanish wild boar, and for this reason the wild Spanish pig shares this haplotype [28].5. ConclusionsAll analyses and the expected level of genetic similarity of mtDNA showed moderate haplotype diversity and low nucleotide diversity among the Pillareño Creole pig population and indicate that the populations of Creole pigs from Ecuador have directly descended from an Iberian ancestral population from Spain. Over the time, these populations then thrived and became stable with the correct animal breeding, and this genetic inheritance has been preserved throughout the years.The evolutionary linkage of the Pillareño Creole pig with the Iberian pig suggests the population is an important source of sustainable richness and has the genetic base to follow the Iberian pig model of production according to the quality of the products and with respect for the environment.Pillareño Ecuadorian Creole populations from rural communities in Ecuador were grouped in nine haplotypes with Clade EI and EII, which have not previously been reported for Creole populations; hence, this study could be considered a model for future research into other Creole pig populations of the American continent.

animals : an open access journal from mdpi

10.3390/ani13081358

PMC10135182

Animal welfare policy regarding husbandry practices in sheep in Australia differs between states and territories. This dis-uniformity of the legislature can be confusing and limit the application of the law, particularly with growing pressure from the local and global community to improve animal welfare. The influence of scientific evidence contributing to the development of these policies is unclear. This article explores the Australian animal welfare legislature and the scientific evidence informing husbandry practices commonly performed at lamb marking.

The development and substance of animal welfare policy is subject to a range of social, cultural, economic, and scientific influences that commonly vary within and between countries. Discrepancies in policy can create confusion and mistrust among stakeholders and consumers and limit the ability to create a uniform minimum level of requirements to safeguard animal welfare, as well as create a level ‘playing field’ for farmers when trading with other jurisdictions. The livestock sector is receiving growing scrutiny globally for real and perceived violations of animal welfare, for example, the practice of mulesing in Australia. This article explores animal welfare legislation within Australia and how it reflects the scientific evidence surrounding routine husbandry practices in sheep, including tail docking, castration, and mulesing. While there is some variation between state and territory legislation, the most notable concern is the lack of enforceable recommendations surrounding the evidence-based use of analgesia and anaesthesia for painful husbandry procedures. The age at which these procedures are recommended to be performed is relatively consistent across Australian jurisdictions, but there is a marked difference compared to international legislation. The global context of animal welfare legislation, public perception, and producer perception of these procedures are also discussed, highlighting the difficulty of creating robust animal welfare legislation that promotes a good standard of welfare that is respected worldwide whilst being practical in an Australian setting given our unique geography and climatic conditions.

1. IntroductionThe farming of animals, once widely accepted by society, is now under growing scrutiny as animal production becomes more intensive and social attitudes toward the use of animals changes. This scrutiny comes from numerous groups of differing social, scientific, and political backgrounds. These groups include animal rights organisations, farming stakeholders, companies reliant on trade markets, the public, animal welfare scientists, veterinarians and animal health professionals, and politicians representing local, state, or federal interests [1]. The influence of these groups on the overall welfare of the individual animal can be substantial and may be beneficial or detrimental. Balancing the interests of these groups and the welfare of the relevant animals can be delicate and fraught with conflict. Legal frameworks for animal welfare protection should provide the scaffolding upon which a consensus can be reached. This consensus should optimise animal welfare based on available scientific evidence and meet socially respected requirements to promote a high standard of production and welfare and maintain a social licence to operate. The reality is often somewhat different and can depend largely on prevailing economic and political interests at the time. It is worth noting that the very existence of an animal welfare legislative framework supposes a utilitarian approach to the use of animals in society [2]. Animal use for human benefit is allowed, provided there are conditions in place to minimise suffering and promote welfare. However, it is the balance between what level of suffering is ‘reasonable’ or ‘necessary’ that may be contested, and factors that play into this equation are not purely based on animal outcomes but may be human-centric such as practicality and economic feasibility [2,3].Notwithstanding the need for policymakers to balance multiple, often competing interests, it is generally considered (and stated) that legal provisions have a basis in evidence. In considering issues of animal welfare, it would be assumed that this evidence is derived from animal welfare science [4]. However, there has been little examination of the extent to which Australian legislatures incorporate animal welfare science into policy, the extent of the uniformity of this incorporation across Australian jurisdictions, and how this contrasts with international policy. The latter has become of greater importance of late with the creation of trade agreements, for example, the Australia–United Kingdom Free Trade Agreement, where, in the animal welfare context, a country’s treatment of their animals may be highlighted on the world stage [5]. The recent signing of this agreement caused controversy in the UK due to the perceived lower standards of animal welfare in Australia owing to the continued practice of mulesing in Australia. In addition, several surveys in Australia and internationally have highlighted inconsistencies between consumer and farmer perceptions of mulesing [6] and other husbandry procedures and the relevant legislation [6,7,8,9]. A similar concern may arise with one of the world’s biggest trading blocs, the European Union (EU), in the forthcoming trade agreement [10].In this article, we tackle the question of the linkage between science and animal welfare policy using a case study approach based on the practice of lamb marking. We do this by examining the available science around specific aspects of lamb marking to understand the extent of the weight of this evidence. The subordinate legislation related to this practice across the states and territories is then sourced for relevant provisions and to assess uniformity across these jurisdictions. Policy from selected international jurisdictions is also examined as a comparator. We then discuss the extent of the linkages of policy principles identified with the established science. We also explore the perceptions of farmers surrounding these procedures, as this is a driving influence on compliance with any legislative changes or evidence-based recommendations on husbandry procedures. Finally, we briefly discuss the challenges associated with assimilating science into policy and how this might practically be achieved. We conclude with the extent to which the Australian system appears to have achieved this in the case of lamb marking.2. What Is Lamb Marking?Marking is the common term used to describe practices to identify young stock and perform early procedures aimed at maintaining flock health and productivity. The procedures performed vary depending on the market, management style, tradition, culture, and the environment. In Australian systems, lambs are typically ear tagged with property ID and vaccinated against a variety of infectious diseases depending on the management system. Surgical procedures may also be performed at these times according to the production style and farmer preference. These procedures most commonly include tail docking, with or without mulesing, and castration in males not intended for breeding. Tail docking is the amputation of part of the tail and can be performed by applying a tight rubber ring to the tail, which induces ischaemic necrosis and sloughing of the tail; cutting the tail at the desired length between the vertebrae; or cutting the tail with a heated sharp knife to cauterise the wound after incision. Mulesing is the removal of skin around the perineum and tail. This technique is only performed in Australia and most commonly involves using a sharp knife or mulesing shears [11]. Castration is the removal of the testicles by either applying a tight rubber ring to the neck of the scrotum or using a clean, sharp knife to incise the scrotum and remove the testicles. Immunocastration is a newer alternative to traditional methods of castration that does not cause pain [12,13]. This method blocks the normal functioning of the hypothalamic–pituitary–gonadal axis by administering two doses of vaccine against gonadotrophin-releasing hormone (GnRH). Immunisation against GnRH results in the suppression of testosterone production and spermatogenesis [12,14]. While this technique has been widely adopted in pigs, there is currently no licenced product in sheep [13,14,15].Castration has been recorded as early as the 3rd millennium BC, while tail docking is less frequently described historically but thought to have become more widely used with the selection of sheep for longer and finer fleeces, which were more prone to accumulation of faecal matter and urine staining. Archaeological evidence of docking exists from the 13th century, and the procedure is thought to have become routine by the 18th and early 19th centuries; the agricultural revolution and popularity of the Merino breed played a major role in the widespread use of the technique [16].The reasons for performing these procedures are historically much the same as they are today; castration is typically performed to prevent unwanted breeding, reduce aggression, improve stock person safety, and improve meat quality [12,17]. Castration techniques that remove the scrotum can also reduce the risk of flystrike and carcass contamination due to faecal matter building up on the scrotum [18]. Tail docking and mulesing are primarily performed to reduce dag formation (accumulation of faecal matter around the tail and hindquarter or breech) and urine staining in an effort to reduce the risk of breech flystrike [19]. Cutaneous myiasis, commonly known as flystrike, is the infestation of a wound by maggots and flies; it is a very painful condition and can result in death or significant morbidity [19]. These procedures can cause significant pain and distress associated with physical tissue injury, handling stress, and temporary separation from the dam (mother of the lamb) [20,21]. The age at which these procedures occur, the technique, and the analgesic or anaesthetic regimen vary between production systems and farms both within and between countries. All of these factors will influence the duration and severity of pain experienced by the animal. Our current understanding of the degree of influence these factors have on pain experience is limited. There is growing evidence from the human and rodent literature [22] suggesting longer-lasting effects that we have not appreciated as yet and have not been considered in legislative decisions. In the following sections, we will discuss the scientific evidence on the impact of different methods of performing common husbandry procedures, the ages at which they are performed, and associated pain mitigation strategies.3. Animal Welfare Legislative Framework in AustraliaAustralia is a federation of six states and two territories (The Commonwealth of Australia Constitution Act 1900 (The Constitution)), with laws at federal, state, and local government levels. The Australian Constitution (s 51) is silent on animal welfare, and thus, it is considered a residual power for which the eight Australian state and territory governments are responsible. The only exception to this is when animal welfare may be considered as part of an aspect of trade or biosecurity under the Federal government’s exclusive powers around trade and commerce and quarantine (s 51 (i), (ix))—a key example being regulation of the Australian live export trade.As a result of these constitutional limitations, the Australian animal welfare legal framework consists of primary state and territory acts and delegated legislation. The former are overarching and general and provide the key offences. The main offences are a prohibition on being cruel to animals and the creation of a duty of care for owners of animals to provide for their welfare [23]. Subordinate legislation in the form of regulations, codes of practice, and standards are then used to provide greater technical detail on a species, type of production practice, or controversial issues [23]. Provisions written into regulations are usefully directly enforceable, with offences being directly associated with the provisions. These documents, therefore, have a greater legal weight than the so-called “soft” law or quasi-delegated legislation represented by Codes of Practice or Standards. Codes of Practice have a lower legal weight and ability to enforce, and their legal status varies considerably across jurisdictions. Their legal enforceability is also dependent on whether they are a compulsory or voluntary code of practice. For example, in South Australia (SA), a breach of a prescribed Code of practice provision is directly enforceable and subject to a penalty via Reg 5 (Animal Welfare Regulations 2012). Alternately, in some states, e.g., Victoria, compliance with a POCTA code merely provides a defence to a prosecution for cruelty under the enabling act. Voluntary codes in all states work similarly by assisting in the defence or prosecution of a cruelty charge in court. Codes may be incorporated into law by a variety of means. The most common method is either via direct referral in the regulations or by being listed as a prescribed code via a schedule (usually to the regulations, see, e.g., SA). A less common way of making them the law is through administrative means by referral in licence conditions around certain businesses. This method is commonly used in the regulation of animal slaughter [24]. From this brief background, the reader might already get the sense of how the different legal weight placed on these Codes can create a disparity between the jurisdictions with a scenario potentially existing where states are using the same document, but its enforceability varies due to its method of incorporation into the legislative framework.3.1. History of Delegated Legislation around the Livestock IndustriesDuring the 1800s, the states and territories introduced laws on animal welfare and animal cruelty offences based on equivalent regulations in Britain. At Federation in 1901, the states retained responsibility for those functions by virtue of the signing of the Constitution [25]. The 1960s saw the rise of animal rights advocacy, and the public’s attention was drawn to the conditions of animals used in intensive farming systems. The practice of mulesing lambs, debeaking chicks, and tail-docking piglets without pain relief was widely publicised [26]. The Australian export wool trade grew during the 1980s but saw a backlash from wool garment manufacturers and consumers against sheep that were mulesed. Australia’s sheep regulatory agencies aimed to develop consistent standards to reflect changing attitudes towards farm animal welfare [20,27].Model Codes of Practice for the Welfare of Animals (MCOPs) were developed in the early 1980s with a focus on livestock. Their development was driven by the desire to provide consistent husbandry guidelines for all farm animals so that both domestic and international markets were assured of the welfare considerations made during the production of animal-sourced commodities [23]. However, in spite of good intentions to harmonise, each jurisdiction’s approach to the use of the Codes differed; some adopted them in their entirety, others modified them, whilst some chose not to adopt them at all [26].These Codes were updated during the 2010s into the Australian Animal Welfare Standards and Guidelines (for all livestock species), overseen by the Primary Industries Ministerial Committee (PIMC) and in conjunction with each State’s department responsible for the Animal Welfare Act. Whilst the standards and guidelines are usually presented as one document, there is an important distinction between a standard and a guideline in these documents. The standards are the basis for developing consistent legislation across Australia and use the word “must”; hence, provided adopted by the states, they represent the legal requirements. Guidelines are recommended practices to achieve good welfare, and non-compliance will not constitute an offence under the law. Rather than eight different animal welfare regimes, the aim of these documents was to have [27]: “national standards of livestock welfare that are consistently mandated and enforced in all states and territories.” This national approach was to provide quality assurance and cost benefits for the primary industries and reflect modern animal welfare expectations from consumers [27].In 2018, the Australian Productivity Commission (PC) issued a report on the regulation of Australian agriculture, including the management and welfare of farm animals [28]. It proposed the establishment of a federal Australian Animal Welfare Agency (AWAC) to oversee a nationally consistent approach to farm animal welfare and noted the importance of looking to scientific evidence and ethical values in setting industry standards. The Productivity Commission recommended the formal adoption of the Animal Welfare Standards and Guidelines endorsed by the Primary Industries Management Committee within each state and territory through the incorporation of these standards into their respective animal welfare laws [26]. However, this recommendation was not adopted federally, with the government’s response being to reiterate that the responsibility for animal welfare regulation, compliance, and enforcement fell to the state and territory regulators [28]. As a result, state Codes continue to vary in their recommended practices and currency.3.2. Animal Welfare Laws and Codes of Practice for SheepEach state and territory has its own animal welfare or prevention of cruelty to animal acts [29], which enable either general or specific regulations and any compulsory Codes of Practice.Subordinate legislation in the form of regulations is enabled under each act and is updated by the relevant responsible government department as necessary. As described earlier, these documents get their legal force via different mechanisms: direct referral or attached to schedules. Some states have elected to put Code or Standard provisions into their regulations directly to increase their legal weight. This is the scenario in SA where the sheep Standards and Guidelines have been incorporated into the regulations. New South Wales (NSW) and the Australian Capital Territory (ACT) have adopted the Standards and used the actual document, i.e., not amended it. Whilst Queensland (Qld) bases its own Code on the Standards. The Northern Territory (NT) does not have a code of practice for sheep. It is also worth noting the legal status of these documents. In the ACT, Victoria (Vic) and Tasmania (Tas), these are voluntary or advisory documents that are not directly enforceable but may be used for evidentiary purposes in court (Table 1).4. Tail Docking and Castration—Science and PolicyTail docking and castration are common husbandry practices in Australia [18]. The utility of tail docking is largely environment- and breed-dependent, and the need to carry out docking should be assessed based on unique climate and management conditions [36]. Similarly, the need for castration is based on management conditions and may not be required when lambs are marketed for slaughter prior to puberty, which typically occurs at 3–6 months of age [18,37].There is conflicting evidence linking undocked sheep with an increased risk of breech flystrike [36,38,39]. Scobie et al. [40] found that dag formation was dependent on seasonal and management factors, and the length of the tail did not alter the risk of flystrike. Watts and Marchant [41] compared lambs docked either at the third palpable tail joint or as short as possible, finding that flystrike was far more common in short-docked sheep. Flystrike is more common in warm and wet weather, particularly in breeds with wrinkled skin and wool- or hair-covered breech [19,42]. Other factors, such as parasite burden and nutritional imbalance leading to diarrhoea, can increase the risk of flystrike [38].Management of these factors plays an important role in the prevention of flystrike. In regions where there is a higher risk of flystrike, tail docking at the third palpable joint has been associated with the least amount of dags and urine staining in ewes [40,43]. Tail docking at the third palpable joint, which is equivalent to the length of the vulva, is also widely recommended as the optimal length to reduce the risk of vulval cancer, bacterial arthritis in lambs [44], and rectal prolapses and to maintain rectococcygeal muscle integrity [43,45].Tail docking and castration are commonly studied in conjunction and are discussed together. Surgical castration and tail docking are rarely used in Australia. In 2016 only 3% and 6% of surveyed Australian sheep producers still used a sharp knife for castration and tail docking, respectively [46]. Surgical methods are associated with a higher risk of complications, such as haemorrhage, compared to bloodless methods, such as the use of rings. There is also evidence of a greater physiological stress response with surgical castration and tail docking, with these procedures causing a greater and more prolonged increase in cortisol compared to rubber ring castration and tail docking [47,48,49]. Mellor et al. [50] conducted a review of castration and tail docking techniques, using cortisol response to rank the severity of commonly used techniques, including surgery, rubber ring, and hot iron tail docking with and without analgesia. They recommended that surgical methods be phased out in preference of ring methods, ideally with local anaesthetic instilled prior to ring application [50]. In contrast to this, a number of studies assessing behavioural signs of acute pain following husbandry procedures indicate that castration and tail docking with rubber rings causes a greater and more prolonged negative welfare impact compared to surgical methods [21,51,52,53,54,55]. Lomax et al. [54] used nociceptive threshold testing to compare wound sensitivity in 6–12-week-old Merino lambs that had been surgically castrated with or without topical anaesthesia, revealing significant primary and secondary hyperalgesia for at least 4 h after the procedure in the castrated lambs that had not received analgesia. Allodynia around the castration site was not identified in any of the lambs. In this study, lambs that were tail docked with a sharp knife and received no analgesia developed allodynia at the tail wound site 4 h after the procedure, and all surgically docked lambs, regardless of analgesia, developed primary hyperalgesia at the wound site. Lamb’s tail docked with a hot iron showed no evidence of primary hyperalgesia or allodynia up to 4 h after docking. The application of a local anaesthetic to the hot knife wound reduced tail wound sensitivity from baseline levels [54]. Analysis of acute pain-related behaviours between groups of lambs that were either surgically castrated and tail docked, surgically castrated and hot knife docked, or rubber ring castrated and tail docked demonstrated a marked increase in behaviours in the ring group that dominated their experience to the point where nociceptive threshold testing could not be performed. There was no significant difference in pain-related behaviours between handled control lambs and lambs that were surgically docked or docked with a hot iron and had local anaesthetic applied at the time of the procedure [54].Other bloodless methods of castration include use of various clamping instruments to crush the spermatic cords and testicular blood supply, inducing ischaemic necrosis. One of the commonly studied castrators is the Burdizzo. The use of this device in combination with the rubber ring reduced the length and duration of behavioural signs of pain and cortisol response in lambs compared to ring and surgical castration [50,56,57]. Despite strong evidence that the combination of Burdizzo castrators applied proximally to rubber rings reduces pain following castration, they have not been adopted widely because they are technically difficult to use, there is a higher risk of procedural failure, and they increase the time taken for marking [58].There is strong scientific evidence that local anaesthetics injected subcutaneously or applied directly to the wound reduce behavioural and physiological sings of acute pain following tail docking and castration in lambs from 2 days up to 12 weeks of age [59,60,61,62]. Non-steroidal anti-inflammatories (NSAID) have also been shown to reduce some pain behaviours and physiological signs of pain following castration and tail docking [63,64]. Small et al. [65] found significantly lower lamb mortality from marking to weaning in ring-docked and castrated lambs treated with the NSAID meloxicam compared to those that received no form of analgesia. This study did not have a handled control group, and the causes of lamb mortality were not recorded, which limits the conclusions that can be drawn. However, lamb losses are a significant welfare issue and economic burden in the sheep industry, and further investigation of this finding is warranted.Both local anaesthetics and NSAIDs reduce acute pain but do not completely ameliorate it, and they do not address chronic pain associated with tail docking and castration [66,67]. Hyperalgesia at the tail docking site following hot knife docking can last for at least 3 months [67]. Currently, there are no pain mitigation options addressing the chronic component of pain associated with these tail docking and castration.A 2016 survey of Australian sheep farmers found that 97% of producers use rubber rings for castration. The same survey found that the selection of tail docking method changed depending on the production system, with most wool producers electing to use a gas knife (78%), whereas meat producers tended to prefer rubber rings (65%). However, it is likely that there are also some state-by-state differences, with gas knives being more commonly used in WA and SA (74% and 75%, respectively) compared to other Vic, Tas, Qld, and NSW (45%, 59%, 33%, and 49%, respectively). Qld farmers reported the highest proportion of farmers using a sharp knife (28%) [46].4.1. Tail Docking PolicyIn considering policy around tail docking, there is inconsistency across the jurisdictions around the need for a certain length of tail. This may reflect the difference in minimum standards and guidelines advised by the AHA Animal Welfare Standards and Guidelines [30]. The Qld and SA codes state the required minimum standard, which is to leave a tail stump of at least one palpable joint, whereas the Tas, WA, Vic, and NSW codes require tail length to be long enough to cover the vulva in ewes and be a similar length in males, which is equivalent to three palpable joints. This fits with the scientific evidence around tail docking length and is in the guidelines advised by AHA [30]. There is also inconsistency in the recommended age for procedure performance, with some states not providing any guidance on this other than the age at which anaesthesia must be used. The guidelines recommended by AHA advise tail docking to be performed as early as possible and before 12 weeks of age. Of particular interest in relation to this is that SA and Qld, states that have adopted the Standards and Guidelines, appear to have made a deliberate omission of this recommendation in their laws. The recommended age for tail docking across most of the states is between two and twelve weeks and not until at least 24 h old to allow for parental bonding. It is also worth noting that the newer standards and guidelines endorse the performance of this procedure at an earlier age (from 24 h) in comparison with the older state codes from Vic and WA. Pain relief and/or anaesthesia are only required for lambs over six months of age (Table 2). The guidelines recommend that suitable pain relief is used when practical and economically feasible despite strong scientific evidence that tail docking is painful, even in very young lambs [51], and that analgesia mitigates acute pain following tail docking [52,54,60,63]. Other than the requirement of anaesthesia in sheep over 6 months of age, there is no recommendation to use analgesia for younger lambs in any of the state codes (Table 2).4.2. Castration PolicyMost codes recommend castration take place as early as possible, generally between 24 h and 12 weeks. Analgesia or anaesthesia is required only if the ram is over six months. Recommended methods are either by rubber rings or cutting, although the Standards and Guidelines suggest an “appropriate tool that causes the least pain” (Table 3). This flexibility in the choice of the method provided by policy likely reflects the controversy around the relative welfare impact of the methods, with no method conclusively being shown to have less animal impact. South Australia’s Regulations do not specify a recommended age or method, again an interesting observation since this State has adopted the Standards and Guidelines which are not silent on these matters.5. Mulesing-Science and PolicyMulesing was developed in Australia in the late 1920s by a grazier called John Mules. The procedure involves surgically removing the wool-bearing, wrinkled skin around the perineal region to enlarge the bare area of the breech and prevent the build-up faeces and urine in the wrinkles, thus reducing the risk of breech flystrike [20,68]. At the time of development, the Australian Merino sheep industry was struggling with a significant increase in flystrike-associated morbidity and mortality due to the breed’s wrinkled breech, favourable Australian weather conditions for flies, and the introduction of the fly Lucilla cuprina [20], which accounts for at least 90% of all strikes [69]. Mulesing was a cheap, fast, and effective method of reducing the risk of breech strikes, and the popularity of the procedure gradually grew, with 70% of Australian Merino producers mulesing their ewe lambs in 2017 [70]. Flystrike remains a significant issue for the Australian sheep industry, costing just over $323 million AUD in prevention, treatment, and production losses annually [69]. It is worth noting that this is a particular issue with the prevalent Merino breed used in Australia for wool production due to the amount of wrinkling. If other breeds were used, the problem would no doubt be reduced. The procedure has received global scrutiny for its negative impacts on welfare and is banned in most countries, with our close neighbours New Zealand banning the procedure in 2018 [71]. Phasing out mulesing in New Zealand was largely industry-led and took roughly 5 years [72]. Differences in the Australian climate, wool industry, predominance of the Merino, and larger enterprises compared to the New Zealand wool industry have substantially delayed the phasing out of mulesing in Australia [72]. Nevertheless, Australian livestock industries are working towards phasing out mulesing through research into alternative ways of preventing flystrike, including breeding programmes to reduce wrinkle scores [21,68] and developing extension strategies to educate and support producers transitioning to non-mulesing operations [72]. Flystrike remains a major concern, and the risk of flystrike is expected to increase with the emergence of chemical resistance, limiting the efficacy of chemicals used for prevention and treatment [68,69]. It has also been suggested that the distribution and abundance of the fly population may increase with climate change [68], thus increasing the risk of flystrike. This highlights the importance of continued support for research investigating flystrike prevention and treatment to foster a sustainable sheep and wool industry that is able to meet consumer demands and maintain the social license to operate [72].Producers commonly use mulesing shears to remove the skin around the breech and on either side of the tail, leaving an open wound that heals by secondary intention leaving a wool- and wrinkle-free area [20]. This procedure is widely known to cause considerable pain that can persist for days to weeks [73,74,75,76]. Surgical mulesing elicits marked changes in physiological and behavioural markers. Behavioural indicators of pain after mulesing include statue standing, hunched posture, reduced lying behaviour, longer time to mother up and feed, reduced grazing behaviours, and an aversion to the handler [55,74,75,77,78,79,80]. Significant elevations in cortisol have been demonstrated in lambs aged from 5–40 weeks of age [73,74,75,76,81]. Other physiological markers of inflammation and pain, including haptoglobin, neutrophil/lymphocyte, and beta-endorphins increase dramatically [73,74,75]. Likewise, numerous studies have demonstrated a reduction in average daily weight gain for the week following mulesing [75,76,77]. While the use of analgesics reduces the physiological effects and behavioural aberrations associated with surgical mulesing, they do not abolish them and do not address any chronic effects [82]. A combination of a non-steroidal anti-inflammatory drug (NSAID) and topical anaesthetic and antiseptic has been shown to provide the most effective pain relief [83]. Registered products available for mulesing in Australia are Metacam®â, an injectable form of the NSAID meloxicam, Buccalgesic®, an oral formulation of meloxicam, and Tri-Solfen, which is a gel-based spray containing lignocaine, bupivacaine, adrenaline, and cetrimide. Administration of Tri-Solfen® and either form of meloxicam at the time of mulesing significantly reduced pain-related behaviours over the first 24 h post-procedure [80,83]. A multimodal approach to analgesia has been shown to be superior to the use of sole agents [81,83,84].Various non-surgical alternatives to mulesing have been trialed with little or no improvement in welfare or flystrike prevention over the surgical mulesing technique. These include clips to induce ischaemic necrosis around the breech and later sloughing of the tissue, injection of chemical agents (sodium lauryl sulphate, cetrimide) to induce scar formation around the breech or application of liquid nitrogen to the breech resulting in necrosis of the skin [75,76,77,78,85]. Other strategies of flystrike control, such as breeding for reduced breech wrinkling, preventing scouring, management practices including regular crutching and appropriate use of insecticides, and managing the fly population are more viable long-term options that will maintain the social licence and the marketability of Australian wool and meat products globally [19].Most states require lambs to be over 24 h old before mulesing and recommend between two and twelve weeks of age (Table 4). In spite of the common recommendation for the procedure to be performed between 2 and 12 weeks, it is noteworthy that all states essentially allow the procedure in animals up to the age of 12 months, with WA having no upper cap on age. This large window for performance may reflect the practicalities of performing this procedure, for example, in acquiring the services of accredited mulesing contractors. In some states, there is guidance around operator competency with detailing around knowledge/experience required or supervision needed. Only Vic requires that operators have received some form of accredited training in order to perform the procedure. Victorian guidelines also require analgesia for all sheep, plus anaesthetic if over 6 months, although it is important to remember that this is a voluntary code, so this may not actually be the routine practice in this state (Table 4).6. Veterinary Legislative FrameworkWhilst the veterinary legislative framework is not focussed on welfare, it may provide useful guidance around the perceived severity of these procedures. These procedures are listed within the veterinary legislative framework in all states (with the exception of Vic) in the context of defining an act of veterinary medicine or surgery (Table 5). In all states, the performance of an act of veterinary medicine without being a registered veterinary professional constitutes an offence under the Act. Across the states, these two frameworks are generally consistent in terms of legality, e.g., if a person complies with one framework, they are unlikely to fall foul of the other, perhaps with the exception of SA (see table for detail). In general, the veterinary frameworks are less conservative with respect to age limits on procedures; there is a broader range of early ages considered before they become acts of veterinary surgery. This is not unsurprising as these documents are not aimed at farmers and do not dictate routine husbandry practices. However, they show broad consistency across the states with the proposition that castration and tailing of sheep over 6 months and mulesing over 12 months represent a procedure that should be performed by a veterinarian. This points to an appreciation that by this age, a more advanced level of surgical skill may be required, and of course, access to analgesic or anaesthetic drugs.7. DiscussionRegularly reviewing the need for these procedures and available alternatives will benefit not only animal welfare but also the economics and efficiency of the relevant farming enterprises. This is particularly important in the current climate, where the welfare impacts of these procedures are being challenged by consumers and the wider public. The long history and culture surrounding these procedures result in skills and techniques being passed down across generations, sometimes with little change or consideration of newer techniques or improvements. A survey of Australian sheep farmers found that 20% of farmers stated that their father had played a substantial role in their attitude towards animal welfare [6]. This transfer of skills is highly valued within farming communities and should be respected and considered as recommendations and standards are updated. It is important to also consider a science-based approach to guideline formation, and indeed transparency of this approach, i.e., it is clear to reviewers what the scientific basis of the recommendations is. This is a key component of clinical practice guideline formation in evidence-based medicine, and it is surprising that clear referencing of the science has not been more extensively adopted into the often-controversial animal welfare policy space. A brief discussion based on our observations on the linkage between science and law in this policy area follows.7.1. Age at MarkingRecommendations on the age at which husbandry procedures should be performed in lambs vary moderately across Australian state and territory legislation. However, recommended age limits globally are considerably different [94]. This is likely to lead to some confusion among producers and the public, particularly in relation to international trade. There are three ages that are commonly cited in legislation internationally as limits for performing painful husbandry procedures: less than 7 days old, less than 12 weeks old, and less than 6 months old. For example, rubber ring tail docking is only permitted up to 7 days of age in England [95] and Wales [96], whereas in Canada, it is recommended to be performed before 7 days of age and prohibited over the age of 6 weeks [97]. In Scotland, tail docking can be performed up to 3 months before veterinary oversight is required [98]. New Zealand recommends tail docking be performed before 6 weeks of age [71]. This variation appears to reflect the prominent production system in the relevant regions but fails to reflect the scientific reasoning behind such choices. Consideration of the production system is, of course, vital to creating recommendations of practice, as often these systems have developed over the course of centuries and techniques have been learnt and passed down through generations in response to the local environmental and cultural factors. A 2016 survey of Australian sheep farmers found that most lambs were castrated and tail docked at an average of 6.5–6.7 weeks. No farmers surveyed reported docking over 6 months and only 5% of farmers reported docking lambs over 3 months [46]. This reflects the common Australian practice of mustering all ewes and their lambs together for marking at one timepoint after the end of lambing when lambs are all at least 2 weeks of age, thus reducing the stress of repeat handling. Here, we discuss the research exploring the influence of age on response to husbandry procedures and how this may inform the legislation listed in the previous section.Research comparing tail docking and castration in lambs of different ages has repeatedly demonstrated a significant increase in pain behaviours and physiological measures of pain across all studied age ranges [47,50,51,66,99]. Kent et al. [47] and Molony et al. [51] measured cortisol and behavioural changes acutely after rubber ring, surgical, or rubber ring and burdizzo castration in 5-, 21-, and 42-day-old lambs. In all ages, all methods caused a significant increase in cortisol from baseline, with the peak occurring earlier in surgical and ring with burdizzo methods and roughly 10 min later in rubber ring castration and tail docking. The change in cortisol was significantly greater in the 5-day-old lambs after rubber ring castration compared to the older lambs. Kent et al. [66] compared active pain behaviours, and scrotal lesion width and degree of swelling following castration with a rubber ring in 2-day and 28-day-old Suffolk or Dorset cross lambs and 42-day old Scottish Blackface lambs to assess chronic inflammatory responses and long-term pain of this procedure. In the younger lambs, the scrotal lesions were smaller and healed more rapidly than in the 42-day-old lambs, and they were less likely to become septic. There was a significant relationship between the increase in active pain behaviours and the change in lesion score and size only in the 42-day-old lambs and not the 2-day and 28-day-old lambs [66], suggesting that the older lambs tended to suffer from larger and more painful lesions than the younger lambs. Electroencephalography has provided another method of assessing pain in lambs following husbandry procedures. Using electroencephalography while lambs were under halothane anaesthetic, Johnson et al. [100] and Johnson et al. [101] found that older lambs had a more pronounced cerebro-cortical response to castration with a rubber ring than younger lambs. In the first 10 days of life, the magnitude of the cortical response to noxious stimuli (castration with a rubber ring) increases rapidly [101], suggesting that the perception of noxious stimuli in younger lambs is different and likely less pronounced compared to older lambs. However, these studies only assessed male lambs and pain perception, and the consequences of early life pain may differ between sexes [102,103]. Whilst it generally appears that procedure performance at an earlier age is beneficial, there is a growing body of research demonstrating longer-term effects of painful procedures during early development in a range of species, including lambs [104,105,106], humans [107], and rodents [103,108]. These findings point towards the need to consider pain relief in animals of all ages to avoid later negative consequences.Our understanding of the perception of pain in infancy in humans has changed significantly over time. Historically, there was a general acceptance that infants felt pain, and efforts were made to alleviate that pain by ancient physicians and philosophers; this social dogma changed to a denial of the clinical significance of infant pain which lasted throughout most of the 20th century [109]. It was thought that babies and young animals did not perceive or suffer from pain in the same way adults do due to an underdeveloped nervous system. Additionally, the risk of anaesthetics and analgesics in these patients was seen to outweigh the seemingly limited benefits. Consequently, numerous painful surgical procedures were performed without any form of anaesthetic or analgesic [110]. Further research has revealed poor pain management during early life can have a range of negative repercussions on pain sensitivity, cognition, social interaction, mood, and stress resilience in later life [94,109]. There is also evidence of intergenerational effects of early-life pain in rodents [111] and sheep [106]. Considering these negative consequences of early life pain identified predominantly in rodents and humans, it is worth investigating further in other species. Farmed species, such as sheep, are exposed to numerous painful procedures very early in life and are often at a relatively high risk of infection and inflammation due to their outdoor or intensively housed environment. Theoretically, the negative consequences of early-life pain and inflammation would be expected to be present in these populations. There are some studies investigating the influence of painful husbandry procedures in sheep and cattle on later-life pain and productivity. Clark et al. [106] demonstrated increased pain behaviours during parturition in 2-year-old ewes that had been exposed to lipopolysaccharide (LPS) at 48–72 h old to simulate a mild infection and ewes that had been tail docked at 72–96 h old compared to controls. Those ewes exposed to LPS also had a significantly longer inter-birth interval than control ewes, suggesting some influence on birth ease through currently unknown mechanisms. This study went further to measure nociceptive thresholds of lambs from these ewes during tail docking at 3 days of age, finding lambs from the LPS treated group had significantly higher mechanical nociceptive thresholds across 2 days compared to lambs from tail docked ewes and controls. Altered pain perception later in life following injury or illness at an early age was also demonstrated by McCracken et al. [104], who found that male lambs castrated with a rubber ring at 1 day old displayed significantly greater pain behaviours at tail docking with rubber ring at 26–34 days old, compared to lambs that had been castrated at 10 days of age. These studies in sheep are consistent with findings in the human and rodent literature, suggesting that painful procedures early in life negatively affect pain perception and may make affected individuals less resilient to later life stressors. This leads us to question the relevance of the legislation implying that if painful husbandry procedures are performed at an early age (which varies depending on location), pain mitigation strategies are deemed unnecessary. Granted, the methods used to perform tail docking, castration, and mulesing are best performed at a younger age to reduce the size and developmental complexity of tissue affected [66], but this does not override the need for appropriate analgesia to be provided in all cases.From a practical approach, the current analgesic options available to farmers are somewhat limited as they have a relatively short duration of action (30 min to 72 h [82,112,113]), peri-operative analgesia or anaesthesia requires prior administration and double-handling, they can also be impractical or difficult to administer during marking, and they can be cost prohibitive. There is also evidence that education on the use of pain relief is lacking, as a number of surveys show that producers are not always using available analgesics appropriately [112,114]. For example, a survey of Australian sheep producers found that of the 30% of producers using pain relief for rubber ring castration, over half (58%) reported using Tri-Solfen®, which is an unsuitable analgesic for this method and indicates a misunderstanding of the mode of action of this product [112]. Additionally, the use of suitable multimodal analgesia (NSAID and appropriate local anaesthetic), which is the current best practice [82,83], was used by less than 10% of producers for castration and tail docking (1% and 7.7%, respectively) [112]. Education programmes covering the recognition of pain in animals, the detrimental effects of pain, and the use of pain relief are clearly an important part of promoting the appropriate use of pain relief for husbandry procedures [8,45,112]. Effective dissemination of new scientific findings and legislative changes or recommendations is crucial for constructive development within the farming sector. Government departments and agencies are not always seen as trusted sources of information [45,115]. Whereas experienced farmers are seen as trusted advisors within farming communities [45], and knowledge and experience are often passed down between farmers and families through informal training [45,46]. These factors should be taken into consideration when developing intervention and education strategies to effect real change.In spite of this discussion, it is, however, heartening to see that whilst only 8.4% of Australian wool producers who routinely mules their lambs and provide pain relief reported using optimum combination analgesia, 92% of producers who mules do use some form of pain relief [112]. This is despite policy only requiring it over 6 months. This finding serves to remind us that the law is merely there to set a minimum standard, a level playing field, as it were. Farmers can, and clearly do, practice at a higher standard spurred on by industry guidance or incentivisation via assurance schemes.7.2. Farmer PerceptionFarmer perception of routine husbandry procedures often differs significantly from the public’s [9,116]. Soriano et al. [9] surveyed Brazilian sheep producers and the public about their impression of animal welfare issues in sheep farming and their knowledge of animal protection laws. Only 3.7% of the farmers that performed tail docking used an anaesthetic for the procedure, but 45.7% of the surveyed farmers stated that this was a form of animal maltreatment. This contrasts with 88.9% of citizens who thought this was maltreatment. Another interesting finding from this survey was the limited awareness of animal protection law; only 5.9% of farmers knew of the laws, but they could not cite any, whereas significantly more citizens (17%) knew of the animal protection laws. Similarly, Woodruff et al. [45] found that a lack of awareness of the recommended tail docking length was a major factor driving docking practices in 57% of surveyed Victorian sheep farmers that docked tails shorter than three palpable joints. Knowledge and implementation of current legislation and guidance on farming practices among the farming community appear to be a major barrier globally [7,9,45,46]. This leads us to query the efficacy of enforced legislative changes over other methods of knowledge dissemination and changing practice, such as education programmes for farmers and other stakeholders. Ultimately, a combination of legislative change and stakeholder-led education programmes is more likely to create sustained and widespread improvements in animal husbandry. The disconnect between industry and citizen viewpoints also poses a key challenge for legislators who must balance multiple viewpoints and priorities when making public interest laws (such as animal protection laws).7.3. The Science-Policy Interface in Animal LawIt is generally considered that the drafting of law takes into consideration the prevalent scientific evidence. Law reform bodies also commonly commit to driving evidence-based law reform processes as well as enhancing the decision-making around policy inclusions considering both transparency and democratisation of the processes [117]. This inclusion of science into law has clear potential benefits for all major stakeholders: for the animals, it is hoped that the use of welfare science will ensure policy that at least safeguards their welfare, if not improves it; for citizens and consumers, it should serve to reassure them of this welfare protection, and for the industry, it should provide uniformity of practice across jurisdictions creating a level playing field which is particularly relevant in matters of trade. However, it is naïve to think that science will completely inform the content of written law; instead, the law likely reflects a delicate balance between competing interests, viewpoints, and topical societal opinions. In the area of food law, three approaches have been used to describe regulation in this area. These approaches likely hold similarly for animal welfare law. These have been labelled “political—democratic”, “economic”, and “scientific” [118]. In a political–democratic approach legal content is determined by the support that the majority will lend to a certain opinion, i.e., the public determines the law. As the name suggests, in the economic approach, the law is driven by economic forces, whereas the scientific approach leaves the experts (scientists) to decide the legal provisions. In reality, these approaches likely overlap in the law drafting and consultation phases. However, at their intersection, there is often an inherent tension between scientists who tend to pursue objectivity and the elimination of bias and the process of policymaking. The latter requires consideration of objective information (guided by science) and subjective value judgements (such as the nature of the desirable outcome or the balance of competing interests). This often lends to the scenario when people may agree on a common set of facts but disagree on the appropriate policy response [118]. It is also worth noting that, at least in respect of animal welfare law, science may play a greater or lesser role depending on the nature of the subject matter and the positioning in the regulatory framework. As an example, there is probably a greater opportunity for incorporation of science into technical material on animal husbandry and use in delegated legislation, for example, around tail docking. However, prevailing societal viewpoints may play a greater role when considering broad provisions around animals in general, for example, around the inclusion of sentience or whether certain practices are justified. In consideration of methods of driving law reform, it is also important to consider that law reform does not occur in a vacuum; laws generally follow community attitudes rather than shaping them, and as a result, legal reform needs support from a broad community base [119,120].Notwithstanding the need to balance science against other societal and economic considerations in legal drafting, a further challenge for policymakers is how to source and evaluate the welfare science available and its value for inclusion. Jurisdictions typically approach this using different methods, which may be influenced, at least in part, by resources available. It is possible that it is the reliance on different methods of assessing and critiquing the relevant science that contributes to the diversity in legislation in this area.The European Union utilises a multi-step approach to the incorporation of science into law. The EFSA (European Food Safety Authority) is an agency of the EU with its core activity being to collect, appraise and integrate scientific evidence to address risks [121]. One of EFSA’s panels is dedicated to Animal Health and Welfare. This committee is made up of European scientists with expertise relevant to animal welfare and health. A key feature of the EFSA groups is their commitment to independence with strict working practices to reduce conflict of interest as well as members being vetted for any conflicts of interest, which may include the provision of advice or services to any industry covered by EFSA’s work. Typically, this group produces substantial reviews of the scientific literature on the topic of interest, incorporating a risk of bias assessment and assessment of certainty in the evidence. The culmination of this work can then be used by policymakers at both the EU and state level to feed into law reform [122] via a standard democratic process involving consideration of stakeholders generally achieved via representation of the member states in the EU Parliament [123]. Additionally, as is common with the law-making process, if any policy is expected to have a considerable impact economically, socially, or environmentally, an impact assessment will need to be prepared to gauge the impact on stakeholders [123]. The EU system is arguably unique in providing considerable resourcing of scientific expertise to contribute to the law-making and implementation process. The focus on the provision of independent and non-biased advice, along with the assessment of certainty in the scientific evidence based on established principles of evidence-based practice, is also laudable. Other jurisdictions adopt facets of this model. For example, when the new Standards and Guidelines were generated in Australia, a review of the literature was used in development of the Australian Animal Welfare Standards and Guidelines for Pigs. Whilst this review was funded by the Australian Pork Industry, it was subject to independent peer review [27]. A comprehensive independent review of Cephalopod Molluscs and Decapod Crustaceans was also recently commissioned by the UK government prior to recommending the inclusion of these species as “animals” in animal welfare law [124]. However, it appears that in most jurisdictions, the approach to performing reviews of the literature is somewhat ad hoc and perhaps based on perceptions of risk due to public interest in the area. There is also variability in the types of reviews performed with varying use of systematic methods to incorporate an evaluation of certainty in the evidence to guide policy-makers.Whilst outside the scope of this article, it is also worth mentioning that law-making in this area may be subject to regulatory capture [125]. This is defined when a regulatory agency is acting in the interests of the industry it is regulating and, in doing so, is creating an inconsistency with the public interest. This may particularly be a risk when there is an overrepresentation of industry in standards/code development and when there is industry control over the direction and reporting of welfare science conducted through channelling of funding [125]. The EU process of independent expert review of the science may go some way to avoid regulatory capture but is unlikely to have full effect; this likely requires considerable procedural change at both the law-making and enforcement levels.8. ConclusionsThere is broad consistency across the Australian jurisdictions in relation to specific provisions around procedures at lamb marking, for example, recommended age ranges at the performance of procedure and the use of anaesthesia in animals over 6 months old. Recommendations for the use of pain relief in animals less than 6 months are non-specific or absent in most states. The scientific evidence surrounding marking procedures indicates that castration, tail docking, and mulesing cause pain acutely and for at least two days post-procedure, regardless of age. This pain can be mitigated to some extent by NSAIDs and local anaesthetics that are licensed for use in sheep in Australia. There is a clear disconnect between the relevant legislation and scientific evidence. Legislation is lacking in traceability back to the science through no direct referencing. It also remains unclear from documents in the public domain what the scientific basis was and the process for assimilating the science of sheep husbandry during the creation of the new sheep standards and guidelines.In spite of calls for national harmonisation of requirements around farm animal welfare, there is obvious dis-uniformity between the states and territories around provisions related to sheep. This has arisen through inconsistent incorporation of the new Standards and Guidelines into the state’s welfare frameworks, with some states either retaining their own document or modifying the published version. Moreover, the enforceability of the Code varies considerably across the states. Vic provides an excellent example of this with a Code that arguably is the most welfare friendly by requiring all mulesed animals to have pain relief and for operators to be formally trained in the procedure. However, this code is a voluntary code of practice in this state, and therefore, there are no direct ramifications for failing to adhere to these provisions. In comparison, NSW, having adopted the AHA Sheep Welfare Standards and Guidelines, requires that pain relief be used in sheep that are mulesed between 6 to 12 months of age. These standards are mandatory, and violation of them may be used as evidence of an offence in court [126].Whilst resourcing is likely to be an issue, there is a need for Australia to address how well its animal welfare policy documents reflect current scientific evidence, as well as the transparency of this incorporation. A consideration of the processes and people involved in the making of delegated legislation in this area of public interest law is needed. This need is especially urgent given enhanced global trade networks, and current scrutiny of our practice brought about through recent free trade agreements.

animals : an open access journal from mdpi

10.3390/ani12010004

PMC8749667

Equine asthma shares similarities with human asthma. The aim of the study was to evaluate whether within breath analysis improved the sensitivity of oscillometry at detecting subclinical airway obstruction in horses with asthma in remission of clinical signs. From this study, we can conclude that the within-breath oscillometry is sensitive in discriminating horses with severe asthma in clinical remission of the disease from control horses. Additionally, oscillometry allowed to identify the increase in expiratory reactance similar to that due to expiratory flow limitation observed in human asthmatic patients with airway obstruction.

Oscillometry is a technique that measures the resistance (R) and the reactance (X) of the respiratory system. In humans, analysis of inspiratory and expiratory R and X allows to identify the presence of tidal expiratory flow limitation (EFLt). The aim of this study was to describe inspiratory and expiratory R and X measured by impulse oscillometry system (IOS) in horses with severe asthma (SEA) when in clinical remission (n = 7) or in exacerbation (n = 7) of the condition. Seven healthy, age-matched control horses were also studied. Data at 3, 5, and 7 Hz with coherence > 0.85 at 3 Hz and >0.9 at 5 and 7 Hz were considered. The mean, inspiratory and expiratory R and X and the difference between inspiratory and expiratory X (ΔX) were calculated at each frequency. The data from the three groups were statistically compared. Results indicated that in horses during exacerbation of severe asthma, X during expiratory phase is more negative than during inspiration, such as in humans in presence of EFLt. The evaluation of X during inspiration is promising in discriminating between horses with SEA in remission and control horses.

1. IntroductionHorses can spontaneously develop equine asthma, a non-infectious chronic lower airway disorder of adult horses, which shares several similarities with human asthma [1,2]. Based on the severity and the clinical presentation, the disease is classified as mild-moderate or severe equine asthma. Severe equine asthma (SEA) is characterized by coughing, exercise intolerance, and recurrent episodes of increased respiratory effort at rest, representing the exacerbation of the condition, alternated with periods of remission of the clinical signs [3]. The gold standard for the diagnosis of SEA is the cytological examination of bronchoalveolar lavage fluid (BALf) in horses with compatible clinical signs, that shows the presence of a marked neutrophilic inflammation [4]. Nevertheless, BALf collection is a relatively invasive procedure, that requires the sedation of the patient and the instillation of a large volume of fluid in the lungs of the horse. Moreover, during the remission of the clinical signs, BALf cytology may not allow to discriminate between healthy and affected horses [5]. In humans, the gold standard for the asthma diagnosis is the detection of alterations in pulmonary function testing [6]. In horses affected by SEA, conventional lung mechanics allows to identify the presence of airway obstruction [7,8,9]; however, this technique requires the use of an esophageal balloon and shows a low sensitivity for mild obstruction [10], therefore it is currently performed only in research settings. For this reason, in the last 30 years the attention of the researchers has been focused on oscillometry, a technique currently used in humans for the evaluation of asthmatic patients. As described by Dubois et al. [11], oscillometry allows to measure the mechanical properties of the lung (i.e., resistance, R, and reactance, X), evaluating the response of the respiratory system to external forcing over-imposed to spontaneous breathing. The Impulse Oscillation System (IOS) is a method based on a repetition of impulses generated from a loudspeaker and applied to the respiratory system that allows the determination of R and X across multiple frequencies [12]. Van Erck et al. [13,14,15,16] first reported on the use of IOS in horses with severe asthma, in which they evaluated the frequencies from 5 to 20 Hz. They reported that the results of IOS and conventional lung mechanics were well correlated. Moreover, IOS was more sensitive than standard mechanics during bronchoprovocation tests. Lower airway obstruction was characterized by negative frequency dependence of R, positive frequency dependence of X and negative X values throughout the frequency range. In 2006, Klein and colleagues evaluated the results of IOS at frequencies of 1, 5, and 10 Hz, that were considered the most representative of equine lower airways [17]. For the first time, the within-breath analysis was performed, and inspiratory and expiratory R and X were reported. Additionally, the within-breath analysis of asthmatic horses with subclinical inflammation, showed higher R values and lower X values when compared to those of controls [18]. Recently, other authors reported an association between R measured by IOS and histopathological findings of the airways of SEA horses [19]. To date, there is no report of IOS values of SEA when horses are in remission of the clinical signs.In humans, the within-breath analysis of X allows to calculate the parameter Delta X (ΔX), defined as the difference between the inspiratory and expiratory reactance at each frequency. It has been shown that this parameter allows the detection of tidal expiratory flow limitation (EFLt) in COPD and asthma. This measure has an important diagnostic value [20,21], but it has not been applied for the evaluation of asthmatic horses. The aim of the present work was therefore to describe the results of within-breath analysis, including ΔX, measured by IOS in horses with severe asthma when they are in the exacerbation and in the remission phases of the disease.2. Materials and Methods2.1. Sample SelectionTo perform the study, seven horses with SEA in exacerbation of the clinical signs (4 geldings and 3 mares, age of 11.9 ± 3.4 years), seven horses with SEA in clinical remission (1 gelding and 6 mares, aged 16.4 ± 5.0 years) and seven age matched healthy controls (7 mares, with a mean age of 13.1 ± 3.5 years) were studied. The horses with SEA were selected from a well-characterized population of asthmatic horses of the research herd of the Equine Asthma Laboratory, Faculty of Veterinary Medicine, University of Montréal. Horses in asthma exacerbation were kept in stable and fed hay; horses in remission of the clinical signs were kept at pasture 24 h/day for at least 6 months and fed pelleted hay when needed. Control horses were from the teaching herd of the Faculty of Veterinary Medicine of the University of Montréal and were considered free from respiratory diseases based on history and clinical examination. All horses had been previously trained to IOS measurement. The study was approved by the Animal Care Committee of the Université de Montréal (Protocol Rech-1324) and conducted in compliance with the guidelines of the Canadian Council on Animal Care.2.2. IOS MeasurementHorses were restrained in stock and underwent IOS measurement by Equine IOS MasterScreen (Jaeger, Würzburg, Germany), as previously described [13]. Briefly, the system consisted in a plastic mask adapted to fit on the muzzle of the horse, sealed by a rubber tape. The mask was attached through a tube to a loudspeaker that produced the impulses, and to a pneumotachograph placed directly in front of the face mask. The pressure and flow response of the respiratory system to the impulses superimposed to the animal spontaneous breathing were measured. Prior to each experiment, the system was calibrated by means of a 2-L calibration syringe, forcing known volumes of air through the pneumotachograph. At least three measurements of 30 s each were performed, and the mean value of the three measurements was studied.The data collected by LabManager (version 4.53, Jaeger, Würzburg, Germany) was then analyzed using Fast-Fourier transformation (FAMOS imc, Meβsysteme, Berlin, Germany). The mean total, inspiratory and expiratory R and X and the corresponding coherence (Co) of the respiratory system at all frequencies of impulses (from 0.1 to 20 Hz) was obtained. For this study, only values at 3, 5, and 7 Hz were studied, as they were the only frequencies with Co considered adequate (Co > 0.85 at 3 Hz and 0.9 at 5 and 7 Hz). Co reflects the quality of the measurement [17]. The ΔX, measured as the difference between the mean inspiratory and expiratory reactance at each frequency, was also calculated.2.3. Statistical AnalysisThe mean inspiratory and expiratory R and X and the ΔX were calculated at each frequency for the three groups and collected on an electronic spreadsheet (Microsoft Excel, Redmont, WA, USA). Data distribution was evaluated by means of Shapiro–Wilk normality test. If data were normally distributed, the comparison between the three groups was performed by means of one-way ANOVA and Dunnett’s multiple comparison test. If data were not normally distributed, the comparison was performed by Kruskal–Wallis test and Dunn’s multiple comparison test. Statistical analysis was performed using a statistical software (Prism Graphpad 9.1.0 for MacOs; San Diego, CA, USA). Statistical significance was set at p < 0.05.3. ResultsThe Shapiro–Wilk normality test showed a normal distribution for all the parameters, except for inspiratory R at 3 Hz (R3i) and ΔX at 7 Hz (ΔX7). Results of IOS measurement are reported in Table 1.Statistical comparison between groups showed significant differences between SEA horses in exacerbation and control horses for R at 3 Hz, for mean (R3, p = 0.0002), inspiratory (R3i, p = 0.0011) and expiratory (R3e, p = 0.0008) parameters.For X, significant differences were present between horses in exacerbation and control horses at each frequency for mean (X3, p < 0.0001; X5, p < 0.0001; X7, p < 0.0001), inspiratory (X3i, p < 0.0001; X5i, p < 0.0001; X7i p = 0.0007), and expiratory parameters (X3e, p < 0.0001; X5e, p < 0.0001; X7e, p < 0.0001). Between control horses and asthmatic horses in remission, differences were present for mean X at 7 Hz (X7, p = 0.0173) and for inspiratory X at 3, 5 and 7 Hz (X3i, p = 0.009; X5i, p = 0.0017; X7i, p = 0.012).The ΔX values were significantly higher in horses in exacerbation of severe asthma than in control horses at 3 and 5 Hz (ΔX3, p = 0.0029; ΔX5, p = 0.001), indicating a worsening of the airway obstruction during the expiratory phase of breathing.4. DiscussionThe present study represents the first report on IOS measurements and ΔX in asthmatic horses in remission of the disease. Horses in remission of severe asthma are of particular interest, because they can be used as a model for subclinical airway obstruction. A previous study, in fact, demonstrated the presence of a residual bronchoconstriction even after one year of treatment with inhaled corticosteroids or strict antigen avoidance [5].Concerning the measurement technique, IOS generated a spectrum of frequencies ranging from 0.1 to 20 Hz. Nevertheless, it has been demonstrated that the frequencies lower than 10 Hz are the most representative of the lower airways in the equine species [17]. Moreover, in a previous study reporting data obtained in horses by means of forced oscillations (FOT), the frequencies considered as the most sensitive were 1, 2, and 3 Hz [22]. For this reason, we decided to evaluate only the results at 3, 5, and 7 Hz; we excluded the data at lower frequencies because the oscillations generated by the IOS could interfere with higher harmonics of spontaneous respiratory frequencies, and therefore the quality of data could be negatively influenced [23]. For the same reason, and as suggested previously [17], only impedance data showing high values of coherence (>0.85 at 3 Hz and >0.9 at 5 and 7 Hz) were included, in order to optimize the quality of data. Other studies on IOS values in horses with asthma did not report the coherence values [13,14,15,16,18], and therefore the comparison with our data is not possible.In agreement with previous reports, IOS identified several differences between horses with SEA in exacerbation of the clinical signs and controls [13,14,15,16]. Differences in R were found only at the frequency of 3 Hz for the whole breath and for inspiratory and expiratory R. This result is similar to what reported in a recent study [19]. In human medicine, the increase in R at low frequencies is indicative of the presence of lower airway obstruction during clinical exacerbation of asthma [24]. The absence of differences at higher frequencies is also coherent with previous reports, as SEA horses are characterized by negative frequency dependence of R [16].In the present study, horses in asthma exacerbation also showed significant lower values of X compared to controls, at all frequencies and for all the phases of breathing. This finding agrees with previous reports [15,19]. Negative values of X at low frequencies reflect peripheral airway obstruction [12]. In humans, X is decreased in the presence of various obstructive respiratory diseases, including asthma, chronic obstructive pulmonary disease (COPD), and emphysema [25].Moreover, our results showed that the values of ΔX at 3 and 5 Hz in the SEA group in exacerbation were significantly higher than in controls, meaning that the expiratory X was significantly lower than the inspiratory X. Similar findings have been observed in human patients with COPD, but specifically only in patients where the airflow during expiration did not increase despite increasing efforts of the patient. This conditions, called tidal Expiratory Flow Limitation (EFLt), is due to the narrowing of some airways (chock points) consequent from the dynamic compression of peripheral airways [26]. During an IOS measurement, the oscillations cannot penetrate through the choke points and, therefore, impedance data represents the mechanical properties of the part of the lung between choke points and airway opening only. As most of lung compliance is located in the lung periphery (i.e., between chock points and alveoli), when choke points develop, the expiratory reactance drops [20]. In a similar way, exacerbation of SEA is characterized by an early peak of expiration, and a consequent decrease in the expiratory flow [27]. This also may be due to the presence of some choke points that cause a drop in expiratory X and a consequent increase in ΔX. This supports the presence of EFLt in SEA, which may contribute to pulmonary hyperinflation and exercise intolerance, as reported in humans [28].Finally, IOS allowed to identify significantly lower X7, X3i, X5i, and X7i in SEA horses in remission compared to controls. This is the first report of the sensitivity of IOS in discriminating between healthy and horses in remission of severe asthma. This finding is surprising because negative values of inspiratory reactance are suggestive of restrictive diseases, such as interstitial lung disease, more than obstructive [29]. Moreover, horses with SEA in clinical remission of the clinical signs have normal lung function when evaluated using standard lung mechanics, despite the presence of a residual airway obstruction has been demonstrated [5]. It could be hypothesized that this residual bronchospasm does not interfere with the measurement of expiratory reactance by IOS. Nevertheless, it has been reported that asthmatic horses suffer from a chronic remodeling of the airways, that involves not only the smooth muscle mass [5], but also the vessels [30], the epithelium and the interstitial tissue [31], that is only partially improved by treatment or antigen avoidance. As the presence of pulmonary fibrosis and emphysema induces lower values of inspiratory X in humans [32], it could be speculated that the presence of a persistent subepithelial fibrosis and hyperinflation in horse with SEA in clinical remission [31] could have contributed to the decrease in inspiratory X.5. ConclusionsThe within breath analysis of IOS measurement showed some differences between control horses and SEA horses in remission, that could be a promising result for the identification of asthmatic horses in absence of clinical signs. Moreover, the parameter ΔX suggests the presence of EFLt and dynamic airway compression in SEA horses in exacerbation of the clinical signs.

animals : an open access journal from mdpi

10.3390/ani11082320

PMC8388488

Some of the equipment used in equine dentistry is difficult to clean and disinfect. Since it is vital to avoid the spread of infections in equine healthcare it is important to develop practical and easy-to-follow methods for cleaning and disinfecting dental equipment. The aim of this study was to investigate hygiene in equine dentistry. Dental equipment and the head support, where horses rest their head during dental care, were sampled for the amount of bacteria between each patient before and after dental care as well as after cleaning and/or disinfecting. The amount of bacteria was, in general, high on dental equipment and the head support after dental procedures. Bacteria were found in different amounts on most of the dental equipment after cleaning or disinfecting, which indicates a risk for spreading infections when using the equipment. For the head support, cleaning and/or disinfecting generally resulted in a reduced amount of bacteria, indicating a lowered risk for spreading infections. There is a great need for evidence-based guidelines on hygiene in equine dentistry to decrease the risk of transmitting infections between patients, facilities, and stables.

Equine dentistry has developed immensely and human dental equipment, such as handpieces, are often used. Measures to avoid the spread of infectious microorganisms are important, but this is challenging since handpieces are difficult to decontaminate. Thus, it is necessary to develop effective IPC measures in equine dentistry. The aim of this study was to contribute to the evidence needed for future evidence-based guidelines on IPC by investigating hygiene in equine dentistry. Used handpieces and dummies (i.e., handpieces not used during dental procedure, reflecting environmental bacterial contamination) and the head support were sampled each day before the first patient, for each patient after treatment, and after decontamination. All equipment was sampled with 3M TM Swab Samplers and the head support additionally sampled with dip slides. After dental procedures, the detected bacterial load was often high on used handpieces, dummies, and the head support. After decontamination, handpieces did not meet the criteria for high-level disinfected equipment. In all but one case decontamination of the head support resulted in a lowered bacterial load. There is a great need for evidence-based guidelines on hygiene in equine dentistry, including IPC measures, to decrease the risk of spreading infectious microorganisms between patients, facilities, and stables.

1. IntroductionEquine dental care is carried out at veterinary hospitals, clinics, and mobile practices. Equine dental health is a rapidly growing area in veterinary clinical practice. The frequency of treatments and types of dental procedures have increased during the last decades. The advancement of equine dental care in Sweden has made it common to use human dental equipment both for routine and advanced procedures. Handpieces, e.g., low-speed handpieces (LSH), surgical low-speed handpieces (SH), and high-speed handpieces (HSH), are used for both simple procedures, such as decreasing enamel ridges, and more advanced procedures, such as endodontic treatments. Even though protocols for cleaning, disinfecting, and sterilizing handpieces are in place for use in human dentistry there is a lack of knowledge regarding the cleaning, disinfecting, and sterilizing routines needed to ensure low risk for spreading infectious microorganisms between equine patients.Measures to avoid the spread of infectious microorganisms between patients, and to staff, is common clinical practice in both human and veterinary medicine. This aspect of equine dentistry is challenging as handpieces are difficult to decontaminate, especially in mobile practices. Thus, there is a great need for effective infection prevention and control (IPC) measures in equine dentistry. The advanced dental procedures also entail a risk for transmission of infectious microorganisms between equine patients. For example, in a North American study, respiratory pathogens such as Equine herpes virus 1 and 4 (EHV-1, EHV-4), Equine influenza virus (EIV), Equine rhinitis B virus (ERBV), and/or methicillin-resistant Staphylococcus aureus (MRSA) were detected in 22% of healthy horses presented for routine dental care [1]. Furthermore, bacterial contamination of external and internal surfaces of handpieces has been shown after human dental procedures [2,3,4,5]. In addition, a higher degree of contamination of the environment and, thereby, an increased risk for patients when using high-speed devices, compared to using low-speed devices, has been shown [6,7]. There are, to our knowledge, no studies on contamination of handpieces or the surrounding environment in equine dentistry.Based on use and hygiene, requirements for medical equipment, including dental handpieces, are categorized as non-critical, semi-critical, and critical [8]. The different categories require different levels of cleaning, disinfecting, and sterilizing (see Table 1). Handpieces are classified as semi-critical equipment, as long as they are used for non-sterile procedures, under Swedish and US hygiene guidelines for human dentistry as well as Swedish guidelines on IPC in equine healthcare and in small animal dentistry literature [8,9,10,11]. There is, however, no international consensus on criteria for the expected cleanliness for high-level disinfected equipment. Guidelines in human dentistry on how to clean, disinfect, and sterilize handpieces differ between countries. Several studies on human dental equipment demonstrate the challenges to meet criteria for both high-level disinfected and sterile equipment for handpieces. Surface disinfection of the external surface of handpieces resulted in failure to meet the criteria for high-level disinfection [12]. In another study, one of four cleaning devices intended for handpieces, the washer-disinfector (WD), provided an acceptable test result [13]. In one study, type N steam sterilizers failed to provide sterile handpieces whilst type B steam sterilizers provided sterile handpieces [14]. According to Pusterla et al. [1], equine dental equipment used for routine dental procedures, not expected to expose the pulp (i.e., semi-critical equipment), is rarely cleaned and disinfected between patients. Even though the equipment used for routine dental procedures can differ between countries, the conditions for cleaning procedures and risk of spreading infections can be assumed to be comparable. Moreover, IPC routines for equipment used in equine dentistry are not listed in the syllabus of equine dentistry courses in Sweden and in the European specialist program in equine dentistry [17,18]. In our experience, a commonly used decontamination method for handpieces in equine dentistry is surface disinfection with an intermediate disinfectant (a disinfectant with effect on most vegetative bacteria, some mycobacteria, some fungi, some enveloped and non-enveloped viruses [19]) without previous cleaning. However, no decontamination procedure in equine dentistry has yet been evaluated or published. Guidelines for IPC procedures in equine dental practice, based on solid data, are needed. The overall aim of this study was to contribute to the evidence needed for future guidelines on IPC procedures for equine dentistry by investigating hygiene in equine dentistry, specifically by: (1)assessing the bacterial load on handpieces and the patient environment during equine dental care;(2)assessing if manual cleaning of handpieces with detergent or disinfection with surface disinfection is sufficient to meet the Swedish criteria for high-level disinfected equipment; and(3)determining the bacterial load on the immediate surroundings after surface disinfection, or cleaning followed by surface disinfection.2. Materials and MethodsThe study was carried out in the dental practice of a veterinary hospital during the autumn of 2020. The veterinary hospital’s patient load consists of approximately 9000 patients per year and approximately 500 of them are dental patients. Sampling was carried out during two working days, with a total of 11 horses submitted for dental care.2.1. SamplingEquipment and surfaces to be sampled for bacterial load were selected based on a pilot study carried out in the dental practice of another veterinary hospital (for details from the pilot study see Table S1: Bacterial load pilot study). The veterinary hospital’s patient load consists of approximately 5000 patients per year and approximately 500 of them are dental patients. Based on the pilot results, sampling of handpieces and the head support was standardized (for details about sampling methods tested, see Table S2: Sampling methods tested). The sampling surfaces of the equipment are illustrated in Figure 1 and the sampling protocol is illustrated in Figure 2. Make and model of used handpieces can be found in the figure text in Figure 2. Handpieces were sampled with 3M TM Swab Samplers (3M TM Swab Sampler, Saint Paul, Minnesota, USA) with letheen broth, an already established method in the food industry for control of hygiene [20]. The 3M ™ Swab Samplers Method was modified to enable sampling of the different surfaces LSH, SH, and HSH, see Table 2 [21]. All samplings with 3M ™ Swab Samplers were carried out by the second author and the first author held the handpieces, wearing non-sterile nitrile gloves, during sampling. The samples were then analyzed for total aerobic colony count (ACC).The samples from the head support were also analyzed for total ACC, using two sampling methods; 3M TM Swab Samplers with letheen broth (both study days) and dip slides (Envirocheck ® Dip Slide Disinfection Control (DC), 9,4 cm2, Orange, USA) with TSA agar/TSA agar with a neutralizer, neutralizing several disinfectants (one of the study days). A particular 10 × 10 cm surface of the head support, a cushioned device supporting part of the horse’s lower jaw, was repeatedly swabbed at each sampling with the 3M ™ Swab Samplers Method, see Table 2 [21]. Also, an adjacent specified surface of the head support was sampled with dip slides. The surface chosen for sampling can be contaminated by fluid from the mouth during dental examination and treatment. The dip slide was pressed firmly to the surface for 15 s, then turned over and the opposite side of the slide was pressed against the adjacent surface for another 15 s [22]. All sampling with dip slides was carried out by the first author. For each sampling day and sampling method, one or two negative controls (unexposed dip slide and swab sampler) were applied. The controls were put in the dental care room just before sampling started for the day, approximately 10 min before the day’s first patient, and the controls were stored in the room until the gathering of sampling material after the last sampling of the day.2.2. Cleaning and Disinfecting MethodsFive protocols for cleaning and disinfecting handpieces and the head support were used (for details see Table 3). Protocols 1 and 2 consisted of the veterinary hospital’s own protocol for surface disinfection with an intermediate disinfectant of handpieces and the head support. Protocol 3 consisted of manual cleaning of LSH and HSH and protocol 4 consisted of manual cleaning of SH. Protocol 5 consisted of manual cleaning of the head support followed by surface disinfecting with an intermediate disinfectant. LSH and SH were lubricated after every cleaning or disinfecting with a lubricant (PANA SPRAY Plus, NSK / Nakanishi inc., Kanuma, Japan) containing ester oil, ethanol butane, and propane. The HSH was lubricated using lubricating oil (MD-30 Advantage Dental Handpiece Oil MD-30, iM3, Sidney, Australia) containing synthetic hydrocarbon oil and ester oil after the last patient of the day. All handpieces were put back, out in the open, on a metal tray adjacent to the patient after cleaning or disinfecting and lubricating. The veterinary technician and the first author carried out every other cleaning or disinfection and lubrication of handpieces and the head support. During the period of sampling the dental care room and dental equipment were used three days a week. When protocols 1 and 2 were used, the dental care room and equipment had been unused for four days and when protocols 3–5 were used the dental care room and the dental equipment were used the day before. 2.3. Bacteriological AnalysesAll 3M Swab Samplers and dip slides were taken to the laboratory at the Swedish University of Agricultural Sciences on the day of sampling. 3M™ Swab Samplers were vortexed and 1 mL broth was drawn from the sampling tube and put onto a 3M Petrifilm TM aerobic count (AC) Plate (3M PetrifilmTM Aerobic Count Plate, Saint Paul, Minnesota, USA), see Figure S1: Petrifilm TM negative control, as further described in the 3M TM Swab Sampler Method [21]. Samples were incubated aerobically in 30 ± 1 °C for 48 ± 2 h. Dip slides were incubated in 37 ± 1 °C for 48 ± 2 h. The colonies were counted manually by the first author, as described in the interpretation guide [23]. All 3M Petrifilm TM AC Plates and dip slides were photographed for documentation. 2.4. Data ManagementMicrosoft ® Excel ® 2016 (16.0.5134.1000) (Microsoft Corporation, Redmond, Washington, USA) was used for data management and descriptive statistics. 3. ResultsThe study included 80 samples from handpieces, 42 samples from dummies, 24 samples from the head support, and two negative controls using 3M TM Swab Samplers. In addition, dip slides were used in 14 samples from the head support and for two negative controls. No bacterial growth was detected in the samples from negative controls.3.1. HandpiecesBacterial growth was detected in all samples from HSH, both after dental procedures and after cleaning or disinfecting (see Table 4). In all samples from external surfaces from LSH and SH, bacterial growth was found after dental procedures, and after cleaning or disinfecting bacterial growth was still detected in all but one sample. After dental procedures, bacterial growth was detected in all samples from the coupling of LSH and in 6 of 10 samples from the shaft in SH. After cleaning or disinfecting, no bacterial growth was found in couplings from LSH while bacterial growth was found in 2 of 10 samples from the shaft of SH. 3.2. DummiesBacterial growth was detected in all samples from external surfaces of dummies before the first patient of the day and in 11 of 12 samples from the external surface of dummies after dental procedures compared to 5 of 11 after cleaning or disinfection (see Table 5). In 2 of 3 samples from couplings of dummies, bacterial growth was detected before the first patient of the day. In addition, bacterial growth was detected in 2 of 11 samples from the coupling of dummies after cleaning or disinfecting. 3.3. Head SupportIn both samples from the head support before the first patient of the day bacterial growth was detected, and in one of them the CFU/cm2 were too numerous to count. The bacterial load detected on the head support was high (usually too numerous to count) after dental procedures. Both cleaning and/or disinfection reduced the bacterial load as seen in most of the samples (see Table 6). 4. DiscussionTo the best of our knowledge, this is the first study of hygiene in equine dentistry. According to Swedish guidelines in human dentistry, all handpieces should meet criteria for high-level disinfected equipment, and handpieces used for surgical procedures should be sterile [9,15,16]. In equine dentistry, the risks of spreading blood-borne diseases, such as those that are important in human dentistry [8,24], are not seen as a major risk. In equine dentistry there are, however, risks of spreading various pathogenic and resistant microorganisms [1]. According to the Swedish guidelines on IPC in equine healthcare, dental equipment that comes in contact with mucus membranes, but not penetrating sterile tissue, shall meet criteria for high-level disinfected equipment and surgical dental equipment shall be sterile [10]. According to the American Animal Hospital Associations’ dental care guidelines for dogs and cats, all dental instruments shall be cleaned and sterilized after each use [25] whilst World Small Animal Veterinary Association guidelines states all dental equipment shall be cleaned, disinfected, and/or sterilized based on the equipment’s intended use [26] Also small animal dentistry literature recommends semi-critical and critical instruments to be sterilized after each use [11]. All the recommendations are brief and reflect hygiene recommendations in human dentistry and the differences in guidelines on how to clean, disinfect, and sterilize dental equipment reflects the differences found between countries in human dentistry. All these recommendations are very brief and no clear criteria is given for handpieces. It is important to take into account both the differences and the similarities in risks between human and equine dentistry and to have knowledge of, e.g., the microbial contamination in equine dentistry when developing guidelines on IPC measures in equine dentistry. In this study, equipment and the close patient environment were highly contaminated after dental procedures. The bacterial load in the immediate environment was lower after cleaning and/or disinfecting, but handpieces did not meet the criteria for high-level disinfected equipment after cleaning or disinfecting. It can be assumed that a high bacterial load is an indicator of the presence of potentially pathogenic microorganisms. In a study by Adams et al. [27] investigating the occurrence of Staphylococcus aureus in a human intensive care unit, most Staphylococcus aureus were detected on heavily contaminated hand-touch sites. This study has shown that dental procedures, using handpieces, contaminate the equipment and the surrounding environment, with a potentially increased risk of transmission of pathogens between patients, staff, and facilities. As HSH is used for endodontic treatment, transmission of microorganisms can have severe consequences since the pulp is exposed, i.e., there is an increased risk for infection. To classify HSH as critical equipment could emphasize its importance to improve IPC measures in equine dentistry. For semi-critical equipment as LSH and SH, when not used for endodontic treatment, it can be discussed whether the strictest definition of high-level disinfected equipment is needed in equine dentistry. An important factor for considering to use the less strict definition of high-level disinfected equipment (i.e., free from pathogens and occurrence of occasional vital microorganisms, see Table 1) for semi-critical LSH and SH is that it is important to identify an achievable, and measurable, threshold value for manual cleaning and disinfection. It is difficult to estimate the risk of infection transmission if the less strict Swedish definition of high-level disinfected equipment would be applied for handpieces. The results of our study indicate that even the less strict criterion may require more meticulous routines for cleaning and disinfection.In this study, the effect of surface disinfection of handpieces was similar to the results reported by Pinto et al. [12] in a study in human dentistry, where handpieces did not meet even the less strict criteria for high-level disinfected equipment in human dentistry [9]. The couplings in both LSH and HSH can be regarded as a bridge between external and internal surfaces. The couplings can, if the IPC measures are ineffective, serve as a vector spreading infectious agents between patients. Infectious agents may occur in horses without clinical symptoms of infection, for example, Pusterla et al. [1] reported respiratory pathogens in 22% of healthy horses submitted for routine dental care.If the upper limit for bacterial load on external contact surfaces of 2.5 CFU/cm2, as suggested in studies on human hospital cleanliness [28,29,30,31], is used as the limit of acceptance for the head support (a non-critical piece of equipment), most samples in this study meet the criteria after cleaning and/or disinfecting. Limits of the study: The bacterial load on the shaft of the SH after dental procedures in this study diverges from results reported by Smith et al. [3], in median 1000 CFU/surgical gear compared to up to 30 CFU/shaft in this study. Smith et al. [3] sampled the surgical gear which can be assumed to be more highly contaminated compared to the shaft sampled in this study. Smith et al. [3] also used a better sampling method and a culturing method enabling identification of a wider range of bacteria. If the surgical gear would have been sampled in this study using the same method as Smith et al. [3], it can be assumed the bacterial load would have been considerably higher. Petrifilms TM were incubated in 30 ± 1 °C which means environmental flora is probably dominating and potential pathogenic bacteria may have been overgrown. The amount of potential pathogen bacteria would probably have been more accurate if Petrifilms TM had been incubated in 37 ± °C, which is optimal for most mammalian pathogens [32]. Data on length of time in contact with dental tissue was not collected; this data could possibly have provided important information about the degree of contamination after different lengths of time in contact with dental tissue. More research will be needed to evaluate how manual cleaning followed by chemical disinfecting of external and internal surfaces of handpieces can result in the less strict Swedish definition of high-level disinfected handpieces, i.e., free from pathogens and occurrence of occasional vital microorganisms. Other topics for future research should be to develop evidence-based guidelines by (1) design and test IPC routines for equine dental procedures and (2) investigate what level of hygiene is needed to minimize the risk of transmission of infectious agents between patients in equine dentistry. High contamination of the equipment and the close patient environment, combined with handpieces not meeting criteria for high-level disinfected equipment after manual cleaning or disinfecting, indicates an urgent need for evidence-based guidelines on hygiene in equine dentistry. Based on the study results, protocols for assessment of contamination level could be developed. Larger series of data from several clinics as well as mobile practice should be collected as a basis for such guidelines. Categorization of dental equipment as critical, semi-critical, and non-critical equipment, and a clear definition of hygiene criteria for such equipment are also needed. In addition, guidelines on how to clean, disinfect, and, in some cases, sterilize dental equipment are necessary. Methods for monitoring each step are also needed.5. ConclusionsThe detected bacterial load on the equipment and in the close patient environment was often high after dental procedures. Handpieces did not meet the criteria for high-level disinfected equipment after cleaning or disinfecting. In most cases cleaning and/or disinfecting of the head support resulted in a lowered bacterial load. This implies there is a need for evidence-based guidelines on IPC procedures for equine dentistry. In addition, data to support appropriate threshold levels are needed.

animals : an open access journal from mdpi

10.3390/ani11082410

PMC8388748

Long-distance transport in the global swine industry is more the rule than the exception. We tested the impact on the rates of temperature change and air enthalpy on the stress response and muscle pH in pigs subjected to long-distance travel from Spain to Portugal performed in the summer and winter. We found that winter journeys are more adverse for the animals because during the journey, abrupt variations in rates of temperature change and air enthalpy caused a marked physiological stress response and effects on the meat pH after 45 min. These results indicate the need to develop new environmental control strategies that mitigate abrupt temperature changes during travel to attenuate the biological cost of such long-distance transport on the animals.

Current legislation in the European Union places limits on live pig transport according to outside temperature, but less is known about the effects of sudden changes in the thermal microenvironment in trailers, particularly during long-distance transport. In this study, we measured the temperature and relative humidity inside livestock vehicles carrying 1920 Spanish finisher pigs (live weight 100 kg and 240 animals per journey) during eight long-distance (>15 h) commercial journeys to slaughter from northern Spain to Portugal in the summer and winter. Here, we report the rate of change in the air temperature (°C × min−1) and air enthalpies in the transport vehicle (kg water kg dry air-1). At sticking, blood samples were taken for to measure cortisol, glucose, and creatine kinase (CK) as stress response indicators, and the meat pH after 45 min and the pH after 24 h were also determined. The rate of change in the air temperature and enthalpy was higher inside the livestock vehicle during the winter months and was positively related with higher cortisol and glucose levels and lower pH after 45 min (p < 0.05). It is proposed that the rate of temperature change and air enthalpy represent useful integrated indices of thermal stress for pigs during transport.

1. IntroductionPig production in the European Union is increasingly industrialized and specialized [1], with the EU being the second largest pig producer in the world, with 24.1 million tons of pork produced in 2019 [2]. Spain is one of the most important pig producers in Europe and almost a third of national production is exported [3]. To maintain this high level of competitiveness, the pig sector depends on road transport as one of its strategic components in the European pork supply chain [4]. The high demand for pork meat in some member countries has stimulated intracommunity trade involving the long-distance transport of live pigs [5]. Even under favorable conditions, long-haul transport can cause different degrees of stress to animals, ranging from discomfort and aversion to death [6]. Extreme ambient temperatures during long-distance journeys are considered as one of the most important risk factors for dead on arrivals, non-ambulatory animals, skin lesions, and carcass downgrading, especially when microclimate conditions are outside of the optimal thermal comfort zone for pigs [7]. These thermal conditions may be complex and result from the interaction of several factors such as external climatic conditions, heat and water production from the animals, ventilation regimes, distribution and flow rates, and additional external sources of heat and/or moisture [8].The temperature humidity index (THI) forms the basis for calculating ventilation on farms [9]. The Livestock Weather Safety Index (LWSI) is derived from the THI and is widely applied across species, as described by Eigenberg et al. [10]. The LWSI has been related to mortalities during pig transport in Denmark [11]. Barbosa-Fihlo et al. [12] have compared using enthalpy with the Black Globe and Humidity Index (BGHI) proposed by Curtis [13] in the assessment of thermal comfort in broilers. Enthalpy was deemed the better index and the same authors [14] later applied an Enthalpy Comfort Index (ECI) to poultry during transport, reporting that hostile thermal conditions are well predicted by this index. Enthalpy, a concept that combines temperature and relative humidity, is the heat energy of the air and is the major determinant of the dry and latent heat loss to the environment. It can be calculated using simple tools (i.e., a thermometer and hygrometer) and mathematical models [15]. Advances in this area will underpin improvements in handling and the transport of livestock, especially under more extreme climate conditions.Current legislation in Europe: European Union Council Regulation 1/2005 [16] defines temperature limits during the transport of livestock on journeys over 8 h in duration. However, the legislation does not specify any corresponding limits for humidity or water vapor content in the air. Similarly, there is no consideration of the effects upon the animals regarding the rate of change of the thermal conditions. According to EC 1/2005, the upper temperature limit for the long-distance transport of livestock on long journeys is 30 °C with a tolerance of 5 °C, meaning the absolute limit is 35 °C. Although integrated indices of temperature and humidity are used extensively in other areas of animal production to predict their impact on production and welfare, this approach has not been applied to animal transport in the European legislative context, and few studies have considered the potential application of enthalpy as an integrated index of thermal load. In this study, the aim was to develop and compare psychrometric charts as well as rates of change in the temperature and enthalpy from long distance (>8 h) journeys carrying pigs across the Iberian Peninsula from northern Spain to southern Portugal. In addition, the potential use of rates of change of temperature and enthalpy as non-invasive indicators and their impact on blood stress indicators and meat pH in pigs were evaluated.2. Materials and MethodsA total of eight long-distance commercial journeys from a pig finishing farm located in the town of Campanas in the autonomous community of Navarra in Spain (42.69 N, 1.65 W, and 575 m.a.s.l.) to a EU licensed abattoir at Vila Franca do Rosario in the Lisbon region in Portugal (38.97 N, 9.25 W, and 88 m.a.s.l.) were studied. The distance for all of the journeys was 936.5 km with an average duration of 14 h and 30 min. Campana’s climate is Cf2b according to Köppen–Geiger’s climate classification; warm and humid with cold winters and mild summers with rainfall throughout the year, except for two relatively dry months. The annual average temperature is around 12 °C with an average monthly temperature of 5 °C in the coldest month (January) and 20.9 °C in the warmest month (August), attending to the typical climate data [17]. Pigs were carefully handled during the pre-slaughter period: they were handled at unloading and in lairage using plastic paddles only. They were loaded and unloaded by the workers hired by the farm and the abattoir and were kept together in familiar groups during transport and lairage. All procedures were conducted in accordance with the guidelines for the ethical treatment of animals in applied animal welfare studies [18].2.1. Study DescriptionThe eight long-distance journeys monitored the transport of a total of 1920 Spanish finisher pigs (240 animals per journey) in the winter (n = 3) and in the summer (n = 5). All of the pigs were Large White/Landrace × Duroc pigs with a mixed group males and females who were six-months-old (with an approximate average live weight of 100 kg). Pigs were off feed for 12 h before transport. Loading was usually conducted at approximately 05:00 a.m. by three farm operators, avoiding mixing between unfamiliar pens. The farm operators went to one pen at a time to drive the pigs toward the loading platform using plastic bags and plastic boards. The loading procedures lasted approximately 2 h per journey. The animals were loaded and transported from Campanas (Spain) to a commercial abattoir in Vila Franca do Rosario (Portugal). This journey duration is compliant with the overarching transport regulation, EC 1/2005, which prescribes a maximum journey time for adult pigs of 24 h when undertaken on higher standard vehicles and with constant access to water. For each journey from the farm to the abattoir, members of the research team accompanied the truck to ensure and verify that the journey met the objectives of the study. All journeys always took the same route and had the same lorry and driver.The vehicle that was used was an articulated lorry with a tractor unit (MAN, Munich, Germany) towing a trailer (Carrozzeria Pezzaioli, Montichiari, Italy). The trailer had three floors with six compartments per floor (each compartment measured 220 cm long × 245 cm wide and 84 cm high), giving a total surface area of 5.39 m2 per compartment and an average stocking density of 0.42 m2/pig). The total loading capacity of the truck was about 27,000 kg and was equipped with suitable drinking systems with nipples to provide water during the journey. The trailer had both natural and mechanical ventilation systems, which consisted of twelve automatic fans per floor or six per side. The fans, nine in each truck, were 225 mm in diameter with a 11,700 m2/h flow in compliance with EC 1/2005. The trailer had a hydraulic controlled elevator for loading and unloading and provided anti-slip floors with incorporated side guards.2.2. Enthalpy AssessmentData on temperature and relative humidity were collected inside the livestock vehicles during loading, transport, and unloading using Hobo data loggers (Hobo H8 loggers, Onset Computers, Bourne, MA, USA). Prior to loading, two loggers were placed on the lorry at the same level as the pigs in the middle floor of the trailer, and the loggers had an inside that was specifically designed perforated metal tubing to let air in while avoiding contact with the animals. Sensors were pre-programmed to record temperature and relative humidity at regular 5 min intervals and were fitted and removed by a member of the research team before and after each journey. Approximately 20 mm of wood shavings were placed on each floor of the vehicle as bedding. 2.3. SlaughterThe abattoir operated from Monday to Friday (from 06:00 a.m.to 15:00 p.m.) with a slaughter capacity of 2000 head/day at a rate of 220 heads/h. On arrival at the abattoir, pigs were unloaded with an adjustable-slope metal ramp with an anti-skid floor. After unloading at the abattoir, the pigs were showered for 15 in the winter or for 30 min in the summer, and the pigs were kept in lairage pens without mixing the groups on arrival and were given access to water through nipple drinkers. The lairage time for all animals was at least 12 h from arrival, including overnight rest and slaughter the following morning. At the end of the lairage period, pigs were stunned using a CO2 chamber with 70% CO2 atmosphere for approximately 60 s in a one-gondola dip-lift system. Following stunning, pigs were horizontally exsanguinated. Carcasses were then eviscerated and split before being placed in a chiller set at 4 °C for 24 h.2.4. Physiological AssessmentBlood samples were taken at the time of slaughter to evaluate physiological stress from 20 pigs per journey (one 10 mL tube per animal, with anticoagulant, EDTA-K3), totaling 160 sampled animals. Once all of the samples were collected for each journey, the samples were refrigerated for 10 h until they were centrifuged at 1300× g for 10 min to obtain plasma. The parameters measured in the plasma were cortisol, glucose, and creatine kinase. Plasma cortisol was assessed by ELISA. Calibrators were prepared with vials of cortisol in PBS and BSA and lyophilized at the concentrations of 0, 10, 30, 100, 300, and 900 ng/mL. For the colorimetric reading of samples, the blank and calibrators were performed within 20 min from the end of the assay using a spectrophotometer (Hitachi 717®) at 405 (for concentrations below 30 ng/mL) and 450 nm (for concentrations between 30 and 900 ng/mL). Plasma glucose was determined by the enzymatic colorimetric method (GOD/PAP). All of the solutions were pipetted into a cuvette and were incubated for 20 min at room temperature (15–25 °C). The absorbance of the samples and the standard absorbency was read against the blank using a spectrophotometer (Hitachi 717®) at 505 nm. CK levels were measured using a Roche/Hitachi 717 Chemistry Analyzer (Roche Diagnostics, S.L., Sant Cugat del Valles, Spain) with Boehringer Mannheim reagents. 2.5. pH MeasurementsMuscle pH was measured on the carcasses from the 160 animals sampled during the slaughter. After slaughter and dressing but before carcass cooling the initial pH of the loin was measured (pH 45 min) using a portable pH meter (HANNA, mod. HI9125) with temperature compensation. The electrode was inserted in the Longissimus dorsi muscle (LD) between the 13th and 14th intercostal space, perpendicular to the midline of the left held carcass, at an average depth of 2.5 cm. Afterward, the ultimate pH was measured from the same animals at 24 h post-mortem at the same location on the carcass. 2.6. Enthalpy Models and Statistical AnalyzesPsychrometric graphs were obtained for each of the eight journeys using data collected by the sensors placed at animal height inside the vehicles. The graphs were obtained based on the ASBE model, which included temperature, relative humidity, absolute humidity and enthalpy. The psychrometric data ASAE D271.2, defined in April 1979 and reviewed in 2005 (ASABE 2006, ST. Joseph, MI, USA), were used to calculate the psychrometric properties of the air surrounding the pigs. The temperature gradient was calculated using the Savitzky–Golay algorithm for one dimension, tabulating the data. The numeric derivatives were calculated using the Savgol routine in Matlab version 7.0 (Mathworks Inc., Natick, MA, USA). A polynomial routine was also used to test the neighboring data around each point. The points were processed by replacing them with the value of the polynomial. The derivatives were presented after programming the derivatives of the polynomials of each point. A window of 21 points with a fifth order polynomial was used. After that, we calculated and graphed the speed of temperature change (i.e., temperature gradient °C/s) and the apparent temperature in each case [19,20].Enthalpy (h) is a thermal comfort index that expresses the heat amount in 1 kg dry air in kJ and is determined by the equation as seen in Barbosa-Fihlo et al. [21]. H=(6.7+0.243t+((RH100)·10[7.5t237.3+t]))4.18 where: H = enthalpy (kJ/kg dry air);t = temperature (°C);RH = relative humidity (%).All of the statistical analyses were performed using the statistical program SAS/STAT (Statistical System Institute Inc. Cary, NC, USA. 2000). The data for temperature and humidity were analyzed using repeated measures, while the data on cortisol, glucose levels, and meat pH were analyzed using PROC MIXED. The experimental unit was each journey. Averages were compared by the least significant distance, with a level of significance of 5% (p < 0.05).3. ResultsIn the eight journeys that were studied, there was no mortality or non-ambulatory animals. There were five journeys that corresponded to the winter season (January–February), and the remaining three occurred in the summer season (June–August). Table 1 summarizes the average temperatures and relative humidity for each journey. The average inside temperature during winter journeys was 13.8 ± 3.9 °C (CV = 28.3%) and was 28.9 ± 4.1 °C (CV = 14.0%) in the summer. Figure 1 shows the changes in temperature inside of the truck over time for both the summer and winter journeys. During most journeys, the temperature increased as the transport progressed, with two journeys in summer surpassing 30 °C. Figure 2 shows the psychrometric graph for each journey, with more data points for the summer journeys in the upper right quadrant (higher temperatures and higher water content in the air). The average humidity for the winter months was 5.5 g water/kg dry air and was 9.8 g water/kg dry air in the summer. Figure 3 summarizes the speed of temperature change or gradient for the summer and winter journeys. The average speed of change for the winter months was 11.8 H/min−1 and for the summer months, it was 12.8 H/min−1. On average, plasma cortisol levels (±SD) were significantly higher (p < 0.05) in the winter (57.8 ± 11.7 nmol/L) than in the summer (28.8 ± 4.3 nmol/L). Plasma glucose was also significantly higher (p < 0.05) in the winter (309.7 ± 37.9) than in the summer (60.2 ± 26.6). CK exhibited no significant differences among the seasons (p > 0.05) and averaged 4887.3 ± 2649.1 U/L. The pH (±SD) of the LD muscle after 45 min was significantly lower (p < 0.05) in the winter (6.08 ± 0.24, range 5.29–6.56) compared to in the summer (6.24 ± 0.24, range 5.33–6.8). However, the pH after 24 h was not significantly different between the seasons (p > 0.05) and was 5.56 ± 0.13 (range 5.34–5.94).4. DiscussionSince the beginning of the 20th century, pig production has continued to undergo massive intensification and specialization in most industrialized countries, leading to larger and fewer farms and abattoirs with increased distances between them [22]. Long-distance transport has been reported to be physically, metabolically, and emotionally very demanding for animals [23]. Thermal stress is one of the key factors that can exacerbate the effects of long-distance transport on pig health and welfare [24]. In this context, the current study shows that winter transport is thermally more unstable, with abrupt changes in temperature and enthalpy compared to during the summer. These microclimatic conditions have a clear impact on animal-based welfare indicators such as cortisol and glucose as well as on pH after 45 min, but this difference disappears between the seasons after 24 h. Overall, the results suggest that enthalpy during transport can be a useful non-invasive indicator of animal welfare.Thermal stress is defined by the inability to maintain a constant body temperature by behavioral and physiological adaptation alone. This inability can result in heat stress or cold stress and, in extreme or prolonged cases, welfare consequences can lead to multi-organ failure and death [25]. The European Commission [16], has established a maximum temperature at which livestock, including pigs, must be transported on long journeys (i.e., 35 °C), but there are no accompanying limits for relative or absolute humidity. The thermoneutral zone of 100 kg pigs is centered around 20 °C [26], but the results from the current study show that temperature variations during transport vary much more widely, from about 5 to 35 °C. The coefficients of variation and gradients in the relative humidity and enthalpy were also high, especially in the winter, suggesting sudden shifts in the thermal environment around the animals. The results from the physiological parameters and the meat quality measurements suggest that pigs subjected to sharper enthalpy gradients had poorer welfare. The enthalpy ranges reported here are similar to a previous study by the research group that analyzed long-distance pig transport from the UK to Spain [15]. The psychrometric graphs underline the large differences between winter and summer journeys, with higher temperatures and lower humidity in the former and the opposite in the winter, as also seen in Seedorf et al. [27] and Lucas et al. [9]. Psychrometric graphs are not commonly used to model the microenvironment around livestock during transport although they can be used to calculate the absolute humidity and thus provide a better idea of the effort that animals need to make to lose heat to the environment. Indeed, the THI index can be graphed onto the psychrometric graphs themselves to designate danger zones. Using GPS data, we could then backtrack and find where along the way the thermal microenvironment may be more challenging on-board. Extreme ambient temperatures during live transport are considered to be one of the most relevant risk factors for injuries (both ambulatory and non-ambulatory) and deaths on arrival rates, especially as transport generally occurs when microclimate conditions are outside the ideal thermal comfort zone of the pigs [28]. However, the results presented here suggest that even though there was no mortality among the 1920 pigs that were transported, some winter journeys with high variation in temperature may be pushing the coping abilities of the animals. Other authors [29] have observed marked mortality and carcass defects in pigs transported in the winter compared to those transported in the summer in Spain.During long-distance transport pigs are exposed to stressful events such as a new environments, new smells and noises, loading and unloading, mixing with unknown animals, deprivation of food, among others factors [30]. Elevation of plasma cortisol and glucose concentrations are considered to be more sensitive and reliable indicators to reflect the intensity of the stress response during transport [31]. The results showed that the animal-based measurements of the stress response, cortisol and glucose, showed significant differences between the seasons, with the winter being more stressful than the summer. These physiological measures validate the results concerning enthalpy rates. However, for the plasma CK enzyme variable, no significant differences were found between the summer and winter journeys. Although the average values we found for this enzyme are three times higher than those reported in pigs of a similar category during journeys of less than one hour in the Iberian Peninsula reported by Oliván et al. [32], CK is an important biochemical marker used to measure muscle exhaustion and fatigue during pig transport [33,34] because of the greater the amount of muscle microtrauma and the greater passage of this enzyme to the extracellular environment [7]. It is possible that the high CK levels found are the result of the interaction between thermal and environmental conditions (sensory stimuli, social interactions, density), the duration of the journey, and lairage time.Acute thermal stress immediately before slaughter accelerates muscle glycogenolysis, increases lactic acid concentration, and produces a rapid decrease in muscle pH early post-mortem while the carcass is still hot [35]. In pigs, this results in pale, soft, and exudative (PSE) meat characterized by a lower water holding capacity. In contrast, animals subjected to chronic heat stress have reduced muscle glycogen reserves, leading to lower production of lactic acid and dark, firm, and dry (DFD) meat characterized by high ultimate pH and greater water holding capacity [36]. The results show that the pH after 45 min was affected in the pigs that were transported in the winter, however the average values remained within normal ranges. Surprisingly, at 24 h, this effect disappeared, which possibly related to the good conditions of the vehicles and the logistics implemented for these types of journeys. This may be due to the fact that European Legislation provides a series of guidelines that have substantially improved animal transport in the region [18]. It is possible that the same type of journey, without forced ventilation, could have greater heat stress effects on product quality [37].5. ConclusionsOur results correspond to journeys under commercial conditions in specialized trucks, with a modern pre-slaughter logistics chain and following European regulations governing pork transport. Under these conditions, long-distance journeys during the winter in the Iberian Peninsula presented abrupt variations in the rates of temperature change and air enthalpy. These abrupt changes were reflected in higher values of cortisol and glucose, but not in CK. Muscle pH was affected at 45 min although at 24 h, these effects were not observed. Our study has shown that mitigation strategies to avoid thermal stress should be aimed at controlling abrupt changes in rates of temperature change and air enthalpy during long-distance transport.

animals : an open access journal from mdpi

10.3390/ani13101716

PMC10215913

This study aims to investigate the behavior and ecology of nutria (Myocastor coypus), a semi-aquatic rodent that was introduced to South Korea for commercial farming and subsequently damaged aquatic ecosystems after its release. How the behavioral ecology of introduced nutria changes over time and across seasons remains unclear. Twenty-four adult nutria (twelve males and twelve females) were radio tracked in 2015–2016 to identify their home range size and activity patterns in the Macdo wetland, South Korea. This study found that the nutria home range size varied seasonally, with males having larger home ranges than females. Additionally, nutria showed crepuscular and nocturnal activity patterns throughout the year, with no significant difference between sexes. The findings of this study provide crucial information on the home range and activity patterns of introduced nutria in the Macdo wetland, which can guide management efforts to mitigate their impacts on the ecosystem. It is the first quantitative analysis of the home range and activity patterns of introduced nutria based on radio tracking data in the Macdo wetland.

Nutria (Myocastor coypus) are semi-aquatic rodents that were introduced in South Korea for commercial farming but significantly damaged aquatic ecosystems. Understanding nutria ecological behavior is essential for developing effective control and eradication strategies to mitigate their impacts. Thus, this study aimed to investigate the home range and activity patterns of 24 nutria (12 males and 12 females) in the Macdo wetland in South Korea from 2015–2016 through radio tracking. The average minimum convex polygon home range of the nutria was 0.29 ± 0.55 km2, with a 95% kernel density estimation (KDE) home range of 0.43 ± 0.85 km2 and a 50% KDE home range of 0.05 ± 1.1 km2. The home range of males was larger than that of females; however, the winter home range of females was as large as that of males. The home range also varied seasonally, with the smallest observed in winter. The nutria showed crepuscular and nocturnal activity patterns throughout the year, with no significant difference between sexes. The activities in spring, summer, and autumn showed no significant differences, but the activity in winter was significantly different from that in the other seasons. This study may serve as a basis for developing appropriately timed and scaled management strategies to mitigate the impacts of nutria on ecosystems. In conclusion, several environmental and biological factors contribute to the behavior of nutria in South Korea.

1. IntroductionInvasive alien species are a major contributor to the current global biodiversity crisis, impacting native biodiversity communities through competition, ecological changes, economic costs, and health risks [1,2]. Among invasive vertebrates, nutria (Myocastor coypus) are semi-aquatic rodents native to subtropical and temperate regions of South America that have now spread to every continent except Australia and Antarctica. They are used for meat and fur production [3,4,5]. Non-native nutria has caused substantial ecological and economic damage by destroying cultivated crops, aquatic vegetation, and trees. Hence, the nutria is listed as one of the 100 most invasive alien species by the International Union for Conservation of Nature [3,6].Nutria were introduced to South Korea in 1985 to boost the local economy through breeding farms; however, the demand for nutria products did not meet expectations, causing a decline in the farming business [6]. Consequently, nutria, like many other mammalian species, were intentionally released or escaped into the natural environment, establishing populations in several areas, including Jeju Island [7,8]. By 2014, they were present in 19 administrative districts, posing threats to agricultural and irrigation systems because of aggressive foraging and burrowing [9]. Nutria also have a negative impact on public health as they transmit zoonotic diseases, such as leptospirosis and toxoplasmosis [10,11].Nutria are difficult to eradicate because of their high fecundity [12]. Lethal control through trapping and hunting has been effective at the local level in Britain, the Delmarva Peninsula in the United States, and South Korea [3,13]. In response to public requests regarding increased agricultural damage, the Korean government launched a 5-year “Nutria Eradication Project,” in which 27,487 nutria were captured between 2014 and 2018 [6]. Although their distribution range has been reduced to 14 local administrative districts, nutria persist in the Nakdong River Basin [9]. If effective control measures are not implemented, the nutria population may spread further to earlier distribution sites and across the entire nation.Effective management of nutria requires a comprehensive understanding of their ecology and behavior [14]. Specifically, their home range and activity patterns must be assessed to develop suitable control and eradication methods and to optimize the spatial and temporal aspects of control initiatives. Very-high-frequency (VHF)-based radio tracking systems are widely used to monitor nutria [15] owing to their low operating cost and high efficiency and their ability to be detected under water [16].Several studies have focused on the ecology, diet, distribution, climate change-induced habitat suitability, and impact of nutria on agriculture and aquatic ecosystems [8,9,17,18]. However, a detailed investigation of their home range and activity patterns remains lacking to date. Thus, this study aimed to evaluate the home range and activity patterns of nutria according to sex and season by using a VHF-based radio tracking system. The results of this study are expected to provide valuable information to the government regarding the legislation of management policies that can effectively control and eradicate nutria at federal and national scales.2. Materials and Methods2.1. Study SiteThe Macdo wetland (126°34′30″–126°39′00″ E, 37°15′00″–37°160′30″ N) is situated in Busan Metropolitan City in the southeast corner of the Korean Peninsula. It covers a rectangular area of 2.58 km2 with a length of 6.90 km (Figure 1). The lowest and highest monthly mean temperatures at the study area, based on Busan weather station data, are −3 °C and 25 °C, respectively. The primary vegetation types in this region include Bromus japonicus, Alisma orientale, Setaria faberi, and Oenanthe javanica. This wetland is one of Korea’s most representative seasonal habitats for migratory birds, making it a haven for bird watchers. The wetland is also an important nutria habitat, with records dating back to 2009 [6].2.2. Capturing and HandlingLive traps were placed in areas with nutria activity in October 2015, with apples and carrots used as bait. The traps were checked daily. Captured nutria were anesthetized with alfaxalone (0.4 mL/kg) administrated intravenously, and basic information such as sex and weight was recorded. A matched sample of 24 adult nutria (12 males and 12 females) were selected for this study. Each nutria was fitted with a VHF radio transmitter (R2030, ATS Inc., Minneapolis, MN, USA) attached to its neck and released at a site 100 m from the capture point (Figure 2).One week after release, the individuals were radio tracked using a truck with a VHF radio collar antenna (ATS Inc., Minneapolis, MN, USA) attached to a handheld receiver (ICOM Inc., Osaka, Japan) to obtain details regarding their movement and activity patterns. The location of each individual was verified by direct observation after homing in on the signal. The survey was conducted once a month for three consecutive days and nights, and the data collected included GPS positions, such as latitude, longitude, date, and time. The strongest signals were used to access the individual to minimize bias. The ethical guidelines published by American society of mammologist for conducting research involving animals was followed [19]. During the survey period, 12 surveys were conducted, and the number of locations varied from 12 to 195 owing to field conditions and individual behavior variability.2.3. Data AnalysisHome Range EstimationWhen estimating the home range of each individual, we considered only locations with statistical independence based on the Schoener index [20]. We estimated the home range of each nutria based on 100% minimum convex polygon (MCP), 95% fixed-kernel density estimation (KDE95), and 50% KDE (KDE50) [21,22] by using the Home Range Tool extension in ESRI ArcMap 10.1 (ESRI, Redlands, CA, USA). MCP is a simple method often criticized for overestimating home range [23]. KDE utilizes a nonparametric probability density function to account for the nonlinear curved outlines of the home range [24]. KDE50 reflected the core area of the home range of each nutria. The least-squares cross-validation estimator was used to calculate the smoothing factors for the fixed-kernel estimators [25]. We compared the average and seasonal home range sizes between sexes using Welch’s t-test [26], whereas analysis of variance was used to compare seasonal differences in the average home range. The chosen p-value for significance was set at p < 0.05. 2.4. Activity PatternsTo determine the activity patterns of the studied species, we considered the locations obtained during sampling at 1–2 h intervals for three consecutive days and nights each month. We then evaluated differences in the probability of a nutria being active (each nutria location was binary coded: 1 = active, 0 = inactive) in relation to the period of the day, sex, and season. Periods were defined by sunlight: diurnal, activity during the day; nocturnal, activity at night; crepuscular, activity during twilight; and cathemeral, no differences in activity between the three time periods [27]. The Wald chi-square test was used to examine differences in the probability of a nutria being active in relation to the period of the day, sex, and season.3. Results3.1. Home Range of NutriaBetween October 2015 and September 2016, we recorded 2336 independent locations from the 23 adult nutrias (11 males and 12 females) fitted with radio transmitters (Table S1). The transmitter of nutria #N18 was destroyed; thus, its activity was not monitored. However, the data from 23 individuals were monitored sequentially throughout the survey period. During the 12-month survey period, we estimated the home ranges of individuals in 12–195 locations. The 100% MCP analysis revealed an average home range of 0.29 ± 0.55 km2 (range 0.03–0.88 km2) over the entire tracking period. The mean KDE95 and KDE50 were 0.43 ± 0.85 km2 (range 0.01–0.71 km2) and 0.05 ± 1.1 km2 (range 0.004–0.18 km2), respectively (Table 1). No significant difference in seasonal home range was found between males and females in winter (t = 2.29, p = 0.064), and the winter home range estimated by KDE95 was similar for both sexes (Table 1). However, when the entire study period was considered, males had a significantly larger home range than females (t-test, t = 2.26, p = 0.03; see Table 1). Nutria home range showed seasonal changes regardless of the estimator used (MCP = 3.31, p = 0.021; KDE95 = 2.64, p = 0.006; KDE50 = 3.80, p = 0.005). The smallest nutria home range was observed in winter among all seasons (Table 1). Typical home ranges (MCP and KDE) for nutria #N1 and #N3 are shown in Figure 3. A summary of the seasonal home ranges for each sex and their test statistics are presented in Table 1.3.2. Activity Patterns of NutriaWhen the entire period was considered, the nutria showed unimodal crepuscular activity (with approximately 39.6% of locations at dawn and dusk) and nocturnal activity (on average, 46.2% of locations were at night) peaks (Figure 4). Less than 14% of all nutria locations were recorded between 06:00 and 18:00, indicating minimal diurnal activity. Nutria activity significantly varied along the circadian period, with the highest activity during twilight and the lowest activity during the day (χ2 = 10.02, p = 0.015). We did not detect different activity patterns between sexes (χ2 = 3.21, p = 0.06), but females had greater activity intensity in almost every season than males (Figure 4). No significant difference in nutria activity was observed between spring, summer, and autumn; however, the activity in winter was significantly different from that in the other seasons (Table 2). Although winter had the lowest number of nutria trap locations (n = 252), the activity curve displayed evidence of activity during certain times of the day.4. DiscussionAn in-depth understanding of ecological behavior is essential for effectively managing alien and invasive mammals that have been successfully introduced, established, and spread in new environments [28]. The behavior of such species is influenced by various factors, including local environmental factors, such as habitat size and habitat type; biological factors, such as capture and killing; and physical factors, such as dams and walls in the area they inhabit [29]. However, specific behavioral information on nutria in South Korea is currently unavailable. Even within the introduced range, such information is scarce and often only available in the native Korean language. This is partly due to the difficulty in continuously recording individual behavior in the field, considering that these animals spend most, if not all, of their time in or near aquatic habitats. This study utilized radio telemetry to extract behavioral data on the home range and activity patterns of nutria, aiming to guide the implementation of the nutria eradication project. In the present study, the average MCP home range was 0.29 ± 0.55 km2, and the KDE95 and KDE50 were 0.43 ± 0.85 and 0.05 ± 1.1 km2, respectively. The range observed in our study is similar to their native South American range [30], but larger than that (95% MCP of 0.043 km2, KDE95 of 0.085 km2, and KDE50 of 0.018 km2) in similar habitats along the Miryang wetland in South Korea [31]. This difference in home range may be associated with the available habitat. For instance, home ranges are smaller in small wetlands and larger in large wetlands. The Miryang wetland is narrow (0.21 km2) and bounded by roads and fences, creating a hostile matrix that is difficult for nutria to travel across. By contrast, our study area is a broad riparian habitat (2.58 km2) adjacent to the Nakdong River. Food and adequate feeding and nesting platforms may spread over greater distances, resulting in a broader home range. Such large home ranges have also been observed for species inhabiting open areas [32].Our study also revealed sex differences in home range, with male nutria exhibiting a home range that is 0.23 km2 larger than female nutria on average. Sex differences in home range have also been observed for nutria in introduced [31] and native ranges [30,33]. This phenomenon is consistent with the general trends observed in various rodents [34,35]. The observed differences between sexes are often attributed to differences in body size and, consequently, differences in their energy requirements. Because of the difference in energy requirements between sexes, males would cover larger foraging areas than females [36]. Significant differences in body weight between sexes were observed in the present study. Although nutria are not sexually dimorphic, males are heavier and larger than females [37], which could partly explain the observed differences in home range between sexes.Males and females differ in reproductive behavior. Males are typically more aggressive and engage in more territorial behavior than females, creating hierarchies prior to the mating season [38] and mating with multiple females [12]. Reproductive behavior also influences differences in home range between sexes, with males searching larger areas for mates [39] and females reducing their home ranges to fulfill the energy needs of reproduction. In many species, the spatial organization of males is strongly influenced by the distribution of females, as the reproductive success of males is often determined by their ability to find and defend mates [40,41]. The mating system in nutria is a resource defense polygyny, wherein dominant males compete to monopolize key resources (territory and foraging areas) that are then used by reproductive females [12,42]. Therefore, by occupying a large home range, males can maximize their chances of mating and fertilize more females, leading to greater reproductive success [43].The ecological functions of rodents are related to their activity and space use patterns, which may exhibit seasonal variations [44]. In the present study, the home ranges of nutria varied seasonally and were considerably smaller in winter than in the other seasons (Table 1). Winter survival necessitates physiological and behavioral modifications [45] because movement costs can be exceptionally high owing to low temperatures. Furthermore, the reduced availability of food resources during winter may account for small home ranges. In response to these challenges, rodents tend to minimize locomotion and spend more time in nests or resting areas to conserve energy, particularly during winter [46,47]. Nutria exhibit predominantly nocturnal and crepuscular activity patterns, with some individuals displaying diurnal activity during winter. The activity patterns of the nutria analyzed in the present study were similar to those of other nutria populations in Argentina [48], Italy [49], and South Korea [31]. Other similar competing species sometimes present similar circadian activity patterns, with increased activity during twilight and nighttime, although marked differences have been found between sexes [50]. For instance, Zschille et al. [50] found sex differences in American minks. Further comprehensive studies may reveal sex differences in nutria activity that were not evident in the data collected in this study.In the present study, no seasonal differences in nutria activity patterns were found during spring, summer, or autumn. However, the activity in winter differed significantly from that in the three other seasons (Table 2). Small bouts of diurnal activity occurred in winter, when the night was colder, and nutria foraged during day. The local absence of wild predators or control plans may have favored the activity patterns of nutria during the day. This interpretation is supported by previous findings that nutria may become diurnal during cold months [51,52]. This adaptation to daylight hours is presumably a response that minimizes energy loss and maintains adequate food intake at low environmental temperatures [53]. The freezing of water surfaces at night can prevent nutria from accessing food sources, such as aquatic vegetation [54,55]. Accordingly, food provided to captive nutrias throughout the 24 h cycle restores the crepuscular and nocturnal behaviors of this large rodent [53,56].Radio-tagged nutria in Germany are diurnal and never detected during crepuscular or nocturnal hours [57]. However, in our study area, nutria activity peaked after sunset and continued until sunrise. The highest activity during twilight and nighttime could be associated not only with rising temperatures during the day but also with stress conditions produced by human activity. Our study site is located adjacent to Busan (the second largest city in South Korea), which has a population of over 4316 inhabitants/km2. During the day, people frequently use this area and outdoor facilities, often in family groups or with dogs. This increased human activity during the day may restrict the activity of nutria, as has been proposed in suburban areas where they have been introduced [49]. Furthermore, retaliatory killing/capturing following the eradication program [6] has created a “landscape of fear” for nutria, most of which have adopted crepuscular and nocturnal behavior in our study area. Several individuals were captured/killed from 2014 to 2018 in South Korea (including our study sites), which might have increased nutria activity during the darkest hours. During the implementation of the nutria eradication project in South Korea, several individuals were caught or killed during daylight [6]. Considering this information, we predict that these diurnal hunters would have placed substantial pressure on nutria, leading to the evolution of crepuscular and nocturnal activity patterns to reduce their probability of encountering humans. Many species undergo rapid and significant behavioral changes owing to strong human harvest selection [58]. Although the hunting pressure is currently low for the nutria population under study, the temporal activity patterns that evolved in response to earlier selection pressures may still be present. Behavioral plasticity has been observed in nutria, which can adapt to a wide range of environmental conditions, including natural ponds, swamps, and riverine habitats. In South Korea, introduced nutria have successfully settled in the Nakdong River Basin [4]. Various studies have predicted that suitable habitats are confined to midstream and downstream riverine areas of basins that experience relatively mild winters [9,59]. As a result of ongoing global warming, temperatures in the basin are expected to increase, particularly in the upstream regions [60]. If proper control measures are not implemented, nutria will retain their current distribution, and further expansion is likely to occur gradually from south to north through the water channel [9]. Hence, eradication efforts should consider dividing this broad Nakdong River Basin into two distinct management zones (i.e., middle and lower Nakdong Basins) which can be treated as individual units and are large enough to minimize the chance of reinvasion from adjacent zones. Isolation of nutria populations is likely to improve the effectiveness of eradication efforts because it reduces adaptability through loss of genetic diversity or inbreeding, which can ultimately lead to extinction [61].Poisoning, shooting, and trapping were traditionally employed to mitigate the impacts of nutria. In South Korea, the use of rodenticides is restricted because of their consumption hazards to non-target species, and shooting is prohibited. Trapping is commonly used for controlling nutria populations because of its low cost and effectiveness [13,62,63]. Considering that females are prolific breeders, trapping females within their home ranges should be considered as a reference to delineate control actions. Thus, any control plan that aims to reduce nutria populations in areas similar to our study area should involve placing traps on every kernel home range of 0.39 ± 0.2 km2. Furthermore, nutria activity peaked from dusk to dawn, suggesting that trapping to capture individuals for eradication may be most effective between 18:00 and 06:00. Multiple capture cage traps (MCTs) could be a good option for capturing nutria in urban and rural habitats. The ability of MCTs to capture multiple individuals of all size ranges is particularly important when dealing with species such as nutrias, which form social groups [64].Our study focused on the Macdo wetland, which is only a small portion of the large Nakdong River Basin. The microhabitat of nutria could vary in different regions of the river and connected wetlands. The findings of this study may not fully represent the overall nutria habitats in South Korea. Therefore, further studies are required to better understand the activity pattern and ecological impact of nutria on the aquatic ecosystem. Future data collection efforts should focus on the use of microhabitats, mating/reproductive strategies, dietary preferences, and dietary item variations that may drive shifts in home ranges and activity patterns. The potential impacts of nutria control on nesting bird species and other native wildlife that may be present should also be considered.5. ConclusionsOur study described the seasonal home ranges and activity patterns of nutria introduced in South Korea, and the results showed significant differences in home range size between sexes and seasons. Introduced nutria showed more intense activity during crepuscular and nocturnal hours and a relatively lower activity intensity in the afternoon. This study provides crucial information for controlling nutria in the study area and other regions with similar characteristics. It is the first quantitative analysis of the home range and activity patterns of introduced nutria based on radio tracking data in the predator-free Macdo wetland in South Korea. Future studies should consider investigating larger areas and take into account other variables that may impact the behavior of nutria in order to develop more comprehensive management strategies for controlling the ecological impact of nutria in South Korea and other regions.

animals : an open access journal from mdpi

10.3390/ani11082242

PMC8388468

Tail docking is a procedure practiced on millions of lambs all over the world. The objective is to prevent fecal soiling on the lower part of the tail, reduce soiling of the breech, and thereby lessen the risk of blowfly strike. Docking can be done with a knife or a clamp, but applying a latex ring round the tail, cutting off the blood supply so that the tail drops off a few weeks later, is the most popular method. All methods cause acute pain which diminishes substantially after the first hour. The present trial determined whether local anesthetic delivered by a prototype Numnuts® device, a novel, dual-function applicator, would reduce this pain in two to four-week-old lambs. Comparison of lambs that were sham handled, lambs that underwent ring tail docking and a third group of lambs that underwent ring tail docking and that were injected with lignocaine using the dual function device was conducted. All lambs were returned to their pen with their mothers and videoed for three hours for behavioral signs of pain. Every five minutes for the first hour and then every ten minutes, each lamb’s posture, movement and feeding behavior was classified and quantified, and the data subjected to statistical analysis. It was concluded that applying lignocaine using the novel device greatly reduced the degree of pain observed.

Docking the tail of lambs is a standard husbandry procedure and is achieved through several techniques including clamps, hot or cold knives and latex rings, the last of which is the most popular. All tail docking methods cause acute pain which can be reduced by application of local anesthetic, however precise anatomical injection for optimal efficacy requires considerable skill. This pen trial evaluated the ability of local anesthetic (LA) delivered with a dual function ring applicator/injector to alleviate acute tail docking pain. Thirty ewe lambs were assigned to one of three treatment groups (n = 10 per group): ring plus local anesthetic (Ring LA), ring only (Ring) and sham handled control (Sham). Lambs were videoed and their behavior categorized every five minutes for the first hour and every 10 min for the subsequent two hours after treatment. There was a significant effect (p < 0.001) of treatment on total active pain related behaviors in the first hour, with Ring lambs showing higher counts compared to Ring LA or Sham. Ring lambs also displayed a significantly higher count of combined abnormal postures (p < 0.001) than Ring LA or Sham lambs. Delivery of 1.5 mL of 2% lignocaine via the dual action device abolished abnormal behaviors and signs of pain in Ring LA lambs. However, lambs in the Ring LA group spent less time attempting to suckle compared to Ring and Sham lambs, suggesting that some residual discomfort remained.

1. IntroductionDocking the tails of lambs by applying a vasoconstrictive latex rubber ring is a widespread practice used in many countries. The procedure causes acute pain and stress that lasts for over an hour [1,2,3]. Trials have shown that when local anesthetic (LA) is injected into the tail prior to the ring being applied, pain can be greatly alleviated [1]. The vast majority of farmers would prefer to cause as little pain as possible when docking their animals [4], however injection of LA is slow and cumbersome, and requires considerable technical skill for accurate location of the injection site [5]. To administer LA and a rubber ring currently requires two tools: a syringe and needle to inject the LA, and a set of marking pliers to fit a ring on the tail. Furthermore, injection with a syringe and needle risks needle stick injury. It is therefore unsurprising that very few commercial-scale farmers use local anesthetic for tail docking.To address these issues of logistics, ergonomics and operator safety, a novel dual function marking instrument (Numnuts®) was developed by Senesino Ltd., (Glasgow, UK), that allows the operator to fit a latex docking ring around a lamb’s tail and then inject local anesthetic into the tail adjacent to the ring (www.numnuts.store/the-development/, accessed on: 17 July 2021). The Numnuts® device provides accurate and consistent local anesthetic application without a requirement for detailed knowledge of animal anatomy or extensive operator training. The trial described here examined the degree of pain relief provided by docking the tail of lambs using a late-stage prototype of the Numnuts® device. 2. Materials and MethodsThe animal phase of this study was carried out at the Moredun Research Institute, Midlothian Scotland, under UK Home Office License PPL 70/8075, and the data analysis was carried out at the Commonwealth Scientific and Industrial Research Organization (CSIRO), Armidale New South Wales, Australia.2.1. The Applicator Device A protype version of a novel dual-function marking tool was developed. The device allows the operator to rapidly fit a rubber ring around a lamb’s tail and then inject 1.5 mL of local anesthetic past the ring into the tail, just cranial (proximal) to the constriction site. When the rubber ring contracts over the tail, it temporarily holds the prongs of the device in a fixed position around the tail. This temporary fixation enables the injection mechanism to consistently deliver a metered 1.5 mL volume of local anesthetic subcutaneously into the tissue of the tail beneath the ring.2.2. Design of the Efficacy TrialThe efficacy of local anesthetic (1.5 mL 2% lignocaine hydrochloride, Troy laboratories, Australia) injected using a late-stage prototype of the Numnuts® applicator (Senesino Ltd., Glasgow, UK) was examined in 2 to 4-week old, Greyface cross Texel ewe lambs. Thirty single-born lambs (5.8–11.8 kg) were assigned to three treatment groups (n = 10 per group): ring plus local anesthetic (Ring LA), ring only (Ring) and sham handled control (Sham) groups were balanced by stratified randomization on weight. Lambs were individually identified by large colored numerals sprayed on their flanks. The lambs were housed as ewe-lamb pairs in group pens (8 × 5 m) with deep straw bedding over concrete floors at the Moredun Research Institute, Bush Loan, Edinburgh, Scotland (Figure 1). Pens housed 7–10 lambs each. Activity in each pen was recorded by two video cameras connected to digital video recorders positioned on opposite sides of the pen, and footage was captured by a video management software (Huawei Technologies Co., Ltd., Reading, UK). Treatment application took 1 min to undertake. During treatment application, lambs were restrained in dorsal recumbency in a marking cradle. Ring lambs had rubber rings (Elastrator Brand) applied using the prototype applicator without an injection, Ring LA received an injection of 1.5 mL lignocaine via the prototype applicator at the time of ring application. Sham controls had their tail manipulated without application of a ring or injection. After the procedure, the lambs were returned to their pens, with each pen containing a mix of treatment groups. Due to operator error, although all treatments were represented in each pen, treatments were not fully balanced between pens.The responses of the lambs were videoed for analysis of active pain avoidance behaviors. Behaviors and descriptions used in this study (Table 1 and Table 2) have been previously validated as pain related behaviors in response to ring castration and tail docking [6,7]. Personnel quantifying the behaviors were blinded for the treatment group, having not been present at treatment administration. Video resolution was not sufficient to identify which lambs had rings and which did not. Postures were classified and scored at 5-min intervals for the first hour and at 10-min intervals for the second and third hours, as shown. Active pain related behaviors were classified every 5 min for the first hour and were summed to give a total count. The time that behaviors were recorded was based on the lamb’s treatment time, as time zero for each individual. Teat seeking behavior was also classified during the scoring of active behaviors.2.3. Analysis of the ResultsCounts for postures were summed over two intervals: the first hour, and hours 2 and 3 combined, so that 12 counts were recorded per animal for hour 1 and 12 counts for hours 2 and 3 combined. In hours 2 and 3, the number of observations for each lamb varied between 7 and 12. In this interval, some observations were missed when a lamb was obscured by other animals in the pen. For animals available for observation on fewer than 12 occasions, scores for each posture were rescaled to 12. Analysis of variance (ANOVA) was performed on total counts in the first hour, and a repeated measures model was fitted to examine the change in a variable between the two time blocks, hour 1 and hour 2 and 3. Residuals from the repeated measures models could not be normalized by data transformation. Data for the first hour were suitable for analysis without transformation. Liveweight was tested as a covariate and fitted when significant (p < 0.050). Sham handled lambs were not present in all pens, so the pen (i.e. group) was not fitted in the analysis as the pen was confounded with treatment. Data for hours 2 and 3 were not normalized by transformation, and were analyzed by a Kolmogorov–Smirnov non-parametric test. Active pain related behaviors were analyzed two ways. For each animal, total active pain behaviors (Table 1) were summed at each of the 12 observation points during the first hour and the change over time was analyzed in a repeated measures model. Data was log transformed for analysis. Secondly, total active pain behaviors for each animal were summed across the 12 observation points and analyzed by ANOVA. Total count of teat seeking activity, eating at the trough and sucking in the first hour, and sucking in hours 2 and 3 were analyzed by ANOVA. Eating at the trough in the first hour was log transformed for analysis. Post-hoc contrasts between treatments were performed by univariate F tests. Eating at the trough in hours 2 and 3 was analyzed by a Kolmogorov–Smirnov non-parametric test. Analyses were performed in Systat version 9. Plotted data are least squares means ± standard error, except for back transformed values where error bars are not plotted.3. Results3.1. Behavioral PosturesThere was an effect of treatment on the combined count of total abnormal postures in the first hour (p < 0.001, Figure 2b) Ring lambs displayed a significantly higher count of combined abnormal postures than Ring LA or Sham lambs (p < 0.001). There was no difference in abnormal postures between Sham and Ring LA lambs (p = 0.716). Data could not be transformed to normality for repeated measures analysis of change in total abnormal postures between time intervals. In hours two and three, there was a low count of combined abnormal postures in all treatment groups (Figure 2a).Subsequent analyses on individual types of abnormal postures examined the two time periods independently. For the first hour, there was an effect of treatment on abnormal walking (p < 0.002). Ring lambs displayed higher counts of abnormal walking than Ring LA or Sham in the first hour (p < 0.002, Figure 2c). There was no difference in abnormal walking between Sham and Ring LA lambs (p = 0.763, Figure 2c). There was an effect of treatment on abnormal lying (p < 0.001). Ring treatment displayed higher counts of abnormal lying than Ring LA or Sham (p = 0.001). There was no difference in abnormal lying between Sham and Ring LA lambs (p = 1.000) (Figure 2d). There were few instances of abnormal standing in the first hour and an effect of treatment was not observed (p = 0.293, Figure 2e). For hours two and three, there were no differences between treatments for total abnormal postures or for the individual postures of abnormal standing, abnormal walking or abnormal standing.3.2. Active Pain Behaviors in the First Hour, Eating and Sucking There was an effect of treatment (p < 0.001), time (p < 0.001), and a treatment by time interaction (p < 0.001) on total active pain behaviors in the first hour. The count of active pain behaviors was higher in Ring lambs than Ring LA lambs at 5, 15, 25 and 30 min (Figure 3a) and approached significance at 10 and 20 min. There was no difference in active pain behaviors between sham and Ring LA lambs at any time point. For total active behaviors summed across the 12 time points, the effect of treatment was highly significant (p < 0.001, Figure 3b). Ring lambs displayed a higher count of active pain-related behaviors (p < 0.001) than Ring LA or Sham lambs. There was no difference in active pain behaviors between Sham and Ring LA lambs (p = 0.861).There was an effect of treatment on teat seeking (p = 0.045). Applying the ring tended to reduce teat seeking behavior in Ring lambs (p = 0.08), and decreased teat seeking in Ring LA lambs (p = 0.016, Figure 3c), however Ring and Ring LA lambs did not differ (p = 0.465). Treatment tended to reduce sucking behavior in the first hour (p = 0.054, Figure 3d). There was no effect of treatment on eating at the trough (p = 0.191) in the first hour, but the count for this activity was very low (Figure 3e). There was no effect of treatment on eating at the trough or sucking in hours two and three.4. DiscussionThe aim of the current study was to look at the efficacy of a novel dual-function marking tool and its ability to reliably provide a single dose of local anesthetic at the time of tail-docking. The current trial showed that injection of 1.5 mL local anesthetic into the tail using the prototype device at the time of ring application abolished abnormal behaviors and signs of pain in the first hour after tail docking. However, the lambs in the Ring LA group spent less time attempting to suckle, suggesting that some residual discomfort may have remained. These results corroborate those of a subsequent field trial, where using the Numnuts® device to apply the ring and lignocaine suppressed the degree of pain observed following tail docking [8], and align with several other studies which showed the benefits of local anesthetic delivered by syringe and needle [1,9,10].In this study the application of the local anesthetic lignocaine using a dual function marking tool was able to reduce the acute pain experienced by lambs undergoing ring tail docking. Lignocaine has been reported to provide consistent pain relief to lambs in the first hour following ring castration and tail docking, with reduced cortisol response and reduced display of pain related behaviors in lambs receiving lignocaine following tail docking compared to lambs that did not receive lignocaine [11,12,13]. Although pain related behaviors were reduced in the lambs that received lignocaine in the current study, lambs in this group still had reduced teat seeking and sucking behavior, indicating some residual pain. As lignocaine is a fast-acting short duration analgesic, only the acute pain phase in response to tail-docking was examined. There has been extensive research into the acute and chronic effects of ring tail-docking and castration [7,14,15,16,17]. The acute pain phase of ring tail-docking has been reported to last up to an hour [18], with measures of chronic pain in lambs being hard to observe and unvalidated making it difficult to determine differences in the long term. Previous work has found no difference in chronic pain responses or in growth rate in lambs castrated and tail-docked with rubber rings that were applied with and without local anesthetic [13]. However, prevention of secondary hyperalgesia has been reported in lambs receiving local anesthetic following ring castration and tail docking compared to those without [19]. Long term effects were not looked at in this study, as the effects of local anesthetic at relieving tail docking pain are well reported and scope of this study was to look at the device’s efficacy at providing a single dose of local anesthetic. As most differences in treatment (with and without local anesthetic) following ring castration and tail docking are observed in the acute pain phase [13,18,19], only this phase was examined in the current study.The provision of pain relief for lambs undergoing painful husbandry procedures has increased over the last few years. Producers now have access to registered, easy to use products such as Tri-Solfen [20,21,22] and Buccalgesic [23,24,25] for surgical procedures such as mulesing, castration and tail-docking. Producers have faced limitations in their ability to provide feasible acute pain relief provision for ring tail docking [8]. Previous research has looked at the use of Tri-Solfen in providing pain relief for lambs undergoing hot knife tail docking (the lambs were concurrently ring castrated without the application of LA), however the topical formulation had minimal impact on behaviors when applied to the open wound on the tail in lambs that were concurrently castrated with a ring [26]. There has also been work that has looked at coating rubber rings in lignocaine as a method for delivering pain relief [9]. The lignocaine-coated rings ameliorated some of the pain in response to ring castration when compared to normal rings, however they were not as effective as injections of lignocaine [9]. Absorption of lignocaine through intact skin is limited, and delivery of pain relief via this route is slow and does not adequately address the acute pain phase of ring castration and tail docking. In the present study, application of a metered dose of lignocaine using the prototype Numnuts® device significantly reduced the acute pain response in lambs that underwent ring tail docking. 5. Conclusions The present study demonstrated that a novel dual-function marking tool can provide a measured 1.5 mL dose of lignocaine to the site of tail docking. This led to immediate pain relief for the acute pain phase of ring tail docking in lambs that received the local anesthetic compared to those that were not provided with pain relief. The present results are a positive step towards providing sheep producers with a device that would allow a rapid, practical and safe method for providing large scale relief of pain caused by tail docking.

animals : an open access journal from mdpi

10.3390/ani11041101

PMC8070519

This study investigated the types of materials targeted by cats eating non-nutritive materials (‘pica’), at about 6, 12 and 18 months of age, as reported by owners. Pica was most common at about 6 months, as compared to the older age categories. Most cats targeted a single type of material, with plastics and other materials being chewed or eaten more commonly than wool or other fabrics. The factors associated with the occurrence of “chronic pica” (pica exhibited at all three timepoints) in cats were also investigated. Moving to a new house, renting rather than owning a home, and living in a household without a dog(s) were factors found to increase the odds of a cat displaying chronic pica.

The prevalence and cooccurrence of pica towards different target materials were investigated using prospective data from three questionnaires completed by owners participating in a longitudinal study of UK pet cats. Pica towards one or more material types was reported in 42.9% (229/534), 32.0% (171/534), and 30.9% (165/534) of cats aged approximately 6, 12, and 18 months, respectively. At all timepoints, it was most common for only one material type to be targeted. Associations between potential explanatory variables and “chronic pica” (pica exhibited at all three timepoints) were also explored. Multivariable logistic regression revealed moving to a new house when the cat was aged approximately 6–12 months, renting rather than owning a home, and living in a household without a dog(s) when the cat was aged 2–4 months increased the odds of chronic pica occurrence. This study provides novel data from a cohort of UK pet cats and it is hoped this will increase the understanding of pica and provide direction for areas for future research.

1. IntroductionThe term “pica” is used to describe the ingestion of non-nutritive items. It is known that cats target a range of items including fabrics (made of wool, cotton, or synthetic materials), shoelaces, rubber, plastics, paper, cardboard, wood, and metals [1,2,3].Bradshaw and others broadened the definition of pica in cats to include chewing and/or sucking of non-nutritive items [1]. Whether these three behaviours (ingestion, chewing and sucking) should be grouped together or examined separately is debatable. Some suggest that chewing and/or sucking are kitten or infantile behaviours that are only retained by some into adulthood, therefore, the motivations for the behaviour may be different to that of ingestion [4,5]. Additionally, Borns-Weil and others reported that the age of onset of ingesting, chewing, and sucking differed by material type [2]. In this study, the term pica will be used to describe chewing of non-nutritive items with or without ingesting.The short and long-term impacts on the health and welfare of cats that exhibit pica are largely unknown and require further investigation. However, theorised significant impacts include gastrointestinal problems (such as intestinal obstructions), nutrition absorption problems or inbalances, a reduced intake of food, feline infectious peritonitis, pyruvate kinase deficiencies, and wear or damage to teeth and gums [4,6,7,8,9,10]. It should be noted that the definition of pica used by the majority of the authors of these studies is limited or not provided, and in some of the studies the number of cats with pica was very low (i.e., <5 cats). Welfare impacts are unknown.Although pica may be unlikely to be a direct cause of relinquishment to shelters, it has the potential to affect the human–animal bond. Owners of cats exhibiting pica may need to be vigilant to try and prevent ingestion of non-nutritive items, primarily for the welfare of the cat, but also to prevent damage to household items. Also, it could be hypothesised that an owner preventing access to a pica-targeted item might unwittingly cause frustration or distress to the cat, impacting on welfare.There is limited research exploring the prevalence of pica in cats and the factors influencing onset and occurrence. Bradshaw and others suggested that the onset of pica could happen at any point during the first 4 years of life [1]. They found that onset within their study population most commonly occurred between 2–4 months of age and also noted that onset frequently occurred in the first 2 months following rehoming. This led the authors to theorise that separation from the mother and siblings and/or introduction to an unfamiliar environment could be associated with onset, but also recognised several factors could confound this, so this could potentially be a correlational association rather than a causal one. Another previous study reported that age of weaning appeared to be associated with pica in Birman cats [2], but no evidence of an association has been reported yet for other breeds [2,3].Bradshaw and others also reported that for some cats within their study population the onset of pica occurred between 6–18 months of age, and in these cases, onset could not be linked to rehoming, so the authors suggested that sexual maturity or territorial behaviour could be associated with onset [1]. However, no evidence of an association between pica and neuter status, or pica and the sex of the cat, were reported in two other previous studies [1,3]. Bamberger and Houpt reported more male cats (21/32) than female cats exhibited pica in their study [11], but due to a relatively small sample size and lack of control population, those findings may be less robust than the studies with larger sample sizes. To the author’s knowledge, no longitudinal data on pica in cats has been reported. Whether pica is predominantly exhibited by kittens and is subsequently more or less likely to be retained as cats age would be useful to explore to increase the understanding of this behaviour.Several studies of pica have focused on Oriental breeds [1,2]; this is perhaps due to anecdotal and clinical experience that suggested an increased problem in these breeds [12,13], but little evidence of associations exists between breed and pica.Factors such as boredom and lack of social interactions have been speculated to be contributing factors for pica, however, a previous study reported pica did not appear to be a result of a suboptimal environment [3]. This is an area, however, that requires considerable exploration.We hypothesise that pica is likely to be influenced by multiple factors, some of which may occur months or years before the owner considers the behaviour to be a problem. Whilst cross-sectional studies can identify associations, they cannot establish causality. Studies using pre-existing data generally include little environmental information. A better approach to elucidate the relative influence of environmental factors on behavioural outcomes is to recruit a cohort of kittens before these problems occur and follow them through life. Therefore, to provide a better understanding of pica and factors associated with the behaviour, this study used data collected prospectively to: summarise the prevalence and cooccurrence of pica towards different target materials in cats aged 6, 12, and 18 months as reported by their owners, and;identify and quantify early-life risk factors for the occurrence of pica exhibited by cats at all three timepoints, which we term ‘chronic’ pica. Exploring pica, which has been retained over time and become maladaptive, allows differentiation from “normal” or transient kitten behaviour. 2. Materials and Methods2.1. Study Design and ParticipantsData for this study were collected prospectively as part of the ‘Bristol Cats Study’ (BCS)—a longitudinal study of pet cats within the United Kingdom (UK). Between May 2010 and December 2013 (inclusive), 2203 cat owners were recruited to the study using a variety of advertising methods [14]. To be included in the BCS, the participants were required to: (1) live in the UK, (2) be aged 18 years or more, and (3) own a kitten (or kittens) aged 8–16 weeks at the time of registration.BCS participants were asked to complete self-administered questionnaires (either online or via postal paper copies) when their cat reached specific ages. The data for this analysis were obtained from the first four questionnaires, which were issued between May 2010 and April 2015 [14]. Questionnaire 1 (Q1) was issued to owners of cats aged 2–4 months, Questionnaire 2 (Q2) at 6.5–7 months, Questionnaire 3 (Q3) at 12.5–13 months, and Questionnaire 4 (Q4) at 18.5–19 months.The questionnaires consisted of mostly “closed questions” with multiple-choice answers. Data collected included the demographics of the owner, characteristics of the cat, and information relating to the management of the cat. All data from respondents were anonymised prior to analysis. The study was approved by the University of Bristol ethics committee (Reference UIN/13/026).2.2. Outcome VariablesTo investigate the occurrence of chronic pica within the BCS cohort, in Q2–4, owners were asked whether, at the time of questionnaire completion, their cat chewed with or without ingestion each of the following four materials: woollen fabrics, other fabrics, plastics, or other materials (Table S1 in Supplementary Materials).For the risk factor analysis for pica, the two outcome categories were defined as: cats whose owners had reported chronic pica towards one or more of the material types (woollen fabrics, other fabrics, plastics, or other materials) at all three time points (Q2–4); andcats whose owners had reported never observing pica towards any of the material types (woollen fabrics, other fabrics, plastics, or other materials) at all three time points (Q2–4). Cats reported by their owners to have exhibited pica intermittently toward one or more of the material types at one or two of the time points (Q2–4) were excluded from analysis.2.3. Potential Explanatory VariablesSummarised in Table 1 are variables assessed for association with chronic pica, and included breed, acquisition age, sex, neuter status, appetite, and outdoor access. To enhance the statistical power of the analysis, especially when variables had categories that contained few data points, univariable analysis was utilized to justify combining categories that had similar associations with the outcome and where merging categories was judged to be logical.2.4. Study SizeThe study size was determined by the number of cats within the BCS study whose owners had completed Q1–4 (inclusive) and had completely answered the questions on pica (see Table S1 in Supplementary Materials) in Q2–4. If owners had responded “do not know” to the pica questions, they were excluded from analysis as they could not be placed into either of the outcome categories. To remove any effects of clustering at the level of the household, if an owner had registered more than one cat onto the BCS, one of their cats was randomly selected for inclusion in this analysis using a random number generator, and their other cat (or cats) were excluded. The study had 80% power to detect an odds ratio of ≥3 with a 95% confidence level assuming 50% of controls were exposed to the variables of interest (Epi-Info 7, CDC, www.epitools.ausvet.com.au/ -accessed on 30 June 2020).2.5. Descriptive StatisticsThe prevalence and co-occurrence of pica towards the four different materials (woollen fabrics, other fabrics, plastics, or other materials) when the cats were aged 6, 12, and 18 months were summarised.2.6. Statistical Analysis of Pica toward Material Types and Cooccurrence between TimepointsCochran’s Q tests were run to determine if there were differences between the type of behaviour shown (pica and no pica) and the three time points for each of the four materials. To reduce the chances of a type I error being caused as a result of multiple testing, the Bonferroni correction was used. The critical p-value required was 0.0125. McNemar tests were used to provide post hoc analysis of variables included in Cochran’s Q tests where p < 0.0125.A chi-square test was used to test for an association between pica reported in early life (Q2 and/or Q3) and subsequent pica reported in Q4.2.7. Statistical Analysis of the Potential Risk Factors Associated with Chronic PicaThe statistical package IBM SPSS Statistics for Windows (Version 26) (IBM Corp: Armonk, NY, USA) was used for univariable and multivariable logistic regression analyses. In the univariable analysis, there was some variation in sample size for the potential explanatory variables due to some owners having not completed all questions within the four questionnaires.Where variables had a category that contained no cases, one control was randomly selected using a random number generator (Research Randomizer—http://www.randomizer.org/ - accessed 30 June 2020) and recoded as a case so that the univariable model could be fitted to the data. After each alteration, the data were restored to the original format.Variables found to have a univariable p-value of < 0.2 were included in the building of a multivariable model using the backward elimination technique. To facilitate comparison of models, cats with missing data for any of the eligible variables were excluded from the dataset. If two variables were found to be highly correlated (|r| > 0.9), one variable was excluded based on the creation of two models, each including one variable, and the model with the highest log-likelihood was selected. At the final stage of model building, all variables with p-value of < 0.05 were retained in the final model. The Hosmer and Lemeshow test was used to assess the fit of the model to the dataset.3. Results3.1. Prevalence and Cooccurrence of Pica within the Bristol Cats Study Cohort Reported in Questionnaires 2, 3 and 4The number of cats recruited to the BCS was 2203, 64.6% (n = 1423) of their owners completed Q1–4 (inclusive). There were 889 cats that were excluded due to non-completion or partial completion of the pica question in Q2–4, leaving data from 534 cats eligible for descriptive analysis. For the risk factor analysis, a further 250 cats were excluded due to not meeting the outcome category criteria as pica was intermittently reported at just one or two of the time points (Q2–4) and thus could not be classified as chronic pica. This left 284 cats that were eligible for inclusion in the univariable analysis. To enable comparison of multivariable models, 113 cats were excluded because of missing data for variables that were to be included in the multivariable analysis. The resulting sample available for analysis consisted of 171 cats.Table 2 shows the prevalence of owner-reported pica, and the Cochran’s Q tests results reveal highly significant associations between the age of the cat and the behaviours exhibited towards all four material types. Post hoc tests revealed that the prevalence of owner-reported pica significantly decreased between Q2 and Q3 (i.e., between approximate ages 6 months and 12 months) for all types of materials. In contrast, the prevalence of owner-reported pica was not significantly different between Q3 and Q4, although the prevalence of owner-reported pica for all four types of materials was significantly lower at Q4 when compared with Q2.A chi-square test revealed a highly significant association between pica in early life (Q2 and/or Q3) and subsequent pica reported in Q4 (Table 3). Of the 165 cats that exhibited pica in Q4, 81.2% (n = 134) were also reported to show the behaviour in Q2 and/or Q3. Also, importantly, of the 280 cats that showed pica in early life, 52.1% (n = 146) did not show pica at Q4.The cooccurrences of pica towards different material types are summarised in Table 4. In Q2, Q3 and Q4, 42.9% (229/534), 32.0% (171/534), and 30.9% (165/534) of cats were reported to express pica, respectively. At all three timepoints, it was most common for only one material type to be targeted as 47.2% (108/229), 55.0% (94/171), and 58.2% (96/165) of cats targeted only one material type in Q2, Q3, and Q4, respectively.3.2. Early-Life Risk Factors for Pica3.2.1. Univariable AnalysisOf the 534 cats for which pica data from Q2–4 were available, 53.2% (n = 284) were eligible for inclusion in the risk factor analysis due to meeting the criteria for the two outcome categories. Of these 284 cats, 21.5% (n = 61) exhibited pica towards one or more material types at all three time points and 78.5% (n = 223) did not express pica at all three time points. Table S2 in Supplementary Materials summarises the univariable logistic regression. There were 27 variables with a p < 0.2 identified for inclusion in the multivariable model building process, however, seven variables were excluded due to being highly correlated. These variables were: single or multi-cat household, cat receives food treats, and frequency with which household members played with the cat per week. Breed and neuter status were not found to be significant at the univariable analysis.3.2.2. Multivariable AnalysisThree variables were retained in the final multivariable model (Table 5). Cats that lived in a rented home had increased odds of exhibiting chronic pica (reported in Q1), OR (95% CI) = 3.41 (1.45–8.03), compared to cats living in homes owned by their owners. Also, cats belonging to owners who had moved to a new house (reported in Q3) had increased odds of displaying chronic pica, OR (95% CI) = 13.95 (1.41–138.25) compared to cats whose owners had not reported moving house in Q3.Cats living in a household without dogs (reported in Q1) had increased odds of being reported by their owners to exhibit chronic pica, OR (95% CI) = 4.86 (1.24–18.95) compared to cats living in households where dogs were present.The final multivariable logistic regression model for the chronic pica was found to correctly classify 82.5% of cases; the Hosmer and Lemeshow test provided evidence that the model was a fair fit for the data (0.269).4. DiscussionThis study has presented descriptive data on the prevalence of pica towards non-nutritive items exhibited by cats at three data collection points (Q2, Q3 and Q4 when cats were aged 6.5–7 months, 12.5–13 months, and 18.5–19 months respectively). To the authors’ knowledge, this is the first study to examine the prevalence of pica within a longitudinal study of UK-owned pet cats. Most existing research in this field has explored pica within populations that were potentially subject to selection bias as the authors purposefully recruited cat breeds to their studies that were thought to be inclined to exhibit pica.Bradshaw and others reported that onset most commonly occurred between 2–4 months of age, and also between 6–18 months of age [1]. The Cochran’s Q tests in this current study revealed highly significant associations between the cat age and pica exhibited towards all four material types. Post hoc McNemar tests revealed pica was significantly more likely to be reported in early life (Q2 than in Q3 and Q4). For all four of the material types, pica was most commonly reported at Q2 (6.5–7 months of age). Also, a chi square test revealed a highly significant association between pica in early life (Q2 and/or Q3) and subsequent pica reported in Q4. However, more than half (52.1%) of the 280 cats that showed pica in early life did not show pica at Q4. These statistical results suggest that pica declines in prevalence after initial onset, although some cats appear to retain the behaviour into adulthood.This study investigated the cooccurrence of pica towards different material types. At all timepoints, it was most common for only one material type to be targeted, and the most commonly targeted material type was plastics. These findings are contradictory to those of Bradshaw and others who reported it was most common (34.2% of 152 cats) for three types of materials to be targeted [1]. Bradshaw and others reported a preference for fabrics as 93% of cats in their study targeted wool, 64%—cotton, 53%—synthetic fabrics, and only 22% targeted rubber or plastic materials [1]. However, in our study, and the study of Demontigny-Bédard and others [3], fabrics were not the preferred target item. Demontigny-Bédard and others reported that “shoelaces or threads” and “plastics” were the two most commonly ingested items, and plastics were the most chewed material type of the 73% of 100 cats that chewed objects [3]. There could be a number of explanations for the apparent preference for plastics observed in this current study. This could indicate a difference in material preference between populations of cats and/or availability of the material to the cats. Alternatively, the difference could have arisen because of the nature of the study: In this prospective study, owners were asked specifically to look for and report signs of chewing, and the evidence of chewing behaviours might be more noticeable on plastics than on fabric or other items. For example, teeth marks on a hard plastic item will probably be permanent, whereas chewing on fabric may not leave a visible mark unless a hole was made. Additionally, it is possible that the prevalence of pica towards “other materials” was under-reported, as no explicit examples of “other materials” were provided with the question and it was left to the participants to interpret.The multivariable logistic regression analysis revealed that stability of the environment in which the cat lived appeared to influence the expression of chronic pica. Cats belonging to owners who had moved to a new house (reported in Q3 for the previous 6-month period when cats were aged 12.5–13 months) had increased odds of displaying chronic pica, than cats whose owners had not reported moving to a new house in Q3. Previous studies have suggested that stressful events could influence pica [1]. Novelty maybe be stressful [15,16] and a new environment could provide many stimuli that could induce stress. Why moving to a new home should have a larger impact on a cat aged approximately 12.5–13 months than a cat aged 6.5–7 months is unknown. However, it is speculated that an older cat may be more affected due to being more established in the original home than a younger cat. Further work would be useful to explore this association, particularly due to the large confidence interval and the variation in effect according to age of cat.Cats with increased odds of expressing chronic pica were found to belong to owners who rented their home (reported in Q1), rather than owners who owned their home. A potential explanation for this is that owners may be more likely to react to their cat chewing items in a rented property compared to a property they owned. If the owner tries to distract the cat from expressing pica by interacting with it, this could have a reinforcing effect [17]. It is also possible that renting or owning a property could be a proxy for socio-economic factors or this could be confounding from other variables. This warrants further investigation.Finally, the presence of dogs within the cats’ environment was found to influence the reported presence/absence of chronic pica. Cats living in households without dogs (reported in Q1—aged 6.5–7 months) had increased odds of exhibiting chronic pica, compared with cats living in households with dogs. This could suggest that either a familiar dog or dogs within a household have a protective effect on the expression of pica (for example, due to increased opportunity to show social behaviour and/or few periods of time without company), or factors within the environment prevent the cats from displaying pica, or pica being observed by owners (for example, avoidance of areas occupied by the dog (or dogs)). Without information on the relationship between the cat and dog (or dogs) in the household, this can only be speculated on and more research is required. It should be acknowledged that data on the presence of dogs within the cat’s household as reported in Q1 was analysed. Changes may have occurred within the household regarding dog ownership, so this finding should be interpreted with caution.It should be acknowledged that owners were not asked how frequently their cats exhibited pica, as data on pica were collected as part of a questionnaire collecting information on many aspects of the cats’ lives at that timepoint. Therefore, the frequency of the behaviour shown by cats classified as chronically exhibiting pica will vary, and some cats that showed the behaviour may have done so infrequently and potentially not to the extent of clinical or behavioral concern. Also, it is possible that owners were subject to panel conditioning due to becoming more aware and/or looking for signs of pica following answering questions about the behaviour in the BCS questionnaires. However, the data presented here is a useful addition to existing research and can be used to direct future work into pica.5. ConclusionsThis study found pica was most commonly reported by owners in Q2 (6.5–7 months) and declined thereafter. This potentially indicates that pica is a kitten behaviour that is not necessarily continued with increasing age in all cats. Awareness of this finding might provide owners with reassurance should they be concerned by seeing their cat exhibiting pica when a kitten. Also, awareness of the factors associated with chronic pica reported in this study could help owners observing pica in young cats to potentially address the cat’s environment and the stability of that environment and reduce the odds of the behaviour becoming chronic. Other factors not explored in this study, such as how the owner responses to the cat exhibiting pica behaviour may be of great importance to subsequent behaviour, and this would be a valuable future area of research. This study moves forward understanding of the complexities of pica in cats and we hope provides direction for future research.

animals : an open access journal from mdpi

10.3390/ani11102899

PMC8532608

This article considers ethical views concerning animals of research participants working in animal tourism and conservation who identify as Māori, the indigenous people of Aotearoa New Zealand (hereafter New Zealand). Field work interviews and discussions revealed views about the environment and about the spirit and spiritual connection of people, animals and nature. Understanding the views held by Māori people is important in New Zealand, as it is in any society with an indigenous people, but especially because of Te Tiriti ō Waitangi, one of the founding documents of New Zealand. This partnership agreement between Māori and the British Crown requires and supports a greater understanding of Māori knowledge and culture and accounting for this in our ethical and legal thinking. Our results show that there are factors that the Māori participants consider integral to animal care and management that are different from standard Western views and that it is necessary to reshape how the relationships between humans and animals are considered. We offer ways in which these ethical views of local indigenous community members may be included in policy and laws relevant to animal welfare.

This article considers the complexity and diversity of ethical concepts and beliefs held by Māori, the indigenous people of Aotearoa New Zealand (hereafter New Zealand), relating to animals. A combination of interviews and focus group discussions were conducted with individuals who identify as Māori and were working with wildlife, primarily in an eco-tourism and conservation context. Two main themes emerged from the data: ethical concepts relating to the environment, and concepts relating to the spiritual relationships between people, animals and the environment. These findings highlight that the connections between humans and animals through a Māori lens are nuanced in ways not typically accounted for in Western philosophy. This is of particular importance because of the extent to which standard Western thought is embodied in law and policy related to human treatment of animals and the environment. In New Zealand, relationships and partnerships are informed by Te Tiriti ō Waitangi, one of New Zealand’s founding documents. Where these partnerships include activities and environments involving human–animal interaction, policy and legislation should account for Māori knowledge, and diverse of thought among different hapū (tribal groups). We conclude by exploring ways of including Māori ethical concepts around animals in general, and wild animals in particular, in law and policy, providing a case study relevant to other bicultural or multicultural societies.

1. IntroductionAnimal welfare and human–animal relationships have evolved from concepts discussed by philosophers, ecologists and ethologists to topics of public debate [1]. Māori (the indigenous people of Aotearoa New Zealand) are a case in point of this, developing their own knowledge and values (mātauranga Māori) about animals and their relationships to people. Accompanying this, there is growing demand and support for giving greater respect to the knowledge and values of indigenous peoples across the world [2]. The relationship between mātauranga Māori and animal welfare may be one that is distinct from that found in typical Western thought. This article will cover a case study from Aotearoa New Zealand (hereafter New Zealand) and how mātauranga Māori might explain and progress animal welfare and human–animal relationships. The data contained within this article will be of specific use within a New Zealand context but may find parallels with other indigenous cultures around the world particularly those in the Pacific islands.Within New Zealand, animals play a considerable role in both society and the national economy. Dairy farming and other agricultural industries contribute significantly to the New Zealand economy, as does the tourism industry, much of which depends on the country’s native wildlife to attract overseas visitors [3,4]. Animals can be affected both positively and negatively by these activities, and they and other practices are increasingly driven by values-based decision-making. To date, frameworks and values of Western origin have dominated animal welfare discourse (e.g., models such as the Five Domains and three circles approach) and this is reflected in policy in New Zealand, as elsewhere in the Western world [5,6].New Zealand is a society that is committed to biculturalism as underpinned by Te Tiriti ō Waitangi, one of the founding documents of New Zealand signed in 1840 between the British Crown and more than 500 Māori chiefs [7]. Māori identify through whakapapa (genealogy, lineage or descent) from their whanau (family) to their hapū, and iwi affiliations. The term Māori is used to refer to the indigenous people of New Zealand collectively. Hapū refers to kinship group, clan, tribe, subtribe or groupings of extended families. Iwi refers to a collective of hapū forming an extended kinship group, or tribe. It often refers to a large group of people associated with a territory, descended from a common ancestor.Like the other aspects of mātauranga Māori, the Māori language, te reo Māori, is nuanced and complex. There are many concepts that te reo Māori refers to that are unique and have great importance to the way Māori perceive and understand the world. These concepts are such that it is beyond the scope of this article to provide complete English language definitions of all terms. The limited definitions used in this paper relate to the material discussed herein and are not intended to encompass all aspects of the concepts they refer to.This Treaty means that there is a need for policy and ethical frameworks that are informed by, or arise from, mātauranga Māori [8]. As part of the framework of New Zealand society it gives political, legal and ethical reasons to understand Māori culture and tradition particularly within specific tribal contexts. Māori settled in New Zealand centuries before Europeans and have a distinctive culture, language, knowledge system, and traditions. This includes a well-developed system of laws, values, beliefs and practices that have developed over time and are deeply embedded in the social context, this is known as tikanga Māori [9]. Tikanga Māori has implications for both what we may consider to be the right way to live and be, and with regard to animals and their state of being, for example what it means for an animal to live a good life [9,10,11].Given that both animal–human relationships and Māori culture are important to New Zealand, it is crucial for relationships with animals to be understood from a Māori perspective in addition to the currently dominant Western views; the latter have long been the default in animal management contexts particularly within agriculture and fisheries. Ethical consideration of animals can operate in many ways. One can consider animals from various perspectives, at one end of the spectrum animals can be viewed as a species, a population, or a herd, or conversely on the other end of the spectrum, they can be individuals. There is a wide array of literature and research regarding Māori understandings of, and relationships with, the natural world and associated resources, which includes animals. Much of the literature does not have a specific focus on animals as individuals, but instead focuses on them at the level of populations or species [12,13,14,15,16].A central element of a Māori understanding of the natural world is that humans are a part of it, not in control of it. The whakapapa relationship between Māori and the environment is one that ties them deeply to it, further it establishes that neither animals nor the rest of the natural world, a category that includes humans, exist for the purpose of being exploited and extracted for human use.Two concepts considered integral to Māori understandings of and relationships with the environment are kaitiakitanga and mana whenua. Kaitiakitanga may be understood as guardianship or stewardship but also encompasses concepts such as resource management stressing the balance of human, material, and non-material elements. Mana whenua is the ultimate and paramount power and authority regarding a territory of land (whenua), derived from the gods. Local whānau and hapū hold mana whenua responsibilities. This transcends legal ownership, and obliges them to manage and protect the whenua in their territories. Both concepts are generally not discussed within policy settings, nor academically, in relation to obligations to individual animals, but rather in terms of species, populations of animals, the environment or the ecosystem as a whole [10,12]. For example, when eel (tuna—Anguilla dieffenbachii and Anguilla australis, species of freshwater eel indigenous to New Zealand) are mentioned with reference to kaitiakitanga it is most commonly in discussions about the body of water they live in, and the population of tuna as a whole [17,18]. Such holistic views of the environment are reflective of a concern for overall environmental well-being as opposed to imbalances that may arise from a focus on singular species or entities.Likewise, there are a number of academic accounts of Māori health, an important component of animal welfare [19], but not specifically of the health of animals. Both Te Whare Tapa Whā, the four cornerstones model of Māori health and well-being, and Te Wheke, the octopus model of Māori health and well-being, have a human focus [20,21]. However, some of the elements in these accounts may suggest applications to animal welfare from a Māori perspective, insofar as, from that perspective, humans and animals may be similar for the purposes of health and welfare.Two valuable accounts of welfare that find common use within animal-focused industries are the Five Domains model and the three circles model [5,6,19]. These models serve as valuable tools for assessing the welfare of animals, especially in ways that can be measured or contrasted against one another. The Five Domains model consists of four physical domains relating to the animal’s physical state and interactions with its environment and other animals (nutrition, environment, physical health, and behavioral interaction) and one mental domain. Observable evidence of impacts collated in the four physical domains informs understanding of positive and negative affective states in the mental domain that are considered most relevant to the animal’s overall welfare state [22]. The Five Domains model itself does not provide criteria for what is a good or acceptable life but is predicated on the notion that individual animal’s affective experiences matter to them and thus are of key importance for understanding their welfare states [23].The three circles model consists of three overlapping circles akin to a Venn diagram [5]. The three circles are physical health and functioning, affective states and natural living, which represent fundamentally different value positions about what a good life for animals consists of. This model proposes that welfare science cannot resolve these differences, and any assessment of animal welfare must consider all three (like the three circles model, the Five Domains model implicitly considers the physical health and functioning, and natural living states of the animal but only in so far as they influence affective states). Whether these accounts would, and could, be compatible with a Māori understanding of animal welfare is an open question which this research aims to address.To elucidate Māori perspectives with regard to animal welfare and human–animal relationships, we conducted a series of interviews and focus groups. Direct conversations with Māori individuals and groups from both the tourism and conservation sectors provided insight into how animals are viewed. The context and detail garnered from these conversations may be used to develop an understanding of the relationships between Māori and animals, with implications for ethics and welfare.The aim of this research is to examine the views of Māori with regard to human–animal relationships and use this data to contribute to an articulation of Māori views about human–animal relationships and animal welfare and how these views may align with commonly accepted animal welfare thought of Western origin. We should note that any reference to a body of thought such as “Western” and “Māori” or “indigenous” risks mischaracterizing it as homogenous, when each reveal diverse theories, concepts, and values. In all cases where we use these terms, we are referring to thought arising from people who are of these origins, or which derives from established concepts with that origin, and do not mean to make or imply any broader generalizations about Western, Māori or indigenous thought.2. Materials and Methods2.1. Rationale for Research ApproachThis research is grounded in a constructivist epistemological position; it aims to gain knowledge about the world through others experiences and beliefs, in this case the relationship between Māori and animals [24]. Alongside the existing academic literature, this method is the most likely to develop a nuanced understanding of animal welfare; it is influenced by detailed Māori experiences expressed through participants’ narratives.We have sought to limit the way our own knowledge may influence these data through the use of informed grounded theory in the data analysis [25], but we acknowledge that this data cannot be truly objective. The narratives have been gathered and interpreted through an academic research process. Nonetheless, we note that all researchers reflected on this potential subjectivity throughout the data collection and analysis process. In order to do this, Informed Grounded Theory methodology was used and the research was conducted in a way that aimed to respect and nurture the unique cultural identity of Māori.2.2. Study DesignThis research project has been informed by both written and kanohi ki te kanohi (face to face) consultation with the Ngāi Tahu (the principal iwi (tribe) of the South Island of New Zealand, also known as Kāi Tahu) Research Consultation Committee, at the University of Otago. Ethical approval was gained through the University of Otago Human Ethics Committee, Application 20/012. The focus group and interviews were conducted between the dates of 19 June 2020 and 26 June 2020 with individuals from Māori backgrounds who were working, or had recently worked, in wild animal tourism and conservation.Members of the focus groups and interview participants were recruited via snowball and convenience sampling. Individuals known to the researchers as having extensive interactions with animals were approached, as they were deemed to be ideal participants in the study.Inclusion criteria were: (1) the participant must identify as having Māori ancestry, and (2) the participants must have significant interactions with animals or other ways of relating to animals that have given them reason to reflect on animal welfare. Exclusion criteria included non-Māori, those living outside New Zealand; those under 16 years of age and those who were unwilling to have their views recorded and potentially published.Six participants contributed to our research over two individual interviews and one focus group discussion undertaken in personal meetings that enabled engagement that was kanohi ki te kanohi. These participants whakapapa (trace their ancestry) to the Kāi Tahu, Kāti Māmoe, Wāitaha, Rāpuwai or Kāti Hāwea iwi and hapū.2.3. Interview StructureThe interviews and focus group lasted between 60 and 90 min and followed semi-structured questions to guide the narratives around participants’ views and beliefs regarding animal welfare and their relationship with animals. This method allowed for discussion to develop organically and explore detailed understandings. The focus of the researchers in the interviews and focus group was upon an individual’s personal interactions with animals in professional workspaces and their reflections on individual beliefs or philosophies. This enabled participants to express and reflect on their views and understandings of animal welfare and their relationships with animals with insights into whether, and in what form, their understandings were informed by te ao Māori (the Māori worldview), or mātauranga Māori. The interviews were digitally audio recorded and transcribed.2.4. Data AnalysisThematic analysis of the transcripts was conducted using NVivo qualitative analysis software. Informed Grounded Theory (IGT) was used to analyze the transcribed data. IGT is a data analysis methodology based on the principle of using Grounded Theory (GT) methods informed by the existing research literature and theoretical frameworks [25]. GT methods are designed for investigating areas where there has been previously little to no research performed (e.g., the nature of the relationship between te ao Māori and the welfare experiences of individual animals). IGT expands on these basics by incorporating the literature research through a neutral theoretical stance that allows for clear conclusions to be drawn from the obtained data without allowing previously known theories to taint or obscure these conclusions.2.5. Positioning the ResearcherAs a multicultural research team, many of whom do not have Māori ancestry, there are a bevy of ethical considerations that must be taken into account [26]. There have historically been a number of cases where indigenous cultures were exploited or misrepresented through research, and this has understandably fostered a sense of mistrust towards prospective researchers in some cases [27]. One of the research team members is of Māori descent and guided our research process with particular focus on issues of tikanga to ensure cultural respect. Every effort was made to ensure that the data obtained through this research were both accurate and appreciative of the complexities of the rich and detailed Māori worldview.Efforts were made to translate concepts between Māori and English as accurately as possible, however, there may still be nuanced elements of Māori concepts that may be lost in translation and cannot be fully explained in English—they can only be truly expressed in te reo Māori (Māori language). None of the research participants were native speakers of te reo Māori but all were at various stages of learning. The literature reviewed for the project was also written in English and thus may suffer from similar issues. Additionally, as an oral tradition some Māori knowledge was likely not available for us to access.3. ResultsAnalysis of the transcripts obtained over the course of the interviews and focus group discussions conducted revealed several key themes that were mentioned across multiple transcripts. Three themes identified in our analysis were kaitiakitanga, mauri (the life force or vital essence of a thing), and spiritual connections.3.1. Kaitiakitanga and the Natural State of ThingsParticipants expressed strong support for the protection and promotion of indigenous New Zealand species, as well as efforts that enable this through habitat preservation. Participants saw an integral relationship between animals and the environment. One participant who worked in penguin conservation said the following:“Wearing my kaitiaki [guardian] hat looking at the blue penguin I use them as an indicator species because they don’t travel too far. They indicate to me how healthy the marine environment is.”This participant viewed individual animals as elements relating to a greater environment and that this link was important when viewing things from a kaitiakitanga perspective. There was a common notion amongst the participants that improving the health of the environment would improve the welfare of animals within it.When discussing conservation, there was a significant focus on pest control and the removal of invasive species. Emphasis was placed on the importance of a natural environment, one that was ‘untainted’ by both invasive species and destructive human interaction. A trend among participants was to advocate for a strategy of limited intervention with indigenous species whenever possible, with the exception of ensuring that their environments remained free from invasive predators and other outside influences. As one participant said “Human intervention is a big mistake”, this highlighted the view that human intervention should be avoided unless there is no other acceptable alternative.Action was justified to encourage the survival of particularly threatened species, but any intervention would be limited as much as possible. Overall, a natural environment was seen as something desirable, at least with regard to those animals not equipped to deal with the foreign elements that may have been introduced.A participant who had ties to native bird conservation placed value on providing an environment where animals could flourish free of ‘unnatural’ predators (e.g., rats, stoats, possums and other creatures that had been introduced by humans). In describing some of the sanctuaries where she worked, she said: “We don’t have rabbits, goats, pigs, stoats, ferrets. We don’t have any of the mustelid family. They are killing machines.” She stressed that these introduced animals were eliminated due to the damage they do to an environment not evolved to account for them. A priority was placed on maintaining what could be called the ‘purity’ of the natural world, keeping it free of foreign elements that might disturb or damage it.This went further than merely keeping introduced or invasive animals out of the environment, however. It extended even into the interactions between humans and native animals. One of our conservation-focused participants, when asked about their policy with injured animals, said: “The natural life should not be interfered with too much; sometimes we cause more trouble than we mean to… What would happen if we weren’t there? If you remove us from the equation, they usually can look after themselves. It is whoever is the fittest. When there are three bright orange beaks coming out of the nest, the parents just aim for the brightest color that they can put the food into, and those birds will trample over each other to get to the top. It is the strongest one that will survive, and that is what makes the next generation strong because only the strong survive. It is a hard lesson for some people to learn.” Participants emphasized the importance of native animal species living in a natural environment and engaging in natural behaviors, including predation. It was said that:“If a bird falls out of the nest and a weka [Weka—Gallirallus australis, a flightless bird endemic to New Zealand] comes along and takes off with it, it’s part of the natural world; it’s our Serengeti”.This is seen as beneficial for animals, allowing for the strongest to survive and contribute to the next generation, thus allowing them to be strong as well. Human interference with this process was seen by one participant as detrimental and should be limited when possible. This relates to the value placed on preserving species for the future; what is beneficial for animals is not directly beneficial for the individual animal but rather for the survival and flourishing of the species as a whole.The interviews also explored the notion of the value of species. The idea that different species of animals are valued differently and thus the welfare of the less valuable could be sacrificed to further that of the other. This was most apparent between the treatment of invasive species such as rats or stoats and the treatment of indigenous wildlife. Invasive pest and predator species were universally valued less than the indigenous wildlife, with campaigns to remove them from, or curtail their actions within, protected areas being encouraged and supported. “We are doing all this work to protect our native species. Long term, we will see cats being an inside pet. You will have to be a registered breeder to be able to move them and that process will be so arduous that there aren’t many breeders, and we would see the population drop to about 5% over about ten years.” This is in direct contrast to indigenous animals, where engaging in their natural behaviors was encouraged and considered important to their welfare.There is a greater element to this, however, than merely the inherent value a species has through its status as native to New Zealand. There is also the value that is given to species based on what they are able to provide or do for us. A participant, when questioned about why they felt the conservation of muttonbird (titi—Puffinus griseus, also known as the sooty shearwater or muttonbird) was important, said: “I think it is as simple as I like eating them, and it is no different than a European farmer for thousands of years ensuring that sheep are around”. So, it is not only the inherent native status of a species that may cause it to be valued but the use that can be obtained from that species. Animals that can be harvested or can provide some kind of service have a value of their own. As one participant said. “Everybody wants to see the wildlife in this pristine environment. It is unique and people just love to see the freedom. We are the visitors there. The wildlife and the forest are paramount, and we are just privileged to live in that environment.” The value of species and individual animals can stem from the relationship individual members of the species have with humans. This value may be derived from direct interaction with animals or from less tangible benefits gained from their presence.The considerations intertwined in kaitiakitanga also play a role in how the welfare of animals might be thought about or prioritized. Many participants reported being directly involved in conservation efforts in order to encourage the survival of indigenous species. They played a direct role in the preservation of native species within New Zealand. One participant said that the conservation work they were doing today was especially important for future generations and the relationship they would have with the environment.“[I want] for them to tell the story of success, hand those stories down. That is how we work, that is how I work. Hand those stories down. They are beautiful stories for our mokopuna’s [grandchild or grandchildren]. I grew up with my grandparents. […] My mum would say to me today it is not about you; it is about the mokopuna. It is about us doing our best for our mokopuna that we can.”The above comment highlights that for this participant, the impetus behind conservation efforts and the values of guardianship that kaitiakitanga encourages is to allow mokopuna and future generations to experience the environment that is available now, ideally in a better condition than it is currently. This participant held views that there is value to be found in interacting with the environment and the indigenous wildlife that live within it. Further, it suggests that this value may be thought of as valuable beyond their personal experiences. It can be seen as something that should be valued by others in the future. The value of preserving the environment lies not only in one’s personal interactions with that environment, but the interactions one’s descendants will have down the line.Another participant considered that the land itself has intrinsic value, regardless of whether humans are present or not.“I think the humans will die off before the animals do. We have to ensure that our ethos that comes from ourselves as Kai Tāhu is to leave the whenua [the land] in a better condition than when the responsibility was passed to us. That includes everything that is living upon it.”This goes beyond the perspective that wildlife should be preserved for future generations, it suggests that value may not only be found in the environment through experiencing it, but that there is some inherent value in leaving it in a better condition than it was before. This leads into one of the other major themes we identified, the mauri of the environment.3.2. The Mauri of ThingsMauri was another concept that underpinned many of the things we discussed. One participant described mauri as the following: “It is a lifeforce, an essence of life. Another way of thinking is the spirit in te ao Māori is the wairua, and that is something beyond death and can interact with the afterlife, but the mauri is something that is “life”, and if it dies, it dies.” Mauri was seen by participants as an essence that was present in all living things and in the world around them. There is inherent value in mauri, and enhancing the mauri of a place is something to be encouraged and desired. To some participants mauri was not seen as something that dissipated when a living creature died, but rather something that changed forms and remained within the environment. Although the mauri of the creature is gone it is also in some ways passed on. One participant described this as a cycle: “When I think of Ki uta ki tai which is mountains to the sea, that really is about mauri because you have that rain that comes onto the mountains, eventually coming down the rivers, going out into the ocean and then going back up onto the mountains. It is a closed circle, and you are impacting on that mauri by discharging things into that environment or, when you are harvesting things, killing more and leaving that lying in the ground and contaminating the ground or contaminating the waters.” This relates to the value that is placed on animals living in a natural environment; by allowing them to live in their natural state, their mauri with is not impacted upon as it might be if there was regular intervention. The elimination of pest and predator species also works towards this goal, limiting their impact upon the mauri of the environment and allowing the mauri to flourish as it would without their presence.Due to the interconnected relationship between wildlife and the environment, the acknowledgment of mauri within the environment can be seen as directly beneficial to the welfare of animals within that environment. When discussing involving themselves in an advocacy space for wildlife one participant expressed the following: “If we have enough mauri within ourselves to come to those tables, then that will then come back to protecting our wildlife.” This highlights a belief that if the life force within them is strong enough to drive them to advocate for the land and the wildlife, then that mauri works to restore and improve the mauri of the land.Furthermore, the mauri of the environment can allow it to be seen as a living entity in its own right. In our discussions, we heard many references to the idea that the land itself was alive and needed to have its own voice. One participant spoke of their personal experiences of advocating on behalf of the environment in the Environment Court, a New Zealand court that hears issues arising under the Resource Management Act 1991 [28]: “I have stood up in Environment Court and talked on behalf of the trees, because the trees aren’t considered an entity…” This ties into the idea that Māori may act as representatives for the lands for which they hold mana whenua; they act not as owners, but as advocates for entities that play an important role in their lives and beliefs. “This has happened only in the last two decades through people doing it, standing up and saying ‘I put this submission on behalf of the trees that are going to be affected by the activity involved’…” Thus, the mauri of things is thought not to be separable; it is an intrinsic quality that must be respected, acknowledged, and advocated for. This connection to mauri is one that exists on a spiritual level for Māori, informing the final major theme about the spiritual connections between things.3.3. Spiritual Connections and WhakapapaThe spiritual and familial connections between participants and animals was another theme evident in the research. Two major categories were present within this theme: the spiritual and familial connections between participants and the land, and the spiritual and familial connections between participants and wildlife.3.3.1. The LandParticipants said that the land has tremendous spiritual significance. The health and management of the land were considered to be of great importance. This, in turn, was seen to be directly related to being spiritually connected to the wildlife that lived on that land. Caring for the wildlife that lived on the land was seen as the same as caring for the land itself. When asked why it was important to continue improving the welfare of one of their taonga species, one participant said: “Because it is a part of who we are, and it is really important for us as Māori to keep connecting to the land.” This participant evidently saw no distinction between looking after the land and looking after the species that live on it. The relationship with nature was holistic—by being spiritually connected to the land, they are spiritually connected to the wildlife that lives there. The land they consider to be their ancestral home is a part of them, with deep connections that go back many generations.“She was brought up in that lighthouse, and her whenua [placenta—also used to refer to the land or domain] is buried underground. My grandmother’s whenua was buried underground.”The generations that have lived on traditional lands have left physical and spiritual impressions that are significant to their descendants. This is reflected in pepeha, a way of introducing oneself in Māori in a process that shares connections to people and places that are important to you and where you come from, and other aspects of Māori practices where they express the connection to the land, particularly landscape features that are viewed as ancestors through whakapapa. “We understand in that whakapapa […] the land and the animals are our ancestors, and, in that relationship, we are subservient to the land and the animals.” The familial relationship between participants and the land is one where they consider the land to be part of their ancestry and feel the same obligations to it that they feel to their ancestors. Participants said that those who have been denied access to this land feel they can replenish well-being and heal by rebuilding their connections to it.3.3.2. The AnimalsA connection to animals was also reported through whakapapa, where some animals connected through common ancestors of Māori. In a discussion about management rights being restored, one participant said:“We have been cut off from our land and our taonga [treasured] species for a long time.”The context around this is again holistic—the same familial obligation that is felt towards the land extends to the animals as well. Being separated from those elements may be just as harmful as being separated from one’s human family and ancestors according to several Māori participants’ understandings.As mentioned before, the spiritual connection to wildlife is deeply intertwined with the spiritual connection to the land. Often, connecting with one is also connecting with the other. “I believe that the albatross and the titi and the penguins, so ko tangata toroa, ko tangata titi, ko tangata kororā are waiting for us to come back home, come back to here [to the land] and look after them properly.” This view is indicative of the value of kaitiakitanga where those who have spiritual and familial ties to the land can truly be the kaitiaki who look after the natural inhabitants. That without these ties, there is something that may be missing that can contribute to the welfare of the wildlife. When the healing that can be gained from the land and wildlife was discussed, it was asked if the wildlife can benefit from this healing as well. “Yes, I do think so. One of the things I have been noticing is that they need more protection and advocacy space. The protection of their fishing grounds, the protection of their environment in things like the Port Otago Mana Whenua consultation group and things like that. If we have enough mauri within ourselves to come to those tables, then that will then come back to protecting our wildlife.” An almost symbiotic connection is suggested, where the participants benefit from the presence of the land and the wildlife and the wildlife benefit from the kaitiaki who advocate and speak on the behalf of the natural environment and animals within.4. DiscussionThe views of participants covered a range of different concepts when discussing the ethical relationship between Māori and animals. In this paper those views will now be discussed according to two themes—the natural world or the spiritual world—noting that both worlds are seen as interconnected according to Māori tikanga and practices such as wāhi tapu (a place sacred to Māori in the traditional, spiritual, religious, ritual or mythological sense). Each of these themes has its own sub-themes. The natural world refers to elements of kaitiakitanga and the natural environment, while the spiritual world refers to the relationship between animals and Māori, the spiritual health of animals, and the concept of mauri.4.1. The Natural WorldThe importance of the natural world was a common topic within the discussions. The natural world held great significance to participants as their ancestral lands containing various natural taonga, to whom they have kaitiaki and genealogical relationships, associated with cultural values and tikanga.4.1.1. KaitiakitangaAs previously mentioned, one of the most prevalent themes distinguishing the relationship between Māori and animals was the concept of kaitiakitanga. Kaitiakitanga is not simply a set of behaviors or actions one must perform, rather it extends to a philosophy or way of thinking about the natural world (human and non-human, living and non-living) that is greater than this [12,13]. This obligation of guardianship and caring for the environment was a core concept that participants expressed when discussing their views and goals for the environment, particularly those participants with connections to conservation efforts. They stressed the importance of caring for the environment and working to keep it healthy and flourishing.Given the environmental focus of kaitiakitanga, which tends to emphasize groups, populations, species, ecosystems and includes the non-living elements of the environment as well as the living, it can seem that kaitiakitanga does not focus on the welfare of individual animals as such. However, this is arguably a mischaracterization that overlooks the holism of te ao Māori; from a Māori perspective, individual welfare may not be achievable in isolation from others. Patterson stated that “the welfare of the whole depends upon that of each individual; the welfare of each individual depends on that of the whole”, clearly establishing that welfare is an interconnected principle [15]. It is dependent on other related parties also having a high degree of welfare and contributing to the welfare of others in turn.This seems to align with the views of participants; those with commitments to conservation emphasized the importance of the population as a whole over individual animals, at points advocating against intervening to improve the situation of individuals as this was perceived ultimately to be harmful. Kaitiakitanga has a holistic focus rather than an individualistic one, and if welfare is also conceived of holistically, it may also be provided for.Kaitiakitanga includes recognition that many individual animals have their own mana (power or status; mana is a supernatural force accompanied by status and power) and mauri—there are numerous accounts of Māori traditions where animals are taonga species and may take on the role of kaitiaki or guardians for humans or natural areas. For example, Schwimmer (1963) discussed the role of individual animals inhabited by atua (gods) that were guardians of Ngāti Wai iwi in the Northland region [29]. As atua, these animals have mana (and in Whangaruru are referred to using the term mana) which Schwimmer explains expresses the belief within Ngāti Wai that these animals both have mana, and are also the source of the mana that people have.Kaitiakitanga exists at a nexus of the spirit, the environment and the human [12]. A tendency we observed within our discussions was that, from a Māori perspective, the health of the wildlife in an area would often be treated synonymously with the health of the land. Some participants regarded improving the health of the land and improving the health of the wildlife upon that land as essentially synonymous. This has specific implications for the way Māori may view animal welfare as it suggests that from their perspective, work that improves the health of the land feeds back into improving the health of the animals, and consequently animal welfare.The Māori worldview is primarily holistic [30]. This, however, highlights one of the intricacies of kaitiakitanga as a concept when applied to something such as animal welfare. Kaitiakitanga is a traditional and sustainable approach to environmental and resource management. As exemplified in our findings, acts of kaitiakitanga are holistic in character. It can justify care for populations of animals and species, while at the same time advocate for the care of individual animals within them. Because this care is holistic, focusing attention at the group level will benefit both the group and the individuals within it; in turn, focusing on any individual will also benefit the group, and ecosystem.From a contemporary Western perspective, welfare is not often conceived of holistically. Although animal welfare may be assessed on a population-wide level, this is not generally considered the primary focus of welfare. Welfare, as defined by Fraser et al. (1997), is something individuals have, and this may be aggregated to determine the population welfare [19]. While this links individual and population-level welfare, it is possible, from this view, for a population to have a good total or average welfare, while some individuals within the population have poor welfare. In fact, the welfare of the population may, over time, be improved by some individuals faring poorly, since this means there will be selection pressure against them and any disadvantageous traits they may possess, in favor of fitter individuals. This is different from a kaitiakitanga approach, which appears to emphasize a unity of individual and population levels of welfare, rather than them being two separate and different things that can be in conflict with each other.4.1.2. The Natural EnvironmentThe natural environment emerged as an important element of the relationship between Māori and animals. This is in line with the emphasis that Māori ethical concepts put on respecting the natural world. As part of that, several participants advocated limiting human interference with the natural way of the world. Some participants suggested that non-intervention is important for species to allow them to develop naturally and that by interfering in their development through actions such as medical intervention, the ability of future generations to survive could be negatively impacted. It would appear that the ability to sustain and provide for oneself is considered important to the welfare of an animal from a Māori perspective. This may be similar to how adaptations being used and natural behaviors being expressed is important within a natural living orientation to animal welfare [19,31].Interestingly, this non-intervention was disregarded in cases where the survival of an indigenous species was sufficiently threatened, suggesting that while it may be important to welfare, it is of lesser importance than the survival of the valuable population or species. Some participants stated that interactions that were harmonious with and not disruptive to those animals’ own behaviors and environment were permissible, therefore non-intervention was perhaps advocated primarily in preference to disharmonious intervention that could otherwise occur. This is, in some ways, a mirror of Māori traditions that focus on achieving this objective in other contexts, such as the traditional Māori method of selecting trees to construct a waka (traditional canoe) through careful and respectful ritual; this is demonstrated in the tale of Rata and his canoe [30].It should be noted however, that the term ‘natural’ can be interpreted in many ways. There is the commonly accepted meaning that refers to the environment absent of human constructs, the portion of the world that has not been created by humans or significantly redeveloped by them. This is the view expressed in nature-culture dualism, where ‘culture’ refers to all human artifacts (e.g., cars, buildings, tracks through the forests made by people) and ‘nature’ to everything else not produced through human agency [32]. However, among participants we noticed a further, or alternative, division of what was ‘natural’ and what was ‘unnatural’. There was a perception that the category of ‘unnatural’ extended beyond things merely created by humans, to things such as species that humans had played a role in introducing. With regard to the New Zealand environment the ‘natural’ environment was thought to consist of those plants and animals native to New Zealand prior to colonization by Pākehā (Non-Māori, foreigner, New Zealand European).In particular, a distinction was perceived to exist between native and non-native species, with the former natural, while others were not. Those species such as rats, stoats and possums were thought to be ‘unnatural’ in that the native environment of New Zealand was not adapted to them. Removing them from areas within New Zealand and preventing them from preying on native wildlife was seen as protecting or restoring the ‘natural’ environment.4.2. The Spiritual WorldThe spiritual world is ingrained in Māori thought and tradition [33,34,35]. Humans, animals, plants and all living entities have a wairua, a spirit or soul that persists beyond death [35]. Interviews with participants did not involve discussion about whether there was a spiritual world or how it interacted with the material world; for some it was expressed as integral to their understanding of the world.4.2.1. Connections between Māori and AnimalsAt the forefront of Māori relationships to animals is the wider setting of the surrounding landscape, as one participant stated: “…it is a part of who we are, and it is really important for us as Māori to keep connecting to the land.” This is recurrent in Māori creation tradition, te orokohanga o te ao (the beginning of the world), that tells the origins of the land. The land itself is an ancestor, known as Papatūānuku (the mother earth in the Māori account of creation, the first female element of the universe and the giver and sustainer of life. Mother of the first atua—the first gods) in Māori cosmology, one that they are connected to and must honor as they do the human ancestors who have come before them [13].The recitation of whakapapa, one’s genealogy back across the generations to where one is related to the gods, is a concrete example of how Māori identify as being inseparable from nature. Whakapapa extends beyond genealogy to provide an understanding of how there are many interconnections with the natural world—calling back to the land, traditional gods, ancestors and distant relations. Animals can be thought of as kin, both in the sense that there can be an emotional bond between animals and Māori but also in the sense that some Māori believe that animals are part of their ancestry [10,13,36]. One participant told a story of their mother and grandmother visiting them in the form of an albatross on their mother’s birthday. The emotion the participant shared when recounting this story did much to convey the significance the experience had to them and the importance they gave the spiritual relationship they had with both their ancestors and the albatross.The bonds between Māori, animals and the land are thought to contribute to the welfare of animals, where interconnections mean the health of one influences the health of the others. On one level, this seems common sense, as animals living in an environment that is not healthy seem likely to fare worse than those in a healthy environment. This seemed to extend beyond the physical into the spiritual nature of animals and was deeply intertwined with the land and consequently with Māori who have ancestral ties to the land, as tangata whenua (people born of the land, and who therefore whole hold mana whenua). This is demonstrated in the reference to land in whakapapa where Māori often refer to their hapū or iwi’s mountain and waterway. It was said by a participant that “when you are connected with the land, I believe they [animals] connect with us”, this was in the context of spiritually important events taking place between Māori people and animals. This is particularly relevant to taonga species, those species attributed with particular cultural or sacred characteristics or values, which have special importance for Māori.4.2.2. The Spiritual Health of AnimalsAnother major theme evident in our research is the emphasis on spiritual health. Spiritual values have incredible potency within Māori culture and traditions and were of visible importance to participants. In his definition of wairua, Barlow (1994) says that Māori understand that all things have a spirit, not merely humans but “birds, animals, fish and even the earth itself” [35].Finally, Durie (1998) and Pere (1991) conceptualized models of health informed by Māori values and knowledge—Te Whare Tapa Whā and Te Wheke—that strongly emphasize the importance of spirituality to the health and welfare of individuals [20,21,36,37]. Although these are accounts of human health, they may be helpful in understanding animal welfare. Many Western philosophical conceptions of animal welfare can be found to draw from accounts of human welfare, so it is a plausible option for a Māori understanding of animal welfare to do the same. It is already known that the environment is thought to have a form of spiritual health so it is possible that animals on both individual and population levels are also influenced by spiritual health.4.2.3. MauriThe concept of mauri, of vital energy, arose frequently in our research. Mauri is a vital part of the very existence of things within te ao Māori [38]. Barlow (1994) defines mauri as what merges the physical and spiritual parts of an entity together [35]. The emphasis on the value of enhancing the mauri of a place was a common sentiment expressed by participants. One participant defined mauri as “a lifeforce, an essence of life” and it was a common theme arising within discussions for the mauri of a place or area to be considered important. However, it goes beyond merely being an animating force; mauri makes it possible for a thing to exist within the bounds of its own creation [35]. It is not merely about life but about the essence of a thing, and the enhancement and support of that essence is thought to be a thing to strive for.Enhancing mauri is most often achieved when restoring the natural state of an environment. Damage to, or contamination of, the environment is damage to or loss of the environment’s mauri [16,39]. When participants discussed the concept, it was with regard to how they might improve the mauri of an area, such as a stretch of coastline or a waterway. Although animals were referenced in these discussions, the focus was on the environment as a whole (including animals) rather than on the animals as individuals. However, as living creatures, the wildlife undisputedly has mauri of its own and is also considered part of the environment in which it lives [13,15]. As noticed by a participant in reference to “ki uta ki tai”, there is a cyclical relationship within the environment where mauri interacts with animals, plants and other components. Enhancing the mauri of the environment in such a way that it also enhances the mauri of animals within that environment is valuable and beneficial for them.Possession of mauri is commonly used as a reason why entities should be respected, but how does this relate to the welfare of animals? One of the interpretations of mauri that Patterson (1998) highlights is that it can be thought of as character or essence in addition to lifeforce [15]. Patterson explains that respecting mauri involves understanding distinctive qualities of creatures (and other things in nature) and respecting them for what they are [15]. Fraser (1997) describes one account of welfare (natural living) that values an animal’s ability to engage in natural behavior and use their natural capabilities [19]. The alignment of these two views is striking.Both views suggest similarities to the Aristotelian concept of telos. Telos is the ultimate aim or purpose of a thing. In relation to animal welfare this has been interpreted as the behavioral needs of an animal as a characteristic member of its species (for review of accounts of telos, see [40,41]). On this view, one must understand the distinctive behavioral qualities of animals, usually characteristic of members of their species in general, in order to know what a good life for them is, and determine whether they are leading a good life. The opportunity to use and develop characteristic behaviors is constitutive of a good life and any instance of such behaviors contributes intrinsically to the welfare of the animal. For example, a good life for birds is one in which they can fly, unless they are a flightless species. This potential similarity with Aristotelian thought will be revisited in our discussion of ethical implications of this research.Overall, it is apparent that there are many aspects of Māori thought that provide ways to consider the lives of animals that are distinct from typical Western understandings. The spiritual links between Māori and animals and between animals and the environment could have significant implications for animal welfare practices in New Zealand.5. Conclusions5.1. Implications5.1.1. Implications for Law and PolicyMāori values play an important role in policy development within New Zealand stemming back to the establishment of the Te Tiriti ō Waitangi [7]. This is reflected to varying degrees in legislation, for example the New Zealand Resource Management Act 1991 explicitly incorporates some Māori concepts, as does the New Zealand Conservation Act 1987 [28,42]. However, the primary piece of animal-centric legislation within New Zealand, the Animal Welfare Act 1999 (AWA) [43] does not have any reference to Māori concepts. This is also the case for other related legislation such as the Wildlife Act 1953 and the National Parks Act 1980 [44,45]. The Marine Mammals Protection Act 1978 has some small mention of Māori consultation but no mention of Māori concepts outside of this [46].Among participants there was call for the continuation and implementation of certain animal-related policies, such as pest-control policies, from which the natural environments they worked in can benefit. Importantly, through Te Tiriti ō Waitangi settlements the customary uses of animal species are often recognized within resulting Acts of legislation, e.g., Ngāi Tāhu Claims Settlement Act 1998 [47]. Participants were individuals from eco-tourism and conservation backgrounds. They all held similar views regarding government policies that affected their relationship to wildlife, such as those around customary use, protecting native species or controlling the spread of invasive species.Given the bicultural political foundation of New Zealand, laws relating to the treatment and welfare of animals, such as the Animal Welfare Act 1999, the Wildlife Act 1953, the National Parks Act 1980, the Veterinarians Act 2005, the Biosecurity Act 1993, and the Conservation Act 1987, ought to be reviewed for their inclusion of relevant Māori ethical concepts and values, and the degree to which they accommodate or facilitate Māori approaches to customary use, and animal care, use and management [42,43,44,45,48,49]. Our research has highlighted that there are factors that Māori consider integral to wild animal care and management that are different from the standard Western views. The inclusion of these values within relevant legislation would further the goal of protecting animal welfare, and also support integrating Māori concepts and values into policy and practice. There are also instances where customary use of certain wildlife species occurs, for example kiwi feathers used in raranga (weaving), and tītī (muttonbird) used for kai (food). Legislative reviews and resulting management would ideally not interfere with such customary use without full engagement with those hapū and iwi affected.5.1.2. Implications for Models of Animal WelfareThe Five Domains model and the three circles model represent a standard Western understanding of welfare and are broadly accepted and used to perform animal welfare assessments in a wide range of areas. The need to account for Māori concepts and allow for practices of tikanga Māori, and indigenous thinking more generally, within such assessments raises the question of how these models might align with Māori or indigenous understandings or be revised to do so.The Five Domains model does not explicitly include any Māori concepts. Key concepts that may be relevant for inclusion are the wairua and mauri of animals which may correlate with domains within the model. For example, if an animal with weakened mauri or wairua means that it will be unable to express natural behaviors, or be in good health, and/or will experience negative affective states, then this may be accounted for by the model, since these are present in it.However, the Five Domains model is dependent on the sentience of an animal in order for it to be applied. Its emphasis on affective states as integral to welfare presupposes that animals are capable of experiencing these states. Aspects of mauri or mana however, do not depend on sentience; when an animal dies, parts of its body may be seen to have mana or mauri of their own. Or it may be that non-sentient life has its own mauri and thus welfare. The Five Domains model could not account for these circumstances. Certainly, this does not diminish its usefulness, the majority of cases that require animal welfare considerations likely deal with sentient animals and for this purpose the model is still useful. However, outside of this it may struggle to adapt to a Māori conception of welfare.The three circles model appears to have similar difficulties as the Five Domains model with regard to spiritual elements of welfare. In order to integrate a Māori view of welfare into the three circles approach, it may be necessary to incorporate elements of spiritual welfare in one or more of the circles. Alternatively, there may be some possible commonalities; the spiritual emphasis on connections to the environment appears to mesh well with the natural living circle. This circle covers most of the functional components of living in one’s natural environment and may also be thought to include the spiritual connections and dependencies an individual might have to the land they live in. Fraser’s (1997) position that welfare has an element of natural behavior to it supports that mauri may be encompassed by this circle [19]. The greatest difficulty lies in the notion of a purely spiritual element of health that is unrelated to the environment, however, the holistic view te ao Māori takes of the world means that this may be avoided through the connections such a state may have to other aspects of health.There is, therefore, potential for these models of animal welfare to be revised to incorporate Māori ethical concepts. This may smooth the process of introducing such concepts into welfare practice and assessment.5.1.3. Ethical ImplicationsThere is significant debate about the ethics of the various uses of animals that are the subject of this paper, such as animal tourism [50,51] and farming [52,53,54] including whether and how they should be reformed, or whether they are ethically permissible in any form. All are based on valuing animals instrumentally, while not in all cases precluding the fact that they can also be valued intrinsically (any acceptance that animal welfare matters for its own sake values animals with the capacity for welfare intrinsically to that extent).We do not have enough data on these applied normative views to engage in any in-depth, direct, engagement with this debate in this paper. The participants in our research are engaged in animal tourism, and their views imply acceptance or endorsement of at least some of the fundamentals of some of these activities, most notably for present purposes animal tourism and associated conservation. However, this should not be taken as implying that the Māori ethical concepts and relationships we discuss do not suggest a normative approach to animal use, indeed, our discussion has covered many normative implications of Māori ethical concepts.Most notably, Māori ethical concepts as they relate to animals found ethical regard for animals in a kinship relation—that of whakapapa. This relationship between humans and animals of mutual descent from the atua Papatūānuku and Ranginui means not just ethical duties to animals, but also a holistic identification, with all being part of the natural environment. This suggests a relational animal ethic.Clare Palmer (2010) has advanced a relational ethic toward wild animals that is apposite to mention here [55]. We can only note some initial similarities and differences between the views of participants in this paper. Notably, Palmer’s relational ethic justifies an intuition that wild animals ought in many cases not to be assisted by people (i.e., where the requisite moral relations between people and animals are absent). We describe apparently similar endorsement of non-intervention to assist wild animals in some cases. However, we see evidence that this is based in a view that it is ultimately better for animals—the individual animal and the population as a whole, due to the holistic account of welfare we have described—if they are not assisted in those cases. Palmer rejects this view after finding counterexamples that show that human assistance would benefit the animal. It is notable for our purposes that Palmer considers only individualist accounts of welfare in her argument, which are the norm in Western philosophy of welfare. In the present paper the purported inconsistency that concerns Palmer is reconciled by the holistic linking of individual and group welfare that we describe, a view she does not consider. This reveals an alternative justification for a relational ethic that similarly supports the intuition that we often lack obligations to assist individual wild animals.Tikanga Māori has also been characterized as a form of Aristotelian virtue theory [56]. The similarity that we describe of some aspects of mauri to the concept of telos found in virtue theory further supports this view. On this Aristotelian account, being moral is part of the telos of human life. When we are virtuous, we flourish—thus intimately linking human welfare with ethics. A virtue theoretic approach to animal use has potentially radical implications for current practice, particularly in farming, requiring farming practices and conditions be at least altered to allow animals to express their natures [57]. Although there is a virtue theory analysis of tourism, focusing on developing the moral character of tourists [58] its application to animals in tourism is so far absent [59]. We lack data here to do more than suggest that application and invite further research building on this.5.2. Final ThoughtsThis research is a preliminary study. There are limitations with the methodology as the field work was very much concentrated on local participants with iwi and hapū affiliations in the Otago/Southland region of Aotearoa New Zealand. It is not possible to generalize the findings, nevertheless the research provides a starting point for comprehending how Māori ethical concepts and values may inform and influence relationships with animals. This research represents the views of individuals from conservation and ecotourism backgrounds, further research is needed to represent the views of other stakeholders.Informed by the interviews with Māori participants from particular iwi detailing how they value animals we have been able to identify important themes that align with views described in the literature [10,12,13,29]. Significant value was placed on native life that exceeded that of introduced species. Further, the well-being of these animals was thought to be linked to Māori concepts that are not present in the influential Western accounts of animal welfare we consider, although there may be some areas of compatibility nonetheless, as discussed. The majority of these concepts focused on the spiritual health and mauri of animals (and people as kaitiaki) in addition to their physical health. Finally, a holistic relationship between Māori and natural environments, including animals, was apparent. A lot of the care that goes into improving the welfare of animals is focused on the collective, landscape well-being rather than on distinct individuals. These views have both interesting similarities and differences to relational and virtue ethics as they apply to animals.In New Zealand, the strengthening of Te Tiriti ō Waitangi relationships in conservation spaces has seen a valuing of mātauranga Māori and te ao Māori alongside Western ways of knowing, although much more progress is possible. Such ways of knowing contribute to our understanding of the connections between humans and animals, including, from an animal ethics context, what it means for an animal to live a good life and how animals ought to be treated.This is, we hope, the beginning of a kōrero (conversation, discussion) about the welfare of animals, and our obligations to them, in this academic context. It is not, and not intended to be, a definitive account of these. This kōrero engages with one which is long and ongoing within te ao Māori, and we are very grateful to those within it who shared their time and knowledge with us.

animals : an open access journal from mdpi

10.3390/ani13050789

PMC10000203

Skeletal injuries are common in athletic horses. This literature review covers over three decades of research focused on preventing bone-related injuries and demonstrates how research develops over time. In an initial study evaluating the role dietary silicon can play in racehorse injuries, an observation of mineral loss from the cannon bone was observed after the commencement of training. Subsequent work revealed the loss was associated with horses being removed from pasture and placed into stalls, resulting in decreased mechanical loading on the skeleton. As bone responds to the load placed upon it, continued research focused on housing and exercise requirements to prevent such bone loss. Only short sprints are needed to maintain or increase bone strength. Conversely, endurance exercise, without high-speed exercise, fails to cause bone to become stronger. Exercise can be either forced or voluntary but having free access to exercise does not guarantee that animals will perform it. Thus, horse behavior needs to be taken into consideration. While proper nutrition is critical for bone health, it does not guarantee it without appropriate exercise. Pharmaceuticals impact various factors associated with bone health. Many items influencing equine bone health can also be applied to humans.

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.

1. IntroductionFor many decades, the high incidence rate of dorsal metacarpal disease (commonly known as bucked shins [1]) 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 [2]. This ailment is similar to shin splints which has commonly plagued human athletes, and, in particular, runners [3]. 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 [4].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 [5]. 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 SiliconThe initial project began with the examination as to whether supplementing a bioavailable source of silicon (Si) could decrease injury rates in equine athletes [6]. The work was inspired by earlier reports on the essentiality of dietary Si and the implications for new bone development [7,8,9,10]. 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 [6]. 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 [6] 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 [11]. Likewise, with broodmares, trends (p < 0.10) for altered bone resorption were observed in postpartum supplemented mares compared to controls [12]. 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 [13]. 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 [14].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) [15]. 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 [16].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 [17], skepticism accompanied these claims. To investigate this, 44 two-year-old Standardbreds were radiographed to identify osteochondrotic defects in the fetlock and hock joints [18]. 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 [19]. 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 [20]. 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 [19], 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-HousingIn 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) [21]. 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) [22]. 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 [23]. While not as precise as techniques such as computed tomography [24], which has been shown to be strongly correlated to bone ash weight [25], 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 [26] and, when used with digital radiographs, the need to use unprocessed images has been recognized [27]. 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 (Figure 1) [21]. 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 [28]. 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 [29] would be sufficient once new bone began to be deposited. Given that shin soreness in humans had been linked with low Ca intake [30], 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 [31]. They were fed diets balanced to meet NRC requirements [29]. 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 [29] and another group that received them at higher concentrations [32]. 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 [33].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 [32] as they did in the study with the longer adaptation period [31].Reflecting on the initial study in which bone loss had been observed in the 53 horses in race training [21], 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 [31,32] 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 [34]. 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 [35]. 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 [29], 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 [36]. 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 [37]. 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 [38].4. Role of Exercise in Bone DevelopmentWhile it was assumed that the differences in bone associated with pasture- versus 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 [39]. 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 [40]. Using the RBAE technique [22,23], 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 [41]. 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 [39,40,41], 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 [42]. 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 [43].Further, bone responds more to mechanical strain (the amount of bending) than it does the number of times it is bent [44]. 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 [45]. In an avian model, it was determined that only four consecutive cycles prevented bone loss, and 36 cycles increased new bone formation [46]. 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 [47]. 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 [48], 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 [49]. 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 [39,40], 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 [49], 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 [50]. 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 [51]. 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 [52]. 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 FactorsBesides 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 [53]. 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 [54].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 [55]. 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. ConclusionsMany 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 [56]. 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 [57]. 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 [58]. 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 [59]. Granted, this is based upon the assumption of adequate Ca intake and other nutrients, and this is especially true after menopause [60]. 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 [53]. Additionally, potential analgesic effects should be of great concern whether with pharmaceuticals such as bisphosphonates or corticosteroids [61]. 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 [62]. 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.

animals : an open access journal from mdpi

10.3390/ani11041158

PMC8073562

Air-breathing fish constitute a broad evolutionary group of fish, which are generally characterized by distinctive phenotypical plasticity. These fishes usually inhabit waters where oxygen deficiency occurs periodically, which is why they have developed a variety of accessory respiratory organs (AROs) that may be used in an obligatory or a facultative manner. Knowledge of the structure of these organs is important for both the breeding and the conservation of these fish species. The aim of this study was to conduct a comparative histological analysis of two types of AROs found in the Anabantoidei suborder and the Clariidae family, both of which are freshwater fish taxa of high ecological and commercial importance.

Accessory respiratory organs (AROs) are a group of anatomical structures found in fish, which support the gills and skin in the process of oxygen uptake. AROs are found in many fish taxa and differ significantly, but in the suborder Anabantoidei, which has a labyrinth organ (LO), and the family Clariidae, which has a dendritic organ (DO), these structures are found in the suprabranchial cavity (SBC). In this study, the SBC walls, AROs, and gills were studied in anabantoid (Betta splendens, Ctenopoma acutirostre, Helostoma temminckii) and clariid (Clarias angolensis, Clarias batrachus) fishes. The histological structure of the investigated organs was partially similar, especially in relation to their connective tissue core; however, there were noticeable differences in the epithelial layer. There were no significant species-specific differences in the structure of the AROs within the two taxa, but the SBC walls had diversified structures, depending on the observed location. The observed differences between species suggest that the remarkable physiological and morphological plasticity of the five investigated species can be associated with structural variety within their AROs. Furthermore, based on the observed histology of the SBC walls, it is reasonable to conclude that this structure participates in the process of gas exchange, not only in clariid fish but also in anabantoids.

1. IntroductionFish, like other animals, breathe oxygen, and its availability is crucial for their survival. An aquatic environment offers a much lower oxygen content compared to atmospheric air, which is why fish evolved special anatomical structures that enable efficient oxygen uptake from the water. Evolutionally, the ability to breathe atmospheric air was first achieved among vertebrates by fish, which occurred around 400 million years ago [1]. This ability arose independently in different evolutionary lines of fish, and the manner of air-breathing which developed in Sarcopterygians was inherited by the tetrapods. On the other hand, in the Actinopterygii clade, air-breathing mechanisms continue to be supported by dissolved oxygen uptake from the water. While, in some species, this ability has regressed with time, in others, it either remains active to the present day or was reacquired after the preceding regression [2,3]. In the latter group of fish, oxygen uptake takes place not only through the gills, a special form of respiratory organ equivalent to the lungs of terrestrial vertebrates, but also through accessory respiratory organs (AROs), which are frequently found in fishes living in poorly oxygenated waters throughout the world [4]. Some of the most well-known representatives of air-breathing fish are species belonging to the suborder Anabantoidei (order: Anabantiformes) and the family Clariidae (order: Siluriformes).The Anabantoidei, commonly called labyrinth fish, consist of either three or five families, depending on the methodological approach of the taxonomists. Anabantidae, Helostomidae, and Osphronemidae are the main families, as indicated by genetic-based research [5,6,7]; however, in classical descriptions, the subfamilies Luciocephalinae and Belontiinae were distinguished as separate families from the Ospronemidae [5,8,9,10]. The natural habitats of labyrinth fishes are found in the tropical waters of Africa and South Asia, in various aquatic ecosystems characterized by constant or periodic low oxygen content [11]. All of these species are characterized by high phenotypic and morphological plasticity, which, during the life of the fish, allow it to adapt to changing environmental conditions [12]. This developmental plasticity is mainly driven by environmental conditions, such as the content of dissolved oxygen in water, and not by genetic determinants [13,14].The Anabantoidei develop a labyrinth organ (LO) late during their growth, sometimes even after the attainment of sexual maturity [15], until which the gills remain the leading respiratory organ. The LO is located in the suprabranchial cavity (SBC), which is fully separate from the buccal cavity (unlike in its sister suborder Channoidei [7,16]), and is a plate-based organ covered by a heavily vascularized respiratory epithelium. The entire structure is flexible and may be supported by loose connective tissue and cartilage and/or bone. In some fish species, muscle tissue also occurs within the organ. The location and morphology of the LO also depend on the overall size of the body, as well as on the structure and size of the skull [1]. The origin of the LO has been investigated before, and the first hypothesis was that this organ evolved from a gill-derived structure [17]. It was later verified by Hughes and Munshi (1973) [18] that pillar cells in the AROs are not of the same type as in the gills, which probably indicates morphological differences between these organs.A structure similar to the LO, the dendritic organ (DO), is found in members of the Clariidae family, commonly known as air-breathing catfish, which belong to the Otocephala clade and are significantly distinct from the Anabantoidei in terms of phylogeny (clade Euteleostei) [19,20,21]. Therefore, this is an indication that these catfish evolved their own type of ARO following the mechanism of parallel evolution, while the secondary loss or reduction of this organ in some clariid genera was described as an ecophenotypic variation [22]. The Clariidae use the DO to absorb atmospheric oxygen during the seasonal drying of African and Asian tropical swamps and rivers [20,21]. These fish are only facultative air-breathers, in contrast to Anabantoidei; however, as suggested by Damsgaard et al. (2020) [8], air-breathing in Clariidae may be associated with a variable metabolic rate. With circadian changes in its metabolism, the African sharptooth catfish (Clarias gariepinus, Burchell, 1822) breathes more intensively during the night, and this increases simultaneously with the rise in its metabolic rate. The efficiency of this organ is associated with the presence of highly vascularized structures occurring not only in the DO itself, but also in the wall of the suprabranchial cavity chamber [23].The DO forms near the second and fourth gill arches and has a structure which bears similarity to a tree or shrub [24]. This organ is located at the posterior of the gill arches, with the smaller fragment located on the second gill arch and the larger on the fourth. The general structure of the dendritic organ resembles that of the LO in the Anabantoidei—there is a strongly vascularized, respiratory epithelium, with pillar-like cells and mucocytes, all supported by connective tissue [25]. The DO is connected to the gills through the same cartilage from which the branches of gill arches deviate [20]. The presence of a cartilaginous core ensures the relatively high durability of the DO during drought, in contrast to the gills, which collapse in the absence of water, due to being non-rigid [26].A thin, respiratory epithelium completely covers both the LO and the DO, where it rests on a basal plate, and is generally similar to the gill epithelium, which is beneficial for the diffusion of gases to and from the capillaries [26,27]. The epithelial layer also includes mucocytes responsible for: (a) providing optimal conditions for gas exchange, (b) protecting the delicate respiratory epithelium from all kinds of impurities, and (c) preventing particles suspended in water or air from interacting with the gills or the superficial cavity, possibly causing micro-injuries [26].The respiratory mechanism of the discussed fish species is not limited only to the gills, LOs, or DOs, because additional parts of these organs have also specialized to support this process. The walls of the gill and suprabranchial cavities are constructed similarly due to their common embryological origin during craniofacial development. Furthermore, the suprabranchial chamber is not only the location of the respiratory organs, but, in some fish species, this structure also actively participates in the processes of hearing [28], digestion [29], or breathing [1,8,23,30,31,32].Hitherto, there is a lack of histological comparison between the AROs of Asian and African Clariidae and representatives of the Anabantoidei. The need to obtain knowledge about the anatomy and physiology of these fishes appears to be crucial for two major reasons. Firstly, some of these fish species are of great local (and sometimes global) economic importance, either in aquaculture or in the ornamental fish trade. Secondly, in an era of globalization and the prevalent lack of clear and transparent international provisions regarding animal transportation and introduction, the invasiveness of some of these air-breathing species will increase significantly, especially more so during a climate catastrophe. It is therefore crucial to expand our basic knowledge about the anatomy and physiology of such fish species, so that, in the future, it can be used effectively to improve means of protection, breeding, and reproduction of aquatic wildlife. Therefore, the aim of this study was to compare the histological structure of the LO, DO, and SBC wall and expand the current knowledge about these air-breathing fishes. This comparison could allow for a better understanding of the structure of the AROs and may possibly allow us to learn more about the mechanisms of evolutionary solution that determined the ecological plasticity of fish and their adaptation capacity to the ever-changing environmental conditions.2. Materials and MethodsThis study was carried out in accordance with the guidelines provided by the 2nd Warsaw Local Ethics Committee for Animal Experimentation, residing at the Warsaw University of Life Sciences.Post-juvenile individuals of five fish species were used in the study: the Siamese fighting fish (Betta splendens, Regan, 1910), the leopard bushfish (Ctenopoma acutirostre, Pellegrin, 1899), the kissing gourami (Helostoma temminckii, Cuvier, 1829), the Angolian walking catfish (Clarias angolensis, Steindachner, 1866), and the Philippine catfish (Clarias batrachus, Linnaeus, 1758). For each species, the material was taken from five randomly purchased individuals (Table 1).Fish were slaughtered by decapitation with prior stunning. Whole fish were fixed in 4% neutral buffered formalin (NBF) and decalcified in Leica Decalcifier II (Leica Biosystems, Nussloch, Germany). Samples were subjected to a standard paraffin procedure [33] and were cut longitudinally and sagittally with the Leica RM2265 (Leica Biosystems, Nussloch, Germany) microtome at 5 µm thickness. Standard procedure was conducted to stain the slides with hematoxylin and eosin (HE), and, based on initial results, additional stainings were carried out. The collagens and intracellular matrix components were stained with Mallory trichromate (Supplementary Data 1). To visualize the mucosa cells and blood vessels, a modification of the standard AB/PAS (pH 2.5) procedure was performed (Supplementary Data 1). The slides were analyzed with a Nikon Eclipse NI-E microscope with a Nikon DS-Fi3 camera and NIS Elements AR software (Nikon, Tokyo, Japan).Morphometric analysis of the epithelial height was performed in all investigated fishes. For each of the four studied locations (the cranial, dorsal, and caudal parts of the SBC wall, as well as the LO/DO), a total of 50 measurements per group were conducted. Statistical analyses were performed with Statistica 13.3 software. Differences between parts of the SBC were analyzed for significance with a t-test (p < 0.05) and displayed as means with their standard deviation (±SD).3. Results3.1. Gills and Gill CavityThe gills of all the investigated individuals were located in gill chambers (Figure 1A,B), with the cores of the gill arches (bony in Claridae, cartilaginous–bony in Anabantoidei) covered by a thin layer of loose connective tissue, along with primary and secondary lamellae (Figure 1C,D).In all investigated individuals, different types of gill rakes were presented, with the most compound structures (acting as a filtration organ) found in the kissing gurami fish. The filtratory rakes were structured similarly to gills, with their cartilaginous core, connective tissue, and single-layered epithelium clearly visible. The lanceolate-shaped, branched gill rakes were observed as having a bony core and acidic mucosal cells. In the secondary lamellae, the highly vascularized, single-layered epithelium contained mucosal cells with acidic, neutral, and mixed mucus (Table 2; Figure 2A,B).3.2. Labyrinth Organ (LO) and Suprabranchial Cavity of Anabantoidei SpeciesThe LOs of all the studied fish species had a mixed, cartilaginous–bony core. Each of its elements was surrounded by loose connective tissue, covered by a single-layered epithelium (Figure 1C). In this organ, as in the case of gills, mucous cells were identified. Both the LOs and gills were characterized by a distinct network of blood vessels (Figure 2C). The LOs of C. acutirostre and B. splendens were similarly structured, but the cartilaginous core of these two species was characterized by a more compact structure than in H. temminckii, indicating partial ossification (pink arrow in Figure 2C). In the epithelium of the LO and the suprabranchial cavity, both acidic (blue) and neutral (magenta) mucous cells were identified (Figure 2C). In the suprabranchial wall of B. splendens, cavity-like structures were observed, with multiple mucosa cells found at their bottom, and without any visible blood vessels surrounding these structures (black arrows for caveolae, green for mucosa cells, Figure 2E,G). The complexity level of these caveolae was species-specific (the most complex were found in B. splendens) and varied depending on their localization. In the anterior and posterior parts of the suprabranchial wall, the caveolae were the most complex, while in the dorsal part they were the least complex (Figure 3C–E,G–I,K–M). The mean epithelial height values in the cranial, dorsal, and caudal parts of the SBC wall were significantly different (p < 0.05) in all investigated anabantid species (Table 3).Furthermore, in the suprabranchial cavity walls of all three Anabantoidei species, small, extension-like structures (similar to gill lamellae) were observed, as distinguished from the caveolae. The structure and length of these protrusions/extensions varied in terms of location, but a pronounced network of blood vessels was always found in their immediate proximity (Figure 3C–E,G–I,K–M).3.3. Dendritic Organ (DO) and Suprabranchial Cavity of Clariidae SpeciesThe DOs of both Clariidae species were divided into two parts; the smaller one was located in the gill cavity, while the larger and more widespread part was located within the suprabranchial cavity and located caudo-dorsally in relation to the gills (Figure 1B,D). The structure of the DOs was similar between species, regardless of the studied location. Elastic cartilage formed the core, covered by a thin layer of loose connective tissue and lined with a strongly vascularized, respiratory epithelium (Figure 1D and Figure 2D). In some of the sections of the DOs, a different kind of cartilage was noticed, with irregularly shaped cells and centrally arranged nuclei. In addition, the amount of basic substance in-between the chondritic territories was noticeably less than that of typical hyaline cartilage. Both cartilage types were surrounded by loose connective tissue as described above (Figure 2D). In the epithelium covering the DO fans, there were mostly acidic mucous cells, stained dark-blue with Alcian blue (Figure 2D). The walls of the gill cavity were structured differently than in the Anabantoidei species. Moreover, morphological differences were also observed depending on the location of the observed epithelia. In the anterior parts of the wall, instead of cavities as in Anabantoidei, a thin respiratory epithelium was observed, with numerous tongue-like capillaries and mucous cells (Figure 2E–H). In the centripetal and caudal parts, mucous cells were present, and the epithelium was similar to that of the oral cavity or esophagus. Folded extensions, similar to the shorter ones observed in the Anabantoidei in the anterior and caudal parts of the suprabranchial cavity membrane, were observed in the caudal area in Clariidae (Figure 3K–N). The mean epithelial height values in the cranial, dorsal, and caudal parts of the SBC wall were all significantly different (p < 0.05) in both investigated clariid species (Table 3).4. DiscussionThe ichthyofauna of oxygen-poor ecosystems developed several evolutionary solutions in order to improve the efficiency of gas exchange [1]. For instance, numerous modifications of hemoglobin and other blood components facilitated their adaptation to different oxygen levels, both in air and water [34,35]. Other physiological adjustments included various metabolic strategies which allowed them to withstand endogenous ammonia toxicity during emersion [36]. Meanwhile, histoanatomical solutions are associated with the presence of the respiratory epithelium not only in the gills but also in the gill-derived AROs [25,37], or even in the posterior intestine [38]. This epithelium is usually single-layered, with abundant mucous cells, and is, overall, a delicate structure that is fixed to its basal lamina and underlying connective tissue (rich in collagen), both of which provide proper mechanical support and necessary elasticity of the whole membrane [39]. On a larger scale, the gills are arches located in the gill cavity with a cartilaginous or bony core, with numerous extensions in the form of filaments and lamellae to maximize the gas exchange surface [40]. Likewise, some parts of the AROs may also have a core made of cartilage, bone, or an intermediate type of skeletal tissue, but the exact tissue type depends on the species. For instance, in fish from the Clariidae family, the presence of a hyaline cartilage core, as was the case in both species in this study, is normal [41]. Furthermore, fishes also developed muscular elements in their AROs, which set these structures in motion (fully or at least to some extent) and therefore impact the air ventilation of their respiratory chambers [42]. However, few researchers pay specific attention to the structures of the gill and suprabranchial cavities, both of which may serve a prominent respiratory function, as hypothesized for fishes belonging to both Clariidae [43] and Anabantoidei [31]. The extensions presented on the DO’s surface for both Clariidae species are thin-walled structures which, when exposed to air, assume shapes described as finger-like [26], balloon-like [31], tongue-like [20], or globular [26]. Due to the inflow of blood into these structures through the capillaries, their shape changes, increasing the surface area accessible for gas exchange. Meanwhile, when the gills are able to maintain sufficient blood oxygenation on their own, the blood flow through the DO capillaries is reduced, restricting the ability of red blood cells to reach the tips of these finger-like lamellae, which is why these structures enter a “resting state”, lying flat on the surface of the DO [26]. Such a situation was observed in this study, in the dorsal and caudal sections of the suprabranchial membrane of the two Clariidae fish species. This resting mechanism provides a barrier between atmospheric air in the suprabranchial chamber and blood in the respiratory lamellae in the DO [26]. Considering the fact that in Clariidae, the anterior part of the gill cavity, along with the entire surface of the suprabranchial cavity wall, contains numerous structures similar to the gills and identical to those of the DO; it can be assumed that the suprabranchial cavity’s epithelium itself may also participate in gas exchange.Presumably, the outgrowths found in the wall of the suprabranchial cavity of the studied Anabantoidei species could also have a respiratory function, similarly as the structures found in Clariidae fish. The highest complexity among these structures was found in the cephalic and caudal segments of the cavity of the Siamese fighting fish and kissing gourami. The cells presented in these protuberances were not only erythrocytes but also pillar-like cells similar to those co-forming the respiratory epithelium of the gills [44,45]. Therefore, these numerous extensions in the suprabranchial cavity wall correspond to the ”islets” described by Munshi (1968) [46] as locally occurring structures resembling lamella II of the gills. However, this similarity is justified further, as analogous structures have been demonstrated in the respiratory islets of the Clarias and Heteropneustes genera [8]. Therefore, it may be assumed that this membrane, similar to that of Clariidae, probably also participates to some extent in gas exchange.Mucous cells were present in large numbers in the both of the studied ARO types because they are key producers of the surface surfactants responsible for enhancing gas exchange. Surfactants, as in alveoli in tetrapod lungs, reduce surface tension, which promotes more efficient gas exchange [47]. They also have a protective effect by coating the respiratory epithelium with a protective layer, reducing the adhesion of foreign particles [48], and acting as an antimicrobial and antiradical cover [49]. Depending on the chemical composition of the secretions, mucins can stain with both Alcian blue and Schiff’s reagent. In the present study, mucous cells presented in the suprabranchial cavity epithelium in all investigated individuals were characterized by a differentiated response in AB/PAS staining, although cells with acidic or mixed secretions were predominant. The amount of acidic mucins in the mucus correlates with the degree of hydration of the respiratory epithelium, due to its high water-binding capacity. A histochemical analysis of the DOs of African catfish [25] demonstrated mucous cells producing acidic and mixed mucins, similar to the examined C. batrachus and C. angolensis. Meanwhile, neutral mucus has a lower viscosity compared to acidic mucus and provides excellent protection against physical damage [50], but has a lower preventive potential against parasites and microorganisms [51].Mucous cells were also present in the described cavities/caveolae in the Anabantoidei, with few blood vessels in close proximity. A similar structure was described in the study of Olson et al. (1994) [24], which identified two types of structures of a respiratory and non-respiratory nature in the buccopharynx wall in Channa punctatus. The respiratory structures consisted of squamous epithelium with erythrocytes and mucous cells “protruding” above the epithelial line, while the non-respiratory part was characterized as a “papillated surface”. All sections of the suprabranchial cavity of the Anabantoidei fish were organized in a similar manner—as alternating or continuously occurring epithelial tissue fragments with or without protuberances, and with or without mucous cells. Blood vessels were present underneath the individual respiratory sections, as in C. punctatus [32], indicating the convergent evolution of similar adaptations in different organs, with differences in the LOs being a feature likely correlated with respiratory performance and habitat occupation, rather than taxonomy [15].5. ConclusionsIn conclusion, to date, morphological research has not indicated that the suprabranchial cavity wall in investigated labyrinth fishes directly participates in air respiration, a feature which was discovered in Clariidae [25]. Acknowledging the fact that its structure and structural organization partly resembles both the LO and the DO in Clariidae, with the current state of knowledge, it seems highly probable that such an active respiratory role of the suprabranchial epithelium occurs also in the Anabantoidei. In the future, it appears plausible to carry out further analyses which take into account the detailed structure of the suprabranchial cavity wall and its potential physiological importance for these fish.

animals : an open access journal from mdpi

10.3390/ani11113062

PMC8614321

The cloning technique is important for animal husbandry and biomedicine because it can be used to clone superior breeding livestock and produce multipurpose genetically modified animals. However, the success rate of cloning currently is very low due to the low developmental efficiency of cloned embryos, which limits the application of cloning. The low developmental competence is related to the excessive cell death in cloned embryos. Interleukin 17D (IL17D) is required for the normal development of mouse embryos by inhibiting cell death. This study aimed to investigate whether IL17D can improve cloned pig embryo development by inhibiting cell death. Addition of IL17D protein to culture medium decreased the cell death level and improved the developmental ability of cloned pig embryos. IL17D treatment enhanced cloned pig embryo development by regulating cell death-associated gene pathways and promoting genome-wide gene expression, which is probably via up-regulating the expression of a gene called GADD45B. This study provided a new approach to improve the pig cloning efficiency by adding IL17D protein to the culture medium of cloned pig embryos.

Cloned animals generated by the somatic cell nuclear transfer (SCNT) approach are valuable for the farm animal industry and biomedical science. Nevertheless, the extremely low developmental efficiency of cloned embryos hinders the application of SCNT. Low developmental competence is related to the higher apoptosis level in cloned embryos than in fertilization-derived counterparts. Interleukin 17D (IL17D) expression is up-regulated during early mouse embryo development and is required for normal development of mouse embryos by inhibiting apoptosis. This study aimed to investigate whether IL17D plays roles in regulating pig SCNT embryo development. Supplementation of IL17D to culture medium improved the developmental competence and decreased the cell apoptosis level in cloned porcine embryos. The transcriptome data indicated that IL17D activated apoptosis-associated pathways and promoted global gene expression at embryonic genome activation (EGA) stage in treated pig SCNT embryos. Treating pig SCNT embryos with IL17D up-regulated expression of GADD45B, which is functional in inhibiting apoptosis and promoting EGA. Overexpression of GADD45B enhanced the developmental efficiency of cloned pig embryos. These results suggested that IL17D treatment enhanced the developmental ability of cloned pig embryos by suppressing apoptosis and promoting EGA, which was related to the up-regulation of GADD45B expression. This study demonstrated the roles of IL17D in early development of porcine SCNT embryos and provided a new approach to improve the developmental efficiency of cloned porcine embryos.

1. IntroductionSomatic cell nuclear transfer (SCNT) technique, also called cloning, is important for animal husbandry, biotechnology, biopharmacy and biomedicine because it can be used to clone superior breeding livestock [1,2] and produce multipurpose genetically modified animals [3,4,5]. However, the developmental efficiency of SCNT embryos is extremely low [6,7,8,9], which limits the practical application of cloning.So far, a broad spectrum of research has characterized the biological, molecular and epigenetic determinants of mammalian SCNT embryo development. The afore-indicated determinants involve: (1) the origin of nuclear donor cells [10,11,12,13,14], (2) the quality of nuclear recipient oocytes determined by the parameters related to meiotic, epigenomic and cytoplasmic maturity [15,16], (3) the methods used to stimulate the embryo-specific developmental program of enucleated oocytes [17,18,19,20], (4) the incidence of programmed cell apoptosis in in vitro-cultured nuclear donor cells and cloned embryos [21,22,23], (5) the intergenomic communication between nuclear DNA and mitochondrial DNA in cloned embryos [24,25,26,27,28,29], and (6) the capacity of donor cell nuclei to be reprogrammed in cloned embryos [30,31,32,33].Apoptosis is one of the major reasons that affects cloned embryo development. Many studies have shown that the cell apoptosis level in SCNT embryos is higher than that in fertilization-derived counterparts [34,35,36]. Treatment with anti-oxidative molecules, such as vitamin C [37], vitamin E [38], and melatonin [39,40], improve cloned embryo development by inhibiting apoptosis. Cytokines, such as IGF1 [41] and CSF2 [42,43], also regulate apoptosis-related genes to increase the developmental rate of SCNT embryos. In addition, small RNAs, such as miR-101-2 [44] and miR-449b [45], decrease apoptosis to enhance the developmental efficiency of SCNT embryos.Interleukin 17D (IL17D) is a cytokine that plays critical roles in mouse early embryo development by regulating apoptosis-associated pathways [46]. In fertilization-produced mouse embryos, IL17D expression is naturally up-regulated during 2-cell to 4-cell stage, and the inhibition of IL17D transcription impairs embryo development by increasing apoptosis. Meanwhile, supplementation of IL17D rescues embryonic developmental defects caused by the inhibition of IL17D expression.The purpose of this study was to investigate whether IL17D plays roles in regulating pig SCNT embryo development. We demonstrated that the addition of IL17D to the culture medium increased the developmental ability of cloned porcine embryos via modulating apoptosis-relevant pathways and global gene expression.2. Materials and Methods2.1. Ethics StatementThis study was performed in accordance with the “Guidelines with Respect to Caring for Laboratory Animals” issued by the Ministry of Science and Technology of China. The animal experimental protocol was approved by the Institutional Animal Care and Use Committee of South China Agricultural University. All efforts were made to minimize the suffering of animals tested.2.2. Medium and ReagentsAll the chemicals used in this study were procured from Sigma–Aldrich Company (MO, USA) unless otherwise mentioned.2.3. In Vitro Oocyte MaturationPorcine ovaries were collected from a local slaughterhouse and transported to the laboratory in 0.9% (w/v) NaCl solution supplemented with penicillin-G (100 IU/mL) and streptomycin sulfate (100 mg/L) at 30–35 °C. Follicular fluid containing cumulus–oocyte complexes (COCs) was aspirated from 3–6 mm diameter antral follicles by using an 18-gauge needle and syringe. COCs with at least three layers of compact cumulus cells and a homogenous cytoplasm were selected from follicle fluid and incubated with in vitro maturation medium [47] at 38.5 °C with 5% CO2 for 42–44 h. Matured COCs were freed from cumulus cells by repeated pipetting in Dulbecco’s phosphate-buffered saline (DPBS) (Gibco, Grand Island, NY, USA) containing 1 mg/mL hyaluronidase. Oocytes with uniform cytoplasm, round cell morphology, and clearly visible polar body were selected as mature oocytes for subsequent experiments.2.4. In Vitro FertilizationPorcine semen was purchased from a local animal husbandry company. The sperm density was adjusted to 2 × 107/mL with modified Tris-buffered medium(mTBM) [48] for capacitation. The in vitro mature oocytes were washed with mTBM and transferred to a four-well plate with 40–50 oocytes and 500 µL mTBM per well. The capacitated semen (50 µL) was added to obtain 1 × 106 sperm per well. After incubating at 38 °C for 6 h, the fertilized eggs were washed with DPBS supplemented with 1 mg/mL BSA to remove the sperm on the surface. The eggs were then transferred to PZM-3 medium [49] and cultured in an incubator at 38.5 °C and 5% CO2. Cleavage rate, blastocyst rate, and total cell number of blastocysts were calculated at 48 and 144 h.2.5. Somatic Cell Nuclear TransferPorcine fetal fibroblasts were cultured in DMEM with 10% of FBS at 38.5 °C and 5% CO2. When the cells reached a confluence of 90–100%, they were digested with trypsin and resuspended in in vitro manipulation medium for later use. Mature oocytes were denucleated by blind aspiration. One donor cell was injected through the enucleated incision into the perivitelline space of each enucleated oocyte. Reconstituted embryos were cultured in PZM-3 at 38.5 °C for 4 h and 5% CO2. The reconstructed embryos were activated in the fusion solution [1] through two direct current pulses of 150 V/mm for 50 ms. The reconstituted embryos were transferred to PZM-3 and cultured in an incubator at 38.5 °C and 5% CO2. The numbers of cleaved embryos and blastocysts were calculated at 48 h and 144 h post-activation, respectively. Blastocysts were defined as embryos that contain a fluid-filled blastocoel and have at least 20 cells.2.6. IL17D TreatmentHuman IL17D protein (Cat no. CG96, Novoprotein, Fremont, CA, USA), which has 98% homology to porcine IL17D in amino acid sequences, was added to the PZM-3 culture medium of porcine IVF and SCNT embryos at 1-cell stage (right after fertilization of IVF embryos or activation of SCNT embryos) or 4-cell stage (48 h post fertilization or activation of SCNT embryos). After culturing for 48 h, the medium containing IL17D was replaced by PZM-3 medium. The experimental design and groups of IL17D treatment is shown in Table 1.2.7. Transcriptome SequencingTranscriptome sequencing was performed as previously described [50]. Twenty to thirty four-cell stage embryonic cells of the same treatment group were mixed as one sample and collected into a 1 mL tube containing 3 µL of lysis buffer with RNase inhibitor. Complementary DNA (cDNA) was generated and amplified for sequencing based on smartseq2 method. After cDNA amplification and purification, the overall quality of the initial cell was performed with an Agilent 2100 Bioanalyzer. We determined the degradation of the sample according to the RNA integrity number by Agilent Bioanalyzer. The clustered library preparations were then sequenced on an Illumina Nova platform, and 150-bp paired-end reads were generated. Raw read data were the original RNA-Seq reads which used Trimmomatic to filter low-quality reads and trim the linker sequence [51]. Clean read data were then aligned to the susScr11 reference genome using STAR [52]. Reads aligned to genes are counted by cufflinks (v2.2.1). The fragments per kilobase of exon model per million mapped reads (FPKMs) are normalized using cuffnorm. Differentially expressed genes are calculated using cuffdiff [53]. Differentially expressed genes between two groups (p < 0.05) were filtered by [log2 FoldCharge] > 1 and FPKM > 5. Raw RNA-Seq data for 2-cell and 4-cell stage IVV Duroc embryos, SCNT Duroc embryos, and SCNT Laiwu embryos, were obtained from the Gene Expression Omnibus (GEO) datasets (accession no. GSE125706) [54].2.8. Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR)Total RNAs were extracted from 4-cell stage embryos and blastocysts by using the Qiagen AllPrep DNA/RNA Micro Kit (Qiagen, Gaithersburg, MD, USA) according to the manufacturer’s instructions. cDNA was generated by the PrimeScript RT reagent kit with gDNA Eraser (TAKARA, Shiga, Japan). The synthesized cDNA was used for qRT-PCR by PowerUp SYBR Green Master Mix (Thermo Fisher Scientific, CA, USA). qRT-PCR was performed on QuantStudio™ 7 Flex Real-Time PCR System (Thermo Fisher Scientific) by following the parameters recommended by the manufacturer. The reactions were run and performed in triplicate as previously described [55]. GAPDH was used as the house-keeping gene, and the relative mRNA expression was calculated using the 2-ΔΔCt method. The primer sequences are shown in Table 2. The amplification efficiency of each pair of primers at different cDNA template concentrations was validated to be >95% (Figure S1 and Table S1), following a previously reported method [56].2.9. Terminal Deoxynucleotidyl Transferase (TdT)-Mediated 2′-Deoxyuridine-5′-Triphosphate-Digoxigenin Nick-End Labeling (TUNEL) AssayBlastocysts were analyzed by the TUNEL apoptosis detection kit (YEASEN, SH, China). The embryos were fixed in 4% paraformaldehyde and permeated for 30 min in 0.5% Triton X-100. The embryos were incubated in TdT incubation buffer at 38 °C for 1 h. The nuclei were stained with Hoechst33342 at room temperature for 5 min. The stained embryos were placed on slides, and photos were taken under the microscope.2.10. Plasmid ConstructionThe coding sequences (CDS) of porcine GADD45B gene (GenBank no. XM_005654701.2) was synthesized and inserted between the NheI and HindIII sites at the multiple cloning sites of the pcDNA3.1(+)-EGFP vector to generate the pcDNA3.1-GADD45B-EGFP vector.2.11. MicroinjectionMicroinjection of the porcine GADD45B expression plasmid into cloned pig embryos was performed using a micropipette driven by a Piezo (Eppendorf). Ten picoliter of plasmids or water was injected into the cytoplasm of each embryo after activation of SCNT embryos. After microinjection, the embryos were transferred to PZM-3 and cultured in an incubator at 38.5 °C and 5% CO2.2.12. Statistical AnalysisSPSS software version 20 was used for statistical analysis. A Chi-square test was performed to determine differences in cleavage rate, blastocyst rate, and percentage of apoptosis cells. One-way ANOVA was performed to evaluate differences in gene expression level and total cell number of blastocysts.3. Results3.1. IL17D Expression Is Abnormal in Cloned Pig Embryos Compared to That in In Vivo-Fertilization-Derived Pig EmbryosAnalysis of a published transcriptome sequencing data set showed that the expression of IL17D mRNA is up-regulated by approximately eight folds in in vivo fertilization-derived (IVV) porcine embryos while it is unchanged in cloned porcine embryos during the 2-cell to 4-cell stage (Figure 1). This suggests that up-regulation of IL17D expression during early stage is required for normal porcine embryo development and the expression of IL17D in cloned pig embryos is abnormal.3.2. IL17D Treatment Improved the Developmental Competence of Porcine SCNT and IVF EmbryosThe activated cloned embryos at the 1-cell stage were treated with 5, 25, 50 and 100 ng/mL IL17D for 48 h to examine the effects of IL17D on porcine SCNT embryo development. Treatment with 50 ng/mL IL17D significantly enhanced the blastocyst rates of porcine SCNT embryos (Table 3). Treatment with 25 and 100 ng/mL IL17D did not significantly improve but tented to improve the blastocyst rate of porcine SCNT embryos (Table 3). The addition of 50 ng/mL IL17D at 1-cell stage for 48 h also significantly enhanced the blastocyst rate of the treated porcine IVF embryos (Table 4). However, treatment with porcine SCNT embryos with 50 ng/mL IL17D at 4-cell stage for 48 h did not significantly affect the developmental efficiency (Table 5).3.3. IL17D Treatment Inhibited the Apoptosis of Porcine SCNT and IVF Embryos at Blastocyst StageTreatment with 50 ng/mL IL17D at 1-cell stage for 48 h also significantly increased the total cell number of porcine SCNT and IVF embryos at the blastocyst stage (Figure 2A,C). The number and proportion of apoptotic cells at the blastocyst stage significantly decreased in porcine SCNT embryos treated with IL17D (Figure 2B,D,E). At the blastocyst stage of IL17D-treated porcine SCNT embryos, the mRNA expression of the pro-apoptotic gene BCL2L11 was down-regulated and the transcription of the anti-apoptotic gene BCL2 was up-regulated. The mRNA abundance of four other tested apoptosis-related genes was not significantly changed (Figure 2F). These results indicated that IL17D treatment inhibited apoptosis in porcine SCNT embryos.3.4. IL17D Treatment Increased the Expression of Global Genes in Porcine SCNT Embryos at 4-Cell StageWe observed that porcine SCNT embryos treated with 50 ng/mL IL17D at 1-cell stage for 48 h exhibited higher developmental rate than the control group at 4-cell stage (Figure 3A). This observation, together with the result shown in Table 5, suggested that the added IL17D in the embryo culture medium exerted or started to exert its effects before the 4-cell stage of porcine SCNT embryos. Therefore, 4-cell stage SCNT-IL17D and SCNT-NC embryos were collected for transcriptome sequencing to compare their differences in gene expression patterns. The PCA results showed that three samples in the SCNT-IL17D (IL) group were separated from the three other samples in the SCNT-NC (NC) group (Figure 3B). This finding implied that the two groups of embryos had different gene expression patterns. The mRNA expression level of transcriptome sequencing-detected genome-wide genes was significantly increased in the IL group compared with that in the NC group (Figure 3C). The analysis of differentially expressed genes (DEGs) showed that in IL embryos, the number of up-regulated genes (175) was higher than the number of down-regulated genes (30) (Figure 3D). Hence, IL17D treatment up-regulated the expression of global genes in porcine SCNT embryos at the 4-cell stage. To confirm the transcriptome sequencing data, we randomly selected eight genes including FOS, JUN, BAX, BAD, MAP3K8, MYC, BCL2L11, and GADD45B for qRT-PCR verification. The mRNA expression levels of the eight genes examined by transcriptome sequencing matched with those measured by qRT-PCR (Figure 3E,F).3.5. IL17D Treatment Improved Porcine SCNT Embryo Development by Regulating Apoptosis-Related PathwaysAmong the top 10 significantly enriched KEGG pathways for DEGs between IL and NC groups, three pathways including apoptosis pathway, MAPK signaling pathway, and NF-kappa B signaling pathway are involved in the IL17 signaling pathway (Figure 4A) [57]. The three IL17-related pathways are also relevant to apoptosis. The transcription levels of most DEGs enriched in the three apoptosis-associated pathways, including pro-apoptotic and anti-apoptotic/pro-survival genes, were up-regulated in IL group compared with those in NC group (Figure 4B) [58]. These results suggested that IL17D signal through the IL17 pathway to act on apoptosis-related genes to enhance cloned porcine embryo development.3.6. The Effects of IL17D on Improving Porcine SCNT Embryo Development Might Be Related to the Up-Regulation of GADD45B ExpressionThe above results suggested that IL17D enhanced pig SCNT embryo development by inhibiting apoptosis and promoting EGA. Among the genes whose expression was up-regulated at 4-cell stage in IL17D-treated pig SCNT embryos, GADD45B was noticeable because it not only has anti-apoptotic functions [59,60], but also plays important roles in EGA [61]. To examine whether the effects of IL17D on enhancing pig SCNT embryo development is related to the upregulation of GADD45B expression, we constructed a porcine GADD45B expression plasmid (Figure 5A) and injected it into cloned pig embryos. Expression of the EGFP marker gene that was linked to the porcine GADD45B gene was observed at 2-cell stage of injected pig SCNT embryos (Figure 5B). This suggested that GADD45B was overexpressed in injected pig SCNT embryos. Injection of GADD45B expression plasmid showed no effect on the total cell number at blastocyst stage of cloned pig embryos (Figure 5C). However, injection of 100 ng/µL of GADD45B expression plasmid increased the blastocyst rate of cloned pig embryos and injection of 50 ng/µL and 10 ng/µL of GADD45B expression plasmid tended to improve the blastocyst rate of cloned pig embryos (Table 6). This suggested that IL17D improved porcine SCNT embryo development probably by up-regulating GADD45B expression.4. DiscussionIL17D enhanced porcine SCNT embryo development by inhibiting apoptosis. The functions of IL17D observed in porcine SCNT embryos are consistent with those reported in mouse IVF embryos [46]. Our transcriptome data showed that the expression levels of pro-apoptotic DEGs such as BCL2L11 [62], PMAIP1 [63], and DDIT3 [64] were up-regulated with anti-apoptotic DEGs such as GADD45B [60], MCL1 [65], and DUSP1 [66], in SCNT-IL17D embryos compared with those in SCNT-NC embryos. However, the ratio of the expression level of pro-apoptotic genes to that of anti-apoptotic genes might be lower in SCNT-IL17D embryos than in SCNT-NC embryos. This phenomenon might result in the overall anti-apoptotic effect in IL17D-treated SCNT embryos, which is supported by the data of this study.In early mouse embryos, transcription of IL17D is positively associated with the expression of its promoter-associated noncoding RNA (pancil17d), which is oppositely transcribed from the bidirectional IL17D promoter and is indispensable for EGA of mouse embryos [46]. This suggested that IL17D is involved in EGA. SCNT embryos are associated with defects of embryonic genome activation (EGA) [54,67]. IL17D also promoted EGA in treated porcine SCNT embryos because it increased the expression of global genes at 4-cell stage, which is the major EGA stage for porcine embryos [68,69]. The promotion of EGA in porcine SCNT embryos by IL17D might be related to the up-regulation of the expression of GADD45B, which is functional in inhibiting apoptosis and promoting EGA [59,60,61]. More importantly, overexpression of GADD45B enhanced the developmental ability of cloned pig embryos.IL17D transcription was naturally increased by about eight folds in porcine IVV embryos during 2-cell to 4-cell stage, suggesting that the up-regulation of IL17D before the 4-cell stage is required for normal porcine embryo development. This finding is consistent with our observation that the developmental ability of porcine SCNT embryos was improved by supplementation of IL17D at 1-cell stage but not at 4-cell stage. Nevertheless, the addition of IL17D at the 4-cell stage of mouse IVF embryos rescued the developmental failure induced by the suppression of IL17D expression. The difference in the action time point of IL17D in porcine SCNT embryos and mouse IVF embryos might be related to their differences in species and type of embryos.We also tried to knock down IL17D expression by RNA interference to examine its effects on cloned porcine embryo development (data not shown). However, only one siRNA with high specificity for targeting porcine IL17D mRNA was predicted by professional siRNA designing software due to the high homology of the IL17D gene to a gene called EEF1A lysine methyltransferase 1 in the pig genome. This potential IL17D-targeting siRNA could not decrease the porcine IL17D transcript level in transfected porcine cells and injected porcine SCNT embryos. Therefore, in the future, other strategies, such as IL17D gene knockout, should be employed to investigate the effects of inhibiting IL17D expression on porcine embryo development. In addition, future studies should test whether IL17D also participates in regulating the development of the SCNT embryos of other species, and whether IL17D can also improve the in vivo full-term developmental competence of cloned embryos.5. ConclusionsIn summary, IL17D improved the developmental competence of cloned porcine embryos by suppressing apoptosis and promoting EGA, which probably was related to the up-regulation of GADD45B expression. This study not only elucidated the functions of IL17D in early development of porcine SCNT embryos but also provided a new way to increase the developmental efficiency of cloned porcine embryos.

animals : an open access journal from mdpi

10.3390/ani11071939

PMC8300243

In the freezing process of boar sperm, there are obvious differences in freezability between individuals. Studies suggest that specific freezability markers might be useful in good (GFE) and poor freezability ejaculate (PFE) selection prior to cryopreservation. Therefore, we performed UHPLC-qTOF-MS analysis to explore the difference in the metabolic level of seminal plasma between boars with differential freezability, and the results showed that the content of D-aspartic acid, N-acetyl-L-glutamate (NAG), and inosine were significantly different. These findings present new insights into the role of metabolism in sperm freezability and provide research directions for exploring potential biomarkers of freezability.

Some potential markers of boar sperm freezability have been found in spermatozoa, but little attention has been paid to seminal plasma. The seminal plasma is composed of secretions from the testis, epididymis, and accessory sex glands. The exposure of spermatozoa to small molecules such as metabolites can affect sperm function. However, details and significance of the seminal plasma metabolome related to boar sperm freezability are unknown. Therefore, the main aim of this study was to explore the differences in the metabolic level of seminal plasma between boars with differential freezability and to explore the candidate biomarkers of semen freezability. A total of 953 metabolites were identified in boar semen plasma by UHPLC-qTOF-MS analysis, and 50 metabolites showed significant change between the GFE group and PFE group. Further, twelve metabolites were subjected to metabolic target analysis, and three metabolites (D-aspartic acid, N-acetyl-L-glutamate (NAG), and inosine) showed differences. In conclusion, there is significant difference in the metabolome of seminal plasma between GFE and PFE individuals. D-aspartic acid, NAG, and inosine in seminal plasma may be potential markers for assessing sperm cryopreservation resistance in boars.

1. IntroductionArtificial insemination has been widely used in pig production worldwide. However, frozen–thawed boar semen accounts for less than 1% of the semen used for insemination [1]. On one hand, boar spermatozoa in general presents low freezability because of its high cold shock sensitivity [2,3]. On the other hand, the quality of frozen-thawed boar semen shows strong variability in freezability between individuals [4]. Therefore, it is meaningful to distinguish between high and low freezability individuals before cryopreservation procedures and to select high freezability individuals for cryopreservation to improve the efficiency of artificial insemination utilizing post-thawed sperm. To solve this issue, researchers are engaged in a lot of work to distinguish good (GFE) and poor freezability ejaculates (PFE) [5,6,7].Previous research on boar ejaculate freezability biomarkers mainly focused on proteomics. Numbers of proteins from sperm or seminal plasma, such as heat-shock protein 90 (HSP90AA1) [8], acrosin-binding protein (ACRBP) [9], triosephosphate isomerase (TPI) [9], and fibronectin 1 (FN1) [10], have been reported as markers for predicting boar ejaculate freezability [11]. In addition, a study demonstrated that genomic differences existed between good and poor freezers in the sequences of polymorphism restriction fragments of 16 candidate genetic markers [6]. Other freezability markers include patterns of sperm motile subpopulations in extended semen [12], specific kinetic parameters evaluated at the cooling step [13], and acrosin activity [14,15].Sperm freezability is a complex phenotype and it cannot be accurately predicted based solely on conventional parameters [1,13]. Current knowledge implies that the seminal plasma is much more than a nutrient medium. Seminal plasma is composed of secretions from the testis, epididymis, and accessory sex glands. Seminal plasma contains a variety of substances, such as proteins, ions, and metabolites including amino acids, lipids, nucleosides, minerals, electrolytes, and steroid hormones [16,17]. As metabolites are the final products of metabolism, changes in their composition and content can reflect the state of the sperm and individual metabolic timeliness [18]. A recent study shows that metabolites play a role in sperm energy production, motility, pH control, and regulation of metabolic activity [19]. Furthermore, metabolites in seminal plasma may affect downstream and complementary changes in gene/protein expression [20]. Thus, we hypothesized that particular metabolites in seminal plasma could be considered as markers for sperm freezability.Therefore, the aims of this study were to compare the metabolome of seminal plasma between GFE and PFE as well as identifying potential metabolites as biomarkers of freezability. We used an Ultra-high Performance Liquid Chromatography-Quadrupole Time-of-Flight Mass Spectrometry (UHPLC-qTOF-MS) based metabolomics approach to obtain the metabolic profile of seminal plasma from boars with good and poor sperm freezability. Furthermore, the potential metabolites were confirmed by targeted metabolomics analysis. Findings in the present study will provide a new perspective for boar sperm freezability prediction.2. Materials and Methods2.1. Sample Collection and Preparation of Seminal PlasmaThe boars (n = 10) were chosen based on production records over 2 years. All boars were Landrace, and they were raised under the same management conditions and received the same nutrition. Semen was collected using the gloved hand method. The semen collection rhythm was twice a week, and one single ejaculate per boar was used in this study. After collection, the spermatozoa-rich fraction of each ejaculate (80–100 mL) was filtered through gauze and subsequently divided into two aliquots of equal volume. The first one was used for seminal plasma separation from spermatozoa through centrifugation at 500× g and 4 °C for 30 min. Seminal plasma preparations were then examined using phase microscopy to ensure no spermatozoa remained. Clean seminal plasma samples were then stored in liquid nitrogen. Another spermatozoa-rich fraction aliquot was diluted in Androhep Plus (Minitube co., ltd., Hauptstrasse, Germany) at 2 × 10 [8] and then used to cryopreserve.2.2. Cryopreservation and Thawing of Sperm SamplesFirstly, the semen samples were stored at 17 °C to cool, then centrifuged at 500× g for 10 min. Soft sperm pellets were subsequently diluted to 2 × 10 [9] spermatozoa/mL in Androstar Cryo Plus (Minitube, Germany) containing 20% egg yolk. Then, the spermatozoa were cooled slowly to 5 °C for 5 h and subsequently diluted to 1 × 10 [9] spermatozoa/mL with a freezing medium containing 6% glycerol (Sigma-Aldrich, St. Louis, MO, USA) at 5 °C. Afterward, sperm samples were packed in 0.5 mL labeled plastic straws (Minitube, Germany). The straws were then transferred to a programmable freezer (CryoMed 7457 (Thermo Fisher, Waltham, MA USA)). The cooling ramp was as follows: wait at 4 °C → 2 °C/min to 2 °C → hold for 1 min at 2 °C → 35 °C/min to −30 °C → hold for 1 min at −30 °C → 35 °C/min to −150 °C → hold for 4 min at −150 °C. The straws were finally plunged into liquid nitrogen and stored before use.2.3. Assessment of Sperm QualitySperm motility parameters obtained were those described by Yeste et al. [21]. Sperm motility assessment was carried out utilizing a commercial computer assisted sperm analysis (CASA) system (CASAS-QH-III, Tsinghua Tongfang Co., Ltd., Beijing, China). After evaluating three replicates per sample (a minimum of 1000 spermatozoa were counted per replicate), the corresponding mean standard error of the mean (SEM) was calculated.2.4. Sample Classification into GFEs and PFEsTo classify seminal plasma samples into two groups (GFEs vs. PFEs), spermatozoa were cryopreserved and thawed; sperm quality assessments were carried out at three different points: pre-freeze, refrigerated semen at 17 °C, and frozen–thawed spermatozoa at 30 min post thawing. To distinguish seminal plasma samples between two groups of good (GFE) and poor (PFE) freezability, boar sperm was characterized by a reduced sperm motility.2.5. Ultra-High Performance Liquid Chromatography-Quadrupole Time-of-Flight Mass Spectrometry (UHPLC-qTOF-MS) Data AcquisitionAnalysis data were acquired using a UHPLC-high definition quadrupole time-of-flight MS instrument (UHPLC-qTOF SYNAPT G1 HD-MS system, Waters Co., Ltd., Milford, MA, USA), equipped with a TripleTOF 6600 (Q-TOF, AB Sciex). A binary solvent method consisting of eluent A (25 mM NH4Ac and 25 mM NH4OH in water pH = 9.75) and acetonitrile (B) was carried out with an elution gradient as follows: 0 min, 95% B; 0.5 min, 95% B; 7 min, 65% B; 8 min, 40% B; 9 min, 40% B; 9.1 min, 95% B; 12 min, 95% B, delivered at 0.5 mL min−1. The Triple TOF mass spectrometer was used for its ability to acquire MS/MS spectra on an information-dependent basis (IDA) during an LC/MS experiment. In this mode, the acquisition software (Analyst TF 1.7, AB Sciex) continuously evaluated the full-scan survey MS data as it collected and triggered the acquisition of MS/MS spectra depending on preselected criteria. In each cycle, 12 precursor ions, whose intensity was greater than 100, were chosen for fragmentation at a collision energy (CE) of 30 V (15 MS/MS events with product ion accumulation time of 50 ms each). ESI source conditions were set as follows: ion source gas 1 as 60 psi, ion source gas 2 as 60 psi, curtain gas as 35 psi, source temperature 600 °C, Ion Spray Voltage Floating (ISVF) 5000 V or −4000 V in positive or negative modes, respectively.2.6. Multivariate Data (MVD) AnalysisUHPLC-qTOF-MS data were analyzed using SIMCA 13 software (Umetrics, Umea, Sweden) and interactive XCMS (version 3.2). Before exporting the data to SIMCA for visualization and biomarker selection, the LC-MS raw data were first processed (noise elimination, peak picking, alignment, and retention time correction) with MarkerLynxTM software (version 4.1, Waters Corporation, Milford, MA, USA). The following parameters were used for data processing: retention time (Rt) range of 2.5–11 min, mass range of 100–1000 Da, mass tolerance of 0.02 Da, and an Rt window of 0.2 min. The data matrix obtained from MarkerLynxTM processing was then exported into SIMCA 13 for PCA and OPLS-DA analysis. The data were Pareto-scaled, and no transformation was used. For the XCMS analysis, the MassLynxTM raw data (.raw) were converted to NetCDF format using the DataBridge application in MassLynxTM (Waters, Co., Ltd., Milford, MA, USA). The converted data (NetCDF format) were then used in XCMS for processing, statistical analysis, visualization, and biomarker identification as described by Chang et al. [22]. The parameters were as follows: feature detection set as centWave method, minimum peak width = 5, maximum peak width = 20, retention time correction set as Obiwarp method, Profstep = 1, alignment set as m/z width = 0.015, min fraction = 0.5, and bw = 5; statistics were set as statistical test = unpaired parametric t-test (Welch t-test), paired t-test, and post hoc analysis with the threshold p-value = 0.01 and fold-change = 1.5.2.7. Relative Distribution and Statistical AnalysisTotal intensity values (integrated area under the peak) from MarkerLynxTM XS software (Waters Corporation, Manchester, UK) pre-processed data matrixes were used for univariate statistical analyses. SPSS software (IBM SPSS Statistics for Windows, Version 22., Armonk, NY, USA; IBM Inc., Chicago, IL, USA) was used for such descriptive statistics. Here, Univariate Analysis of Variance (ANOVA) was performed as two-tailed complete randomized blocks and used to compare the non-treated with the different time points of treated cells. ANOVA was followed by the Bonferroni post hoc test where differences between the means were considered significant at p < 0.05 and indicated in the box-and-whiskers plots.2.8. Targeted Metabolomics AnalysisTargeted metabolomics analysis was performed using QTRAP 5500 (AB SCIEX). The target metabolomics metabolite extraction method is the same as that of UHPLC-qTOF-MS data acquisition. We performed absolute quantification of candidate differential metabolites based on standard products, and the standard products were purchased from Yuanye Biological Technology Co., Ltd. (Shanghai, China).3. Results3.1. Classification of Boar Ejaculates into GFE and PFE GroupsThe semen collected from the selected boars showed similar sperm motility. Fresh sperm with motility higher than 75% was processed for freezing. The sperm motility of fresh sperm, kept at 17 °C and then thawed, was analyzed (Figure 1A). The difference in the sperm freezability of these boars was evaluated based on the ratio of thawed motility to fresh motility (the relative sperm motility) (Figure 1B). Five GFE and five PFE semen were chosen.3.2. Metabolomic Analysis Based on UHPLC-qTOF-MS TechnologyA total of 953 metabolites were identified after UHPLC-qTOF-MS analysis of these seminal plasma samples, regardless of group. Metabolites were identified and categorized according to their major chemical classes, including carboxylic acids and derivatives, organooxygen compounds, amino acids, peptides, analogues, fatty amides, fatty acyls, benzene and substituted derivatives, purine nucleotides, pyrimidine nucleotides, glycosyl compounds, fatty acids, and conjugates (Figure 2). A total of 534 (POS, 298; NEG, 236) features could be mapped to current databases. According to the classification of metabolites, it was found that in the POS mode the main metabolites were organic acids and their derivatives, which contains 68 metabolites, accounting for 24% of all metabolites detected. The remaining metabolites were carboxylic acids and their derivatives (10%); nucleosides, nucleotides, and analogues ranked third (8%); followed by the organic oxygen compounds, organoheterocyclic compounds, lipids and lipid-like molecules, benzene and its substituted derivatives, benzenoids, and the other 14 metabolites (Figure 2). In the NEG mode, 87 kinds of organic oxygen compound metabolites were detected, accounting for 37% of all metabolites detected, followed by nucleosides, nucleotides, and analogues (10%), carboxylic acids and their derivatives (10%) ranked third, followed by organoheterocyclic compounds (8%), lipids and lipid-like molecules (8%), fatty acyls (4%), and 12 other metabolites (Figure 2).3.3. Identification of Potential Freezability BiomarkersTo identify potential biomarkers in seminal plasma associated with sperm freezability, PCA and OPLS-DA models were applied to the classification of the GFE and PFE groups. The quality of the OPLS-DA model was checked by seven-fold cross-validation (Figure S1). The model’s information was shown in Table S1. The two groups of samples have not been processed in any way, so the PCA analysis has not been clearly distinguished, which does not affect the subsequent analysis (Figure S1A,B). There was a clear separation between the two groups in both positive (Figure S1C) and negative ion mode (Figure S1D) in the OPLS-DA score plot. We shuffled groups in permutation tests to construct the OPLS-DA model randomly, as results show that the overall R2Y and Q2 of the original model are higher than the R2Y and Q2 of the model constructed by the replacement test, indicating that the original model grouping is not over-fitting (Figure 3E,F).Based on the analysis of the OPLS-DA method, the calculated VIP and p value is shown in Figure 3. According to the OPLS-DA and volcano plot, metabolites with a VIP score greater than 1 and a p-value less than 0.05 were identified and considered as candidate freezability markers. The cluster analysis of each candidate metabolite is shown in Figure 4A,B. Finally, a total of 50 metabolites showed significant differences between the GFE and PFE groups (Tables S2 and S3).3.4. Pathway Analysis of MetabolitesWe analyzed the differential metabolic pathways via KEGG analysis (Table S4). These differential metabolites were mainly enriched in amino acid biosynthetic metabolic pathways such as alanine, aspartic acid, glutamic acid, arginine, proline, cysteine, and methionine biosynthetic metabolic pathways. Some of the metabolites are enriched in purine metabolism, pyrimidine metabolism, terpenoid backbone biosynthesis, aminoacyl tRNA biosynthesis, and other metabolic pathways (Figure 5A,B).3.5. Confirmation of Freezability Biomarkers by Targeted Metabolic AnalysisAmong the 50 candidate metabolites obtained from the above analysis, 3-methylhistidine (p = 0.036), phenethyl caffeate (p = 0.049), S-adenosyl-L-homocysteine (p = 0.049), D-aspartic acid (p = 0.031), L-methionine (p = 0.018), DL-2-aminoadipic acid (p = 0.014), L-glutamine (p = 0.009), N-acetyl-L-glutamate (NAG) (p = 0.009), cytidine (p = 0.023), inosine (p = 0.012), quercetin (p = 0.034), and norethindrone acetate (p = 0.024) were chosen and further verified by targeted metabolism. The result reveals that D-aspartic acid, NAG, and inosine showed significant differences between GFE and PFE (p < 0.05) (Figure 6).4. DiscussionCryopreservation of sperm is important for the preservation of the boar sperm. In general, 40–50% of the sperm population cannot survive after cryopreservation, even when “optimized” cooling/thawing protocols are used [1,23,24]. There is a considerable variability between ejaculates in their ability to withstand cryopreservation procedures. Mammalian seminal plasma is mainly formed by secretions of the epididymis and accessory sex glands [25]. Seminal plasma contains large spectra of metabolites; the current concept states that seminal plasma can modulate sperm function. Previously, metabolites have been identified in bull and boar seminal plasma, and attempts were made to explore the candidate biomarkers of fertility [26]. The current study aimed to find a specific metabolomics signature in the seminal plasma of high freezability boars. Our study is in fact the first to compare the seminal plasma metabolome of boars between GFE and PFE. This model gives a global view of the metabolites in boar seminal plasma with both high and low freezability. Moreover, we confirmed the candidate metabolite biomarkers by utilizing the targeted metabolome method. In general, the main compounds in boar seminal plasma in the present study were carboxylic acids and derivatives, organonitrogen compounds, amino acids, peptides and their analogues, fatty acyls, purine nucleosides, pyrimidine nucleosides, and fatty acids and their conjugates. The changed 50 metabolites were enriched in amino acid biosynthetic metabolic pathways, purine metabolism, pyrimidine metabolism, terpenoid backbone biosynthesis, aminoacyl tRNA biosynthesis, and other metabolic pathways. The results implied that amino acid metabolism plays an important role in the regulation of freezability. Based on functional analysis such as KEGG, we chose 12 metabolites that could be confirmed by targeted metabolic analysis. Finally, the results confirm that D-aspartic acid, NAG, and inosine were lower in the GFE group than the PFE group. It is interesting that the level of D-aspartic acid was higher in the GFE group than the PFE group. Previous studies reported that D-aspartic acid occurs in human seminal plasma, and the concentration of D-aspartic acid was significantly reduced in oligoasthenoteratospermic individuals [27]. Then, studies on Leydig cells and spermatogonia in vitro demonstrated a direct effect of D-aspartic on the steroidogenic pathway and spermatogenesis. Therefore, D-aspartic mainly functions as a modulator of spermatogenesis in mammals [28]. Further, D-aspartic treatment can increase the motility of sperm [29]. However, attempts at using D-aspartic to improve the reproductive activity in animals of commercial interest have yielded mixed results. The higher concentration of D-aspartic acid in seminal plasma might impede the sperm cryotolerance ability. NAG is synthesized from acetyl-CoA and glutamate by N-acetyl glutamate kinase, which catalyzes the key regulatory step in the pathway to arginine biosynthesis. Moreover, in mammals, NAG is an allosteric catalyst of carbamoyl phosphate synthase-I (CPS-I) [30]. Carbamoyl phosphate and ornithine are catalyzed by CPS-I to produce citrulline [31]. Therefore, the NAG is the essential co-factor of CPS1 in the urea cycle [32]. Mammalian NAG is found in the mitochondrial matrix of cells in the liver and intestines [33]. The lower NAG level might result from the low enzyme activity of N-acetyl glutamate kinase. There was a study that reported the existence of ionotropic glutamate receptors and glutamate transporters in sperm [34]. The study also indicated that glutamate receptors and transporters might have functions other than neurotransmission in sperm [34]. The different level of NAG between the GFE and PFE groups implies that the amino acid biosynthesis is related to sperm freezability. Inosine is the main substance in the pathway of uric acid metabolism. Inosine has good permeability to the cell membrane and can directly enter the cell, convert it into nucleotides, and then further become ATP to participate in metabolism [35]. Exogenous inosine could accelerate the differentiation of rat intestinal epithelial cells [36]. However, there are only few studies reporting inosine in seminal plasma [37]. It seems that the inosine level was significantly higher in the seminal plasma of oligozoospermic and azoospermic than normozoospermic men [35]. Inosine in seminal plasma might activate pyruvate oxidases, increase the activity of coenzyme A, and stimulate metabolism in sperm. The negative correlation between inosine levels and sperm freezability indicates the role of nucleosides metabolism in sperm cold shock sensitivity. In the targeted metabolic analysis, some metabolites showed no significant differences between the two groups. However, they might also affect other functions of sperm. For example, 3-methylhistidine in urine has been shown to be correlated with protein catabolism in skeletal muscle [38]. It can be speculated that 3-methylhistidine in semen plasma can indicate the body’s metabolic state and sperm function. The functions of these metabolites in semen plasma and sperm need to be further analyzed in the future.In summary, for the first time, we found that there are significant differences in metabolomic profiles between GFE and PFE individuals. Furthermore, some candidate metabolites were confirmed by the targeted metabolic analysis. It can be inferred that one indicator alone may not be able to accurately evaluate and that multiple markers may be needed to predict sperm freezability. It will be meaningful to evaluate sperm freezability in combination with genome, proteome, metabolome, and epigenome data.5. ConclusionsThis study, for the first time, investigated the metabolome profile of boar seminal plasma with high and low freezability. Fifty metabolites show significant difference between the GFE and PFE groups. The carboxylic acids and derivatives, amino acid, peptides and analogues, organooxygen compounds, and fatty amides are the main components of these changed metabolites. Moreover, our results indicate that D-aspartic acid, NAG, and inosine might be the potential markers associated with freezability. Further studies would be required to investigate the mechanism underlying the relationship between metabolites and sperm freezability.

animals : an open access journal from mdpi

10.3390/ani11061670

PMC8228460

Tail biting in pigs is an expression of suboptimal animal welfare. The search for causes and solutions is difficult. Recent studies have shown that injuries to the tail occur with considerable frequency, even without the intervention of other pigs, and that the injuries are not confined to the tail but can also be present in ears, teats, claws, coronary bands, heels, soles, and other body parts. This review summarizes the existing findings on a new syndrome introduced as swine inflammation and necrosis syndrome (SINS). This paper will present clinical alterations and gather evidence to better understand the underlying mechanisms. It concludes by presenting methods to combat the syndrome through improving pig husbandry and feeding and by selecting less-susceptible breeding animals.

Tail biting is a prevalent and undesirable behaviour in pigs and a major source of significant reduction in well-being. However, focusing on biting considers only one part of the solution, because tail damage can be found with a high prevalence without any action by other pigs. The lesions are not limited to the tail but can also be found in the ears, heels, soles, claw coronary bands, teats, navel, vulva, and face. Environmental improvement alone often fails to overcome the problem. This review addresses a new inflammation and necrosis syndrome in swine (SINS). It shows the clinical signs and the frequencies of occurrence in different age groups. It compiles scientific evidence from clinical and histopathological studies in newborn piglets that argue for a primary endogenous aetiology of the disease. Bringing together the findings of a broad body of research, the possible mechanisms leading to the disease are identified and then discussed. This part will especially focus on microbe-associated molecular patterns in the circulation and their role in activating defence mechanisms and inflammation. Finally, the methods are identified to ameliorate the problem by optimizing husbandry and selecting a suitable breeding stock.

1. IntroductionInflammation and loss of tail integrity can seriously impair animal welfare in pigs (European Food Safety Authority (EFSA) [1,2]. One of the most studied causes is tail biting, a very prevalent undesirable behaviour that has particularly been identified in growing pigs [3,4,5,6,7].A number of external factors are well-accepted triggers for the problem, including: insufficient activity and boredom due to poor environmental enrichment and comfort; failure to satisfy natural behaviours; stress of any kind; low-quality air, food, and water; too much sunlight; excessive temperature; regrouping; excessive housing density; inappropriate pen structure, leading to confrontation between animals or the inability of all animals to feed simultaneously; disease; malnutrition; stimuli such as blood; and many more [7,8,9,10]. Internal factors such as aggressiveness, frustration, and genetic causes have also been identified as triggers for tail biting, and the multifactorial nature of the problem cannot be overcome by focusing on individual components [7,9]. Even with intensive use of the available measures, 25–70% of animals may have damaged tails (e.g., [11,12,13]). While earlier studies focused on tail-biting as a behavioural disorder attributable to the barren environment and housing conditions, a prevalence of tail biting between 14% and 20% was recorded in 2004 under extensive outdoor conditions, as is common practice in Switzerland [14].Tail lesions are of special interest, not only due to their severe direct impact on animal welfare but, also, because tail docking is still used as the major preventive measure in most countries [15]. Tail docking, however, further increases damage, pain, and animal welfare concerns, without completely eliminating the problem and leaving the underlying causes unresolved [16]. Despite rising demands to ban tail docking in the EU (EU directive 2008/120IEC), field observations and scientific studies have shown that discontinuing tail docking under current practical conditions can seriously increase the prevalence of tail lesions [12,17].Evidence from research and practice suggests that tail lesions might be caused not only by tail biting but, also, by inflammation and necrosis, which can occur without any action from other pigs [18,19,20,21,22,23,24,25,26]. In 50% of litters, up to 75% of piglets [19] can be affected. These lesions are also not limited to the tail but can be observed in the ears, heels and soles, claw coronary bands, teats, navel, vulva, and face [21,22,23,24,25,26]. Most affected piglets have lesions in more than one body part. The syndrome-like combination of different body parts and the clinical domination of inflammation and necrosis in these areas led to the term swine inflammation and necrosis syndrome (SINS; [22]).The simultaneous occurrence in such disparate body regions as tail, teats, claws and others [22,24]; the evidence that SINS can be triggered before birth, when biting and mechanical irritation (e.g., from the floor) are excluded [25]; and the histopathological evidence for vascular-associated inflammation in neonatal piglets with (still) intact epidermis [23,24] all argue for a primary endogenous cause of the syndrome.There is much to suggest that inflammation and lesions strongly affect animal welfare in swine, that they affect much more than just the tail of the animals and that tail biting and mechanical irritation due to technopathies alone are not sufficient to comprehensively address and combat the issue. The aim of this review is to summarize the current knowledge on SINS, to offer a hypothesis and mechanisms on the development of the syndrome and, derived from these, starting points to overcome the disease.2. Inflammation and Necrosis Syndrome in Swine (SINS)SINS is a newly identified, distinct syndrome resulting from the combined presence and signs of clinical inflammation and dead tissue in the acral areas. It particularly affects the tail base, tail tip, ears, coronary bands, heels, soles, claw walls, teats, navel, and face (Figure 1) and can be observed in suckling piglets, weaners, and finishing pigs [21,22,23,24,25].The signs generally start with a loss of bristles, followed by swelling and redness. At later stages, exudation and, finally, necrosis can be detected. Several studies found a loss of bristles in 30–90% of the piglets, mainly at the tail base and ears. Swelling and redness were reported at a slightly lower prevalence in the tail base, tail, and teats. Exudation and necrosis were rare in newborn piglets, with 0.7% of piglets being affected at the tail, ear, and teats [24], rising to 2–10% in three-day old piglets [23,25]. The number of affected individuals increased from suckling piglets to weaners and decreased again in fatteners but was still present at up to 6.8% (loss of bristles), 10.7% (swelling), and 10.7% (necrosis) in the tail, ear, and teats in fatteners. The claw walls, soles, and heels were also affected. Other clinical signs of the presence of local inflammation were vein combustion on the ears, teats, and veins of the hind limbs. The relationship between the total SINS score and the first appearance of signs in different body parts was shown by the authors of [25]. According to them, the first signs that can be found are a loss of bristles at the tail base and ears, redness of heels, swelling of tail base, tail, teats, and coronary band inflammation. Bleeding, exudation, necrosis, and ring tail at the tail tip were the clinical signs that developed last and only in severe cases of SINS. This differentiation is important for the early diagnosis of SINS [23]. However, the mild signs are easier to overlook.3. SINS Diagnostics and Differential DiagnosticsSINS signs are relatively easy to clinically diagnose. It is important to be aware of the meaning of the clinical signs and to clean the animals, e.g., in the area of the claws, in such a way that alterations can be detected. It is also important for scoring to be conducted by an experienced person and to expect variations between the results of different observers. However, no statements are available yet on the magnitude of the deviations that should be expected.In SINS scoring, the base of the tail, the rest of the tail, including the tail tip, the ears, the teats, the navel, the coronary bands, the claw wall, the soles, and heels are considered [21,22,23]. The face and vulva can also be recorded. To facilitate scoring with a high repeatability of results and low stress to the piglets, the body parts to be assessed should be photographed. The actual scoring can then be done based on the photos [24,25].Although it would be possible to score the individual body parts semi-quantitatively, binary scoring has proven to be effective so far. Here, an unaffected physiological state was scored with 0 and deviation with 1. This is simpler than semi-quantitative scoring and allows for a more comprehensible evaluation. Nevertheless, a good differentiation can be achieved by collecting the binary scores for all possible findings of the respective body parts (e.g., loss of bristles (0/1), swelling (0/1), reddening (0/1), rhagades (0/1), exudation (0/1), bleeding (0/1), necrosis (0/1), and ring-shaped constrictions (0/1)) for the tail (see below).The tail and tail base are usually scored separately, considering the loss of bristles, swelling, reddening, rhagades, exudation, bleeding, clinical signs of necrosis, and the occurrence of ring-shaped constrictions at the tail (Figure 1A–I (top)). If, for example, all these clinical signs could be observed in the tail base, the tail base score would be 8. Ear scoring is focused on the loss of bristles, congestion of ear veins, and clinical necrosis. Necrosis can include any part of the ears and, also, the ear tips (Figure 1A–D (middle top)). Teats are scored for scab formation, swelling, reddening, clinical signs of necrosis, and the congestion of blood vessels. The navel is scored for redness and swelling. The face is scored for oedema around the eyes and nasal oedema. Each claw is individually scored for wall bulging, wall bleeding, sole reddening, detachment of sole from heel, reddening of heel, heel cracks, heel bleeding, detachment of heel, redness of coronary band, exudation from the coronary band, and clinical signs of necrosis in the coronary band (Figure 1A–D (middle bottom and bottom)). The total claw score should then be divided by 8 to give a score comparable to that of the other body parts. Field experience shows relatively good agreement between the findings of different claws. However, detailed data on this subject are not yet available. Additionally, congestions of the inner thigh veins can be recorded.The different studies show some variation in the consideration of individual body parts and clinical variations. A precisely standardized procedure is not yet available. More studies are needed to define an optimal standard.The resulting binary scores can be presented by the organ system as a percentage of the affected piglets. All recorded binary scores can be summed to form an organ score for the individual piglet, and all organ scores can be summed to form the total SINS score. To ensure equal weighting of the organ scores, they can first be z-transformed and then added together. Again, exact standardization has not yet been performed, because possible weighting functions for the individual organ scores cannot yet be reasonably estimated. In addition, it is likely that optimal weighting functions will differ in different herds. In any case, the SINS score corresponds to the normal distribution [24,25].Lesions to tail, ears, skin, teats, and claws are generally highly prevalent in pig production systems [27,28,29]. Thus, other primary diseases must be considered differential diagnostically. This can be difficult under practical conditions, especially in older piglets and fatteners, where mixed forms of bites, mechanical irritation due to technopathies, infection, and SINS might occur [23]. Tail biting has particularly been identified in growing pigs [3,4,5,6,7]. Tail biting cannot be responsible for tail lesions in piglets at birth, and suckling piglets have never been reported to bite their tails and, especially, not the tail base, where lesions are common in SINS [21,22,23,24,25]. The literature does not describe how annular tail lesions could result from bites or technopathies, nor how technopathies could lead to inflammation and lesions in the tail base area. Studies that describe the occurrence of tail base lesions failed to discover any evidence of such external causes despite thorough monitoring of the piglets [21,22,23,24,25]. In addition, bites usually leave typical injuries that are easily diagnosable if the pigs are monitored and promptly bonitized.Inflammation and necrosis of the ears of pigs are also commonly found [30,31,32,33,34]. For example, Pejsak et al. [33] described an ear necrosis syndrome in weaners and fatteners and discussed the possible association with (sub-) clinical infections with Mycoplasma suis, as well as Staphylococcus hyicus and Streptococcus suis. Park et al. [32] associated ear necrosis with environmental factors and infections with Staphylococcus aureus and Staphylococcus hyicus. In these previous studies, it remained, however, unclear whether the pathogens were causative or secondary invaders. Papatsiros [31] associated ear necrosis (porcine necrotic ear syndrome) in weaners with Porcine Circovirus Type 2 (PCV-2) infections and recommended to intensify vaccination control. Earlier, Pringle et al. [30] associated necrotic ear lesions with Treponema socranskii. In contrast, Weissenbacher-Lang et al. [34] described a comparable syndrome, denoted PENS (porcine ear necrosis syndrome), in 5 to 10-week-old pigs. The authors investigated the prevalence of infectious agents in the herd, such as Streptococci, Staphylococci, and Mycoplasmata, but could not detect any of these agents in the affected animals. The authors proposed that other (non-infectious) causes, such as mycotoxin exposure or stress factors, should be considered as primary causes of PENS. lndeed, recent investigations showed that the exposure of pregnant sows to mycotoxin DON (deoxynivalenol) resulted in typical necrotic tail lesions even in neonatal piglets [35].Alopecia, swelling, coronary band injuries, and swelling and haemorrhaging into the claw corium were described in suckling piglets by Mouttotou et al. [36] and KilBride et al. [37]. The authors suppose that some of these lesions are associated with a reduction in suckling and active behaviours and a slower growth rate because of the pain associated with such injuries [38]. The authors attributed the alterations solely to mechanical irritation by the floor. Recently, however, clear evidence for an additional internal component in such lesions was provided by histopathological findings in piglets [23]. Even in newborn piglets [24], 20.5%, 65.1%, 76%, and 82.9% of the individuals were found with the swelling of heels, inflammation of coronary bands, redness of heels, and wall bleeding, respectively, directly at birth. An ongoing study in these piglets is disclosing the histopathological findings in the claws and ears (Wenisch, pers. communication) consistently with the findings at the tail base [24].Several results provide evidence that the expression of individual clinical signs of SINS is clearly modified by environmental effects. Pigs with SINS react more sensitively to unfavourable barn floor conditions than those with healthy claws due to the primary load in the area of the heels, soles, and claws. Ears seem to be particularly affected by SINS in pigs with insufficient ability to regulate their body temperature [21]. This explains why the signs of SINS can vary significantly depending on the existing environmental factors in different herds and why the correlations between organ scores can be relatively low [26]. However, in a cohort of 646 piglets, none was completely free from signs of SINS, and of the seven body parts examined, including the tail base, tail tip, face, ears, teats, navel, and claws, 3.8 ± 1.07 of body parts (mean ± SD) were affected simultaneously in an individual [25]. In this study, forty percent of piglets were affected in at least five of the seven body parts.4. SINS as an Endogenous DiseaseThree main observations support the assumption that SINS is primarily an endogenous disease, even though it may be modified by technopathies and other mechanical stressors: (1) The simultaneous occurrence in such disparate body parts as the tail, teats, claws [22,24,25]; (2) evidence that SINS can be expressed before birth [24]; (3) evidence that inflammation originating from blood vessels can be present before birth when biting and mechanical irritation (e.g., from soil) are excluded and in piglets with (still) intact epidermis [23,24].Clinical signs at the tail, claws, and ears were confirmed histopathologically. Vasculitis, thrombosis, intimal proliferation, oedema, and hyperaemia were detected together with intact epidermis [23]. Bristle loss is associated with inflammatory processes in the deeper parts of hair follicles [23,24]. Significant proportions of neonates may be affected. In the study by Kuehling et al. [24] on a conventional farm, 40–80% of neonatal piglets were affected by haemorrhages of the claw wall, coronal inflammation, redness of heels, bristle loss, and redness of the tail and ears. The inflammation could be characterized by granulocytes in considerable numbers, macrophages, and lymphocytes, indicating an onset of inflammation at least 4 days before birth [39], while the piglets were not older than 2 h. Thus, SINS must be assumed to have developed in utero.Inflammation and necrosis have often been considered to result exclusively from biting and mechanical irritation. However, the above studies demonstrated that these cannot be the sole causes. Similar conclusions were already reached by Penny et al. [18]. The authors suspected that inflammations and necroses were the result of circulatory disturbances, which were triggered by vasoconstriction and aggravated by further circumstances. This hypothesis was confirmed by the above-mentioned findings of vasculitis, intimal proliferation, and thrombus formation at the base of the tail, directly cranial to the clinical lesions with inflammation and necrosis [24]. A second corroboration was the demonstration of a sharp drop in temperature from the affected base of the tail to the tip of the tail in piglets with SINS using an infrared thermography device [21]. In addition, several studies showed that inflammation of the tail tip (outside of biting events) is always associated with alterations at the tail base, while piglets with intact tail bases never show signs at the tail tip [23,24].5. Hypothesis and Background of the Pathogenesis of SINSBased on the histopathological findings and the data from infrared thermography, compiled in the previous paragraph, Reiner et al. [23], Ringseis et al. [26], and others suggested that SINS must be attributed to the local inflammatory processes in association with blood vessels. According to Van Limbergen et al. [35], necrotic tail lesions in newborn piglets can be associated to the exposure of the sow to deoxynivalenol (DON). Additionally, the necrosis of tails, ears, and coronary bands in suckling piglets might be directly associated with mycotoxins and LPS (lipopolysaccharides) from sows’ milk [34,35,40,41,42,43]. To date, no accurate dose-response studies are available that allow precise quantification of the relationship between ingested mycotoxin and LPS levels and clinical consequences in piglets and pigs of different ages and against a background of different mixing ratios and cofactors. Nevertheless, among others, mycotoxins and LPS could be directly or indirectly implicated in causing the inflammation of blood vessels in affected body regions. The following section collects findings that support this hypothesis and, thus, represent a possible starting point to explain the pathogenesis of SINS (Figure 2).5.1. Microbe-Associated Molecular Patterns (MAMPs) from the IntestineOne of the most conclusive papers in this regard is that of Nordgreen et al. [44]. The authors suggested that problems in the microbiota, gut barrier, housing environment and hygiene, immune activation, mycotoxins, psychological stress, nutritional status, and feed composition can synergistically lead to inflammation directly or indirectly in association with LPS.A major source of LPS and other microbial components with similar effects, collectively called microbe-associated molecular patterns (MAMPs), is the intestine (for a review, see reference [35]). Under physiological conditions, endotoxins always flood from the intestinal tract to the liver, where they are inactivated by Kupffer cells [45], whose clearance capacity can normally eliminate the endotoxins completely. Abnormally high levels of degradation products from the gut are observed in the presence of increased microbial proliferation, intestinal disease, a high protein-to-crude fibre ratio, and disruption of the blood–intestinal barrier [46]. They may also result from coprostasis that occurs as a consequence of fever, overfeeding, or an excessively high ambient temperature [47,48,49,50,51]. By overcoming the blood–intestinal barrier, MAMPs can reach the circulatory system. The blood–intestinal barrier is complex, oxygen-dependent, and sensitive to disruption. In pigs, damage to the blood–intestinal barrier directly results in increased LPS influx [47,48]. The blood–intestinal barrier in pigs is particularly susceptible to heat stress [49,50,51] and reduced gut perfusion with relative water deficiency and increased water requirements for thermoregulation [50,51] when contact cooling fails on dry concrete or plastic floors [52]. Mycotoxins (DON and similar substances) disintegrate the tight junctions of the blood–intestinal barrier and increase the LPS uptake in pigs [53,54]. They also directly cause intestinal and liver inflammation in pigs and develop synergistic effects with LPS [53,54,55,56,57]. By disrupting the blood–intestinal barrier, mycotoxins and LPS potentiate their mutual uptake.Microbiota dysbiosis and intestinal barrier impairment are also associated with a number of chronic inflammatory disorders and systemic diseases in humans (for review, see references [58,59], and the pathogenic involvement of endotoxins is well-described [60,61,62].5.2. MAMPs and the LiverMAMPs are translocated from the gut to the liver via the portal vein. Since the liver acts as a barrier to degrade MAMPs due to the activity of macrophages (Kupffer cells) and other intrahepatic immune cells [63,64,65,66], the liver largely prevents MAMPs from entering the systemic circulation. However, in increased intestinal microbial proliferation, or if the intestinal barrier is disrupted (“leaky”), the liver is confronted with high concentrations of MAMPs. In this case, MAMPs are recognized not only by hepatic immune cells but, also, by hepatic parenchymal cells, which are abundantly equipped with specific MAMP recognition receptors, such as Toll-like receptors (TLR). Upon the recognition of MAMPs by TLRs, various inflammatory and stress signalling pathways, such as nuclear factor-kappa B (NF-κB), are activated. In addition, the c-JUN N-terminal kinase (JNK) and endoplasmic reticulum (ER) stress-induced unfolded protein response (UPR) are stimulated. This results in liver inflammation and impaired organ functionality [67]. First, the evidence for the induction of the inflammatory processes in the liver of piglets with SINS was provided by Ringseis et al. [26]. Proinflammatory genes and genes involved in the stress response, e.g., TNF, HP, ICAM1, SOD1, and CRP, were induced in piglets with SINS. This induction was accompanied by broad alterations of the metabolic pathways [26]. As a result of transcriptomic and metabolomic examinations, the metabolic processes dealing with lipid and fatty acid metabolism were shown to be particularly involved. This could be explained by the crosstalk between inflammatory signalling (NF-κB, UPR) and hepatic lipid metabolic pathways in inflammatory liver diseases [68,69,70,71].5.3. Further Sources for MAMPs and Elevated CytokinesPathogens, particularly of the digestive tract and respiratory tract, are common sources of MAMPs and frequent causes of elevated cytokine levels in pigs (reviewed in [44]). Poor hygiene, dust, LPS, and high levels of ammonia in the air are regularly present in pig houses and lead to activation of the inflammatory cascade via the respiratory tract [72,73,74,75]. Pigs are exposed to massive stressors, including high housing density, lack of pen structure, regrouping, lack of opportunities to eat at the same time, disease, poor housing air, etc. [10,76,77]. A number of studies demonstrate that cytokine activation comparable to that elicited by MAMPs is induced in humans [78], rodents [79], and, also, in pigs [80,81,82,83].5.4. Mode of Action of MAMPs, e.g., LPSThe pathogenicity of endotoxins in animals arises primarily from the release of endogenous mediators and is, thus, secondary [84]. The goal of this mechanism is to contain, control, and eliminate the bacteria. However, the excessive or systemic activation of mediators by endotoxins can result in severe disease and possibly even death as a consequence of the mediator effect. Target cells for endotoxins are neutrophils, macrophages, and platelets. Major mediators secreted in response to endotoxins include tumour necrosis factor-alpha (TNF-α), interleukin 1β (IL-1β), IL-6, and IL-8. This mode of activation was also confirmed in pigs as the trigger for post-partum dysgalactia syndrome (PPDS) [85,86].IL-1 activates the vascular endothelia and lymphocytes. This leads to improved access for effector cells but, also, to tissue destruction. It also promotes the production of IL-6 and the development of a fever. IL-6 increases fever and stimulates the production of acute-phase proteins. The latter include, for example, C-reactive protein. They bind to bacteria and opsonize them or trigger a complement reaction. Elevated levels of acute-phase proteins were found in piglets with SINS [87]. IL-8 represents a chemotactic factor for leukocytes and improves the access of effector cells to the damaged tissue. TNF-α is the main trigger of inflammation (pain, redness, swelling, and increased temperature at the site of infection). In was elevated in the liver tissue from piglets with SINS [26]. Redness and warmth set in due to the increased vessel diameters, and swelling occurs due to increased permeability. The histopathological findings in piglets with SINS include hyperaemia and oedema [24]. Thus, there is an overall increase in the influx of immunoglobulins and complement proteins, as well as immune cells, and lymphatic drainage is promoted. Through the induction of adhesion molecules on the endothelia, monocytes and granulocytes are slowed down, docked, and infiltrated into the tissue (extravasation). The induction runs via the expression of selectin. These changes also result in increased blood clotting; small vessels are relocated, and pathogens are prevented from spreading further in the organism. Thrombosis in the tail base of piglets with SINS was found in the histopathological study by Kuehling et al. [24]. These essential properties of TNF-α to limit local inflammation can have disastrous consequences once an infection becomes systemic or LPS enters the blood in larger quantities. Systemic vasodilatation can then lead to shock and disseminated intravascular coagulation (DIC). In addition to the cytokines already mentioned, biogenic amines (e.g., histamine and serotonin), oxygen radicals (NO and H2O2), and, mediated by cyclooxygenase-2 (COX-2) activity, various arachidonic acid derivatives, especially PGE2 but, also, PGF2α, PGI2, and thromboxanes, are released [88].The release of mediators occurs through activated defence cells (macrophages, Kupffer cells, etc.). Pathogens or their components invading via the intestine, urogenital tract, or fissures or wounds of the claws or skin are recognized by their MAMPs via specific receptors called Toll-like receptors (TLRs) in reference to the corresponding Toll receptors in Drosophila. LPS is primarily recognized by TLR-4 in several species [89,90]. To activate the TLRs, LPS must first bind to a lipopolysaccharide-binding protein (LBP). This complex, in turn, binds to CD14 receptors on the membrane of macrophages and is then coupled to TLR4 in an MD-2-dependent manner. The formation of this receptor complex activates a series of factors that introduce the signal that has arrived at the cell membrane into the cell and transfers it to the nucleus (overview in reference [91]). The final step is the formation of transcription factors—in particular, NF-κB, which starts their expression by binding in the promoter region of specific genes involved in inflammation, immune communication, and immunomodulation [46,92].5.5. MAMPs and Consequences to the PeripheryThe cytokines now expressed exert their effects locally in the tissues of the gut and liver. The binding of PGE2 to prostaglandin receptors controls the cyclo-AMP-mediated function of the ion channels of smooth muscle and peripheral and central neurons, thereby modulating their activity. This leads to a reduction in tone and slackening of smooth muscle of blood vessels and the intestine. A drop in blood pressure or stasis are possible consequences [93]. The permeability of the blood vessels is increased. Prostaglandins have an activating and potentiating effect on macrophages and the immune system. Increased pain sensitivity develops at the nerve endings of pain fibres, and afferent fibres of the vagus nerve are irritated. IL-1 and TNF-α act synergistically in this process. Excessive production of the mediators results in the following symptoms: general depression, fever, impaired motility of the smooth muscles of the intestine (up to stasis) and the urogenital tract, and leukopenia, followed by leucocytosis, metabolic disturbances, and disturbances of the circulatory system up to shock. In this context, the aforementioned symptomatology can be reproduced by endotoxins but, also, by TNF-α alone [88,94].5.6. Central Effects of MAMPsThrough cytokines via the blood pathway [95] and through prostaglandins (PG) and cytokines via the vagus nerve [96], there is a reporting of inflammatory processes to the central nervous system [97,98]. This can lead to systemic reactions such as depression, anorexia, and fever. The flooding of LPS from the intestinal tract leads to activation of the complement system. Subsequently, C5 receptors on Kupffer cells in the liver are activated. Their stimulation induces the expression of cyclooxygenase-2 (COX-2) and, thus, the rapid formation of PGE2. PGE2 travels via the blood pathway to the preoptic anterior hypothalamus (POA), where it triggers a fever via prostaglandin receptors.PGE2 action can also be directed to the POA by the stimulation of vagus terminals in the liver via the ventral noradrenergic bundle. The vagus signals are received by the medulla oblongata and neuronally transmitted to various brain centres. Activation of corresponding centres of the area praeoptica by noradrenaline causes an induction of COX-2 there and, thus, also PGE2-mediated initiation of a fever. Cytokines from the bloodstream can reach specific cytokine receptors in the area of the circumventricular organs, which do not have a blood–brain barrier. Here, they again trigger the formation of PGE2, which is the actual stimulus for the central effects. PG receptors in the paraventricular nucleus activate the hypothalamic–pituitary axis, those in the area praeoptica activate the fever centre, while disease-specific sensations and behaviours arise in the limbic system. Within minutes after the recognition of bacterial components in the organism by macrophages, fever is formed [99,100]. The purpose of fever is to promote the immune system and to inhibit numerous pathogens. The latter is realized both directly by the high temperature and by indirect effects, such as the lowering of various metal plasma levels required by bacteria at high temperatures. However, with increasing temperature, there are also more and more disruptions of important endogenous functions.Disease sensations and disease-specific behaviour (sickness behaviour) eventually develop through the activation of PG receptors in the limbic system, with specific actions on ion channels [101]. The disease-specific sensation and behaviour that develop are not directly due to the fever but arise in parallel. These are highly organized behavioural changes after defence/immune stimulation, which are essential for dealing with pathogens, for example, in the sense of reserve conservation. The most important symptoms are dullness, reduced participation in the environment, secretion, sensitivity to pain, anorexia, and adipsia [88]. In this way, inflammatory processes are associated with mental illness in humans and rodents (reviewed in reference [44]). There is much to suggest that tail biting in pigs may also be influenced in this manner [44].6. Genetic Effects6.1. Effects of the BoarPractical experience from pig farms with uniform sow base regularly shows evidence of boar or boar line effects on progeny SINS scores. Demonstrating a genetic basis of SINS would be an important milestone in combating the syndrome, as husbandry improvement measures, often insufficient on their own, could be supported by targeted selection of less sensitive boars. It would make control more effective and sustainable. This background led to a study with 19 boars (4 Duroc and 15 Pietrain boars) from 8 different breeding companies participating in the market in Germany [25]. The study was conducted on 39 sows, with each sow inseminated simultaneously with two different boars to increase the significance of the study. A total of 646 piglets were paternity tested and scored for signs of SINS on their third day of life. More than 70% of the piglets were affected at the tail base, ears, coronary bands, and heels. Bristle loss, swelling, redness, venous congestion, and claw wall bleeding occurred most frequently.Offspring from Duroc boars had significantly lower SINS scores (4.87 ± 0.44) than offspring from Pietrain boars (10.13 ± 0.12). Within the Pietrain breed, SINS scores of offspring were significantly affected by the boar. Replacing the Pietrain boars by Duroc boars resulted in a 59% reduction in the SINS scores of their offspring under the given husbandry conditions. The cumulative percentage of three-day-old piglets from Duroc boars affected by SINS signs at the tail base was 43.5%. The corresponding values for the average Pietrain progeny were 165%. The progeny of the best Pietrain boars and the poorest Pietrain boars had values of 92.1 and 199.6, respectively. Exudation and necrosis occurred only in progeny of average (4.4%) and poor Pietrain boars (20.1%), but not in progeny of Duroc boars and best Pietrain boars. The effects on teats were even more pronounced.Total SINS scores in the offspring of the best Pietrain boars were almost 40% lower than that of offspring in the poorest Pietrain boars. These findings confirm considerable genetic effects on the outcome of SINS under a given husbandry. The results clearly show that individual breeding companies had boars with both favourable and unfavourable distribution of SINS in their offspring. It has to be assumed that the expression of the SINS signs is significantly influenced by husbandry and feeding. It can be suspected that the absolute differences between boars might be weaker or stronger under more favourable or less favourable conditions.However, the decisive results of the study were the clear differentiation between Duroc and Pietrain boars and the pronounced variation within the Pietrain boars that were included in the study. Such findings are currently being taken seriously by numerous breeding companies in various countries. Further studies are needed to characterize the genetic background of these effects and to make them useful to combat the syndrome.6.2. Effects of the SowThe genetics of the sow can also have significant effects on the SINS scores of the offspring [22]. Based on 20,000 pigs on 19 farms, it was found that more than 60% of fatteners from one of four sow lines (sows from four different relevant commercial breeding lines) had inflamed tails, while only 20–30% of fatteners from the other lines were affected. The same effect was observed for inflammation of the ears (40% vs. 0–13%). The fact that the sow line whose progeny showed high prevalence of tail and ear inflammation at the same time showed the least signs of biting, underlines the syndrome character of SINS on the one hand and proves on the other hand that tail lesions due to SINS or due to biting have to be strictly distinguished from each other. Of course, genetic differences a priori cannot be expected between all lines and populations.7. Opportunities to Improve SINS by Environmental OptionsImproving water and raw fibre supplies is generally accepted to have a positive impact on intestinal health and the improvement of animal welfare in swine [7,102,103,104]. Insufficient water uptake is a major risk factor for the development of postpartum dysgalactia syndrome (PPDS), a widespread disease in postpartum sows that is also related with inflammation triggered by MAMPs [105]. The resulting constipation is accompanied by exuberant bacterial growth in the intestine and flooding of endotoxins (MAMPs) [106,107]. This can be worsened by a lack of fibre [108,109], with the addition of fibre being “probably the most-cited factor to reduce PPDS” [110]. Constipation (coprostasis) is a leading sign and cause of PPDS [111,112,113]. Bacterial colonization of the endometrium, the bladder and the mamma were identified as further sources for MAMPs in PPDS [111,112,113,114,115]. Further resources for MAMPs were detected in injuries and fissures in teats and claws as well as laminitis in the sows [107]. Thus, sows with intact teats, claws, and skin, and which are free from coprostasis should have a lower burden on their piglets to develop SINS.To test the role of water, fibre, and sow quality, including coprostasis, on the development of SINS in offspring, SINS scores for the offspring (120 suckling piglets, 120 weaners and 120 fatteners) from 40 sows were examined. Out of 123 sows, the 20 sows with the least alterations in teats, claws, and skin were contrasted to those 20 sows with the most severe alterations [23]. Sows were screened at day 50 of gestation. The offspring of 10 sows of good quality and 10 sows of poor quality were studied under standard housing conditions in a first run and under improved housing conditions in a second run, which included good crude fibre and sanitized water from open drinkers being continuously available to all ages. Coprostasis occurred exclusively under standard housing conditions, without improved water and fibre supply (R2 = 0.74), and more frequently in sows with poorer quality in skin, claws, and teats.SINS scores in suckling piglets, weaners, and finishers of low-quality sows under standard housing conditions were highest but decreased significantly when housing conditions were improved. Sow quality had direct significant effects on inflammation and necrosis of suckling piglets and weaners under standard housing conditions. Offspring from sows with coprostasis had significantly higher SINS scores at every age.Improved housing resulted in a 39, 56, and 81% decrease in SINS symptomatology in suckling piglets, weaners, and finishers, respectively. That the effects were detectable up to the fattening phase must be emphasized. The strongest effect of housing was the reduced incidence of coprostasis in sows. Thus, the husbandry effect could be split into a direct effect on the sows’ offspring and an indirect effect by provoking coprostasis in the sow. Husbandry improvements (fibre and water), sow quality, and coprostasis explained 57%, 67%, and 45% of the variance in SINS scores in suckling piglets, weaners, and finishers, respectively.In conclusion, the use of fibre, sanitized water from open drinkers, and sows with healthy claws, healthy teats, and intact skin can have positive effects on the prevalence of SINS in suckling piglets, weaners, and fatteners. Avoiding coprostasis in the sow seems to play a particularly important role [23].8. ConclusionsFocusing on biting is only one part of the solution to control tail lesions in swine and includes three major concerns: (1) Tail damage can be found without any action by other pigs and up to 75% of piglets can be affected. (2) The lesions are not limited to the tail. They can also be found in ears, heels, soles, claws, coronary bands, teats, navel, vulva, and face. (3) Environmental improvement alone often fails to overcome the problem.Recognizing a primary endogenous syndrome as the cause of clinically detectable inflammation and necrosis at the aforementioned body parts leads to the second part of the solution: SINS. The syndrome can be triggered before birth and can be detected with considerable prevalence in neonates, suckling piglets, weaners, and fattening pigs.The assumption that SINS is primarily an endogenous disease, even though it is modified by technopathies and other mechanical stressors, is supported by three findings: (1) The simultaneous occurrence of signs in disparate body parts such as the tail, teats, claws, and others; (2) the clinical expression of the syndrome before birth; and (3) pathohistological signs of vascular-associated inflammation with vasculitis and thrombosis, together with intact epidermis in newborn piglets, where biting and mechanical irritation (e.g., from the floor) can be excluded.The idea of underlying circulatory disorders is supported by clinical and pathohistological results. A huge number of published findings support the hypothesis that these disorders might be due to microbe-associated molecular patterns (MAMPs), particularly from the intestine, that activate the defence cascade. The role of feed composition, nutrition, mycotoxins, gut microbiota, gut barrier, housing environment, hygiene, immune activation, and even psychological stress has recently been elaborated in detail. The resulting expectation of liver inflammation with massive switch of metabolism from anabolism to acute phase and inflammation was demonstrated.Using sows with intact claws, mammaries, and skin and which are free from constipation can favourably and sustainably influence inflammation and necrosis in their offspring, from suckling piglets to finishing pigs. The use of crude fibre and sanitized water from open drinkers across all ages resulted in a further massive decrease in SINS signs at every age. Significant differences between offspring of Duroc and Pietrain boars and offspring of different Pietrain boars suggest the possibility of a sustainable improvement of SINS by breeding.

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