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ARS Home Southeast Area Stoneville, Mississippi Warmwater Aquaculture Research Unit Research





- Waldbieser, Geoffrey - Geoff
- Evans, Jeffrey - Jeff
- 2023
- Publications
- Food Animal Production (101)

Research Project: Biophotonics - Emerging Imaging Technologies for Food Animal Research **Location:
                            Warmwater Aquaculture Research Unit** 2023 Annual Report **Objectives**
                                

1. Develop approaches for cellular and tissue biophotonic imaging using nanoparticles and/or photon emitting technologies.
1.A. Improve gamete and embryo quality using biophotonics and nanoparticles.
1.B. Develop animal model organoid systems related to reproductive and nutrient transferring tissues to better model in vivo physiological and biochemical properties.

2. Investigate paternal contributions and/or environmental variations in conceptus developmental and subsequent offspring survivability, health, and growth.
2.A. Evaluate the effects of seminal plasma uterine priming on post-natal calf growth and metabolism. 
2.B. Determine the effects of a compromised bovine vaginal microbiome at parturition on dam performance and neonatal outcomes. 
2.C. Mitochondria patterns in gametes and implications to embryo-fetal development.

3. Improve animal health using biophotonic sensors to monitor physiologic processes and disease resurgence, and develop nanoparticles as an alternative to antibiotic use.
3.A. Livestock semen quality improvement.
3.B. Bioluminescence monitoring of intravaginal drug delivery to reduce incidence of calving difficulty in cattle.
3.C. Photonic imaging for rapid diagnosis of disease.
3.D. Photonic imaging for improved reproductive efficiency. **Approach**
                                

The project will improve gamete and embryo quality by characterizing the molecular events governing oogenesis and folliculogenesis in mammals using new in vivo and in vitro approaches. Animal model organoid systems related to reproductive and nutrient transferring tissues will be developed to better model in vivo physiological and biochemical properties. The project will evaluate the effects of seminal plasma uterine priming on post-natal calf growth and metabolism. We will determine the effects of a compromised bovine vaginal microbiome at parturition on dam performance and neonatal outcomes. We will identify investigate maternal and paternal mitochondrial patterns in gametes and implications to embryo-fetal development. The project will utilize novel compounds as antibiotic alternatives to improve livestock semen quality. Bioluminescence monitoring of intravaginal drug delivery will be used to reduce the incidence of calving difficulty in cattle. Finally, photonic imaging such as near-infrared spectroscopy will be used for rapid diagnosis of disease and improvement of reproductive efficiency. **Progress Report**
                                

In recent years, we developed a unique approach to perform ovarian intrafollicular injection of bio-substances in living mares, followed by aspiration of follicular fluid (FF) and biopsy of follicular fragments (FWB) for ex-situ research. Within Sub-Objective 1A, this approach was used for two primary goals: conduct intrafollicular interventions to improve female fertility and understanding of Extracellular Vesicles in ovarian follicle growth and seasonal variations. This preliminary test was achieved with intrafollicular injection of red fluorescent liposomes (in living mares) to visualize binding to follicular cells and non-fluorescent liposome-encapsulated doxorubicin (in cultured porcine ovarian follicles), to enable visualization following binding to cell nuclei. After 24h of leaving mares at the farm, aspirated FF and FWB were examined under fluorescence microscopes (Biophotonics). Injected follicles revealed fluorescent FF and follicle cell membranes (granulosa and theca cells). Similar results were observed with porcine samples, and doxorubicin fluorescence appeared within the nuclei of granulosa and theca cells. Together, these results indicated the possibility of, in situ, influencing folliculogenesis and oogenesis in living animals that may tremendously impact assisted reproduction outcomes of females.

Within Sub-Objective 1B, we examined various organoid culture preparations for bovine mammary glands. Briefly, bovine mammary tissue was harvested from the udder of an Angus heifer at slaughter. Following dissection, washing, and digestion tissue fragments underwent successful culture in a Matrigel matrix with organoid medium. Organoid media was changed every 2 to 3 days and organoids were expanded between days 7 and 10. Growth and confluency were evaluated by hour using a Cytosmart live cell imaging.

Under Sub-Objective 2A, we sought to determine the effects of seminal plasma uterine priming on postnatal body weights, carcass ultrasound measures, and indices of body composition. To realize these objectives, embryo-recipient cows randomly received 0.5mL of pooled seminal plasma or no treatment (control) at estrus and underwent embryo transfer seven days later. Indices of body composition, carcass ultrasounds and bioelectrical impedance analysis (BIA), were collected pre-weaning at an average 209 days of age. Weaning weights and hip heights were collected at an average of 212 days of age. A treatment by day interaction was not observed for pre-weaning body weight. However, a main effect of treatment was observed for pre-weaning body weights, which was decreased in seminal plasma calves compared with controls. Weaning weights and hip height were not different between treatments. Interestingly, previously reported data from this project show offspring from seminal plasma treated cows had decreased embryo size at days 35, 40, and 45 of gestation, and decreased birth weights. It appears seminal plasma uterine priming prior to embryo transfer negatively impacts offspring growth pre-weaning, however, by weaning this difference is diminished. It is less clear why pre-weaning weights differ due to similar body composition among treatment and control calves; however, these measures may have been collected after the period of compensatory gain occurred.

Under Sub-Objective 2B, we aimed to elucidate the microbial community composition of the bovine dam vaginal and calf nasal microbiota post-partum after utilizing betadine lavages (BL). The dam vaginal and calf nasal microbial communities and immune responses were evaluated at 0-, 15-, 30- and 60-day post-partum. Microbiota composition of the dam haircoat, udder, and IgG in the colostrum/calf sera were also evaluated at day 0. Serial BL prior to parturition did not alter the alpha diversity of the dam-vaginal microbiota but did alter the calf-nasal microbiota at parturition. Dams receiving BL prior to calving had increased colostrum IgG concentrations compared to CON dams. These results suggest physiological insults (BL) prior to parturition led to an increased immune response which altering dam colostrum IgG. Thus, neonatal colostrum consumption could drive immune responses against inoculating bacteria resulting in differing nasal microbial communities between treatment groups. The beta diversity of the calf nasal microbiota was significantly different at day 0 compared to all other timepoints. The calf nasal beta diversity at day 15 was similar to day 30 but significantly different compared to day 60. There was no effect of time on altering the alpha or beta diversity of the dam vaginal microbiota. The calf nasal microbiota was different from the dam vaginal microbiota at all timepoints post-partum, regardless of treatment. At day 15, the alpha and beta diversity of calves was altered compared to day 0, suggestive of a reinoculation timepoint between 0 and 14 days of age. Together, this data contributes to the paucity within beef cattle dam-calf microbiota literature and provides directionality for future research objectives within this field.

Within Sub-Objective 3A, we started isolating extracellular vesicles (EVs) from bio-fluids for further characterization and use as nanoparticle cargo carriers to improve livestock gamete quality in the last two years. In this preliminary approach, we extracted EVs from raw semen harvested from commercial fertile boars and equine follicular fluids to conduct comparative studies to understand their biological relevance for further enhancement of gametes' quality. Preliminary results indicate specificities of EVs and contents (e.g., miRNAs and proteins) that may positively impact livestock fertility through gamete quality improvement. 
Within Sub-Objective 3B: Dystocia contributes to increased risk of cow and calf respiratory and digestive disorders, mastitis, metritis, and retained placentae leading to significant financial losses to producers. Dystocia, or difficult calving, caused by feto-pelvic disproportion is most observed in first-time calving heifers and is dependent on fetal birthweight and maternal pelvic size. MSU scientists collaborated with a colleague at Emory University in Atlanta, GA (Dr Santangelo Philip) to construct a synthetic mRNA emitting light (H2 relaxin-Nanoluciferase mRNA) that enables visualization of protein expression. This construct possesses a secretion signal permitting transfection to eukaryotic cells. The preliminary approach consisted of in vitro transfection of bovine vaginal epithelial cells and cervical mucosa and in vivo transfection of Holstein cows by aerosolizing the vagina/cervical wall with the H2 mRNA construct. Light emission (bioluminescence) detection in exposed in vitro and in vivo cells and tissues, respectively, indicated translating the incorporated mRNA construct into luciferase and relaxin proteins. Both light emission and relaxin expression were detected in cell lysates (cell lines, vaginal and cervical cells, and uterine tissues), cell culture supernatants, and cervical-vaginal secretions. This pioneering study provides evidence to support the potential use of H2 relaxin mRNA therapy as a novel approach to reducing the incidence of dystocia in heifers.
For Sub-Objective 3C, the overall goal is to develop spectroscopic based technologies that can be performed in situ, and in vivo bio-imaging of biological processes in whole animal systems and in real-time. Near Infrared (NIR) spectroscopic profiles for biological systems of interest (biofluids, tissues, excretions) can map stress or pathogen induced physiological responses and the degree of physiological change when calibrated and validated. We have three major studies in Biophotonics disease detection under Sub-Objective 3C. The first is development of rapid in-situ analysis of bovine respiratory disease (BRD) caused by either bacterial or viral pathogenic insult in cattle and subsequent development of predictive modeling of disease presence using Near Infrared spectroscopy and metabolomics based NMR. The first trial focused on Bovine Syncytial Respiratory Virus and was conducted in April and May utilizing 14 male calves over a 12 day period of sample collection. Biological fluid samples were obtained along with clinical parameters, ultrasound images and trans-tracheal washes were collected to test for viral load. Fluid samples are being analyzed for spectral signatures along with biochemical markers to delineate unique viral disease profiles and generate prediction models. Prediction models for each biofluid using PCA-LDA to compare Accuracy, Sensitivity, and Specificity for breath condensate (78.5%, 70%, 85%) and nasal secretions (78.6%, 72%, 86%) collected from animals infected with BRSV when directly compared to non-infected control. We are also using Near Infrared spectroscopy to develop photonic signatures and predictive modeling for fungal and bacterial and fungal diseases in catfish species used in aquaculture. To this end, we have developed means for spectral collection in live catfish to discriminate species with predictive modeling across a multi-model approach that utilizes machine learning algorithms. Once methodology is established for non-mutable traits such as species and sex, more dynamic and continuous traits, such as disease and reproductive state, can be examined in live animals. **Accomplishments**
                                

1. Slick-haired Holstein cattle physiology during lactation. Through cooperative agreement with an ARS researcher in Stoneville, Mississippi, Mississippi State University, and University of Puerto-Rico, Mayaguez, researchers examined thermotolerance capabilities, blood metabolites, and mammary gland physiology of Puerto Rican slick-haired Holstein cattle during lactation. Heat stress threatens dairy cattle production putting at risk food security, farmers’ income, and animal welfare. The identification of traits in thermotolerant animals will allow producers to optimize selection of replacement animals that are better adapted to adverse environmental conditions. For this study we provide a thorough account of Puerto Rican slick-haired Holstein cattle exposed to heat stress conditions. Slick-haired Holsteins showed longer solar radiation exposure associated with longer grazing time, while maintaining a lower body temperature and increased blood supply to the mammary gland which translated into greater milk production compared with their wild type-haired counterparts. Lastly, we identified multiple biomarkers for milk production and mammary blood flow that were increased in the slick-haired Holstein, which provides broader impacts for the environment, farmers, and the scientific community. **Review Publications**
                            *Baloch, A., Feugang, J.M., Rodriguez-Osorio, N. 2023. Genomic and epigenomic applications in animal and veterinary sciences. Frontiers in Veterinary Science. 10. Article 1167079. https://doi.org/10.3389/fvets.2023.1167079.*
                                *Dlamini, N.H., Nguyen, T., Gad, A., Tesfaye, D., Liao, S.F., Willard, S.T., Ryan, P.L., Feugang, J.M. 2023. Characterization of extracellular vesicle-coupled miRNA profiles in seminal plasma of boars with divergent semen quality status. International Journal of Molecular Sciences. 24(4):3194. https://doi.org/10.3390/ijms24043194.*
                                *Gebremedhn, S., Gad, A., Ishak, G.M., Menjivar, N.G., Gastal, M.O., Feugang, J.M., Prochazka, R., Tesfaye, D., Gastal, E.L. 2023. Dynamics of extracellular vesicle-coupled microRNAs in equine follicular fluid associated with follicle selection and ovulation. Molecular Human Reproduction. 29(4). Article gaad009. https://doi.org/10.1093/molehr/gaad009.*
                                *Ishak, G.M., Feugang, J.M., Pechanova, O., Pechan, T., Peterson, D.G., Willard, S.T., Ryan, P.L., Gastal, E.L. 2023. Follicular-fluid proteomics during equine follicle development. Molecular Reproduction and Development. 89(7):298-311. https://doi.org/10.1002/mrd.23622.*
                                *Feugang, J.M., Ishak, G.M., Eggert, M.W., Arnold, R.D., Rivers, O.S., Willard, S.T., Ryan, P.L., Gastal, E.L. 2022. Intrafollicular injection of nanomolecules for advancing knowledge on folliculogenesis in livestock. Theriogenology. 192:132-140. https://doi.org/10.1016/j.theriogenology.2022.08.032.*


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