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United States Department of Agriculture

Agricultural Research Service

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Research Project: Biophotonics - the Application of Novel Imaging Methodologies to Livestock Production Research

Location: Warmwater Aquaculture Research Unit

2011 Annual Report

1. Profiling the global proteome of the porcine ovarian follicle. The mammalian oocyte progressively acquires its maturation during its intra-follicular growth. This follicle contains the follicular fluid (FF) nourishing the oocyte and whose composition changes throughout folliculogenesis and significantly affects the developmental competence of the oocyte. Scientists at Mississippi State University performed a proteomic analysis of FF derived from three different follicle developmental stages (small, medium, and large non cystic). Approximately 2,000 individual proteins were found in each category of non-atretic FF. Each FF origin was characterized by several qualitative and quantitative differences. Data comparisons indicated proteins appearing specifically in one or another FF type, while approximately 1,300 proteins were found in all three FF. These proteins are suitable candidates for the study of oocyte and follicle maturation. Furthermore, we tested the developmental impacts of relaxin found in the follicular fluid on porcine oocyte and pre-implantation embryo. Relaxin had beneficial effect on oocyte nuclear maturation and total cell number of blastocysts, and affected gene expression of its own receptors in oocytes.

2. Elucidating spermatozoan markers that may influence developmental processes. Mammalian spermatozoa are highly specialized cells shaped to transport and deliver not only the paternal genome to the fertilized oocyte, but also other molecules. Although there are clear indications on mRNA delivery and roles during fertilization and early embryo development, our knowledge on protein movements are still undetermined. Scientists at Mississippi State University profiled the global proteome of frozen-thawed boar spermatozoa. Approximately 3,000 individual proteins were detected in frozen-thawed boar spermatozoa with 65% of total proteins fully annotated. To our knowledge, these data are the first reported in pig spermatozoa, and constitutes a large pool of proteins from which candidates could be selected for further studies aiming at defining their short (fertilization, first embryo cleavages) or long-term (embryo quality) developmental effects.

3. Quantitative bioluminescence imaging of functional estrogen receptor activity within intact porcine ovarian follicles in vitro. Activated estrogen receptors (ER) in response to estrogen bind to specific sequence called estrogen response elements (ERE) and induce transcription. The objective of these investigations were to evaluate whether the estrogen induced ER binding activity in granulosa cells of antral ovarian follicles is determined by bioluminescence imaging system, and how estrogen concentrations in follicular fluid affect the ER binding activity. Through this research, scientists at Mississippi State University have demonstrated the development of a new methodology for measuring functional and ligand activated estrogen receptors in intact porcine ovarian follicles in vitro using a bioluminescence molecular imaging system. This research will lead to a greater understanding of folliculogenesis in the context of the whole follicle, which may clarify ways to improve reproductive performance (e.g., greater follicle numbers may equal more eligible follicles for ovulation which may increase litter size).

4. Elucidating the relationship between L-arginine and mammalian reproduction. This research is evaluating how L-arginine (identified in other studies as beneficial to fertility and reproductive processes) may affect the developing vascular structures of the placenta and fetus, and how L-arginine may affect the uterus during the peri-implantation period. This project has already demonstrated that an important developmental gene, vascular endothelial growth factor receptor-2 (Vegfr2), can be monitored non-invasively in living fetoplacental tissues using bioluminescent imaging technology. This first report established the methodology for a subsequent study which evaluated the effect that dietary L-arginine supplementation has on mammalian reproduction and fetoplacental Vegfr2 gene expression using bioluminescent imaging. The next step in this research project is to further this knowledge by conducting in vitro cell culture experiments which will evaluate other potential pathways affected by L-arginine in mammalian reproductive tissues. Overall, it is expected that information gained from this project will aid in the development of strategies intended to improve reproductive performance in animals of agricultural importance as well as other mammalian species.

Review Publications
Greene, J.M., Dunaway, C.W., Bowers, S.D., Rude, B.J., Feugang, J.M., Ryan, P.L. 2011. Invivo monitoring of fetoplacental vegfr2 gene activity in a murine pregnancy model using a vegfr2 -luc reporter gene and bioluminescent imaging. Reproductive Biology and Endocrinology. 9:51.

Feugang, J.M., Rodriguez-Munoz, J.C., Willard, S.T., Bathgate, R.A., Ryan, P.L. 2011. Examination of relaxin and its receptors expression in pig gametes and embryos. Reproductive Biology and Endocrinology. 9(1):10.

Feugang, J.M., Greene, J.M., Willard, S.T., Ryan, P.L. 2011. In vitro effects of relaxin on gene expression in porcine cumulus ooxyte complexes and developing embryos. Reproductive Biology and Endocrinology. 9(1):15

Ryan, P.L. 2011. Horse species symposium pathogenic and reproductive dysfunction in hourses. Journal of Animal Science. 89(5):1538-1540.

Ryan, P.L., Christiansen, D.L., Hopper, R.M., Walters, F.K., Moulton, K., Curbelo, J., Greene, J.M., Willard, S.T. 2011. A noval approach to monitoring pathogen progression during uterine and placental infection in the mare using biophotonic imaging technology and lux modified bacteria. Journal of Animal Science. 89:1-11.

Borazjani, A., Weed, B., Patnaik, S., Feugang, J.M., Christiansen, D., Elder, S., Ryan, P.L., Liao, J. 2011. A comparative biomechanical analysis of term fetal membranes in human and domestic species. American Journal of Obstetrics and Gynecology. 204(5):365.e25-365.e36

Ryan, P.L., Greene, J.M., Christiansen, D., Hopper, R.M., Walters, F.K., Leblanc, M.M. 2010. Emerging diagnostic approaches for evaluation of fetal and pregnancy well-being in the mare. Society for Theriogenology Newsletter. 2(2):149-161.

Mochal, C.A., Miller, W.W., Colley, A.J., Linford, R.L., Ryan, P.L., Rashmir-Raven, A. 2010. Ocular findings in quarter horses with hereditary equine regional dermal asthenia. Journal of the American Veterinary Medical Association. 237(3):304-310.

Burdick, N.C., Carroll, J.A., Randel, R.D., Willard, S.T., Vann, R.C., Chase, C.C., Lawhon, S.D., Hulbert, L.E., Welsh, T.H. 2011. Influence of temperament and transportation on physiological and endocrinological parameters in bulls. Livestock Science. 10:1016.

Last Modified: 8/19/2014
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