Location: Warmwater Aquaculture Research Unit2011 Annual Report
1a. Objectives (from AD-416)
The objective of this research area is to develop novel imaging technologies aimed at confronting critical issues facing production animal agriculture by monitoring, in real-time, cellular and molecular processing in the context of the living organism. Specific research projects will cover a broad range of research in the cellular and molecular biological sciences, disease-environment interactions, animal-plant interfaces, and growth and developmental physiology with applications aimed at understanding physiological mechanisms with a specific emphasis on enhancing production performance in livestock.
1b. Approach (from AD-416)
As part of this initiative, novel technologies which utilize the photon (light), thermal signatures (heat), spectroscopy and fluorescence will be adapted to cellular- and molecular-based strategies to permit physiological processes to be monitored in a dynamic fashion at the levels of single, living cells to entire organisms in vivo. These non-invasive technologies (e.g., biophotonics, using light as a quantitative indicator beacon ofmolecular events) will enable the expression of genes, the invasiveness of bacteria, the breakdown of plant or dietary components, or hormone-receptor interactions to be visualized in living systems both in the laboratory and field, and under traditional livestock production environments. Faculty with expertise in functional imaging will interface with collaborating scientists working in the animal, plant and veterinary sciences to develop these novel systems aimed at addressing specific hypothesis-driven and production-based questions. Results from this initiative will not only develop new models to advance scientific progress in reproductive biology, food safety, disease, plant-animal interactions, and environmental physiology, but will also develop technological advancements that will address experimentally critical questions which heretofore have not been addressable in living systems. Finally, we will expand the use of biophotonic-based technologies to address physiological questions in animals with potential application to field-based monitoring systems.
3. Progress Report
Biophotonic imaging was advanced from in vitro to in vivo in highly technical models, most notably mastitis monitoring in dairy cattle. After overcoming significant regulatory hurdles, we successfully pioneered in vivo imaging in dairy cows in the field to assess the progression of experimentally induced mastitis and to monitor real-time bacterial pathogenesis. Additionally, molecular approaches in bio-marker identification, new uses of doppler sonography, and continued development of gamete and embryo systems for developmental monitoring were all significantly advanced. New laboratory animal models were initiated to speed development of biophotonic paradigms for livestock-based applications. New amphibian model paradigms permitted use larger egg and embryo systems to test feasibility of developmental monitoring. Another focus was a model of biophotonics to improve food safety of beef products through pre- and post-harvest intervention. Specifically, we demonstrated the larvae of the filth fly were capable of acquiring Escherichia coli and transmitting it when emerging from the pupae stage. Studies are underway to develop an intrafollicular ovarian microinjection method to transfect living follicles in vivo to monitor estrogen-regulated genes during the growth of follicles on the ovary, and we have successfully achieved intrafollicular transfection and gene detection in these systems. Biomarkers are also being identified for oocytes and embryos using proteomic approaches, protein-receptor studies and gene transcription-biophotonic paradigms. In collaboration with industry partners, we are using biophotonic modalities to study wound infection model systems and developing evidenced-based veterinary diagnostics to determine the best possible drug combinations (antibiotics and immuno-modulators) to treat mares presented with uterine infections during late gestation. These novel research directions may facilitate new clinical avenues for biophotonic-based research. Thermal imaging technologies are being applied to the monitoring of (1) reproductive function in livestock; (2) mammary physiology in dairy cattle; and (3) measures of thermal comfort (e.g., energetics) and/or stress-related responses among mammals and avians in the production or natural environment. We have cooperated in mammalian (bovine and equine) research to determine the relevance of thermography in providing meaningful end-points of importance to production parameters. One study investigated the use of gradual weaning methods on behavior of calves during the weaning process compared with calves that continued to nurse. Thermography and remote locomotion monitoring devices proved instrumental in demonstrating that gradual weaning methods had no major influences on behavior or physiological parameters compared to continuous nursing behaviors. In summary, our accomplishments are yielding new research tools which have the potential to develop translatable technologies for an enhanced understanding of physiological processes to improve agricultural livestock production, health and/or overall profitability.
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.
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.