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United States Department of Agriculture

Agricultural Research Service


Location: Warmwater Aquaculture Research Unit

2013 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:
New methodology was developed for measuring transgene expression in intact porcine ovarian follicles in vitro using a bioluminescence molecular imaging system. In addition Mississippi State University (MSU) scientists have developed a new bio-imaging tool for monitoring gamete interactions using nanoparticles. Thermography and remote locomotion monitoring devices proved instrumental in elucidating that gradual calf weaning methods had no major influences on behavior compared to continuous nursing behaviors. Thermal imaging methodologies were also used in cooperative studies of avian energetic models that impact catfish pond predation (pelicans and cormorants), heat stress applications in non-domestic species, and environment x genetic x temperament and stress interactions in beef cattle. Using photonic Escherichia. (E.) coli MSU scientists have been able to demonstrate that the larvae of the filth fly are capable of acquiring E. coli and transmitting E. coli when emerging from the pupae stage. MSU scientist has also demonstrated that the utilization of purge as a sampling protocol for detection of E. coli O157:H7 is currently not a feasible alternative to current USDA protocols. MSU scientist developed a pre-harvest tool for monitoring shedding patterns of E. coli O157:H7. After overcoming significant regulatory hurdles, MSU successfully initiated the in vivo imaging in the dairy cow to assess the progression of experimentally induced mastitis. These were the first to take biophotonic imaging to the field in the dairy cow, and for the monitoring of bacterial pathogenesis as it occurs. MSU scientist also successfully assessed antibiotic efficacy in real-time in vivo. These studies will establish a novel animal model for assessing therapies in cattle, as well as provide new avenues for research in mastitis. Lentiviral transfection methodologies were optimized and employed, and transgenic embryos were produced. The imaging systems and dual reporter technologies were effective, however embryo viability became a continuing problem. Therefore we shifted to a new system for working with transgene incorporation and monitoring in gametes using nanoparticles – this research has been highly successful and has established new directions for research in the bioimaging of reproductive processes and gamete interactions. MSU scientists have identified relationships between the hormone relaxin and its receptors associated with the oocyte and embryo which will be used in further studies as a biomarker for developmental progression. MSU scientists have further tested the developmental impacts of relaxin found in follicular fluid on porcine oocyte and pre-implantation embryos, and the role of relaxin in fertilization processes. MSU found that relaxin had beneficial effects on oocyte nuclear maturation and total cell number of blastocysts, and affected gene expression of its own receptors in oocytes.

4. Accomplishments
1. Elucidating spermatozoan markers that may influence developmental processes. Using porcine semen from predicted “good” and “bad” semen freezers (as determined by post-thaw viability), molecular markers are being identified to try and predict the freezability status of boars, as MSU have accomplished in stallions in the past, which would enhance the widespread application of cryopreservation in the swine industry.

2. Developing new biophotonic models. Quantitative Bioluminescence imaging of functional estrogen receptor (ER) activity within intact porcine ovarian follicles in vitro. The objective of these investigations were to evaluate whether the estrogen induced ER binding activity in granulosa cells of antral ovarian follicles could be determined by bioluminescence imaging system, and how estrogen concentrations in follicular fluid affect the ER binding activity and photonic detection. Through our research, MSU 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. In addition, through techniques aimed at DNA transfection in these systems, MSU have stumbled upon a new method to possibly stimulate occyte maturation; these results are being followed up on currently to assess the potential application of this technique to in vitro fertilization research, etc.

3. Developing new biophotonic models. Self-illuminating quantum dots are nanoparticles that are less than 100 nanometers in diameter. Their coating with the light-emitting protein Renilla luciferase forms complexes which have promising applications in in vivo imaging. These complexes can be further combined to specific tags such as antibodies or peptides for various in vitro studies. Especially in reproduction, these conjugates may contribute to a better comprehension of molecular events associated with fertilization and beyond. To this end, MSU are continuing to evaluate the ability of mammalian spermatozoa to harmlessly incorporate nanoparticles. MSU's research has expanded to comparative studies among animal classes and species to ascertain morphological and carrier-specific differences among spermatozoa types which may influence the use of these particles for gene/DNA delivery.

Review Publications
Sanchez-Rodriguez, H.L., Vann, R.C., Youngblood, R.C., Baravik-Munsell, E., Christiansen, D.L., Willard, S.T., Ryan, P.L. 2013. Evaluation of pulsatility index and diameter of the jugular vein and superficial body temperature as physiological indices of temperament in weaned beef calves: relationship with serum cortisol concentrations, rectal temperature, and sex. Reproductive Biology and Endocrinology. 151:228-237.

Greene, J.M., Feugang, J.M., Pfeiffer, K.E., Bowers, D., Ryan, P.L. 2013. L-arginine enhances cell proliferation and reduces apoptosis in human endometrial RL95-2 cells. Reproductive Biology and Endocrinology. 11:15-26.

Feugang, J.M., Youngblood, R.C., Greene, J.M., Fahad, A.S., Monroe, W.A., Willard, S.T., Ryan, P.L. 2013. Application of quantum dot nanoparticles for potential non-invasive bio-imaging of mammalian spermatozoa. Reproductive Biology and Endocrinology. 10:45-53.

Last Modified: 09/19/2017
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