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United States Department of Agriculture

Agricultural Research Service

Research Project: BIOPHOTONICS - THE APPLICATION OF NOVEL IMAGING METHODOLOGIES TO LIVESTOCK PRODUCTION RESEARCH
2010 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
Specific accomplishments from 2009-2010 include advancements in molecular approaches to bio-marker intensity for enhanced detection of novel end-points and the beginnings of a transition from in vitro systems that optimize our imaging systems to in vivo systems of practical importance and proof-of-concept testing in production-based models. New models have been developed and tested ex vivo for monitoring bacterial pathogens in the mammary gland and reproductive tract (uterus) of the bovine, and are shifting to laparoscopic approaches for infusing and/or monitoring bacterial pathogens under development for technology transfer to in vivo bovine research models (reproductive tract and mammary gland). We are also exploring new laboratory animal models to aid in the development of biophotonic paradigms and speed technology development for livestock-based applications. Studies are underway to develop an intrafollicular ovarian microinjection method to transfect the 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. Studies are also using biophotonic gene regulation modalities for studying dietary restriction influences on gene regulation as related to the developing fetus and ovarian function, as well as development of transgenic (photonic) embryos for constitutive expression of target genes using biophotonic detection systems. 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 livestock species in the production environment. In summary, accomplishments under the Biophotonics Initiative as applied to agricultural livestock, and some peripheral applications for model development, 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.


4.Accomplishments
1. Elucidating Oocyte and Embryo Markers. Relaxin secretions are found in the vicinity of oocytes and embryos, and exert its effects through membrane receptors RXFP1 and RXFP2) which have not yet been described in porcine embryos. We have determined the presence of RXFP1 and RXFP2 receptors in porcine gametes and embryo, and evaluated the developmental effects of porcine relaxin. Collectively we found that the addition of porcine relaxin to embryo cultures significantly increased the mean cell number within blastocysts in all experiments, and concluded that the pig embryo expresses RXFP1 and RXFP2 receptors which may facilitate a role for relaxin during oocyte maturation and embryo development in the pig. We will utilize this relationship to investigate biomarkers for embryo development and ligand-receptor interactions as monitored by biophotonic imaging of tagged reagents.

2. Developing New Biophotonic Models. Quantitative bioluminescence imaging of porcine antral follicles in vitro Determining the relationships between optimal imaging time, plasmid DNA dose and luciferase expression for quantitative bioluminescence imaging is critical to the development of new biophotonic paradigms. We have analyzed the time course of luminescent signal emitted from transfected porcine whole follicle units following cationic lipid transfection with increasing doses of plasmid DNA (pGL4) encoding a luciferase reporter gene. The signal intensity reached a peak at 1 to 1.5 min after the injection and declined gradually afterward. The luciferase expression level of follicles in the 3 µg DNA group tended to be greater than the 1 µg group, but did not differ from the 2 µg group. Overall, a higher level of luciferase expression was observed in follicles transfected with 3 µg of pGL4, with an optimal time for quantification at 1.5 min after luciferin injection. These data are the first to demonstrate luciferase detection and quantification, indicative of transcription, within whole antral follicle cultures in vitro. These optimizations will facilitate additional study of the ovarian follicle in vitro, with future studies aimed at in vivo transfection and imaging to study follicular dynamics in situ.

3. Improving Biophotonic Paradigms. Acquiring highly stable photonic plasmids in our transformed bacterial models for biophotonic bacterial tracking in vivo is critical to experimental paradigm development. We have studied plasmid differences in stability within transformed Salmonella Typhimurium (S. typh-lux) using three different plasmids and characterized their respective photonic properties. Photonic emissions were positively correlated with bacterial concentrations for the plasmids pAK1-lux, pCGLS-1 and pXEN-1. When stratified by high, medium and low density bacteria concentrations, photonic emissions for high density populations containing pAK1-lux, pCGLS-1 and pXEN-1 resulted in differences of photonic emissions across a range of bacterial concentrations among plasmids; i.e., plasmid stability and emission characteristics differed in a bacterial concentration-dependent manner. These data have implications for improving photonic detection using the most optimal constructs for transformation of bacterial models.

4. Applications of Thermography: Assessment of Thermal Signatures of Nose-clip Weaned Calves Using Digital Infrared Thermography. Anti-suckling nose-clips are devices used for two-stage, low-stress weaning of calves. Several different nose-clip types are available including an adjustable size or a “one-size fits all”, non-adjustable nose-clips. We used thermography to investigate whether muzzle temperature differences might exist between calves weaned by different methods, and found differences between the devices associated with inflammation along with effects on calf behavior. This suggests that thermography may be useful in evaluating effects of nose clips on nasal tissue as these “low-stress” weaning methods are further evaluated.

5. Applications of Thermography. Using Thermography to Monitor Energetics in Cormorants. Avian predators on aquaculture ponds have an impact on production operations. Understanding the energetics of these avian predators may further enhance our ability curtain seasonal predation, or at least understand the contribution of aquatic livestock to their diets. To this end, cormorants were captured in winter night-roost sites in the delta region of Mississippi for a bio-energetics study. These birds were transported to a captive research facility and individually housed in 1m x 2m x 1.5m metabolism cages. Each morning for 6 days, thermography was used to record daily eye, chest, back, and feet temperatures for each bird. Mean eye and foot temperatures were 24.9 C (0.4 SE) and 31.1 C (0.56 SE) respectively. These thermography data will be incorporated in an energetic model to refine the impact of cormorants to the aquaculture industry.

6. Applications of Thermography: Thermal Assessments of Temperament and Serum Biomarker Changes in Livestock Models. The effects of animal temperament on endotoxin (lipopolysaccharide; LPS)-induced changes in body temperature and the secretion of cortisol and epinephrine in cattle was investigated in Purebred Brahman bulls selected based on temperament score. Temperament differentially affected the response of the adrenal medulla, but not the adrenal cortex, to endotoxin challenge. These data suggest that temperament affects the degree of endocrine response to an endotoxin challenge. As temperament affects the endocrinologic (epinephrine) and physiologic (temperature) response elicited by an exogenous activator of the innate immune system, the cohesive immuno-endocrine mechanisms that prevent infection may be compromised during periods of eustress as well as periods of stress. The use of thermography is an important tool for monitoring temperature differences when studying behavior and temperament in cattle as it provides a real-time, non-invasive measure that requires no contact or association with the animal model at the time of measurement.


5.Significant Activities that Support Special Target Populations
Mississippi and other southern states have unique challenges related to their livestock production environments (e.g., heat stress in dairy cattle), disease, and pre-harvest food safety issues (e.g., Salmonella in swine). Unfortunately, many of our experimental models have not been able to take the next step toward solving longstanding problems in the livestock industries. Moreover, many of our new molecular and biotechnological initiatives have not addressed matters that can be translated back to the live animal in a production setting. To this end, there is a critical need for technological innovations that will permit production-based questions to be asked and answered in the context of the living animal.

In Mississippi, agriculture is the number one industry, with the poultry, catfish, dairy and meat animal (swine and beef) industries contributing upwards of $2 billion statewide. The costs associated with, for example, infections of the mammary gland in dairy cattle (mastitis results in a $2 billion loss to dairy producers nationwide), the spread of Salmonella in swine (livestock diseases cost our economy $17.5 billion dollars nationwide), or the causes of early embryonic mortality in beef cattle (a loss of $1.4 billion to cattle producers nationwide) are significant. Through this initiative we will develop new (likely patentable) means with which to monitor physiological processes in real-time in the living animal; which may off-set economic losses due to disease or production inefficiencies by the development of non-invasive early warning systems for application to real-world settings.


Review Publications
Burdick, N.C., Carroll, J.A., Hulbert, L.E., Dailey, J.W., Willard, S.T., Vann, R.C., Welsh Jr, T.H., Randel, R.D. 2010. Relationships Between Temperament and Transportation With Rectal Temperature and Serum Concentrations of Cortisol and Epinephrine in Bulls. Livestock Science. 129:166-172.

Curbelo, J., Moulton, K., Willard, S. 2009. Photonic Characteristics and Ex Vivo Imaging of Escherichia coli-Xen14 Within the Bovine Reproductive Tract. Theriogenology. 73:48-55.

Sykes, D.J., Couvillion, J.S., Martin, J.M., Althen, T.G., Rude, B.J., Crenshaw, M., Gerald, P., Ryan, P.L. 2010. Comparison of Ground Raw Soybean and Soybean Meal Diets on Carcass Traits of Gilts. Journal of Muscle Foods. 21:509-518.

Moulton, K., Ryan, P., Lay Jr, D.C., Willarad, S. 2009. Photonic Plasmid Stability of Transformed Salmonella Typhimurium: A Comparison of Three Unique Plasmids. BMC Microbiology. 9:152-159.

Ryan, P.L., Christiansen, D.L., Bagell, C.A., Vaala, W.E. 2009. Evaluation of Systemic Relaxin Blood Profiles in Horses as A Means of Assessing Placental Function in High-Risk Pregnancies and Responsiveness to Therapeutic Strategies. Annals of the New York Academy of Sciences. 1160:169-178.

Last Modified: 12/20/2014
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