Physiology Research Program

 To improve reproductive efficiency and facilitate the use of artificial insemination (AI) and embryo transfer (ET) in beef cattle.

Major Activities Underway:

  Estrus synchronization, AI and ET research is being conducted using Angus cows in the Virginia Tech Research Herd at the University's Kentland Farm. The herd consists of registered (80) and commercial (40) Angus cows. All cows and replacement heifers are used in estrus synchronization research and registered cows are bred exclusively using artificial insemination. The commercial cow herd serves as recipients for an ET program. Testing heat detection methods, evaluating estrus synchronization and re-synchronization methods, and comparing methods of freezing and transferring bovine embryos are the current research emphases. The VT Research herd is also engaged in a 3-yr study to evaluate alternative methods of controlling bovine leukosis virus (BLV).

Progress (past 5 years):

Research conducted during the past 5 years has demonstrated the importance of controlling development of the ovulatory follicle in addition to controlling the lifespan of the corpus luteum in order to achieve high conception rates following estrus synchronization and AI. Ultrasonographic monitoring of follicles developing under prolonged periods exogenous progestin administration has documented that ovulation of a "persistent" follicle lowers fertility. This information has been utilized to modify existing estrus synchronization programs in an effort to improve the synchrony of estrus and fertility of cows bred following estrus synchronization.

 Efforts to improve the synchronization of estrus and ovulation in ET recipient cows have been successful. Methods for synchronizing recipients for use in ET without heat detection and a reliable method for re-synchronization and re-use of recipients that fail to become pregnant has been demonstrated.  Other research involving ET has revealed the importance of embryo placement in achieving acceptable pregnancy rates.

Cooperative Work:

  Research on ET has been conducted in conjunction with Dr. Randall Hinshaw of Ashby Herd Health, Harrisonburg, VA. Work with Dr. Hinshaw, a private practitioner, has been performed at Kentland Farm and on the private farms in Virginia that have cooperated in our research.

 The research on BLV is being conducted in cooperation with Dr. Kevin Pelzer and other faculty in the Virginia-Maryland Regional College of Veterinary Medicine.

 As a faculty member in a commodity department, I have major responsibilities in the areas of teaching and research.  Both my research and teaching responsibilities complement one another.  The research has both basic and applied components, with the ultimate goal of improving animal production efficiency.  The goal has been to involve students at the graduate, undergraduate and high school level in research.  Finally, the research tends to be collaborative recognizing that research questions are generally multidisciplinary, and involve the input and expertise of more than one person.

Major Activities Underway:

 My research is directed towards three areas.  The first is investigating the neurochemical regulation of food intake in poultry.  The second involved a collaborative effort with the late   Dr. Kornegay in APSC and Dr. Grabau in Plant Pathology and Weed Science investigating methods to decrease phosphorus excretion in both turkeys and chickens.  The final area is the role of dietary lipids in the maternal diet and their influence on embryonic development.  It appears that the lipid composition of the maternal diet affects embryonic mortality and bone development.

Progress (past 5 years):

 While difficult to ascertain, this is probably best indicated on the vita accompanying this report.  The various publications, theses, and dissertations indicate our progress.  Briefly, in the area of neurochemical regulation of food intake, studies have shown the CRF, histamine, glutamate, and leptin act within the brain of chickens to inhibit food intake.  While glutamate appears to stimulate food intake when injected into the brain of mammals, the opposite appears to happen in birds.  In the area of phosphorus waste, an extensive series of experiments have shown that phytase, when added to the diet of either chickens or turkeys, is very effective at liberating phytate phosphorus in the diet.  Since phytate phosphorus is liberated, it is possible to formulate diets with decrease amounts of inorganic phosphorus and thus decrease the amount of phosphorus passing through the animal as non-absorbed phosphorus.  Furthermore, phytate can be fed as a diet supplement, or it is equally efficacious when provided in the form of transformed seeds such as soybean seeds containing a foreign gene allowing for the synthesis of phytase.  Regarding the role of dietary lipids in the maternal diet, inclusion of Menhadden fish oil compared to chicken fat or soybean oil into the diet of broiler breeders results in increased embryonic mortality.  In addition, the bones of chicks from hens fed fish oil appear to be shorter and weaker than those from hens fed the other lipids.

Cooperative Work:

 My research has always involved considerable cooperative activity.  I currently have cooperative efforts with faculty members on campus including J. Wilson, K. Webb, H. Veit, E. Grabau, and J. Lee.  I also have projects with researchers at USDA in Beltsville,
Dr. Furuse in Japan, and Dr. Flory at Hollins College.  Collaborations are essential as the scope of our projects expand, and the need for more sophisticated techniques increases.

Future Plans:

I plan to continue in my current role and remain active in teaching, research, and advising.  Each of these roles is essential to the overall mission of the department, and they clearly collaborate one another.  Regarding areas of research, I will continue to study food intake regulation, and we are beginning a series of studies to determine if the lipid composition of the diet of breeder males affects semen characteristics and fertility.

The general goal of current research projects is to optimize production of human therapeutic pharmaceutical products utilizing swine as animal bioreactors.  Inherent within this general goal are the specific goals of examining ways to optimize transgene expression and production of transgenic swine which express human therapeutic blood clotting proteins (Factor VIII, Factor IX, fibrinogen, von Willebrand’s factor) and enhancing fundamental understanding of the relationship between transgene production, regulation, and expression.  A long range goal of the transgenic research is to develop pigs that are efficient, safe, cost-effective systems for obtaining the complex human therapeutic proteins needed to maintain hemostasis of pro- and anti-clotting factors.  There is currently a billion dollar U.S. market for these proteins. The goal of a second project is to examine methods to optimize production of porcine relaxin and to explore its effects on up-regulating the expression of collagenases that remodel extra-cellular matrix components of connective tissue.  Since remodeling of connective tissue is critical in the treatment of several diseases (such as scleroderma, fibromyalgia, and glaucoma), there is potential for utilizing porcine relaxin produced by a pseudopregnant, superovulated porcine model that we are studying for these therapies.

Major Activities Underway:

 Several current projects are on-going in support of the goals described above.  1) Groups of gilts which have been estrus synchronized, superovulated, and inseminated are flushed for collection of embryos, the embryos microinjected with the various genes of interest, and the microinjected embryos transferred to recipients.  Several recipient gilts are pregnant from these transfers of the “new” gene constructs under investigation.  2) Transgenic gilts (expressing either Factor VIII, Factor IX, or fibrinogen) produced from earlier gene constructs are lactating and colostrum and milk are being collected from them for laboratory purification and analysis of the protein expressed.  We are also examining methods of handling and storing the milk following collection in order to optimize stability of the proteins.  3) One trial has been completed and a second one begun to compare the quantity of relaxin harvested at day 100 of pseudopregnancy from the corpora lutea of control gilts compared with superovulated gilts.  4) Various tissues are being investigated for the presence of relaxin receptors as a preliminary to utilizing relaxin therapeutically.  5) We are also investigating new ways to purify relaxin, employing expanded bed chromatography and immunoaffinity chromatography methods.

Progress (past 5 years):

In brief, we 1) have evaluated various systems for collection of porcine zygotes for DNA microinjection and transfer and for culture of microinjected embryos, and refined those procedures to optimize efficiency within our research program, 2) have generated transgenic pigs expressing recombinant human protein C (rhPC, an anti-clotting factor), Factor VIII, Factor IX, and fibrinogen, 3) have obtained information on the rate limitations of post-translational modification (PTM) of rhPC by incrementally increasing expression levels by using different whey acidic acid (WAP) promoters and genomic verses cDNA hPC gene constructs, 4) have used immunoaffinity and subsequent ion exchange chromatography to isolate a variety of post-translationally mature and biologically active populations from partially PTM and low activity rhPC populations, 5) have discovered inefficiencies in proteolytic processing and ?-carboxylation at high levels (>0.1 g/l) of expression and demonstrated differences in the glycosylation of rhPC compared to hPC, 6) examined genotypic and phenotypic stability of four lines of transgenic swine expressing rhPC in their milk and found that single chain content was not dependent on expression level and was consistent within each transgene line but varied among lines; therefore suggesting that native swine genetics may play a role in selection of production herds with optimal PTM proteolytic processing capability, 7) found that the presence of consistently high levels of rhPC may slightly alter the mammary gland to increase transcytosis of plasma proteins and reduce the length of lactation, hence high expression levels may not always be optimal,
8) determined that hormonal induction of lactogenesis in transgenic virgin gilts expressing recombinant human fibrinogen could successfully identify those pigs likely to express recombinant human therapeutic proteins during a natural lactation, thus providing a means for early identification of transgenic gilts to be selected for use in large-scale pharmaceutical production, 9) have extensively characterized that effects of machine milking of sows on milk yield, litter weights, and milk composition, 10) have verified the efficacy of utilizing superovulated, pseudopregnant gilts as a model for production and harvesting of porcine relaxin for possible human therapeutic applications.

Cooperative Work:

I am the supervisor of the Swine Biotechnology Unit.  All of the above described work involving transgenic swine represents the cooperative efforts of several scientists, graduate students, and support personnel working within our multi-disciplinary Transgenic Animal Research Group.  This group includes Virginia Tech personnel in the departments of Animal and Poultry Sciences, Chemical Engineering, Dairy Science, and the College of Veterinary Medicine.  We also work collaboratively with personnel of the American Red Cross (ARC) in Rockville, MD.  The ARC has been our major financial sponsor for most of our research projects.  I also work cooperatively with personnel in Chemical Engineering and the College of Veterinary Medicine (a somewhat different group compared with those involved in the transgenic research) on the studies involving relaxin.  In addition, Dr. Allen Harper, located at the Tidewater Agricultural Research and Extension Center, and I are in the process of completing a project (not discussed previously) examining the effects on conceptus development and reproductive performance in sows of supplementing diets with an organic source of folacin.

Future Plans:

Future plans are focused on continuing research studies that will facilitate the accomplishment of the goals delineated above relative to 1) the utilization of transgenic swine as animal bioreactors for the production of human therapeutic proteins; and 2) on examining porcine relaxin as a possible product for human diseases that involve remodeling of the extra-cellular matrix and connective tissue.

 The overall objective for my research program is to find new ways of improving or augmenting immunity or disease resistance to enteric pathogens in commercial poultry.  Inherent in this objective is the goal of improved efficiency and profitability of poultry production through improved bird health and productivity and improved product safety with regard to foodborne pathogens.  I feel important elements of these objectives include the education of graduate students, the introduction of undergraduate students to science, and the dissemination of research findings to the scientific and poultry producing communities.

Major Activities Underway:

 Research currently being conducted towards these goals is multi-focused.  A major portion of my research is to identify and characterize the responsiveness of the immune cells in the gastrointestinal tract of chickens to enteric pathogens and determine the specificity and role of these responses in the development of complete or partial immunity.  I am particularly interested in the potential involvement of two somewhat enigmatic cells of the avian immune system, mast cells and eosinophils, in early neonatal immune responses.  The second focus of my research is modulation of these immune responses for improved disease resistance and vaccination.  Several options being investigated include a dietary additive derived from peppers and timing of vaccination.

 Progress:

 In the initial stages of these projects, studies have identified the responsiveness of two distinct immune system cell types, mast cells and eosinophils, to Salmonella enteritidis or Eimeria tenella infections in neonatal chickens.  The influx of these cells into the lamina propria of the distal ileum and ceca apparently contribute to an inflammatory response.  As mast cells and eosinophils are largely overlooked for involvement in mechanisms of immunity in chickens, these observations are individually significant in suggesting these cells play a larger role in gut immunity to enteric pathogens in the chicken.  In a recently accepted publication, we described the measurement of correlates of intestinal anaphylaxis, or secretory responses to pathogens, in chickens immunized against Eimeria species, which based on mammalian studies, would correlate with the presence of mast cells.  These studies have resulted in several research collaborative arrangements as well as invited presentations at scientific meetings nationally and internationally.

 More recently, we have demonstrated a heightened response of mast cells and eosinophils to Salmonella enteritidis or Eimeria tenella in the ceca of broilers during the second week of neonatal life, as compared to the first week of life.  This suggests a potential development associated effect of the host response of the chicken to these enteric pathogens and could have important implications on the timing of vaccination.

 Initial investigations with dietary administration of capsaicin, the extractable pungent component of peppers, in poultry feed have demonstrated increased resistance to Salmonella in birds consuming the treated feed.  I have just established a collaboration with several scientists who have been investigating rodent repellent characteristics of this compound.  We have planned studies to apply the disease resistant effects and rodent repellent effects of the compound for application in commercial poultry feed for decreased feed loss from rodent consumption and decreased exposure to and resistance to diseases, particularly Salmonella, carried by the rodents for poultry.

Cooperative Work:

 This research has lead, and continues to lead, to several very productive and beneficial collaborations.  I currently have a specific cooperative agreement ($10,000) in place with Dr. Harry Danforth of the USDA/ARS Livestock and Poultry Sciences Institute, Parasite Biology and Epidemilogy Laboratory in Beltsville, Maryland for three years.  Paperwork to initialize additional funding ($13,000) from Dr. Danforth is currently being prepared in his agency.
Dr. David Caldwell (Department of Poultry Science, Texas A&M University, College Station, Texas) is also involved integrally involved in this project and contributes through the conducting of experiments in his facilities that we do not have the capability to conduct at Virginia Tech.  Dr. Caldwell has finished one M.S. student on a portion of this project and currently has 2 other M.S. students involved in our studies.  I have received 3 invitations to international meetings (Mexico) as a result of these projects and a project is being planned with Dr. Guillermo Tellez at the Universidad Nacional Autonoma de Mexico.  I have submitted a grant in collaboration with Dr. Joseph Dunn (President and Director of Research, Snyder Seed Corp., Buffalo, NY.) and Dr. Dick Austic (Cornell University, Department of Animal Science) for the nutritional and dietary additive investigations of immune response modulation and disease resistance.  This collaboration has just recently been initiated and we plan on submitting two additional grant proposals in the next month.  I am also working with Dr. Paul Siegel in the department to investigate the genetic aspect of the immune response to these pathogens by using his genetic lines of birds.

Future Plans:

  My major focus of research will continue to be on finding new ways to improve or augment disease resistance and immunity in poultry.  The projects currently underway, which I have described above, have just been planned and initiated in the last 2 years, so there is a vast amount of research that waits to be performed.  I will have a M.S. student beginning in the fall (2000) and at least one undergraduate with a research project that will each undertake a portion of these studies for their research.  I plan to continue conducting research that can improve bird health and productivity for commercial producers while at the same time answer basic questions concerning the immunity of the bird.