Reproductive Physiology and Management Research Program

1. Reproductive Physiology and Management Research Program Robert Dailey, Jorge Flores, Keith Inskeep, Marlon Knights, Paul Lewis and Matthew Wilson

2. Projects/Support Hatch 427/NE1007 - Ovarian and Environmental Influences on Embryonic/Fetal Mortality in Ruminants 10/1/02-9/30/07 State 149 (Animal Health Funds) - Effect of Peptidoglycan-Polysaccharide (PG) Complex of Streptococcal Cell Wall on Reproductive Efficiency and Mastitis in Sheep, 10/1/00 – 9/30/02 - Dailey State 170 (Animal Health Funds) – Pattern of Cytokines in Uterine and Vaginal Tissue and Secretions during Discrete Stages of the Estrous Cycle of Sheep, 10/1/02 – 9/30/04 - Dailey

3. Projects/Support con’t. State 140 (WV Legislature/WV Department of Education)- WV Sheep Improvement Project – Increasing Farm Income by a Strategic Shift in Breeding Time for Ewe Flocks, 7/1/98 – 6/31/07 Lewis and Inskeep NRSP-7 – Effectiveness of Intravaginal Progesterone Inserts… and FSH … Ewes, 1998-2003 - Inskeep, Lewis and Knights USDA-NRI 2001 -35203-10982 Investigation of the Benefits of Early Gestational IGF-I in Sheep, 9/1/01-8/31/04 – Wilson

4. Support from industry and other sources Pharmacia Animal Health Nat. Inst. Occup. Safety & Hlth. InterAg Division of DEC International The Ohio State University Vetrepharm Dept. of Pathology, W V U Elysian Fields Farm (PA) WV Sheep Federation University of Michigan Colorado State University ARS Forage Unit, Beaver, W V Impres Medical Corp. Intervet Dept. of Immunology, W V U Dept. of Biology, W V U Jeff Lawson Farm (VA) USDA Sheep Exp. Station, DuBois ID Dale Evans Farm (MD) Allen Pysell Farm (MD) Richard Blamble Farm (MD) Michigan State University University of Wyoming 20 W Va Sheep Producers Antonio Lopez S. - Spain

5. Philosophical Approaches Conduct basic research into factors that limit reproductive efficiency and management of sheep in West Virginia and the Appalachian Region. Conduct applied research to test concepts generated by basic research, to enable wide application of new concepts, and to identify other problems deserving basic studies. Collaborate to maximize understanding and output.

6. Research Areas Regulation of Luteal Function Induction & Synchronization of Estrus Early Embryonic Mortality Late Embryonic and Fetal Mortality - Progesterone Metabolism Mastitis and Reproduction Embryonic and Postnatal Growth Regulation of Follicular Dynamics

7. Regulation of Luteal Function Current and Proposed Studies: Changes in gene expression of the prostaglandin synthesis and endothelin systems, and of prostaglandin dehydrogenase activity in corpora lutea. Signal transduction mechanisms mediating actions of endothelin and prostaglandin in luteal cells: - Changes in concentration of intracellular Ca++ - Role of protein kinase C isozymes, especially .

8. Induction & Synchronization of Estrus Anestrous Ewes (Knights) Three studies evaluated the CIDR-G (to deliver progesterone) and single injections of FSH for out-of-season breeding. One goal was to maximize conception rates. The second goal was to increase ovulation rate and litter size (to 2.0). (1392 ewes) Conclusions: Progesterone increased pregnancy rates to two services from 45% with ram introduction alone to 62%. Treatment for 5 days was as effective as treatment for 12 days. FSH marginally increased ovulation rate, but did not consistently increase litter size. These data have been reported to USDA-NRSP7 for submission to FDA for approval of the CIDR-G for anestrous ewes. Three papers published.

9. Induction & Synchronization of Estrus Ewes during the breeding season(Knights and Dixon) Three studies were conducted with CIDR-G to synchronize estrus during the breeding season. Treatment with 2 CIDR-G for 12 days yielded pregnancy rate to first service and prolificacy of 90% and 1.9 vs 79% and 2.0, respectively, (ns) in control ewes. 401 ewes Treatment with CIDR-G for 5 days with PGF2α 24 h before insert removal increased pregnancy rate to first service (63%) compared to ewes that received PGF2α only (44%; P < .05). 164 ewes Treatment with CIDR-G for 5 days with PGF2α at insert removal increased pregnancy rate to first and second services (71 and 72% P < .05) compared to ewes that received PGF2α only (58 and 61%; P<.01). 1014 ewes

10. Breeding Anestrous Ewes Using Ram Effect and GnRH Ram is a powerful tool to induce ovulation in anestrous ewes. - It can be enhanced by injection of progesterone at ram introduction. - Estrus can be synchronized with subsequent injection of PGF2α (day 12-16), but pregnancy rates to two services usually do not exceed 70%. Objectives: Will more ewes ovulate if ram introduction is followed by injection of GnRH? Will GnRH enhance pregnancy rates in response to the ram effect and PGF2α? In two trials to date, GnRH has not increased estrous response or pregnancy rate.(Jordan, Wurst and Knights)

11. Induction & Synchronization of Estrus Ewe lambs during anestrus (Knights and Baptiste) Fall born ewe lambs were treated in July. Groups were: Control Used CIDR-G device (progesterone) Used CIDR-G device and ram introduction Used CIDR-G device, ram introduction and 25 g EB 24 h after CIDR withdrawal Ram introduction alone increased LH pulse frequency, follicular development and ovulation. Progesterone alone did not. Progesterone and EB combined with ram introduction increased estrous response.

12. Late Embryonic & Fetal Mortality Studies in Ewes :

13. Anestrous Ewes That Experienced Pregnancy Loss From Day 25 to Lambing _________________________________________________ 1998 1999 Synchronization Studies - Knights

14. Determining Late Embryonic and Fetal Loss in Ewes Summer 2000 and 2001 (N = 1400)Breeding - May to September 1. Embryos / Fetuses on each day of pregnancy diagnosis 2. Lambs at term d25 45 65 85 Term Loss Loss Loss Loss

15. Effect of Face Color On Embryo or Fetal Loss During Several Stages of Gestation a, b,cColumns with different letters are different (P < 0.05)

16. Ewes Losing Embryos Present at Day 25 of Gestation By Type of Loss Multiple Partial Single Complete Multiple Complete

17. Corpora Lutea (Ovulations) Not Represented By Lambs Born in Ewes Treated With Three Dosages of FSH Knights et al., 2002

18. Effect of Mastitis on Reproductive Efficiency Bacteria Conception/ Pregnancy Failure Mastitis Inflammatory Mediators Cell Wall Components (peptidoglycan, lipopolysaccharide)

19. Mastitis and Reproduction in Dairy cowsAmanda Barker – Tennessee data Mastitis Caused: • Increased Number of Services • Increased Interbreeding Interval • Increased Days Open • Increased Calving Interval

20. PEPTIDOGLYCAN Cell Wall of Gram-Negative and Gram-Positive Bacteria Hurlbert, 1999

21. Sheep Model for Effects of Mastitis on Reproduction (Amanda Stewart) Rationale: • Bacteria promote inflammation and infection after the host digests the bacterial cell wall • Major component of cell wall is peptidoglycan (PTG) • Majority of embryonic loss in cows is by days 6- 8 Method: Inject PTG in ewes on day 5 post-breeding Results: - Induced transient fever - Reduce progesterone - Increased cortisol - Increased prostaglandin metabolite (PGFM) - Decreased pregnancy rate

22. Effect of Blocking Inflammatory Increase in PGF2a on Pregnancy Rate (Amanda Stewart) Rationale: PTG increased PGFM. Did increased PGF2 decrease pregnancy rate? Objective: • Does blocking PGF2 following PTG challenge result in increased embryo survival? • Are other inflammatory mediators involved? Treatments: 30 g PTG/kg body weight on day 5 plus saline (n = 10) PTG plus flunixin meglumine (fm) at 0, 6, and 12 h (n = 10) Saline plus fm (n = 4) Saline alone (n = 5) Jugular blood:Progesterone, Inflammatory mediators: TNF-, IFN-, TGF-, IL-1, IL-2

23. Results - Pregnancy Rates Peptidoglycan plus saline 60% Peptidoglycan plus FM 50% Saline plus FM 75% Saline 80% Conclusion: Increased PGF2a not solely responsible for pregnancy losses.

24. Effect of Peptidoglycan on Reproductive Efficiency and Mastitis in Sheep (Holásková) Primary objective was to determine if immunization prevented: 1. fever at bacterial challenge 2. early embryonic loss, and 3. mastitis in ewes. Five days after breeding, sheep were injected s.c. with PTG.

25. Results Immunization did not: prevent fever on day 5, enhance pregnancy maintenance, prevent mastitis after weaning. In fact, increased dosage of PG-PS lowered the probability of successful pregnancy. This result fits earlier finding by Coleman and Dailey that reproductive disorders occurred more commonly in cows that had previously experienced the disorder. Mastitis tended to occur in fewer immunized ewes.