Reproductive Physiology and Management

Research Program

Robert Dailey, Jorge Flores, Keith Inskeep, Marlon Knights, Paul

Lewis and Matthew Wilson

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

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

Support from industry and other sourcesPharmacia Animal Health Nat. Inst. Occup. Safety & Hlth.InterAg Division of DEC International The Ohio State UniversityVetrepharm Dept. of Pathology, W V UElysian Fields Farm (PA) WV Sheep FederationUniversity of Michigan Colorado State UniversityARS 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 Wyoming20 W Va Sheep Producers Antonio Lopez S. - Spain

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.

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

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 .

Induction & Synchronization of EstrusAnestrous 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.

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

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)

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.

Late Embryonic & Fetal Mortality

Studies in Ewes :

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5

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% E

we

s

1st service

2nd service

Anestrous Ewes That Experienced Pregnancy Loss From Day 25 to

Lambing

1998 1999_________________________________________________

Synchronization Studies - Knights

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

Determining Late Embryonic and Fetal Lossin Ewes

Effect of Face Color On Embryo or Fetal Loss During Several Stages of

Gestation

a, b,c Columns with different letters are different (P < 0.05)

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% E

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RY

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BLACK WHITE MOTTLED

DAY 25 TO TERM DAY 45 TO TERM DAY 85 TO TERM

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Ewes Losing Embryos Present at Day 25 of Gestation By Type of Loss

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25 45 65 85 105 125 TERM

DAY OF GESTATION

% O

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Multiple Partial

Single Complete

Multiple Complete

Corpora Lutea (Ovulations) Not Represented By Lambs Born in Ewes Treated With Three Dosages of FSH

Knights et al., 2002

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FSH (mg)

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Effect of Mastitis on Reproductive Efficiency

Mastitis

Bacteria

Cell Wall Components (peptidoglycan, lipopolysaccharide)

Inflammatory Mediators

Conception/

Pregnancy

Failure

Mastitis and Reproduction in Dairy cowsAmanda Barker – Tennessee data

Mastitis Caused:

– Increased Number of Services

– Increased Interbreeding Interval

– Increased Days Open

– Increased Calving Interval

PEPTIDOGLYCAN

Cell Wall of Gram-Negative and Gram-Positive Bacteria

Hurlbert, 1999

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

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

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.

Primary objective was to determine if immunization prevented:

1. fever at bacterial challenge

2. early embryonic loss, and3. mastitis in ewes.

Five days after breeding, sheep were injected s.c. with PTG.

Effect of Peptidoglycan on Reproductive Efficiency and Mastitis in Sheep (Holásková)

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.

Results