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W112: Reproductive Performance in Domestic Ruminants

Annual/Termination Reports (SAES-422): [07/15/2002] [08/28/2003] [08/17/2005] [08/18/2006]

Date of Annual Report: 07/15/2002

Report Information:
  • Annual Meeting Dates: 05/30/02 to 05/31/02
  • Period the Report Covers: 06/2001 to 05/2002

    • Participants:
    Brief Summary of Minutes of Annual Meeting:
    The meeting was called to order at 8 am on Thursday, May 30, 2002 by Chair Tim Rozell (KS). Meeting participants introduced themselves and 2 new members, Andrea Cupp (NE) and Derek McLean (WA), were welcomed. Colin Kaltenbach (AZ), administrative advisor, explained the new electronic format for the annual report which is due within 60 days of the meeting. Due to the limited character field available for the minutes, the group decided that the annual report should include a brief summary of the minutes excluding details regarding collaborations for the upcoming year.

    Deb Hamernik (USDA-CSREES) provided news and updates regarding personnel, budgets and funding opportunities at USDA. She also explained her role as National Program Leader for Animal Physiology. Earlier receipt deadlines for FY 2003 NRI applications were noted, including December 1 for Animal Reproduction.

    The business meeting began at 8:50 am with the election of Gary Moss (WY) as Member-at-Large for 2003. On behalf of the group, Chair Rozell (KS) expressed appreciation to Dale Holcombe (NV) for making local arrangements for the meeting in Reno. Dennis Hallford (NM) moved that the 2003 annual meeting be held in Reno, NV. The motion was seconded by Jerry Reeves (WA), and passed. The meeting will be held May 29-30, 2003. Dale Holcombe (NV) volunteered to gather information about possible meeting locations in the Reno area. This information will be disseminated to the group later in the year.

    The group was reminded to send assay methods and procedures that may be of interest to the group to Jim Berardinelli (MT) so they can be compiled on the W112 web site (www.repro.montana.edu/W112). The purpose of this list is to provide an accessible archive of assay procedures that will not be lost as people retire.

    The manuscript on estrous synchronization authored by Mike Day (OH) and Tom Geary (MT) was discussed briefly. Tom offered to contact Mike to check on the status of the paper. Troy Ott (ID) suggested Professional Animal Scientist as an appropriate journal for publication of this manuscript.

    Members discussed news at each of their respective stations. Several position openings were noted, including department chairs at Colorado State, Michigan State, and Texas A&M. Jerry Reeves (WA) pointed out that Derek McLean had recently been hired at Washington State and plans to become a member of W112.

    The remainder of the meeting was devoted to presentations and discussions of individual station reports and discussions of and arrangements for collaborations for 2002-2003. Plans for collaborations were recorded in writing and submitted to Secretary Adele Turzillo (AZ) who will compile them and make them available to the group.

    Accomplishments:
    OVARIAN BIOLOGY

    1. Follicular Development

    Better methods to synchronize estrus and ovulation will require the ability to control ovarian follicular development and atresia. Results in heifers indicated that atresia of dominant bovine follicles is not caused by altered expression of oxidative stress response proteins. Oxidative stress does not appear to mediate follicular atresia in cattle.

    Growth/differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15) are ovarian growth factors. Immunization of ewes against GDF9 and BMP15 enhanced ovulation rate and may provide an alternative to current superovulation procedures.

    Binding of follicle-stimulating hormone (FSH) to its receptor on granulosa cells initiates follicular development and differentiation. Two functional variants of the FSH receptor that appear to have unique structural characteristics and biological activities were found in bovine and porcine granulosa cells. Determining the functionality of these variants may increase our understanding of ovarian follicular development.

    Cystic ovaries have a negative impact on reproductive efficiency. In lactating dairy cows, high peripheral concentrations of LH did not cause development of cysts. Pulsatile infusion of LH decreased the interval to first ovulation in postpartum cows.

    2. Sexual Behavior

    Sexual behavior and improvement of breeding capacity was studied in rams. Selecting ewes is unlikely to effect sexual behavior or orientation of their male offspring. Conditions for monitoring sexual behavior need to be considered carefully to make sound decisions on sexual performance. The naloxone challenge test cannot discriminate between asexual and sexually active rams. Sexual performance in Polypay rams did not alter reproductive performance of Polypay ewes. Serving capacity tests should be used without emphasis on level of sexual performance to obtain 60% lambing rate. The moderate heritability and high repeatability for ram sexual performance imply that 1 screening for this trait is adequate and favorable response to selection for serving capacity can be expected. Selection for ram libido score may enhance fertility despite small genetic correlations of libido score with number of lambs born.

    Inhibin is a gonadal protein that exerts negative feedback on FSH secretion. A sensitive, specific 2-site immunoassay for ovine and bovine inhibin B was developed. In rams with detectable inhibin B concentrations, secretion was pulsatile. This assay may facilitate a better understanding of male reproductive physiology and development of strategies to assess testicular function in rams and bulls.

    3. Ovulation

    Follicular rupture and subsequent oocyte release are prerequisites for pregnancy. The preovulatory LH surge initiates proteolytic degradation of the follicle wall preceding ovulation. Matrix metalloproteinases (MMP) degrade extracellular matrix components. The LH surge increases MMP-1 and MMP-13 in bovine preovulatory follicles. MMP-1 and MMP-13 may mediate degradation of the follicle wall prior to ovulation. LH surge-induced elevations in intrafollicular prostanoid synthesis are required for follicle rupture in dairy cattle.

    4. Luteal Function

    The endogenous diazepam endozepine was detected in ovine luteal tissue and was 6 times higher in large luteal cells compared to small luteal cells. Since endozapine stimulates steroidogenesis, endozapine may mediate high constitutive production of progesterone by large luteal cells. These results suggest potential development of pharmaceutical products that would reduce embryonic wastage caused by luteal insufficiency.

    5. Environmental Control

    The role of thyroid hormones in seasonal anestrus was investigated in ewes. Thyroid inhibition with large dosages of propylthiouracil (PTU) decreased serum T4, inhibited onset of anestrus and extended the breeding season. PTU induced hypothyroidism in pregnant ewes. Decreasing sunlight and solar radiation appear to play little role in onset of cyclicity once puberty is attained in Rambouillet ewes in southern New Mexico.

    Season and steroid hormones have important effects on GnRH and LH secretion. Anti-gonadotropic decapeptide (AGD) is a potent inhibitor of GnRH secretion. Experiments in ewes suggest that AGDs inhibition of GnRH secretion is predominant during anestrus. In another study, non-genomic effects of estradiol (E) were demonstrated in ovine pituitary cells. Rapid inhibitory actions of E on luteinizing hormone (LH) secretion were mimicked by membrane impermeable forms of E, suggesting that the inhibition occurred at the level of the plasma membrane. These results could lead to development of novel methods to regulate LH secretion.

    Systemic inflammatory stress activates gene expression pathways in the hypothalamus that may play a role in neurobiological regulation of the stress response and mediate negative effects of stress on reproduction. Results indicate that stress, or stress-like levels of cortisol, interrupted follicular development and ovulation in ewes. The anti-gonadal effect of cortisol is due in part to reduction in pituitary GnRH receptor expression.

    Several studies focused on relationships between nutrition and reproduction. Prepartum diets high in linoleic acid had no effect on subsequent reproductive performance in beef heifers. However, postpartum supplementation with dietary oils high in linoleate may influence reproduction in heifers by altering the synthesis and/or secretion of PGF. Feeding an ionophore antibiotic (Lasalocid) prior to calving and during the postpartum period increased calving rate. Because whole cottonseed reduced body weight gain, body condition score, antler growth, and testis growth in Red Deer stags and Fallow bucks, supplemental feeding of whole cottonseed to deer should be avoided. Elevated concentrations of progesterone in ewes fasted during the luteal phase may result from decreased metabolism reflected by changes in thyroxine and insulin.

    Adipose-derived leptin has been implicated in the regulation of energy metabolism, feeding behavior, and reproductive hormone secretion. Peripheral concentrations of leptin were positively correlated with scrotal circumference and serum testosterone in growing bulls and with back fat and body condition scores in heifers. Brahman cows tended to have greater concentrations of metabolic hormones than Angus cows and this difference may be sensitive to forage quality. High levels of leptin in milk early in lactation may provide a mechanism for thermoregulation, satiation, and homeostatic endocrine control in the neonate. Cows with higher concentrations of leptin had shorter postpartum interval. Leptin concentrations were positively correlated with IGF-1, body weight and body condition score, and negatively correlated with postpartum interval.


    UTERINE BIOLOGY

    1. Maternal Recognition of Pregnancy

    The bovine uterus is exposed to conceptus-derived interferon-tau (IFNt) during early pregnancy. IFNt is anti-luteolytic and stimulates several genes including the ubiquitin paralog ISG17. Induction of ISG17 is a pregnancy-specific response not observed with components of the ubiquitin/proteasome-dependent degradation pathway. PI3K appears to have a role in IFNt signaling in bovine endometrial (BEND) cells to reduce PGF secretion.

    Mx is another IFNt stimulated gene expressed during diestrus and early pregnancy in ovine endometrium. Mx may be involved in pregnancy establishment and maintenance and co-regulated by steroids. Uterine Mx expression may be a general phenomenon during early pregnancy in domestic ungulates and is found at the fetal/maternal interface throughout gestation. Manipulation of Mx expression may reduce early embryonic wastage. Assaying for Mx or other interferon induced proteins in blood may provide a method for early detection of bred, non-pregnant, ruminants allowing for faster re-breeding and fewer days open.

    2. Placental Development

    Regulation of endometrial and placental progesterone secretion was investigated. LH appears to stimulate endometrial PGF secretion in nonbred ewes and prevent luteolysis 15-50 d post breeding. Uterine/placental PGF secretion is not reduced until the end of the first trimester, and thus may play a role in the 30-40 percent of pregnancies lost during this time. Caruncular/placental secretion of PGE2 appears to be regulated by PSPB after d 50 post breeding.

    Exposure of pregnant ewes to hyperthermia (HT) during maximal placental development causes placental insufficiency and intrauterine growth restriction (IUGR). Ultrasonography was used to monitor placentome development in IUGR pregnancies, and Doppler velocimetry provided a good index of umbilical and fetal hypertension. These methods could be used to study maternal nutritional influences on fetal and placental development. HT alters fetal placental angiogenesis, similar to the response to hypoxia. Further study of these 2 environmental insults on placental and fetal development will help differentiate mechanisms of placental insufficiency and IUGR.

    3. Fetal maturation

    A study was designed to characterize conceptus development in in vivo- and in vitro-derived bovine pregnancies. Results suggest the importance of placental development in the birth of large calves derived from in vitro-produced embryos. High birth weights may be a consequence of aberrant placental development due to disruption of placental restraint on fetal growth toward the end of pregnancy.

    ANIMAL DISEASE

    1. Mastitis control

    The effect of post-weaning fast and intramammary antibiotic infusion on hormone profiles during the drying off period in ewes was investigated. Fasting animals for 2 d at weaning influenced concentrations of IGF-I, GH, insulin, T3 and T4. Intramammary antibiotic infusion affected serum insulin, GH, T3 and T4 concentrations during the drying off period. These data suggest that hormones associated with lactation are influenced by both fasting and intramammary antibiotic infusion following weaning.

    REPRODUCTIVE TECHNOLOGY

    1. Immunocastration

    Aggressive behavior was evaluated in bulls, steers and bulls actively immunized against GnRH. Data indicate active immunization against GnRH is an effective alternative to physical castration. Immunocastration consistently lowers aggressive behavior and is likely to facilitate handling and management of male cattle on the ranch and feedlot.

    A sterilization vaccine against GnRH is being developed as an alternative to surgical spaying of feedlot heifers. Ovalbumin-GnRH fusion protein was the best antigen for immunization. Results indicate that GnRH immunocastration in bulls decreased testosterone (as indicated by body weight gains compared with surgically castrated steers) and scrotal size compared to intact bulls. A double hormone target sterilization vaccine (LH beta subunit and GnRH) is being developed to overcome the problem of non-responding animals.

    2. Assisted reproductive technologies

    Widespread use of artificial insemination (AI) in beef cattle on the Western range will require simple and efficient methods to synchronize estrus. Select Synch and CO-Synch are current protocols that use exogenous hormones to synchronize estrus. Results in beef heifers indicated that injection of GnRH 1 wk prior to CO-Synch did not increase pregnancy rate. Since ovulation of smaller follicles may result in higher embryonic mortality, protocols may be improved by assuring adequate follicular growth before ovulation. Addition of MGA at the time of PGF during Select Synch delayed estrus and ovulation and resulted in ovulation of larger follicles. However, pregnancy rates of cows receiving a bolus of MGA at the time of PGF with CO-Synch and timed AI were similar to control cows. The MGA/PGF protocol increased estrus synchrony and pregnancy rates in beef heifers compared to Select Synch and MGA/Select Synch. In postpartum anestrous cows, a progesterone-releasing CIDR induced and synchronized estrous cycles but MGA for 7 d had no effect. Fertility was improved following termination of pregnancy at 35 d of gestation compared to d 25 or during the luteal phase in non-pregnant cows.

    Success of farming muskoxen for qiviut (underwool) in Alaska requires husbandry tools specific for this species. Importance of the bull in seasonal ovarian activity and his effect on estrous synchrony were tested. Introducing bulls in August truncated the breeding period to 2 wk (4-6 wk shorter than normal). A radiotelemetric system was found to be an excellent tool for estrus detection. This information will support development of management strategies to increase reproductive efficiency in reindeer and muskoxen.

    Importance of the bull in the onset of ovarian activity was also investigated in beef cows. The effect of bulls on resumption of estrous cyclicity in first-calf beef cows was independent of type of suckling. Cows exposed to bulls later (d 55) during postpartum anestrus responded faster to the biostimulatory effect of bulls. Exposing cows to bulls or their excretory products did not alter breeding performance in first-calf restricted suckled beef cows using a modified CO-Synch protocol, but presence of a bull may enhance breeding performance of cows bred at timed AI using CO-Synch.

    In red deer hinds, electronic estrus detection using the Heat Watch System was not as reliable for estrus detection as marking by stags.

    Bovine spermatogonial stem cells were transferred to nude mouse testis and colonized as round cells that appear to be spermatogonial in nature.

    PLANS FOR THE COMING YEAR

    Many new and on-going collaborations among participating research stations are planned for 2002-2003. Space limitations prohibit detailed description of these collaborations in this report. A complete list may be obtained from the Secretary (Turzillo) or Administrative Advisor (Kaltenbach).



    Impact Statements:
    1. A bulletin is the final stages of preparation that summarizes the pros and cons, pluses and minuses of various estrous synchronization methods that have been developed and/or tested by participants in the W-112 project. This information will allow producers to make informed judgements as to whether to use estrous synchronization and if so which method is the most appropriate for their particular situation.
    Last Modified: unknown

    Date of Annual Report: 08/28/2003

    Report Information:
  • Annual Meeting Dates: 05/29/03 to 05/30/03
  • Period the Report Covers: 06/2002 to 05/2003

    • Participants:
    Brief Summary of Minutes of Annual Meeting:
    The meeting was called to order at 8 am on Thursday, May 29, 2003 by Chair Adele Turzillo (AZ). Meeting participants introduced themselves, and the minutes of the last meeting were approved. Colin Kaltenbach (AZ), administrative advisor, pointed out that we were in the middle of the current project cycle. He also further discussed the electronic format for the meeting minutes, and following general discussion, it was decided that a full-set of minutes would be posted on the W112 list-serve. Deb Hamernik (USDA-CSREES) provided a handout with news and updates regarding personnel, budgets and funding opportunities at USDA. Included in this information is the plan for the NRI to move to electronic submission and the 5-year strategic plan. Dr. Hamernik also discussed the CSREES budget and the recent request for supplemental applications by the NRI.The business meeting began at 9:00 am, by thanking Dale Holcombe for making the local arrangements in Reno, NV. Andy Roberts (USDA-ARS, Miles City, MT) was elected as the Member-at-Large for 2004. Discussion then focused on where to hold the 2004 meeting. Sites that were suggested included Reno, NV, Las Vegas, NV, Tucson, AZ and Davis, CA. After considerable discussion, it was moved (Dennis Hallford) and seconded (Todd Hansen) that the meeting be held in Las Vegas, NV. Following additional discussion about the dates, it was voted on and passed that the 2004 W112 meeting will be held May 24 and 25, 2004 in Las Vegas, NV. Adelle Turzillo volunteered to take charge of the local arrangements.

    As old business, Mike Day (Ohio) discussed the status of the estrous synchronization training manual. As a training manual, it is complete but does not include any discussion on the costs of the various treatment protocols. After discussion, it was decided to include a range of cost for each protocol, and the document would be complete. Dr. Day asked the W112 members to review the document as is, such that after a final edit the document could be placed on the list-server. Additional discussion focused on where to publish the document. It was decided that this training manual on estrous synchronization was best suited for electronic publication.

    In additional old business, Jim Berardinelli (MT) discussed the server problems he has been faced with in operating the W112 web site. These problems should soon be rectified, allowing the web site to be updated into a useful format.

    The remainder of the meeting was devoted to presentations and discussions of individual station reports. Discussions and arrangements for station-to-station collaborations during the 2003-2004 year were made. These planned collaborations were recorded and submitted to Secretary Russ Anthony (CO), who will compile them and make them available to the members via the list-server.

    Accomplishments:
    Ovarian Biology:
    1. Gonadal Development
    Bovine testicular cells cultured for up to 3 weeks as dispersed cells on a bovine embryonic fibroblast feeder layer can colonize recipient nude mouse testes suggesting spermatogonial stem cell survival. Bovine testicular tissue explants cultured for up to 3 weeks have increased germ cell proliferation compared to controls. Germ cells from bovine testicular explants can colonize recipient nude mouse testes and the number of colonization competent cells increases from 1-3 wk of culture.

    Ovarian maturation is accelerated in heifers that are weaned early and fed a high concentrate diet, resulting in increased diameter of dominant follicles and precocious onset of puberty. Weaning at 3 months of age did not accelerate puberty relative to heifers that were suckling their dams to 7 months; if body weight gains were similar.

    2. Follicular Development
    When estradiol is used to initiate follicle atresia, timing of emergence of the next wave of ovarian follicles is dependent upon dosage of estradiol administered, rather than timing of atresia. During estradiol induced atresia, rapid reductions in intrafollicular estradiol concentrations are accompanied by reductions in mRNA for aromatase, and to a lesser extent for 17a hydroxylase and mRNA for LHR, FSHR, Fas and Fas  Ligand are unchanged at 24 h after estradiol treatment.

    Fasting prior to proestrus influenced ovarian follicular dynamics and the ability of
    Developing follicles to secrete estradiol. Feeding fats high in linoleic acid did increase the formation of ovarian follicles in postpartum anestrous cows.

    Premature ovulation of a dominant follicle results in decreased ovulatory size, reduced luteal function and compromised conception rates, compared to animals induced to ovulate a larger, more mature dominant follicle and in animals that spontaneously ovulate.

    3. Sexual Behavior
    The volume of the ovine sexually dimorphic nucleus (oSDN) corresponds to the type of sexual partner preference exhibited by rams. SDN volume for female-oriented rams (FORs) is greater than for male-oriented rams (MORs) and neurons for the SDN express high levels of aromatase mRNA with greater expression in FORs than in MORs and ewes. This further implies that the brains of MORs are incompletely masculinized and support the hypothesis that naturally occurring variations in sexual partner preference are determined by underlying biological mechanisms.

    The LH surge mechanism was operational in 4 of 4 ovariectomized ewes, 3 of 4 castrated male-oriented rams (MORs) and 0 of 4 castrated female-oriented rams (FORs). Peak serum LH concentrations, latency, or duration of the estradiol-induced surge response were not significantly different between ewes and MORs. However, LH in ewes and MORs were greater than in FORs during the interval of the surge. These results indirectly support the hypothesis that MORs are incompletely androgenized/masculinized with the MORs exhibiting a neuroendocrine LH response similar to ewes. The response of MORs to estrogen might serve as the basis for future development of a practical test that can be used by producers to identify these animals.

    Restraint stress increased cortisol and decreased testosterone similarly in both low and high libido rams and consequently does not provide a method of differentiating such rams by level of libido.

    Based on behavioral observation data alone, low libido rams were approximately 50% as efficient and male-oriented rams approximately 75% as efficient as high libido rams at mating ewes in a competitive mating environment.

    Present findings show that active immunization against an inhibin a-subunit peptide neither advanced puberty nor increased sperm production in ram lambs. On the contrary, it decreased LH and testosterone secretion and delayed the onset of puberty. A better understanding of underlying inhibin physiology may lead to the development of an inhibin immunogen and immunization protocol that consistently increases FSH secretion in ram lambs. If so, the approach would need to be reassessed for effects on the onset and rate of sperm production.

    4. Ovulation
    Short-term fasting increased body weight loss and time from PGF2a to ovulation, suggesting an influence of short-term fasting on time of ovulation.

    5. Luteal Function
    Our data suggest nitric oxide (NO) is not luteolytic in cattle and that estrous synchronization affects response of bovine luteal tissue to agonists or antagonists of prostaglandin secretion indicating a better understanding of the effects of estrous synchronization on luteal function are needed to optimize positive effects of estrous synchronization.

    Messenger RNA encoding induced nitric oxide synthase (NOS) differed, and endothelial NOS tended to be altered, in response to PGF2a, suggesting that PGF2a may regulate amounts of mRNA encoding NOS in the bovine CL.

    Overexposure of the developing bovine corpus luteum to LH is detrimental for luteal production of oxytocin and progesterone. It is possible that initial stimulation of small luteal cells by LH causes these cells to produce a paracrine factor that acts upon the large luteal cell to impair their function.


    6. Environmental Control
    Our finding that the EBA agent does not appear to be able to produce abortions in sheep, should be of use to sheep producers whose grazing ranges contain Ornithodorus coriaceus. Their sheep production is not affected by the presence of the tick, even if a portion of endemic ticks are infected with the EBA agent. Probes and methods developed to detect the presence of the EBA agent in animal tissues and ticks are reliably able to detect the EBA agent in ticks.

    The results of studies conducted during the past year indicate that stress, or stress-like levels of cortisol, interrupt follicular development and ovulation in sheep. The anti-gonadal effect of cortisol is due, at least in part, to cortisol- dependent reduction in GnRH receptor expression in pituitary tissue.

    Transportation stress results in an increase followed by a gradual suppression of anterior pituitary secretion of ACTH accompanied by a decrease in anterior pituitary responsiveness to corticotropin releasing factor and arginine vasopressin stimulation.

    Testicular and epididymal sperm content and daily sperm production of tropically-adapted Bos taurus bulls are more similar to tropically-adapted Bos indicus bulls than to temperate Bos taurus bulls raised in the subtropics during late spring and summer.

    Temperament of an animal can have an influence on reproductive performance. Measuring exit velocity from a chute seems to be a better measurement than temperament scoring systems. The temperament of the dam is strongly associated with temperament of the calf.

    Brahman cows tend to have greater concentrations of metabolic hormones relative
    to Angus cows and these concentrations may be sensitive to seasonal dynamics of
    forage quality in the Chihuahuan Desert.

    Uterine Biology:

    1. Maternal Recognition of Pregnancy


    Sheep oviduct specific protein (OSP) mRNA is expressed only in cells of the ampulla (AMP) but is present in the apical portions of cells of the isthmus (IST). Surprisingly, we found sOSP in the AMP and IST, and mRNA for sOSP in the AMP of prepubertal ewe lambs.

    The bovine ISG15 E1 has been partially sequenced to confirm identity. This enzyme is the first of several that dictate specific conjugation of ISG15 to targeted proteins.

    2. Placental/Fetal Development

    While alterations in uterine blood flow and oxygenation of the pregnant uterus may play a role in the etiology of hyperthermia-induced placental insufficiency leading to fetal growth restriction (FGR), our results indicate that hypoxia alone cannot account for the observed changes in placental vascularization.

    The results of experiments with nuclear transfer (NT)-cloned bovine pregnancies strongly suggests placental involvement in the birth of large calves for both IVF and NT procedures. Heretofore, the embryo/fetus itself is a primary focus of the problem.

    Modulators of cellular calcium signalling pathways given alone do not affect placental progesterone secretion. Progesterone receptor-mediated events appear to suppress placental PGF2a secretion and progesterone receptors may play a role in placental secretion of progesterone in cattle. PGE2 does not appear to regulate placental progesterone secretion in cattle.


    3. Mastitis Control

    A subclinical mastitis infection caused by S. aureus does not influence systemic concentrations of IGF-1 and T3, although the local infection increased serum cortisol and decreased T4 concentrations within the first 36 hours following infection. Serum IgG was not affected by either amoxicillin or Mastfrigao treatment or S. aureus infection. In contrast, milk IgG concentrations were greater in NCG compared to all other treatments by hour 48, indicating that treatments used for subclinical mastitis had a direct effect on milk IgG concentrations.

    Reproductive Technology:

    1. Immunocastration

    Immunization against recombinant fusion proteins of both LHb and LHRH was not beneficial in inducing sterility in beef heifers. Results of our most recent experiment would suggest focusing on the LHRH antigen alone.

    A field study in Brazil with an LHRH immunocastration protocol is in progress. The vaccine causes a significant reduction in scrotal circumference when compared to intact controls. One hundred days after the last immunization seventy-three bulls from one of the ranches were sent to slaughter. Intact bulls had higher carcass weights and muscle percentage compared to the other two groups. Castrated and immunized animals had greater marbling and fat percentages than intact bulls.

    2. Assisted Reproductive Technologies

    Using a week-long MGA feeding period prior to a GnRH-PGF synchronization system resulted in a relatively high pregnancy rate relative to previously reported results for timed A.I. trials

    Progestins differ in their ability to induce estrous cycles in postpartum anestrous beef cows. Administration of progesterone for 7 days with a CIDR induced ovulation and estrous cycles in postpartum anestrous beef cows, but neither the normal dose (0.5 mg/d) nor a high dose (4 mg/d) of MGA induced ovulation or estrous cycles.


    Exposing cows to bull or excretory products of bulls did not alter the breeding performance of first-calf restricted suckled beef cows using a modified CO-Synch protocol. However, physical presence of a bull may enhance breeding performance of cows bred at timed AI in this protocol.

    Ewes receiving a CIDR on d 0 have the same P4 on d 12 to 14 as CIDR-treated OVX ewes suggesting premature demise of the corpus luteum which may adversely impact conception rate. Progesterone from CIDRs rapidly enters the circulation (within 1 h) at the time of insertion and is rapidly cleared after CIDR removal (< 1ng/mL by 1 h). After 2, 15-d periods in situ, CIDRs were no longer able to increase serum P4 concentrations.

    Early weaning fails to improve reproductive performance of red deer females. This may be related to the strong seasonality of this species and the possible male effect from the stag.

    Our research demonstrates the effectiveness of using modified cattle CIDRs for estrous synchronization in muskox cows. In addition, our results illustrate an interaction of bull exposure and CIDR in the timing of the synchronized estrus.

    Our group has previously shown radiotelemetry to be a reliable and accurate means of detecting estrus in muskoxen but our recent results indicates radiotelemetric technology is less effective with reindeer.

    Impact Statements:
    1. 1. A training manual for estrous synchronization of ruminants has been completed and is ready for electronic publication. This manual presents the pros and cons of various estrous synchronization protocols that have been developed and/or tested by participants in the W112 project. This manual will provide producers an easy-to-use guide on estrous synchronization, allowing them to make informed production decisions.
    2. The participants in the W112 project published 66 refereed journal articles during the last reporting period, many of which were derived from collaborative efforts initiated through the W112 Regional Research Proj
    Last Modified: unknown

    Date of Annual Report: 08/17/2005

    Report Information:
  • Annual Meeting Dates: 06/13/05 to 06/14/05
  • Period the Report Covers: 06/2004 to 06/2005

    • Participants:
    Brief Summary of Minutes of Annual Meeting:
    Chair Gary Moss called the meeting to order at 8 a.m. on June 13, 2005. Participants were introduced, minutes from the last meeting approved, and appreciation expressed to Charlie and Yoshi Weems for arranging meeting facilities.

    Colin indicated a new number would be assigned to the renewed project and that the project renewal needs to be completed by January 2006. Deb Hamernik discussed updates and changes in USDA granting programs and led discussion concerning changes being considered regarding federal funding (formula funding vs competitive funding).

    Business items included the unanimous election of Derek McLean, Washington State University as Member at Large and selection of Reno as the site for the 2006 meeting on May 22-23. Dennis Hallford was selected to lead the organization and arrangement of accommodations for the 2006 meeting. Announcements and news from individual stations were given. No discussion of old business. Attendees then discussed aspects of the current project that remained to be completed. The business portion of the meeting concluded with discussion of the new objectives, development of a schedule and selection of individuals to lead the writing of the project renewal. Andy Roberts will provide summary of past and current accomplishments and publications. Troy Ott, Derek McLean, Kim Vonnahme and Tom Geary will prepare remaining aspects of the project renewal. Andrea Cupp will edit the first draft. Deadlines for the rewrite are: Sept 1, 2005 for individual membership submission of research brief; Oct 1 for first draft; Nov 1, for second draft; and Dec 1 for final draft. Members were encouraged to recruit new faculty from within their stations during the renewal process. After a great deal of discussion, the following objectives were recommended.

    1. Determine the basic physiological, immunological, metabolic and genetic systems that contribute to optimum reproduction in domestic ruminants with emphasis on: - gonadal development and function -establishment of pregnancy, placental function and fetal development - pubertal, seasonal and post-partum anestrus

    2. Develop tools, techniques and management strategies to allow producers to control reproductive efficiency including development of : - markers for reproductive health and disease - chemical and immunological means of regulating fertility

    The remainder of the meeting was dedicated to presentations of individual reports and identification of collaborative projects for the ensuing year. The meeting adjourned at noon on June 14. Collaborative Efforts:

    Collaborators: Alaska, Missouri. Project. Evaluation of IGF-1 and Leptin in muskoxen and reindeer Collaborators: Alaska, USDA (Miles City), Washington. Project. Evaluation of LHRH vaccines Collaborators: Colorado, New Mexico. Project: PGF production by CL. Collaborators: Colorado, Wyoming Project: Study genomic markers in hearts from steers with high altitude disease to see if these genes are associated with hypertrophy in hearts from fetuses out of undernourished dams. Collaborators: Colorado, Wyoming Project: Effects of maternal undernourishment on preproghrelin system in fetus. Collaborators: Colorado, Wyoming. Project: Study cases of hypertrophy in hearts from fetuses in response to maternal under nutrition. Collaborators: Colorado, Idaho. Project: Continue development of test for non-pregnant beef and dairy cows, and examine ISG15/MX response in cows and fetuses infected with BVDV. Collaborators: Colorado, Idaho, Missouri, Ohio, USDA (Miles City). Project: Examine ISG15 mRNA in blood from pregnant cows. Collaborators: Colorado, Texas. Project: Examine ISG15 mRNA in blood from pregnant deer and Brahman cows. Collaborators: Hawaii, Texas. Project: Growth factor influences on bovine caruncular secretion of PGE1. Collaborators: Hawaii, Colorado. Project: Effects of PGE1 or PGE2 on mRNA for LH receptors in CL and caruncular endometrium. Collaborators: Idaho, Ohio, Colorado Project: Determine if uterine characteristics contribute to decreased fertility in response to a short proestrus period. Collaborators: Idaho, USDA (Miles City) Project -ISG in peripheral blood following AI in beef cattle. Collaborators: Idaho, WSU and USDA Idaho Project: Genetic scan for genes associated with cryptorchidism. Collaborators: Kansas, Wyoming Project: Regulation of FSH-Receptor variants. Collaborators: Kansas, Nebraska Project: Evaluate FSHR and VEGF expression in ovary and testis cultures. Collaborators: New Mexico, USDA (Miles City, Montana). Project. Characterize factors influencing longevity of replacement females. Collaborators: New Mexico, Wyoming, USDA (Miles City, Montana). Project. Effects of feed restriction on ovulatory follicle developments and oocyte viability. Collaborators: Nebraska, WSU. Project. Role of VEGF in bovine testis grafts. Collaborators: Nebraska, WSU. Project. Spermatogonial germ cell transplantation Collaborators: Nebraska, Oregon. Project. Evaluate vascular development in fetal gonads from sheep. Collaborators: New Mexico, North Dakota. Project. Monitor T3, T4, IGF-I in fetal and maternal blood from ewes fed Se or undernourished during pregnancy. Collaborators: North Dakota, USDA Idaho. Project. Selenium project and BW data. Collaborators: North Dakota, Minnesota. Project. Effects of estrous synchronization with natural service on calving distribution. Collaborators: North Dakota, Wyoming. Project. Evaluate uterine/placental function in nutrient-restricted cows. Collaborators: Texas, USDA (Miles City). Project. Feed efficiency in developing heifers Collaborators: WSU, USDA (Miles City). Project. Determine differences in pituitary gene expression in cycling and postpartum anestrus cows.

    Accomplishments:
    Objective 1. Investigate molecular, cellular and endocrine mechanisms that limit or control reproductive efficiency in domestic animals.

    Hypothalamic-Anterior Pituitary Gland Physiology.

    Research has provided knowledge concerning which estrogen receptor, alpha (ERa) or beta (ERb), is involved in mediating actions of estradiol on gonadotropin secretion. The ability of estradiol to induced a preovulatory like surge of gonadotropins in ovariectomized ewes was determined to require activation of both ERa and ERb. Inhibitory effects of estrogen on FSH secretion were determined to be mediated by ERa, but not ERb. This observation demonstrates the potential of using ERa agonist to modify secretory patterns of LH without altering secretion of FSH, which may have application in improving regimens for superovulation. Stimulation of either ERa or ERb leads to stimulation of GnRH receptors in cultured ovine pituitary cells. Thus, the type of estrogen is not important for stimulating an increase in sensitivity of the pituitary to GnRH.

    A novel membrane receptor has been identified for progesterone. It is likely that previously reported rapid actions of progesterone are mediated via this receptor.

    Results demonstrate for the first time that the PRL gene is expressed in the medial preoptic area of the hypothalamus, amygdala and frontal cortex of the ovine brain. Expression of the PRL gene is greater in the MPOA and amygdala in male-oriented rams than in heterosexual rams and ewes. Expression of the PRL gene in the MPOA and amygdala during sexual differentiation of the brain may account in part for the display of male-oriented sexual behavior in the adult ram.

    Ovarian Physiology.

    Completed research indicates that progesterone is able to prevent an oxytocin induced influx of calcium into large luteal cells. It is hypothesized that this effect may be important in regulating maintenance of the corpus luteum during early pregnancy.

    Preovulatory regulation of tissue plasminogen activator and TIMP-4 is dependent on increased intrafollicular prostanoid synthesis indicating such molecules may be key intrafollicular mediators of the ovulatory process.

    The angiogenic factor, VEGF appears to be important in progression of follicular development from primordial to later stages of follicular development. In addition, levels of expression of the different isoforms of VEGF appear to be differentially regulated during final stages of preovulatory period.

    Indomethacin and/or atrigel were ineffective in delaying or preventing luteolysis in the bovine CL.

    Prostaglandin F2-alpha activates Ca2+/PKC and MEK/ERK signaling pathways to induce Egr-1 expression and apoptosis during luteal regression.

    Oocyte Physiology. Messenger RNA abundance for follistatin and bB and a subunits of inhibin/activin is increased coincident with zygotic genome activation in cattle. Follistatin mRNA abundance is positively associated with competence of bovine oocytes.

    Use of exogenous control RNAs represents a viable approach to control for variation in RNA recovery and efficiency of reverse transcription in real time PCR studies of mRNA abundance in oocytes and early embryos. Ribosomal protein L-15, cyclophilin-A, phosphoglycerokinase, ²-glucuronidase, glyceraldehyde 3-phosphate dehydrogenase, ²-actin and histone H2A mRNAs are not constitutively expressed during oocyte maturation and early embryonic development, but rather are dynamically regulated. Thus, contrary to reports in the literature, they should not be used for normalization of RNA abundance for genes of interest during bovine oocyte maturation and early development.

    Testicular Physiology.

    Five cryptorchid families consisting of a total of 92 members plus 26 controls have been generated for the purpose of studying the genetic mechanisms associated with this trait.

    Research demonstrates that VEGF may be working through the PI3 kinase pathway to elicit its effects on vascular development and cord formation during testis morphogenesis.

    Feeding of MGA to bulls during the pre- and peri-pubertal period appears to decreased body weight and circulating concentrations of testosterone.

    Bovine testis grafts on the backs of nude mice produced elongate spermatids 6 months after grafting. VEGF treatment of grafted tissue improves the percentage of seminiferous tubules supporting the differentiation of germ cells into elongate spermatids. Culture of testis tissue prior to grafting does not improve the number of seminiferous tubules supporting the differentiation of elongate spermatids. Cryopreservation of testis tissue prior to grafting does not negatively effect the production of sperm by grafts and suggests tissue can be stored for long periods and be used to produce sperm. This technology will lead to new approaches to generate transgenic animals and preserve genetic material from bulls.

    GeneChip array analysis of gene expression in bovine testis tissue and embryonic fibroblasts provides a valuable dataset of information about the factors involved in the establishment of spermatogenesis in bulls and spermatogonial stem cells biological activity. These data will be submitted to the NCBI Gene Expression Omnibus (GEO) website database for public access. Uterine-Placental-Fetal Interactions. Alterations in uterine gene expression during the ensuing estrous cycle in cows that experienced decreased preovulatory estradiol concentrations in response to manipulation of proestrus may be responsible for lower fertility and/or attenuated luteal function observed in previous studies in cows with a shortened proestrus.

    Periattachment factor has been identified in a number of species during the critical window during early pregnancy when the conceptus is rapidly growing and establishing attachment to the uterine lining. Completed research provides methodology to use RNA interference to examine the importance of this nuclear protein in vivo.

    Cloning of the ovine Mx1 (oMx1) promoter/enhancer revealed the presence of several different transcription factor-binding sites. During telophase, oMx1 was shown toco-localize with remnants of mitotic spindles at intercellular bridges between dividing cells, which is consistent with a role for Mx1 as a fission molecule in uterine cells and indicates that oMx1 may be involved in cell division. These results expand knowledge concerning mechanisms that regulate expression of this gene and the physiological function of the gene.

    Experimental results suggest that progesterone can act nongenomically in a dose-dependent manner to interfere with the binding of oxytocin to its receptor in the plasma membranes of ovine endometrium. Progesterone interference with binding of oxytocin to its receptor impairs downstream signal transduction via phosphoinositide hydrolysis. Progesterone produced by the conceptus may, during a critical period of gestation, act in part on a local basis to suppress activation of oxytocin receptors and thus ensure survival of the embryo/fetus.

    Fetuses die when the ISG15 system is disrupted through genetic deletion of one of the enzymes. Dysregulation of ISG15 and its conjugates may have a detrimental impact on implantation-placentation, which then causes fetal death. The hinge region cysteine 80 contributes to instability of rISG15, which leads to formation of dimers and loss of secondary structure and function in interacting with its initiating enzyme (E1). Instability of rISG15 affects structure, protein-protein interaction and function as an ubiquitin homolog.

    Nutritional effects during gestation. Similar to previous findings with the vascular endothelial growth factor family, placental expression of angiopoitens are altered in a gestational age related fashion in growth restricted pregnancies, again highlighting that changes in vascular growth factors during early to mid-gestation set placental development on a different functional trajectory, as evidenced by altered umbilical Doppler velocimetry and fetal blood pressures.

    Management system under which an ewe (and maybe a cow) is selected was shown to have profound effects on her ability to protect her fetus from intrauterine growth restriction in the face of maternal insults such as inadequate nutrition.

    Fetal intrauterine growth restriction resulting from inadequate nutrition during the first half of gestation in ewes and cows has significant negative health effects on their offspring after birth. These health effects are not alleviated even if females are realimented during the second half of gestation such that birth weights are normal.

    In the ewe, the conversion of placentomes from A to D type increases their ability to delivery nutrients to the fetus, thereby protecting the fetus from intrauterine growth restriction. This conversion normally occurs during late gestation in response to the exponential growth of the fetus, but some animals have the ability to convert their placentomes during early gestation in the face of a maternal stressor such as inadequate nutrition. This ability for early placentomal conversion appears to be related to pre-gestational ewe selection, and thus may be epigenetic in nature.

    Hypertrophy of fetal left ventricular heart in response to maternal inadequate nutrition is associated with changes in angiotensin receptors, which may have longer-term implications in the development of cardiovascular disease during post-natal life.

    Nutritional and metabolic effects in non-pregnant animals.

    Both acute and chronic nutrient restriction decrease LH pulse frequency, and increases the suppressive potency of E2 on LH pulse amplitude.

    Increased propensity for fat deposition and ADG were shown to have positive influences on attainment of puberty whereas variations in efficiency of gain or residual feed intake did not. These finding indicate that producers should be cautious when selecting against fat deposition. In contrast, selection for feed efficiency does not appear to have detrimental effects on capacity of heifers to reach puberty in adequate time to be bred as yearlings.

    The relationships identified among prepubertal serum concentrations of IGF-1 and thyroid hormones and date of puberty warrant further investigation.

    Supplementing postpartum beef cows with a fibrolytic enzyme improved average daily gain but when cows are grazing high quality forages may not improve lactation or reproduction traits.

    Bos indicus cows which were higher in milk production had shorter postpartum intervals to estrus while in Bos taurus cows there was the expected finding that higher milk production resulted in more time from calving ton conception, as expected.

    Feeding a high-concentrate diet to heifers weaned at 3-4 months of age results in precocious puberty through accelerating the peripubertal decline in the negative feedback of estradiol on pulsatile secretion of LH.

    Divergent selection of heifers for high or low IGF-I altered characteristics of follicular growth and FSH concentrations, and concentrations of progesterone during the mid-luteal phase. The physiological significance of this variation in follicular and endocrine characteristics and the potential impact on reproductive competence is currently unknown.

    Supplementing cows with high-linoleate safflower seeds increased plasma and oviduct fatty acid concentrations as well as serum PGFM, but MBH and uterine fatty acid concentrations were unaffected by lipid supplementation during the early postpartum period.

    Nutritional status of beef cows during early lactation was not influenced by lipid supplementation. Supplemental fat provided during the first 60 d of lactation did not influence levels of IGF-I or NEFA, and did not improve conception rates or calf performance.

    Beneficial effects of feeding safflower seeds on puberty in ewes lambs appears to be more likely due to fat supplementation than the presence of phytoestrogens, as safflower seeds did not possess as much estrogenic activity as a beet pulp/soybean meal based control supplement.

    Behavior. It was determined that melengestrol acetate (MGA) will not suppress rut associated aggression in male reindeer.

    Measurement of exit velocity from a squeeze chute can be a valuable tool for both assessment of cattle temperament and a predictor of stress responsiveness to future handling events.

    Objective 2. Develop and improve assisted reproductive technologies to enhance sustainability of production systems for domestic ruminants.

    Estrus, estrous synchronization and AI.

    The biostimulatory effect of bulls does not appear to involve a unique social bonding mechanism analogous to the cow-calf bond. The mechanism may operate through some other sensory pathway linked to the reproductive neuroendocrine-endocrine system of the postpartum cow. This sensory pathway may either directly attenuate the negative effect of the cow-calf bond or directly stimulate the neural centers responsible for altering GnRH release from the hypothalamus

    Inducing a prenatal hypothyroid state in ewe lambs does not alter seasonal reproductive patterns.

    It is concluded that SMB and a CIDR may alter the ratio of PGE2: PGF2a secreted by the bovine endometrium, and that this alteration could interfere with implantation.

    Concentrations of progesterone in liver and skeletal muscle did not differ between ewes receiving CIDR for 5 or 14 d and untreated control ewes. These results contribute towards knowledge required by FDA as part of the approval process for using CIDR in sheep.

    Ovulation of smaller ovulatory follicles results in lower pregnancy rates in beef cows and heifers. While induced ovulation of smaller follicles resulted in a higher incidence of embryonic mortality in cows, induced ovulation of smaller follicles in heifers did not affect pregnancy maintenance. Reduced subsequent progesterone synthesis may be one mechanism by which induced ovulation of smaller follicles may lead to reduced pregnancy rates and greater embryonic failure in cows. Future ovulation synchronization protocols and management practices that ensure ovulation of fully developed follicles may be improve pregnancy establishment and maintenance. Exogenous estrogen in ovulation synchronization protocols may improve survival of embryos derived from ovulation of smaller follicles.

    The biostimulatory effect of bulls can be used to increase the number of cows cycling before the implementation of estrous synchronization protocols. Analyses of a 3-yr longitudinal study indicated that timed AI pregnancy rates may be improved by exposing primiparous beef to bulls before and during an estrous synchronization protocol that included GnRH, PGF2a, and fixed time AI. However, overall AI pregnancy rates using this type of protocol for synchronization of estrus and fixed-time AI are not improved by the presence of bulls. In other studies, bull exposure in combination with a 7d MGA feeding with GnRH on d1 and PG on d7 did not increase pregnancy to AI or total pregnancy over cows given the short MGA synchronization protocol in the absence of bull exposure.

    Studies conducted during the past year demonstrate that recombinant gonadotropins can be used to induce precocious puberty in sheep and may have broader application in ovulation induction protocols.

    Decreasing the interval from the first GnRH injection to PGF2± from 7 to 5 days increased pregnancy rate in 2 yr-old cows when timed AI was used with a CO-SYNCH + CIDR protocol. These data suggest that the 7-day interval used in most standard synchronization protocols may not be ideal to achieve optimal pregnancy rates in timed AI programs.

    Pregnancy detection and maintenance. Efforts to identify a reliable endocrine factor that is indicative of pregnancy in reindeer have ruled out the use of estrone sulfate.

    Stressors applied to heifers and cows approximately 14 days after breeding result in increased PGFM concentrations in serum suggesting that PGF is also elevated during this time period and may interfere with pregnancy establishment. Administering flunixin meglumine to cattle approximately 14 d after AI appears to increase pregnancy establishment in beef cattle subjected to stress. However, improvements in pregnancy rate were not observed in the absence of stress.

    Results on IVF calves help to understand how in vitro manipulations of early embryos result in deviations from normal prenatal development.

    Results indicate that components of the innate immune response are activated in PBMC during the period of pregnancy recognition signaling, and indicate that Mx1 and Mx2 are differentially regulated during this time.

    Pregnancy-associated gene expression has been identified in blood that may be useful markers for a viable embryo.

    Immunocastration. Antisera that target defined epitopes on bioactive peptides may prolong the functional life of the peptide and, thereby, enhance the physiological response.

    Disease. Interferon-induced genes are among several other gene products that have been shown to be up regulated in calves that are persistently infected with BVDV.

    Impact Statements:
    1. Results obtained from research conducted by W-112 participants advances knowledge of basic mechanisms that regulate reproduction in domestic animals. This knowledge is requisite for development of new and modification of current methods for regulating and enhancing reproductive processes. Crucial to the sustainability of production systems for domestic ruminants are the use of feed resources, improved reproductive performance, and production of high quality products.
    2. . Elucidation of mechanisms by which nutrition impacts reproduction is central towards optimizing feed resources and determining beneficial and detrimental impacts of nutrition on livestock and, potentially, humans. Advances in the knowledge of mechanisms that mediate puberty, estrous cycles, ovulation and pregnancy provide the basis for continued development and testing of regimes to control timing of, and improvement of, ovulation rates, conception rates and birth of viable offspring. Indic
    3. Participants in W-112 published 76 refereed journal articles during the past reporting period, many of which were derived from collaborative efforts initiated through the W-112 Regional Research Project. Collaborative effort has also resulted in the publication of a Handbook of Estrous Synchronization on the world wide web (www.oardc.ohio-state.edu/estroussynch) Collaborative projects planned for the coming year are listed below.
    Last Modified: unknown

    Date of Annual Report: 08/18/2006

    Report Information:
  • Annual Meeting Dates: 05/22/06 to 05/23/06
  • Period the Report Covers: 05/2005 to 05/2006

    • Participants:
    Brief Summary of Minutes of Annual Meeting:
    Chair Andy Roberts called the meeting to order at 8 a.m. on May 22, 2006. Participants were introduced, minutes from the last meeting approved, and appreciation expressed to Dennis Hallford for arranging meeting facilities and food. Colin indicated that the W112 renewal was approved by the Western region without any requests for additional information or revisions. Colin also indicated that this was his first-ever experience with a renewal project that made it through the review process without any requests for modifications. He also expressed thanks to the writing team for crafting a project that was in good shape. The new number for the project will be W1112 (number was changed after the meeting). The official start date of the W1112 project is October 1, 2006 and it will terminate on September 30, 2011. Deb Hamernik discussed the new W1112 five year plans and reminded participants to pay attention to assurance statements when submitting their CRIS projects at their institutions. Valid approval for the use of recombinant DNA or animals (with approval dates) must be included on the Assurance Statement. There must be one year remaining on the IBC and IACUC approvals so that studies can be initiated during the first year of the project. On the CRIS AD-416 form, enter the objectives for the W1112 project rather than your own individual station objectives. Participants are encouraged to provide more information regarding impact statements. Impacts are defined as: measurable changes in economic social, health or environmental conditions to the discipline. Potential impacts can be discussed for basic projects. She also indicated changes in personnel within USDA CSREES. Applications to NRI must adhere to the published guidelines (budget caps, pages limits, etc.) or they will be returned without review. SF424 R&R application forms are now going to be used across the federal government. These forms are available on the CSREES website and will be used for NIH, NSF, DOE and NRI. Electronic submission of applications to NRI and SBIR will be required Oct 1, 2006 through grants.gov. Deb presented Jim Berardinelli an award because he was the first individual to submit an NRI application electronically through grants.gov. He was funded so it was in fact a success story. Congrats Jim!!!

    Business items included the unanimous election of Dean Hawkins as Member at Large and selection of Fort Collins, Colorado for the next W1112/W112 meeting on May 21-22, 2007. Tod Hansen was selected to lead the organization and arrangement of accommodations for the 2007 meeting. Announcements and news from individual stations were given. There was no discussion of old business. Individual state announcements occurred with members talking about events, job openings, etc that had happened since our last meeting.

    The remainder of the meeting was dedicated to presentations of individual reports, and identification of collaborative projects for the ensuing year. A lengthy discussion occurred determining how we can translate the research our group is currently involved in to the producer or producer-oriented groups. We discussed having a day symposia for producers, veterinarians, extension educators, etc on either nutritional effects on reproduction, oocyte quality, heifer development, etc. The meeting adjourned at noon on May 23, 2006.

    Over several subsequent e-mails the W1112/W112 group decided to have the 2007 meeting in Fort Collins, CO from June 5-6, 2007. The meeting date was changed to be prior to the Beef Improvement Federation (BIF) meeting that will be held from June 6-9th. We also would add a 4 hour session directed to the BIF membership and producers at the end of the W1112/W112 meeting that would address fetal development. We would close our meeting at noon on June 6th and then start the special session at 1pm. Six potential speakers were identified from this group. Tod Hansen and Terry Nett were selected from the membership to organize this special session.

    Accomplishments:
    Objective 1. Investigate molecular, cellular and endocrine mechanisms that limit or control reproductive efficiency in domestic animals.

    Estrous cycle Physiology/ CL function

    1.) Intraluteal administration of meloxicam, a COX-2 inhibitor, increased the length of the ovine estrous cycle and length of time at the end of the luteal phase for serum progesterone to decrease to less than 1 ng/mL. 2.) Steady-state concentrations of mRNA encoding ODC and eNOS vary during the ovine luteal phase and are altered in response to PGF2a. 3.) Antisera that target defined epitopes on bioactive peptides may prolong the functional life of the peptide and thereby, enhance the physiological response. Therefore, these bioactive peptides may be useful in superovulation, synchronization protocols, or experiments to determine ovarian function.

    Hypothalamic-Anterior Pituitary Gland Physiology

    1) Microarray analysis identified several genes that exhibited differential levels of expression in anterior pituitaries that were associated with the transition from anestrous to cycling status in postpartum cows. The roles that many of these genes have in regulating reproductive function have not been previously considered, and several of the transcripts represent as of yet uncharacterized genes. Thus, further verification of these results will undoubtedly result in the identification of novel mechanisms involved in regulating reproduction.

    2) Identification of sheep fetal brain tissues expressing steroid receptors provides valuable information as to the roles of gonadal steroids in regulating sexual differentiation of the brain and potential causes for unique adult sexual behaviors of rams. 3) Recombinant gonadotropins can be used to induce precocious puberty in sheep and may have broader application in ovulation induction protocols. 4) The fact that estradiol receptors associated with the plasma membrane modulated numbers of GnRH receptors may provide the opportunity to develop novel treatments to regulate pituitary sensitivity to GnRH without influencing other cellular mechanisms regulated by the nuclear estradiol receptor. 5) Identification of a functional G-protein coupled receptor for progesterone in sheep provides a putative explanation for the rapid effects of progesterone on cellular activity. A better understanding of the physiological role of this receptor may enhance our ability to regulate individual biological actions of progesterone. 6) The KiSS-1 peptide was shown to modulate LH secretion in the ewe exclusively by modulating secretion of GnRH without any effect on the pituitary gland. Effects of KiSS-1 were not influenced by presence of estradiol and progesterone. This peptide may provide a novel means of regulating LH secretion independent of the actions of estradiol and progesterone. 7) Antisera that target defined epitopes on bioactive gonadotropin peptides may prolong the functional life of the peptide and, thereby, enhance the physiological response.

    Ovarian Physiology.

    1) Ovulation of smaller ovulatory follicles results in lower pregnancy rates in beef cows and heifers. All mechanisms by which ovulation of smaller follicles reduces pregnancy rates have not been elucidated, but at least a portion of this reduction in fertility appears to be due to reduced subsequent progesterone synthesis. It makes sense that endogenous estrogen secretion is also indicative of follicular maturity and subsequent progesterone synthesis. Results of these studies should improve the ability to develop ovulation synchronization protocols and management practices that ensure fertile ovulations. 2) Treatment of bovine cortical cultures with VEGFR-TKI (8uM) inhibited follicle activation and resulted in more follicles at earlier stages of development. Thus, inhibition of VEGF signal transduction can inhibit follicle progression. This is similar to data in the rat. Using rat perinatal ovaries both VEGF164 (recombinant) and an antibody to the inhibitor VEGF isoforms had similar effects with increases in vascular density and follicle progression to advanced stages of development. While inhibition of the VEGFR2 receptor surprisingly increased vascular development but inhibited follicle progression. Understanding the regulation of VEGF may allow for better manipulation of follicle progression and development in domestic livestock. 3) An inhibitory isoform, VEGF164b has been isolated and sequenced in the bovine. Expression of mRNA for angiogenic isoforms VEGF164, 120 and inhibitory isoform VEGF164b are differentially regulated prior to the LH surge. Both angiogenic isoforms are reduced while VEGF164b is increased at 18 hrs after prostaglandin. Only VEGF164 is increased after the LH surge. Understanding why these isoforms are expressed prior to and after the LH surge will allow us to manipulate ovulation and potentially also alter formation of the CL. 4) Granulosal cell CART mRNA and follicular fluid concentrations of the CART peptide are negatively associated with health status of bovine follicles collected at multiple defined stages of follicular development. 5) CART treatment of bovine granulosal cells inhibits FSH induced influx of extracellular calcium in a Go/Gi dependent manner suggestive of a role for regulation of [Ca2+]i in CART signal transduction. 6) Alternative Expression of FSH Receptor Transcripts: We have previously determined that alternate transcripts of the FSH receptor are produced differently at important stages of follicular development in the cow. To better understand the precise role of each form of the FSH receptor on granulosa cell differentiation and development, stable granulosa cell lines have been obtained with produce either FSHR-1 or FSHR-3, and no other forms of the FSHR. Using these cell lines, a series of experiments is planned to characterize patterns of expression of both mRNA and protein for each of these forms of the FSHR, as well as to validate techniques to enhance our ability to detect each form of the FSHR from bovine tissues or primary cultures of granulosa cells. Oocyte-embryo Physiology.

    1) JY-1 (a novel oocyte/embryo specific gene) mRNA transcripts present in early embryos are of maternal origin and appear to be required for early embryonic development. A cDNA encoding for a putative human JY-1 ortholog has been cloned from human ovary and ES cell RNAs. 2) 1756 RNA transcripts of oocyte and cumulus cell origin representing genes co-regulated and differentially regulated within the two cell compartments during meiotic maturation were identified. 3) A novel algorithm has been developed and applied to characterization of components of the oocyte transcriptome using data derived from 2 channel microarray experiments. 4) A negative association of cumulus cell cathepsin mRNA abundance with oocyte competence and development to the blastocyst stage has been established. Treatment with a cathepsin inhibitor during meiotic maturation enhanced blastocyst development. 5) Bovine/Porcine stem cell project: We have focused on inserting marker genes into pig umbilical cord cells with subsequent injection of cells into blastocyst-stage embryos. If these cells can integrate into various tissues of the developing embryo, it would confirm their identity as pluripotent stem cells.

    Testicular Physiology.

    1) Immunoneutralization of inhibin holds potential as an approach to increase sperm production. Results of study one show that the magnitude of change in sperm production is directly proportional to the inhibin antibody titer achieved. Inhibin appears to be involved in the regulation of the number of primary spermatocytes. 2) Results of study two indicate that inhibin free "-subunit has little if any effect on pituitary LH secretion. Thus, the immunoneutralization of endogenous inhibin free "-subunit likely does not account for the decrease in LH secretion that occurs in ram lambs actively immunized against inhibin free "-subunit. This information will be used in the development of immunogens designed to stimulate gametogenesis. 3) Treatment of indifferent testes with VEGF164 increased vascular density by 57% (P < 0.05) and perturbed seminiferous cords similar to results with another isoform, VEGF120. We determined VEGF164b and 188b mRNA were not expressed in the testis during cord formation and testis morphogenesis, but both were present in the ovary at the same time point. Furthermore, mRNA for Flt1 is expressed in the ovary and not in the testis. We speculate that negative regulators of VEGF, VEGF b isoforms and Flt1, may inhibit mesonephric endothelial cell migration in the ovary where their absence allows for endothelial cell migration in the testis resulting in testis-specific vascular development. 4) VEGF treatment increased graft weight and the percentage of seminiferous tubule cross-sections with elongating spermatids at the time of graft removal. Cultured testis tissue grafts were smaller, and had fewer seminiferous tubules per graft. However, there was no difference in the percentage of seminiferous tubule cross-sections that contained any germ cell type between groups. These data indicate for the first time that bovine testis tissue can be manipulated to better support germ cell differentiation in grafted tissue. 5) We generated data demonstrating that both donor age and grafting period may be important factors regulating the maturation of bovine testis xenografts, indicating that intrinsic differences exist within testis tissue at these donor ages. These data provide the framework for further study of bovine spermatogenesis using ectopic testis xenografting. 6) Examination of gene expression in testis tissue grafts using real-time RT-PCR indicated that Sertoli cell specific genes were lower in 8 week donor tissue grafts compared to the other aged donors. Additionally, c-kit, a germ cell specific gene was nearly absent in grafted tissue. Microarray analysis of donor tissue identified several genes that are involved in angiogenesis or tissue growth that were differentially expressed in 2, 4, and 8 week old bovine testes. Expression of these genes (angiogenin, transgelin, thrombomodulin, early growth response 1, insulin like growth factor 2, and insulin like growth factor binding protein 3) decreased over time. This research identified several factors that could contribute to efficiency of ectopic testis tissue grafting. This knowledge could lead to exogenous treatements for improved ectopic testis grafting techniques. 7) Cryopreservation adversely effected growth, testosterone production, and germ cell differentiation of mouse grafts. Cryopreservation did not appear to influence growth, testosterone production, or germ cell differentiation in pig grafts. These data indicate that cryopreservation is a feasible method for long-term testis tissue storage prior to ectopic testis xenografting. 8) FSH and testosterone contribute to the spermatogonial stem cell niche to regulated stem cell biological activity. 9) Using DNA parent verification there are considerable differences in serving capacity of individual bulls in a multiple sire breeding herd and this variation is not correlated with scrotal size. Uterine-Placental-Fetal-Nutritional Interactions during Pregnancy.

    1) More than one agonist may regulate caruncular endometrial PGE secretion for implantation. 2) In the ewe, the conversion of placentomes from A to D type increases their ability to delivery nutrients to the fetus, thereby protecting the fetus from intrauterine growth restriction. This conversion normally occurs during late gestation in response to the exponential growth of the fetus, but some animals have the ability to convert their placentomes during early gestation in the face of a maternal stressor such as undernutrition. This ability for early placentomal conversion appears to be related to pre-gestational ewe selection, and thus may be epigenetic in nature. These data suggest that the management system under which a ruminant female is selected can have profound effects on her ability to protect her fetus from intrauterine growth restriction in the face of maternal insults such as undernutrition. 3) Fetal intrauterine growth restriction resulting from undernutrition during the first half of gestation in ewes and cows have significant negative health effects on their offspring after birth, including insulin resistance, hyperphagia, obesity, decreased skeletal muscle mass, hypertension and infertility . These health, fertility and body compositional effects of early maternal undernutrition are not alleviated even if females are realimented during the second half of gestation, and are born at a normal birth weight. 4) It was demonstrated that concentration of estradiol during the preovulatory period did not alter total uterine IFNÄ content in pregnant heifers on d 15.5 of gestation but level of nuclear progesterone receptor mRNA in the uterine endometrium tended (P < 0.10) to be greater in females with greater estradiol concentrations during proestrus. 5) The presence of estrogen receptor in plasma membranes of the ovine endometrium can be used to examine the nongenomic rapid actions of estrogen during various stages of the estrous cycle and gestation as related to establishment and maintenance of pregnancy. Ability of progesterone to inhibit oxytocin-induced cellular function in transfected COS-7 cells may serve as a model system to further examine the mechanism by which this steroid acts to maintain pregnancy in ruminants. 6) Adaptive responses to fetal nutrient deprivation have a greater impact on fetal ²-cell development and function than other endocrine cell types within the pancreas. Insulin is an important anabolic hormone in the fetus and fetal ²-cells respond to nutrient concentrations during the second half of gestation; thus, making ²-cells important for coordinating the rate of fetal growth with the maternal nutrient supply to the fetus. We show that the disparity in fetal hypoglycemia and hypoxia increase over the final 30% of gestation, and the ²-cell responsiveness also decreases due to lower ²-cell mass and impaired islet physiology and metabolism. In addition the IUGR fetuses develop a greater sensitivity to insulin, which might be required to respond to the lower ²-cell responsiveness. These adaptations will promote fetal survive during nutrient deprivation, but if prenatal compensatory mechanism persist after birth they could result in complications in animal health, production, or market value. 7) Impaired placental function is the primary cause of fetal growth restriction. We have demonstrated that the development of functional placental insufficiency in sheep is associated with changes in placental expression of the insulin-like growth factors and their binding proteins, increased placental apoptosis, and differential changes in endothelial nitric oxide abundance in the cotyledon and umbilical artery. The observed changes help explain the failure of the placenta to develop normally, and why there is impaired oxygen and nutrient provision to the fetus. 8) Impaired fetal development has been associated with altered postnatal growth and health of the offspring. We demonstrated that prenatal androgen exposure not only reduces size at birth, but also results in changes in left ventricular size and concentration of endothelial nitric oxide and insulin receptor indicative of long-term physiological changes in adult sheep which may impact their health and longevity.

    Nutritional and metabolic effects in non-pregnant animals.

    1) Short-term (5 d) feed deprivation prior to ram introduction during proestrus results in an approximate 30% reduction in lamb crop. Provisions need to be made to ensure that adequate feed resources are available during winter breeding in ewes to prevent feed deprivation that can occur as a result of adverse environmental conditions. 2) In reindeer, seasonal body weight changes and pregnancy status will be correlated with seasonal changes in plasma leptin, IGF-1 and GH and these data combined with retrospective data collected in 2002/03. 3) We have determined that nutrition may affect expression of VEGF isoforms in the ovulatory follicle. There was an increase in VEGF120 isoform in heifers fed whole soybeans for 167 days during heifer development that were aspirated 60 hours after synchronization with prostaglandin compared to controls. These differences were not correlated with changes in progesterone or estrogen. Also there was not difference in VEGF164 isoform. We are presently evaluating VEGF164b isoform. 4) Heifers which are more efficient in feed utilization, low relative feed intake, have less body fat than less efficient heifers. 5) Heifers with low relative feed intake reached puberty at similar rates as their less efficient (but fatter) herdmates.

    Behavior.

    1) Both adrenal cortical and medullary function are influenced by temperament in response to typical animal handling procedures, including transportation. 2) Weaned calves with poor temperaments are less competent immunologically than those with calm temperatures. 3) Breed type of cattle influenced anatomic parameters of the hypothalamic-pituitary-adrenal axis whereas temperament influences endocrine function of the adrenal cortex and medulla. 4) Cattle with poor temperaments have reduced fat deposition compared with calm cattle.

    Objective 2. Develop and improve assisted reproductive technologies to enhance sustainability of production systems for domestic ruminants.

    Estrus, estrous synchronization and AI.

    1) In reindeer, we found a difference in gestation length between the EARLY and LATE groups (P = 0.002) and a negative correlation (r = -0.754; P = 0.002) between gestation length and breeding date. There was no difference in the birth weight of the calves. 2) We are analyzing reindeer milk for %fat, %protein, %lactose and % solids-not-fat at 3 stages of lactation (15, 45, and 75 d post-calving) from 12 female reindeer. Milk component data from each cow will be correlated with their calfs growth rate and survival. 3) Use of Modified CIDR-b for Estrous Synchronization in Reindeer Bovine CIDR modifications (following manufacturers directions) produced systemic progesterone levels consistent with luteal phase progesterone and had 100% retention rate. Pregnancy rate following synchronization was 88%. 4) Radiotelemetric Estrous Detection in Reindeer. Placement of radiotelemetric transmitters lower on the females rump, coupled with shaving only the top 1.5 cm of hair, were tried in an effort to improve estrous detection in reindeer with this technology. 5) Modifications of the standard CO-Synch + CIDR estrous synchronization program that included: a. shortening the interval from the initial GnRH injection to CIDR withdrawal/PGF2± from 7 to 5 d, b. administering two PGF2± doses 12 h apart and c. increasing the interval from PGF2± to the second GnRH from 60 to 72 h; increased timed-AI pregnancy rates in postpartum beef cows by 13.3%. 6) Development of dominant follicles occurs early after calving and lack of follicular development does not delay return to estrus. It seems to be obligatory for most cows to have a silent ovulation with formation of functional luteal tissue before return to normal estrous cyclicity. 7) Early weaning at the start of a synchronized breeding season increased AI pregnancy rates and cow weights at the time of normal weaning. The magnitude of improvement in AI pregnancy rate is greater than has been reported with temporary calf removal (48 h) indicating the effects of permanent calf removal may be manifested through improved pregnancy maintenance. Early weaning may be a viable alternative to culling cows during periods of low forage production in semi-arid rangelands. We are evaluating the fertility of heifers developed under early and normal weaned strategies. 8) Liver and skeletal muscle progesterone concentrations did not differ from control values in ewes receiving CIDR for 5 or 14 d. Therefore, the levels of progesterone in CIDR treated ewes were within limits allowable for slaughter and increased the chances of CIDRs being approved for utilization in regulation of sheep estrous cycles. 9) Fenceline contact of cows with bulls can accelerate resumption of ovarian cycling activity but it is not as effective as close physical contact between cows. The nature of the biostimulatory effect of bulls appears to be related to the intensity of the pheromonal stimulation. Limiting the intensity of the biostimulatory stimuli of bulls directly limits that biostimulatory effect of bulls on primiparous cows. Thus, a major factor involved in the mechanism of the biostimulatory effect of bulls is intensity of the stimuli (frequency of exposure, duration of exposure, and quantity of stimuli) provided by bulls. 10) Continuous presence of bulls stimulates resumption of luteal activity and is coincident with increased cortisol concentrations. We hypothesize a possible association between adrenal activation and the biostimulatory effect of bulls in postpartum anestrus cows. This may be a critical mechanism for this effect. The biostimulatory effect of bulls alters the progesterone response in an ES protocol that included GnRH, PGF2±, GnRH and TAI, and that progesterone concentrations after the first GnRH injection may determine the success rate of GnRH-based ES protocols in primiparous beef cows. 11) Comparison of CIDR to MGA in a 7-11 Co-synch protocol with timed insemination. Previous research has shown that the 7-11 Cosynch protocol using melengestrol acetate (MGA) is effective in synchronizing beef heifers. This study compared a vaginal insert containing progesterone (CIDR) to MGA in the 7-11 Cosynch protocol on beef heifers. No difference in pregnancy rate was observed between the CIDR (46%) and MGA (47%) treatments.

    Puberty.

    1) Attainment of puberty in early-weaned heifers can be hastened through feeding a high-concentrate diet from approximately 4 to 6.5 mo of age in a manner similar to that of heifers that are continuously fed the high-concentrate diet after early weaning. 2) Early postnatal concentrations of metabolic hormones are related to growth characteristics and may therefore be related to age at onset of puberty. 3) Determined that dried distillers grains has a positive effect on conception to AI when fed during heifer development and therefore is an effective method to provide protein to heifer development diets with no negative effects on reproductive performance.

    Pregnancy detection and maintenance. 1) Detection of low ISG15 mRNA levels in blood on days 17-25 provides an accurate indicator of cows that are not pregnant. This might be used as a management tool to help identify nonpregnant cows 18 days following AI so that they can immediately be re-synchronized and inseminated. 2) The results of these experiments suggest that if intraluteal concentrations of progesterone can be maintained during the period of normal luteolysis that it should be possible to prevent luteolysis and perhaps reduce the 20-35% early embryonic wastage that is attributable to faulty mechanisms associated with maternal recognition of pregnancy. 3) Pregnancy induces many of the same blood and endometrial genes and several of these are involved with interferon and antiviral responses. Many pathways are also uniquely induced in blood, when compared to the endometrium, and may function in maintaining early pregnancy in the cow. 4) The decidualizing uterine stromal compartment, in addition to the ovarian corpus luteum and placenta, is a site of steroid synthesis during early gestation, and the embryo provides signaling cues to tightly regulate steroid biosynthesis at the maternal:embryo interface. Similar mechanisms probably occur in the cow. 8. Maternal nutrient restriction alters gene expression in the fetal heart, which may affect sensitivity of the steer heart to stress by 15 months of age. Differential programming of right ventricular gene expression in the fetus during early gestation may be detrimental to animal health, particularly at high altitude.

    Immunocastration.

    One of the problems with development and approval of a sterilization vaccine for cattle has been the stigma against Freunds complete adjuvant. We have evaluated a bacterial DNA immunostimulant with an ovalbumin-LHRH fusion protein and found the bacterial DNA to be superior to Freunds in stimulating LHRH antibody production.

    Disease.

    1) Persistent infection with BVDV results in antiviral responses in blood cells which includes induction of type 1 interferon-induced genes, chemokine-mediated immune responses and bone remodeling with a concomitant suppression of extracellular remodeling, adhesion and T-cell-mediated responses. 2) Development of a single or multiplexing diagnostic for calves that are persistently infected with BVDV is probable.

    Effort.

    Participants in W-112 have published 68 refereed journal articles, 2 book chapters and one genbank sequence during the past reporting period, many of which were derived from collaborative efforts initiated through the W1112/W112 Regional Research Project. Collaborative projects planned for the coming year are listed below.

    Collaborative Efforts for the Coming Year:

    Collaborators: Alaska, Montana. Project. Determine Glucocorticoid levels in reindeer. Collaborators: Alaska, New Mexico. Project. Determine Thyroid hormone concentrations in blood from reindeer. Collaborators: Alaska, Missouri. Project. Determine Leptin- IGF-1 and GH in blood from reindeer experiments. Collaborators: Alaska, Montana State. Project. Evaluation of cortisol concentrations in reindeer during photoperiod cycle. Collaborators: Arizona, Michigan. Project. Determine the impact of placental insufficiency and IUGR on ovary development to elucidate the potential impact in female reproductive performance. Collaborators: Colorado, Minnesota. Project. Examine the ability of alpha subunit of inhibin to regulate LH secretion in sheep. Collaborators: Colorado, New Mexico. Project: Evaluate the role of luteal PGs in demise of the CL in cattle and sheep. Collaborators: Colorado, Wyoming Project: Undernutrition in fetal development. Collaborators: Colorado, Wyoming, Ohio, Montana. Project: Evaluate blood and endometrial ISG15. Collaborators: Colorado, Penn State Project: Pregnancy induced gene expression in bovine and ovine blood and endometrium. Collaborators: Colorado, Idaho, Missouri, Ohio, USDA (Miles City). Project: Examine ISG15 mRNA in blood from pregnant cows. Collaborators: Colorado, Texas. Project: Examine ISG15 mRNA in blood from pregnant deer and Brahman cows. Collaborators: Hawaii, Texas. Project: Growth factor influences on bovine caruncular secretion of PGE1. Collaborators: Hawaii, Colorado. Project: Regulation of mRNA for enzymes in the PG synthesis pathways in sheep uterus. Collaborators: Idaho, Ohio, Colorado Project: Determine preovulatory estradiol and uterine expression of ISG-15, MX and other proteins. Collaborators: Kansas, Nebraska Project: Evaluate FSHR and VEGF expression in ovary and testis cultures. Collaborators: Michigan, Wyoming. Project: Determine factors affecting oocyte quality. Collaborators: Montana State and USDA (Miles City, Montana). Project: Utilization of the biostimulation effect of bulls to improve estrus synchronization response and fixed-time AI in postpartum cows. Collaborators: Nebraska, Arizona. Project: Determine effects of nutrient restriction in lambs on testis development and VEGF production. Collaborators: Nevada, Wyoming. Project. Differential gene expression in placentomes as they progress from type A to type D. Collaborators: Nebraska, WSU. Project. Role of VEGF in bovine testis grafts. Project. Spermatogonial germ cell transplantation Collaborators: Nebraska, Oregon. Project. Evaluate vascular development in fetal gonads from sheep. Collaborators: Ohio, Michigan State. Project. Examine CART expression in granulosa cells of follicles following estradiol induced atresia. Collaborators: Oregon, Wyoming. Project. Collaborate on MOR and FOR ram analysis. Collaborators: Texas, USDA (Miles City). Project. Feed efficiency in developing heifers Collaborators: Texas, USDA (Miles City, Montana). Project. Examine amygdallas characteristics associated with high and low EV bulls and heifers after puberty. Collaborators: WSU, USDA (Miles City). Project. Determine differences in pituitary gene expression in cycling and postpartum anestrus cows. Project. LHRH immunization. Collaborators: USDA (Miles City), Ohio. Projects. Effect of early weaning on age at puberty, 5 vs 7 d Co-synch + CIDR, estrogen and fertility at timed AI. Collaborators: USDA (Miles City), California. Project. Evaluation of gonadotropin analogs. Collaborators: USDA (Miles City), Colorado. Projects. Early Pregancy gene expression related to ovulatory follicle size/maturation. Progestin/Progesterone affinity for different progesterone receptors. Collaborators: USDA (Miles City), Missouri. Projects. Reciprocal ET of cows ovulating large or small follicles into recipients ovulating L/S follicles. Collaborators: Washington, Nevada. Projects. Gene expression in developing testis. Collaborators: Washington, Minnesota Projects. Inhibin A- immunized rams and effects on spermatogenesis. Collaborators: Wyoming, California, Nebraska, Arizona. Teaching Collaborations. Sharing of notes, lectures, case studies for Mammalian Endocrinology course.

    Impact Statements:
    1. Advances were made in understanding how nutrition impacts fetal development, placental efficiency, heifer development, estrous cycles, development of more viable offspring.
    2. Significant progress in understanding how behavior, exposure to male biostimulation, and disease may affect general overall health, feed and reproductive efficiency.
    3. Progress was made in identifying factors that can be commercialized for pregnancy detection, and immunocastration techniques continue to be fine tuned to be more producer friendly
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