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W1112: Reproductive Performance in Domestic Ruminants

Annual/Termination Reports (SAES-422): [07/24/2007] [10/15/2008] [08/31/2009] [06/16/2010] [07/14/2011]

Date of Annual Report: 07/24/2007

Report Information:
  • Annual Meeting Dates: 06/04/07 to 06/05/07
  • Period the Report Covers: 10/2006 to 09/2007

    • Participants:
    Brief Summary of Minutes of Annual Meeting:
    Chair Andrea Cupp called the meeting to order at 8 a.m. on Monday June 4, 2007. Participants were introduced and the minutes from the 2006 meeting were approved without discussion. Tod Hansen welcomed everyone to the Animal Reproduction and Biotechnology Lab (ARBL) and the Equine Reproduction Lab (ERL) at CSU. Tod provided historical comments and a description of ARBL and the ERL, the CSU Foothills Campus and a description of research and training program. The group thanked Tod, Terry Nett and the rest of the CSU group for local arrangements. Registration fee for the meeting was $50 per attendee.

    Colin Kaltenbach reported that we are completing year 1 of the W1112 multistate project and the termination date is September 30, 2011 and encouraged participants to submit their reports in a timely manner. All participants need to keep the email list up-to-date by making an email address changes on the NIMS system. The group asked Colin for an update on efforts to transition USDA Hatch or Experiment Station funds from the current system into a competitive funding system. Deb Hamernik will cover this in her comments. Colin reported that there is no effort or plan to stop multi-state projects or committees because these have a high priority. Deb Hamernik welcomed Clay Lents from the University of Georgia to the group and noted that Matt Cannon from Oregon State University asked to join the project. Everyone was reminded when submitting a Hatch project to make sure assurance statements are valid before submission into the CRIS system. The biosafety and IACUC approval dates must be valid or the submission will be returned to the Experiment Station. Deb described some of the legislative activity that may affect extramural research funding through the Farm Bill. There is a proposal to merge ARS and CSREES administration offices in Washington DC to eliminate administrative duplication. The CREATE-21 and NIFA programs have been proposed in an attempt to double the funding for agricultural research. In the USDA Secretarys proposed 2007 Farm Bill, new funding is proposed for specialty crops, bioenergy/bioproducts, and organic agriculture (primarily crops); however, no new funding for animal production was proposed. The budget for fiscal year 2007 was unusual because Congress did not pass the budget until after 10/01/2006. Therefore the same amount of money was appropriated in 2007 as 2006. Agricultural earmarks were eliminated from the USDA FY2007 appropriation. In FY2007, only the Department of Homeland Security or the Department of Defense received earmarks. The NRI received a small increase in budget in 2007 and expects a similar small increase in 2008. All NRI grants require electronic submission. Applications to NRI must adhere to the published guidelines (budget caps, pages limits, etc.) or they will be returned without review. All NRI submissions are electronic using the SF424 R&R application forms available on grants.gov. Deb also updated personnel changes within USDA CSREES. Deb was questioned about the size of the awards for NRI grants and she answered that the agency is targeting approximately $100,000 (direct costs) per year. Last, Deb updated the group about the bovine and porcine genome sequencing projects. The cattle genome project is starting the annotation phase of the project and is interested in having scientists join the project to link function with gene prediction. The pig genome project will start annotation next year.

    Business Andrea Cupp explained the rotation and responsibilities of the member at large and the chair of the group prior to asking for nominations for the member at large. Mike Day was elected member at large by a unanimous vote.

    Old business Tod Hansen explained attempts to coordinate the W1112 meeting with the BIF meeting in Fort Collins. The purpose of having the meetings at the same location would provide members of W1112 to present a program on fetal development as an outreach activity. Support from local extension personnel was positive, however, the idea did not get support from BIF organizers so the attempt was dropped. The group expressed appreciation to Tod and Russ Anthony for the effort. Although the symposium with BIF did not work out, several members of the group said they enjoyed holding the meeting at a station.

    Dennis Halford presented an update on his work with the Food and Drug Administration to provide sheep tissues as part of the process for CIDR approval in sheep. The tissues have been submitted to the FDA for analysis.

    Announcements and news from individual stations were given. Individual state announcements occurred with members talking about events, job openings, etc that had happened since our last meeting. New business Laramie, Wyoming was selected as the site for the 2008 W1112 meeting. The meeting will follow the Western Section of ASAS meeting on June 24-26. The W1112 meeting will start in the afternoon on June 26 and finish on June 27. Gary Moss agreed to serve as the local host for the meeting. A comment was made that in the future the group try to have the W1112 meeting in late May to avoid conflicts with meetings that occur later in the summer. Late May 2008 was not an option due to conflict with the 2008 SSR meeting.

    The group continued a discussion from previous years about ways to increase the exposure of the multi-state project to extension personnel or cattle producers. It was suggested that one or two members of the W1112 group participate in the Beef Symposium during the Western Section ASAS meeting in 2008 in Laramie. This would need to be coordinated with the WSASAS Program Committee. Dennis Halford volunteered to discuss this at the 2007 WSASAS meeting and request to be on the agenda for the 2008 meeting. Milan Shipka volunteered to lead a subcommittee to work with WSASAS to develop a symposium or incorporate W1112 members into a symposium at the WSASAS meeting every 3rd year. Several other ways to improve outreach were mentioned including having a symposium and inviting extension specialists to highlight management research, interaction with the Beef Reproduction Task Force and to have a symposium at the WSASAS meeting every third year to highlight research. Colin read the outreach section of the W1112 project, which is available on NIMS, to the group and emphasized that each member of the group already does the type of outreach described in the project. He suggested that each member include these activities in their report. Dean Hawkins suggested a W1112 poster be made that could be sent to members and used at meetings. A new poster could be made each year and the each new poster would highlight a different objective of the project. The group agreed that a poster should be made and Derek McLean and Dean Hawkins will work on the poster. Members were asked to submit material for the poster. Similar to this idea, Milan suggested that when members do a presentation as an outreach activity, they share the powerpoint file with the other members of the group. The group discussed the use of the W1112 website as a way to share information and powerpoint presentations. It was suggested that in the future a My Space or Sharepoint type of format be used for the website so each station can submit materials including research and teaching resources. Jim Berardinelli updated the group on the website and said the website would need to be set up so that each member can sign in before submitting material to keep the site secure. Jim will work with the information technology person at MSU to update the website.

    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 11:00 am on June 6, 2007.

    Collaborative Efforts: Collaborators: Nebraska, Wyoming Project: Analysis of testes from rams treated with RU486 from 4-8 weeks. Project: Evaluate behavior, size, and VEGF expression in MGA prepubertal bulls treated with RU486 at 4-8 weeks. Project: Evaluate VEGF isoforms in tissues from fat ewes and malnourished ewes.

    Collaborators: Montana State, Fort Keogh (Miles City, USDA) Project: Evaluate breeding performance of primiparous suckled beef cows exposed to bulls using a CID7 estrous synchronization protocol and TAI.

    Collaborators: Arizona, New Mexico Project: Utilization of sheep for research projects

    Collaborators: Montana, New Mexico Project: Cytology studies of main olfactory epithelium, neuro-sensory epithlia, and mucal odorant binding proteins in pre-ovulatory beef heifers

    Collaborators: Colorado, Minnesota Project: Fetal tissue collection for steroid transfer

    Collaborators: Washington, Minnesota Project: Inhibin assay of bovine grafts in mice

    Collaborators: Wyoming, Minnesota Project: Fertility testing of inhibin immunized ram lambs

    Collaborators: Colorado, Hawaii Project: Utilizing mRNA for LH receptors in the ovine corpus luteum and effects of PGE, PGE2, and endocannibinoid receptor agonists for CB1 and CB2 receptors in vivo in ewes on luteal function.

    Collaborators: Hawaii, Texas Project: Effects of endocannibinoid receptor agonist for CB1 and CB2 receptors on bovine luteal function in vitro.

    Collaborators: Alaska, Colorado Project: Anti-GnRH in reindeer bulls

    Collaborators: Alaska, Montana Project: Mid-cycle bull exposure and cortisol levels

    Collaborators: Georgia, Texas, Wyoming Project: Study the influence of feed efficiency on beef cow reproduction

    Collaborators: Arizona, Nebraska Project: Effect of IGR on testis development

    Collaborators: Arizona, Michigan Project: Impact of IUGR on ovaries

    Collaborators: Kansas, Nebraska Project: Evaluate FSH receptors in testes and ovaries during development Project: Quantitation of FSHR3 and FSHR1 at different stages of embryonic development in male and female rats.

    Collaborators: Nebraska, Washington Project: Effects of inhibitory VEGF isoforms, VEGF, and GDNF on spermatogenesis

    Collaborators: Michigan, Kansas Project: Evaluate CART signaling in stable cell lines over expressing FSH Receptors 1 and 3. Project: Sharing cell lines. Look at FSHR1 and FSHR3 expression and signaling at different stages of follicular development

    Collaborators: Michigan, Washington Project: Search for CART and JY-1 mRNA expressions in bull testis tissue.

    Collaborators: Kansas, New Mexico Project: Effects of energy and protein on expression of FSH-R variants at different stages of follicular development.

    Collaborators: Washington, Kansas Project: Differences in bovine and ovine FSHR-3 and FSHR-1 during development

    Collaborators: Wyoming, Arizona Project: Islet isolation and function in vitro

    Collaborators: Colorado, Wyoming Project: Determining fetal response to maternal infection with BVDV.

    Collaborators: Colorado, Texas Project: Determining early pregnancy in deer

    Collaborators: Colorado, The Pennsylvania State University Project: Optimizing pregnancy detection in cattle.

    Collaborators: Colorado, The Ohio State University Project: impact of reproductive steroid hormone concentrations on uterine mechanisms regulating embryonic survival in cattle

    Collaborators: WSU, USDA (Miles City). Project. Determine differences in pituitary gene expression in intact, LHRH immunized and castrated bulls.

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

    Estrous cycle Physiology/ CL function 1. Both mRNA for nitric oxide and polyamine synthetic enzymes and corresponding product or enzyme activity fluctuate during the ovine luteal phase and in response to PGF2±.

    2. Nitric oxide is not luteolytic when infused chronically intrauterine in ewes, but instead may be luteotropic or antiluteolytic via alteration of the PGE:PGF2± ratio.

    3. Endothelin-1 (ET-1) is not luteolytic in ewes, but instead may be luteotropic or antiluteolytic via alteration of the PGE: PGF2± ratio in inferior vena cava plasma.

    Hypothalamic-Anterior Pituitary Gland Physiology 1. The non-genomic effect of estradiol (E2)-BSA, delayed the beginning and the length of the massive release of LH, probably by targeting only the pituitary gland. However it was not able to obliterate the robust effects of E2 induced by acting at both, the hypothalamus and the pituitary gland.

    2. Estradiol-BSA stimulated an increase in the number of GnRH Receptors (GnRHR) in cultured ovine pituitary cells comparable to E2 and that stimulation of GnRHR was mediated by a membrane action. 3. The ovine membrane progesterone receptor (mPR) specifically binds progestins as only progesterone, 20±-hydroxyprogesterone and 17±-hydroxyprogesterone significantly displaced binding of 3H-Progesterone to membrane fractions from CHO cells expressing ovine mPR.

    4. Tandem linkage of the genes encoding the a, FSHb, and LHb subunits is leading to the construction and expression of an ovine single chain triple-domain chimeric gonadotropin. The ability to construct dually active single-chain analogs could permit more effective control of the half-life and duration of gonadotropin actions. This should aid in the design of analogs that could be used to promote superovulation and/or out-of-season breeding in sheep.

    Ovarian Physiology 1. The CART peptide is a potent inhibitor of LH stimulated androstenedione production by thecal tissue collected during the early stages of a follicular wave in cattle. These results further support a stage specific local role for CART in regulation of follicular steroidogenesis.

    2. The inhibitory effects of CART on FSH induced estradiol production are potentially mediated via regulation of ERK1/2 and Akt activation and (or) activity. CART treatment stimulates expression/activation of phosphatases that in turn terminate FSH-induced Erk1/2 signaling. CART-induced acceleration of dephosphorylation of pErk1/2 and termination of the FSH-induced Erk signaling pathway is mediated, at least in part, by increased tyrosine phosphatase and serine/threonine phosphatase activity that is transcription and translation dependent.

    3. 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.

    4. Luteolysis in the ewe appears to involve two stages. Prostaglandin F2a (PGF2a)) decreases progesterone production and an influx of calcium initiates apoptosis. Oxytocin induces an increase in intracellular calcium levels in small luteal cells that may lead to apoptotic cell death, that normal luteal concentrations of progesterone prevent the oxytocin and perhaps the PGF2a -induced increase in the number of small and large luteal cells which respond to these hormones with increased intracellular levels of calcium, and that intraluteal secretion of PGF2a was required for structural luteolysis although steroidogenesis was decreased in the corpus luteum. 5. VEGFxxxb antibody may have greater effects at stimulating follicle progression than treatment with either angiogenic isoform VEGF164 or VEGF120. Higher doses of VEGFxxxb antibody are needed in the ovary in E13 organ cultures to have effects on vascular density when compared to the testis. This would make sense since there are greater amounts of VEGF164b in the ovary.

    6. VEGFR-TKI signal transduction inhibitor (inhibits FLT1 and KDR receptors) inhibited both vascular density and follicle progression while the KDR specific inhibitor, V1, inhibited only follicle progression.

    7. VEGF188 is greater during late embryonic ovarian development (E16), while the VEGF120 and 164 angiogenic isoforms were greater after birth- P0 to P3 at the time that primordial follicles are forming and progressing to later stages of development. Expression of VEGFxxxb isoforms are in oogonial clusters, and later during ovarian development in all stages of follicles in pre-granulosa, granulosa and theca cells and in vasculature.

    8. We have determined there is sex-specific expression of VEGF164b with 4 fold higher levels in the ovary during sex-differentiation than in the testis through real-time PCR for VEGF164b. We believe increased expression in the ovary inhibits endothelial cell migration and allows for vascular to develop from the ovary. While in the testis lower levels of VEGF164b allows for endothelial cell migration and development of vasculature from the adjacent mesonephros.

    9. Ovulation of smaller ovulatory follicles results in lower pregnancy rates in beef cows and heifers. A portion of this reduction in fertility appears to be due to reduced subsequent progesterone synthesis.

    Oocyte-embryo Physiology. 1. JY-1, a novel bovine oocyte/embryo specific protein can regulate function of ovarian somatic/granulosa cells and is required for early embryogenesis.

    2. Follistatin treatment of early bovine embryos accelerates time to first cleavage and enhances blastocyst development and cell allocation in favor of trophectoderm cells.

    Testicular Physiology 1. Immunoneutralization of inhibin holds potential as an approach to increase sperm production. Results show that mean daily sperm production per gram testis was increased 26%. Results also indicated that active immunization against inhibin altered testes weight. Total sperm production was more than doubled in those ram lambs that had heavier testis. Evidence did not support the possibility that LH-stimulated testosterone secretion was decreased following immunoneutralization of inhibin.

    2. The adjuvant Emulsigen with the addition of bacterial DNA as an immunostimulant may promote a greater immune response than Freunds adjuvant in bulls. The enhanced immune response may be advantages in the immunoneurtralization of reproductive hormones. Immunization of bulls against a multi-antigenic peptide (MAP) did not increase the immune response over that of a peptide-carrier protein conjugate.

    3. Treatment of E13 rat testes with VEGF164 and VEGF120 increased vascular density by 57% (P < 0.05) and perturbed seminiferous cords.

    4. We determined that an antibody to the VEGFxxxb isoforms increases vascular density by approximately 100% in the testis and also perturbs cord formation thus; inhibiting VEGFb isoforms may be more affective than adding angiogenic isoforms.

    5. VEGFxxxb isoforms are expressed in sub populations of germ cells during early testis development and later are expressed in Sertoli, and interstitial cells.

    6. We have injected VEGFxxxb antibody into KDR/LacZ male mice that are adult and prepubertal and are evaluating effects on spermatogenesis. Preliminary data suggest that VEGFxxxb antibody treatment (which prevents actions of VEGFxxxb isoforms) destroys spermatogenesis in certain stage seminiferous tubules.

    7. Determined protein expression of VEGF protein with a pan-VEGF antibody (both angiogenic and anti- or inhibitory isoforms) during testis development. All three VEGF isoforms- 120, 164 and 188 are present during cord formation.

    8. VEGF has a functional role in promoting germ cell survival and differentiation in bovine testis. Uterine-Placental-Fetal-Nutritional Interactions during Pregnancy. 1. Investigation of pancreatic endocrine formation in sheep determined that mature beta cell replication was occurring concurrently with endocrine differentiation in the fetal pancreas. The highest rates of beta cell proliferation were found during the first half of gestation and then declined to a stable rate in the final trimester. Our data in sheep show that beta cell differentiation and proliferation occur simultaneously during pancreas development.

    2. Fetal adaptations of glucose utilization (GUR), insulin action, and glucose production (GPR) in IUGR fetal sheep were investigated. The results indicate that insulin responsive fetal tissues (liver and skeletal muscle) adapt to the hypoglycemic-hypoinsulinemic IUGR environment by developing mechanisms that promote glucose utilization, particularly for glucose storage, including increased insulin action, glucose production, shunting of glucose utilization to glycogen production, and maintenance of glucose transporter concentrations.

    3. Sheep fetuses with hyperthermia-induced PI-IUGR are hypoxic, hypoglycemic, hypoinsulinemic and fetal oxygen content is negatively associated with norepinephrine (NE) concentrations. Catecholamines increase with fetal hypoxia and inhibit pancreatic beta-cell insulin release in PI-IUGR fetus.

    4. Uterine venous catheters, maternal femoral artery catheters, and uterine artery flow probes were used to investigate early placental development in fetal growth restricted sheep pregnancies. Animals were assigned as either thermoneutral (TN) controls or to hyperthermic (HT) conditions. The data collected so far indicate that while there may be some reduction in uterine blood flow during the first 15 days on treatment, this is countered by elevated maternal arterial pO2 such that oxygen uptake by the uterus is increased.

    5. Vascular endotheial growth factor (VEGF) expression is upregulated in the placenta and may not be mediated through the Hif-1 pathway. A 5x5 microarray experiment identified other pathways that may result in induction of angiogenic growth factor expression. 6. Alterations in maternal endocrine status may have influenced maternal transport of nutrients to the developing fetus, resulting in the observed fetal growth restriction. Further, mammary gland wt may have been impacted by maternal endocrine patterns during pregnancy.

    7. It appears that nutritional restriction any time during mid- to late pregnancy impacts placentome number, and that cellularity of the placentome can be altered by Se or mid-gestational diet regardless of nutritional levels during late pregnancy.

    8. It appears that placentome types do not differ in vascularity or caruncular or cotyledonary angiogenic factor expression.

    9. 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 occurring in offspring of nutrient restricted mothers are not alleviated even if females are realimented during the second half of gestation, and offspring are born at a normal birth weight.

    10. 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. 11. Maternal obesity from conception through mid-gestation results in a marked increase in the size of the fetal pancreas and the numbers of insulin secreting ²-cells that it contains per unit area. Because the ²-cell population is derived from pluripotent cells which are partially differentiated and can under go only a limited number of cell divisions, a premature acceleration of ²-cell mitoses could have detrimental impacts on pancreatic function in later life.

    Nutritional and metabolic effects in non-pregnant animals. 1. In reindeer, changes in body weight over the entire season were not different between groups bred early and late. Plasma will be analyzed for leptin, IGF-1, GH, progesterone, and estrogens.

    2. Genetic markers in reindeer are being identified to associate DNA polymorphisms with milk yield, milk composition and calf growth rate. Milk samples were collected from reindeer cows and analyzed for % protein, % fat and % lactose. All calves were weighed at birth and weekly thereafter until weaning and average daily gain was calculated.

    3. Estradiol (E2) clearance is decreased during nutritional restriction in sheep. This suggests that dietary alterations in SHBG expression and/or enterohepatic recirculation are also involved in the decreased E2 clearance during fasting or nutrient restriction.

    4. LH pulse frequency was suppressed in fasted relative to fed wethers even when fed and fasted animals had uniform circulating E2 concentrations. This result supports the postulate that energy restriction enhances the negative feedback potency of E2.

    5. Changes in estrogen clearance associated with nutritional status markedly affect the dynamic pattern of LH secretion in sheep LH is affected by GnRH produced in the pituitary.

    6. Feed restriction during post-weaning development of replacement heifers improved efficiency of feed utilization. However, this improved efficiency was accompanied by a 5% reduction in pregnancy rate. Harvested feed requirements per pregnant heifer were reduced by 22% during the 140-d development period, representing a major potential for cost reduction.

    Behavior 1. Acclimatization to facilities and protocols for 48 hr is necessary for intensive blood sampling in primiparous, postpartum, suckled beef cows.

    2. Bull exposure did not alter mean concentrations of cortisol in cows; however, bull exposure changed temporal patterns of cortisol concentrations by decreasing the frequency, increasing the duration of pulses and synchronizing the pulsatile rhythm of cortisol in anestrous, suckled beef cows.

    3. Bulls did not appear to influence systemic glucose and thyroid hormones in primiparous postpartum suckled beef cows. However, it appears that adipose metabolism in postpartum beef cows may be affected by the presence of bulls.

    4. Exit velocity can be measured reliably as early as 21 to 24 days after birth. Calf exit velocity is associated with serum cortisol concentrations but not with temperament of the cow.

    5. Progesterone appears to facilitate sexual behavior in rams

    6. Expression of the progesterone receptor in hypothalami of rams may be important for sexual differentiation of behavior.

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

    Estrus, estrous synchronization and AI 1. Switchback breeding trial of variability of gestation length in reindeer showed that gestation length was negatively correlated with day of breeding for both years indicating earlier bred cows have longer gestation length. In addition, gestation length did not differ for cows carrying female calves compared to cows carrying male calves, calf birth weight did not differ between males and females and there was no significant correlation between dam age and gestation length.

    2. The relationship of dams body weight, milk components, and milk energy density to reindeer calf growth rate in early and late calving reindeer showed that calf ADG did not differ between groups.

    3. Recently, an intravaginal progestogen product, Eazi-Breed CIDR, was approved for estrus synchronization of beef heifers. Comparison of CIDR to MGA in a shorter-term time-breeding program demonstrated that a greater percentage of heifers were pregnant in the CIDR treatment (55%) than in the MGA treatment (33%).

    4. Ultrasound analysis can be used to identify reliably (phenotype) cattle with consistently high follicle numbers and low circulating FSH concentration or low follicle numbers and high circulating FSH concentrations during the follicular waves of estrous cycles and such animals differ in their response to superovulation.

    5 Progesterone is rapidly metabolized by ovine liver but is stable in skeletal muscle of sheep during long term freezer storage. These data should allow FDA to finalize a decision on whether to approve CIDR use in sheep. 6. Application of intravaginal progesterone during the early postpartum period in spring lambing ewes failed to induce return to estrus.

    7. Use of an intravaginal progesterone insert in prepubertal ewe lambs failed to induce puberty at 6.5 mo of age.

    8. The use of CIDR as a progestin source in a 14-d progestin, PGF2a, and timed AI and GnRH estrous synchronization protocol is as effective as the use of MGA to synchronize estrus and generate AI pregnancies in beef heifers.

    9. Using a CIDR for 7 d with PG given upon of removal of CIDR yields a superior estrous synchronization response compared to that of using a CIDR for 14 d followed 17-d later by PG. However, both CIDR protocols yield similar and acceptable AI pregnancy rates when combined with TAI and GnRH in primiparous beef cows.

    10. MGA was a useful tool for shortening length of the lambing season, and although PG600 enhanced ovulation rate, it had no beneficial effects on ewe productivity.

    Puberty 1. MGA when fed prior to puberty can affect testis size and alter testosterone production in yearling beef bulls. This increase in size is not an alteration in testis composition but a result of a larger testis. Effects on spermatogenesis need to be conducted; however, this may be a viable way to increase testis size and optimize sperm production in male seedstock.

    2. Initial investigation on linseed meal (LSM) supplementation during early lactation does not appear to impact onset of puberty in heifer calves. However, it appears that LSM supplementation may enhance wt gain without influencing BCS of heifer calves.

    Pregnancy detection and maintenance 1. Maternal viral infection during pregnancy programs fetal development and induces a differential type I interferon response.

    2. Pregnancy loss in Isg15 null mice is a maternal phenotype that is manifest during implantation

    3. Deletion of Isg15 gene results in differential gene expression and pregnancy loss between 7.5 and 12.5 dpc

    4. Periattachment factor (PF), a nuclear protein, was first identified in the bovine conceptus at markedly greater concentrations at d 17.5 than d 15.5 of gestation. The robust increase in PF mRNA during a critical time point in early trophoblast expansion, along with its identification in other expanding/regenerating epithelium, may suggest an important role in conceptus development.

    5. Regulation of Receptor transporting protein-4 RTP4 during early pregnancy may contribute to the complex signaling cascade associated with pregnancy recognition in this species.

    6. Ovine(o) Mx2 expression, like oMx1, is regulated by IFN tau and is expressed in distinct cells of the uterus compared to oMX1. The functional significance of the different cellular localization between oMx1 and oMx2 is not clear, but may suggest that these related proteins possess distinct cellular functions.

    7. The oMX1 promoter and enhancer regions were cloned and characterized. Identifying positive and negative regulatory regions in MX1 promoter may suggest a complex regulation of MX1 during early pregnancy in ruminants.

    Immunocastration 1. Luteinizing hormone-releasing hormone immunization alters pituitary hormone synthesis and storage in bulls and steers. Synthesis and storage of LH and FSH, as measured by pituitary LH and FSH content and expression of the LH ²-subunit and common ±-subunit genes, was suppressed by LHRH immunization.

    2. A Bacterial oligodeoxynucleotide immunostimulant is superior to Freunds Adjuvant for LHRH sterilization vaccine in heifers. Treatment with 3.4 mg ova-LHRH in CpG ODN 2006/w-o per immunization is recommended for LHRH immunization in heifers.

    3. A longevity study in male rats shows that immunization with LHRH-ovalbumin fusion protein did not shorten the lifespan of the rats, and in fact may have increased the lifespan by several months. This gives some comfort to the vaccine not being detrimental to other systems other than the reproductive system in the animals body.

    4. The use of a LHRH vaccine in white tailed deer achieves 80% contraception after 4 years. Simliarly a single shot porcine zona pellucida (PZP) vaccine in white-tailed deer achieved 80% contraception after 5 years.

    Disease 1. Evidence for steroid hormone regulation of components of the uterine antiviral defense could lead to the development of more effective strategies to block or treat sexually transmitted viral infections.

    2. Pronounced antiviral response in maternal and fetal ncpBVDV infection is accompanied by up regulation of ISG15.

    3. Development of a single or multiplexing diagnostic for calves that are persistently infected with BVDV is probable.

    Animal Growth 1. Calm calves have heavier weaning weights than excitable calves. Calves with medium or high serum protein 24 hours after birth have heavier weaning weights than calves with low serum protein. Calves nursing calm or normal temperaments cows had higher serum protein than calves nursing excitable cows.

    2. Hauling stress has negative effects on body composition traits, specifically % intramuscular fat and rib fat.

    3. Exit velocity measured at feedlot entry failed to be indicative of feedlot performance of steers.

    4. We continue to work with the estrogen and phytoestrogen (SDG from flax) model. Previous data indicate that SDG may be anti-estrogenic with increased lengths of feeding. We have found that lactational exposure to SDG in beef cows does not impact puberty, but may influence growth in heifer calves.

    Immune Function 1. Excitable calves had lower serum IgA concentrations 24 hours after birth compared with calm calves. Serum IgG and IgM concentrations were negatively correlated with serum cortisol concentrations. These results suggest that temperament can influence passive immunity in the newborn calf.

    2. Serum protein at 24 hours of age is a better predictor of weaning weight than plasma protein or serum IgG, IgA or IgM.

    3. Temperament and thus increased body temperature of calves can negatively affect the response of calves to vaccination.

    Impact Statements:
    1. Advances were made in understanding how nutrition impacts fetal development, placental efficiency, heifer development, estrous cycles, and development of more viable offspring
    2. New image analysis programs have been developed to investigate vascular development and follicular growth
    3. Significant progress in understanding how behavior, exposure to male biostimulation, and disease may affect general overall health, feed and reproductive efficiency.
    4. Progress has been made in identifying factors that can be commercialized for pregnancy detection, and immunocastration techniques continue to be fine tuned to be more producer friendly.
    Last Modified: 24-Jul-2007

    Date of Annual Report: 10/15/2008

    Report Information:
  • Annual Meeting Dates: 06/26/08 to 06/27/08
  • Period the Report Covers: 10/2007 to 09/2008

    • Participants:
    Brief Summary of Minutes of Annual Meeting:
    Chair Derek McLean called the meeting to order at 1 p.m. on Thursday, June 26, 2008. Participants were introduced and the minutes from the 2007 meeting were approved without discussion. Doug Hixon, Chair, Department of Animal Sciences, University of Wyoming, welcomed everyone to UW in the Mary Mead Meeting Room at the UW Livestock Center.

    Colin Kaltenbach indicated that the summary report for W-112 needs to be completed in 60 days and stressed that the impact statements contained in the individual reports are of increasing importance.

    Brenda Alexander discussed the arrangements for dinner and lunch the following day. The registration fee for the meeting was $60 and it was arranged that Colin would collect this fee at the first break.

    The group thanked Steve Ford, Brenda Alexander and Gary Moss for local arrangements.

    Deb Hamernik reviewed a handout for the USDA update. Vacant positions at USDA and grant writing workshops were highlighted. She drew attention to the high priority research and extension areas listed in the handout as a result of the Farm Bill authorization. Deb reviewed organizational changes and acronyms. FRI replaces NRI, with similar priority areas with the additions of bioenergy and conventional plant breeding. Funding allocations were discussed with 60% to fundamental research, not less than 30% to multidisciplinary efforts, not less than 40% for applied research with funding increased to 700M/year. She also indicated that indirect costs increased to 22%. The establishment of the Research, Education and Extension office (REEO), and the establishment of NIFA to replace CSREES was described. The intent is the raise the profile of agricultural research and will be led by a director that is appointed by the President for a 6 year term. Deb shared a series of graphs which profiled the changes in competitive and state funding since 1985, changes in earmarked funds, funding level by animal species, and number of faculty and PhDs being trained in animal sciences. Funding level by state for research with beef cattle was discussed. Proposed NRI funding budgets and statistics for funding were discussed. Talked about impacts and impact statements, and Deb reiterated the importance of impacts. G. Smith questioned Deb about the potential delay in the RFA with the new NIFA organization. D. Hallford asked about the NRSP7 program and Deb indicated that there will still be some money for this program next year.

    Business

    Gary Moss was nominated for member at large by T. Geary and Kim Vonhame was nominated by Andy Roberts. Gary Moss was elected as the new Member at Large.

    Three sites for 2009 were nominated: Reno by Hawkins; New Mexico by Cupp; and Overton by Weems. (Voted to hold the 2009 meeting in Reno the following day). Dennis Hallford agreed to coordinate meeting arrangements for Reno.

    J Berardinelli made a motion to have the meeting May 18-19, 2009, this was seconded by T. Geary. These dates were approved by acclimation.

    Old Business:

    The creation of a poster to advertise W-112 to constituents was discussed by the group. The poster would highlight one of three main areas for the group, and would rotate between these areas every 2-3 years. The poster will be distributed electronically between members so that it can be edited as segments highlighted as appropriate. Derek volunteered to build the first poster and then circulate it to the membership.

    The W-112 website is with J. Berardinelli on his computer. Jim discussed the design of the page, some of the options that can be used, indicated he is working on the web page and that some revisions will be posted in August, 2008.

    New Business:

    None brought forward. Members shared open positions at their institutions.

    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 4:00 PM on June 27, 2008.

    Collaborative Efforts:

    AZ/WY Examine undernourished ewe tissue for ghrelin and ghrelin receptor.

    HI/TX/CO Implant CL with vehicle, PGE1 or PGE2 to examine mRNA for LH receptor, bound and unbound LH receptors, luteal weights and progesterone profiles. Examine lysophospahtidic acid (LPA), agonists and antagonists of estrogen receptors (alpha and beta) and agonists and antagonists of LPA receptors LPA2 and LPA3 on progesterone, PGE, PGF2a secretion by luteal tissue in vitro.

    CO/WY Examine gene expression in CL from pregnant and nonpregnant sheep.

    CO/PZ Provide antibodies against ISG15 for MX studies.

    CO/OH Examine ISG15 expression in CL collected on d 218 following high or low estradiol exposure.

    CO/TX Examine endocrine function of IFN-tau on the CL using uterine vein infustion.

    CO/TX/MT (USDA) Continue work on utility of low expression of ISG15 as a marker of nonpregnant ruminants.

    CO/ID Work on purification of gonadotropes and knock down genes involved in second messenger pathways involved in membrane bound steroid receptors.

    CO/NM Evaluate estrogen receptors in tissues from preovulatory beef heifers.

    MT/NM Determine metabolic hormone profiles of postpartum suckled primiparous beef cows in response to bull biostimulation.

    MT/NM Analyze IGF-1 in free ranging elk and postpartum cows.

    MT/MI Cooperate on embryo transfer protocol to evaluate in vivo viability of JY-1 altered embryos.

    MT/AK Cooperate on graduate student training.

    MT/MT (USDA) Cooperate on graduate student training.

    ND/WY Cooperate on IUGR studies in sheep and cattle.

    ND/AZ Collaborate on Ghrelin studies.

    NE/WA Examine role of VEGF in testis function; Microarray analysis of persistent follicles.

    NE/WY Evaluate testicular tissues of rams treated with RU486; Evaluate effects of undernutrition on VEGF expression in oviductal and endometrial tissues.

    NE/KS Expression of FSH receptor (3) isoforms in bovine follicles.

    OH/MT (USDA) Determine dose and duration of progesterone required to induce onset of estrus and prevent initiation of luteal phase.

    TX/GA Continue work on feed efficiency and reproduction interactions.

    UT/WA/MT (USDA) Collaborate on statistical analysis of bovine Affymetrx data.

    WY/ND Work on nutrient restriction studies in pregnant cattle and sheep.

    WY/MT (USDA) Analyze pituitaries of from fasted animals for effected gene products.

    WA/KSU Expression of FSHr3 isoforms in prepubertal pig testis.

    WA/MT (USDA) Gene chip analysis of bovine array data.

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

    Estrous cycle, physiology/CL function

    1. Recent data suggest that PGE1 is a more potent antiluteolytic agent than PGE2 by increasing mRNA for LH receptors and occupied and unoccupied LH receptors on luteal and caruncular endometrium.

    2. IFN-Ä also has an endocrine role in protecting the CL from luteolysis, stabilizing production of progesterone, or programming immune cells such that the CL survives in response to pregnancy.

    3. Flax fed ewes also had more (P < 0.01) omega-3 fatty acids and total fatty acids in plasma. Reports of increased pregnancy rates in dairy cows fed flax may relate to P4 metabolism.

    4. Ghrelin and its receptor (GHS-R1a) are expressed within the reproductive tract of dairy cattle and, thus, may be involved in the metabolic regulation of reproduction.

    5. Slight nutritional differences between low quality forages fed for a short period of time, had no effect on follicular waves, dominant follicle size or cycle length or neuroendocrine function in cyclic beef heifers.

    Hypothalamic-Anterior Pituitary Gland Physiology

    1. E2-BSA does not impact secretion of GnRH when administered to ovariectomized ewes. We infer that this means that E2-BSA does not cross the blood-brain barrier.

    2. There was no surge of GnRH or LH in any of the ewes treated with E2-BSA. This indicates that E2-BSA does not dissociate into E2 and BSA in vivo.

    3. It will be possible to design regimens to alter secretion of LH only at the level of the anterior pituitary gland without influencing secretion of GnRH. This may be used to develop novel mechanisms for synchronizing the LH surge in estrous (ovulation) synchronization schemes.

    4. The chimeric ovine gonadotropin expresses both LH and FSH activity in sheep. Moreover, the ovine-based single chain gonadotropin induces profound and long lasting stimulation of follicle growth. This persistent expression of bioactivity suggests that the recombinant proteins have an extremely long functional life.

    5. The single chain gonadotropins induce an immune response that may preclude their use in treatment protocols that require repeated administration.

    6. The single chain hFSH analogs have a long functional life in sheep. The long-lived nature of the analogs is likely due, at least in part, to the additional glycosylation that is permitted by the incorporation of the CTP segment into the single chain gonadotropin motif.

    Ovarian Physiology

    1. The CART peptide is a potent inhibitor of follicular estradiol production in vivo. , intrafollicular CART injection resulted in a ~ 50% decrease in follicular fluid estradiol concentrations (P < 0.01; Figure 4) and a 10-40 fold decrease in granulosa cell aromatase mRNA abundance 24 h after treatment administration (P < 0.02). Results demonstrate a potent negative regulatory role for CART in regulation of estradiol production in vivo.

    2. Inappropriate regulation of VEGF may also affect primordial follicle formation and progression of follicles. Therefore, VEGF is critical to follicle development and manipulation of this gene may allow us to develop better synchronization regimes or IVF procedures to ensure successful pregnancy outcomes.

    3. VEGF inhibitory isoforms may be involved in sex-specific vascular development and gonadal morphogenesis. In appropriate regulation of VEGF during sex differentiation could lead to abnormal development of vasculature or seminiferous cords and would affect life-long fertility of animals.

    4. VEGF isoforms are increased in estrogenic follicles. Therefore, VEGF isoforms may be a potential marker for viable follicles/oocytes to be used in IVF or embryo transfer procedures

    5. Results indicate that circulating concentrations of IGF-1 during the peripartum period were better indicators of rebreeding performance of first calf heifers than either BCS or BW. Associations of IGF-1 and date of second calving were stronger when IGF-1 was measured prior to, rather than after first calving.

    Oocyte-embryo physiology

    1. Injection of follistatin siRNA 2 did not affect proportion of early and late cleaving embryos (21% vs 19% and 41% vs 37%) and total cleavage rate (80% vs 81%). However, injection of follistatin siRNA 2 decreased the proportion of embryos reaching the 8-16-cell stage (41% vs 59%) and percent blastocyst development (12% vs 27%, P < 0.05).

    2. Experiments were repeated and effects of follistatin siRNA 3 determined (25-30 embryos per treatment, n = 4 replicates). Similar results were obtained as for follistatin siRNA 2 injection. Results support a requirement of endogenous follistatin for bovine early embryogenesis.

    3. Results of the present studies conclusively demonstrate a positive contribution of follistatin to regulation of bovine early embryogenesis and blastocyst cell allocation in vitro thus establishing follistatin as a functional contributor to bovine oocyte competence.

    4. Results support a potential requirement of JY-1 for normal progression through embryonic genome activation. Additional studies will be required to determine if the requirement of JY-1 for normal embryonic transcription is global or JY-1 regulation is restricted to specific genes/gene families and their functional significance.

    5. Indices of embryonic genome activation are impeded in embryos subjected to siRNA mediated ablation of JY-1.

    6. MX1 was present in the membrane fractions of the sucrose gradient, and the protein moved to the lower (more soluble) fractions of the gradient when exosomes were pre-treated with detergent. Finally, CHMP6, a protein associated with MVBs, co-localized with MX1 in oGE cells. These results support a mechanism in which MX1 regulates secretion via MVBs in oGE cells.

    7. Results from a single experiment showed that reduced levels of MX1 caused cells to proliferate at a faster rate than control cells treated with the 5-MIS-MOA or medium only, regardless of IFN tau treatment (p < 0.05). These experiments support the hypothesis that MX1 interacts with TUBB in vitro and provide evidence that levels of MX1 protein may affect oGE cell proliferation.

    8. Results suggest that three splice-variants of MX1 mRNA are present in sheep, and that, unlike MX1a and MX1b, MX1c is highly abundant and is not regulated by IFN tau.

    9. A linear relationship (P < 0.05; r2 = 0.36) between peak estradiol concentrations and total number of nuclei per embryo (grade 1 to 4) was observed. In conclusion, decreasing the duration of elevated estradiol exposure did not affect fertilization rate or embryo development, suggesting that oocyte quality and oviductal function was not impaired in these animals.

    10. IFN-Ä acts in a paracrine manner to affect release of prostaglandin F2±. However, we detect significant release of IFN-Ä in the uterine vein and expression of interferon stimulated genes in corpora lutea from day 16 pregnant sheep. Also, we demonstrate that 24 h infusion of IFN-Ä into the uterine vein in non pregnant sheep induces IFN-stimulated genes in the corpus luteum. We conclude that IFN-Ä also has an endocrine role in protecting the CL from luteolysis, stabilizing production of progesterone, or programming immune cells such that the CL survives in response to pregnancy.

    11. Using mice as a gene deletion model for cattle, we have demonstrated that ISG15 is a molecular sentinel that functions to assist mothers when coping with environmental stressors imposed on pregnancy.

    12. The window of time during early ruminant pregnancy during which the conceptus is rapidly elongating and becoming apposed to the uterine lining, is the time frame in which significant pregnancy loss occurs and coincides with the expression of PF. While the function of this nuclear protein has yet to be determined, the use of lentiviral-mediated RNA interference clearly demonstrates that PF is required for normal conceptus elongation and survival. Additionally, these studies demonstrate the usefulness of lentiviral-mediated RNA interference to study the function of genes expressed in the ruminant placenta.

    Testicular Physiology

    1. In comparison to baseline expression (0-hr), the expression of KDR and Flt-1 were significantly induced (p<0.05) at 3 hrs, regardless of treatment. However, VEGF treatment did significantly increase the expression of Flt-1 and KDR mRNA at 24 hrs when compared to controls. These data support the conclusion VEGF is important in germ cell survival in the developing bovine testis.

    2. At three days of SSC-peritubular myoid cell co-culture, testosterone treatment resulted in a significant increase (p < 0.05) in stem cell colonization following transplantation supporting our hypothesis that peritubular myoid cells and testosterone are important components of the SSC niche in the adult testis. 3. Development of methods to control reproduction of overabundant wildlife and feral species should provide tools to reduce populations of problem species and reduced damage done to agricultural crops and natural habitats of competing species, and reduce the spread of disease to domestic livestock. 4. Development of methods for SSC isolation, propagation and cryopreservation will allow earlier and more wide-spread use of superior males genetics.

    5. SSC cryopreservation will allow fertility to be restored follow treatments (chemo therapy) that deplete adult stem cell populations.

    6. Identification of Y-specific genes or markers associated with increased fertility will allow for enhanced genetic screening of breeding stock and identification of superior males earlier in life.

    7. VEGF inhibitory isoforms may be involved in sex-specific vascular development and gonadal morphogenesis. In appropriate regulation of VEGF during sex differentiation could lead to abnormal development of vasculature or seminiferous cords and would affect life-long fertility of animals.

    8. Examination of bovine testis with in situ hybridization revealed that the bCDYL and bCDYL2 transcripts were found mainly in spermatids, though the amounts of transcripts varied among genes/variants. In addition, antisense transcripts were detected in bCDYL variants 2/3, and 4, and the bCDYL2 gene.

    9. Expression analysis of the bDDX3Y gene in 12 bovine tissues by RT-PCR showed that both transcripts are predominantly expressed in the bovine testis.

    10. An association study among bulls from four groups of high fertility (high non-return rate), low fertility (low non-return rate), sub-fertility, and infertility has indicated that these SNPs and their haplotypes are significantly correlated with bull fertility. Out data suggest the bovine DAZL SNPs can be potentially used in marker-assisted selection (MAS) for male fertility in cattle breeding programs.

    11. Overall, these data demonstrate that GDNF up-regulation of Bcl6b, Erm, and Lhx1 expression through SFK signaling is a key component of the intracellular mechanism for SSC self-renewal.

    12. Results implicate Leydig cells and possibly myoid cells as contributors to the SSC niche microenvironment and add CSF-1 to the list of niche factors influencing SSC self-renewal.

    13. Using magnetic activated cells sorting technology we have been able to isolate a6-integrin, Thy1, and Ep-CAM expressing cell fractions from bull testes. We are now assessing whether these cells fractions are enriched for SSCs compared to the total testis cell population based on expression of other SSC markers and the functional ability of bovine SSCs to colonize the testes of recipient mice following transplantation.

    14. Studies demonstrated that ± L-PGDS reacts with oocytes and also spermatozoa, resulting in increases of in vitro sperm-oocyte binding and inhibition of fertilization, and may have a possible role in bovine fertilization.

    15. Interactions of BSP A1/A2 and 30 kDa and osteopontin with the sperm membrane undergo modifications dictated by the oviductal fluid. BSPs are thought to modulate cholesterol and phospholipid movement from the sperm membrane and help sperm binding to the oviductal epithelium. Furthermore, our model suggests that OPN participates in sperm-oocyte interaction, affecting fertilization and early embryonic development.

    16. Results of two experiments confirm that in ram lambs active immunization against I-inhibin peptide increases sperm production by approximately 26%. The increase may be advantageous in the use of winter-born ram lambs during the following fall-breeding season.

    Uterine-Placental-Fetal Nutritional Interactions during Pregnancy

    1. Across treatments, peripheral progesterone concentrations were decreased (treatment x time, P < 0.05) and steady-state concentrations of PGR mRNA were greater (P < 0.05) in the non-pregnant than pregnant heifers. In summary, peripheral progesterone concentrations, conceptus development and IFNt production were not affected by reducing preovulatory estradiol concentrations.

    2. In sheep fetuses with placental insufficiency induced IUGR, functional changes in pancreatic ²-cells come prior to declines in ²-cell mass and mitosis, indicating insulin might be a primary initiator of fetal growth restriction. Strikingly, these studies indicate that the major impairments in ²-cell development and function take place during the final third of gestation when nutrient and oxygen disparities double due to impaired placental transport. 3. In lambs from IUGR pregnancies, we found an early overcorrection in first phase insulin secretion. The greater insulin secretion responsiveness was superimposed onto persistently higher insulin sensitivity; thus making insulin secretion and insulin action discordant as a result of fetal nutrient and oxygen deprivation.

    4. In the IUGR lambs the greater insulin sensitivity resulted from increased peripheral glucose uptake rather than central insulin sensitivity because hepatic glucose production was similar. These data indicate that a skeletal muscle and adipose tissues are primary targets for the improved insulin sensitivity.

    5. Ghrelin likely affects early embryonic development based on its pattern of expression within the reproductive tract (highest in the ampulla).

    6. The nested factorial probe-level statistical model can be used to assess differential expression in future studies where large sample sizes (number of biological replicates on microarrays) are not practical, as in bovine NT studies.

    7. The statistical tools developed here can be used to study a possible genetic (and subsequently pathway or ontology) basis of successful (vs. failed) NT pregnancies without requiring large numbers of biological replicates.

    8. In the ewe, the conversion of placentomes from A to D type increases their ability to deliver nutrients to the fetus, thereby protecting the fetus from intrauterine growth restriction. This conversion normally occurs during late gestation (>120 days) 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.

    9. The effect of maternal obesity on the ratio of estrogen-receptor (ER) ² to ER± in the fetus may affect the propensity for obesity in offspring by influencing the appetite stimulatory NPY and AgRP

    Nutritional and metabolic effects in non-pregnant animals

    1. Live animal carcass composition is highly correlated with BW and DMI, but not RFI. Parameters of live animal carcass composition may not be useful predictors of feed efficiency in limit fed beef heifers.

    2. The ovulatory response to acute nutritional restriction was invariable in this experiment and not related to RFI. The effects of acute nutritional restriction on ovulation in beef heifers are probably mediated by many factors but the influence of RFI appears to be minimal.

    3. Selection for low residual feed intake may increase the age at puberty in Brahman bulls. Bulls with low residual feed intake are more efficient but do not have as much body fat as their less efficient (high residual feed intake) herdmates.

    4. Limit feeding, a fixed percent of body weight, may be more appropriate for Brahman bulls than ad libitum feeding to establish feed efficiency.

    5. The correlation between prepubertal residual feed intake and postpubertal residual feed intake in heifers was 0.48 (P < .0001). Residual feed intake measured before puberty is only a moderate predictor of variation in this trait during the postpubertal period.

    6. Our efforts in the selenium studies continue to determine how selenium being fed impacts maternal and fetal tissues during pregnancy. Data indicate maternal nutrition impacts circulating hormones, as well as maternal and fetal placental tissues from the offspring. Further data are being analyzed for growth parameters and carcass composition of the offspring born from dams fed supranutritional levels of selenium as well as differing global levels of nutrition. Interestingly, both over- and under-nutrition impact colostrum yield and IgG production.

    7. Work with the estrogen and phytoestrogen (SDG from flax) model indicate that SDG may be anti-estrogenic with increased lengths of feeding. Estrogen and linseed meal impact vascularity of the GI tract in ewes and therefore may impact nutrient uptake in pregnant ruminants.

    8. Results indicate that circulating concentrations of IGF-1 during the peripartum period were better indicators of rebreeding performance of first calf heifers than either BCS or BW. Associations of IGF-1 and date of second calving were stronger when IGF-1 was measured prior to, rather than after first calving.

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

    Estrus, estrous synchronization and and A.I.

    1. In various studies that have included 1700 postpartum cows, timed AI pregnancy rate was 69.8% for cows that received the 5d CO-Synch + CIDR program with the 2XPGF treatment (n=586; Table 2). In cows receiving a 7d CO-Synch + CIDR program or the 5d program with either the 1XPGF or 1XCLP treatment (n = 745) timed AI pregnancy rate was 56.8%. We have achieved the overall aim of this area of study by increasing timed AI pregnancy rate from the 50 to 60% range, up to approximately 70%. We have also concluded that to take advantage of the enhanced timed AI pregnancy rate that is achieved with the 5d program in postpartum cows, two PGF or CLP doses are necessary.

    2. In anestrous cows, GnRH1+ cows had greater follicle growth from d-5 to 0 (P<0.01) compared to GnRH1- but similar growth rate from d-2 to 0 (P>0.10), while CIDR+ cows had faster growth rate from d-2 to 0 (P<0.05) compared to CIDR- cows but the growth rate was similar from d -5 to 0. In both experiments, follicle growth was faster (P<0.05) from d-5 to d0 in cows ovulating large (>11mm) compared to small follicles.

    3. Milk progesterone in non-pregnant dairy goats treated with an intravaginal insert containing progesterone was similar to values quantified in milk from control does over a 22-d period.

    4. Intravaginal inserts containing progesterone do not result in more progesterone in milk than that expected in normally cycling or pregnant does.

    5. Treatment with bovine somatotropin at a synchronized estrus results in increased serum IGF-I and decreased triiodothyronine and progesterone compared with control values.

    6. A hormonal method to induce estrus, ovulation and CL formation in seasonally anestrous ewes was tested. The method did not use a gonadotropin or the isolation of rams before breeding. Elimination of these steps would reduce cost and the separate housing of rams and ewes. The method, which utilizes progesterone delivered via a CIDR-G and an i.m. injection, has been reported to be highly effective in a study conducted in a middle-east country. Present results, however, demonstrated that under Minnesota conditions the method was ineffective. Our findings may preclude Minnesota sheep producers from committing resources to try the published method.

    Puberty

    1. Precocious puberty was induced in 60, 41 and 0% of HIGH-S, LOW-S and CONT treatments respectively. Concentrations of insulin in response to feeding did not differ between the HIGH-S and LOW-S treatments but both of the high energy diets resulted in greater insulin concentrations during the 6 hours after feeding than in the CONT treatment.

    2. Criollo heifers may reach puberty earlier and at lighter body weights than British breeds.

    Immunocastration

    1. One vaccine currently available which may prove useful in a wildlife disease management strategy is GonaCon". This contraceptive vaccine, directed at gonadotropic releasing hormones, has been proven safe and effective in a variety of species. Regulatory approval is expected soon for the injectable form of GonaCon", and efforts are underway to develop an oral form of this vaccine which would greatly expand its potential usefulness in disease management strategies.

    Pregnancy, pregnancy detection and maintenance

    1. Identification of failed pregnancies at 18 days after insemination allows for rapid re-synchronization and re-insemination of open cows every 21 days. This should substantially reduce the calving interval in the dairy industry.

    2. The negative relationship described here between gestation length and the time of conception is very robust and evident from our earliest dataset. This relationship adds to the growing body of information that challenges previously held concepts of gestation length being a species specific, genetically fixed parameter. The magnitude of the negative relationship (- 0.37) is consistent with findings recently reported in Finnish reindeer, Iberian red deer, and New Zealand red deer.

    3. The lack of association between gestation length and the sex or birth weight of calves, both in the switchback study and the larger dataset was unexpected. While this is similar to findings in red deer, it differs from the Finnish reindeer study. In the latter, late conceiving reindeer produced calves of lighter birth weight and significantly more females. Although we found a significant tendency for birth weight of males to increase with gestation length, this wasnt true for females and the sex of the calf didnt vary with gestation length. In fact, there was preponderance (non-significant) of females arising from early conceptions, in contrast to the Norwegian study.

    Animal Growth

    1. 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. This lends support to the idea of epigenetics, whereby environmental factors such as maternal malnutrition and stress can alter maternal gene expression patterns through alterations in methylation or acetylation of DNA.

    2. Maternal obesity from conception through mid-gestation accelerates fetal growth, and results in a marked increase in the size of the fetal pancreas and the numbers of insulin secreting ²-cells that it contains. Because the ²-cell population is derived from pluripotent cells which are partially differentiated and can under go only a limited number of cell divisions, a premature acceleration of ²-cell mitoses could have detrimental impacts on pancreatic function in later life, which will be evaluated.

    3. Undernutrition may impact embryo survival by influencing the expression of oviductal genes that regulate intracellular processes and synthesis of IGF-I in the endometrium.

    4. While it has been reported that when adolescent ewe lambs are impregnated by transfer of embryos derived from mature ewes and placed on a high plane of nutrition throughout gestation fetal growth restriction ensues, this does not appear to happen when the ewe lambs are naturally mated. This may infer that the earlier reports result from embryo x nutrition interaction that does not occur in naturally mated ewe lambs.

    Disease, immune function

    1. Persistent infection with ncpBVDV induces a chronic type I interferon response. Because type I interferon acts as a growth suppressive cytokine, this long-term upregulation may contribute to the IUGR seen in persistent BVDV and in other viral infections during pregnancy.

    2. Bovine fetuses infected with BVDV prior to development of the fetal immune system do not recognize the virus as foreign. This failure of recognition of the virus is believed to be caused, in part, by an inability to mount a strong innate immune response.

    3. Infection with ncpBVDV induces a vigorous type I IFN response in acutely infected animals. Presence of a fetus persistently infected with ncpBVDV causes down-regulation of SDF-1/CXCR4 signalling in the blood of the dam. This could have deleterious consequences on fetal development and the immune response.

    4. Alteration of lymphocyte function is important for rescue of the bovine corpus luteum during maternal recognition of pregnancy.

    Behavior

    1. Maximum rectal temperature is reached within 20 minutes of onset of transportation. Exit velocity is correlated (0.62; P < 0.01) with rectal temperature during transportation. Temperamental cattle have increased cortisol and epinephrine concentrations as well as increased rectal temperature during transportation.

    2.Temperament affects the degree of response of the bovine adrenal medulla but not cortex to a lipopolysaccharide (e Coli) challenge. Calm animals had higher concentrations of epinephrine following the challenge than temperamental animals.

    3. Temperament does not affect growth of the suckling calf; however, temperament does influence postweaning growth traits of calves.

    4. Bull exposure decreased cortisol pulse frequency and lengthened cortisol pulse duration before ROA. The results indicate that changes in characteristics of cortisol concentrations patterns precede the biostimulatory effect of bulls to induce resumption of ovulatory activity in postpartum, anovular, suckled beef cows.

    5. Androstadienone, administered oro-nasally to postpartum cows did not alter mean, baseline, and pulse amplitude of cortisol. However, androstadienone exposure appeared to decrease pulse frequency and increase the duration of pulses of cortisol. These preliminary results indicate that androstadienone may be a putative pheromone involved with the biostimulatory effect of bulls on resumption of ovulatory activity in postpartum, suckled beef cows.

    6. A phase of testosterone-responsive sexual development occurs in rams at 6 to 8 weeks of age. Progesterone antagonist administered during this phase perturbed subsequent testosterone production but did not overtly influence postpubertal sexual behavior.

    7. Progesterone appears to be more important for the expression of sexual investigative behaviors than consummatory behavior in adult rams.

    8. While reindeer are affected by male presence and can be estrous synchronized, the male effect does not cause luteal demise followed by a return to estrus. Introduction of the reindeer bull during mid-estrous cycle does not explain the occurrence of repeated short estrous cycles reported by other researchers.

    Impact Statements:
    1. Our increased understanding of pituitary, testis, oocyte, follicle and corpus luteum function/development enables us to more efficiently manipulate ovulation, estrus, puberty, pregnancy and production of sperm.
    2. New image analysis programs and statistical procedures have been developed to investigate vascular development, follicular growth and microarray data.
    3. Progress has been made in identifying factors that can be commercialized for pregnancy detection, and immunocastration techniques continue to be fine tuned to be more producer friendly.
    Last Modified: 15-Oct-2008

    Date of Annual Report: 08/31/2009

    Report Information:
  • Annual Meeting Dates: 05/18/09 to 05/19/09
  • Period the Report Covers: 10/2008 to 09/2009

    • Participants:
    Brief Summary of Minutes of Annual Meeting:
    Chair Dean Hawkins called the meeting to order at 8 am on Monday, May 18, 2009. Participants introduced themselves and minutes from the 2008 meeting were approved without discussion. The group thanked Dennis Hallford and Mike Teglas for making the arrangements for the meeting in Reno.

    Colin Kaltenbach indicated that the summary report for W-112 needs to be completed in 60 days and stressed that the impact statements for the committee were of increasing importance. A handout was provided that gave some guidance as to the desired content of impact statements. It was stressed that we should have 2-3 key impacts to highlight each year and that during the meeting the next day, we would have a session to identify these key impacts. Mark Mirando indicated that impacts should address exactly this, not reiterate results and for fundamental research, impacts should identify the critical knowledge gaps that were closed as a result of the research.

    Mark Mirando provided a handout regarding USDA Programs. The National Institute of Food and Agriculture (NIFA) was created in the last farm bill and will be led by a director appointed by the president.

    " A budget page from Mark that included NRI/AFRI funding was reviewed. Appropriations for 2009 were discussed. Grants.gov appears to be working well for proposal submission. Noted that strict deadlines are being enforced, and validation of a submission could take up to 48 hours, therefore submit proposals early! " The new Integrated Solutions program was discussed. With the new program, integrated proposals will no longer be included in the Animal Reproduction program. Bill Weiss is the panel manager for integrated solutions and Pat Johnson is manager for the animal reproduction. The integrated proposals can request up to 1 million dollars/proposal. A committee of 6 reviewed the pre-proposal letters of intent and approximately 25 proposals were requested for this program in 2009. " It was indicated that eligibility for equipment grants have been restored to previous levels. " Grant workshops were announced and the post-award meetings for USDA-NRI in animal reproduction were discussed. " Mark indicated that Adele Turzillo is new national program leader in animal production systems. " Funding statistics for the NRI last year were discussed. Size of awards is expected to stay relatively constant and it was indicated by Mark that more detailed budget justifications are needed in some proposals.

    The meeting site for next year was discussed.

    Overton, Fargo, Reno, Davis were all nominated and seconded. It was decided to defer the final decision until the next day.

    Dates for the meetings in 2010 were discussed and May 17-18, 2010 was suggested as consistent with the current year. It was unanimously decided to meet in 2010 on these dates.

    Announcements were made by each station regarding new faculty, positions available, changes, etc.

    Mike Teglas indicated he had arranged a Basque meal for the evening for those that wish to attend and will provide directions later in the day.

    Some major announcements were that Niswender retired, Bondurant and Anderson retired, Hamernik left CSREES and is now at UNL, Nett won Hartman award and Reeves the Casida award and Hawkins had moved to WTAM. New members of W112, Jim Pru and Mike Teglas were in attendance.

    Station reports proceeded until 5:30 pm and adjournment for the day.

    On Tuesday morning, presentations continued from 8:00 until 9:45 am.

    Discussion was reopened for the location of the next meeting. Votes were taken for Fargo, Overton, Reno, Davis and after a runoff between Davis and Fargo, it was determined that the meeting will be held May 17-18, 2009 in Davis. It was suggested that the meeting be held in Sacramento to reduce costs, but consensus appeared to favor Davis as the meeting location.

    A discussion of collaborations between stations ensued, which is summarized below.

    Impact statements were discussed, and it was suggested that we submit 2-3 this year. A statement regarding the importance of residual feed intake from Ron Randel, an estrous synchronization statement incorporating primarily work by Mike Day and Tom Geary, and a third impact regarding twinning and lambing rate in ewes that would be provided by Gary Moss were discussed. It was considered that this third impact may be submitted in the following year. Randel and Day were to submit these impacts to Colin Kaltenbach in the next 3 weeks.

    A general discussion ensued about impact statements.

    Mark Mirando indicated that he and Adele Turzillo will alternate as our representatives. Mark indicated that if AFRI grants are awarded, it is import for the awardee to move quickly to finalize animal protocols and receive funds in a timely manner.

    Tod Hansen asked when the deadline will be for next year? Mark hopes to move this deadline back to November in increments over a two year period.

    A motion by Jim Berardinelli to adjourn the meeting was seconded by Charles Weems.

    The meeting adjourned at 11:30 am on Tuesday, May 19, 2009

    Collaborative Efforts for the coming year:

    CO/OH ISG mRNAs in maternal blood and IFNt in conceptus

    CO/WA , use mouse ISG15 knock out model to understand role of ISG15 in bovine uterus

    CO/NM thyroid hormones in heifers carrying BVDV infected fetuses

    CO/HI/TX PGEs and CL function using intraluteal implants

    CO/WY CL gene expression in pregnant vs nonpregnant ewes

    AK/MT/WY Male exposure and induction of estrus

    AK/WY/MN Influence of progesterone on sexual behavior

    AZ/CO Influence of gestational nutrition on pancreatic function

    AZ/WY Influence of gestational nutrition on insulin

    FL Antiviral assay available for members

    FL/AK Polymorphisms in caribou and muskoxen

    MI/FL Extended oocyte/embryo cultura

    MI/MT Oocyte quality and follicle diameter

    OH/MT/WA Estradiol influences on fertility

    OH/PA MX and IFNt in cattle

    MT/MI Oocyte quality

    NE/WA VEGF and gonadal function

    NE Collaborate on using obesity markers to identify variants in cattle

    NV/TX Stress hormones and abortion in cattle and sheep

    NM/KS Metabolic hormones and reproduction

    NM/WY/MT Gender for twins and effect on reproduction

    NM/OH Influence of IGF-1 on precocious puberty

    TX/MN Inhibin concentrations and temperament

    WTAM/NM Development in intact males

    ND/WA Fetal programming

    WY/MT/NE Progesterone and sexual behavior

    WY/UT SNPs in sheep

    AR/NM Pasture systems and reproduction

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

    Estrous cycle, physiology/CL function

    Endocannabinoid type 1 and type 2 receptors may play a role in luteolysis. In addition, PGE1 and PGE2 may be anliluteolysins in the ewe to prevent luteolysis.

    Alteration of lymphocyte function is important for rescue of the bovine corpus luteum during maternal recognition of pregnancy.

    Significant release of IFN-Ä in the uterine vein and expression of interferon stimulated genes in corpora lutea were detected in day 16 pregnant sheep and 24 h infusion of IFN-Ä into the uterine vein in non pregnant sheep induces IFN-stimulated genes in the corpus luteum; supporting an endocrine role for IFN-Ä in maternal recognition of pregnancy.

    Expression of LHR and mTOR depends on the stage of luteal development, differentiation, and regression, and PGF regulates LHR and mTOR expression during luteolysis.

    Hypothalamic-Anterior Pituitary Gland Physiology

    Polymorphisms within the promoter region of the bovine FSH gene are associated with NEFA and hormone concentrations of Brahman influenced cows.

    A polymorphism in the FSH promoter region of Angus-Brahman influenced bulls are associated with bull immune responses.

    It was suggested that FFA, especially long chain unsaturated fatty acids, impair GnRH-induced LH secretion.

    E2 acting by a non-genomic mechanism induces a slight but significant increase in the number of GnRH receptors and this action occurs directly on the pituitary gland.

    Augmentation of the non-genomic actions of E2, through high doses of E2BSA, interferes with the onset, duration, and magnitude of the surge of LH induced by E2.

    The increase in the number of GnRH receptors induced by activation of ESR1 can account for the slight increase in secretion of LH, observed following administration of a selective ESR1 agonist.

    The presence of readily detectable amounts of two forms of ±-inhibin-like immunoreactivity in bull and ram plasma and non-detectable amounts of ²-subunit indicates that substantial amounts of free inhibin ±-subunit exists in ovine and bovine plasma. In cattle a portion of the free ±-inhibin-like immunoreactivity appears to come from extragonadal sources. Presence of ±-inhibin-like material in plasma likely underlies some of the variation in responses to active immunization against inhibin.

    Passive immunization against inhibin at two-day intervals resulted in a 2- to 3-fold sustained increase in FSH concentrations during the treatment period.

    Ovarian Physiology

    CART binding sites are present on bovine granulosa cells and granulosa cell CART binding is increased in response to FSH treatment.

    VEGF isoforms may be indicators of follicle status and oocyte competence in beef cattle.

    Dietary intake in donor ewes has a substantial effect on oocyte quality, which results in lower rates of cleavage, and morula and blastocyst formation after IVF in sheep.

    Oocyte-embryo-uterine physiology

    MX1 was associated with a distinct population of exosomes. Exosomal proteins are implicated in angiogenesis, immune modulation, apoptosis, and cell signaling. Determining the function of MX1 in the secretion of exosomes will improve understanding of the biology of early pregnancy.

    Many of the same genes regulated in the endometrium during early pregnancy are also regulated in the peripheral blood leukocytes (PBL) and some of these genes may be under the influence of progesterone. Alteration in gene expression in PBL during early pregnancy appears to be extensive and raises the question of the roles these genes play not only in pregnancy establishment, but also in affecting the maternal circulating immune system. Spermatozoa from some bulls and oocytes may associate with SPP1, suggesting a facilitory role on in vitro fertilization and embryo development. It is suggested that RCA-1 lectin enhances binding of UTF-derived SPP1 to bovine oocytes, resulting in increased sperm-egg binding and fertilization in vitro and a possible role in fertilization. Antibodies to SPP1, L-PGDS and BSA react with oocytes incubated with ODF and inhibit sperm binding, fertilization and embryonic development in vitro, suggesting a potential role of these proteins in these events

    SPP1 improved the efficiency of bovine in vitro embryo production and influenced sperm capacitation.

    Five ovine trophoblast cell lines were generated from day 15 sheep conceptuses that include both mononucleated cytotrophoblasts and binucleate cells that are being used to investigate the mechanism of action of Periattachment Factor (PRR15).

    Using mice as a gene deletion model for cattle, it was demonstrated that ISG15 is a molecular sentinel that functions to assist mothers when coping with environmental stressors imposed on pregnancy.

    At least four FGFRs preside in pre- and peri-attachment bovine conceptuses. Moreover, conceptuses express at least three candidate FGFs during elongation, the time of peak IFNT expression.

    FGF2 may signal through a downstream PRKCD to positively regulate IFNT expression in bovine trophectoderm.

    There was no effect of addition of FGF2 on competence of embryos to develop to the blastocyst stage although addition at Day 0 hastened the onset of hatching.

    Dispase digestion and FACS is a useful approach for obtaining enriched populations of BNCs and MNCs from midgestation bovine cotyledons.

    Cumulus expansion and progression to metaphase II, and fertilization and cleavage rates following in vitro fertilization are reduced following siRNA mediated ablation of JY-1 in oocytes and such effects can be rescued by addition of recombinant JY-1 to culture medium.

    The common SMAD (SMAD 4) of maternal origin is required for bovine early embryogenesis.

    Preliminary results suggest that the effect of follicle size on fertility may be on oocyte maturation or early embryonic development (before d 7). Uterine angiogenesis, manifested by increased capillary cell proliferation, enlargement of capillaries and increased expression of factor VIII, is initiated very early in pregnancy.

    Localization of PrPC to specific cells and structures within embryos, and in uterine and placental tissues suggests that PrPC has a significant, but unknown, role during the estrous cycle and during developmental processes of early pregnancy.

    Changes in angiogenic factor expression of CAR and FM tissues during early pregnancy are associated with dramatic increases in capillary cell proliferation and capillary size.

    Supplementation of LSM during late gestation does not appear to impact growth rate in calves or the onset of puberty in heifer calves. Testicular Physiology

    An approach was developed that when combined with the Y chromosome BAC tiling paths and BAC fluorescent in situ hybridization (FISH), will facilitate the clarification of the MSY region in cattle.

    It was demonstrated that the Thy1+ cell population in pre-pubertal bull testes is enriched for proliferating spermatogonia indicating that Thy1 is a conserved marker of SSCs in the bull.

    TGFBeta1 does not appear to affect alternative splicing of the VEGF gene which may indicate that the VEGF gene is regulated differently in cancer cell lines versus the gonad. We are sorting through effects of IGF-1. While Activin appears to increase cord formation in the testis there does not appear to be any effects on expression of the VEGF isoforms.

    Gonadal morphology in VEGF Sertoli and Granulosa cell KO mice is altered and their gonads are smaller which may result in reduction in fertility (1 less pup than controls) plus abnormal structures on the ovaries.

    VEGF anti-angiogenic isoforms may increase germ cell apoptosis therefore factors that increase amounts of these isoforms may negatively affect spermatogenesis and male fertility.

    Findings supported the conclusion that Sertoli cells undergo critical maturation events by 2-wks of age in the boar testis.

    Findings supported the hypothesis that peritubular myoid cells and testosterone are important components of the SSC niche in the adult testis.

    Uterine-Placental-Fetal Nutritional Interactions during Pregnancy

    Fetal desensitization of adrenergic receptor responsiveness resulting from chronically elevated catecholamine exposure in utero could promote a fetal programming response that leads to dyslipidemia.

    Thus, Chronic, mild, pulsatile hyperglycemia causes impaired insulin responsiveness, dysregulation of ROS, and oxidative stress in the late term sheep fetus, indicating that oxidative stress may be a mechanism for the adverse effects of diabetic pregnancy on offspring.

    Through examination of fetal cardiac gene expression, it was apparent that the timing of the environmental insult to the fetus impacted the number of genes with altered expression as well as the degree to which their expression was altered.

    Early to mid-gestation hypobaric-hypoxia appears to have its greatest impact on gene expression within the right ventricle of fetal sheep.

    Both dam nutrition and replacement heifer development impact feed costs and retention of young cows in the herd. Heifers developed on restricted diets from dams wintered on restricted diets had greater retention rates to their 5th breeding season.

    Nutritional plane and Se supply play a significant role in mammary and colostrum composition in pregnant ewe lambs as well as passive immunity of their offspring.

    It is suggested that nutritional plane and Se supply during gestation affect subsequent ewe milk production and composition, even when ewes are fed to nutritional requirements during early lactation.

    Data suggest that FGF2 and receptor FGFR2 IIIc have a role in regulating early placental growth.

    It is proposed that FGF2 plays a role in regulating uteroplacental vascular and nonvascular growth during early pregnancy.

    Maternal nutrition affected expression of FSHR in fetal ovaries, and therefore may play a regulatory role in fetal, and perhaps postnatal, follicular development.

    Fetuses of ewes made obese at conception and continuously fed above NRC requirements throughout gestation, exhibited marked increases in glucose and fatty acid uptake and adiposity by midgestation. At birth, lambs from these obese mothers had an increased % body fat and a reduced pancreatic function.

    Female offspring born to ewes nutrient restricted 50% from day 28 to day 78 exhibited reduced ovarian P4 secretion and decreased reproductive performance as adults.

    Ewes born twin to a male lamb tended to have lower lifetime productivity than ewes born twin to a female lamb.

    Statistical Methodology

    The nested factorial probe-level statistical model can be used to assess differential expression in future studies where large sample sizes (number of biological replicates on microarrays) are not practical, as in bovine NT studies.

    Statistical tools were developed that can be used to study a possible genetic (and subsequently pathway or ontology) basis of successful (vs. failed) NT pregnancies without requiring large numbers of biological replicates.

    Nutritional and reproduction

    One third of heifers with elevated plasma GHR concentrations became anestrus although no direct correlations between circulating GHR concentrations and reproductive parameters were detected. Heat stress increases plasma ghrelin concentrations with measurable changes in ghrelin concentrations limited to the peri-prandial period.

    Serum urea nitrogen is elevated in heifers grazing overseeded ryegrass in the spring, but declines prior to the time of breeding.

    Perturbations in energy balance may alter the responsiveness of the hypothalamic-pituitary axis to the stimulatory effects of kisspeptin.

    Limit feeding replacement beef heifers reduces the variation in RFI. Nonetheless, sufficient variation in RFI still exists to identify animals with differences in feed efficiency.

    Excessive UIP may actually enhance reproductive performance in mature cattle as well as increase calf weaning weights.

    Extending grazing in heifer development systems decreases costs without compromising production.

    Selection for low residual feed intake, more feed efficient animals, can result in about 14 days earlier return to estrus after calving. Bos indicus, Bos taurus and crossbred cattle differ in circulating concentrations of insulin-like growth factor I breed type. All animals with Bos indicus genetics had higher concentrations of IGF- I than their Bos taurus counterparts. Residual feed intake was not related to concentrations of IGF- I. Measuring concentrations of IGF- I is not a reliable physiological marker for selecting more feed efficient cattle. Cortisol response to an ACTH challenge is not a reliable physiological marker for selecting more feed efficient cattle. Metabolic effects of fasting on reproduction may be mediated by the expression of gastrin-releasing peptide (GRP) and FSH in the ovine anterior pituitary gland.

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

    Estrus, hormones, estrous synchronization and A.I.

    Timing of a 30 day period of energy supplementation or type of supplement did not affect serum blood urea nitrogen or glucose concentrations and did not alter pregnancy rates to timed AI or overall pregnancy rate.

    Recombinant single chain analogs of ovine gonadotropins can be used to induce a superovulatory response in sheep and may have broader application in ovulation induction protocols.

    Using a CIDR for 7d with PG given on the day of CIDR removal results in an improvement of the estrus synchronization response compared to that of a CIDR for 14d followed 17d later by PG but both programs yielded similar overall AI pregnancy rates when combined with TAI and GnRH in postpartum beef cows.

    AI pregnancy rates are not altered by exposing heifers to bulls during an estrus synchronization protocol that included CIDR for 14 d, followed 18 d later with PGF2± (PG), and, timed AI (TAI) and GnRH.

    Pregnancy rates in low antral follicle count heifers are reduced when compared to high follicle count cattle. There are also correlations with ovarian weight, reproductive tract score and antral follicle count which may be predictors of reproductive longevity in the herd.

    Pregnancy rates in ewes were not adversely affected by applying progesterone via a CIDR on the day of estrus.

    Treatment of ewes with bovine somatotropin at a synchronized estrus resulted in decreased conception rate and reduced weights of offspring.

    Serum thyroid hormones and prolactin change substantially near the time of estrus and ovulation. Cyclic activity was induced but not maintained in prepubertal Rambouillet ewe lambs by progesterone and PMSG administration.

    The 5 d CO-Synch + CIDR program for timed AI in postpartum beef cows results in an 11% increase in timed AI pregnancy rate relative to the traditional 7 d program. The requirement for 2 injections of PGF that is inherent to the system cannot be circumvented by postponing timing of AI by 12 h.

    Relative to the traditional 7 d CO-Synch + CIDR program, the 5 d program results in ovulation of follicles of similar diameter that tend to produce greater peak estradiol concentrations during the proestrus.

    Puberty

    It was demonstrated that in contrast to rodents, transiently induced hypothyroidism during the neonatal period of Sertoli cell proliferation in rams negatively effects sperm production at puberty and emphasizes the importance of neonatal nutritional requirements for establishing the foundation of sperm production in adult animals.

    Genetic selection for age at puberty impacts postpartum anestrous interval in beef cattle and could potentially influence timing of conception in postpartum cows. No influence of residual feed intake on age or weight at puberty in Brahman bulls was detected.

    Precocious puberty is induced with both high and low starch diets that support accelerated weight gains. Occurrence of precocious puberty was associated with increased systemic concentrations of IGF-1 in heifer fed two diets that supported similar ADG.

    Immunocastration

    The SpayVac" and IVT-PZP vaccines presented in an emulsion form with AdjuVac" produce a single-shot immunocontraceptive vaccine lasting up to 7 years. Two doses of GonaCon prevented pregnancy of elk cows for at least 3 yr therefore its use for population management of elk warrants consideration as part of a strategy to control brucellosis.

    One-injection and two-injection formulations of GonaCon and GonaCon-B produced multi-year contraception in adult female white-tailed deer.

    Long-term contraception of mustang mares with a single shot of either the SpayVacTM or GonaConTM vaccine is possible.

    Results of immunocontraception studies suggested that White-tailed does possess an FSHRH peptide secreted by the hypothalamus which modulates follicle development, and resulting estrogen production and reproductive behavior.

    Disease, immune function

    Persistent infection with ncpBVDV induces a chronic type I interferon response perhaps contributing to the IUGR seen in persistent BVDV and in other viral infections during pregnancy.

    Bovine fetuses infected with BVDV prior to development of the fetal immune system do not recognize the virus as foreign as a result, at least in part, to the inability to mount a strong innate immune response.

    Infection with ncpBVDV induces a vigorous type I IFN response in acutely infected animals. Presence of a fetus persistently infected with ncpBVDV causes down-regulation of SDF-1/CXCR4 signaling in the blood of the dam which could have deleterious consequences on fetal development and the immune response.

    An experimental inoculum using infected splenocytes from SCID mice has been a quantifiable and predictable method of infection to produce EBA induced abortions over 2 years. This has led to the production of a murine origin inoculum to be tested for use as a "stop gap" method of vaccination and protection. Heifers appear to be more adept at coping with and recovering from an immune challenge compared with bulls. A sexually dimorphic innate immune response to an endotoxin challenge was found for tumor necrosis factor-06, interleukin 6 and interferon gamma.

    Behavior and Stress

    A component of the biostimulatory effect of bulls involves altering follicular wave dynamics by shortening the inter-wave interval and increasing the dominant follicle diameter, providing new insight into the mechanism whereby bull exposure accelerates resumption of ovulatory cycles in anestrous postpartum cows.

    Bull exposure affects temporal patterns of leptin concentrations in anestrous postpartum cows. Adrenal cortical function is influenced by both breedtype and gender. Angus cattle routinely have greater circulating concentrations of cortisol than do Brahman cattle and steers and heifers routinely have greater concentrations of cortisol than do bulls. When animals are challenged with an endotoxin a sexually dimorphic adrenal cortical response was found with heifers having a greater response than bulls. There were no differences in adrenal medullary function found. Initial expression of adult male sexual behavior appears to be influenced by developmental effects of progesterone. Subsequent expression, however, appears independent of progesterone influence.

    Producers are aware of differences in ram libido that would affect flock performance they have not incorporated breeding performance as an important criteria for ram selection.

    Usefulness/Impact Statements:

    Overview The data obtained during the past year by W1112 participants yielded substantial advancements in basic and applied reproductive mechanisms. The breadth and depth of W1112 projects is immense and covers areas as diverse as estrous synchronization, pregnancy failure, testis function to genomic modulation. Data collected over the past year have increased our understanding of pituitary, testis, oocyte, follicle and corpus luteum function/development that may enable us to more efficiently manipulate ovulation, estrus, puberty, pregnancy and production of sperm. Furthermore, advances were made in understanding how nutrition impacts fetal development, placental efficiency, heifer development, estrous cycles, and development of more viable offspring. Members continue to develop novel approaches to investigate basic and applied aspects of reproductive efficiency. New image analysis programs and statistical procedures have been developed to investigate vascular development, follicular growth and microarray data. Recombinant proteins have been developed to manipulate reproductive function. Members of our group have also made significant progress in understanding how behavior, exposure to male biostimulation, and disease may affect general overall health, feed and reproductive efficiency. Also progress has been made in identifying factors that can be commercialized for immunocastration and in fine tuning these approaches to be more producer friendly. Participants in W-1112 have published 98refereed journal articles many of which were derived from collaborative efforts initiated through the W1112 Regional Research Project.

    Impact Statements:
    1. Previous and ongoing research by W-1112 members served as the basis for the design of a new estrous synchronization program that results in increased pregnancy rates by artificial insemination of 10 to 15% relative to traditional programs. Implementation of this approach with 10% of the 40 million beef and dairy cows in the USA on an annual basis, would result in a 12.5% increase in fertility with enhanced profitability of approximately $31 million through reduction in days from calving to conception and an additional savings of $40 million due to due to a reduction in cow replacement rate. When this value is amplified 5 to 10 fold through the various industries allied with beef and dairy production, the total economic benefit of this technology resulting from W-1112 findings would exceed $400 million per year for the cattle industry (beef and dairy) in the USA.
    2. Selection for low residual feed intake (feed efficiency) would increase returns to producers by approximately $20 per cow which would be significant considering that nations beef cow inventory is approximately 32 million head.
    Last Modified: 31-Aug-2009

    Date of Annual Report: 06/16/2010

    Report Information:
  • Annual Meeting Dates: 05/17/10 to 05/18/10
  • Period the Report Covers: 10/2009 to 09/2010

    • Participants:
    Brief Summary of Minutes of Annual Meeting:
    Chair Mike Day called the meeting to order at 8:30 am on Monday, May 17, 2010.

    Minutes from the 2009 meeting were approved without discussion. Tom Adams was thanked for making arrangements for this year's meeting.

    Colin Kaltenbach requested an update of the membership list for NIMSS files and noted that that the project terminates in approximately 15 months. A renewal of the project is due by January 15, 2011 and needs to be submitted to the Western Directors in March, 2011. To meet those deadlines, the re-write of the project must be completed by December 15, 2010. Dennis Hallford, Tracy Davis, Mike Teglas, and John Stevens agreed to develop new objectives for presentation to the group on Tuesday morning.

    Adelle Turzillo provided a handout regarding new developments at NIFA. We were encouraged to submit stakeholder comments about AFRI programs and to review AFRI-RFAs carefully. Four of the seven AFRI-RFA programs contain an animal component that may fit with our research programs. The trend for new grants is towards large multi-disciplinary projects rather than multiple small grants.

    Last year's election of Kim Vonnahme as Member-at-Large for 2009-2010 was confirmed. Tracy Davis was selected as the Member-at-Large for 2010-2011.

    Hawaii was selected as the site for next year's meeting on May 16-17, 2011.

    Updates and announcements were made by each station regarding new faculty, positions available and ongoing changes. Registration costs requested for meeting attendance were $60.00.

    The remainder of the day was dedicated to station reports. The meeting adjourned at 5:00 p.m. for the day.

    Tuesday May 18, 2010. The meeting was called to order at 8:30 a.m.

    The first business of the day concerned the re-write of the project for the 2012-2017. New objectives for the re-write were presented, adapted, and approved to read as follows:

    Objective 1. Examine molecular, metabolic, genomic, endocrine, and immunologic mechanisms influencing testicular and ovarian function, reproductive behavior, conception rate, embryo and fetal development, attainment of puberty, and effects of climate/season on reproductive patterns of domestic ruminants.

    Objective 2. Development and application of methodologies to improve animal health, well-being, and reproductive efficiency of domestic ruminants in order to allow sustainable operation of production systems in the western United States.

    Subsequent discussion focused on procedures and formation of a writing committee for the project's renewal. Tom Geary will be solicited by Mike Day to prepare the summary of accomplishments for the past five years of the project. All participants will be asked to submit a paragraph of their research plans for the next five years as they pertain to the newly adopted objectives. These paragraphs will be due to Gary Moss by September 15, 2010. Gary Moss, Mike Teglas, and Terry Nett and nearby participants (esp. at WY and CO) will coordinate the collation of those paragraphs into a readable document for submission by December 15, 2010.

    The remainder of the morning was dedicated to the completion of station reports and discussion of collaborative research plans. The meeting was adjourned at 12:00.

    Collaborative Efforts for the coming year:

    NM/MT. Metabolic hormone patterns in ewe lambs with differing RFIs.

    NM/CO. Nutrient restriction vs. metabolic hormone patterns

    NM/CO. Thyroid involvement in BVD infected cows.

    NM/AR. Metabolic hormone profiles in cows consuming endophyte infected fescue.

    NM/OH. Follicular fluid IGF.

    WY/MT/NM. Meta-analysis of birth type on ewe reproductive performance.

    CO/OH ISG mRNAs in maternal blood and IFNt in conceptus.

    CO/WA use mouse ISG15 knock out model to understand the role if ISG15 in bovine uterus.

    CO/HI/TX PGEs and CL function using intraluteal implants

    AK/WY/MN Influence of progesterone on sexual behavior

    AZ/CO Influence of gestational nutrition on pancreatic function

    AZ/WY Influence of gestational nutrition on insulin

    MI/FL Extended oocyte/embryo culture

    MI/MT Oocyte quality and follicle diameter

    OH/MT Follicle age influences on fertility

    OH/PA MX and INFt in cattle

    MT/MI Oocyte quality

    NV/TX Stress hormones and abortion in cattle and sheep

    TX/MN Inhibin concentrations and temperament

    WT AM/NM Development in intact males

    ND/WA Fetal programming

    WY/MT/NE Progesterone and sexual behavior

    WY/UT SNPs in sheep

    AR/NM Pasture systems and reproduction

    WA/NE Role of VEGF in Spermatogonial Stem cell development and differentiation.

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

    Estrous cycle, physiology/CL function:

    Dimeric inhibin is present in the plasma of ram lambs and yearling ewes. In addition to dimeric inhibin, both ram lambs and yearling ewes appear to have an excess of monomeric inhibin ±-subunit. Attempts to detect dimeric inhibin in ovine plasma using a two-site ELISA format have been unsuccessful. Present findings confirm that dimeric inhibin is indeed present in ovine plasma. Plasma ±-inhibin in ram lambs consists of two ±-subunits, 47 and 17 kDa, and one ²-subunit of 14 kDa. The 47 kDa ±-subunit is most prominent. This larger ±-band is consistent with non-biologically active free inhibin ±-subunit. The smaller 17 kDa ±-band and the 14 kDa ²-band are consistent with biologically active dimeric inhibin. Thus, the ±RIA, which detects both the 47 and 17 kDa forms of the inhibin ±-subunit, does not necessarily reflect biologically active dimeric inhibin.

    Plasma ±-inhibin in yearling ewes consists of 46 and 14 kDa ±-subunits and a single 14 kDa ²-subunit. The inhibin forms are either very similar or identical to those in ran lambs. The intensity of immunostaining of the 47 kDa ±-band in ram lambs was greater than the 46 kDa ±-band in yearling ewes. Concentrations of ±-inhibin increased decreased immediately following CIDR removal and then increased.

    Dynamic differences exist in the gene expression profile and accompanying functional gene categories and biological pathways regulated by PG in bovine CL that are responsive versus refractory to the luteolytic actions of PG. Such differences are linked to candidate transcription factors that may potentially mediate the stage specific gene expression response to PG associated with luteolysis.

    PGE1 or PGE2 prevent luteolysis in Angus or Brahman cows by preventing loss of message for LH receptors and occupied and unoccupied LH receptors and alter mRNA for prostanoid receptors on luteal tissue. Analogues of PGE1 and/or PGE2 may be viable approaches to prevent loss of luteal progesterone in cows and to decrease pregnancy loss during the first trimester of pregnancy.

    Hypothalamic-Anterior Pituitary Gland-Ovarian Physiology

    Using a lentiviral-based siRNA knockdown strategy, it was established that PGRMC1 is necessary for mediating the anti-apoptotic actions of progesterone. PGRMC1 deficiency was found to reduce tumor growth and enhance sensitivity to chemotherapy using an immunocompromised mouse xenograft model.

    Gene expression for proteins regulating folliculogenesis was higher in the Suffolk ewes compared to the Polypay. There were no observed differences in the number of preantral and antral follicles between the two breeds. Recombinant single chain analogs of ovine gonadotropins can be used to induce a superovulatory response in sheep and may have broader application in ovulation induction protocols.

    Gonadal morphology in VEGF Sertoli and Granulosa cell KO mice (using pDMRT1) is altered and their gonads are smaller which may result in reduction in fertility plus abnormal structures on the ovaries. Potential differences in ovarian reserve in the females and alteration in endocrine profiles in both sexes (high androgen in males and reduced estrogen in females) are being analyzed.

    Gonadal morphology in VEGF granulose cell KO mice (using AMHR2) is similar to that of VEGFXDMRT1 mice in the ovaries are smaller and have less follicles. The VEGFAMHR2 cre positive mice have reduced body size and have more dramatic reductions in estrogen over cre negative control mice. Steroidogenic enzymes to determine if there are differences that may result in reduced estrogen concentrations in blood plasma are being evaluated.

    Eliminating a co-receptor for VEGF, neuropillin-1, decreases ovarian size and reduces the number of follicles on the ovary. The data is preliminary, but suggests that angiogenic VEGF isoforms are necessary for appropriate granulosa cell function and follicle development.

    Oocyte-embryo-uterine physiology:

    Preovulatory concentrations of estradiol did not influence amount of uterine mRNA for ISG-15 and Mx2 or embryonic mRNA for CSH-1 and IFNt on d 17.5 of gestation/estrous cycle.

    Glucocorticoid exposure during the second half of pregnancy has deleterious effects on pregnancy outcomes. Our findings over the past year indicate that the embryo signals within the endometrium shift toward local production of glucocorticoids. It is hypothesized that activation of GRs within the endometrium results in establishment of a barrier that precludes access of maternal immune cells to the embryo through increased expression of adhesion molecules.

    Through the use of conditional knockout mice, it was established that b-catenin signaling in the stromal compartment of the endometrium is essential for coordinating epithelial responses to steroid hormones (i.e., mesenchymal-epithelial interactions). It was established through microarray analysis and validation by RT-PCR and in situ hybridization that the embryo induces endometrial expression of an as yet uncharacterized gene referred to as decidual restricted gene 1. It is hypothesized that Drg1 is functionally required for the establishment and maintenance of pregnancy.

    It is possible that synchronization of estrus of beef heifers with prostaglandin F2± may impair function of the corpus luteum in the ensuing estrous cycle. This may be of physiological significance relative to early embryo survival.

    The effect of follicle size on fertility may be manifested primarily through estradiol effects on the uterus since cows induced to ovulate while serum estradiol concentration was low had reduced ability to maintain a pregnancy to d 28. Also, d 28 serum concentration of PAGs may be indicative of pregnancies that will not be supported to term. Differential methylation patterns may help explain large offspring syndrome in calves born to IVM-IVF and cloning procedures and could serve as a model for study of a similar condition in humans. JY-1 induced regulation of cumulus expansion is dependent on presence of additional oocyte- secreted factors and potentially mediated, at least in part, by regulation of PGE2 production and expression of genes linked to stabilization of the hyaluronan rich extracellular matrix.

    Fibroblast growth factors (FGFs) stimulate IFNT production in bovine trophectoderm. Several FGFs are expressed in bovine endometrium and conceptuses. FGF2 promotes endoderm development in bovine embryos. Also, FGF2 and 10 stimulate trophectoderm migratory activity. Endogenous FGFs are important for normal bovine embryonic development. Blocking FGR activity with specific kinase-domain inhibitors does not affect blastocyst formation in vitro but compromises subsequent development and function. FGFs also promote oocyte competency. Embryo development in vitro is improved by supplementing FGF2 or 10 during in vitro maturation. Also, treatment with FGFR inhibitors during maturation limits subsequent embryo development.

    A FACS-based approach was developed to obtain enriched TGC samples for study of the events that lead to late embryonic and fetal losses in cattle so that new schemes can be developed to limit the severity of these losses. Current work focused on developing ways to study trophoblast giant cells (TGCs) from bovine placentae.

    Ovine trophoblast cells exhibit phenotypic changes, when cultured on Matrigel matrix, similar to what is observed by mouse trophoblast stem cells and first trimester human cytotrophblasts, exhibiting migratory and invasive behavior. This may suggest that the phenotype of trophoblast cells may not differ significantly between non-invasive ruminant conceptuses compared to invasive human and mouse conceptuses, inferring that the differences in placentation result from functional differences in the uterus.

    Culture of ovine trophoblast cells on Matrigel matrix upregulates the expression of PRR15, allowing a more robust system to examine its function and regulation.

    IFNT has endocrine effects on the CL between Days 13 and 14 of pregnancy and may protect the CL through mechanisms that are complementary, yet independent to its paracrine effects on the OXTR endometrial pathway.

    CXCL12/CXCR4 pathway is activated during implantation and placentation in sheep and is likely playing a role in the communication between trophoblast cells and the maternal endometrium.

    When added to culture media, bovine ghrelin significantly reduces bovine embryonic development

    Testicular Physiology:

    Effects of VEGF on testis development and spermatogenesis were evaluated to improve our understanding of factors critical for spermatogonial stem cell differentiation and maintenance in cattle.

    Testis morphology in VEGF Sertoli and Granulosa cell KO mice is altered and their gonads are smaller which may result in reduction in fertility plus the testes appear to have less numbers of sperm.

    VEGF anti-angiogenic isoforms may increase germ cell apoptosis and may, therefore, negatively affect spermatogenesis and male fertility.

    VEGF anti-angiogenic isoforms may increase germ cell apoptosis therefore factors that increase amounts of these isoforms may negatively affect spermatogenesis and male fertility.

    Uterine-Placental-Fetal Nutritional Interactions during Pregnancy

    The application of a powerful image analyses program has allowed vascular development to be quantified in the sheep placentome, and to determine if morphology of the ovine placentome impacts vascularity. Moreover, impacts of maternal diet on placental vascular function are being assessed.

    Early- to mid-gestation maternal undernutrition in twin sheep pregnancies, followed by realimentation appears to set the fetus on a more rapid growth trajectory during late gestation, which may be carried forward into the postnatal period. Maternal undernutrition may enhance the pregnancy induced insulin resistance, such that upon realimentation, maternal uterine artery glucose concentrations are significantly elevated, even when compared to the control fed ewes receiving the same diet.

    While uterine artery, umbilical vein and umbilical artery glucose concentrations do not differ between control and continuously undernourished twin pregnancies, the umbilical vein:umbilical artery difference in glucose concentration is significantly less in continuously undernourished pregnancies. This may suggest that even with normal glucose delivery to the fetus, that fetal metabolism is still reduced. Reductions in metabolic set points within these offspring may well impact postnatal growth rate, feed efficiency and body composition.

    Models of both maternal undernutrition and maternal obesity have been established to allow evaluation and comparison of the impacts of each condition on fetal growth and development, as well as offspring health, growth efficiency, glucose-insulin dynamics and reproductive function. Results from these studies will shed light on the specific physiological mechanisms involved, as well as lead us to management practices to optimize offspring health and production efficiency. This is especially pertinent when one considers that in the human, both maternal undernutrition and maternal obesity lead to the birth of offspring which exhibit an increased incidence of obesity, insulin resistance, type 2 diabetes and cardiovascular disease in later life. By studying both maternal under and overnutrition simultaneously, we can elucidate any similarities on fetal growth and development as well as on offspring postnatally. Optimizing the uterine environment to facilitate the birth of healthy, growth-efficient offspring is important for livestock production

    Female offspring born to ewes nutrient restricted (NR) during the first half of gestation exhibited a marked reduction in fertility. This decreased fertility was associated with decreased progesterone concentrations in systemic blood during the estrous cycle of adult ewes born to NR mothers. Corpora lutea recovered from the adult female offspring of these NR ewes exhibited markedly decreased progesterone content during the midluteal phase of an estrous cycle. CLs are currently being evaluated both histologically and for selected mRNA and protein expression differences to determine the reason for this difference.

    Chronically elevated norepinephrine, without hypoxemia, continually suppresses plasma insulin concentrations in fetal sheep. Termination of the norepinephrine infusion results in hyper-insulin secretion in response to glucose and arginine, showing chronic suppression by NE causes ²-cell compensation. Insulin secretion from MIN6 cells is suppressed by the adrenergic receptor ±2, similar to rat and mouse islets. Therefore, MIN6 cells provide a useful culture system to examine alterations in insulin secretion responsiveness following chronic catecholamine suppression.

    In IUGR fetuses, islet vascularity is reduced to a greater extent than ²-cell area. These findings begin to link disruptions in endothelial cell function and islet blood flow as mechanism for decrease islet function in utero.

    Statistical Methodology:

    Additional statistical models (and preprocessing methods) were assessed using spike-in data, specifically FIRSTP (Rubin 2009) and RMANOVA (Xu and Cui 2008), under both RMA and GCRMA. An appropriate sampling distribution of test statistics from the nested factorial model was determined, using permutation methods. This allows computation of traditionally reported P-values for each gene. The developed NFM method was found to be competitive against other statistical models, and was applied to the motivating bovine NT data (4 clone vs. 3 control arrays, in cotyledon). The resulting 584 significant genes were found to be biologically relevant. The developed NFM method was implemented in R code (affyNFM), now freely available on the project leaders website (www.stat.usu.edu/~jrstevens).

    Nutrition and Reproduction:

    Steroidogenic capacity of dominant follicles was not influenced consistently by diets designed to induce precocious puberty.

    Studies continue to determine how selenium being fed impacts maternal and fetal tissues during pregnancy. Data indicate maternal nutrition impacts circulating hormones, as well as maternal and fetal placental tissues from the offspring. Further data are being analyzed for growth parameters and carcass composition of the offspring born from dams fed supranutritional levels of selenium as well as differing global levels of nutrition. Both over- and under-nutrition impact colostrum yield and IgG production. Milk production in ewes that are underfed continue to be lower than control fed ewes. Ewes that are overfed will have decreased colostrum, but will have similar milk production 3 to 4 days after parturition.

    Level of harvested feed provided to cows during the winter appears to impose a uterine programming influence that alters circulating concentrations of IGF-1 and reproductive performance in their daughters when they are subsequently reared on restricted levels of nutritional intake. Heifers developed on restricted diets from dams wintered on marginal levels of feed had greater concentrations of IGF-1 from precalving through 2nd prebreeding and greater retention rates to their 5th breeding season than contemporary heifers that were out of dams provided adequate supplemental forage throughout the winter. Residual feed intake did not influence resumption of luteal function during the transition into the breeding season or temporal patterns of progesterone and metabolic hormones. Ewes classified as efficient RFI gained more weight during ram exposure than ewes classified as efficient and not exposed to wethers.

    Behavior and Stress:

    Research related to objective identification of temperament, and the coupling of stress-responsiveness with immune function, provide added impetus to effort to develop gene-based methods to select for Calm, stress-tolerant, immune-competent, disease-resistant cattle.

    The biostimulatory effect of rams in ewes accelerated resumption of luteal function during the transition into the breeding season. More importantly, this effect appears to include changes in certain metabolic hormones that may be involved with the physiological mechanism of this biostimulatory effect during the transition into the breeding in sheep.

    Exposing primiparous, postpartum, anovular, suckled, beef cows to mature bull urine for 12 h daily does accelerate resumption of ovulatory activity but it does appear to improve artificial insemination pregnancy rates. Bull urine per se may not be the medium that carries the biostimulatory pheromone(s) that accelerates resumption of ovulatory activity in the bovine. However, bull urine may carry an additional pheromone(s) that is associated with fertility in the bovine.

    Peinatal exposure of male rats to progesterone or RU486 decreased initial sexual behaviors and numbers of serotoli cells.

    Differences in gene expression in LP rams as identified by micro-array analysis were not confirmed by quantitative PCR. Further analysis may reveal a panel of genes that may help identify LP rams but no overt changes were identified.

    Objective2. Develop and improve reproductive technologies to enhance sustainability of production systems for domestic ruminants.

    Estrus, hormones, estrous synchronizations and AI: The 5 d CO-Synch + CIDR program for timed results in an 11 percentage point increase in timed AI pregnancy rate relative to the traditional 7 d program. The requirement for 2 injections of can be circumvented by giving two doses of PGF at the time of CIDR withdrawal. This program provides a substantial increase in timed AI pregnancy rate and similar convenience to the traditional 7 d program.

    Pregnancy rates in ewes were improved by administering 10 mg of PGF2a at CIDR removal.

    Exposing heifers to bulls during an estrus synchronization protocol that included a CIDR for 14 d, followed 18 d later with PGF2± (PG), and, timed AI (TAI) and GnRH can substantially improve AI pregnancy rates. It appears that combining bull exposure with a estrus synchronization protocol that include controlled internal drug release devices (CIDR) for 14 d, followed 18 d later by PGF2± (PG), and, timed AI (TAI) and GnRH would be beneficial for improving reproductive performance of yearling beef heifers. Thus, inclusion of a bull in progesterone-based synchronization protocol would be an important, sustainable management practice to improve reproductive efficiency in beef cattle herds.

    The ovine adrenal gland has been reported to be a source of extragonadal inhibin. Present findings provide evidence that in wether lambs extragonadal inhibin does not exert a significant inhibition of FSH secretion.

    Cows culled from the herd at different ages may have altered follicular gene expression which is not optimal for follicular development and may affect fertility of that animal.

    Recombinant single chain analogs of ovine gonadotropins can be used to induce a superovulatory response in sheep and may have broader application in ovulation induction protocols.

    Puberty:

    Sex of lamb, type of birth, and age should be considered when evaluating serum hormone profiles in rapidly growing lambs. Scrotal circumference is highly correlated with dam milk production, but direct preweaning gain is only lowly correlated with scrotal circumference and postweaning gain is not correlated with scrotal circumference. Thus, how bulls are fed during development postweaning should not affect sperm production. No in utero effects on age of puberty or any spermatozoa morphological characteristics were detected indicating that purebred breeders should not realize a benefit in fertility of bulls by over conditioning their cows. Immunocastration

    The GnRH vaccine has proven to be an effective means of contraception in elk but it does not interfere with pregnancy existing at the time of immunization. GnRH antibodies are effectively transferred to the neonate via colostrums, but they do not interfere with normal development/function of the hypothalamic-pituitary gonadal axis in the offspring.

    Disease, immune function:

    Maternal thyroid deficiency during pregnancy might reflect a pregnancy with a PI fetus for BVD and could compromise fetal development and maternal health

    An experimental inoculum using infected splenocytes from SCID mice was used successfully to induce abortion in susceptible cattle. This method of transmitting infection to cattle is a quantifiable and predictable method of infection that has produced EBA induced abortions consistently through the past 2 years. Data generated from the past 2 years has lead to the production of a murine origin inoculum that that is being developed for use as a "stop gap" method of vaccination and protection in susceptible heifers and pregnant cows. The first year using this vaccine has shown it to be safe (none of the vaccinated heifers became ill or had abortions induced) and effective. Vaccinated animals did not abort when challenged by experimental infection as did the control group of animals. The development of a safe and effective method of protecting cattle from EBA is necessary for producers raising cattle in EBA endemic area of the West. It will also be of utility (or even more so) for producers outside of the area were EBA is known to occur that plan to move their animals into endemic regions in order to utilize new pasturing opportunities.

    It appears that even up to 3 year following an abortion cattle maintain enough immunity against the agent of EBA to protect against subsequent infection. The upcoming final year of this study will be used to expand the numbers of animals test to see if immunity 3 years post infection is consistently strong enough to protect cows from subsequent abortion. This information will be of use to cattle producers because it can potentially give them guidelines for determining which cattle to breed for the following year and help producers utilize pasture resources in pajahuello tick endemic areas with greater confidence.

    Behavior and Stress:

    Plasma ghrelin concentrations are differentially regulated in heat-stressed vs. pair-fed dairy cattle during hyperinsulinemia-euglycemia.

    The identification and use of high-sexually performing rams with desired genetic traits is a requisite to the timely incorporation of those traits into a flock. High-sexually active rams appear to exhibit more exploratory-type behaviors than low-sexually performing rams.

    The adrenal cortex stimulation test does not identify feed efficient cattle.

    Exit Velocity (EV; the rate at which calves exit from a handling chute) is a useful, viable and objective indicator of temperament classification of beef calves. The increased cost associated with the potential for more excitable or temperamental cattle to injure themselves or workers and to damage facilities, in addition to the negative effects of temperament on growth, immunity and carcass characteristics, motivates producers to select against more temperamental cattle.

    Although temperamental calves may be objectively identified as early as 21 days of age, temperament may be more easily identified prior to weaning by use of the Exit Velocity trait. This new management technique may enhance the ability of producers to select against temperamental animals.

    Transportation per se may not be as stressful as previously presumed, and it may be that the handling, loading and unloading of cattle may be more stressful than the transportation event itself. This knowledge may impact procedures to transport cattle as well as impact subsequent product quality.

    Selection pressure against temperamental cattle results in beef with improved sensory traits which should lead to increased consumer satisfaction which would permit Texas to have a greater market share of the meat industry.

    Pregnancy:

    Use of several parameters (especially ultrasound and estrous detection) after breeding allows for early diagnosis of pregnancy (d 19) which should improve our understanding of (and prevention of) early embryonic losses and may yield identification of blood components capable of accurately identifying pregnancy establishment.

    Nutrition:

    Selecting beef heifers for low residual feed intake (RFI), a feed efficiency trait, results in earlier return to estrus and improved conception rate when forage conditions are adequate.

    Plasma insulin-like growth factor-I (IGF-I) is not a physiological marker for feed efficiency in Brahman cattle.

    Impact Statements:
    1. Epizootic bovine abortion can cause abortions in susceptible herds of up to 60%, leading to estimated losses of 10 to 15 million dollars to beef cattle producers in California alone. Research indicates that natural immunity following experimental abortion by the agent of EBA will last for 3 years following abortion. This information allows cattle producers to better manage their herds in EBA endemic sites. The first year of research has been completed on the development of a stop-gap vaccine against the disease that has been shown to be safe and effective for use in open cattle. Further testing and field trials will be conducted in 2010-2011.
    2. Cows left in presence of bull (or bull urine) show an increase in A.I. pregnancies of up to 20% . The bulls have a similar effect on pregnancy rates (increase of up to 18%) if kept in with cows from the time of synchronization. Producers can use this information to increase pregnancy rates and decrease their calving intervals translating to increased profits from their herds.
    3. Lead efforts for FDA approval of CIDR use in sheep for synchronization of breeding were completed. This approval provides sheep producers in the U.S. with a method to better manage breeding in their flocks by shortening lambing intervals and increasing rates of pregnancy.
    4. . Selecting beef heifers for low residual feed intake (RFI), a feed efficiency trait, will return approximately $50 more per cow per year than their less feed efficient herd mates. If Texas had 1 million cows selected for this trait, it would be worth $50 million dollars per year to the Texas beef industry.
    5. Temperamental calves have 5.6% lower weaning weights than calves with a calmer temperament. Calm calves outgain temperamental calves by 60 pounds during the initial 45 days following weaning which would yield a 15-pound advantage in body gain in stocker calf operations. Following weaning, temperamental calves have an increased secretion of cortisol, the stress related catabolic hormone released by the adrenal gland, following weaning, and they have a less robust immunoglobulin response to vaccinations provided at weaning which may place such calves at a disadvantage if they encounter respiratory or digestive system pathogens when they enter a feedlot operation. Thus, temperament and stress-responsiveness traits may affect health status, cost of sustaining wellness, growth efficiency and ultimately product value.
    Last Modified: 16-Jun-2010

    Date of Annual Report: 07/14/2011

    Report Information:
  • Annual Meeting Dates: 05/30/11 to 05/31/11
  • Period the Report Covers: 09/2009 to 09/2010

    • Participants:
    Brief Summary of Minutes of Annual Meeting:
    Chair Gary Moss called the meeting to order at 8:30 am on Monday, May 30, 2011.

    Kim Vonnahme, Secretary, was not present at the meeting. A motion was made by Dave Kreider and second by Terry Nett to select Tracy Davis, Member-at-Large for 2010-2011, to be Secretary for this meeting. There were no objections.

    Minutes from the 2010 meeting were approved without discussion.

    Colin Kaltenbach requested that those present provide update for the membership list for NIMSS. Colin also announced that this was his last year serving as the Administrative Advisor for W1112. Colin was thanked for all his years of service and that he will be missed. At this time it is unknown who will serve as the new W1112 Administrative Advisor. It was discussed that the group would prefer an Administrative Advisor with animal experience, but the decision is not ours and a committee will decide.

    Mark Mirando was unable to attend the meeting but did provide a NIFA update presented by Colin Kaltenbach. Director of NIFA, Dr. Roger Beachy, resigned in May 2011. The Acting Director of NIFA is Dr. Chavonda Jacobs-Young. The Undersecretary for Research, Education and Economics (REE) is Dr. Catherine Woteki, former Dean of Agriculture at Iowa State University and first Undersecretary for Food Safety under the Clinton administration. Dr. Woteki also serves as the USDA Chief Scientist. The NIFA budget was decreased and increased for Hatch program for FY2011 Continuing Resolution. A copy of the NIFA Budget report with remaining figures not discussed is included as an attachment. Status of W1112 Project Renewal Colin Kaltenbach commented that our project renewal proposal was submitted in January. There were slight issues with the rewrite that have been addressed. The reviewers considered the second objective to be written more as an outcome rather than an objective. The outcome was rewritten to include Discover or translate. The rewrite for W1112 project has been resubmitted, but has yet to be approved. Gary Moss mentioned that for future rewrites of the project, it would be helpful to keep better track of the collaborative efforts. This can be easily done on a yearly basis with the collaborations listed in the report.

    An impact statement is required on the website for 2006  2011 report. The current project does not end until fall 2011, so there is time before the statement is needed. The impact statement should contain a dollar value because the money impacts of our collaborative research efforts are a measurement to send to congress in support of the budget. Andrea Cupp, Brenda Alexander, Ron Randall, and Dave Kreider will write an impact statement and discuss tomorrow.

    Ryan Ashley was nominated by Andrea Cupp and second by Tracy Davis to be Member-at-Large for 2011-2012. The discussion continued since Kim Vonnahme was not present at the 2011 meeting, she would serve as Secretary for 2011-2012. Tracy Davis would serve as Chair for 2011-2012. All participants were in favor of these motions.

    Brenda Alexander suggested that we table the discussion for next years meeting until Tuesday.

    The remainder of the afternoon was dedicated to station reports for Texas, Arkansas, Colorado, Arizona, New Mexico, Florida, Oregon, Nebraska, Wyoming, and Idaho.

    Fred Stormshak informed everyone that this would be his last meeting with the W1112 project. His participation will be greatly missed.

    Meeting was adjourned at 4:30 by Gary Moss.

    Tuesday, May 31, 2011 Meeting was called to order by Gary Moss at 8:10 a.m.

    Station reports were completed by newest member Dale ZoBell from Utah State University.

    Business: Impact statement was complete and all participants agreed to forward the statement directly to Colin Kaltenbach.

    Registration fees were paid.

    Collaborative projects for 2011-2012 were discussed. Collaborators: Nebraska, Washington Project: Dept of Health & Human Services grant 2009- 2011. Joint publication has been submitted and submission of NIH RO1 grant June 15 titled Role of VEGFA Isoforms on Spermatogonial Stem Cell Renewal.

    Collaborators: Idaho, Washington Project: Evaluate enhanced luteal function on conceptus length and IFNT production in lactating dairy cows.

    Collaborators: Idaho, Colorado Project: Use of new generation of sequencing using isolated gonadotropes.

    Collaborators: Idaho, Colorado Project: Adenoviral delivery of dominant negative dynamin into the ovine pituitary and the effects of estradiol release on LH release.

    Collaborators: Colorado, Idaho Project: RIA for IFNt and Colorado providing reagents for RIA to Idaho.

    Collaborators: Colorado, Hawaii, Texas Project: Continue to analyze the data from the PGE project and write manuscripts.

    Collaborators: Arkansas, Utah Project: Share reproductive and genetic data.

    Collaborators: Colorado, Arizona Project: Develop an assay to evaluate progesterone receptors in mitochondria.

    Collaborators: Colorado, Wyoming Project: Develop a more efficient and cost effective method to synchronize estrus in cattle.

    Collaborators: Arizona, New Mexico Project: Share data about stress response and impact on placental factors and implantation.

    Collaborators: Arizona, Florida Project: Share data from microarray of bovine blastocysts and trophoblast cell line.

    Collaborators: Arizona, Wyoming Project: Insulin secretion in isolated pancreatic islet cells.

    Collaborators: Arizona, Colorado Project: Evaluate vascularization of pancreatic islets.

    Collaborators: New Mexico, Florida Project: Share bovine trophoblast cell line and compare genomic data obtained from bovine placenta and endometrium.

    Collaborators: Montana (Miles City), Michigan, Missouri Project: Investigate the role of follicle size at the time of ovulation on establishment and maintenance of pregnancy.

    Collaborators: Montana (Miles City), South Dakota, Missouri, Washington Project: Investigate the role of estradiol on pregnancy establishment and uterine expression in ovariectomized cows and intact heifers. Collaborators: Florida, Nebraska Project: Examine VEGF in bovine endometrium and placenta.

    Collaborators: Florida, Washington Project: Effect of inhibiting FGF in reproductive tract during early pregnancy in cattle.

    Collaborators: New Mexico, Montana Project: Metabolic hormone patterns in ewe lambs with differing RFIs.

    Collaborators: New Mexico, Colorado Project: Nutrient restriction vs. metabolic hormone patterns.

    Collaborators: New Mexico, Colorado Project: Thyroid involvement in BVD infected cows.

    Collaborators: New Mexico, Arkansas Project: Metabolic hormone profiles in cows consuming endophyte infected fescue.

    Collaborators: New Mexico, Ohio Project: Follicular fluid IGF.

    Collaborators: Wyoming, Montana Project: Meta-analysis of birth type on ewe reproductive performance.

    Collaborators: Colorado, Ohio Project: ISG mRNAs in maternal blood and IFNt in conceptus.

    Collaborators: Colorado, Washington Project: Use of mouse ISG15 knockout model to understand the role of ISG15 in bovine uterus.

    Collaborators: Michigan, Florida Project: Extended oocyte/embryo culture.

    Collaborators: Ohio, Pennsylvania Project: Role of MX and IFNt in cattle.

    Collaborators: Arkansas, New Mexico Project: Pasture systems and reproduction.

    Final Business: Terry Nett informed the participants that a new RIA was developed for IFNt. They have had great success measuring IFNt in cell culture media and serum from blood collected from the uterine vein.

    It was decided that next years meeting will be held May 28  May 29, 2012 in Reno, Nevada.

    The 2011 W1112 meeting was adjourned at 10:12 a.m

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

    Estrous Cycle, Corpus Luteum Function, and Reproductive Performance 1. Endocrine delivery of roIFNT, whether through UV or JV, effectively protects the CL from luteolytic actions of PGF. Also, concentrations of roIFNT as low as 20 mg/day induced ISGs in the CL and provided protection against a challenge with PGF.

    2. AKT, BCL-XL, and XIAP mRNAs are down-regulated within 12h in response to PGF. Endocrin action of IFNT on the CL stabilizes these cell-life genes, which may contribute to luteal resistance to luteolysis during pregnancy.

    3. Microarray analysis confirms endocrine action of pregnancy on the corpus luteum in ruminants, which entails differential expressions of ISGs, chemokines, cell adhesion, cytoskeletal, angiogenic and epithelial to mesynchmal transition genes.

    4. An additional CIDR treatment for 7d (5  12d post-breeding) increased maternal progesterone concentrations by an average 5.5 ng/ml by 1 wk following the 7d harem period in reindeer. There was no effect of progesterone on gestation length.

    5. Genes that are expressed rapidly after PG injection in cattle have been determined to help understand more about CL function and regression. Hopefully this information can be used to understand how we might induce CL regression more efficiency as a method to manipulate estrus cycles in cattle.

    6. PGE1 and PGE2 prevent luteolysis in Angus or Brahman cows by altering angiogenic receptors on luteal tissue as well as preventing loss of message for LH receptors and occupied and unoccupied LH receptors and altering mRNA for prostanoid receptors on luteal tissue. Analogues of PGE1 and/or PGE2 may be viable approaches to prevent loss of luteal progesterone in cows and to decrease pregnancy loss during the first trimester of pregnancy.

    7. Within a larger cattle herd derived from the Texas AgriLife McGregor Beef Cattle Research Center, sub-herds of heifers were identified as having inferior or superior capacity for fertility based on a 5 day Co-Synch + CIDR + artificial insemination protocol.

    Gonadal Development and Function 1. Novel mouse lines where either VEGFA isoforms or the co-receptor to VEGFA angiogenic isoforms have been knocked out in specific cells have been generated to delineate the functions and necessity for appropriate balance of VEGFA isoforms in ovarian and testis development. The actions of these VEGFA isoforms appear to have dramatic impact on male and female reproduction if they are not in appropriate balance. a) Loss of all VEGFA isoforms in Sertoli and some germ cells reduces testis and reproductive organ weight and alters morphology and genes necessary for establishing the spermatogonial stem cell niche are altered compromising spermatogenesis. At six months of age the male mice have 50% reduction in sperm in the epididymus suggesting their reproductive lifespan is compromised.

    b) Loss of NRP-1 in Sertoli cells and deactivation of VEGFA angiogenic isoforms appears to have little effect on organ weights; however morphology of the testis is altered and genes critical for spermatogonial stem cell niche maintenance are dramatically reduced suggesting that too much anti-angiogenic isoform actions are detrimental to reproductive lifespan and male fertility.

    c) Similarly loss of all VEGFA isoforms in the granulosa cells reduces ovarian weight and at eight months of age the KO mice have reduced numbers of CL and 45% less estrogen suggesting that there are less follicles developing and ovulating. Furthermore, genes involved in follicle development and cell survival are also altered suggesting that follicular development is compromised.

    d) If the human VEGFA165b isoform is overexpressed in females then there is a reduction in litter size, a reduction in ovarian weight at 30 days of age and reduced number of primordial follicles as well as a tendency for reduced follicle numbers overall in overexpressing mice compared to controls.

    2. Injection of VEGFA isoforms during spermatogonial stem cell niche establishment affects colonization in recipient males. Angiogenic VEGFA isoform treatment appears to increased numbers of colonies of stem cells suggesting that it increases proliferation and renewal while anti-angiogenic isoforms inhibit colonizationhowever, the length of the colonies are greater. Thus, we are proposing that pro-angiogenic VEGFA isoforms allow for SSC renewal and proliferation while anti-angiogenic VEGFA isoforms stimulate SSC differentiation. Therefore, VEGFA isoforms may be a mechanism to alter or enhance spermatogenesis in males.

    3. We have developed a reproductive longevity X granulosa steroidogenic efficiency model in cattle to determine how both reproductive longevity and granulosa efficiency may affect gene expression in granulosa, theca and cumulus-oocyte complexes as a way to develop markers to select young female to remain in the cattle herd. We have determined that females with lower granulosa steroidogenic efficiency have greater androstenedione in follicular fluid, reduced IGF-1 mRNA and more VEGFA164b in granulosa cells. Furthermore, the increased VEGFA164b also occurs in theca and COC. There is altered gene expression in COC in maternal effect genes and microRNA processing genes suggesting that mRNA stability may be altered which may affect oocyte development and viability. With this model we are elucidating more mechanisms which affect fertility, follicle development and oocyte competence.

    4. Spermatogonial stem cells are sensitive to exposure to alcohol during the establishment and expansion of the spermatogonial stem cell population.

    5. PGRMC1 is important for maintaining a suitable environment in the Seminiferous tubule for germ cell differentiation through actions in Sertoli and Leydig cells. In addition, PGRMC1 participates in the regulation of steroid hormone production in the testis.

    6. Ovulatory follicle size is a good chute side measure of fertility to induced ovulation in beef cows and is indicative of follicle maturity. Increased ovulatory follicle size increases subsequent progesterone production and may actually shorten the lifespan of the resulting CL.

    7. Ovulatory follicle size was negatively associated with cumulus cell cathepsin B and Z expression, strengthening their value as indicators of oocyte competence.

    8. A demonstrated role of the oocyte/embryo specific protein JY-1 in regulation of cumulus cell progesterone production further supports an important potential requirement of JY-1 for fertility in cattle.

    9. Investigation of the role of the CART peptide in regulation of follicular development and specifically dominant follicle selection may ultimately lead to development of new technologies to manipulate the follicular selection process in cattle and potentially enhance efficiency of superovulation.

    Hypothalamic-Anterior Pituitary Gland-Gonadal Physiology 1. The sensitivity of the hypothalamic-pituitary axis to Kiss-10 increases as heifers approach puberty. This appears to be due to estradiol sensitizing the axis to the effects of Kiss-10.

    2. Prenatal critical period for androgen dependent differentiation of the oSDN occurs later than, and can be separated temporally form, the period for development of masculine genitals.

    3. The recombinant single chain analogs of ovine gonadotropins can be used to induce a superovulatory response in sheep and may have a broader application in ovulation induction protocols.

    4. The mouse gonadotrope cell line, alphaT3-1 cells, is not a valid model to study non-classical actions of estradiol via the membrane estrogen receptor.

    Establishment of Pregnancy 1. Intra-uterine administration of the PGF receptor antagonist (AL-8810) did not affect the number of cows diagnosed pregnant at approximately day 30 post-insemination.

    2. Intra-uterine administration of the PGF receptor antagonist (AL-8810) did not affect the incidence of early embryonic loss or subsequent days to next estrus.

    3. An intrauterine dose of fluid (0.5 ml) can be administered 5 days after insemination without compromising pregnancies.

    4. Plasma membrane estrogen receptors in the ovine endometrium are not a rapidly changing population that reflects the contemporary concentration of nuclear receptor or the radioreceptor assay utilized is too insensitive to detect changes in the levels of the plasma membrane receptors.

    5. Increased understanding of regulation and function of the oocyte specific nuclear transport protein KPNA7 may enhance understanding of regulation of the maternal-to-embryonic transition and mechanisms that lead to early embryonic loss and infertility in dairy cattle and reduce efficiency of in vitro embryo production.

    6. Addition of heparin binding protein to sexed, frozen, bull semen failed to improve conception or pregnancy rates. Sire is the major variable affecting fertility of sexed, frozen, bull semen.

    Placental Function and Fetal Development 1. Transcriptional regulation studies have been initiated for PRR15, as have microarrays and pull-down assays. PRR15 is under both positive and negative transcriptional regulation, and it is involved in posttranscriptional regulation through interactions with a variety of nuclear proteins including hnRNPs, and that in its absence tumorigenic pathways are upregulated and integrin-signalling, cell-cycle control, and extracellular matrix interaction pathways are downregulated. We now believe PRR15 is playing a major role in regulating trophoblast differentiation at the time that the conceptus stops its elongation stage and begins apposition with the uterine wall.

    2. Continuation to develop the use of lentiviral mediated transgenesis to alter placental function is still ongoing. In a preliminary study, a 63% reduction in the concentration of oPL mRNA was obtained. While this study was successful, further proving the utility of these approaches, the amount of oPL reduction achieved was unsatisfactory. Accordingly, the targeting vectors are being modified in an attempt to obtain a more robust oPL deficiency. Due to the very high concentration of oPL in normal pregnancies, if oPL expression is adequately reduced, resulting in a phenotypic change in pregnancy, it will provide the proof that essentially any gene express in the ruminant placenta can be altered by this approach.

    3. Expression of CXCR4 (mRNA and protein) in the endometrium from pregnant ewes is increased from day 15 to day 35 of gestation during attachment of the conceptus and placentation.

    4. Expression of mRNA for CXCL12 and CXCR4 is conceptuses increases in a similar pattern during critical times of attachment and placentation in sheep.

    5. Gene expression of CXCL12 is increased in peripheral blood cells from sheep during early pregnancy and it is conceivable that detection of CXCL12 in blood could serve as a pregnancy diagnostic tool.

    6. During early pregnancy in the mouse, the corticosterone biosynthetic enzyme HSD11B1 is up-regulated in response to the implanting embryo. Implications are that the embryo establishes a glucocorticoid barrier around the embryo that may serve to: 1) modulate maternal immune function; 2) regulate nutrient availability; and 3) regulate trophoblast growth.

    7. The murine endometrium harbors a unique population of label retaining cells that display stem cell qualities.

    Uterine-Placental-Fetal Nutritional Interactions during Pregnancy 1. Continuous undernutrition during twin pregnancies is associated with elevated umbilical artery ghrelin concentrations, and increased ghrelin mRNA concentrations. Elevated fetal ghrelin concentrations may provide a signal to the placenta to provide additional nutrient support to the fetus.

    2. No effects of gender on GSIS or b-cell mass were observed.

    3. Fetal insulin secretion is less responsive to physiological glucose concentrations in twins compared to singletons due in part to less b-cell mass.

    4. Repeats bouts of pulsatile hyperglycemia impairs fetal insulin secretion responsiveness and a dysregulation in islet ROS handling.

    5. In IUGR sheep fetuses at 0.7 of gestation, an adrenergic blockade revealed an elevated insulin secretion response to glucose and arginine, but did not affect controls, which is similar to findings in UIGR sheep fetuses at 0.9 of gestation. These findings indicate that hypoxemia via elevated NE cause compensatory insulin secretion responsiveness in IUGR fetuses at 0.7 of gestation prior to appreciable declines in fetal weight.

    6. Surgical Ad restored glucose-stimulated insulin secretion during hypoxemia but reduced it during normoxemia.

    7. After previously reporting the presence of the ghrelin and GHS-R1A receptor mRNAs in the reproductive tract of cattle, it is now evident that the respective proteins are also present throughout the bovine reproductive tract.

    Disease, Immune Function 1. Fetuses persistently infected with ncp BVDV early in gestation induce type I IFN pathways, resulting in chronic up-regulation of ISGs due to persistence of the virus. The innate immune response is not able to clear the virus without contribution from the mature adaptive immune response.

    2. Chronic exposure to the BVDV virus and IFNs can lead to the long-term consequences of disrupted fetal development and impaired postnatal immune responses.

    Nutrition and Reproduction 1. We are measuring phenotypic traits to determine how different supplementation affects reproductive function in beef heifers and are not seeing dramatic effects which may suggest that we need to evaluate differences in supplementation during gestation.

    2. Low residual feed intake (more efficient) animals have a lower insulin response following a glucose challenge than high residual feed intake (less efficient) animals.

    3. More efficient cattle may have a greater population of structural carbohydrate degrading bacteria in the rumen compared to less efficient cattle.

    4. Evaluation of residual feed intake as a heifer may not accurately predit their performance as a gestating cow.

    5. Following acute weaning it requires 10.7 days on the average for calves to learn to eat enough feed to meet intake requirements for maintenance.

    6. Handling and loading of calves resulted in increased body temperatures which returned to normal during transportation. The greatest stressor during transportation is the loading process not the movement during transport.

    7. Female offspring born to ewes nutrient restricted (NR) during the first half of gestation exhibited a marked reduction in fertility. This decreased fertility was associated with decreased progesterone concentrations in systemic blood during the estrous cycle of adult ewes born to NR mothers.

    8. Corpora lutea recovered from the adult female offspring of the NR ewes exhibited markedly decreased progesterone content during the midluteal phase of an estrous cycle. Exposure to maternal NR from early to mid-gestation, programmed increased oxidative stress, and reduced expression of StAR and P450scc enzymes, considered to be rate-limiting in luteal progesterone synthesis. These data suggest epigenetic modification of gene expression in CL of offspring born to NR mothers, which results in decreased progesterone secretion and pregnancy rates.

    Behavior and Stress 1. Ram exposure during the transition into the breeding season alters hypothalamic-pituitary-adrenal axis activity of ewes by permitting an increase in LH pulse frequency which in turn stimulates the ovulatory cascade and accelerates ovulatory activity. The physiological mechanism for this effect is unclear but may be related to relaxation of cortisol inhibition of LH secretion.

    2. Immunization against ACTH23-39 or ACTH1-14 in sheep results in the production of antibodies that recognize and bind specific regions of ACTH and prolong, rather than neutralize, the biological effect of the hormone. This targeted immunization procedure presents a novel means of modulating the activity of endogenous ACTH and may be useful in studying the significance of cortisol secretion in the physiological response to stress.

    3. Acute stress can stimulate indices normally associated with the pro-inflammatory immune response. Assessing the immunological status of an animal at one time point may not accurately depict an animals overall health as it may reflect a response associated with handling.

    4. Temperament is related to the endocrine, behavioral and physiologic responses following an endotoxin challenge. Temperamental animals challenged by an endotoxin utilize nonesterified fatty acids as an energy source while calmer animals utilize glucose.

    5. Temperament alters the reaction to a stressor by increasing glucose and insulin concentrations following a glucose challenge. Temperament clearly modifies metabolic regulatory responses to a metabolic challenge.

    6. Stimuli that increase circulating cortisol may influence expression of cytokines and modulate or prime the immune system prior to a subsequent immune challenge.

    7. Temperament of calves is moderately to highly heritable. Selection for temperament can effectively alter the temperament of a herd or a breed. This research has resulted in the American Brahman Breeders Association adopting the use of pen score to develop a docility EPD.

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

    Estrus, Hormones, Estrous Synchronizations and Artificial Insemination 1. Efficacy of commercial CIDR-g in reindeer was tested. Plasma progesterone concentrations were 2 to 3-fold greater with modified CIDR-b than CIDR-g. Use of commercial CIDR-g in reindeer would simplify estrous synchronization protocols making the technique practical for producers.

    2. Modification of an estrous synchronization protocol was tested in reindeer in an effort to reduce the number of handlings and animal stress. The protocol used frozen-thawed semen, but tests are required to determine if the calves born using this protocol were sired by the AI sire or by the natural service sire.

    Disease, Immune Function 1. Management practices and timing of vaccinations should be evaluated on a herd-by-herd basis. Calves in these herds may be vulnerable to future challenge from BRD viruses unless re-vaccinated. Vaccination at inappropriate times is costly and may provide a false sense of security to producers.

    Behavior and Stress 1. Expression of proceptive behavior in ewes may be important to ensure pregnancy when sexual interest in the ram may be lower.

    2. Expression of sexual behavior in rams with large and extensive lesions in the POA was largely unaffected. More restricted lesions, limited to area near the SDN-POA, had more influence on the expression of sexual behavior. Although conclusions cannot be made in this limited sample size, expression of normal sexual behavior may be dependent on an intact SDN-POA.

    Pregnancy 1. Pregnancy rate in bison determined by fall ultrasonography accurately predicts of calf production at the following roundup. Reproductive loss during gestation occurs in these bison but not at a higher rate than would be expected in cattle and does not appear to be a major factor in determining overall reproductive rate of bison. Examination of the relationship between cow BW, cows that exhibited luteal activity and the dramatic decrease in calf production from 2000 to 2007 one can infer that the number of cows that were anestrus increased, resulting in a decreased in the proportion of cows bred during breeding season and a decrease in the proportion of cows pregnant at roundup. Additionally, normal losses during gestation exacerbated the decrease in calf production. From the historical trends, it would appear that the cause of anestrus during the breeding season is the result of body condition of cows at the NBR, most likely due to range resources or other environmental factors.

    2. Pregnancy rates in Rambouillet ewes were improved by administering 10 mg of PGF at CIDR removal during a fall breeding season.

    3. Administering 5 or 10 mg of dexamethasone to ewes before breeding had no adverse effect on pregnancy rates and the 5-mg dosage may have actually been beneficial.

    4. Use of a CIDR in combination with 500 IU PMSG effectively induced out-of-season cyclicity at 30 days postpartum in lactating anestrous Rambouillet ewes.

    Markers for Reproductive Health 1. An effect of FSH beta subunit promoter sequence on semen morphology (proximal droplets) was observed suggesting that polymorphisms in the promoter regions of this gene may have an effect on semen quality in Brahman influenced bulls and cows. Additional studies are needed to confirm this finding.

    2. No polymorphisms were observed in the FSH beta subunit promoter region of Balancer and Angus bulls in contrast to the variability observed in Brahmans influenced bulls and in Brahman influenced cows.

    Development of Tools 1. A software implementation of a statistical model for small-sample gene expression studies (affyNFM) was validated and published. This was motivated by a small-sample bovine SCNT (clone cotyledons) study and led to meaningful biological conclusions regarding embryonic gene expression in SCNT pregnancies.

    2. Generalized linear models were adapted for complicated experimental designs for next-generation sequencing data, with particular application to understanding the molecular mechanisms governing embryo viability and survival in order to improve the efficiency of assisted reproductive technologies for livestock.

    3. A software implementation was developed to reduce (from two weeks to just a few minutes) the data analysis time for high-throughput genotyping. This genotyping is allowing an efficient assessment of how cloned embryos may be lost because of an immune response mounted by the maternal immune system due to improper expression of certain proteins on some of the fetal cells in the placenta. This assessment will help make the cloning process much more efficient and affordable.

    Impact Statements:
    1. Significant advances have been made which may change how luteolysis or maintenance of the CL of pregnancy in domestic ruminants is viewed. It has been shown that PGE1 and PGE2 prevent luteolysis and IFNT given systemically protects the CL from the luteolytic actions of PGF
    2. Advances were made in understanding how maternal nutrition and obesity impacts growth efficiency, glucose-insulin dynamics and reproductive function of the offspring. Results from these studies shed light on the specific physiological mechanisms involved. These studies provide strong evidence that the optimization of maternal nutrition and the associated uterine environment will contribute to the birth of healthy, growth-efficient offspring.
    3. Male fertility is critical to animal production systems. It has been demonstrated that VEGFA isoforms are important in early testis development to establish the spermatogonial stem cell niche. Furthermore, a stage specific, niche-independent microenvironment exists to regulate spermatogonial stem cells self-renewal in vivo.
    4. Progress has been made to gain further understanding of how normal sexual behavior is dependent upon an intact SDN-POA. Behavior of the animal also impacts reproductive function of the opposite sex, potentially altering the hypothalamic-pituitary-adrenal and/or gonadal axis.
    5. Significant progress has been made to identify proteins that may play a major role in conceptus development and implantation.
    Last Modified: 14-Jul-2011
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