Duration: 10/01/2011 to 09/30/2016

Administrative Advisor(s):

NIFA Reps:

Non-Technical Summary

Statement of Issues and Justification

The W-1112 Regional Research Project was established in 1970 to create a cooperative research group that would combine both basic and applied expertise to determine factors, and develop methods to improve the fertility of domestic ruminants in the Western states. The philosophy and mission for the W-1112 established more than forty years ago continues to be the guiding tenet of our group; that is, cooperative multi-state research, that provides product and technique development and outreach for the benefit of animal producers in the Western region.

The project serves as a forum for the development and conduct of collaborative studies aimed at solving problems that limit the reproductive performance of domestic livestock. Scientists associated with the project collectively possess expertise required to discover basic physiological mechanisms and translate such new knowledge to the management of domestic ruminants. Some stations are best equipped to evaluate the applicability of their results in production situations. In contrast, other stations have the animal resources to test new treatment paradigms arising from the basic studies, but do not have the laboratory facilities (or modern equipment) necessary to perform the basic research studies. These circumstances create an ideal situation for our regional collaborative project. Renewal of the W-1112 Regional Research Project is crucial because interactions among scientists with a broad range of expertise are necessary for the discovery, translation, and transfer of new knowledge to the livestock industry.

Poor reproductive efficiency in domestic ruminants limits profitability and sustainability of animal production systems in the West and throughout the nation. Therefore, we seek to continue our work in this critical area. Participation in the project since its inception has greatly increased in scope and is now comprised of scientists located in the states of Alaska, Arkansas, Arizona, California, Colorado, Florida, Kansas, Michigan, Minnesota, Montana, Nebraska, Nevada, New Mexico, North Dakota, Texas, Utah, Washington, and Wyoming. We believe that the challenges that are shared among the Western states are best addressed by combining the expertise and resources from all the states. The addition of leading reproductive biologists from states outside the West has increased the breadth and greatly strengthened the scientific expertise of W-1112. Renewal of this multi-state project is essential to continue to provide a forum that stimulates the development of new hypotheses, conduct of new collaborative research projects, sharing of resources, and identification and testing of new methods to manage reproduction in domestic ruminants.

The ruminant livestock industry is a critical component of the agricultural industry in the states represented by scientists involved with the project. The dairy, beef, and sheep industries together contribute approximately $63 billion in farm receipts and an estimated overall economic impact on the nation's economy of $332.5 billion. In addition, direct and indirect employment related to the production and processing of these animals or their products supports over 2.3 million jobs (Otto and Lawrence, 2002; Cryan, 2004; Shiflett, J.S. 2008). Over 50 percent of the nation's breeding cows (beef and dairy) and 64 percent of its breeding ewes exist in states represented by participating W-1112 scientists (USDA-NASS, 2010).

W-1112's goals are consistent with the USDA 2010-2015 Strategic Plan's Goals 1, 3 and 4. Specific objects addressed by the project include Objective 1.1 (Enhance rural prosperity), Objective 1.3 (Support a sustainable and competitive agricultural system), Objective 3.2 (Enhance America's ability to develop and trade agricultural products derived from new technologies), Objective 4.1 (Increase access to nutritious food), and Objective 4.4 (Protect agricultural health by minimizing major diseases and pests to ensure access to safe, plentiful, and nutritious food). Our primary stakeholders are famers and ranchers in states represented by scientists participating in the project, but there is broad applicability of our work nation-wide. Our secondary stakeholders are the consumers of animal products that benefit from the reduced prices associated with efficient animal production systems. Our tertiary stakeholders are the citizens of communities whose economies are improved by their proximity to profitable and sustainable animal industries and that benefit from the multiplier effects these industries have on community economies.

Reproductive efficiency is widely regarded as the most limiting factor to profitability in animal production systems. Nowhere is this more evident than in the modern dairy industry. Beef producers also suffer as a result of delayed onset of puberty and extended postpartum anestrus, low fertility, and lighter calves at weaning. In the beef industry alone, the cost of infertility to U.S. producers was estimated to be over $1.06 billion annually (Lamb, et al., 2008). Sheep producers also miss out on the potential for added revenue by not realizing the genetic potential for lambing rates in their flocks. Finally, new challenges are faced by farms and ranches managing domesticated exotic ruminants whose reproductive physiology is relatively unknown.

Sub-optimal reproductive efficiency of domestic ruminants and feed costs associated with producing those animals are major obstacles to maintaining the profitability and sustainability of livestock production enterprises. Up to 70 percent of costs associated with producing viable offspring can be attributed to feed required to maintain their dams during gestation. Likewise, decreased fertility resulting from delayed onset of puberty, prolonged postpartum anestrous intervals, early embryonic mortality, and seasonality of breeding continues limit production. One of the objectives of our work in W-1112 is to provide the scientific and technical expertise that will encourage development and application of science-based management tools to improve the productivity and profitability of livestock producers. In the current project plan we expect to increase our efforts to bring knowledge to producers and help them make decisions based on sound science while at the same time, expanding our understanding of factors that affect reproductive efficiency.

Although artificial insemination and embryo transfer have been widely accepted and adopted by the dairy industry, these techniques are less commonly used in the beef cattle and sheep industries especially in the arid western regions where animals cannot be confined to small pastures for more than short periods of time. However, the improvement in animal health and productivity in the dairy industry has not gone unnoticed and use of these reproductive technologies in beef cattle and sheep is expected to increase during the next decade as methods to increase fertility and reduce costs and labor associated with the techniques are developed and proven reliable. Work from W-1112 members has helped refine, simplify, explain, and standardize many of the estrous synchronization protocols (see http://www.oardc.ohio-state.edu/estroussynch/wester014.pdf), but more needs to be done as is discussed in later sections.

An issue of concern to livestock producers is animal welfare. We are expanding our efforts to develop new management strategies to evaluate and improve animal well-being while maintaining or improving productivity. Producers have been eager to adopt new management strategies that utilize more humane and/or less stressful practices while improving the productivity of their livestock and look to members of this committee to develop these.

To succinctly present the research and activities conducted or planned by members of W-1112, subsequent discussions are subdivided into areas of biological function within animals. Clearly, however, the development and testing of new technologies capable of enhancing livestock industry requires an integrated understanding of those areas.

Related, Current and Previous Work

Most of the stations that are members of this collaborative agreement are located in the arid western states. Due to the relative paucity of vegetation in these states compared to those in other regions of the U.S., very different management techniques are necessary. Therefore, most of the approaches to improve reproductive efficiencies of cattle and sheep in these regions will, of necessity, be different than approaches where these species are maintained in smaller pastures or in confinement as is the case with projects focusing on reproduction in North Central and North East regions.

Related current and previous research by members of W-1112 is based on the premise that applied research experiments stem from a foundation of previous basic research studies. Studies reported herein, therefore, describe the discovery of mechanisms that regulate reproduction and the translation of those results into methods to improve reproductive performance in domestic ruminants. The list of research achievements, publications, and student theses that are the product of the collaborative work of members of the Project is extensive. During the last 5 year period 349 refereed publications and technical bulletins and 7 book chapters were generated. In addition, a symposium of research findings was presented to a nationwide audience of livestock producers at the 2007 Beef Improvement Federation Meetings in Fort Collins, CO. Although it is not possible to detail each significant accomplishment, a summary of the major advances is provided below.


Major Advances:

Biology of the Hypothalamic-Pituitary-Gonadal Axis. Estradiol induces the pre-ovulatory LH surge through genomic actions but the onset, duration and magnitude of the surge are controlled by non-genomic effects of estradiol on numbers of GnRH receptors. KiSS-1 peptide modulates LH secretion by regulating the secretion of GnRH without effects on the anterior pituitary and independent of the actions of estradiol and progesterone. Microarray analysis identified genes that are differentially expressed in the pituitary gland of cows during the transition from postpartum anestrus to estrous cycling. The demonstration that steroid receptors are expressed in sheep fetal brain tissues suggests they may play a role in sexual differentiation of the brain.

Passive immunization against inhibin at 2-day intervals resulted in a 2- to 3-fold sustained increase in FSH concentration. Recombinant single chain analogs of human and ovine gonadotropins with long functional life were produced and can be used to induce precocious puberty and superovulation in sheep and perhaps other ruminants. These gonadotropins induce an immune response with repeated administration which may prevent their usefulness in treatment protocols when used repeatedly.

Ovarian Biology and Follicle Development Research.

Studies focused on vascular endothelial growth factors (VEGF) in ovarian development and follicle number demonstrated the importance of angiogenic VEGF isoforms in granulosa cell function and folliculogenesis. Three variants of the FSH receptor are produced at different stages of follicular development. Stable granulosa cell lines that produce specific FSH receptors were used to characterize precise roles for each receptor variant. Breed differences exist among sheep for genes involved in regulating folliculogenesis.

Estrous Cycle, Physiology, & CL Function.

Luteolysis appears to involve two stages. Prostaglandin F decreases progesterone production by acting directly on large luteal cells where an influx of calcium initiates apoptosis. Oxytocin induced an increase in intracellular calcium levels in small luteal cells. However, normal luteal concentrations of progesterone may prevent the oxytocin- and PGF-induced increase in calcium influx in small and large luteal cells. Intraluteal secretion of PGF is required for structural luteolysis. Endocannabinoid type 1 and 2 receptors may also play a role in luteolysis. PGE1 and PGE2 prevent luteolysis by maintaining LH receptor expression, facilitating binding of LH and altering prostanoid receptor expression. Intra-luteal administration of meloxicam, a cyclooxygenase-2 inhibitor, increased the length of the ovine estrous cycle. Both nitric oxide (NO) and polyamine synthetic enzyme expression and activity fluctuate during the ovine luteal phase and in response to PGF. Neither NO nor Endothelin-1 (ET-1) is luteolytic when infused into the uterus but may be luteotropic or antiluteolytic by altering the PGE:PGF ratio.

Differences in CL gene expression profiles, accompanying functional gene categories, and biological pathways responsive to the luteolytic actions of PGF exist. Such differences are linked to candidate transcription factors that may mediate stage specific gene expression response to PGF during luteolysis. Analogues of PGE1 and/or PGE2 may prevent loss of luteal progesterone production in cows and decrease pregnancy loss during the first trimester of pregnancy.

Oocyte-Embryo-Uterine Physiology.

An oocyte/embryo specific gene (JY-1) was identified. This gene regulates cumulus expansion , is dependent on the presence of additional oocyte-secreted factors, and is potentially mediated in part by PGE2 and genes related to stabilization of the hyaluronan-rich extracellular matrix. Cumulus expansion and progression to metaphase II, fertilization, and cleavage rates following IVF are reduced following siRNA mediated ablation of JY-1 in oocytes. Such effects can be rescued by addition of recombinant JY-1 to culture medium suggesting an obligatory role for JY-1 for pregnancy establishment. Additional studies using siRNA demonstrated an obligatory role for follistatin in regulation of early embryogenesis. A negative association of cumulus cell cathepsin expression with oocyte competence and development to the blastocyst stage was established. Treatment with a cathepsin inhibitor during meiotic maturation enhanced blastocyst development.

Reciprocal embryo transfer and duration of proestrus studies demonstrated that effects of ovulatory follicle size on fertility may be manifested through effects of estradiol on the oocyte and uterus. Preovulatory concentrations of estradiol did not influence expression of ISG-15, MX-2 , embryonic CSH-1 or IFNt on d 17.5 of gestation or the extrous cycle in cattle. However, estradiol tended to increase the expression of endometrial nuclear progesterone receptors. It was demonstrated that the embryo induces endometrial expression of an uncharacterized gene referred to as decidual restricted gene 1 (Drg1). Drg1 may be required for the establishment and maintenance of pregnancy. Endocrine effects of IFNt on the CL between Days 13 and 14 of pregnancy in the ewe may protect the CL through mechanisms complementary, yet independent to IFNts paracrine effects on the endometrial oxytocin receptor pathway. Infusion of IFNt into the uterine vein in non-pregnant sheep induced IFN-stimulated genes in the CL. Conceptus elongation coincides with the expression of peri-attachment factor (PF). The use of lentiviral-mediated RNA interference demonstrated that PF is required for normal conceptus elongation and survival.
The roles of fibroblast growth factors (FGFs) on oocyte competency and pregnancy establishment were elucidated. Embryo development in vitro is improved by supplementing FGF2 or FGF10. At least four FGF receptors (FGF-R) reside in pre- and peri-attachment bovine conceptuses. Conceptuses express at least three candidate FGFs during elongation. FGF2 may signal through a downstream PRKCD to regulate IFNt expression in bovine trophectoderm and promote embryonic endoderm development. Blocking FGF receptor activity with specific kinase-domain inhibitors did not affect blastocyst formation in vitro but compromised subsequent development and function. Treatment with FGF receptor inhibitors during maturation limited subsequent embryo development.

Testicular Physiology.

Studies were conducted to evaluate bovine testis and spermatogonial stem cell (SSC) xenografts. Effects of VEGF on testis development and spermatogenesis were evaluated to elucidate factors critical for SSC differentiation and maintenance. Using magnetic activated cell sorting technology, Thy 1 was identified as a conserved marker of SSC. Peritubular myoid cells and testosterone are important components of the SSC renewal in the adult testis. Three angiogenic and three tissue growth factors that may improve efficiency of ectopic testis tissue grafting were identified.

Active immunization of ram lambs against inhibin increased sperm production 26%. The magnitude of increase in sperm production was directly proportional to the inhibin antibody titer. Inhibin appears to be involved in the regulation of the number of primary spermatocytes in males. Based on DNA parent verification, variation that is not correlated with scrotal size exists in serving capacity of bulls in multiple sire breeding herds.

Uterine-Placental-Fetal Nutritional Interactions.

Evaluation of a large dataset revealed that ewes born twin to a male lamb tended to have lower lifetime productivity than ewes born twin to a female lamb.

Fetal intrauterine growth restriction (IUGR) resulting from undernutrition during the first half of gestation in ewes and cows has negative implications for the health of offspring after birth including; insulin resistance, hyperphagia, obesity, decreased skeletal muscle mass, hypertension and infertility. These health, fertility and body compositional effects were not alleviated even if the dams were realimented during the second half of gestation and offspring were born at a normal birth weight. Greater insulin sensitivity among IUGR lambs resulted from increased peripheral uptake of glucose rather than changes in central insulin sensitivity or liver glucose synthesis. These data indicate that skeletal muscles and adipose tissues are primary targets for the increased insulin sensitivity. In IUGR sheep fetuses, functional changes in pancreatic ²-cells occurred prior to declines in ²-cell mass and mitosis indicating insulin might be a primary initiator of fetal growth restriction. Major impairments in ²-cell development and function appear to occur during the final third of gestation when nutrient and oxygen disparities double due to impaired placental transport. Female offspring born to nutrient restricted (NR) ewes during the first half of gestation exhibited decreased concentrations of progesterone in serum and CLs recovered during the estrous cycle as adults.

Fetuses from ewes 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 obese dams had greater body fat and reduced pancreatic function. 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 neuropeptide Y (NPY) and agouti-related protein (AgRP) neurons.

Impaired placental function was a primary cause of fetal growth restriction. Functional placental insufficiency was associated with changes in placental expression of insulin-like growth factors, their binding proteins, increased placental apoptosis, and differential changes in endothelial nitric oxide abundance in cotyledons and the umbilical artery. These may explain the impaired oxygen and nutrient provision to the fetus. In IUGR fetuses, islet vascularity was reduced to a greater extent than ²-cell area. These studies link disruptions in endothelial cell function and islet blood flow with decreased islet function in utero. Analysis of vascular development coupled with direct measures of blood flow in hyperthermic-induced IUGR pregnancies demonstrated some reduction of uterine blood flow during the first 15 days on treatment, however, reduced flow was countered by elevated maternal arterial pO2 resulting in increased uptake of oxygen by the uterus.

Statistical Methodology.

Statistical tools were generated to advance discovery of important reproductive characteristics. The nested factorial probe-level statistical model can be used to assess differential genomic expression in studies where large sample sizes or number of biological replicates on microarrays are not practical. Additional statistical models were assessed for genomic data 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 competitive against other statistical models and was applied to bovine nuclear transfer data (4 clone vs. 3 control arrays, in cotyledon). The resulting 584 significant genes were biologically relevant. The developed NFM method was implemented in R code (affyNFM), now freely available on a W-1112 members website (www.stat.usu.edu/~jrstevens).

Nutrition and Reproduction.

Level of feed provided to pregnant cows appears exert a uterine programming influence that alters circulating concentrations of IGF-1 and reproductive performance of their daughters when they are reared on restricted levels of nutrition. Heifers developed from weaning to prebreeding on restricted diets from dams wintered on marginal levels of feed had greater concentrations of IGF-1 from precalving through 2nd rebreeding and greater retention rates to their 5th breeding season than contemporary heifers born to dams provided adequate supplemental forage throughout the winter. 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. Feed requirements per pregnant heifer were reduced by 22% during the 140-d development period. Circulating concentrations of IGF-1 during the peripartum period were better indicators of rebreeding performance of first calf heifers than either BCS or BW.

Replacement heifers selected for low residual feed intake experience a 14 d earlier return to estrus after calving. Bos indicus, Bos taurus and crossbred cattle differ in circulating concentrations of IGF- I. Bos indicus animals had higher concentrations of IGF- I than Bos taurus counterparts. Residual feed intake was not related to concentrations of IGF- I. Concentrations of IGF- I are not a reliable physiological marker for selecting more feed efficient cattle. Concentrations of cortisol released in response to an ACTH challenge are not a reliable physiological marker for selecting more feed efficient cattle.

Ghrelin and its receptor (GHS-R1a) are expressed within the reproductive tract and may be involved in the metabolic regulation of reproduction. Hungry heifers with elevated plasma ghrelin concentrations became anestrus although no correlations between circulating concentrations of ghrelin and reproductive parameters were detected. Heat stress increased plasma concentrations of ghrelin during the peri-prandial period. When added to culture media, bovine ghrelin reduced bovine embryonic development.

Behavior and Stress.

Objective identification of temperament, and the coupling of stress-responsiveness with immune function, provided impetus to develop methods to select for calm, stress-tolerant, immune-competent, disease-resistant cattle. Exit velocity from a standard squeeze chute was indicative of stress perception in cattle and can be measured reliably as early as 21 d after birth. Calf exit velocity was associated with serum cortisol concentrations but not with temperament of its dam.

Bull exposure hastened the resumption of estrous cycles in postpartum anestrous cows. Bull exposure decreased frequency, but increased the duration of cortisol pulses in anestrous, suckled beef cows. Bulls did not influence systemic glucose and thyroid hormones in primiparous postpartum suckled beef cows. However, adipose metabolism in postpartum beef cows may be affected by the presence of bulls.

Exposure to glucocorticoids during the second half of pregnancy has deleterious effects on pregnancy outcomes. Embryonic signals result in a shift toward the local production of glucocorticoids. Activation of glucocorticoid receptors within the endometrium may establish a barrier that precludes access of maternal immune cells to the embryo through increased expression of adhesion molecules.

Estrus, hormones, estrous synchronizations and AI.

The optimal timing, dose, and route of administration of progesterone to induce ovulation or estrous cycles was investigated and a protocol developed that increased success to AI by more than 10% in both heifers and cows.

Collaborative efforts by W-1112 members helped gain FDA approval of the EAZI-Breed CIDRs for sheep. Pregnancy rates in ewes were improved by administering 10 mg of PGF at CIDR removal. The administration of bovine somatotropin at a synchronized estrus reduced birth weight of lambs but did not improve fertility. Estrous activity was induced in prepubertal lambs using a combined CIDR and PMSG treatment; however, estrous cycles were not maintained. Actions of thyroid hormones and prolactin were related to fertility during induced estrus and ovulation in sheep.

Modified bovine CIDRs for estrous synchronization in reindeer produced circulating progesterone concentrations consistent with luteal phase progesterone concentrations and had 100% retention rate. Pregnancy rates following synchronization and natural mating were 88%. Reindeer bred earlier during the breeding season have a longer gestation than cows bred later in the year resulting in a very short calving period within a herd.

Puberty.

Scrotal circumference in young bulls is highly correlated with dam milk production; however, direct pre-weaning gain is only lowly correlated with scrotal circumference and post-weaning gain is not correlated with scrotal circumference. Thus, how bulls are fed during development after weaning should not affect sperm production. Over-conditioning of cows during gestation did not exert in utero effects on age of puberty or spermatozoa morphological characteristics. Feeding the progestin, MGA, before puberty increased testis size and altered testosterone production.

Genetic selection for age at puberty impacted the postpartum anestrous interval in beef cattle and may improve rebreeding performance. Precocious puberty is induced in heifers with both high and low starch diets that support accelerated weight gains. Occurrence of precocious puberty is associated with increased systemic concentrations of IGF-1 in heifers.

Immunocastration.

Vaccines containing various GnRH antigens were effective for temporary sterilization in cattle, elk and deer. Vaccination of pregnant elk did not interfere with existing pregnancies and GnRH antibodies were effectively transferred to the neonate via colostrum. The antibodies did not interfere with normal development/function of the hypothalamic-pituitary gonadal axis in the offspring. Long term immunosterilization was also demonstrated using GnRH and porcine zona pellucida (PZP)-based vaccines in deer, elk and wild mares.

The adjuvant, Emulsigen, with the addition of bacterial DNA may promote a greater immune response than Freund's adjuvant. The enhanced immune response may be advantageous in the immuno-neutralization 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.

Disease, immune function. f cattle grazing within regions endemic for the pajahuello tick vector and can result in abortion rates reaching 60%. The pathogen causing EBA as an intracellular bacterium in the class Myxococalesk transmitted through the tick vector. In affected cattle, immunity to subsequent EBA induced abortions occurs but the duration of this protective effect is undetermined. Ongoing efforts indicate that cows are unlikely to abort if exposed to the pathogen in the year subsequent to abortion and that some protection is evident on a herd-wide basis in the second year. An SCID mouse model was developed to serve as a method to raise the etiologic agent of EBA. This development allowed for the consistent transmission of the disease with quantifiable amounts of agent. Ongoing research using the mouse grown agent as a source of antigen to vaccinate cattle herds has shown it to be a safe method of protection against EBA when administered to non-pregnant cows and heifers. Further research is ongoing but preliminary studies have shown the vaccine to provide protection in nearly 100% of the cows and heifers tested.

Bovine viral diarrhea (BVDV) is a widespread problem that can result in abortions, stillbirths, and birth of weak and chronically sick calves that are carriers of the disease. Cows exposed to the virus during early gestation give birth to persistently infected calves. 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 observed in persistent BVDV calves and in other viral infections during pregnancy.

Objectives

1. 1. Discover and translate molecular, metabolic, genomic, endocrine, and immunologic mechanisms that influence 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.
   

Methods

W-1112 collaborators share a common interest in developing methods to optimize reproduction in domestic ruminants but differ in regards to expertise, laboratory facilities, availability of research animals, and basic or applied approaches to research. This combination of abilities provides unique opportunities to discover and translate scientific information crucial for the development of methods to optimize reproduction in domestic ruminants. Examples of such collaborative efforts follow in a later section. Biology of the Hypothalamic-Pituitary-Gonadal Axis. A nearly pure population of ovine gonadotropes will be isolated to study the role of estradiol-17² (E2), and potential interactions of E2 and GnRH, on gonadotrope function. A construct containing the oFSH beta promoter with the extracellular domain of the murine H2Kk molecule will be inserted into the AdEasy Adenoviral Vector System (Agilent Technologies). Once the recombinant adenovirus is made, dissociated pituitary cells will be infected at various MOIs to determine the effective amounts of infectious units. Cells will be incubated with an antibody specific for the murine H2Kk molecule conjugated to biotin followed by the secondary anti-biotin antibody conjugated with magnetic beads. Gonadotrope cells will be selected using a magnetic bead cell separation (MACS) system. Purity will be determined by immunohistochemical analysis of anterior pituitary hormones. After obtaining a purified population of ovine pituitary cells, cells will be treated to determine novel genes expressed in response to hormone stimulation. The genome of the gonadotropes will be sequenced using the SOLiD 3 sequencer located at Colorado State University. This method provides data in developing siRNAs which will be used to determine the importance of the novel genes in gonadotrope function. Potential roles of proteins involved in signaling pathways in gonadotropes will be evaluated using inhibitors developed as a result of protein sequencing. Ovarian Biology and Follicle Development Research. Ultrasonic guided intrafollicular injections, receptor binding assays and functional genomics approaches will be used to determine changes in granulosa cell CART receptor binding that accompanies follicle selection and hormonal regulation of CART binding. Studies will focus on mechanisms and the ability of CART to inhibit IGF1 actions in dominant follicle selection, effects of CART receptor signaling on the formation of multiple, co-dominant follicles, and the molecular identity of the CART receptor. Estrous Cycle, Physiology, and CL Function. The influence of preovulatory events on fertility and molecular events that influence uterine capacity and its ability dto sustain the embryo until attachment will be investigated. Emphasis will be placed on follicular growth rate, follicle size and age at ovulation, oocyte maturation, epigenetic patterns of early embryo expression, peri-ovulatory endocrine patterns, and magnitude of gonadotropic stimulus received preceding the ovulatory LH surge. Endocrine patterns during the early luteal phase and near the time of maternal recognition of pregnancy and their effects on uterine gene expression and oocyte health will be evaluated. Mechanisms through which bulls reduce the duration of postpartum anestrus will be investigated. Urine from bulls presumably contains a pheromone that stimulates the resumption of ovulatory activity by activating the KiSS-10 pathway in the brain which subsequently reduces inhibitory effects of the cow-calf bond and suckling. To evaluate these mechanisms, primiparous, anovulatory beef cows will be exposed to bulls or bull urine. Olfactory epithelial tissue will be obtained to assess changes in functional olfactory signaling proteins and/or their messages. Protein and volatile and semi-volatile components of urine from bulls will be tested for biostimulatory components that alter KiSS expression in concert with changes in LH pulse frequency and ovarian follicular wave development and ovulation. Blood samples will be collected for assay of progesterone to confirm resumption of estrous cycles after biostimulation. Experiments will continue to characterize endocrine and seasonal influences on reproductive behavior, timing of conception and gestation length in Alaskan reindeer. Similar events in muskoxen will be evaluated on an opportunistic basis. Oocyte-Embryo-Uterine Physiology. Research will investigate interactions between gonadotropins, inhibin, follicular steroid production, oocyte viability, uterine function, and early embryonic survivability and development. Effects of diet on oocyte quality assessed by measuring embryo cleavage rate and morphological development after fertilization. Matrix stem cells from Whartons jelly of bovine umbilical cords will be isolated and tested to determine genes necessary for cell maintenance in a pluripotent state. Regulatory elements for Nanog and Oct-4 genes, which are important for early embryo development, will be characterized using promoter constructs driving luciferase and green fluorescent protein reporter genes. Cells from different stages of gestation will be transfected with reporter constructs to determine gene function. The contributions of JY-1 to oocyte regulation of cumulus cell function, successful early embryonic development, and JY-1 regulated signaling pathways critical to its biological actions will be determined using oocytectomy, siRNA microinjection, and immunoneutralization strategies in combination with functional genomics. The biological role of CXCL12/CXCR4 interactions during implantation will be investigated. Specific immune cells expressing CXCR4 and the downstream pathways activated upon ligand binding will be elucidated using approaches that inhibit activation of CXCR4 (i.e., CXCR4 antagonists, antibody neutralization, siRNA knockdown of CXCL12 and/or CXCR4). Early pregnancy loss is estimated at 25% or greater in beef cattle and 45% or greater in dairy cattle. In U.S. beef cattle, this annual loss was estimated to exceed $1.2 billion (Geary, 2005). Most losses are characterized by asynchrony between the blastocyst and uterus or uterine dysfunction. Research will be conducted to develop diagnostic tools useful for the selection of high fertility heifers using unique populations of beef heifers with high and low uterine capacity for early pregnancy. Testicular Physiology. Testicular size and spermatogenic potential of rams and bulls is determined by the number and activity of Sertoli cells. Recent studies indicate that thyroid status can influence testicular development and the duration of mitotic activity of Sertoli cells in sheep. Rram lambs will be treated during the prepubertal period with a long-lived agonist of FSH and a potent TSH receptor blocker to determine if duration and extent of Sertoli cell proliferation can be extended. A deglycosylated analog of oTSH will be developed and administered to block the TSH-receptor to create hypothyroidism without thyroid overgrowth. The SNPs in the first ~600 bp of the promoter 5' UTR (promoter) region of the FSH² subunit gene have been related to reproductive and endocrine measures in bulls. Evaluation of those SNPs will continue in conjunction with examinations of polymorphisms further upstream for additional polymorphisms that may be related to reproductive measures in bulls and reproductive efficiency and function in females. Vascular endothelial growth factor A (VEGFA) supports the survival of germ cells in testicular tissue. Experiments will be conducted to determine if VEGFA stimulates survival and proliferation of spermatogonial stem cells (SSCs), the conversion of pre-spermatogonia into SSCs, or stimulates both events. In addition, whether VEGFA actions are also exerted through somatic Sertoli cells will be evaluated. In vitro fertilization (IVF) methods coupled with characteristics of visually normal sperm cells will be investigated to quantify bull fertility Uterine-Placental-Fetal Interactions. Interactions between embryonic and uterine factors and events that modulate placental and fetal development will be evaluated. Emphasis will be placed on defining actions of uterine-derived glucocorticoids on uterine and embryonic/placental function, effects of exposure of female fetuses to male hormones during critical states of development, and effects of sibling sex on lifetime productivity of ewes. Impacts of maternal nutrition on placental and fetal development and postnatal growth and production traits will be evaluated. Lentiviral-mediated transgenesis in the developing sheep placenta will be used to investigate specific roles of genes expressed within the placenta. Hatched blastocysts will be infected with a lentiviral construct that either knocks-down the expression of the gene of interest, or enhances it, before transfer into synchronized recipients to establish transgenic pregnancies. This technology demonstrated the requirement of proline-rich 15 (PRR15) for normal conceptus development. Transgenic pregnancies will be evaluated to determine the impact on placental function and fetal development. The function of PRR15 in trophoblast cell differentiation and function will continue to be evaluated. Mechanisms whereby maternal undernutrition and over-nutrition results in decreases in pancreatic ²-cell number and insulin secretion by late gestation in their fetuses compared to fetuses of ewes fed to NRC recommendations will continue. Insults, such as nutrient deficiency during pancreas development, disrupts islet organization or b-cell function for life. Regulatory genes (i.e. transcription factors and growth factors) involved in pancreas development and b-cell responsiveness are likely targets to mediate structural, metabolic, or physiological changes to create a lasting effect. The hypothesis that recognition and maintenance of pregnancy is mediated through endocrine action of interferon tau (IFNT) on the CL will be tested by contrasting CL on days 12-15 of the estrous cycle and pregnancy from ewes. Effects of IFNT administered during endogenous release of PGF, the endocrine delivery of IFNT in response to exogenous challenge to PGF, and delivery of IFNT to isolated small and large luteal cells challenged with oxytocin or PGF will also be determined. Studies will also evaluate how specific uterine- and conceptus-derived factors such as influence conceptus development during the first three weeks of gestation. Mechanisms through which progesterone exerts non-genomic effects will be evaluated based on the hypothesis that a membrane progestin receptor (PGRMC1) mediates non-classical actions of progesterone in the gravid uterus. Studies will be conducted to establish a functional role for this progestin receptor in female reproduction. Methods to detect pregnancy as early as 19 d after estrus will be evaluated by combining ultrasonic measures (endometrial echotexture, blood flow to the ovary, and fetal heart beat) with maternal blood characteristics (progesterone, ISG-15, PGFM). Statistical Methodology. Studies to develop and assess statistical and computational methods for genomic technologies to identify and exploit genetic factors for improving reproductive performance will continue. Of particular interest are collaborations involving cloning techniques to recover superior genetics via high-throughput DNA sequencing, next-generation sequencing, and gene expression microarray technologies. Three specific projects in early stages of development are briefly summarized below. High-resolution genotyping of cows and donor cells of cloned bovine pregnancies will be used to identify genetic factors associated with successful cloned bovine embryos. A user-friendly software interface will be created to dramatically reduce the data processing time, ideally to just several minutes. Next-generation sequencing and gene expression microarray technologies are used to identify genetic markers for higher birth weight and pregnancy success for various embryo types; including IVF and scNT-derived. Factorial experimental designs employed suggest the use of ANOVA-type statistical models for continuous data. The discrete nature, however, of next-generations sequencing requires the development and assessment of a Poisson-type statistical model for count data to increase the statistical power to detect genetic markers of interest for the various embryo types. Factorial and repeated measures designs used in next-generation sequencing and gene expression microarray applications lead to a high-dimensional nested hypothesis structure. In this structure, hypotheses are only evaluated when their parent null hypothesis is rejected. Current multiple testing adjustments for high-dimensional genomic data ignore the possibility of such structures. Statistical approaches to exploit this structure will be developed and assessed to reduce type I errors and increase statistical power to detect the genetic markers of interest for the various embryo types. Nutrition and Reproduction. The role global nutrition and specific components of the maternal diet exerts on fetal and placental development and offspring endocrine function ovarian development, and attainment at puberty will be examined. Methods to investigate maternal impacts on uterine and umbilical blood flow is ongoing to determine how fetal and placental development may be hindered, or augmented, with differing maternal dietary treatments. Concentrations of progesterone and fertility of ewes born to over-nourished and obese mothers versus those born the control fed mother will be tested. Impacts of early maternal undernutrition in the cow on maternal hormone concentrations and offspring birth weights of young (3 and 4 year old cows) versus mature (6 and 7 year old cows) pasture fed beef cows will be evaluated. The F1 calves will be followed through weaning to evaluate hormone concentrations, weaning weight and post weaning performance. Ghrelin was implicated in the metabolic regulation of energy balance. The reproductive significance of Ghrelin will be investigated by examining molecular, metabolic and endocrine mechanisms influencing conception rates and embryonic survival in dairy cattle. Projects will be conducted to determine how the diet of a pregnant cow impacts her future fertility and performance of her calf. Effects of various feedstuffs on reproductive processes such as attainment of puberty and postpartum fertility will be monitored. In addition, effects of selection for residual feed intake on reproductive performance of the cow herd will be tested by comparing efficient and inefficient lines of Brahman cows mated to produce efficient and inefficient Herford x Brahman females for lifetime evaluation of productivity. Behavior and Stress. Heat stress during mid-gestation reduces placental efficiency which leads to fetal hypoxia, fetal hypoglycemia and provides a model to study fetal compensatory mechanisms to fetal nutrient restriction. Placental insufficiency induced sheep fetuses and lambs will be used to examine fetal sheep pancreatic development, mechanisms leading to decreased b-cell function, and other postnatal implications. On-farm experiments will be conduced to improve conception rates and fertility of lactating dairy cattle by assuaging effects of heat stress on fertility. Intravaginal data recorders will be used to measure individual body temperatures during breeding and transport after breeding to determine effects of heat stress alone, or in combinations with transport stress on pregnancy rates within a month of breeding. Research will be conducted to examine the interactions between adrenal and immune function in cattle that vary in temperament. Stressors such as transportation will be evaluated for their influence on adrenal and immune function in replacement females and bulls. Interactions between residual feed intake, stress responsiveness, immune function and reproductive performance will be evaluated. The functional relevance of the sexually-dimorphic nuclei of the preoptic area of the hypothalamus will be contrasted in rams that differ in reproductive mating behaviors. In addition, responses of sexually active and sexually inactive rams to ewe vaginal/urine odors will be evaluated. Estrous Synchronization and AI. Investigation of the most appropriate approaches to optimize fertility in timed AI estrous synchronization programs for beef cows and heifers will continue. In addition, experiments will be conducted to evaluate conception rates in CIDR-treated ewes that receive prostaglandin F2a at various time periods near CIDR removal. Efforts to enhance postpartum reproductive performance of ewes will include treatment with CIDRs at various times during the first 30 days after lambing and the benefits of adding a gonadotropin to treatment regimens. Endocrine changes associated with treatments will be performed to provide basic information regarding mechanisms of observed responses. Studies will continue with native reindeer herders to customize and evaluate estrous synchronization protocols and other reproductive management techniques at breeding and calving. Such techniques must be effective, practical to use under some of the extreme logistics presented by ranching in the arctic, and culturally acceptable. Current research plans are to collect and freeze reindeer semen and customize protocols for timed artificial insemination in reindeer that are ranched or farmed or are kept in more traditional reindeer herding situations. A training manual for estrous synchronization of ruminants that provides producers with an easy-to-use guide on the various estrous synchronization protocols was completed by W-1112 members during the previous (2001  2006) funding period with the commitment to update this manual during this funding period. This objective was not accomplished, but we are committed to update it early in the requested funding period (before 2013). Puberty. The hypothesis that high fertility bovine females have a unique gene expression profile will be tested. Genomic and phenotypic indicators of reproductive longevity and impacts of nutrition of the dam and development of the heifer prior to puberty will be used to create early selection tools to indentify replacement heifers with optimal reproductive longevity. Associations between growth, feed efficiency and carcass traits with attainment of puberty, breeding performance and longevity of heifers reared on two levels of feed intake will be investigated. This information will serve as a foundation for studying in utero programming of offspring that make them more efficient, allow the study of epigenetic mechanisms affecting the genetic makeup of individuals, and potentially result in the development of genetic markers for reproductive efficiency in cattle. Mechanisms that result in precocious puberty and its impact on reproductive function will also be studied. Disease, Immune Function. Preliminary data suggests a relationship to innate resistance or susceptibility to bovine respiratory disease in stockers. Polymorphisms in the 5' UTR (promoter) region of both the endothelial (eNOS) and inducible (iNOS) nitric oxide synthase genes have been identified. The role, function, and relevance of nitric oxide genes to innate disease resistance to reproductive diseases, reproductive health and physiological responses in bulls and females will be investigated. Bovine Viral Diarrhea virus (BVDV) has devastating economic impacts on animal agriculture. Estimates of BVDVs economic impact ranged from $15.33 to $20.16 per cow calving (Larson et al., 2002) for a total of $481 to $632.5 million impact to the U.S. beef herd. Fetal infection with ncpBVDV during early gestation results in persistent infection (PI) with chronic viremia and life-long viral shedding, ensuring virus perpetuation in the population. The hypothesis to be tested is that fetal PI alters maternal immune function, compromises fetal growth and immune responses, and results in expression of maternal blood biomarkers that can be used to identify cows carrying PI fetuses. Epizootic bovine abortion (EPA) can cause abortions in susceptible herds of up to 60% and was estimated to cause estimated losses of $10 to $15 million to beef producers in California alone (Teglas, personal communications). The efficacy of a vaccine to protect against epizootic bovine abortion (EBA) will be tested through experimental and field trials in 2010-2011. Its efficacy in protecting against EBA induced abortion will be experimentally evaluated in Angus heifers and in heifers with Angus/Brahma ancestry to assess the potential influence of breed on outcomes. In addition, heifers grazed in pajahuello tick endemic field sites will be vaccinated and monitored for abortion and reproductive success to determine vaccine efficacy. Research will also be conducted to develop an effective ante mortem test to identify previously infected cattle to help producers identify areas of high relative risk prior to the importation of susceptible animals. Collaboration Between Stations. Project members meet annually to present results of completed studies, review the progress of on-going work, discuss industry issues and plan and coordinate the initiation of new studies for the upcoming year(s). We have created a network of collaborating scientists focused on improving the fertility of domestic ruminants in the West. The founding members of the W-112 Regional Research Project established a tradition of cooperation that the current members strive to continue. Indeed, in this era of instant and direct communication, cooperation and collaboration has never been greater or more significant. The extent of collaboration between stations is extensive and may best be described by providing examples of collaborative projects. Collaborative projects in brief include: Determining hormones patterns in ewe lambs with differing residual feed intakes (RFIs; NM, MT); Effects of nutrient restriction on metabolic hormone patterns (NM, CO); Thyroid involvement in BVD infected cows (NM, CO); Metabolic hormone profiles in cows consuming endophyte infected fescue (NM, AR); Follicular fluid concentrations of IGF (NM, OH), Meta-analysis of birth type on ewe reproductive performance (WY, MT, NM); ISG concentrations on maternal blood and IFNt in conceptuses (CO, OH); ISG15 knock out mice to understand the role of ISG15 in the bovine uterus (CO, WA); PGEs and CL function using intraluteal implants (CO, HI, TX); Influence of progesterone on sexual behavior (AK, WY, MN, MT, NE); Influence of gestational nutrition on pancreatic function (AZ, CO); Influence of gestational nutrition on insulin (AZ, WY); Extended oocyte/embryo culture (MI, FL); Oocyte quality and follicle diameter (MI, MT); Influence of follicle age on fertility (OH, MT), Stress hormones and abortion in cattle and sheep (NV, TX); Inhibin concentrations and temperament (TX, MN); Development in intact males (WY, MT, NM); SNPs in sheep (WY, UT); and Role of VEGF in spermatogonial stem cell development and differentiation (WA, NE). Other examples of cooperation include co-advising of graduate students between stations and willingness of member scientists to freely share critical reagents and methodologies with other members of the regional project. Such reagents include antisera, vaccines, tissue and serum samples, RNA and DNA tissue libraries, and novel cell lines and cDNA probes.

Measurement of Progress and Results

Outputs

• The primary output from W-1112 members is peer-reviewed scientific works that represent unique contributions of new knowledge to the fields of reproductive biology.
• Research findings during the previous finding period were presented to a national audience of livestock producers at the 2007 Beef Improvement Federation Meetings in Fort Collins, CO, an activity we plan to repeat during the 2011-2016.

Outcomes or Projected Impacts

• Results of project accomplishments will fill critical gaps in knowledge regarding the reproductive physiology of ruminant animals and facilitate the development and implementation of management strategies that will optimize animal reproduction. " It is difficult to project with certainty the outcomes and impacts of the project because of the long-term nature of many of the basic studies. Regardless, fulfillment of the objective should lead to the development and application of methodologies to improve animal health, well-being, and reproductive efficiency of domestic ruminants in the short-term:
• It is important to recall that the net calf crop for the U.S., based on number of breeding cows that calve annually, was estimated to be 72.7% (Bellows and Lammoglia, 1996). In terms of calves alone this equates to an annual loss that exceeds $5.5 billion. Categories identified with these losses were; animals that failed to become by pregnant by the end of the breeding season (14.6%), losses during gestation (1.6%), losses at or near parturition (5.9%), and losses from birth to weaning (5.2%). Although we are unaware of comparable data for other species, it is reasonable to speculate similar losses occur in sheep, dairy and other ruminants. Because of the magnitude of these losses, we expect research conducted by members W-1112 will continue to lead to new innovations that increase the profitability of livestock production enterprises. " .
• The development of more effective methods of estrous synchronization which will facilitate the use of AI. Previous and ongoing research by members of W-1112 served as a basis for the design of new protocols that increased pregnancy rates by AI by 10 to 15% relative to traditional programs. Implementation of this approach with 10% of the U.S. beef and dairy cows could result in an enhanced profitability of approximately $31 million through reduction in days from calving to conception and an additional savings of $40 million due to a reduction in cow replacement rate. These values represent an overall economic impact of greater than $400 million to the U.S. cattle industry when multiplicative factors of 5 to 10 due to effects on allied industries are considered
• Feed supplied to dams is the single largest expense incurred in production of offspring. Through the use of RFI, animals that use feed resources most efficiently can be identified and should result in an annual savings of $20 to $40 per cow. On a national basis, this technology has to potential of saving cow-calf producers $0.8 to $1.6 billion in annual feed costs. " The development of a vaccine for Epizootic bovine abortion (EBA) is projected to save beef producers in California alone an estimated $10 to $15 million annually. .
• The economic impact of BVDV was projected to range from $15.33 to $20.16 per cow calving. Hence, the development of methods to control this disease would result in an annual savings of f $481 to $632.5 million for U.S. cow-calf producers
• .

Milestones

(2012): The Estrous Synchronization Handbook will be revised by the end of the first year (2012) of the renewed project (http://www.oardc.ohio-state.edu/estroussynch/wester014.pdf)

(2015): A producer symposium on ruminant reproduction will be presented prior to the termination of the project (2015) to facilitate the transfer of new reproductive management protocols discovered as a result of research conducted by W-1112 and also provide a venue for producers to provide input to W-1112 researchers on issues they face in their management systems.

(2016): The development and testing of a vaccine for EBA is expected to be accomplished by 2016

Projected Participation

View Appendix E: Participation

Outreach Plan

We will disseminate information obtained from the proposed research studies to academic and industry leaders by publication in peer-reviewed journals, industry journals, popular press, and other, more widely read technical bulletins. In addition, the information obtained form these collaborative studies will be presented at national and international conferences, industry meetings, and producer field days.

It is also important to note that our representative from MT created and maintains a website (www.repro.msu.montana.edu/W112) that serves as an effective means of communication between stations. The information on the site is regularly updated allowing livestock producers and other stakeholders to follow our progress toward attaining our long term goals. In addition, the ready accessibility of the site by interested scientists, extension personnel and producers makes the website an effective means of obtaining and conveying information and observations among these various constituencies.

Organization/Governance

The members of the W1112 Regional Research Project receive direction from the Executive Committee composed of a Chairman, Secretary and Member-at-Large. Each year a Member-at-Large is elected. In year two, that individual serves as Secretary and then serves as Chairman during year three. The Administrative Advisor, appointed by the Western Directors, also sits on the Executive Committee. Tenure of individuals elected to the Executive Committee is three years.

Literature Cited

Bellows, R.A. and M.A. Lammoglia. 2006. Management decisions to optimize reproduction and profit. Beef 2:5-6. Montana State Univ.

Cryan, R. 2004. The economic impact of the dairy industry. U.S. Dairy Vol. 10 No. 1.

Geary, T. 2005. Management strategies to reduce embryonic loss. Proc. Range Beef Cow Symposium XIX. Univiversity of Nebraska-Lincoln. Pp 69-78.

Lamb, G.C., C. Daheln, and M. Maddox. 2008. What is the economic impact of infertility in beef cattle? The BeefSite. www.thebeefsite.com/articles/1698

Larson, R.L., V.L. Pierce, D.M. Grotelueschen, and T.E. Wittum. 2002. Economic evaluation of beef cowherd screening for cattle persistently-infected with bovine viral diarrhea virus. Bov Pract. 36:106-112.

Otto, D., and J. Lawrence. 2002. Economic impact of the United States beef industry. Ames: Iowa State University, Department of Economics. www.beef.org

Shiflett, J.S. 2008. Sheep industry economic impact analysis. ASI. www.sheepusa.org

USDA-NASS. 2010. National Agricultural Statistics. www.nass.usda.gov/QuickStats

Attachments

Land Grant Participating States/Institutions

AK, AR, AZ, CA, CO, CT, ID, IL, KS, LA, ME, MI, MN, MO, MS, MT, ND, NE, NM, NV, OH, PA, TN, TX, UT, VA, WA, WY

Non Land Grant Participating States/Institutions

USDA-ARS, USDA-ARS/MT