Duration: 10/01/2006 to 09/30/2011

Administrative Advisor(s):

NIFA Reps:

Non-Technical Summary

Statement of Issues and Justification

The W-112 Regional Research Project was established in 1970 with the goal of creating a cooperative research group that could bring both basic and applied expertise to bear on factors that limit the fertility of domestic ruminants in the Western states. Poor reproductive efficiency in domestic ruminants is a growing problem in the West that is limiting profitability and sustainability of animal production systems. Therefore, we seek to continue our work in this critical area and renewal of this multi-state project with the goal of addressing factors that limit the fertility of domestic ruminants in the Western states. This goal is consistent with Strategic Goal 1 (Enhance economic opportunities for agricultural producers,) of the CSREES Strategic Plan (2004-2009). Objective 1.5 under this goal enjoins the contribution of "science-based information, analysis and education to promote efficiency of agricultural production systems". Our efforts in this regard will be evaluated under performance criteria 1.5.7: Increase and improve reproductive performance of animals (CSREES problem area 301); 1.5.11: Improve understanding of fundamental animal physiological processes (CSREES problem area 305); 1.5.13: and Develop and implement comprehensive animal production management systems (CSREES problem area 307). Our primary stakeholders are farmers and ranchers in the Western U.S., but we realize the broad applicability of our work to these industries nation-wide. Our secondary stakeholders are the consumers of animal products that benefit from the reduced prices that are associated with efficient animal production systems. Our tertiary stakeholders are the citizens of communities in the West 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 economic development.

Focusing on reproductive efficiency in the Western states is reflective of importance of Western agriculture to our nation's food supply and the unique features of the West (climate, topography, flora, endemic disease organisms, and demographics) that result in unique management challenges to Western livestock producers. Indeed, the Western and Midwestern states included in the W112 project encompass a land mass that represents more than 50% of the US total and includes more than 55 million cattle and calves and 5 million sheep.

The philosophy and mission for the W-112 established more than thirty five 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.

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 rebreeding, low fertility, and lighter calves at weaning. Sheep producers miss out on the potential for added revenue by not realizing the genetic potential for lambing rates of their flocks. Finally, new challenges are faced by Western farms and ranches managing domesticated exotic ruminants whose reproductive physiology is relatively unknown.

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. However, the improvement in animal health and productivity in the dairy industry has not gone unnoticed and use of these reproductive technologies in the beef cattle and sheep industries is expected to increase during the next decade. Despite recent advances in reproductive technology, cattle producers are still faced with the persistent problem of low fertility.

One of the objectives of our work in the W-112 project is to provide the scientific and technical expertise that will support and encourage development and application of science-based management tools that will improve the productivity and profitability of livestock producers in the Western states. In the current project we will continue and increase our efforts to bring knowledge to the producers to help them make decisions that are rooted in sound science while at the same time, expanding our understanding of factors that affect reproductive efficiency.

The inventory of livestock in the W-112 member states (Table 1) represents a significant fraction of the US total. Indeed, the value of sheep and cattle and their products, produced by farms and ranches in W-112 member states exceeds 44 billion dollars. These figures do not account for the total economic impact of agricultural enterprises on the economies of Western States. For example, recent estimates from California put the total economic impact of the dairy industry in that state at greater than 44 billion dollars. These figures indicate that livestock production is a critical component of the economic health of the Western states and, moreover, that the US supply of livestock and products (meat, dairy, wool, etc.) is dependent on the production efficiency of Western farms and ranches.

Table 1. Cattle and sheep inventory in the W-112 member states a and the value of livestock and products sold (See Attachment #2)


One of the more notable statistics in Table 1 is the growth of the dairy industry in the West. Indeed more than half of the U.S dairy herd now resides in W-112 member states, up nearly 8% between 1997 and 2002. This rate of increase has continued through 2005. For example Idaho recently passed Minnesota and Pennsylvania to become the 4th highest state in total milk production (NASS August 2005). The combined cow herds in only two Western states, ID and CA, account for nearly one quarter of the national herd. In the West, cows are increasingly located on large, modern dairy facilities with average herd sizes approaching 1000 cows, and with many dairies running 5000 cow units. Consolidation and growth in the dairy industry in the West is reminiscent of that which occurred in the poultry and swine industries during the latter half of the twentieth century. While this consolidation has allowed for increased efficiencies and economies of scale, it has created severe challenges for managing reproduction that are unique to the larger dairies in Western states.

The beef industry has failed to take advantage of potential genetic improvements afforded by more widespread use of AI. Estimates of the percentage of beef cattle that are artificially inseminated are below 10%, and this has changed little during the last decade. In fact between 1993 and 2002 the U.S. experienced an approximate 8% decline in sales of beef semen. During this same period, Brazil recorded a 168% increase (NAAB, 2003, ASBIA, 2003). Work from W-112 members has helped refine, simplify, explain and standardize many of the synchronization protocols (see http://www.oardc.ohio-state.edu/estroussynch/western014.pdf ), but more needs to be done.

Another issue of concern in the Western states is animal welfare and we are expanding our efforts to develop new management strategies that will improve animal well-being while maintaining or improving productivity. Producers in this region have been eager to adopt new management strategies that utilize more humane practices and improve productivity of their livestock. Producers look to members of this committee to develop more humane strategies that improve production efficiency.

We believe that the challenges that are shared among the Western states are best addressed by combining the expertise and resources of representatives from all the states. In addition, we have greatly benefited from the participation of experts from states outside the West. Thus, membership in the W-112 project includes leading reproductive biologists from Kansas, Michigan, Minnesota, Missouri, Nebraska, North Dakota, Ohio and Texas.

To effectively address the critical aspects of reproductive biology affecting reproductive efficiency in the Western states the members of the W-112 project have divided into research teams or workgroups. These workgroups place particular emphasis on those components of the reproductive process that limit fertility.

The Gonadal Development and Function Workgroup examines the molecular, cellular, and endocrine mechanisms that control gonadal development, folliculogenesis, spermatogenesis, ovulation, and subsequent luteal function. The members of this group include University and ARS scientists from CA, OR, KS, WA, ID, MT, WY, CO, NM, AZ, AK, NE, ND, TX, MN, MI, OH, and MO.

The Uterine Biology Research Group examines the molecular and cellular aspects of embryo development, implantation, maternal recognition of pregnancy, placentation and fetal growth and development. The members of this group include representatives from CA, WA, ID, ID, WY, CO, MT, OR, OH, HI, ND, TX, and MO.

The Reproductive Technology Research Group includes members from all participating states. The charge to this group is the development and optimization of management protocols, treatment regimens and tools/techniques that will facilitate and encourage the use of artificial insemination and other modern reproductive technologies by cattle and sheep producers in the Western states.

Related, Current and Previous Work

The list of research achievements, publications, and student theses that are the product of the collaborative work of members of the Project is extensive. Indeed, during the last 5 year period 251 refereed publications and technical bulletins and 4 book chapters have been generated by research projects conducted with W-112 regional research support. Although it is not possible to detail each of the significant accomplishments made during the past 5 year period, a summary of the major advances is provided below.

Major Advances:

Biology of the Hypothalamic-Pituitary-Gonadal Axis.
Research characterized the roles of estrogen receptor (ER) alpha and ER beta in mediating differential effects of estrogen on gonadotrope function. The hypothesis that insulin-like binding proteins (IGFBPs) modulate secretion of LH was supported by studies that demonstrated levels of IGFBPs in anterior pituitary gland are inversely associated with circulating levels of LH. A novel membrane receptor for progesterone was identified in hypothalamic, pituitary, uterine and ovarian tissues. In addition, collaborative efforts determined that the volume of the sexually dimorphic nucleus (SDN), levels of aromatase mRNA in the SDN and expression of prolactin in the medial preoptic area (MPOA) of the hypothalamus and amygdal correspond to sexual partner preference exhibited by rams. These results support the hypothesis that naturally occurring variations in sexual partner preference are determined by underlying biological mechanisms.

Ovarian Biology Research
Follicle development. Collaborative efforts by W-112 representatives expanded basic knowledge of mechanisms regulating follicular development, atresia and ovulation. When estradiol was used to initiate follicle atresia, timing of emergence of the next wave of ovarian follicles was dependent upon dosage of estradiol rather than timing of atresia. Diminished estradiol production coinciding with loss of dominance occurred prior to a loss in aromatase mRNA and was associated with enhanced expression of 15 genes involved in apoptosis and a decreased expression of genes that inhibit apoptosis (DICE-1, MCL-1) in granulosa cells (GC). Low estradiol in subordinate follicles was associated with elevated expression of mRNAs for several pro-apoptotic genes in GC and thecal cells. Expression of CART (a potent hypothalamic anorectic peptide) in GC was associated with follicle health status and as a novel regulator of follicular atresia. Three functional variants of the FSH receptor were found in bovine GC, with each form varying with respect to localization and follicle status; indicating differential roles for these receptors in regulating follicular development.

Luteal Function. Research showed that Prostaglandin F2a (PGF) activates Ca2+/PKC and MEK/ERK signaling pathways inducing Egr-1 expression and apoptosis during luteal regression. Progesterone prevented an oxytocin-induced influx of calcium into large luteal cells. Pericytes and smooth muscle cells were shown to regulate tissue remodeling and maintain the integrity of larger blood vessels during early luteal regression. Nitric oxide donors were shown to increase bovine luteal secretion of PGE2, which is both luteotropic and antiluteolytic. Treatment with PGF regulated nitric oxide synthase mRNA. Synchronization of estrus with norgestamet ear implants or a CIDR inhibited luteal and bovine caruncular endometrial secretion of PGE2.

Uterine Biology Research
Research elucidated actions of ISG15 and MX, two uterine proteins postulated to be involved in maternal recognition of pregnancy. Dysregulation of ISG15, and its conjugates may be detrimental to implantation-placentation, resulting in fetal death. The Mx proteins were shown to be expressed in the endometrial luminal epithelium during the periimplantation period in domestic ruminants. In addition, Mx expression was regulated by progesterone; which may have implications for strategies to control sexually transmitted viral diseases. Assays were developed for Mx1, Mx2 and ISG15 in blood as well as a number of other interferon stimulated genes. These assays may provide a method for early detection of failed pregnancies in ruminants. Periattachment factor was identified in a number of species during early pregnancy when the conceptus is rapidly growing and establishing attachment to the uterine lining. A novel mRNA transcript (bPF) was present in bovine conceptus during early expansion but absent after day 30.

Low concentrations of progesterone during maternal recognition of pregnancy may contribute to early embryonic losses. Mechanisms involved in regulating endometrial and placental progesterone secretion include actions and interactions of prostaglandins F and E, oxytocin and its receptor and progesterone receptors. In addition, progesterone can act non-genomically to interfere with the binding of oxytocin to its receptor in the plasma membranes of ovine endometrium. Progesterone produced by the conceptus may act locally during a critical stage of gestation to suppress activation of oxytocin receptors and thus ensure survival of the embryo/fetus. Increasing progesterone during maternal recognition of pregnancy by administering hCG during the early luteal phase may improve pregnancy rates.

Nutrition and Reproduction
Placental insufficiency results in intrauterine growth restriction (IUGR), whereas altered placental function may also lead to excessive fetal growth. Fetal IUGR resulting from inadequate nutrition during the first half of gestation in ewes and cows reduced placental growth and negatively impacted health of offspring after birth. Health effects were not alleviated when females were re-alimented during the second half of gestation even though birth weights were normal. Re-alimentation decreased responsiveness of the cotyledonary bed to angiotensin II, possibly promoting increased vascular perfusion and nutrient uptake. Nutrient restriction during early- to mid-gestation followed by adequate nutrition to term increased fat and plasma leptin concentrations in neonates.

Inadequate maternal nutrition resulted in hypertrophy of the left ventricle/septum portion of the fetal heart and enlarged kidneys; effects suggestive of systemic hypertension. Up-regulation of several genes in the left ventricle of the fetal heart may contribute to ventricular hypertrophy. Left ventricular hypertrophy in response to inadequate maternal nutrition is associated with changes in angiotensin receptors, which may have implications in cardiovascular disease during post-natal life.

In the ewe, alteration in placentome morphology increases their ability to deliver 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. However, some animals have the ability to convert their placentomes when stress occurs during early gestation. This ability for early placentomal conversion appears to be related to ewe selection and may be epigenetic in nature.

Acute nutrient restriction decreased LH pulse frequency and increased suppressive potency of E2 on LH pulse amplitude. Fasting prior to proestrus increased circulating concentrations of progesterone, influenced growth and secretion of estradiol by developing follicles, and increased length of the interval from PGF to ovulation. Fasting during the luteal phase of the estrous cycle decreased lambing rates of ewes and impaired ovulation in cows. Decreased serum concentrations of IGF-I during short-term feed restriction may be responsible for the detrimental effects of fasting on reproduction.

Leptin concentrations in cattle were more dependent on degree of fatness than dietary energy intake. Milk and blood appeared to act as independent leptin depots. High levels of leptin in milk early in lactation may provide a mechanism for thermoregulation, satiation, and homeostatic endocrine control in the neonate. Leptin concentrations were positively correlated with IGF-1, body weight and body condition score, and negatively correlated with postpartum interval. Cows with higher concentrations of leptin had shorter postpartum interval and leptin prevented fasting-mediated reductions in LH pulse frequency in prepubertal heifers and increased the secretion of GH. The stimulatory effects of NPY on GH secretion were attenuated by leptin in cows. Peripheral concentrations of leptin were positively correlated with scrotal circumference and serum testosterone in growing bulls and with back fat and body condition scores in heifers. Brahman cows tended to have greater concentrations of metabolic hormones than Angus cows. Leptin was present in milk and plasma of does in concentrations negatively correlated to kid body weight.

Serum concentrations of IGF-I may be a useful indicator of nutrient and pubertal status in growing cattle and sheep. Serum concentrations of IGF-I and thyroid hormones were associated with puberty onset in ewe lambs. In heifers, IGF-I was positively correlated with relative feed efficiency, feed conversion ratio and dry matter intake. Increased propensity for fat deposition and average daily gain positively influenced attainment of puberty whereas variations in efficiency of gain or residual feed intake did not. Divergent selection of heifers for high or low IGF-I altered characteristics of follicular growth, concentrations of FSH concentrations, and concentrations of progesterone during the mid-luteal phase.

Testes Biology Research
Research determined that vascular endothelial growth factor (VEGF) is involved in both vascular development and formation of the precursor seminiferous tubules (cords) in the developing testis. Ectopic testis tissue xenografting was utilized as a novel technique to investigate bovine testis development and spermatogenesis. Bovine testis tissue grafts produced elongated spermatids 6 months after grafting. Bovine testis tissue was cryopreserved and electroporated to introduce transgenes and produce sperm after grafting. Gene expression and GeneChip microarray analysis of grafted testis tissue and bovine testis tissue were used to identify factors that influence testis development and potential to produce sperm. Culture of bovine spermatogonial stem cells (SSCs) was used to investigate factors that regulate biological activities of these cells. Glial cell line- derived neurotrophic factor (GDNF) stimulated SSC self-renewal. Germ cells from bovine testicular explants were capable of colonizing the testes of recipient nude mice and numbers of SSCs increased over time.
Immunocastration
Research continues in the development of sterilization vaccines as alternatives to surgical spaying of feedlot heifers and castration of males. Immunization of rams and bulls against LHRH resulted in decreased testosterone and scrotal size compared to intact males. Intact bulls had greater carcass weights and muscle percentage compared to castrated or immunized males, whereas castrated and immunized males had greater marbling and fat percentages than intact bulls. Immunization lowered aggressive behavior and would likely facilitate handling and management of intact males.

Estrus Induction and Synchronization
Widespread use of artificial insemination (AI) in beef cattle in the West requires simple and efficient methods to synchronize estrus. Research delineated the optimal timing, dose, route of administration of progesterone to induce ovulation or estrous cycles. Results may improve our ability to get young cows to rebreed and remain productive. Studies of the importance of the bull in stimulating onset of ovarian activity indicate that timed AI pregnancy rates may be improved by exposing primiparous cows to bulls before and during an estrous synchronization protocol.

Premature ovulation of a dominant follicle results in reduced luteal function and conception rates. This resulted in reduced synthesis of progesterone and pregnancy rates in beef cows and heifers. Similarly, cows with a short proestrus exhibited an increased incidence of short luteal phases, decreased concentrations of progesterone during diestrus, and lower pregnancy. Use of a CIDR in an estrous synchronization protocol for natural mating of beef cows tended to reduce the number of days to pregnancy compared to a single injection of prostaglandin. However, benefits of the CIDR treatment were lost due to late embryonic and early fetal losses.

Success of farming musk-ox for qiviut (underwool) in Alaska requires husbandry tools specific for this species. Introducing bulls in August truncated the breeding period to 2 wk (4-6 wk shorter than normal). Other research determined that the timing of a synchronized estrus was influenced by an interaction of bull exposure and using modified cattle CIDRs for estrous synchronization. A radiotelemetric system was found to be an excellent tool for estrus detection in musk-ox, but this technology was less effective with reindeer. Early weaning failed to improve reproductive performance of red deer females.

A training manual for estrous synchronization of ruminants has been completed and is available on line at: www.oardc.ohio-state.edu/estroussynch. The manual, entitled "The Estrous Synchronization Handbook", provides producers with an easy-to-use guide on the various estrous synchronization protocols developed

Objectives

1. Determine the basic physiological, immunological, metabolic and genetic systems that contribute to optimum reproduction in domestic ruminants with emphasis on gonadal development and function, establishment of pregnancy, placental function and fetal development, pubertal, and seasonal and post-partum anestrus.
   
2. Develop tools, techniques and management strategies to allow producers to control reproductive efficiency, including development of markers for reproductive health and disease, and chemical and immunological means of detecting and regulating fertility.
   

Methods

The research by members of the Gonadal Development and Function and Uterine Biology Research Groups will generally address issues most directly relevant to Objective 1. Conversely research conducted by members of the Reproductive Technology Research Group are most likely to conform to the goals detailed under Objective 2. Despite this apparent segregation of effort, it is important to note that Objective 1 and Objective 2 are complimentary. Indeed, we have already demonstrated that much of the work conducted by the Gonadal Development and Function and Uterine Biology Research Groups have practical components that are consistent with Objective 2. Similarly, the applied studies conducted by the Reproductive Technology Research Group are likely to identify new and fundamental questions in reproductive biology that would appropriately fall under Objective 1. Regardless of Objective or Research Group, our unifying long term goal is to apply the latest advances in reproductive biology toward the development and implementation of management strategies and practical tools that will improve the fertility of domestic species in the Western states. Research Methods of the Gonadal Development and Function Workgroups It is clear that the in utero environment can directly impact development of the gonads and have long term effects on fertility in the adult. Consequently members of this workgroup will address factors affecting gonadal development in the male and female. Specifically they will determine the mechanism of vascular endothelial growth factor's (VEGF) actions on primordial follicle activation and progression and vascular development in the ovary. In the testis, they will determine the role of VEGF in sex-specific vascular development and seminiferous cord formation two hallmarks of testis morphogenesis. Testis Biology Research Workgroup: Members will utilize a spermatogonial stem cell transplant model to characterize proteins associated with testis development and the production of spermatozoa in bulls. Expression pattern of proteins associated with Sertoli cell differentiation and formation of the spermatogonial stem cell population will be characterized with the use of grafting. Additional studies will use gene expression data to identify proteins that affect testicular growth, Sertoli and germ cell differentiation and sperm production. Spermatogonial stem cell transplantation will be used to investigate factors that regulate the formation of the spermatogonial stem cell population and how these cells form a niche in the seminiferous epithelium. Finally, techniques to develop effective, repeatable and efficient spermatogonial stem cell transplantation in bulls will be investigated. Ovarian Biology Research Workgroups Follicular Development Workgroup: Assisted reproduction in domestic ruminants is largely focused on regulating the growth and ovulation of healthy follicles. The studies proposed will elucidate the role of inhibin in the selection the preovulatory follicle in cattle. In addition, recent studies revealed a potential role for the CART peptide in regulation of antral follicle steroidogenesis and an association of CART expression with follicle growth development and function. Studies will determine the impact of the difference in preovulatory estradiol secretion by follicles of similar size on embryo development and IFN tau secretion, luteal function, production of targeted proteins associated with pregnancy, receptors for progesterone and estradiol, and gene expression within the uterus. To better understand the function of estradiol in this process, members of this group will also determine the role of each of ER alpha and ER beta in generation of the preovulatory LH surge. Results from these studies will provide critical information on regulation of the development and fate of dominant follicles. Endocrinology and Sexual Behavior Workgroup: While the endocrinology of the estrous cycle of traditional domestic ruminants has been fairly well characterized, our definition of domestic ruminants has expanded to include a number of farmed exotic ruminant species like musk-ox, reindeer and elk. Members of this workgroup will conduct studies to describe the endocrinology of estrus, pregnancy and anestrus in reindeer and musk-ox, including the patterns of steroid hormones, leptin, IGF1, and thyroid hormone. In addition this group will conduct experiments to understand the factors controlling the development and expression of sexual behavior and identify neural centers responsible for normal and aberrant sexual behavior. The long term goals of these studies are to provide tools to manage reproductive cyclicity in all domestic ruminants. Ovulation Workgroup: Rupture of a healthy ovarian follicle and release of an oocyte is a prerequisite for fertility. Members of this workgroup will evaluate and characterize genes exhibiting differential patterns of expression in pituitaries from cycling and anestrous cows. This workgroup will also evaluate novel single chain variants of the gonadotropic hormones and test their efficacy when used to induce estrus and ovulation in sheep and cattle. In addition, studies are planned to determine the effects of isoforms of VEGF on ovulation. A more complete understanding of the cellular and molecular mechanisms involved in follicle rupture will set the foundation that will allow us to develop improved methods to control the ovarian cycle in domestic ruminants. Luteal Function Workgroup: A large portion of early embryonic losses are attributed to inadequate progesterone production by the CL. During luteinization, follicle cells proliferate and new blood vessels grow to support the function of the CL. These processes are controlled by polyamines and nitric oxide. Members of this workgroup will investigate the regulation of L-arginine metabolism towards either polyamines or nitric oxide during CL development. In addition, members of this work group will continue to elucidate relationship between PGE1/PGE2 and ovine luteal LH receptor formation and function. The precise mechanism(s) regulating luteal regression are not completely understood. Members of the luteal function workgroup will test the hypothesis that inhibiting luteal PGF synthesis using short inhibitory RNA (siRNA) to reduce expression COX-2 in the CL will inhibit luteolysis. In addition, experiments will be conducted to determine if normal luteal levels of progesterone inhibit oxytocin-induced calcium influx in small luteal cells and may inhibit PGF-induced calcium influx in large luteal cells. In addition, studies will be conducted to evaluate prostaglandin inhibitors for their ability to improve fertility by increasing pregnancy establishment in beef cattle. A transmembrane receptor for progesterone was recently cloned from Seatrout. Members of this workgroup identified and cloned a similar receptor in sheep. The planned experiments will determine the cellular localization of the putative ovine membrane P4 receptor and identify the ligand(s) it binds, and signaling pathways activated. Similar research will be conducted to determine whether binding sites for estradiol are localized in the plasma membranes of the endometrium of the ewe. Identification of the presence of ER in the plasma membranes of the endometrium will be followed by studies to ascertain the nature of the intracellular signal generated by activation of these receptors. Results from these studies will provide a better understanding of luteal function and regulation of luteal regression. Environmental Control Workgroup: The full benefits of superior genotypes can only be realized when the animal's environment (housing, nutrition, health etc) has been optimized. Research proposed by the Environmental Control Workgroup will examine the impact of stress on reproductive processes in sheep and cattle. This group has developed antisera that recognize various epitopes on ACTH. Using these antisera they will attenuate or enhance the adrenal response to stress. Furthermore, they will use this antibody-based research model to examine the impact of stress-induced cortisol secretion on expression of GnRH receptors in the pituitary and LH receptor and aromatase in the granulosa cell layer of developing follicles. Another focus of this group is to examine the role that nutrition plays in modulating reproductive function. The proposed studies will examine the impact of under-nutrition on expression of GnRH, ER, and markers of neuronal activity in hypothalamic tissue. Members of this workgroup hypothesize that acute nutrient restriction during follicular emergence and selection results in follicular defects and endocrine anomalies that reduce fertility. Experiments will determine the influence of fasting during the luteal phase of the estrous cycle on ovarian follicular recruitment, growth, and development and the influence of fasting on oocyte competence. To address the effects of nutrition on age at puberty, two model systems will be used. The first is a model which involves early weaning and feeding a high concentrate diet. This will be used to determine the interactions between nutritional status and sexual maturation. The second model will involve animals divergently selected for either early or late puberty. The goal is to determine the physiological response to selection in these females which resulted in differences in age at puberty. Research Methods of the Uterine Biology Research Group Embryogenesis and Placental Development Workgroup: Evidence suggests that oocyte RNA transcripts are crucial for oocyte growth, maturation and subsequent early embryonic development. Members of this workgroup will investigate the role of RNA quality on oocyte competence using known models of poor oocyte competence on the gene expression profiles of bovine oocytes. In addition, members of this workgroup will define the specific temporal and hormonal regulatory mechanisms that control expression of potential cumulus and oocyte-derived markers of oocyte competence. These studies should lead to development of systematic breeding programs that are designed to ovulate an oocyte that is competent for establishment and maintenance of pregnancy. Maternal Recognition of Pregnancy Workgroup: Members of this workgroup have identified a novel gene, named Periattachment Factor (PF) that is strongly expressed by bovine, equine, ovine and porcine embryos during a critical window of early conceptus development, but is either not expressed or only weakly expressed in adult tissues. Research will determine the temporal and spatial expression of PF in sheep conceptuses during the peri-implantation period. This will entail generating recombinant ovine PF, making antisera against the protein, and using quantitative real-time RT-PCR, in situ hybridization and immunohistochemistry. The effects of PF on 15 conceptuses will be examined suing short hairpin RNAs (shRNA) to "knock-down" PF expression. Members of this workgroup have demonstrated that a number of pregnancy-stimulated genes in the uterus are also expressed in the peripheral blood immune cells. This workgroup will test the hypotheses that these proteins are markers for a viable embryo, and are critical to the establishment of pregnancy. The sequences will be determined for many of the ruminant mRNAs that are up-regulated in blood and endometrium from day 18 pregnant cows and ewes. Levels of mRNA expression in blood and endometrium will be determined during early pregnancy using RT-PCR. Knock-down of selected mRNAs using RNA interference (RNAi) will be accomplished and extracellular functions of these proteins will be examined in vitro and in vivo using proteomic approaches. Fetal Maturation/Fetal Programming Workgroup: The "fetal origins" hypothesis states that events experienced in utero are the foundation for health and disease in the adult. Members of this workgroup will test this hypothesis using pregnant ewes chronically exposed to elevated ambient temperatures and other treatments designed to impact the growth and development of embryos. One area that has received quite a bit of national attention is the neuroendocrine basis for male-oriented sexual behavior of rams. Members of this workgroup will continue studies to examine the neuroendocrine control of sexual behavior in rams. Specifically, studies will be conducted to determine the role of prolactin produced in the preoptic area of the hypothalamus of these rams. This research has already led to development of potential tools for identification of male-oriented rams so that they can be culled from the breeding population. Members of this workgroup will also study fetal compensatory growth mechanisms to nutrient restriction. This model will use placental insufficiency induced intra-uterine growth retardation (PI-IUGR) to examine fetal sheep pancreas development, function and postnatal outcomes. These studies will test the hypothesis that fetal nutrient deprivation decreases pancreatic endocrine precursor cell and mature b-cell expansion by limiting mesenchymal growth factors that reduce b-cell mass and function. Using the PI-IUGR in sheep, members of this workgroup examine both acute and chronic changes in placental gene expression as the IUGR pregnancy develops. The effects of maternal nutritional status on nutrient partitioning and ultimately growth, development and function of the major fetal organ systems will be examined. Members will investigate the effects of different maternal energy sources on offspring ovarian function, placental development and nutrient flux across the placenta, fetal ovarian development, and the mechanism of angiogenesis and vasoreactivity of the uterine and placental vasculature. Results from these studies will help understand how growth restricted offspring may be "programmed" to exhibit poor post-natal growth and productivity and also to develop diseases later in life. Growth-restricted offspring that survive the neonatal period have a greater incidence of reduced productivity and increased disease. Members of this workgroup will test the hypothesis that fetal growth restriction in lambs and the development of a "thrifty" phenotype predisposes these lambs to altered postnatal growth rates, feed efficiency, fat deposition, and carcass quality. In addition this group will continue to elucidate relationships of nitric oxide and endothelin-1 on luteal, endometrial, and placental relationships and function. Research Methods of the Reproductive Technology Research Group Development of new tools to more effectively manage reproduction in domestic ruminants in the West is the primary goal our members. Ultimately the success of this project will be measured by development and implementation of management strategies and reproductive technologies that will reverse the trend of declining reproductive efficiency in domestic ruminants. All members recognize the critical link between discovery science (often conducted at the molecular level) and translational research. Immunocastration Workgroup: Members of this workgroup will investigate the effects of recombinant LHRH antigens on hypothalamic and pituitary gene expression in bulls. An LHRH fusion protein was developed and studies will evaluate it as means of regulating fertility in both heifers and bulls. Planned studies will involve development of longer-acting carriers and evaluation of new adjuvants to decrease the number of injections required and increase the effective time of the vaccine. The optimal dosage of antigen will also be determined. Members of this workgroup will build on their previous studies defining the endocrine regulation of the estrous cycle in domesticated exotic ruminants to use exogenous chemical and/or immunological means to modify rut-associated aggressive behavior expressed by reindeer bulls and limit seasonal fertility of reindeer bulls without hindering future fertility. Research from this workgroup should provide alternatives to surgical castration and enhance animal welfare along with managing animal behavior to enhance the safety of farmers and ranchers handling large aggressive animals. Marker Assisted Selection Workgroup: Members of this workgroup are taking advantage of the information that resulted from the bovine genome mapping project and our expertise in genomics to identify genetic markers for reproduction that might be rapidly applied to aid in the identification and selection of superior genetics. Studies proposed will involve development of genetic markers for both daughter pregnancy rate and longevity. The basic approaches will include genome-wide screening and candidate gene identification. Assisted Reproductive Technology Workgroup: This workgroup will focus on developing products or protocols to better manage reproduction in both males and females. Members of this workgroup will examine the role of IGF-1 in control of pubertal and seasonal anestrus. In addition, experiments will examine a number of endocrine factors immediately after birth and during the prepubertal period including thyroxine and triiodothyronine. If accurate markers of puberty are available during early-life, ewe lambs and heifers that reach puberty early can be identified quickly and the remainder can be marketed without the expense of management for inclusion in the breeding herd. Members of this workgroup will address the role of the thyroid gland in regulation of seasonal anestrus as well. In the proposed experiments, PTU will be applied to pregnant ewes before they become seasonally anestrus to study early postpartum gonadotropin responses to prepartum thyroid inhibition. Results of these projects will provide basic information on potential markers of puberty onset as well as the role of thyroid hormones in seasonal anestrus. Members of this workgroup will conduct studies aimed at improving superovulation protocols. Studies will be directed at increasing the number and reducing the variability in the number of ova shed. The approach will be to passively immunize beef cows against inhibin during the period of FSH treatment. The timely administration of inhibin-neutralizing antibody may allow more follicles to continue to develop and ovulate. To enhance the use of AI in the beef industry the proposed research will identify factors which are limiting the success of the presently available programs. Initially, members of this workgroup plan to investigate the hypothesis that shortening the interval from follicle wave emergence to regression of the CL (or withdrawal of an exogenous progestin) will increase conception rate to the synchronized ovulation as a result of increased steroidogenesis by the ovulatory follicle and resulting CL. In addition, this workgroup will examine the efficacy of recently developed single chain variants of the gonadotropic hormones to induce estrus and ovulation in sheep and cattle. Additional studies will evaluate the influence of early weaning on reproductive performance of cows and on development and subsequent fertility of heifer calves retained for replacement heifer. Studies are proposed to examine the use of exogenous hormones to advance the onset of seasonal estrous cyclicity and to synchronize estrus in female reindeer and musk-ox. Members of this workgroup will use radiotelemetric mount detectors to describe estrous and breeding behavior in reindeer and musk-ox and conduct experiments to demonstrate management advantages to synchronous and early season calving in reindeer and musk-ox. 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 is best described by providing a few examples of station to station collaboration. Researchers from WY, ID, and industry partners in CO are collaborating on a project that has resulted in development of a cow-side diagnostic test for the early detection of failed pregnancies. This test is being evaluated in cows from farms in MT, CO, WY and ID. Furthermore, collaboration between scientists at ND and WY are determining how nutrition impacts vascularity of the placenta in ruminants. Another example is the multi-state estrous synchronization effort to optimize protocols using CIDRs in beef cows and heifers. Researchers from MN, MT, OH, KS, with industry personnel from MI, MO, and OH and researchers from IL, TN, and IN are identifying the best protocols using CIDRs for synchronization of estrus/ovulation. Cooperative efforts by researchers in MT and WA have been developed to evaluate gene expression in the pituitary glands of cows during the estrous cycle. This information will be used to establish a dataset that will be mined for genes that regulate reproductive efficiency. Other examples of cooperation included co-advising of graduate students between stations and willingness of member scientists to freely share critical reagents 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. Perhaps even more important is the unrestrained exchange of advice and information that occurs at the annual meeting and in countless telephone and email conversations that occur during the interval between meetings. Jointly, the members of this project constitute a vast reservoir of experience, knowledge and technical expertise that is an invaluable resource for all members of the project. Established members of the group work together to help mentor and guide junior faculty members. Finally, members work together to enhance graduate education by identifying superior students and helping to place them in member labs that best fit the students career goals and by hosting students from collaborating labs.

Measurement of Progress and Results

Outputs

• The primary output from W-112 members is peer-reviewed scientific works that represent unique contributions of new knowledge to the fields of reproductive biology. The results will fill critical gaps in our knowledge regarding the reproductive physiology of ruminant animals and facilitate the development and implementation of management strategies that will optimize animal reproduction in the West. One output from the prior funding period that was not accomplished was the organization and delivery of a symposium on ruminant reproduction in the West. We are committed to accomplish this goal early in the proposed project period (before 2008). This will not only allow for more effective communication of our work to producers, but also provide an opportunity for producers to convey their needs to members of our project.
• A cow-side test for the early detection of failed pregnancies.
• An effective immunocontraceptive vaccine.
• A major output from the previous funding period was the publication of the Estrous Synchronization Handbook. We plan to make this a "living document" that will contain information about the latest developments and recommendations from our group.

Outcomes or Projected Impacts

• The success of this multi-state research effort will ultimately be evaluated by examining the impacts on quantitative measures of reproductive performance including; days open, services per conception, pregnancy rates, birth of healthy offspring, and age at puberty. We expect that our collaborative work will result in the development of more effective methods of estrous synchronization and facilitate the use of AI in the West. Our success should be reflected by increased use of AI in the beef industry and reduced services per conception and days open in the dairy industry. Additionally, we expect that our efforts will result in the development of efficient methods of immunocastration that will improve animal welfare and increase production efficiency.

Milestones

(2007): The first and most critical milestone will be the delivery of a producer symposium on ruminant reproduction before 2008. We expect several technological milestones to be achieved during the proposed project including the commercialization of cow-side assay for detecting failed pregnancy in cattle by day 18 after insemination and commercialization of an immunocontraceptive vaccine for ruminants.

(2009): Update the Estrous Synchronization Handbook. This handbook is now widely available to those with internet access and we will convert it from a .pdf document to an .html document to facilitate our ability to keep it current and to make it easier for producers to use.

(2011): A major revision of the Estrous Synchronization Handbook is planned to coincide with the termination of the project

Projected Participation

View Appendix E: Participation

Outreach Plan

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

It is also important to note that our representative from MT has created and maintains a website (www.repro.msu.montana.edu/W112) that has proven to be 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 W112 Regional Research Project receive direction from the Executive Committee, composed of an elected Chairman, Secretary and Member-at-Large. The Administrative Advisor, appointed by the Western Directors, also sits on the Executive Committee. Tenure of each elected officer is one year.

Literature Cited

Attachments

Land Grant Participating States/Institutions

AR, AZ, CA, CO, CT, GA, HI, ID, KS, ME, MI, MN, MT, ND, NE, NM, NV, OH, OR, PA, TN, TX, UT, VA, WA, WY

Non Land Grant Participating States/Institutions

USDA-ARS, USDA-ARS/MT