Duration: 10/01/2021 to 09/30/2026

Administrative Advisor(s):

NIFA Reps:

Non-Technical Summary

Statement of Issues and Justification

The W4112 Regional Research Project was established in 1970 to create a cooperative research group that would combine both basic and applied expertise to identify mechanisms and develop methods to improve the fertility of domestic ruminants in the Western states.  The philosophy and mission for the W4112 established fifty years ago continues to be the guiding tenet of our group; that is, cooperative multi-state research, that provides novel information to aid in product and technique development and outreach for the benefit of animal producers in the Western region and across the nation. 

The project serves as a forum for the identification, 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 basic science questions that may lead to enhancement 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 projects and has been capitalized on by project members, leading to numerous collaborations among many university agriculture experiment station and USDA agriculture research station scientists.  Renewal of the W-112 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 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 Arizona, Arkansas, California, Colorado, Connecticut, Idaho, Illinois, Kansas, Mississippi, Missouri, Montana, Nebraska, Nevada, New Mexico,  Oregon, Pennsylvania, South Carolina, Texas, Tennessee, Utah, and Wyoming.  We believe the challenges of livestock producers are often common among producers and are best addressed by combining the expertise and resources from all multiple states.  The addition of leading reproductive biologists from states outside the West has increased the breadth and greatly strengthened the scientific expertise of W4112.  The W4112 regional project has also welcomed those whose main interests are nutritional or statistical expertise to increase our breath and ability to determine physiological processes and develop tools to enhance reproductive success. 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 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 $81.5 billion in farm receipts and an estimated overall production value of $192 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  56 percent of the nation’s breeding cows (beef and dairy) and 70 percent of the US breeding ewe inventory exist in states represented by participating W4112 scientists (USDA-NASS, 2019). 

W4112’s goals are consistent with the USDA 2018-2022 Strategic Plan’s Goals 2, 4 and 7.  Specific objectives addressed by the project include Objective 1 (Maximize the ability of American agricultural producers to prosper by feeding and clothing the world), Objective 3 and 5 (Facilitate rural prosperity and economic development and provide all Americans access to a safe, nutritious, and secure food supply). Our primary stakeholder is the scientific community. From that community it is our expectation that livestock producers will benefit. Application of basic advances requires time and involvement of extension educators. It is the aim of the W4112 project to effectively transfer gain of knowledge to this audience through scientific publications and presentations to scientific, extension, and producer audiences.

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.  Loss of efficiency also impacts beef producers as a result of delayed onset of puberty, extended postpartum anestrus, low fertility, and consequently 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 to limit production.  One of the objectives of our work in W4112 is to provide the scientific and technical expertise that will encourage development and application of science-based management tools to improve the productivity, efficiency, 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 expanding our understanding of factors that affect reproductive efficiency.  

For the current project, research will be focused in the following main areas: mechanisms of gamete development, ovulation and potential causes of anovulation, establishment of pregnancy, fetal development/prenatal programing that affects fetal development, and male reproduction. 

Related, Current and Previous Work

Many 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., 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 W4112 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 and dissertations that are products of the collaborative work of members of the Project is extensive. During the previous project, 453 publications were generated including: 220 refereed journal articles and book chapters, 217 abstracts, proceedings, and technical bulletins.  In addition, several symposiums of research findings was presented to a National (ASAS-SSR Triennial Reproductive Symposium 2017, Midwest ASAS 2019, 2020) as well as individual invited talks at National and regional meetings. A sheep symposia was planned for 2020 prior to Western section ASAS but it was cancelled due to COVID. 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. The preovulatory estradiol surge influences the establishment and maintenance of pregnancy by an intrafollicular action on granulosa cells to enhance luteinization and (or) an extrafollicular effect on the uterus to promote conceptus elongation and placentation.

Studies conducted within the W4112 community have shown KiSS-1 peptide modulates LH secretion by regulating the secretion of GnRH without effects on the anterior pituitary gland 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 cyclicity.  The higher frequency of GnRH pulses is associated with a higher content of kisspeptin in the hypothalamus during the breeding season, reflecting an increase in kisspeptin input to the preoptic area and mediobasal hypothalamus.

            Ovarian Biology and Follicle Development Research.  Studies focused on vascular endothelial growth factors (VEGF) in folliculogenesis demonstrated that     angiogenic VEGF isoforms can cause increased follicle progression independent of nutritional inputs to beef heifers during development.  Furthermore, antiangiogenic VEGFA isoforms signal differently and can block the actions of angiogenic VEGFA resulting in arrested follicle development. 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. 

Cows with androgen excess (High A4; Androstenedione) have theca cells similar in phenotype to women with Polycystic Ovarian Syndrome.  Gene expression of CYP17 and CYP11 are increased along with GATA6, a transcription factor which can upregulate both CYP17 and CYP11, causing excess androgen to be produced.  Inappropriate expression of CYP17 due to altered FOS may cause increased androgen production in granulosa cells from High A4 cows. Furthermore, granulosa cells from these androgen excess cows appear to be arrested in the cell cycle and do not proliferate appropriately since FSH stimulated cows with androgen excess have 50% reduction in number of granulosa cells within the follicles that develop.  Thus, there appears to be mis-regulation of these granulosa cells which may compromise granulosa cell function and appropriate oocyte development. These cows also appear to have increased inflammation, anovulation and reduced fertility.

            Estrous Cycle, Physiology, & CL Function.   Corpora lutea (CL) function is primarily dependent upon numbers of theca and granulosa cells within the ovulatory follicle that luteinize following ovulation to form small and large luteal cells.  CL function is also dependent on the luteal microenvironment which is affected by immune cells that also secrete cytokines. Studies using microarray analysis of CL collected 4, 2, and 1 hour after Prostaglandin F2-alpha (PGF) administration demonstrated that PGF injection induces expression of immune regulatory genes in vivo. Of particular interest was the prediction of IL-17 signaling and NFκB activation.  It was determined that IL-17, but not PGF, directly activated NFκB signaling in luteal cells and IL-17, in combination with TNF and PGF, reduced LH-stimulated progesterone secretion.  Thus, PGF induced IL-17 and cytokine activation of NFκB signaling contributes to the dampening of progesterone synthesis by inhibiting LH-stimulated progesterone secretion which appears to be critical during CL regression in beef cows. Additional studies evaluated the expression of interferon gamma (IFNg), tumor necrosis factor alpha (TNFa), IL12, IL10, chemokine ligand twelve (CXCL12), and chemokine receptor four (CXCR4) in CL tissue from non-pregnant compared to pregnant ewes. An increase in Th1 cytokines and CXCL12 in CL from pregnant ewes was observed.  The CXCL12/CXCR4 signaling may play a role in regulating the cytokine milieu in the CL thereby influencing CL integrity and function.  This is especially important because CL function is obligatory to pregnancy establishment and CL destruction is obligatory during the estrous cycle to allow a subsequent cycle and another opportunity to become pregnant.

Cows with androgen excess also have sporadic anovulation (do not ovulate two consecutive estrous cycles) and altered metabolic function related perhaps to decreased estrogen carrying capacity in the blood.  These cows also have 17% reduction in calving rate and appear to develop persistent follicles that do not consistently ovulate.  Furthermore, their calves displayed heavier weaning weights which may be due to excess androgens during gestation allowing for increased growth.  Alternatively, this compensatory growth could develop due to feeding high protein supplements either during gestation or early life and be a result of prenatal/neonatal programming.  Current efforts include characterization of liver steroid metabolism and CL response to PGF of androgen excess and normal cows. The incidence of androgen excess cows within herds has been as high estimated to be 20%.

            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 in 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 estrous cycle in cattle.  However, estradiol tended to increase the expression of endometrial nuclear progesterone receptors and estradiol exposure during the periovulatory period was demonstrated to be essential in subsequent embryo implantation associated with pregnancy establishment in the cow.  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 IFNT’s 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 (FGF) 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 FGF 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.

A highly specific radioimmunoassay was developed for ovine IFNT, which has different levels of detection in uterine flushing compared to serum. Unfortunately, this ovine assay may not recognize all forms of bovine IFNT and for this reason a new radioimmunoassay and ELISA for IFNT in bovine tissues and fluids will be developed.

The role of chemokine ligand twelve (CXCL12) and its receptor, chemokine receptor four (CXCR4) exert during early gestation was investigated by delivering a CXCR4 antagonist directly into the uterine lumen during embryo attachment and initial placentation in ewes. A decrease in VEGF was noted in caruncle tissue from treated ewes and alterations in expression of various cytokines were observed. Impaired CXCL12/CXCR4 signaling may lead to poor pregnancy outcomes, such as impaired trophoblast attachment and compromised embryonic growth. A better understanding of CXCL12/CXCR4 signaling during early gestation, may lead to novel applied techniques to improve embryo survival during early gestation in livestock.

Supplementation of protein above NRC recommendations to the dam during gestation increased the ovarian follicular pool of the offspring.

            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.    In conditional KO mice models it was further elucidated that VEGFA angiogenic isoforms appear to enhance SSC renewal while antiangiogenic isoforms either cause apoptosis or allow for SSC differentiation into later stage germ cells.  This information will be applied to bovine or ovine systems to determine effects on stem cell populations in domestic livestock.

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 regulation of the number of primary spermatocytes in males.  Based on DNA parent verification, variation in offspring proportion that is not correlated with scrotal size exists in serving capacity or unknown fertility measures of bulls in multiple sire breeding herds.  Novel biomarkers on sperm associated with fertility have been characterized and removal of abnormal sperm from the ejaculate of bulls prior to extending and freezing of semen for artificial insemination (AI) increased pregnancy rates in cattle.  

Use of short term medroxy progesterone acetate (Depo-Provera) in reindeer bulls reduced aggressive and rut related behavior while maintaining food consumption during the rut reducing rut related weight loss, without blocking fertility in reindeer bulls. The treated bulls behaved as functional castrates. This treatment has implications for the management of males of other species. However, long term treatment of medroxy progesterone acetate may have negative impacts on spermatogenesis and cause reduced sperm density and less mature sperm. Thus, the amount of time the bull is exposed to the progestin is critical for breeding season success.

            Uterine-Placental-Fetal Nutritional Interactions. Significant strides in understanding implications of maternal obesity on offspring health, growth efficiency, glucose-insulin dynamics and reproductive function have been made. Results from studies have led to management practices that optimize offspring health and production efficiency. These studies provide evidence that optimization of maternal nutrition and the associated uterine environment contributes to the birth of healthy, growth-efficient offspring, which is vital to livestock production efficiency in the present and future generations. Manipulation of chorionic somatomammotropin hormone (CSH) influences fetal growth possibly through placental glucose transport implicating this hormone as being essential for normal fetal development while also providing a potential tool to manipulate fetal growth.  Maternal melatonin supplementation increases uterine artery blood flow and influences postnatal growth of offspring.

Intrauterine growth restriction (IUGR) has been associated with metabolic complications that limit performance in offspring. These studies indicate that developmental adaptions to conditions that result in IUGR permanently impair insulin-producing pancreatic β-cells. Growth restricted fetuses have decreased β-cell mass and reduced insulin secretion. A sheep model of placental insufficiency-induced IUGR has similar deficiencies in β-cells. The aim of these studies was to elucidate mechanisms responsible for β-cell dysfunction first by determining gene expression of the islet exome from IUGR fetuses. Near term placental insufficiency-induced IUGR (established by environmental hyperthermia) islets were isolated. IUGR islets had reduced expression of CCL2, CCL3, CCL4, CCL5, CCL21, CXCR4, and a portion of the interleukin family of cytokines including IL-1β, IL-8, and IL-6. IUGR islets expressed greater concentration of CXCL12, IL-6R and GPR75. Placental insufficiency-induced IUGR fetuses have decreased pro-inflammatory chemokines and cytokines that may regulate homeostatic processes in the islets. Previous findings show that IUGR suppresses the inflammatory response in postnatal animals and transiently alters neonatal islet T-helper cells in islets. However, findings also indicate a role for cytokines in β-cell dysfunction because the CXCL12/CXCR4 axis promotes pancreatic β-cell growth and genesis in human and rodent systems.

Impaired placental function was a primary cause of fetal growth restriction.  Undoubtedly the placenta plays a major role in determining fetal growth rate. This is obvious from its role as the interface between the maternal and fetal systems, providing for nutrient transport to the fetus and waste removal from the fetus. Additionally, the placenta is a rich source of hormones and growth factors that likely impact the fetus, mother or both. The existence of the placental lactogens (PLs) has been known for over 50 years, and it has been hypothesized that human and sheep PL modulate maternal and fetal metabolism. Lentiviral-mediated transgenesis within the sheep placenta was used to assess the importance of PL in fetal growth regulation at mid (50 d) or late (135 d) gestation.  A 50% reduction in oPL mRNA and a 38% reduction in placental oPL concentration was achieved using the “knockdown” model. This reduction in oPL was associated with 32% reduction in weight and a 41% decrease in fetal liver weights.  Fetal liver insulin like growth factor (IGF)-1 and -2 mRNA concentrations were reduced 82% and 71%, respectively, with a 62% reduction in umbilical artery IGF-1 concentration. There was a similar reduction in IGFBP-2 (74%) and IGFBP-3 (81%) mRNA.  Thus, in utero nutrition has epigenetic effects on the offspring that influence metabolic and production efficiency of livestock.

            Statistical Methodology. Statistical tools were generated to advance discovery of important reproductive characteristics. The nested factorial probe-level statistical model can assess differential genomic expression in studies where large sample sizes or number of biological replicates on microarrays are not practical using traditional statistical methods.  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 comparable to 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. Development of an LD-based QTL mapping approach that is more powerful and computationally much faster than current methods.  The LD-based nature of this framework requires a two-part null hypothesis (with nulls “no QTL exists” and “QTL is unlinked with marker”), and this bivariate null has required some novel statistical methods.   While these QTL methods have not yet been applied to reproductive genetics data, they deserve mention here due to their origins in this project.

         Behavior and Stress.  Prenatal transportation stress has been established as being a factor which alters the methylation pattern in calves. These alterations result in altered postnatal signaling influencing behavior and stress response of offspring. The impact of even mild stressors during gestation can alter the expression of the calf’s DNA resulting in performance traits which cannot be predicted by gene markers or pedigree analysis.

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.

Prenatal stress may program bull calves to function more normally when presented with a stressor later in life. When stressed at maturity, the prenatally stressed bulls had more frequent LH pulses than control bulls.

Novel management can influence production outcomes. Exercise in pregnant dairy heifers may improve heat tolerance, and improve milk quality during first lactation without adverse effects on gestation or parturition.

Dopamine synthesis and release in the ventral tegmental area (VTA) of the brainstem is credited for pleasurable aspects of certain behaviors such as mating activity and is central to the reinforcement and repeated expression of those behaviors. Following exposure to a putative sexual stimulus, inactive rams had fewer dopamine-synthesizing neurons in the VTA than sexually active rams. Lack of sexual interest in low sexually performing rams may be partially a result of decreased dopamine synthesis in the VTA leading to a less pleasurable experience and attenuated reinforcement of sexual behavior. Expression of ewe behavior does not improve the reproductive success of low sexually-active rams.

          Fertility, estrous synchronization and AI.  The optimal timing, dose, and route of administration of progesterone, PGF, and GnRH to induce ovulation was investigated and a protocol developed that increased conception to AI by more than 10% in both heifers and cows. Studies in sheep have determined administration of 10 mg Lutalyse 2.5 d after ram turn out resulted in a similar proportion of ewes being marked after 5 d of the breeding season when compared to a 12-d CIDR protocol.  Estradiol use during the synchronization of ovulation was shown to increase timed AI pregnancy rates 11 to 27%.  Use of estradiol with synchronization protocols could result in 25% more AI-sired calves, with more calves born early in the calving season resulting in heavier calves at weaning.

 A genome-wide association study was conducted using the EMMAX mixed-model association test to compare genomic differences related to cow fertility. These studies indicate that conceptus growth is not compromised in sub-fertile heifers up to d 14 after estrus. Observed differences in capacity for pregnancy success is manifested between days 14 and 28 when pregnancy recognition signaling and conceptus implantation must succeed for the establishment of pregnancy. Genetic markers identified in this study may be useful to select animals for fertility and enhance our understanding of the physiological pathways governing pregnancy success and loss in mammals.   

A number of circulating exosomal-derived miRNAs were identified that differ in abundance in non-pregnant and pregnant cows.  Future studies are essential to confirm their suitability as biomarkers for assessment of embryonic presence and viability in cattle and facilitate characterization of mechanisms associated with pregnancy failure.

 Late embryonic mortality (> d 28 of gestation) has been reported in both beef and dairy cattle and may vary from 3.2 to 42.7%.  The majority of these losses occur around the time placentomes begin to form (~d 35-40 of gestation). Pregnancy associated glycoproteins at day 31 of gestation may be a potential biomarker of late embryonic mortality and a possible model to help understand this period of pregnancy loss in cattle. 

            Disease, immune function.  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.  Through clarifying how BVDV persists, better biocontainment (detection) and treatment (antivirals) strategies can be implemented.  Identification and selection of breeding animals resistant to viral infection will also be imperative to control this insidious disease.

Objectives

1. Elucidate fundamental cellular, physiological, endocrine, and behavioral mechanisms that regulate gamete development and quality and enhance the management of reproductive function leading to development of translational reproductive biotechnologies.
   
2. Identify impacts of reproductive management, animal management and stress on follicle recruitment, ovulation, corpus luteum function, and pregnancy.
   
3. Determine mechanisms regulating normal embryo development, pregnancy establishment, and maintenance by exploring maternal and paternal factors; including genomics, immune responses, fetal programming, and conceptus/uterine signaling.
   

Methods

W4112 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. 

Presented below are studies that will be conducted during the new project. Collaborations among states are stated with each project.   

Mechanisms of ovulation and cyclicity (objectives 1)

Investigations into the key endocrine events underlying generation of the pre-ovulatory surge will be continued. The underlying molecular mechanisms that allow estradiol to signal the LH surge remain elusive and likely involve both genomic and non-genomic signaling events. Adenoviral vector-directed expression of green fluorescent protein in gonadotropes and fluorescence-activated cell sorting to identify E2-regulated genes in gonadotropes will be used to elucidate these pathways. In the next two years, it is expected that Stoplight Sheep will be established. This animal will have a Cre Recombinase-inducible transgene, as a tool to rapidly identify gonadotropes by gonadotrope-specific expression of Cre Recombinase, and will be used to perform key experiments probing the underlying mechanisms of gonadotrope E2 responsiveness. Scientists at Colorado State University will lead these studies.  

Work to elucidate the roles of FSH and estradiol regulated genes on development of preantral ovarian follicles in cattle. Work will continue to associate in vitro and in vivo methods with the main in vitro methods to include isolation and culture of preantral follicles, and other ovarian tissue. Among in vivo methods, the development of protocols to obtain ovarian biopsies to study of the ovarian response to FSH (and other factors) in real time. Scientists from University of California, Davis, USDA Miles City, and University of Nebraska, Lincoln will participate and collaborate in these studies.

Whole genome sequencing and genotyping of cows with known phenotypes related to how they attain puberty and if they have characteristics of High A4 cows will be conducted. SNPs surrounding FSHbeta, FSHR, AMH, AMHR and any candidate genes using GWAS and high impact SNPs will be evaluated to determine if any combination of genes may predict the puberty classification or High A4 status of these cows/heifers. University of Nebraska-Lincoln will lead these experiments but will collaborate with different aspects of this project with University of California Davis (folliculogenesis and FSH), New Mexico State University (granulosa cell function and FSH, cytokines and VEGF), University of Idaho (puberty), Wyoming (estrous behavior),  metabolism within the CL (Colorado State), comparisons with other high fertility females (University of Missouri and USDA Miles City, Texas A&M), Liver function and metabolism (University of Mississippi).

In vitro models will be used to further study the cell populations of the bovine ovary using the novel single cell RNA sequencing. Scientists from University of California, Davis, University of Nebraska, Lincoln, and Utah State University will participate and collaborate in these studies.

Appropriate ovarian function and subsequent steroidogenesis regulated by pituitary hormones and ovarian signaling molecules such as wingless-type mouse mammary tumor virus (WNT) is required for efficient reproduction. Activation of canonical WNT and FSH signaling pathways decreases steroidogenic enzymes and steroid production in vitro but has not been investigated in vivo. Research evaluating effects of exogenous WNT3A delivery during the follicular phase to impact folliculogenesis, E₂ synthesis and E₂ related events will be led by scientists from New Mexico State University and studies with inhibition of WNT signaling conducted with the High A4 cow model developed at University of Nebraska-Lincoln will be compared with data collected at NMSU.

Behavior and Stress (objective 2)

Studies evaluating the effects of metabolic and heat stress on cattle reproductive performance and fertility will be continued. Scientists at UC, Davis are currently working on a three year project to determine the effect of the slick hair mutation in Holsteins regarding health, production and reproduction. Studies to determine how negative energy balance influences ovarian follicular development will be developed in the next five years. Scientists from University of California, Davis and University of Illinois will participate and collaborate on these projects.

Experiments will be conducted to examine stress mitigation therapies to enhance corpus luteum function in cattle. The observation that steroid metabolic pathways in the corpus luteum are increased in cattle failing to become pregnant has led to further studies. Corpus luteum function will be examined in animals treated intravaginally or intrauterine with antioxidants. It is anticipated that local treatment with antioxidants will enhance corpus luteum function and alter the microbiome of the reproductive tract. These studies could lead to targeted treatment to enhance early pregnancy maintenance. Scientists from Mississippi State University, Montana State University, South Dakota State University, and USDA-ARS Miles City will participate and collaborate on these projects.

Remote identification and monitoring of parturition of individual animals will increase attentiveness, enabling early detection of dystocia in parturition.  Parturition events are marked by subtle changes in animal movement that may be identified through use of developing technologies.  Research evaluating the ability of tri-axial accelerometers attached to an ear tag in sheep to accurately identify and predict parturition-related behavior of ewes in an intensive setting will be ongoing and led by scientists from New Mexico State University.

Establishment of Pregnancy (objective 1)

Pregnancy loss (embryonic mortality, spontaneous abortions, etc.) has been well documented in all mammals, especially in domestic livestock species and humans and is a major cause of reproductive loss. Identifying the embryos that will survive to parturition will be the focus of research that dissect the mechanisms of embryonic mortality in cattle using extracellular derived microRNA and pregnancy associated glycoproteins. Based on these models, animals will be selected to characterize embryonic mortality or maintenance during critical times of gestation. Information gained from this research will fill major gaps in scientific knowledge and aid in the development of strategies to decrease pregnancy loss.

Scientists from Texas A&M, Miles City USDA, and University of Missouri will collaborate and participate on these projects.

The role of IFNT when inducing interferon stimulated genes in the corpus luteum will be further examined.  Clearly IFNT functions to mediate release of prostaglandin F2-alpha from the luminal endometrium.  However, the direct effects of IFNT on the CL and the benefits to sustaining CL function have not been delineated.  Scientists from Colorado State University and the University of Nebraska will collaborate on these studies. 

Recombinant bovine (rb)IFNT and antibodies against rbIFNT have been generated and validated in an ELISA.  This ELISA will be further tested for efficacy in determining pregnancy status and profiling embryo mortality in the next funding cycle. Also, larger clinical studies are envisioned with Colorado State University W4112 collaborators from USDA Miles City Montana, New Mexico State University and Texas A&M University.

Research focused on the genetic regulation of preimplantation embryonic development in the bovine will be initiated during the W4112 project. Research using novel genomic approaches including gene editing will be used to investigate the effect of reproduction-related genes on embryo development. Sires that have high or low ability to produce embryos and/or establish pregnancy will be used to identify key genetic variants associated with pregnancy establishment. This information will be used to select for genetic traits that will improve fertility in cattle. Scientists from University of Missouri will lead these projects.

Studies to elucidate the role of the chemokine CXCL12 and its receptors CXCR4 and CXCR7 will be continued as an imbalance to CXCL12-mediated signaling results in placental dysfunction. A substantial gap in knowledge remains as the contributions of CXCR4 compared to CXCR7 during implantation and placental development remain unclear. This gap in knowledge is because CXCR7 was recently demonstrated to elicit intracellular signaling upon CXCL12 binding. Thus, conclusions drawn from many reports ascribing CXCL12-mediated functions to CXCR4 activation must be reassessed to determine the role(s) of each receptor. To address this gap, a sheep model in combination with in vitro studies and specific chemokine receptor agonists and antagonists will be utilized. A clearer understanding of how CXCL12 secreted by fetal trophoblast cells signals through CXCR4 and/or CXCR7 on trophoblast and maternal cells to help establish pregnancy will be elucidated. It is suggested CXCR7 is principally involved in the multiple pathological events leading to placental dysfunction. Over the next 5 years the hypothesis that CXCL12 induces distinct biological responses through CXCR4 versus CXCR7 will be tested, thereby differentially impacting placental development, function, and fetal growth. Scientists from New Mexico State University, Texas A&M, Mississippi State University, University of Connecticut will participate and collaborate on these projects.

Fetal Development (objectives 2, 3)

Studies will examine maternal factors contributing to conceptus growth and development. Specifically, therapies will be used to target the composition of the bovine vaginal microbiota and its implications in fetal development and early neonatal vigor. These fetal programming projects will involve collaborators from Mississippi State University, Montana State University, South Dakota State University, and USDA-ARS Miles City.

The influence of maternal nutrition during gestation will be continued. Impaired tissue growth during prenatal development can persist into early postnatal growth and through adulthood, thereby hindering the ability to develop optimal muscle mass. Offspring growth, feed efficiency, liver development, metabolism, and epigenetic regulation will be evaluated in a sheep model of poor maternal nutrition (restricted- or over-fed).  The mechanisms by which poor maternal nutrition alters offspring metabolic pathways, influences DNA methylation in offspring liver, and determine the multigenerational effects of poor maternal nutrition on growth, metabolism, and liver development in F1 and F2 male and female offspring will be determined. Scientists from University of Connecticut, Utah State University, North Dakota State University, New Mexico State University, and Mississippi State University will participate and collaborate on these projects.

Further studies in fetal growth restriction (FGR) are planned. Offspring with low birthweights have a greater incidence of perinatal morbidity and mortality, and in those offspring that survive postnatal performance and carcass quality at slaughter are reduced. The role pancreatic islets play during fetal and adult life in FGR offspring will be studied. Studies during the new project will strive to determine programming mechanisms in insulin secretion and action that lower performance in domestic livestock. Scientists from University of Arizona and New Mexico State University will collaborate on these studies.

The regulation of placental endothelial/vasculature development during pregnancy will be investigated. Dysregulation of placental endothelial function leads to reduced utero-placental blood flow and lower placenta efficiency, which is directly linked to adverse fetal outcomes such as fetal growth restriction. A group of miRNAs that mediate growth factors- and cytokines-induced endothelial function and responses in placental endothelial cells have been identified. These miRNAs are hypothesized to be important for the endothelial/vasculature development at the feto-placental-maternal interface. The objective of this research is to determine the role of miRNAs in fetal programming mechanisms that underlying maternal stress associated with adverse fetal outcomes in domestic livestock. Scientist from University of Arizona will lead these studies.

As optimal maternal-placental-fetal interactions are required for a normal pregnancy outcome, research will continue to focus on examining the function of placenta-derived hormones and placental nutrient transporters.  In vivo placenta-specific lentiviral-mediated RNA interference will be used to investigate the physiological ramifications of diminished expression of the hormone Chorionic Somatomammotropin and placental glucose transporters.  This may well be expanded to specific amino acid transporters, and the glucagon receptor.  Our in vivo RNA interference approach, coupled with maternal and fetal catheterization, allows us to interrogate the physiological ramifications of these gene products within the placenta, under non-stressed/non-anesthetized conditions in sheep. Scientists at Colorado State University will lead these investigations.

Studies at University of Idaho will apply the effects of fetal programming to heifer development.  Preliminary data indicate that differences in early gestation nutrition exist under these field conditions.  Parameters investigated will include growth, reproductive tract development, and indicators of ovarian development and maturation. Genomic and epigenetic differences will be determined.

Male Reproduction (objective 1, 2)

Studies will be conducted to identify novel indicators of fertility and improve identification of sires that will improved herd fertility. Scientists from USDA, Miles City will lead these projects.

Factors that drive the expression of male reproductive behavior will continue in the new project. The role the TRPM8 receptor in novel signaling of testosterone will be the focus of research understanding the transient rise in testosterone in sexually active males. Scientists from University of Wyoming, with collaborations from University of Nebraska, will lead these projects.  

Disease, immune function (objective 1, 3)

BVDV is the most important and economically devastating viral-based disease of cattle in the US and costs the cattle industries at least $1.5 billion/year. Transient BVDV infections cause profound immunosuppression, infertility, fetal insults, abortions, stillbirths, weak calves and possibly postnatal immune compromised calves. Transient infection of bovine fetuses on day 175 causes viremia followed by seroconversion and immunoneutralization for non-cytopathic strains of BVDV. Preliminary data suggest increase global methylation of genes in fetal spleen after transient infection with BVDV.  The hypothesis that fetal transient infection with BVDV results in compromised postnatal immune responses will be directly tested through challenge with bovine respiratory disease viral and bacterial agents following weaning.  Scientists from Colorado State University will lead this study.

Diseases associated with Escherichia coli or LPS infusion suppresses estradiol production and follicular development in mammals. Understanding the mechanism linking bacterial infection and ovarian dysfunction remains to be determined. Research investigating distinct cellular events in folliculogenesis and steroidogenesis that may be impacted by infection are ongoing.  Specifically, the impact of disease states induced by LPS on canonical WNT/CTNNB1 signaling during follicle development in vivo will be led by scientists from New Mexico State University.

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 throughout the nation.  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.

Perhaps 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 (eg. invite them to present seminars at their institution, act as reviewers for manuscripts, act as external reviewers on promotion and tenure files, and enhance collaborative research efforts). Sharing of course notes for Endocrinology, Physiology and Anatomy, and Reproduction courses has been a major teaching aide for new members along with ways to develop assessment of learning in courses.  Finally, members work together to enhance graduate education by identifying superior students and help to place them in member labs that best fit the student’s career goals and by hosting students from collaborating labs.

Measurement of Progress and Results

Outputs

• The primary output from W4112 members is peer-reviewed scientific works including abstracts, publications, and presentations that represent unique contributions of new knowledge to the field of reproductive biology. Comments: These results fill critical gaps in our knowledge regarding the reproductive physiology of ruminant animals and facilitate the development and implementation of management strategies to optimize animal reproduction. Refer to attachments for lists of published articles and abstracts.
• Research findings were presented to a national audience of scientists at the 2017 ASAS-SSR Triennial Reproductive Symposium with 3 individuals from W3112 presenting research and one member serving as co-chair of the symposium. Comments: All of these individuals had proceedings papers published in Journal of Animal Science.
• Research findings were presented to a regional audience of scientists at Midwest ASAS meetings in Omaha, NE in 2018 at a Factors affecting Puberty Symposia and in 2019 at a Developmental Programming Symposia.
• A sheep symposia was planned for the 2020 Western Section meeting exclusively from W3112 members, however, due to COVID the meeting was converted to virtual and the sessions and symposia were reorganized.
• An Industry Reproductive Symposia was planned for SSR meetings in 2020 with several members of W3112 invited to speak; however, due to COVID this was postponed to SSR 2021. Comments: If the face-to-face meeting is not held, this Industry Reproductive Symposia will be hosted by W4412 virtually in August of 2021.
• A National Symposium is planned for the 2021 Annual Meeting of the American Society of Animal Science (ASAS) to be held in Louisville, KY. Comments: The symposium will be titled “Factors affecting reproductive performance in ruminants” and will have several members of the W4112 group presenting their latest research.
• Informational newsletters are available from University Beef Website- http://beef.unl.edu/ and the Livestock Range Research Laboratory, Miles City, MT https://www.ars.usda.gov/plains-area/miles-city-mt/larrl/

Outcomes or Projected Impacts

• It is difficult to project with certainty the outcomes and impacts of this 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. The impact those methods have on the sustainability of livestock production systems will depend on their impacts on quantitative measures of reproductive performance including: days open, services per conception, pregnancy rates, birth of healthy offspring, age at puberty, and lifetime productivity. Examples of monetary impacts of selected outcomes include: • The identification of beef females that are less fertile due to either genetic or prenatal development (nutrition interacting with genetics) may allow for sorting these females off the breeding herd or selection against these females for herd replacements. It may also allow for different treatments during gestation to ensure that less of these females are developed in the herd. Currently, it is estimated that sub-fertile females comprise 10% of the national cow population. The economic impact of even reducing infertile females by 5% overall in the US would allow for a reduction in losses of approximately 12 million dollars annually. This moderate estimate would be even more dramatic in dairy animals. • The development of a vaccine for Epizootic bovine abortion (EPA) is projected to save beef producers in California alone an estimated $10 to $15 million annually. • Decreasing embryonic mortality in beef and dairy cows of just 5% would result in an estimated savings of $25 million annually to beef and dairy producers. • The economic impact of BVDV was projected to range from $15 to $20 per cow calving. Hence, the development of methods to control this disease would result in an annual savings of $481 to $632.5 million for U.S. cow-calf producers. • Identification of males with low libido would increase producer profitability by decreasing the number of rams needed during the breeding season. More importantly, however, identifying and using high libido rams would increase the incorporation of desired production traits into the flock while decreasing the risk of having non-pregnant ewes. • Timing and intensity of environmental stress is crucial to subsequent production traits in livestock. Although nutrient stress experienced during gestation leads to glucose dysregulation in adult offspring, transportation stress experienced in utero is protective against stress experienced in later-life.
• In conclusion, 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 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 comparable data for other species is not readily available, it is reasonable to speculate similar losses occur in sheep, dairy and other ruminants. Because of the magnitude of these losses, it is expected research conducted by members W4112 will continue to lead to new innovations that increase the profitability and sustainability of livestock production enterprises.

Milestones

(2026):A symposium on sheep reproduction targeted to extension specialists/ educators and producers will be presented prior to the termination of the project (2026) to facilitate the transfer of knowledge gained from research conducted by W4112 scientists to producers. This symposium would also provide an opportunity for producers to influence research priorities of the W4112 investigators.

(2026):Beef Websites at the University of Nebraska, Lincoln and at the USDA ARS Fort Keogh Livestock and Range Research Laboratory, Mile City, MT will be used as a platform to post project results including reports, podcasts, and monthly newsletters. A similar website targeted to sheep producers will be developed and placed on the University of Wyoming, Agriculture Research Station website.

(2026):Approximately $17.3 million dollars of funding has been secured during the last five years of the project by participating scientists. Collaboration in securing external funding will continue and be monitored as a component of W4112 annual meetings.

(2021):A symposium titled “Factors affecting reproductive performance in ruminants” will be presented at the 2021 Annual Meeting of the American Society of Animal Science. This symposium will combine presentations by 13 members of the W4112 group.

Projected Participation

View Appendix E: Participation

Outreach Plan

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

Organization/Governance

The members of the W4112 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, 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.  2012.  National Agricultural Statistics.  www.nass.usda.gov/QuickStats

Attachments

Land Grant Participating States/Institutions

AZ, CA, CO, CT, FL, IA, ID, IL, KS, MO, MS, MT, NE, NJ, NM, OH, OR, PA, SC, SD, TX, UT, WA, WI, WY

Non Land Grant Participating States/Institutions

USDA-ARS/MT