Duration: 10/01/2017 to 09/30/2022

Administrative Advisor(s):

NIFA Reps:

Non-Technical Summary

Statement of Issues and Justification

The need as indicated by stakeholders. Impaired reproductive performance is a major cause of reduced productivity for ruminants and of reduced profitability for dairy and meat animal producers. The focus of the NE1227 Multistate Project, and its predecessors, NE1027, NE1007, NE161, NE72, NE 41 and NE 1, has been to address nutritional, management and environmental factors that impact ovarian, oviductal, and uterine function, as well as embryo quality, which collectively impact pregnancy rates in domestic ruminants. The long-term goal of the project is to understand how altered ovarian function, impaired oocyte quality, and disruptions of conceptus-uterine interactions contribute to infertility, and devise management strategies that will overcome these factors in order to optimize the chances that animals successfully achieve a pregnancy.  The current proposal will continue this important investigative work, focusing on basic mechanisms that impair or contribute to optimal follicular development, corpus luteum function, oocyte quality, preimplantation embryo development, and uterine-conceptus interactions. Additionally, the feasibility of innovative management practices will be investigated, with development of new strategies based on findings from the basic and applied research. Attaining these goals will positively impact animal producers and the scientific community, as well as indirectly affecting citizens of the region and the nation by promoting sustainable agricultural systems.

The objectives of this proposal align with Strategic Goal 1: Science in the NIFA Strategic Plan (2014-2018). Specifically, they are designed to meet the first sub-goal of Sustainable Agricultural Systems, which “addresses human interaction between science, technology, and agriculture and integrates the biological, physical, and environmental and socioeconomic factors essential to successful production enterprises and viable rural communities”. The success of the project will be measured in part according to NIFA-defined performance measures including (1) Measure 1.1.4 -number of peer-reviewed journal articles, lay publications, patents, and educational activities that increase understanding of biological processes and development of diagnostic techniques; (2) Measure 1.1.10 –behavior change related to factors affecting the decision making process, such as availability of resources and external forces; (3) Measure 1.7.4 –number of college graduates prepared for the professional and technical workforce in the food and agricultural industry; and (4) Measure 1.7.5 –number of graduate students and post-doctorates engaged in NIFA-administered projects and programs with an integrated education component to improve educational opportunities in agriculture.

Importance of work and consequences if it is not done. Improving fertility in ruminants requires fundamental knowledge about internal (e.g. immune cells) and external (e.g. toxins and nutrition) influences on (1) follicle activation, oocyte growth, and oocyte maturation; (2) corpus luteum development, steroidogenesis, and regression; (3) fertilization and preimplantation embryonic development; and (4) conceptus-uterine-ovarian interactions. It is critical to identify the underlying causes of anovulation, fertilization failure, luteal insufficiency, and early embryonic loss in ruminants, which are the leading causes of bovine infertility. By enhancing basic knowledge of the underlying biology surrounding ovarian function and embryonic development, new strategies can be developed for application by producers, veterinarians, and farm consultants. For example, as part of the basic research on follicular development and regulation of corpus luteum function, new reproductive management strategies for first artificial insemination (AI) and rebreeding strategies were developed (Double Ovsynch, optimized voluntary waiting period, extra treatment with prostaglandin F2a during protocols, etc.) and are now in practical use on commercial dairy farms. Studies continue to develop and evaluate management approaches that use already FDA-approved products and novel products in order to provide management strategies that are economical, user- and consumer-friendly, and preserve food quality and safety. Novel nonhormonal strategies, such as supplementation with specific feed additives, have been tested and validated in order to improve reproductive efficiency, reduce pregnancy loss, and improve reproductive health of the animal.

The technical feasibility of the work. The technical members of NE-1227 (molecular biologists, cell physiologists, and animal scientists) are a diverse group of scientists with broad and complementary expertise in ovarian and uterine physiology, oocyte and embryo development, and reproductive management of domestic ruminants. Recent additions of members with expertise in ovarian reserve and activation of follicle growth, preimplantation development of embryos, and maternal recognition of pregnancy will fill important gaps in knowledge in order to more effectively understand factors that cause early embryonic loss in the dairy cow. Previous individual and collaborative productivity of the members are indicative of ability to successfully perform, interpret, and disseminate the specific experiments outlined in the proposal. The diverse nature of the scientists in this project has been one of its strengths, allowing for integration of knowledge to achieve productive management outcomes.

The advantages for doing the work as a multistate effort. Advantages of performing this work as a multistate effort include complementary approaches with collaborative efforts, and technologies that can be integrated and directed toward several objectives simultaneously. The shared experience and data analyses contributed by individuals within the technical group make these interactions more beneficial. Moreover, the combination of basic biological research with innovative applied research more effectively supports outreach programs and engagement, the goal of which is to improve reproductive performance in livestock more rapidly. Using this paradigm, this multistate group has historically been one of the most productive, cohesive, diverse and collaborative multistate research groups nationwide. The commitment of participants to the multistate project is exemplified by numerous collaborative publications, including one in which it was decided to list the project itself as the author, rather than individuals (J. Anim. Sci. 74:1943-1952 [1]). Additionally, members at different stations have contributed to unified animal and cell culture protocols, conducted collaborative experiments, and exchanged samples to take advantage of unique validated procedures and will continue to do so in this project.

Impacts from successfully completing the work. Fulfilling the objectives of this project will provide important new information in order to combat declining fertility among ruminants, and especially dairy cows, in the face of continuous improvement in milk production capability. In the previous project (i.e. NE-1227), proposed research projects were performed using intramural and extramural competitive grant funds garnered by the members (more than $3.6 million). The 15 technical members of NE-1227 published refereed research papers (196), abstracts and conference papers (117), theses and dissertations (27), book chapters and invited reviews (17), technical/extension publications (13), and deposited sequences into the Gene Expression Omnibus (5). In addition, workshops and lectures (13) were presented to producers, veterinarians, and consultants locally and internationally. Based on these outputs, research colleagues investigating reproduction in ruminants around the world developed collaborations and benefited from this multitude of scientific reports and publications.

In addition, veterinarians, consultants, pharmaceutical companies, and breeding organizations who service the agricultural animal industries have been immediate beneficiaries of the work from this project. For example, collaborators have presented annual reports on the project to Select Sires and Genex CRI (Cattle AI organization) and reports at the National Association of Animal Breeders (NAAB). In turn, those groups spread the technology to farm families/producers for implementation, which benefits the on-farm profitability and sustains agricultural production systems that are highly competitive in the global economy.

Student training is another important impact of this project. During the last project period, the 15 technical members submitted more than 80 abstracts to national or international meetings with the bulk of these abstracts presented by graduate students in poster or platform sessions. Furthermore, 37 students completed or are currently pursuing a M.S. or PhD. degree. In addition to graduate students, numerous undergraduate students (98) with an interest in reproductive physiology were introduced to investigative research. These activities represent an important contribution of the project to the education of the next generation of scientists, consultants, and other workers in animal agriculture industries.

Related, Current and Previous Work

ACCOMPLISHMENTS FROM PREVIOUS PROJECT:

This has been an extraordinarily productive period for this regional research project. The group has published almost 200 (196) peer-reviewed scientific publications ranging from very basic reproductive biology to extremely practical reproductive management strategies that are already being applied on dairy farms. These publications appear in some of the top biological and agricultural journals and represent some of the foremost scientific manuscripts from each of the experiment stations represented on this project. Critical and straightforward discussion of the science and practical application of the latest reproductive research has been the hallmark of this project from its inception as NE-1, and has continued to drive the research excellence during the most recent project period. Some of the important accomplishments during this period are highlighted.

Follicular and Oocyte Function:

1. Factors have been defined for the establishment of the primordial follicle pool and for activation of the primordial follicle. Specifically, studies using fetal bovine ovaries strengthened the evidence for estradiol and progesterone (P4) as regulators of follicle formation and capacity to activate and showed that: A) effects of steroids are mediated by nuclear steroid receptors, B) LH, FSH, and estradiol regulate fetal steroidogenesis, C) bone morphogenetic protein (BMP) 4, but not BMP7, promotes follicle activation, D) activin A stimulates follicle formation and activation and regulates fetal ovarian steroid production, and E) the endocrine disruptors BPA and genistein exert differential effects on follicle formation and activation and on steroidogenesis.  
2. Oocyte developmental potential is acutely dependent on sufficient zinc during antral follicular development. Severe fertility problems occurred in females with inadequate zinc.
3. The ovulatory transition involves changes in the Hippo signaling pathway that may be a key regulator of cumulus cell differentiation.
4. Experiments to study a potential role of sonic hedgehog as a maternal effect gene in the oocyte showed that in contrast to rodents, sonic hedgehog is not expressed in the bovine oocyte.  In related work, a cell fate mapping approach was used to identify the cell type within the follicle that is the target of signaling by Indian and desert hedgehog proteins secreted by granulosa cells.
5. Paraoxonase 1 is a negative acute phase protein with antioxidant and antiinflammatory activities and in ovarian follicular fluid of cows it may protect oocytes from local metabolic/oxidative damage.
6. Unlike mouse oocytes, bovine oocytes possess a functional Store Operated Ca2+ entry (SOCE) Ca2+ influx system to refill internal Ca2+ stores required for initiation of Ca2+ oscillations and oocyte activation.
7. A novel oocyte-specific nuclear transporter, KPNA7, was discovered and a number of oocyte specific maternal effect genes, including NOBOX, FIGLA and LHX8 were characterized.
8. The mechanism of CCN1 action on granulosa cells is, in part, mediated by the protein kinase C signaling pathway.
9. A serum-free, bovine granulosa cell (bGC) culture system to study cell growth, differentiation, and sensitivity to cytokine-induced death was developed and used to better understand function of granulosa cells from bovine small follicles (<5mm).
10. Impairment of keratin intermediate filaments within granulosa cells augments surface expression of Fas and Fas-mediated apoptosis.
11. Identified a novel correlation between the microbial ecology of the gut and ovarian inflammation and oocyte transcript abundance.
12. Identified a novel genotype that results in high ovulation rate in cattle. Determined hormonal and follicular growth dynamics and molecular mechanisms that underlie the high ovulation rate.

Corpus Luteum, Oviduct, Uterus, and Pregnancy:

1. Determined the rate of pregnancy loss in lactating dairy cows during the period of maternal recognition using a combination of interferon-stimulated gene expression and pregnancy specific protein B concentrations to confirm the presence of an embryo.
2. Delineated how fibroblast growth factors influence oocyte competency and early embryonic development in cattle. These findings have also been used to develop a ‘cocktail’ of growth factors that promote extraembryonic membrane development in cattle. More recent efforts have identified a new embryo-derived factor that regulates early embryogenesis in cattle.
3. Determined that early plane of nutrition impacts the progression of uterine gland development in heifers, and that this outcome may be mediated by changes in the expression of several local controllers of gland development. These outcomes will help us to better understand how modifications in early management of calves may impact subsequent reproductive potential.  
4. Discovered that the types of T lymphocytes present in the CL depend on the functional status of the CL, and that bovine luteal cells can program T lymphocytes to facilitate luteal function.
5. Evaluated the role of microRNA in a number of different tissues. Discovered that they are involved in major transitional states in the CL, such as the shift from growth to maintenance, and the determination of regression or rescue by an embryo.
6. Determined transcriptomic changes in the uterus and peripheral blood during early pregnancy.
7. Discovered a role for preovulatory estradiol concentrations in conceptus development, uterine gene expression, and subsequent pregnancy loss after AI or embryo transfer.
8. Evaluated the role of CCN1 in the folliculo-luteal transition and in the development and maintenance of the bovine CL.
9. Determined the transcriptomic changes induced by ovarian steroids in oviductal function. Microarray-based transcriptional profiling demonstrated that oviductal epithelial cells had 1563 (ampulla) and 1758 (isthmus) transcripts that differed and the complete dataset was deposited into the Gene Expression Omnibus (National Center for Biotechnology Information, http://www.ncbi.nlm.nih.gov/geo) as accession number GSE63969.
10. Evaluated the role of sperm proteins in bovine early embryonic development.

Improving Reproductive Efficiency in Dairy Cattle:

1. Determined the presence of a growth hormone receptor (GHR) AluI(-) allele in Holstein cows that is associated with increased serum IGF-I concentrations and a shorter calving to conception interval during lactation, and indicator of better fertility.
2. A collaboration has been established among 4 stations in the regional research project with a database of DNA and phenotypic data on fertility that includes 464 cows (to date) for genotyping polymorphisms in candidate genes eg. GHR and IGF-I and comparing with fertility.
3. Determined the impact of pregnancy loss during the period of maternal recognition of pregnancy on luteal function and follicular wave dynamics in lactating dairy cows.
4. Determined specific factors that lower fertility in lactating dairy cows such as: larger uterine size, greater body condition score loss, lack of complete luteal regression, excessive energy content of diet, insufficient P₄ before or after AI, and insufficient magnitude of the GnRH-induced LH surge.
5. Developed strategies to improve fertility by increasing circulating P₄ concentrations before AI (Double Ovsynch), reducing P₄ concentrations near AI (second prostaglandin treatment), and increasing P₄ after AI (Day 5 hCG or GnRH treatment).

 

GENERAL BACKGROUND FOR CURRENT OBJECTIVES:

As evidenced by our previous accomplishments, the individual and collaborative projects of this group have focused on improving fertility in ruminants with an emphasis on dairy cow fertility. Fundamental investigations have been performed of physiologic, cellular, and molecular processes that control ovarian function and establishment of pregnancy in cattle. Further, practical studies have been performed showing that anovulation, decreased insemination efficiency, and early embryonic mortality are the leading causes of open and culled cows in the dairy and beef cattle herds in the United States and that practical solutions are available to improve reproductive management and fertility, providing the potential for billions of dollars in economic benefit for dairy and beef cattle producers [2-4]. Ovarian input into successful embryonic development includes the intraovarian environment associated with follicle and oocyte growth, function of the hypothalamic-pituitary-gonadal axis required for ovulation and oocyte maturation, and development and maintenance of an optimally-functioning corpus luteum. Thus, the overall long-term objective of the group is to use our collective expertise to understand the mechanisms by which internal and external forces modify these important physiological processes and to use this information about core biological processes to rationally develop new and innovative tools to increase cyclicity and breeding efficiency, and reduce early embryonic failures, thereby improving reproductive efficiency of both dairy and beef cattle operations in the United States.

Ovarian Influence on Pregnancy Success: It is well recognized that gamete quality, and in particular oocyte quality, is an important determinant of successful embryo development [5-7]. Indeed, infertility and poor embryo development ensues when oocyte maturation is compromised. The oocyte develops within the context of the ovarian follicle where it is in intimate contact with the surrounding granulosa cells. In fact, the oocyte and granulosa form a bidirectional regulatory loop that ensures adequate expression of factors that sustain growth and development and/or inhibit atresia of the follicle in collaboration with endocrine follicle-stimulating hormone (FSH) [8-10]. Regulation of ovarian follicle growth is a dynamic process during which cohorts of follicles are recruited and grow in successive waves throughout the estrous cycle of the dairy cow [11-13]. Recruitment of each new wave is preceded and triggered by a transient rise in circulating FSH concentrations [14-19]. During each period of recruitment only a single antral follicle is selected to become dominant over the remaining subordinate follicles of the cohort. The dominant follicle then has one of two fates: attain preovulatory status and eventually ovulate, or be eliminated with the cohort of subordinate follicles through follicular atresia. Dynamic changes in circulating hormones, paracrine factors acting within the follicle, and intracellular signaling pathways are involved in follicular growth and selection of an ovulatory follicle. In addition, local interactions and paracrine and endocrine factors play an important role in the development of a high-quality oocyte competent for fertilization and preimplantation development.

The preovulatory surge of luteinizing hormone (LH) causes dramatic changes in the dominant follicle and in the granulosa cells during ovulation. The LH surge is also a stimulus for maturation of the cytoplasmic contents of the oocyte including accumulation and redistribution of organelles, accumulation of essential nutrients, and selective storage and degradation of RNAs and proteins [20, 21]. These changes ultimately lead to the release of the cumulus-oocyte complex into the oviduct and transformation of the ruptured follicle into the corpus luteum, which produces large amounts of P4 [22]. The cumulus cells undergo a process called cumulus expansion, which involves the production of a protein matrix that envelops the cumulus cells and oocyte, and also secrete significant amounts of P4 that may aid in guiding sperm to the expanded complex [23, 24]. Thus, cumulus expansion and luteinization of the ruptured follicle are two key features of terminal differentiation in granulosa cells. In addition, the LH surge stimulates the oocyte to resume meiosis and results in an oocyte terminated at metaphase II [20, 25].

During all of these processes, vascular development is critical, beginning as the activated primary follicle progresses to the secondary stage and continuing as the follicle increases in size and complexity [26-28]. Vascular development proceeds through the process of angiogenesis and is restricted to the region of mesenchymal cells immediately outside the basement membrane that surrounds the granulosa layer of the early follicle. This region eventually becomes the vascularized theca cell layer while the granulosa layer remains avascular. Angiogenesis involves proliferation of endothelial cells to cause expansion of the existing vascular network and maturation of the capillary plexus by association of vessels with mural support cells (pericytes and vascular smooth muscle). A functional vascular network of arterioles, capillaries and venules is established by remodeling [29]. Just prior to ovulation, the basement membrane of the follicle breaks down and there is ingrowth of thecal vessels into the granulosa layer. This initiates the dramatic increase in angiogenesis that contributes to formation of the corpus luteum, one of the most well-vascularized tissues in the body [28]. While a number of proteins or angiogenic regulators critical for vascular development in the follicle and corpus luteum have been identified, the precise interactions among cell types and the signals that coordinate vascular development with development of the follicle and corpus luteum have not been adequately described

Factors that Impair Oocyte Quality and Promote Follicular Atresia: Multiple extrinsic and intrinsic stresses including heat stress (HS) and metabolic dysfunction, cause anovulation, decrease conception rates and compromise the ability to maintain pregnancy in agriculturally important animals. Reproductive dysfunction due to thermal stress is an enormous economic burden and represents a worldwide food security issue. Globally, HS is the primary factor limiting efficient animal protein production for human consumption, and the deleterious effects of HS on reproduction are difficult to mitigate because information on the causative mechanism(s) is scant. Additionally, as climate change continues, seasonal infertility will likely become even more of an economic burden and animal welfare issue in the future. Likewise, metabolic disturbances due to negative energy balance associated with increased milk yield have contributed to decreased fertility in the dairy cow [30]. It is clear from the existing epidemiological data and multiple mouse models, that metabolic dysfunction reduces oocyte quality [31-33]. Indeed, obesity-related metabolic dysfunction has been correlated to mitochondrial dysfunction [31, 34], endoplasmic reticulum stress [35, 36], and increased abundance of oocyte mRNA [33, 37]. Recent studies by our group have demonstrated altered concentrations of circulating NEFA and sex-hormone binding globulin (a liver-expressed protein) and expression of metabolic-related genes in theca cells of beef cows that are sporadically or chronically anovulatory, suggesting that metabolic dysfunction may alter ovarian follicular growth and oocyte quality in beef and dairy cows. What is not clear are the mechanistic links between these external stressors and abnormalities in follicular growth and oocyte quality. Understanding these mechanisms is important in order to circumvent this growing problem

Contribution of the Corpus Luteum to Reproductive Success: P4 is the key hormone released by the CL and is essential for maintenance of pregnancy in all mammals, including ruminants. Circulating P4 concentrations represent a balance between production of P4 by the large and small luteal cells and metabolism of P4, primarily by the liver. In cattle, optimized fertility has been shown to occur when circulating P4 is high before the breeding period [38-40], low near the time of breeding [41-44], and high during luteal development after breeding [45, 46]. Researchers in this regional project have been involved in numerous studies of luteal physiology, including: development of the CL, angiogenesis and blood flow in the CL, luteal steroidogenesis, immune function in the CL, and luteolysis.

A related process, critical for fertility, involves maintenance of the CL in a pregnant animal. The first critical period for luteal maintenance encompasses the time of normal luteolysis or “maternal recognition of pregnancy” that occurs from Days 16 to 25 in cattle [47-51]. During this decisive period, the embryo is dramatically increasing in size through the process of embryo elongation and the CL is maintained in cows with a “healthy” elongation. Alternatively, inadequate communication between the embryo, uterus, and ovary produces failure of luteal maintenance [52] and redirection of the physiology toward luteolysis coincident with a decline in circulating P₄, resulting in loss of the conceptus. Interferon-tau action is integral to all three of the critical processes that occur during this pivotal period of pregnancy: elongation, maintenance of the CL, and immunological protection of the embryo. Nevertheless, the molecular mechanisms involved in these processes, particularly rescue of the CL, are still not completely defined and will be investigated.

Another pivotal period for pregnancy loss or maintenance occurs during the second month of pregnancy (Days 28 to 60). In this period about 12% of pregnancies are lost [53]. Some of the key problems leading to loss of pregnancy include: luteal regression [53], inadequate placentation [54, 55], difficulty in the transition from amniotic to allantoic nutrition [56, 57], alterations in vascularization of the placenta and other systems [58, 59], and underdevelopment of the embryo/fetus [60]. Some of the most dramatic and well-described deficiencies in development during this period occur in embryos that are produced by in vitro fertilization [58, 60] or nuclear transfer [54-57]. The current project proposes to better understand this period in order to reduce pregnancy loss in ruminants.

Uterine Factors that Influence Embryo Survival: Another area of interest is to understand how uterine factors contribute to early embryonic development. The niche for early embryo development remains one of the most fundamental and promising frontiers yet to be completely characterized. In particular, the extended ruminant preimplantation period, (days 4-5 to days 20-21 after fertilization), represents an understudied yet critical phase of pregnancy during which successful early embryonic growth and elongation is completely dependent upon the different components that are contained within the uterine fluid (UF) or “histotroph” [61, 62]. Early attempts to characterize the uterine environment have mostly focused on discovering salts and protein composition [63-69] to uncover buffering and immunomodulatory elements in the UF. Recent studies have utilized mass spectrometry for identifying the different protein components in LUF [70-73]. However, the complete metabolic profile of the UF that would determine energy availability and homeostasis for the early preimplantation embryo is unknown. Secretion of energy substrates such as glucose, lactate and pyruvate is highly regulated by responses of the uterine epithelium [74, 75]. In addition, ruminant metabolism offers unique distinctions in substrate availability and utilization, presenting a system that cannot be extrapolated from other species. Moreover, metabolic stress during high milk production could also be an important consideration for metabolite changes in UF that may contribute to early embryonic mortality. Therefore, knowledge of the content of different metabolic substrates and metabolites in the bovine UF could be important for fertility management. At the crossroads of reproduction and nutrition, new understanding of metabolism in the UF might also allow for nutritional intervention to improve embryo survival in dairy cows.

Finally, it is clear that the early embryo alters the proportions and function of uterine resident immune cells. In other species these changes have been shown to be essential for establishment of pregnancy and to support development of an optimal placenta. Relatively little is known about the changes in uterine immune cells and immune mediating molecules during early pregnancy [76, 77]. However, it is clear that diseases, including subclinical disease, can reduce fertility in dairy cattle [78, 79]. Furthermore, a portion of the nearly 50% reduction in conception rates between heifers and mature lactating dairy cattle may be due to immune dysregulation at the fetal-maternal interface. A focus of this project will be to define the changes in immune cell proportions and expression of immune mediators during early pregnancy and to compare these changes between fertile heifers and subfertile, lactating dairy cows.

Genetic Determinants of Fertility in Dairy Cows: New techniques in molecular genetics and genomics are offering extraordinary possibilities for selection of high fertility dairy cattle and identification of the key genes/processes involved in optimized fertility [80, 81]. Reduced genetic merit for fertility has been linked with unfavorable metabolic status, delayed resumption of postpartum ovulation, and inadequate luteal phase P4 concentrations [82-84]. In addition, reproductive traits have been linked with specific genotypes of tumor necrosis factor α [85], leptin promoter [86], IGF-I [87], and growth hormone receptor [88-90]. Genetic and genomic studies provide unique opportunities for selection of higher fertility dairy cattle and to unravel physiological mechanisms related to genotypes and fertility among high producing dairy cows.

Current Reproductive Management Programs Used with Dairy Cows: Reproductive management programs, including synchronization of ovulation protocols for timed artificial insemination (TAI), are widely used by dairy farms [91, 92]. These protocols can be used to maximize fertility of AI services or to ensure timely insemination of cows that, due to biological or management factors, are not detected in estrus [93-97]. Despite recent advances in our understanding of the reproductive biology of dairy cattle through some of the work conducted by the experimental stations involved in this regional project NE-1227 [95, 98-102] and work conducted by others, the physiological response to synchronization of ovulation protocols remains suboptimal. Ovulatory failure in response to GnRH treatments [96, 98, 103, 104], lack of luteal regression in response to PGF treatments [96, 98, 100, 105], and an inadequate hormonal environment during growth and maturation of the ovulatory follicle [46], contribute to the suboptimal fertility of lactating dairy cows that receive TAI. These issues are exacerbated in previously inseminated cows due to the effect of pregnancy loss on the ovarian function of cows [101]. Therefore, in order to continue improving the fertility of AI services in lactating dairy cows, it is paramount to develop novel treatments that optimize the response to synchronization of ovulation protocols.

Lack of obvious behavioral estrus (which signals that the animal is in the ovulatory stage of the estrous cycle) will impact producer’s insemination decisions, and increase costs associated with increased feeding requirements of presumed inseminated females. Not surprisingly, both HS [106, 107] and LPS [108] impact female estrous behavior and frequency. As in the case of the LH surge, a threshold of E₂ is needed to induce behavioral display of estrus, however the amount required for the latter is thought to be at a lower level [109]. Altered E₂ by HS and LPS may explain the observed impacts on behavioral estrus because E₂ is required for both of these female phenotypic responses.

Another critical aspect of reproductive management programs for dairy cows is nonpregnancy diagnosis, because earlier identification of nonpregnant animals allows immediate reinsemination. A short interbreeding interval for previously inseminated cows is key to reduction of time to pregnancy and to maximize herd profitability [110]. Although traditional pregnancy testing methods (i.e., rectal palpation and transrectal ultrasonography) help to reduce the interbreeding interval when implemented in conjunction with a synchronization of ovulation protocol, monitoring markers of pregnancy in blood or milk may allow earlier identification and rebreeding of nonpregnant cows. For example, expression of interferon stimulated genes (ISG) in white blood cells [101, 111-113] and circulating concentrations of Pregnancy Associated Glycoproteins (PAG) [101, 114-116] allow for identification of nonpregnant cows as early as the third to fourth week after AI. Because these markers may also have value as predictors of the fate of pregnancy they could be used for early identification of cows that will lose their pregnancy [117]. Therefore, research efforts at the NY, PA, VA, and WI experimental stations will also focus on the development of strategies that integrate these early markers of pregnancy into reproductive strategies for second and greater AI service. In addition, understanding and improving Assisted Reproductive Technologies (ART) such as embryo transfer, intracytoplasmic sperm injection (ICSI), and cloning provide opportunities for understanding key aspects of reproduction and improving reproductive efficiency.

STANDARD CRIS SEARCH

A search of the CRIS system was conducted using the following key words: follicular growth, oocyte quality, early embryonic death, corpus luteum function, uterine receptivity, timed artificial insemination, and ruminants. There are 3 current multistate projects studying reproductive performance in domestic livestock. W3171 – Germ Cell and Embryo Development and Manipulation for the Improvement of Livestock aims to understand the biology and mechanisms of gamete development, fertilization, and embryogenesis with an emphasis on production of genetically enhanced animals for livestock production. The main impetus of NC1201 – Methods to Increase Reproductive Efficiency in Cattle is to develop breeding programs and estrous synchronization protocols to increase the efficiency and predictability of sustainable reproductive management programs for cattle. Finally, W3112 – Reproductive Performance in Domestic Ruminants studies the endocrine regulation of reproductive behavior, estrous cyclicity, gamete development, follicular recruitment, ovulation, luteal function, and immune-dependent embryonic loss in order to develop biotechnologies to improve reproductive efficiency in domestic ruminants. It is our view that our proposal complements these other projects with our long-term goal of improving female fertility of ruminants. However, our project will examine novel pathways and mechanisms regulating ovarian function, uterine-embryo cross-talk, and the establishment and maintenance of pregnancy with an emphasis on dairy cattle.

Objectives

1. Determine the Impact of Altered Ovarian Function on Ruminant Reproductive Performance (IA, KY, MS, NE, NH, NY, PA, VA, VT, WI , WV)
   
2. Identify Alterations in Embryo Development and Uterine and CL Function Associated with Declining Pregnancy Establishment in Ruminants – (NY, PA, VA, WI)
   
3. Identify Changes in Genetics and Reproductive Management that Lead to Improved Pregnancy Rates in Ruminants
   

Methods

Comprehensive methods section has been provided in the attachment entitled "NE_TEMP1227 Full Methods.pdf."  Summarized information below.

Objective 1: Growth of the oocyte within the follicular microenvironment, oocyte maturation and ovulation, and the development of a functional corpus luteum (CL) are critical determinants of reproductive success. These collective processes are regulated by a myriad of signaling pathways; however, there remains gaps in understanding the precise mechanisms of action by the signaling pathways or how they are regulated. Furthermore, there are gaps in understanding how negative environmental conditions impinge on these pathways to interfere with follicle growth, oocyte quality and/or luteal function.

1. Determine if Hippo signaling is activated during ovulation to promote GC differentiation. The hypothesis is that Hippo signaling acts as a switch in GCs during ovulation. Primary GC cultures under control or luteinizing conditions will be employed and activation of the Hippo pathway determined by Western blot to detect phosphorylation of the target proteins, YAP and TAZ. Cellular proliferation will be determined using an MTT-based assay and cellular differentiation will analyzed by upregulation of the P4 biosynthetic pathway using real-time PCR, western blot or measurement of P4 accumulation in the media of cultured cells.
2. Identify signaling pathways that regulate Hippo signaling during ovulation. The hypothesis that activation of the PKA and/or MAPK3/1 pathways induces Hippo pathway activation in GCs will be tested, since ovulatory signals detected in GCs due to LH receptor activation involves signaling directly or indirectly through PKA and MAPK pathways. To determine which pathway(s) regulate hippo signaling during ovulation, luteinizing GCs will be cultured with or without specific PKA or MAPK3/1 inhibitors and the status of Hippo pathway activation will be determined by qPCR of Hippo target transcripts and Western blot for pYAP and pTAZ.
3. Determine if ERK signaling enhances GC resistance to apoptosis and follicular atresia. The hypothesis is that activation of the MAPK3/1 pathway induces ERK signaling and GC resistance to FAS-mediated apoptosis will be tested. Genetic manipulation of ERK signaling will be performed to gain insight about ERK signaling roles in immune-mediated apoptosis of GCs and growth selection of bovine follicles; primary cultures of bovine antral follicle GCs will be utilized and several qualitative (immunoblot analysis, confocal microscopy) and quantitative (qPCR, cell death assays) measures of ERK expression and apoptosis assessed. Genetic manipulation of GCs will be implemented to augment/inhibit ERK expression and determine the effects on FAS-mediated apoptosis.
4. Investigate whether hedgehog (HH) signaling plays a role in directing follicle development. We hypothesize that HH signaling regulates the development of vasculature in growing ovarian follicles and CLs. Cortical tissue will be cultured to assess follicle development to primary and secondary stages and associated development of vasculature. HH signaling will be stimulated or inhibited in cultured tissue and effects on follicular and vascular morphology and gene and protein expression patterns associated with vascularization will be determined using qPCR and immunohistochemistry. Cultures of dispersed theca and early luteal cells, containing mixtures of steroidogenic and vascular cells, will be used to examine the development of vascular tubes and the interactions among vascular cell types in response to manipulation of HH signaling.
5. Determine mechanistic regulation of CCN1 expression in GCs during transition to luteal cells. We have previously characterized the regulation of CCN1 mRNA expression by steroid hormones, gonadotropins and prostaglandins in human GC lines (KGN, HGrC1)[209, 210]. Regulation of CCN1 by these same factors in bovine GCs from midcycle cows will be measured by qPCR. Conditioned media will be analyzed for steroid hormones (E2 and P4) and for matrix metalloproteinases by gelatin zymography. Regulation of downstream PGF signaling factors (ERK, RAF, RhoA) and the effect of P4 on CCN1 expression will be monitored in KGN and HGrC1 cells.
6. Investigate roles of SMAD and Hippo pathways in regulating GC function in carriers of the TRIO genotype. GCs will be collected from follicles of carriers and non-carriers of the TRIO genotype using ultrasound-guided follicular aspiration. Pathways will be activated by treatment with activators of SMAD (GDF9, BMP15) and Hippo and activation of SMADs, YAP, and TAZ will be evaluated in the two genotypes by Western blot.

Outcomes (1-6): We anticipate acquisition of in-depth of knowledge related to the specific cellular signaling pathways that regulate GC proliferation and differentiation, theca cell function, and vascular development. The interaction of these pathways defined in the in vitro and in vivo experiments will results in a fuller understanding of how follicular development is regulated in cattle and how this process can be potentially optimized.

7. Determine role of chronic low-level LPS on ovarian function and hepatic clearance of steroid hormones. The hypothesis to be tested is that chronic exposure to LPS, due to compromised intestinal integrity or infection, alters steroid hormone production in the ovary and metabolism of steroid hormones in the liver. Jugular catheters will be surgically placed in 12 multiparous dairy cows to facilitate blood collection and LPS infusion. An infusion pump will be used to deliver chronic LPS (0.02-0.15 µg/kg/h) continuously over 24h for a 7d period. Dominant follicle diameter will be recorded daily 4 days prior to ovulation. At the time of predicted ovulation, follicular fluid will be aspirated for steroid hormone analysis (estradiol and P₄). On the day of predicted ovulation, hepatic biopsies will be obtained and flash frozen for analyses of steroid hormone clearance enzymes.
8. Investigate effects of HS on oocyte quality. Altered insulin and glucose concentrations during HS may be detrimental to the oocyte due to changes in the ultrastructure and/or Ca²⁺ content of the endoplasmic reticulum (ER) which in turn could affect the transcriptome, proteome or epigenome. Lactating dairy cows will be subjected to four sequential treatments: thermoneutral, thermoneutral + hyperinsulinemic hypoglycemic clamp, HS and HS + euglycemic clamp. Oocytes, follicle fluid and GCs will be collected from each cow twice during each treatment. Insulin, glucose, E2, P4 will be measured in the follicular fluid.  Glucose uptake and oxidation will be measured in GCs. Gene expression and ultrastructure analysis of oocytes and GCs will be perforemd.  ER Ca²⁺content will be assessed using Fura-2.
9. Determine impact of fescue on ovarian function. Consumption of endophyte-infected fescue may alter ovarian function and confer direct effects on the ovarian follicle. Cows will be fed either endophyte-infected fescue seed (KY31) or fescue seed largely devoid of endophyte for six weeks; follicular fluid will be aspirated every week for 6 weeks.  Blood samples will be collected throughout the study to evaluate circulating prolactin concentrations. The collected follicular fluid will be added to in vitro oocyte maturation media, and oocyte maturation and embryo development will be monitored. 

Outcomes (7-9): We anticipate that the findings from these experiments will enhance our understanding of the impacts of environmental stressors on ovarian function and fertility and may identify targets for amelioration strategies to improve fertility in dairy cows.  

10. Determine which intracellular compartments contain PTGFR. CLs will be collected from synchronized dairy cows on Days 4 and 11 of the estrous cycle, representing CL that lack or have acquired luteolytic capacity. The CL will be processed for immunoelectron microscopy to visualize the intracellular location of the PTGFR. Preliminary data have been collected that validate the method and the antibody specificity, and have indicated that PTGFR is located inside luteal cells. In addition, tissues from these CL will be processed for immunohistochemistry to determine cellular location of PTGFR and quantify differences in expression and localization in Day 4 vs Day 11 CL.
11. Delineate PGF cellular site of actions. Midcycle CL will be collected and placed into short- or long-term cultures. A PGF-biotin vs. PGF-biotin-streptavidin conjugate will be used to determine if PGF actions require its transport into luteal cells. The PGF-biotin can bind to the PTGFR at the cell membrane or diffuse into cells. However, PGF-biotin-streptavidin is unable to diffuse into the cells. Cells treated with each protein conjugate will be tested for activation of protein kinase C, mobilization of calcium, and inhibition of LH-stimulated P4.
12. Evaluate the effect of estrogen exposure on luteal cell responses to PTGFR. Cultured luteal cells will be obtained as described above. Cells will be treated with estrogen to determine if estrogen promotes translocation of PTGFR to the nucleus of the luteal cells. Cellular localization of PTGFR will be by immunohistochemistry using the antibody that we have optimized for this work.
13. Determine if CL rescue can be achieved by PGE production. Interferon-tau (IFNt) may rescue CL through stimulation of PGE production by the uterine endometrium. IFNt or control protein will be infused into the uterine horn ipsilateral to the CL. After 12 h, cows will be challenged with low doses of PGF administered directly into the uterus. Alternatively, the uterine horn will be treated with or without IFNt and with or without PGE inhibitor. Biopsies of the CL will be taken at 30 min after each PGF pulse for evaluation of mRNA expression (RNA-Seq) and physiological endpoints (e.g. immune cell infiltration).

Outcomes (10-13): We anticipate that these studies will allow understanding of the molecular mechanisms involved in the absence of PGF action in the early CL and the mechanisms that mediate PGF action in the mature CL. In addition, we anticipate that these studies will allow us to further understand the mechanisms involved in rescue of the CL during early pregnancy.           

Objective 2: Early embryonic wastage is a major problem in lactating cattle [53]. A major emphasis is investigating the core processes that control early embryonic development and pregnancy maintenance or loss in cattle. A central concept of this research is that there are two fundamental goals during early pregnancy; initiate, establish, and maintain a healthy pregnancy or in the absence of pregnancy, reinitiate the cyclicity at the earliest possible time. The embryo, therefore, encounters physiological gateways during pregnancy. Likewise, the uterus/dam determines if the current pregnancy continues or is eliminated. These gateways during pregnancy establishment and maintenance involve an intricate and remarkable communication between three distinct structures, the developing conceptus, the uterus, and the CL. However, there are gaps in understanding how this communication occurs.

1. Dairy cows will be synchronized using Double-Ovsynch followed by timed AI All cows will receive GnRH (200 ug) on Day 5 after AI and only cows with a contralateral accessory CL will be used in the study. Synchronized cows with no AI will serve as non-pregnant controls. Ultrasound and blood sampling will be performed daily from d13 to d60 of pregnancy or until CL regression occurs. Day 19, 20, and 21 blood samples will be used to determine interferon-stimulated genes (ISGs) and circulating PGFM and PGEM. Day 22, 24, 26, and 28 samples will be used to determine pregnancy-associated glycoproteins (PAGs). Continuous values (hormone concentrations, follicular/CL sizes) will be analyzed by the MIXED procedure (SAS, version 8.2, 2001). Categorical data will be evaluated by the LOGISTIC procedure (SAS, 2001). Both models will include treatment, parity, and interaction of treatment with parity.
2. Determine potential for altered pregnancy outcome dependent on CL location. We hypothesize that an accessory CL increases fertility if located ipsilateral but reduces fertility if located contralateral to the pregnancy and/or regresses during the period of maternal recognition of pregnancy (Day 16-25). Holstein dairy cows (2,400) will be synchronized (Double-Ovsynch) and blocked by parity and randomized to receive GnRH (200 ug) or saline on Day 5 after AI. Ultrasound will be performed on day of AI (d0) and d5 to detect ovulation. Ultrasound on d12 will be used to determine ovulation and location of accessory CL. Blood samples (n = 400) will be taken on Day 12, 19, and 26 to detect P4, ISGs, and PAGs. Ultrasound will be performed on Days 19, 26, 32, 46, and 60 to determine timing of accessory CL regression and detect pregnancy. Data will be analyzed as described in #1.
3. Determine contribution of uterine factors to early embryonic development. We hypothesize that specific uterine-derived growth factors and cytokines mediate embryo development. These factors may serve as markers for fertility and may also be used as supplements for in vitro-produced embryos. A complete evaluation of uterine fluid (UF) will be performed in synchronized cows sampled on Day 0 (at the time of AI, E2 influence), Day 10 (blastocyst in uterus, P4 influence), Day 14 (IFNt secretion, maternal recognition of pregnancy), and Day 18 (peak IFNt secretion, preimplantation period). Cows will be synchronized together and randomly assigned to two groups (bred or non-bred at Day 0). Embryo survival and mortality will be assessed using values of pregnancy specific protein B (PSPB) and flushing embryos. Comparisons will be made both in series and between the bred and non-bred groups. The sample analysis will use both gas chromatography/time-of flight mass spectrometry (GC-TOF MS) and charged surface hybrid-quadrupole/time-of-flight mass spectrometry (CSH-QTOF MS) for analysis of metabolites.
4. Investigate the immune system contribution to pregnancy failure. While it is clear that the early embryo alters the proportions and function of uterine resident immune cells, little is known about changes in uterine immune cells and immune mediating molecules during early pregnancy [76, 77]. We hypothesize that reduction in conception rates between heifers and mature lactating dairy cattle is due to immune dysregulation at the fetal-maternal interface. We will define the changes in immune cell proportions and expression of immune mediators during early pregnancy and compare these changes between fertile heifers and sub-fertile lactating dairy cows.
5. Delineate how embryonic cell lineages segregate during early development. The formation of definitive epiblast (i.e. embryo/fetal), trophoblast (i.e. placental) and endoderm (i.e. yolk sac) lineages by day 8 after fertilization is crucial for embryo survival. We aim to establish the molecular systems that control the emergence of these lineages and to identify the paracrine/autocrine factors that mediate the relative proportion of each cell type in embryos.

Outcomes: We anticipate that these studies will provide substantial insight into mechanisms associated with maintenance or loss of pregnancy and regression or rescue of the CL during pivotal periods of pregnancy. In addition, we will understand the key changes in UF and uterine immune cells that are associated with early pregnancy and may improve fertility in cattle.

Objective 3: With the development of powerful molecular techniques to identify key genes involved in reproduction and genomic approaches that allow precise selection of specific genotypes, it will be possible during this project to understand and select for higher fertility in dairy cattle. We also intend to develop management strategies that maximize dairy cattle reproductive performance and farm profitability through improved P/AI and a reduction of the interbreeding interval.

1. Investigate associations between fertility outcomes and SNP in candidate genes. We will test the hypothesis that high vs low fertility in dairy cows is associated with individual or combined SNPs in genes linked to reproductive activity. Blood samples from ~1000 lactating dairy cows are currently being obtained from multiple experiment stations for DNA genotyping that will be correlated to pregnancy/fertility phenotype information. DNA will be extracted from blood to genotype GHR, TNFα, and IGF-1 in each cow. The effects of candidate genotypes on pregnancy rates to 1st AI and through 210 days of lactation will be determined.
2. Determine association between genomic evaluation and reproductive phenotypes. We hypothesize that there are specific genomic associations that contribute to reproductive success in cattle. The 2,400 cows in #1 will be subjected to genomic evaluation. Since all cows will be bred using the same reproductive management protocol we expect to determine SNPs that are associated with specific traits, such as circulating P4 concentrations.
3. Increase ovulation and identify non-pregnant dairy cows earlier. First service postpartum. In commercial dairy farms, cows will receive first service TAI after synchronization of ovulation with protocols that vary in the type and dosage of hormonal treatments or interval between treatments. Ultrasound will evaluate ovulation and follicular wave dynamics after GnRH treatments. Hormone profiles (e.g., P4, E2, LH, FSH) will be used to confirm ovulation, determine luteal regression, and characterize the hormonal milieu before, during, and after synchronization of ovulation. Second and greater AI services: Ultrasound or circulating hormone concentrations will be used to assign cows to synchronization protocols based on the type and status of ovarian structures present on the ovaries. The synchronization treatment will be developed following the same premises as for first service. Pregnancy testing: Elevated concentrations of interferon stimulated genes (e.g., ISG15 and MX2) and PAG are predictive of pregnancy in cattle. These technologies will be coupled with synchronization of ovulation protocols for second and greater AI services to identify open cows earlier and reduce the inter-service interval. Circulating ISG and PAG concentrations will be used to determine timing of embryonic and fetal pregnancy losses.

Outcomes: We anticipate an overall improvement in herd reproductive performance and profitability by improving P/AI to all AI services, reducing the interbreeding interval, and reducing reproductive program costs. Given the central role of reproductive performance on the economic viability of dairy farms, optimizing timing of pregnancy in dairy cows will ensure farm profitability and sustainability.

Measurement of Progress and Results

Outputs

• Research data on signaling factors that regulate follicular activation and growth, ovulation, and oocyte quality. Data will identify novel genes, pathways, and mechanisms that affect oocyte quality, CL function, and pre-implantation embryonic development.
• Identify maternal and paternal factors affecting fertilization and establish the role of the oviductal and uterine environment as well as inflammatory factors on successful fertilization and embryo development and implantation. Comments:
• Information that will help producers make informed management decisions to enhance embryonic/fetal survival in ruminants.
• Present research results at professional meetings for animal scientists, veterinarians and other agricultural related professionals. Comments:
• Establish and characterize models that can be used for further research.

Outcomes or Projected Impacts

• Greater understanding of mechanisms involved in reproductive physiology of livestock species.
• New approaches to management strategies (e.g. estrous synchronization) that will enhance embryo survival and pregnancy rates.
• Correlations between molecular phenotypes and measurable markers that can be used to develop herd management strategies that minimize the effects of nutritional/metabolic/heat stress on herd fertility.

Milestones

(2017):Identify novel pathways that influence follicular growth and the quality of oocytes and luteal cells produced after the LH surge which contribute to embryo developmental competence. Measure the impact of metabolic/heat stress on these pathways

(2019):Identify genetic markers of high fertility in the dairy cow and use the basic research data collected to develop and test management protocols for producers.

(2021):Identify new markers based on understanding of ovarian function, oocyte quality and interactions in the reproductive tract that can help to maximize early embryo survival and longevity of animals in the herd. Disseminate these findings to research scientists, clinicians, and producers via extension activities (see outreach plan).

Projected Participation

View Appendix E: Participation

Outreach Plan

Disseminate Information on Reproductive Physiology and Reproductive Management Strategies to Stakeholders in the Dairy Industry

1. Develop and Implement Workshops on Reproduction for Dairy Veterinarians in our states
2. Develop Mechanisms to Disseminate Key Reproductive Physiology to Graduate Students at Universities

 

The research described herein is largely basic. Application of findings from the basic research will be conducted at experiment stations and/or cooperating farms. Results will be communicated in annual reports, in refereed publications, in presentations at national meetings and on the websites of the experiment stations involved. Data will be presented to experiment station advisory boards, and to extension personnel and commodity groups in short courses and at field days.  In addition, some extension specialists and agents will aid in collecting applied data and selecting cooperating farms. Research workers will prepare occasional news releases or participate in interviews.  Through the latter venues, stakeholder input for further work and reactions to work in progress will be obtained.  Thus, an interactive system for dissemination of results and guidance in applications will be maintained throughout the project.

Course: Several institutions will develop a team-taught, graduate course focused on exploring contemporary issues in reproductive biology. The course will be team-taught by faculty at several institutions within NE1227 using teleconference technology. This course is anticipated to provide essential training for the next generation of reproductive physiologists. Students will be taught by the experts in various reproductive biology disciplines across several institutions. Faculty exposure to students also will be increased. Several institutions within NE1227 struggle to achieve the minimum student numbers needed to offer a graduate reproductive physiology course every 1 or 2 years. Reducing teaching load and increasing teaching efficiency will provide several institutions with the opportunity to offer this course more regularly.  

The learning objectives of the course are to: 1) Compare and contrast various reproductive processes that involve the neuroendocrine control of reproduction, gametogenesis, ovarian physiology, early embryogenesis, uterine function, placental biology and fetal programming, 2) Investigate new and emerging reproductive technologies that relate to assisted reproduction and stem cell biology, and 3) Interpret primary reproductive biology literature, theorize new avenues of research that apply to this work, and construct research plans to investigate hypotheses put forth in discussions and with independent exploration that incorporate physiology, endocrinology, cell biology and molecular biology to explain reproductive processes. Participants include Iowa State (Keating), Kentucky (Bridges), Penn State (Pate), Virginia Tech (Ealy), and Wisconsin (Wiltbank).

Organization/Governance

A regional Technical Committee will be organized in accordance with the Manual for Cooperative Regional Research (CSREES-OD-1082, 1977). The voting membership of the Regional Technical Committee shall include at least one representative from each cooperating Agricultural Experiment Station as appointed by the respective Director, a technical representative of each cooperation USDA-ARS research division and other participating organizations. Non-voting members shall consist of the Administrative Advisor and a consulting member representing CSREES.

All voting members of the Technical Committee are eligible for office. A chairperson, a secretary, and a third member of the committee will be elected for 2-year terms to compose an Executive Committee. The Technical Committee will meet at least annually. The chairperson, in consultation with the administrative advisor, will notify members of the time and place of meetings. The chairperson is responsible for the preparation of the annual report of the regional project. The secretary records the minutes and other duties as assigned by the Technical Committee. The Executive Committee may be delegated to conduct the business of the Technical Committee between meetings. Other subcommittees may be named by the chairperson as required.

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