Duration: 10/01/2022 to 09/30/2027

Administrative Advisor(s):

NIFA Reps:

Non-Technical Summary

Statement of Issues and Justification

The need as indicated by stakeholders.  Reproductive performance in beef and dairy cattle is often suboptimal resulting in increased intervals to conception and/or rebreeding failures which collectively reduces farm revenue due to the effect on milk and calf production efficiencies. Causes of pregnancy failure in cows include lack of resumption of cyclicity (anovulation/ anestrus), fertilization failure, poor oocyte quality, suboptimal hormonal environment due to abnormalities in follicle or corpus luteum, abnormalities in embryonic development and uterine-conceptus interactions, and pregnancy loss. Any of these reproductive problems can lead to reproductive inefficiency, increased culling, and substantial economic losses. Reduced fertility in ruminants also affects the consumer because reduced farm efficiencies (i.e., fewer calves per year and reduced milk production efficiency) reduce product supply and/or increase cost.

Importance of work and consequences if it is not done. Improving fertility in ruminants requires fundamental knowledge about endogenous and exogenous influences on 1) follicle activation and development and oocyte growth and maturation; 2) corpus luteum development, steroidogenesis, and regression; 3) fertilization and pre-attachment embryo development; 4) conceptus-uterine-ovarian interactions; and 5) placental development and function.  Understanding ovarian, uterine, and embryo attributes is critical to identify the underlying causes of anovulation, fertilization failure, luteal insufficiency, and pregnancy loss in ruminants, each of which are the critical causes of reduced fertility. This will lead to development of novel and innovative management strategies designed to optimize fertility, and therefore, may lead to management strategies and therapeutics that improve reproductive efficiency and are economical, user and consumer friendly, while still ensuring food quality and safety.

Advantages of performing this work as a multistate effort. We are the first multi-state group established and from its initial inception (NE-1) has been one of the most productive, cohesive, diverse, and collaborative multistate research groups nationwide. Complementary expertise of the group has led to collaborative experimental efforts and incorporation of new technologies that were integrated and directed toward exponential progress of previous objectives. The group uses a variety of animal models that are best suited to address specific molecular and cellular questions associated with tissue function. Additionally, members at different stations have developed best practices for animal and cell culture protocols, conducted collaborative experiments, and exchanged samples to take advantage of unique validated procedures. 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 ¹. In addition to research collaborations, the group developed a multi-institution graduate course in reproductive biology. Furthermore, senior members of the group serve as mentors for junior investigators as they develop internationally recognized research programs and become leaders in the field of reproductive biology.

The technical feasibility of the work. The technical members of NE-1727 (molecular biologists, cell physiologists, reproductive physiologists, animal scientists, and veterinarians) are a diverse group of scientists with broad and complementary expertise in follicle growth, corpus luteum function, oocyte development, conceptus-uterus-ovary interactions, reproductive immunology, and reproductive management of ruminants. Recent additions of members with expertise in ovarian reserve and activation of follicle growth, follicle deviation, genomics of oocyte development and maturation, and pre-implantation embryo development will fill important gaps in knowledge to understand pregnancy loss more effectively in cattle. This shared expertise and wide array of technical capabilities has previously fostered more rigorous and impactful research. Moreover, the combination of basic biological research and innovative applied research more effectively support outreach programs and engagement, the goal of which is to improve reproductive performance in livestock more rapidly. This experimental paradigm will be continued in the newly proposed objectives.

Impacts from successfully completing the work. Specific impactful discoveries from the group include, but are not limited to:

• Roles of pro-inflammatory cytokines and environmental stressors on follicle growth, steroidogenesis, luteal regression, and conceptus-uterine-immune interactions.
• Use of trace mineral supplementation to improve oocyte quality and increase systemic progesterone concentrations.
• Impact of beef cow body condition score and weight on the metabolome of follicular fluid and serum
• Genetic determinants of ovulation and implantation rates
• Endocrine and molecular mechanisms controlling ovulation rate in cows
• Mechanisms associated with preimplantation embryo development and quality.
• Reproductive management strategies that improve reproductive performance in dairy and beef herd management practices.
• Factors that cause pregnancy loss in cattle and development of methods to reduce losses.

 

Data were generated using intramural and extramural competitive grant funds garnered by the members (more than $23 million). From 2017-2021, the group individually and collaboratively published refereed research papers (225), conference papers (70) theses and dissertations (50), book chapters and invited reviews (19), technical/extension publications (9), and deposited sequences into the Gene Expression Omnibus (14). Workshops and lectures (128) were also presented to producers, veterinarians, and consultants locally and internationally. In addition, to dissemination of the data, the findings were used to develop programs to improve synchronization of ovulation and to overcome environmental and nutritional factors that can disrupt reproduction. The group developed new management strategies and technologies to effectively use artificial insemination (AI) and embryo transfer (ET) technologies to improve reproductive performance and thereby immediately impact producers. Indeed, veterinarians, consultants, pharmaceutical companies, breed organizations, and companies serving animal industries benefitted from the work from this project. For example, collaborators have presented annual reports on the project to cattle AI organizations (Select Sires, Genex CRI, Alta Genetics), the National Association of Animal Breeders (NAAB), the American Association of Bovine Practitioners (AABP), dairy industry associations (Minnesota Dairy Association, Vermont Dairy Association), pharmaceutical and technology companies (Zoetis, Merck, Allflex), and extension education organization (PRO-Dairy).  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. The members of NE1727 (NY, IA, MS, OH, OR, PA, KY, MA, NE, TN, VT, WI, VA, and WV) developed a course on Contemporary Topics in Reproductive Biology to improve student understanding of the breadth of reproductive physiology including topics outside their primary area of research. Ninety-seven graduate students enrolled in this course during the last project period. This accounted for a majority of the 103 students who completed or are currently pursuing a M.S. or PhD across the experiment stations. Numerous undergraduate students with an interest in reproductive physiology were also 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

Folliculogenesis: Ovarian follicles progress through dynamic and functionally distinct stages; from growth, selection and dominance to ovulation or atresia. The follicle consists of an oocyte and surrounding granulosa and theca cells that support development and maturation of the oocyte, and their functions are under endocrine and paracrine regulation. Members of the transforming growth factor β (TGFβ) superfamily of ligands activate transcription factors (SMADs) which regulate follicle development and ovulation rate. A novel high fecundity genotype (Trio) was identified in cattle and evaluated extensively as part of this project^2–4. We found that the presence of the Trio allele results in increased abundance of SMAD6 mRNA, which is an intracellular negative modulator of BMP15, in the granulosa cells of antral follicles resulting in decreased granulosa proliferation, reduced follicle growth rate, and selection of three to four dominant/ovulatory follicles instead of just one. How the Trio allele alters follicle growth during the preantral stages of folliculogenesis has not been documented. 

Within human follicles, oxygen concentration is reportedly 1.3% to 5.5% ⁵. Thus, follicle growth and granulosa proliferation occur under hypoxic conditions.  In cancer cells, high levels of O-GlcNAcylation (i.e., hyper-O-GlcNAcylation) promote a metabolic shift from aerobic, oxidative phosphorylation to anaerobic glycolysis (i.e., Warburg effect), which enables cells to continue undergoing rapid proliferation. Despite the knowledge that O-GlcNAcylation occurs in granulosa cells of follicles of different sizes, and it influences granulosa cell proliferation, the existence of and mechanisms responsible for such a switch in granulosa cell metabolism have not been extensively examined.

Steroidogenesis: Theca and granulosa cells produce steroids that coordinate with neuroendocrine hormones to regulate follicle selection and ovulation. Steroid hormones are derived from cholesterol and their synthesis is primarily regulated by pituitary hormones. However, growth factors, cytokines, and androgens/estrogens also modify steroidogenesis via paracrine and autocrine mechanisms. In addition to expression and activity of the steroidogenic enzymes, cholesterol uptake and de novo synthesis by theca cells is also an important determinant of steroid production. This is emphasized by recent studies demonstrating a regulatory loop between LH signaling and sterol regulatory-element binding protein (SREBP-2) which increases cholesterol synthesis, is necessary for optimal steroid production ^7,8. Despite these studies, there are gaps in understanding the paracrine regulation of steroidogenesis.

Oocyte Maturation and Fertilization: Oocyte quality, defined as its ability to be fertilized and support embryonic development, is a pivotal factor in female fertility. Divalent cation homeostasis is required for oocyte, influencing maturation, fertilization, and early embryonic development. For example, zinc (Zn²⁺⁾ is essential for resumption of meiosis and meiosis exit after fertilization. The Ca²⁺ oscillations of fertilization induce egg activation and initiate development. Finally, Mg²⁺ impacts Ca²⁺ oscillations, and Mg²⁺ is essential for embryo development. However, the channel(s) underlying influx of these cations remains unknown in all large domestic species. The TRPM7 locus, which mediates intracellular cation influx, is positively associated with conception rates in heifers ⁹. We hypothesize that TRPM7 not only contributes to bovine fertility, but potentially allows extracellular ion supplementation to affect intracellular ion levels that will positively impact the cellular processes under their regulation and, ultimately, improve fertility.

Corpus Luteum Function and Angiogenesis: After ovulation, the somatic cells of the follicle differentiate into luteal cells, forming a corpus luteum (CL). This structure, which produces progesterone, is essential for establishment and maintenance of pregnancy. In the absence of pregnancy, it undergoes regression and the concomitant loss of progesterone leads to initiation of a new estrous cycle. In addition to differentiation of luteal cells, formation of the CL is accompanied by extensive blood vessel growth or angiogenesis. The building of a rich vascular network requires the coordinated actions of a variety of angiogenesis-related factors. Toward this end, a functional angiotensin system is present in the bovine ovary ¹⁰ and the physiologically active angiotensin II (AngII) regulates the expression of VEGF ¹¹ in the bovine CL. In turn, VEGF and basic fibroblast growth factor upregulate Ang II and steroidogenesis in the bovine CL ¹⁰. In HEK293T cells, Ang II also increases the expression of cellular communication network factor 1 (CCN1) ¹². However, Ang II mechanisms regulating CCN1 expression in steroidogenic bovine luteal cells have not been established.

Environmental Factors Impacting Folliculogenesis and Oocyte Quality: Inherent to the efficiency of reproduction in domestic production systems is the need to overcome an array of management-based, nutritional and/or environmental stressors. Heat-stressed cows have lower circulating glucose concentrations coupled with higher concentrations of insulin ¹³ leading to poor oocyte quality. Chromium propionate improves insulin/glucose dynamics in heat-stressed cows, but it is unclear if and how it will improve oocyte quality.

The environmental contaminant, perfluoro alkylated substances (PFAS) have in vivo half-lives of several years. In humans, PFAS exposures are associated with abnormal ovarian cyclicity, decreased steroidogenesis, increased follicular atresia, and decreased corpora lutea ¹⁴. However, it is unclear how PFAS affects folliculogenesis in ruminants. Additionally, since heat stress alters circulating insulin and insulin regulates chemical metabolizing enzymes in the ovary, a potential for a synergistic effect on PFAS and heat stress exists to hamper fertility.

Sperm capacitation: Efficient fertilization is dependent not only on oocyte quality but also sperm capacitation within the female reproductive tract. Preliminary data indicates that a short incubation with the Ca²⁺ ionophore A₂₃₁₈₇ can induce in vitro fertilizing capacity in sperm from mice sterile knock-out (KO) genetic models and improve embryo development rates in sperm from wild type mice ¹⁶. Consistently, when used with bovine sperm, a temporary increase in intracellular Ca²⁺ ([Ca²⁺]_i) induces significant improvement in in vitro fertilization (IVF) rates and embryo development.

Embryo Development: Genes that are expressed in the oocyte play a key role in mediating initial stages of embryonic development. Most of our knowledge related to maternal genes required for early embryonic development is derived from gene targeting studies in mice. Our understanding of the role of specific oocyte-expressed genes in regulation of early embryogenesis is far from complete, particularly in species such as cattle where the number of cell cycles from fertilization until completion of the maternal-to-embryonic transition (embryonic genome activation) is greater than in mice ¹⁷. Through analysis of the bovine oocyte transcriptome, we have identified several novel oocyte-specific genes (both protein coding and non-coding).

Maternal Recognition of Pregnancy: During the second week of pregnancy, embryonic and extraembryonic tissues, which are essential for producing a conceptus that can signal its presence to the maternal system, are formed. If the pregnancy recognition signal is not sufficiently robust or if the uterus cannot respond correctly to this signal, pregnancy loss will occur. Differentiating between conceptus and uterine receptivity components of fertility remains a major challenge. In the event of failure of adequate signaling and response, the uterus is primed to regress the CL, and reestablish cyclicity for another opportunity to establish a pregnancy.

Uterine Environment and Conceptus-Uterine Interactions: A significant amount of pregnancy failure also occurs during the second and third weeks of gestation in cattle primarily due to insufficiencies within the uterine environment or poor paracrine communication between the conceptus and endometrium. These interactions also involve regulating immune cell function. The early embryo alters the proportions and function of resident uterine immune cells as well as affecting immune cell function in the peripheral circulation ^18,19. In other species these changes were shown to be essential for establishment of pregnancy and to support development of the placenta. However, relatively little is known about the changes in uterine immune cells and immune mediating molecules during early pregnancy in cattle.

Selection of High Fertility Cattle: Techniques in molecular genetics and genomics offer excellent possibilities for selection of high fertility cattle through identification of key genes/processes involved in optimizing fertility. Reduced genetic merit for fertility has been linked with unfavorable metabolic status, delayed resumption of ovulation, and inadequate hormone levels ^20,21. Moreover, reproductive traits have been linked with genotypes of tumor necrosis factor α ²², leptin promoter ²³, IGF-I ²⁴, growth hormone receptor ^25–27, Coenzyme Q9 (COQ9) ^28,29 and paraoxonase-1 (PON1) ³⁰. Genetic and genomic studies provide opportunities for selection of higher fertility cattle and to unravel physiological mechanisms related to genotypes and fertility.

Targeted Reproductive Management is a novel approach that consists of managing subgroups of cows that share biological features or expected performance with programs specifically designed to optimize cow performance, herd profitability, or other outcomes of interest. This approach to management leverages recent advances in high throughput genotyping and phenotyping tools such as genomic testing and automated systems to characterize predictors of cow reproductive and performance outcomes. Multiple sources of biological, management, and performance data are then combined with environmental and economic data to identify subgroups of cows that benefit by tailored reproductive programs.

AI and ET: Improved understanding of the response of cattle to hormonal interventions used in synchronization of ovulation protocols for timed AI and timed ET was accomplished under project NE-1727 and others. This has led to major gains in reproductive and farm labor efficiencies through improved service rates and fertility to AI and ET services. Despite these gains, the response to specific hormonal treatments as part of synchronization of ovulation protocols remains suboptimal ^31,32. Biological conditions observed in cows undergoing synchronization and management conditions of commercial farms which constrain use of more complex interventions hamper responses to some programs. Therefore, research is needed to develop practical mechanisms to circumvent these biological and management limitations for achieving optimal responses and efficiencies.

Project NE-1727, and its predecessors, NE-1227, NE-1027, NE-1007, NE-161, NE-72, NE-41 and NE-1, has a long track record of defining core physiological, cellular, and molecular processes that control ovarian function, oocyte quality, fertilization, embryo development, and conceptus-uterine interactions with the goal of identifying what controls pregnancy maintenance or failure in ruminants. These discoveries have then been used to develop innovative management practices to improve fertility of female ruminants. The current proposal will build upon the outcomes of NE-1727 to further our basic understanding of the physiological mechanisms contributing to ovarian function, embryo development, and conceptus-uterine interactions. It will also continue to develop innovative management strategies based on findings from the basic and applied research. Proposed studies will address the following three objectives.

Objectives

1. Identify Mechanisms that Regulate Ovarian Function and Oocyte Quality during the Estrous Cycle
   
2. Determine Factors Associated with Fertilization, Embryo Development, and Conceptus-Endometrial Interactions that Dictate Pregnancy Success
   
3. Develop and Evaluate Novel Reproductive Management Strategies and Technologies to Improve Reproductive Performance of Ruminant
   

Methods

Obj 1A: Identify basic mechanisms that coordinate oocyte, follicle, and CL function  

1A.1 Oocyte Quality and Fertilization: The TRPM7 locus was positively associated with conception rates in heifers. The hypothesis is that the TRPM7 channel mediates intracellular cation influx that regulates oocyte growth, maturation, and competence. Bovine oocytes will be in vitro matured and Ca²⁺ content, free-Zn²⁺ levels, Zn²⁺ sparks, and free Mg²⁺ levels will be monitored during different stages of development using different dyes (Fura2-Am, FluoZin3 AM and Acid, and Mag-Fura). Expression of TRPM7 in GV and MII oocytes and each stage of pre-implantation embryo development will be determined using a monoclonal antibody ^33,34.

1A.2: Folliculogenesis and Steroidogenesis: Pro-inflammatory signaling alters LH-dependent regulation of cholesterol homeostasis in theca cells resulting in increased estradiol (E2) and androstenedione (A4) levels We hypothesize that E2 and A4 regulate cholesterol homeostasis. Theca cell cultures will be treated with LH, E2 and/or A4, inflammatory factors, pathway inhibitors, and/or estrogen or androgen receptor agonists or antagonists. The importance of lipid uptake receptors, cholesterol synthesis enzymes, and lipid droplet associated proteins will be determined using siRNA transfection, qPCR, and Western blot. Secreted A4 and E2 will be measured in conditioned media. Changes in cholesterol trafficking will be determined using a combination of organelle dependent dyes and fluorescent-tagged cholesterol.

High levels of androgen activate a novel androgen receptor (ZIP9) leading to impaired granulosa cell differentiation and function ³⁵. Thus, granulosa cells will be cultured and expression and localization of ZIP9 will be determined using Western blot and IHC. Gene ablation approaches will determine the ZIP19 dependent effects of androgen on granulosa cell survival, proliferation, and differentiation. Finally, the downstream signaling molecules activated by ZIP9 in bovine granulosa cells will be identified using a combination of protein biochemistry, spectroscopy, and proteomic approaches. These experiments will determine a novel mechanism by which androgen contributes to development of anovulation and cyst formation.

The effect of the nutrient sensor O-GlcNAcylation, on granulosa cell responsiveness to hormone stimulation, metabolism and steroidogenic capacity will be examined, Granulosa cell cultures will be treated with Thiamet-G and OSMI-1 to induce O-GlcNAcylation. Estradiol (E2) and progesterone (P4) will be measured in conditioned media and RNA-seq and proteomic analysis performed using cell lysates.

To determine how hypoxic conditions augment granulosa cell growth, cells will be cultured under low oxygen tension or with a hypoxia mimetic. Changes in protein expression and cell proliferation will be determined using Western blot and MTS assays/ Ki-67 staining, respectively.

The Trio allele is a novel marker of high fecundity. We hypothesize that reduced follicle growth in cattle with the Trio allele is due to increased abundance of SMAD6 and reduced granulosa cell proliferation but not preantral follicle numbers. Ovarian tissue from Trio carrier and non-carrier heifers will be fixed and paraffin embedded. Sections will be stained with hematoxylin and eosin and follicle numbers and dimensions will be determined. Relative abundance and localization of SMAD6 and the cell proliferation marker Ki67 will be determined using immunohistochemistry.

The hypothesis is that a compensatory mechanism exists after unilateral ovariectomy (ULO) and would be evidenced by 1) increased number of antral follicles present in the retained ovary, and 2) an increase in circulating AMH. In addition, we hypothesize that the length required for the intact ovary to undergo compensation in follicular development will be slower in cows carrying the Trio allele. Antral follicle counts, serum AMH, and ovulation rate will be determined before ULO and at multiple times up to 230 days after ULO in each cow.  

1A.3 Corpus Luteum (CL) Function: The hypothesis is that angiotensin II (Ang II) regulates the expression of cellular communication network factor 1 (CCN1) and progesterone biosynthesis in steroidogenic luteal cells. The CL will be collected from dairy cows on day 4 of the estrous cycle, cells dissociated, and then treated with 10^-6 to 10^-9 M Ang I or Ang II for 0.5, 2, 6, 24 and 48 hours. Quantitative PCR will measure expression of CCN1, vascular endothelial growth factor, basic fibroblast growth factor, and the angiotensin receptors, AT1 and AT2. Immunohistochemistry will be used to localize AT1 and AT2, while P4 concentrations will be determined by radioimmunoassay.

Obj 1B: Determine production stressor effects on follicle and oocyte development.

1B.1 Heat Stress: The hypothesis is that hormonal programs used in advanced reproductive techniques (ART), extreme body condition score (BCS), and extreme temperature impact the dominant follicle which subsequently alters oocyte developmental competence. We will model these conditions and collect follicular fluid, granulosa cells, cumulus cells, and the oocyte from pre-ovulatory follicles. Steroids, transcriptome and/or metabolome profiles of mural granulosa cells, cumulus cells, or oocytes will be compared. Matured oocytes will undergo in vitro fertilization and embryo cleavage rates, blastocyst rates, blastocyst transcriptome profiles, and blastocyst metabolome profiles. We expect to unveil reduced metabolic capacity of oocytes or embryos from small pre-ovulatory follicles or low BCS, altered meiotic maturation of oocytes from high temperatures, and that all treatments will result in hormone and metabolic differences in the follicular fluid.

The hypothesis is that tempering hyperinsulinemia/hypoglycemia observed during heat stress ^13,38 with chromium propionate will improve reproductive performance. Dairy cows with clinically normal periparturient periods will be assigned to receive control ration or chromium propionate-supplemented ration. Ovarian structures will be monitored for benchmarks of follicle and CL development.  Plasma concentrations of insulin, glucose, E2 and P4 will be determined.  We expect that cows receiving chromium propionate will have greater circulating glucose and lower insulin concentrations during the summer months, and that this improved glycemic status will be associated with outcomes indicative of improved fertility, especially hastened postpartum resumption of cyclicity. 

1B.2 Micronutrients: Pilot data suggest constant exposure to vitamin A and moderate levels of vitamin D  reduce the number of unfertilized cattle oocytes during in vitro fertilization. We will determine if long-term supplementation with vitamin A and D will improve follicular function in cattle prior to breeding and shorten the window of resumption of ovarian activity post-calving. Gestating beef cattle will be injected with either vehicle control or a 45kIU vitamin D and 300 KIU vitamin A. Females will calve in late winter/early spring followed by artificial insemination 45 days later. At the same time, they will receive another vitamin premix injection at the same dosage. Pregnancy will be determined by ultrasound on GD30.  During the treatment interval and breeding protocols serum progesterone, antimullerian hormone (AMH) and vitamin D will be monitored by validated ELISAs.

Supplemental selenium (Se) is provided to cows which affects both luteal phase and gestational concentrations of P4 ¹⁵. We hypothesize that supplementation with an equimolar 1:1 blend of organic and inorganic forms of Se increases systemic production of P4 via the stimulation of cholesterol uptake by luteal cells and the developing placental interface. CL and placentomes will be retrieved on Day 45 from pregnant heifers and in vitro assays (e.g., qPCR) used to determine mechanisms by which systemic concentrations of P4 are affected by the form of Se provided. By understanding how the form of Se affects the synthesis and secretion of P4 during the establishment and maintenance of pregnancy, recommendations can be made to the producer that will improve fertility outcomes.

1B.3 Toxins: Perfluorooctanoic acid (PFOA; a PFAS member), is detected in human follicular fluid ³⁶ and phenotypically impairs fertility in exposed rats ³⁷. However, mechanisms by which PFAS impairs female fecundity remains ill-defined in livestock animals.  Pre- and post-pubertal pigs will experience either thermal neutral or heat stress conditions in a diurnal pattern, with or without PFOA. After 14 days, liver, ovaries and uteri will be collected and weighed, follicular fluid retrieved, and the ovaries fixed in 4% paraformaldehyde or flash frozen.  Ovarian protein will be isolated, and LC-MS/MS performed to identify global proteomic changes. Serum and follicular fluid will be analyzed by GC-MS to identify PFOA-dependent effects. The contralateral ovary will be sectioned and stained with hematoxylin and eosin for follicle counting. Statistical analysis to determine an additive impact of heat stress on PFOA ovarian endpoints will be conducted. 

Obj 2: Identify factors that improve early pregnancy and develop technologies and management practices to limit conceptus mortality

2.1 Gamete Contribution to Embryos: Successful embryogenesis is dependent on gamete quality. In vitro matured oocytes will be microinjected with small interfering RNA (siRNA) or in vitro transcribed mRNAs. The effect of increased and decreased levels of specific maternal mRNAs on early embryonic development will be determined using IVF and embryo culture. Specific effects on embryonic genome activation, maternal mRNA degradation, and expression of specific embryonic genes will be determined using fluorescent in situ hybridization. Epigenetic effect on the embryonic genome will be measured by immunofluorescence and differential expression of specific embryo genes will be identified using transcriptomics which will be used to identify changes in gene families and developmental pathways.

The effect of changes in [Ca²⁺]_i and metabolism on bull sperm function will be determined. Transient calcium elevation, starvation or a combination of these methods will be used to develop new assisted reproductive technologies ART in the bovine model.

2.2 Conceptus Development and Uterine Interactions: Mechanisms that sustain luteal health during the second month of gestation are critical to maintain pregnancy. We will test the hypothesis that CL maintenance during the second month of gestation is accomplished through an increase in ipsilateral utero-ovarian blood flow resulting in reduced transfer of endometrial PGF_2α to the ovarian artery, thus preventing regression of the CL

To understand key milestones of conceptus development and its interaction with the endometrium, we will: 1) investigate mechanisms of placenta trophectoderm growth and function during conceptus elongation and production of the maternal recognition of pregnancy factor interferon-tau (IFNT), 2) perform functional tests of newly discovered conceptus and/or endometrial secretory factors that likely have important functions during early pregnancy, 3) study physical interactions between the trophectoderm and uterine mucosa that lead to placentation, 4) study the yolk sac to better understand the importance of this extraembryonic tissue during early pregnancy, and 5) identify differences in endometrial gene expression between the ipsilateral compared to the contralateral uterine horn during early pregnancy as it relates to regression of the contralateral accessory CL.

We propose that the ~50% reduction in conception rates between heifers and mature lactating dairy cattle is due in part to immune dysregulation at the fetal-maternal interface. The hypothesis is that conceptus secretory proteins alter immune cell proportions and functions to promote immune tolerance and to facilitate endometrial remodeling and angiogenesis necessary for the formation of a placenta. Changes in immune cells and expression of immune mediators during early pregnancy will be measured and compared between fertile heifers and sub-fertile lactating dairy cows. Chronic uterine catheterization will be used to infuse IFNT, and pregnancy associated glycoproteins, two major conceptus secretory proteins during early pregnancy, alone and in combination to determine their role in regulating immune cell function.

2.3 Artificial Reproductive Technology (ART) In vitro embryo production (IVP) systems, somatic cell nuclear transfer (SCNT) and gene editing technologies will be used to 1) identify oviduct and uterine factors that improve IVP and SCNT embryo development, 2) discover inherent embryonic mechanisms controlling embryonic lineage specification, 3) uncover regulatory mechanisms that are critical for normal embryo development, and 4) examine how interventions occurring during IVP affect post-transfer survival of pregnancies and calf health. Correlations between pregnancy loss and circulating hormones related to IVP and SCNT cattle embryos will be made to better understand early embryo/conceptus mortality related to these ART methods.

2.4 Uterine Contribution to Conceptus Development: Reproduction in ruminants involves complex cellular processes between reproductive cells or tissues which may change over time and biological context. The development of new -omics technologies allow for the identification of physiologically relevant molecules during early reproductive processes that may cause pregnancy failure. Transcriptomic, proteomic and metabolomic approaches will be used to characterize conceptus induced uterine histotroph necessary to support development of the early cow conceptus. The same techniques will test the hypothesis that IVP bovine embryos alter the endometrial transcriptome and uterine histotroph, resulting in a suboptimal uterine environment for pregnancy.  

Obj 3: Identify and develop novel hormonal, genomic, and biomarker-based reproductive management strategies and technologies that maximize reproductive performance and farm profitability through improved fertility, service rates, and labor efficiencies of AI and ET programs for cattle.

3.1 Synchronization Protocols: The first objective is to optimize the response to synchronization of ovulation protocols will increase pregnancy per AI (P/AI) and ET (P/ET). We will develop and test different strategies to improve ovulatory response to initial GnRH treatment during ovulation synchronization protocols for timed artificial insemination (TAI). We will determine the effect of GnRH dose and pre-synchronization using PGF2α (PGF) or PGF and P4 before initiation of a CO-Synch protocol on estrus expression and fertility to TAI. Ultrasonography and hormone profiles will be used to evaluate ovulation, follicular wave dynamics and the hormonal environment before and after AI.

The second objective is to design reproductive management programs that optimize the response to TAI and embryo transfer (TET) protocols to reduce the interbreeding interval. The impetus is to reduce interbreeding intervals and develop methods for earlier pregnancy diagnosis. First, we will evaluate the effect of oxytocin and low dose hCG treatment of non-bred cows on timing of luteolysis. We will subsequently use cows that have previously received TAI. A third experiment will test the overall fertility and reproductive efficiency of dairy cows upon use of ReBreed21. This approach will be tested at multiple experiment stations.

A third objective is to reduce interbreeding interval and develop methods for an earlier pregnancy diagnosis in ET recipients using ReBreed21. This program was previously developed and tested in beef heifers ³⁹. Heifers will be synchronized to receive ET by the traditional method (ET/35 d) or with the ReET21 method (ET/21 d). Fertility and reproductive efficiency from using ReET21 and a traditional Resynch program will be compared. If promising, this ReET21 will be tested at other stations.

A fourth objective is to optimize hormonal responses during a 5-day fixed time ET ovulation synchronization protocol and determine risk factors affecting fertility in dairy cows. We will determine: 1) the effect of GnRH doses on ovulatory response, estrus expression and fertility to ET; 2) the effect of PGF dose and treatment number on luteolysis, estrus expression and fertility to ET; and 3) identify recipient management factors associated with greater fertility and reduced pregnancy loss. We will use ultrasonography and P4 and E2 profiles to evaluate ovarian response.

3.2 Genetic Determinants of Fertility: 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. Thus far, blood samples for DNA genotyping and fertility phenotype data have been collected from 1,060 lactating dairy cows across 7 research stations. Genotypes for GHR, TNFα, IGF-1, COQ9, and PON1, and other related genes will be determined as well as their association with fertility to first AI and pregnancy rates.

Identification of protein biomarkers is hampered due to a lack of tools to deplete high abundant proteins in biological fluids to improve discovery of low abundant proteins, we will develop species-specific reagents to efficiently deplete high abundance proteins in bovine plasma/serum. This strategy will be deployed concurrent with data-independent acquisition proteomics to remove bias for high abundant proteins. This method will enable identification of biomarkers, including low abundant proteins, that predict pregnancy status and detect early embryonic mortality earlier after AI.

3.3 Nutritional Effects on Fertility: Perinatal nutritional environment modulates developmental organization of hypothalamic neurocircuits, which alters programming of the reproductive neuroendocrine axis ^40,41. Pregnant cows will be assigned to low, moderate, or high energy diets to achieve different target BCS. After calving, heifer offspring will be weaned and assigned to a high or low-concentrate diets. At 8 mo. of age all heifers will transition to a common diet until 24 mo. of age and estrus will be synchronized. Heifers detected in estrus will be examined with ultrasonography to characterize the follicular wave dynamics, count antral follicles, and determine timing of ovulation. The effects of peri- and post-natal nutrition on E2/P4 profiles, estrous cyclicity, and pregnancy per AI will be determined. Ultimately, nutritional management recommendations for commercial beef operations will be developed.

3.4 Targeted Reproductive Management (TRM): The objective is to identify predictors for use in targeted reproductive management (TRM) of dairy cattle. Using data collected from cows (n=5,000+) we will determine associations between reproductive and herd outcomes and a) genomic traits; b) behavioral and physiological parameters collected by sensors (e.g., activity, rumination, body temperature, BW, BCS); and c) cow features and historical data (e.g., reproductive and health history). Measures with the greatest predictive value for outcomes of interest will be used to create algorithms with traditional statistical techniques and machine learning to create subgroups of cows for TRM.

Once predictive values have been identified, we will develop a suite of reproductive management strategies for dairy heifers and cows to evaluate the benefits of using TRM. Randomized controlled experiments will be conducted and reproductive performance, herd management practices, and farm profitability measured. The goal is to design TRM programs to optimize AI and targeted hormonal therapy that will increase fertility, optimize the value of offspring (e.g., use of sexed semen), and optimize timing of pregnancy during lactation.

Measurement of Progress and Results

Outputs

• Novel data will be generated and new techniques will be shared amongst experiment stations and with colleagues outside of the multi-state project. Comments: Large datasets from -omics-focused studies will be shared on open access servers. Outcomes of basic research will be used to develop applied studies. Through the latter venues, stakeholder input for further work and reactions to work in progress will be obtained. 
• An additional output is the continued training of undergraduate and graduate students. Comments: Faculty at the extension stations will continue team-teaching the previously developed Contemporary Topics in Reproductive Biology. Graduate student training and introduction of undergraduates to research will continue with opportunities to work collaboratively in the group.

Outcomes or Projected Impacts

• Increased technologies and management strategies to mitigate pregnancy loss and improve animal fertility in the face of climate change. This proposal aligns with Strategic Goals 2 and 7 of the USDA Strategic Plan (2018-2022) which are to “maximize the ability of American agricultural producers to prosper by feeding and clothing the world” and “to provide all Americans access to a safe, nutritious, and secure food supply”. Collectively, outcomes from these studies will close important gaps in knowledge regarding follicle growth and estrous cyclicity, oocyte quality, early embryonic development, conceptus-maternal signaling, conceptus implantation and maintenance of pregnancy in cattle. The complex molecular environments that govern these processes will also be characterized using new omics and single cell technologies. Importantly, the impact of assisted reproductive technologies such as IVP and SCNT on embryo quality will also be elucidated with the goal of bettering these technologies for advancement in food production. Collectively, these outcomes will lead to technologies and/or management strategies that mitigate embryonic/conceptus mortality, and increase pregnancy success. The work proposed also has the potential to lead to housing, management, and nutritional approaches to improve fertility in the face of climate change. In the long-term, continued research by this multi-state group will result in the generation of important new information to enhance the efficiency of reproductive performance in ruminant species which will contribute to meet the growing world-wide demands for animal protein.

Milestones

(2022):All studies will be initiated including tissue collections and design of cross-station animal studies. Raw data will be collected from -omics studies. New techniques will be piloted.

(2024):Analyze and interpret on omics data. New techniques will be shared. Outcomes of studies in 2022-2023 will be disseminated as described in the Outreach Plan.

(2027):Validate -omics data to establish reliable biomarkers of reproductive traits. Interpret the output of basic studies in order to develop new application-based hypotheses. Determine the efficacy of new management programs. Continue to disseminate data as described in the Outreach plan.

Projected Participation

View Appendix E: Participation

Outreach Plan

The basic research described herein will be disseminated in annual reports, peer-reviewed journals, at scientific meetings by faculty and students, and on websites of the experiment stations involved.

Outcomes from applied studies will be presented to each experiment station advisory board and local extension personnel and commodity groups in short courses and at field days. Some extension specialists and agents will aid in collecting applied data and selecting cooperating farms. Research workers will prepare news releases or participate in interviews. 

Members of the group will continue private-public partnerships with cattle AI organizations (Select Sires, Genex CRI, Alta Genetics), the National Association of Animal Breeders (NAAB), the American Association of Bovine Practitioners (AABP), dairy industry associations (Minnesota Dairy Association, Vermont Dairy Association), pharmaceutical and technology companies (Zoetis, Merck, Allflex), and extension education organization (PRO-Dairy).  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.

Organization/Governance

A Technical Committee will be organized with voting membership including at least one representative from each cooperating Agricultural Experiment Station as appointed by the respective Director. Non-voting members shall consist of the Administrative Advisor and a USDA consulting member. All voting members of the Technical Committee are eligible for office. A chairperson, a secretary, and a director 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. 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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Attachments

Land Grant Participating States/Institutions

CT, FL, IA, KY, LA, MA, MD, MO, NE, NH, NY, OH, OR, PA, RI, TN, TX, VA, WI, WV

Non Land Grant Participating States/Institutions